Neuromodulation of Olfactory Learning by Serotonergic Signaling at Glomerular Synapses Reveals a Peripheral Sensory Gating Mech
Neuromodulation of Olfactory Learning by Serotonergic Signaling at Glomerular
Synapses Reveals a Peripheral Sensory Gating Mechanism
by
Monica Li
A Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy
Approved September 2012 by the Graduate Supervisory Committee:
William J. Tyler, Co-Chair Brian H. Smith, Co-Chair Carsten Duch Janet Neisewander Eric Vu
ARIZONA STATE UNIVERSITY
December 2012 ABSTRACT
Sensory gating is a process by which the nervous system preferentially admits stimuli that are important for the organism while filtering out those that may be meaningless. An optimal sensory gate cannot be static or inflexible, but rather plastic and informed by past experiences. Learning enables sensory gates to recognize stimuli that are emotionally salient and potentially predictive of positive or negative outcomes essential to survival. Olfaction is the only sensory modality in mammals where sensory inputs bypass conventional thalamic gating before entering higher emotional or cognitive brain regions. Thus, olfactory bulb circuits may have a heavier burden of sensory gating compared to other primary sensory circuits. How do the primary synapses in an olfactory system "learn"' in order to optimally gate or filter sensory stimuli? I hypothesize that centrifugal neuromodulator serotonin serves as a signaling mechanism by which primary olfactory circuits can experience learning informed sensory gating. To test my hypothesis, I conditioned genetically-modified mice using reward or fear olfactory-cued learning paradigms and used pharmacological, electrophysiological, immunohistochemical, and optical imaging approaches to assay changes in serotonin signaling or functional changes in primary olfactory circuits. My results indicate serotonin is a key mediator in the acquisition of olfactory fear memories through the activation of its type 2A receptors in the olfactory bulb. Functionally within the first synaptic relay of olfactory glomeruli, serotonin type 2A receptor activation decreases excitatory glutamatergic drive of olfactory sensory neurons through both presynaptic and postsynaptic mechanisms. I propose that serotonergic signaling decreases excitatory drive, thereby disconnecting olfactory sensory neurons from odor responses once
i information is learned and its behavioral significance is consolidated. I found that learning induced chronic changes in the density of serotonin fibers and receptors, which persisted in glomeruli encoding the conditioning odor. Such persistent changes could represent a sensory gate stabilized by memory. I hypothesize this ensures that the glomerulus encoding meaningful odors are much more sensitive to future serotonin signaling as such arousal cues arrive from centrifugal pathways originating in the dorsal raphe nucleus. The results advocate that a simple associative memory trace can be formed at primary sensory synapses to facilitate optimal sensory gating in mammalian olfaction.
ii DEDICATION
I would like to dedicate this dissertation to my family, including my parents
Shufang and Kefei Li, who worked tirelessly to give me the best opportunities in life and instilled in me a work ethic to face challenges without backing down. I would also like to dedicate this to my husband, Dox Alim who gave me the support and courage to conquer the difficulties encountered in life.
I would like to dedicate this work to all my teachers, who instilled in me a sense of curiosity for the great unknown and taught me the rewards of learning. I especially am grateful for those mentors and colleagues who have helped me discover the field of neuroscience and taught me how to love this science by doing. Their selflessness inspires me to return their kindness one day in any way
I can, by helping others through scientific and personal acts of kindness.
iii ACKNOWLEDGMENTS
Foremost, I would like to thank my graduate mentor Dr. William J. Tyler.
His excitement for science is contagious and inspired me to immerse myself in study of neuroscience. Leading by example, he has taught me endless lessons on how to do science with passion, care, thoughtfulness and most of all, integrity.
I am deeply indebted to him for believing in me as a novice in the biological sciences and for giving me the opportunity to learn and grow intellectually in his laboratory. Dr. Tyler is more than anyone could hope for in a mentor. He provided the most patient guidance and unwavering support through each step of pursuing my Ph.D.
I would like to thank my doctoral committee: Dr. Brian Smith, Dr. Cartsen
Duch, Dr. Janet Neisewander and Dr. Eric Vu. They have been excellent teachers who have had profound influence on my intellectual growth as an aspiring scientist throughout my graduate school years. First, they have patiently taught me in the classroom, exposing me to fundamental principles in my field and creative ways think about science. They have provided thoughtful input and advice during the development of my thesis project, offered valuable feedback and support near the culmination of my thesis project, as well as during my search for future scientific training.
I also like to thank all the former members of Tyler Lab: Yusuf Tufail, my closest scientific comrade and trustworthy friend, who always gave me encouragement and sound advice. I thank Alex Matyushov, Joey Georges, Anna
Yoshihiro for their help and support as well as Josh Nichols, Zach Gilbert and
Karina Cruz for their help with animal behavior for my thesis project. Finally, I thank Liliana Rincon Gonzales for her advice and faith especially near the end of
iv completing my thesis. Additionally, I thank Veronica Clavijo Jordan and Danielle
Protas for their friendship and support.
v
TABLE OF CONTENTS
Page
LIST OF FIGURES ...... ix
CHAPTER
1 INTRODUCTION: LEARNING AND MEMORY OPTIMIZES
PERIPHERAL SENSORY GATING ...... 1
A peripheral context-dependent sensory gate in olfaction ...... 2
Neuromodulators in learning and memory and sensory gating ...... 7
Olfactory bulb learning and memory: role of neuromodulators ...... 12
Experience-dependent plasticity at the glomerulus…...………….14
Hypothesis: learning-dependent plasticity informs a sensory
gate…………………………………………………………………….18
Overview of Chapters ……………………………………………….18
References……………………………………………………………21
2 ROLE OF NEUROMODULATORS ON LEARNING-DEPENDDENT
PLASTICITY IN GLOMERULI OF RODENTS VERSUS INSECTS
Introduction ...... 31
Insects versus rodents: anatomy and function of the olfactory
periphery ...... 33
Global changes in olfactory experience and plasticity ...... 37
Neuromodulators play key roles in central olfactory circuits ...... 39
Neuromodulators are involved in learning and memory at the
glomerulus ...... 44
Associative learning induced plasticity in antennal lobe versus vi
olfactory bulb………………………………………………………….52
Conclusion: common themes and translation ...... 54
References ...... 58
3 A MODEL TO STUDY LEARNING-MEDIATED PLATICITY AND
SENSORY GATING IN THE MAMMILIAN OLFACTORY SYSTEM
Introduction ...... 71
Methods ...... 76
Results ...... 83
Discussion ...... 91
References ...... 99
4 SEROTONIN MEDIATES OLFACTORY LEARNING PARTIALLY BY
MODULATING EXPRESSION OF 5HT2A RECEPTORS IN
GLOMERULI
Introduction ...... 105
Methods ...... 109
Results ...... 114
Discussion ...... 123
References ...... 137
5 5HT2A RECEPTOR ACTIVATION MODULATES EXCITATORY
GLOMERULAR SYNAPSES
Introduction ...... 146
Methods ...... 148 vii Results ...... 151
Discussion ...... 164
References ...... 173
6 DISCUSSION ...... 176
REFERENCES ...... 190
APPENDIX
A INSTITUTIONAL APPROVAL FOR THE HUMANE USE OF
ANIMALS ...... 216
viii LIST OF FIGURES
Figure Page
1. Olfactory bulb and thalamus represent similar stages in sensory
processing ...... 4
2. Summary of studies on experience-dependent plasticity in the
glomerulus ...... 16
3. The M72-GFP glomerulus is located on the dorsal lateral surface of
the olfactory bulb and varies in morphology across individuals ...... 84
4. The M72-GFP glomerulus is activated by acetophenone,
benzaldehyde and ethyl tiglate ...... 87
5. Mice successfully learn to associate M72 activating odorant
acetophenone with aversive or appetitive unconditioned stimuli .... 89
6. Serotonin, 5HT2AR and 5HT2CR are differentially located in the
olfactory bulb and co-localize differently with GABAergic
interneurons ...... 115
7. Pharmacological blockade of 5-HT2A receptors but not 5-HT2C
receptors attenuates fear conditioned freezing behavior ...... 117
8. Fear associative learning increases density of serotonin puncta and
5HT2AR in the M72 versus control glomerulus ...... 121
9. Reward associative learning increases density of 5HT2AR in the M72
versus control glomerulus ...... 122
10. 5-HT2AR agonist decreases the amplitude of OSN-evoked EPSCs in
both ET cells ...... 153
11. 5HT2AR activation decreases probability of OSN neurotransmitter
release ...... 155
ix 12. 5HT2AR agonist TCB-2 decreases mEPSC amplitude in ET cells and
decreases frequency in PG and ET cells ...... 156
13. 5-HT2AR activation decreases sEPSC amplitude and frequency in ET
cells but does not affect PG cells ...... 159
14. 5-HT2AR decreases the effects of bursting on juxtaglomerular
cells ...... 161
15. Fear associative learning increases excitatory drive in M72-GFP
glomerulus versus control glomeruli ...... 163
16. Proposed roles of serotonergic elements within the glomerular
circuit during learning ...... 185
17. Model: Learning dependent amplitude modulation of excitation and
inhibition in an ON-OFF circuit optimizes sensory gating ...... 188
x Chapter 1
INTRODUCTION: LEARNING AND MEMORY OPTIMIZES PERIPHERAL
SENSORY GATING
Sensory gating is a process by which the nervous system filters out the deluge of information coming from the environment. It is thought to preferentially permit only important information, which is necessary for the animal to function and respond in a behaviorally appropriate manner. As such, sensory gating should be informed by experience and optimized by learning during the lifetime of the organism. In certain kinds of experience, such as learning, the nervous system assigns meanings to cues that were once novel and meaningless.
Circuits responsible for sensory gating learn to tune out unimportant stimuli while placing emphasis on stimuli that are associated with emotionally arousing or salient events. Abnormal sensory gating may be harmful to the organism’s survival. For example, sensory gating that is too easily perturbed may disturb faithful representation of reality. Alternately, a rigid sensory gate that lacks plasticity may prevent an organism from learning associations within an environment. To strike an optimal balance of plasticity is a challenging task for peripheral sensory systems.
In this study, I argue that the optimization of sensory gating requires 1) learning-dependent plasticity, as well as 2) memory formation at incoming sensory synapses. I hypothesize that neuromodulators mediate learning- dependent plasticity, as well as memory formation that informs and optimizes peripheral sensory gating mechanisms. Using the olfactory system as a model, I first provide analyses of existing literature and then present results from a study
1 investigating the role serotonergic neuromodulation in olfactory learning and memory, which I hypothesize informs and optimizes sensory gating at primary glomerular synapses.
A peripheral context dependent sensory gate in olfaction
Olfaction bypasses thalamic gating but implements its own peripheral gating mechanisms
Traditionally, the thalamus is considered to be the master gate of all sensory information. All sensory modalities except olfaction obligatorily pass through the thalamus on their way to higher processing in the cortex. Afferents that carry visual, auditory and somatosensory information congregate in modality specific nuclei in the thalamus and synapse onto relay neurons to the cortex.
Within each thalamic nucleus, cortical feedback, brainstem neuromodulators, as well as intrinsic inhibition provide three different avenues for gating to occur.
These three types of inputs modulate excitatory and inhibitory synapses onto the principal synapse as well as each other (McCormick and Bal 1994, Sherman and
Guillery 2004). With many more modulatory synapses than relay synapses
(Sherman and Koch 1986), it is apparent that the main role of the thalamus is to convey selective attention by amplifying some types information but muting others and thus prevent information overload. The thalamus is the earliest stage at which sensory information interacts with feedback processing from higher centers such as cortex conferring attention or the limbic system conferring emotion. For this reason, some philosophical arguments point to the thalamus as the first place where one becomes consciously and emotionally aware of sensory stimuli, in other words, where sensation becomes perception (Pinault 2004,
Shepherd 2005).In the olfactory system, however, the majority of sensory 2 afferents bypass the thalamus and directly innervate what are referred to the olfactory primary association cortices (anterior olfactory nucleus (AON) and piriform cortex (PF) respectively; (Shepherd 1972, Shepherd, Chen et al. 2004).
The questions then arise: 1) where does sensory gating occur for olfaction? 2)
Where does conscious or emotional perception of odors first occur? The more specific question that is central to my study, however, is: How can learning- dependent sensory gating occur at primary olfactory synapses?
Parallels between olfactory bulb and thalamus: OB as its own sensory gate
Both olfaction and thalamic scholars have proposed the theory that the olfactory bulb is its own thalamus (Shepherd 2005, Kay and Sherman 2007). A detailed comparison of thalamus versus olfactory bulb (OB) circuitry and function reveals striking likeliness and suggest that the two structures may actually be analogous stages in information processing (Shepherd, Chen et al. 2004, Sherman and
Guillery 2004) (Figure 1).
First, the thalamic nuclei corresponding to every sensory modality are organized into glomerular units that are functionally isolated and consist of an afferent driver neuron, a relay neuron and associated interneurons and neuromodulator inputs. Elements within glomerulus do not have myelin separation and thus are co-excitable while the entire glomerulus is sheathed by myelin to isolate it’s activity as functional unit (Sherman and Guillery 2004). The same concept holds true in the olfactory glomeruli where olfactory sensory neurons (OSNs) of the same glomerulus share a myelin sheath and all excite the same set of juxtaglomerular
3
Figure 1. Olfactory bulb and thalamus represent similar stages in sensory processing. Each sensory modality processes different stages of processing. Olfactory information is unique in that it bypasses the thalamus and it does not have extra steps of processing before sensory gating occurs. Both the olfactory bulb and thalamus are the first relay stations equipped with sensory gating mechanisms. These inputs include brainstem/basal forebrain neuromodulators to convey context, cortical feedback as well as strong local inhibition (in OB this occurs at two stations: glomerular station and granule cell station). (OSN) Olfactory sensory neurons, JG (juxtaglomerular includes PG periglomerular and ET external tufted cells), MC (mitral cell), LGN (lateral geniculate nucleus of the thalamus), MGN (medial geniculate nucleus, SON(superior livery nucleus), IC (inferior calculus), VP (ventral posterior nucleus), RF (reticular formation/thalamic reticular nucleus), VTA(ventral tegmental), Ach (acetylcholine), NE (norepinephrine), (5-HT) serotonin, DA (dopamine) (in some species). Figure design and content inspired by Kay and Sherman (2006).
4 and principal output cells (Valverde and Lopez-Mascaraque 1991, Bokil, Laaris et al. 2001). The excitatory and inhibitory elements of the glomerulus not only form strong dendroid-dendritic synapses onto each other but participate in cross activation caused by spill-over GABA and glutamate (Yokoi, Mori et al. 1995,
Murphy, Darcy et al. 2005).
Second, both OB and thalamic relay stations are the first ascending stations to receive feedback from cortical areas as well as from centrifugal neuromodulators. In the thalamus, this includes feedback from sensory modality specific cortical areas onto its own thalamic nucleus and neuromodulators from brainstem (serotonin, norepinephrine) basal forebrain (acetylcholine) and, in some species, midbrain (dopamine) (Fitzpatrick, Diamond et al. 1989, Matsutani and Yamamoto 2008). Similarly, the OB also receives direct cortical feedback inputs from olfactory cortical areas: the anterior olfactory nucleus (AON) and piriform cortex (PF) (Brunjes, Illig et al. 2005, Matsutani and Yamamoto 2008).
OB also has the same sources of centrifugal modulators as the does the thalamus. In both relay stations, these cortical and centrifugal inputs act on both principal relay cells as well as GABAergic interneurons.
Third, the thalamus creates spontaneous oscillations which has a frequency correlated with the arousal state of the animal (von Krosigk, Bal et al.
1993). The OB also has intrinsic oscillations that are state dependent on arousal and olfactory activity (Neville and Haberly 2003, Lagier, Panzanelli et al. 2007).
Oscillations in the thalamus emerge from intrinsic properties of the reticular formation (RF) neurons, which provide strong, phasic GABAergic inhibition to the thalamus. In contrast, slow oscillations within the olfactory glomeruli are largely provided by the external tufted cells (Hayar, Karnup et al. 2004, Hayar, Karnup et
5 al. 2004), while faster gamma oscillations are mediated by granule cell-mitral cell synapses in deeper layers of the olfactory bulb (Lagier, Panzanelli et al. 2007),
Both types of oscillations synchronize glomerular activity and powerfully affect mitral, principal output cell activity. It appears that state-dependent intrinsic oscillations may be a mechanism for sensory gating in both the thalamus and
OB.
Neuromodulators control the oscillations within the thalamus, and thus provides mechanisms for arousal or state dependent sensory gating (McCormick and Pape 1990, McCormick, Pape et al. 1991). Could the same neuromodulators control oscillations in peripheral olfactory systems to sculpt mediate early sensory gating? Thalamic neuromodulators often alter the activity of both inhibitory and excitatory elements of the circuit at once, for example to induce bursting modes or tonic firing modes in the thalamus. In the cat lateral geniculate nucleus (LGN), acetylcholine (ACh) excites M1 receptors in thalamic relay cells but M2 receptors in GABAergic interneurons in the RF each contributing to the common goal of increasing feed forward information flow (McCormick, Pape et al. 1991). These types of mechanisms could also be used by centrifugal neuromodulators in the olfactory bulb for the same purpose (Kiselycznyk, Zhang et al. 2006, Matsutani and Yamamoto 2008)?
From this perspective, the OB acts more like a thalamus than a primary relay station, and may even have some functions of primary cortex. Meanwhile, higher olfactory processing centers such as piriform cortex are more a kin to association cortex or hippocampus for other modalities since they serve roles to integrate inputs from multiple modalities (Haberly 2001). The shared characteristics between the OB and thalamus support the hypothesis about the
6 presence of sensory gating in the olfactory periphery making a hypothesis about sensory gating at the olfactory periphery.
Neuromodulators in learning and memory and sensory gating
Classical models of sensory gating use neuromodulators
Neuromodulators have been found to be essential to the process of sensory gating, as evidenced in several modalities. In the thalamus and reticular formation (RF), these classical neuromodulators modulate feed-forward, feed- back and local inhibitory synapses to alter the thorough-put of information.
Acetylcholine was the classically studied modulator in the cat lateral geniculate nucleus and it has been found to act on thalamic neurons via several mechanisms. First it induces a hyperpolarizing current in the GABAergic neurons of the reticular formation (RF) to aid the transition from bursting mode (drowsy) to single spike mode (awake) (McCormick and Prince 1986). Second, Acetylcholine has dual actions of exciting relay cells while inhibiting inhibitory interneurons via different receptor subtypes to achieve a greater strength of feed-forward throughput of feed forward information as mentioned before(McCormick and
Pape 1988).
The role of dopamine (DA) in thalamic gating is probably the least studied of the neuromodulators. Most species have dopaminergic innervation to the thalamus, although intra-species variation of dopamine fiber density in the thalamus varies. While rodents have very weak presence of DA fibers in the thalamus, strong innervation is found in primates, including humans (Sanchez-
Gonzalez, Garcia-Cabezas et al. 2005, Garcia-Cabezas, Rico et al. 2007,
Garcia-Cabezas, Martinez-Sanchez et al. 2009). Dopamine receptor subtypes:
7 D1 and D2, D4 receptors are found both on relay neurons and RF interneurons in several nuclei. D4 may modulate the GABAergic tone from the Globus Pallidus into the thalamus (Govindaiah, Wang et al. 2010). PET scan studies have shown that release and binding of dopamine is correlated with tasks involving spatial attention in humans (Christian, Lehrer et al. 2006). Meanwhile schizophrenic patients may have less D2/3 binding in that thalamus (Buchsbaum, Christian et al.
2006).
Norepinephrine (NE) and Serotonin (5-HT) have been reported to act in central sensory gating by altering busting properties of RF neurons. For example, NE and 5-HT both induce hyperpolarization activated Na+/K+ current and stops a K+ leak current, possibly mediated by cAMP. This prevents bursting mode and induces single spike model to allow more truthful relay of information during awake states (Pape and McCormick 1989). The actions of NE are modulated by α1 and β1 adrenergic receptors.(McCormick, Pape et al. 1991), while 5-HT’s actions are mainly mediated by 5-HT2 family receptors with a small involvement of 5-HT1 receptors (McCormick and Wang 1991). Using multiple receptor subtypes, 5-HT increases both feed-forward excitation and feed forward inhibition at once in the thalamus.
From a systems level perspective NE and 5-HTare known to act the gating of visual and auditory information in the respective thalamic nuclei. The two modulators have differential effects on several measures of sensory gating such as: receptive fields, signal to noise ratio, sensitivity to weak or peri- threshold stimulus, and sensitivity to signal saturation. For an comprehensive review see Hurley et al. 2004 (Hurley, Devilbiss et al. 2004). Overall, NE tends to increase signal to noise ratio (which could be beneficial ffor event detection but
8 not accurate relay of information), narrow receptive fields, sensitivity to peri- threshold stimulus and at times decrease in responses to avoid saturation.
Meanwhile 5-HT will also sharpen receptive fields, but decrease signal to noise ratio in such a way that would allow for more linear representations of information.
Serotonin has also been well studied as the main modulator involved in sensory gating deficits attributed to positive symptoms of schizophrenia and drugs of hallucination. An excellent review is provided by Geyer et al. (Geyer and
Braff 1987). Serotonergic signaling via 5-HT2 family receptors are involved Pre-
Pulse Inhibition (PPI), a standard test for normal sensory gating in both human and rodent models (Geyer and Braff 1987). Schizophrenic patients show the same trend of disturbed PPI as those suffering from overstimulation of 5-HT2A receptors (Geyer and Braff 1982). A handful of studies support this theory using various models of schizophrenia including actual schizophrenic patients as well as hallucinogenic drugs. These studies implicate 5-HT3, 5-HT5, 5-HT7 receptors in sensory gating deficits, but accumulate the most evidence for 5-HT2A receptors
(Padich, McCloskey et al. 1996, Adler, Cawthra et al. 2005, Galici, Boggs et al.
2008, Mitchell and Neumaier 2008). The specific efficacy of of 5-HT2AR agonizing drugs but not close sibling 5-HT2CR agonists in altering sensory gating properties shows the role of this receptor (Varty, Bakshi et al. 1999). Genetic linkages studies show that polymorphisms in 5-HT2A can be linked to schizophrenia and its related phenotypes (Saiz, Garcia-Portilla et al. 2007, Quednow, Kuhn et al.
2008, Brauer, Strobel et al. 2009). The conclusiveness of these polymorphism studies is reviewed critically, however (Abdolmaleky, Faraone et al. 2004). In terms of therapeutics, 5-HT2AR antagonist co-administration with antipsychotics
9 is useful for providing relieve to positive symptoms of schizophrenia
(Akhondzadeh, Malek-Hosseini et al. 2008). Overall, the involvement of 5-HT signaling, especially via 5-HT2 family receptors, in sensory gating and schizophrenia provides even more of a reason to choose 5-HT as the focus of this study’s investigations.
Classical mechanisms of learning and memory inform my hypothesis
Mechanisms of implicit and explicit memory may reside in different circuits, but have common synaptic and intracellular mechanisms. For example, the well- studied plasticity that contributes to hippocampal spatial learning, involves coincidence of firing between pre and post-synaptic neurons, NMDAR activation, insertion of AMPARs, calcium influx, which even in the short term leads to potentiation of the learning synapse (Bliss and Lomo 1973, Lynch, Larson et al.
1983, Kauer, Malenka et al. 1988, Nicoll, Kauer et al. 1988, Bliss and
Collingridge 1993, Shi, Hayashi et al. 1999, Nicoll, Tomita et al. 2006) (Lynch,
Larson et al. 1983). Downstream signaling cascades include involvement of PKA, cAMP, CamKII, CRE, CREB and post learning changes in gene expression and protein synthesis for memory consolidation (Silva, Paylor et al. 1992,
Bourtchuladze, Frenguelli et al. 1994, Abel, Nguyen et al. 1997). Similarly, the
Aplysia models of implicit learning use the same mechanisms and pathways
(Brunelli, Castellucci et al. 1976, Glanzman, Mackey et al. 1989). Long term plasticity encoding more permanent memories often require changes in external morphology of the presynaptic and post synaptic compartments (Bailey and
Chen 1988).
10 Focus on implicit learning and memory and mechanisms
Implicit types of learning are unique in that they all carry an emotional component in the unconditioned stimulus and involve neuromodulators as a key mechanism. Associative learning is a process by which a neutral stimulus or unconditioned stimulus (CS) is paired with a naturally rewarding or aversive stimuli or unconditioned stimulus (US). The inherently emotional or motivational component of the learning is carried via the US (Pavlov and Anrep 1927). The biochemical signal for the US in simple models are often neuromodulators conferring arousal. For example, Aplysia uses 5-HT to encode for the arousal of the noxious shock US in order to facilitate the motor response of a protective gill- withdrawal reflex. C. Elegans use serotonin to confer the postivie arousal from food reward or negative arousal of noxious chemicals during associative learning
(Sawin, Ranganathan et al. 2000, Zhang, Lu et al. 2005, Ha, Hendricks et al.
2010). Insects use Dopamine (DA) and Octopamine (OA) to convey reward or punishment signals of the unconditioned stimulus during associative learning
(Schwaerzel, Monastirioti et al. 2003). Rodent models of associative fear learning require NE signaling in the basolateral amygdala (BLA) to potentiate sensory inputs carrying the unconditioned stimulus (LeDoux 2000, Johansen, Cain et al.
2011). Rodent reward learning requires modulation of DA synapses in the ventral tegmental area (VTA) by BLA integrator neurons (Ambroggi, Ishikawa et al.
2008, Tye, Stuber et al. 2008, Tsai, Zhang et al. 2009, Stuber, Sparta et al.
2011). Across species, I find common mechanisms: neuromodulators biochemically carry the US signal or help facilitate the integration of information of the US and CS signal to induce plasticity in the learning circuit.
11 Olfactory bulb learning and memory: role of neuromodulators
Centrifugal neuromodulators have been found to play a crucial role in learning and memory in the olfactory bulb (Kiselycznyk, Zhang et al. 2006). A full review of the role neuromodulators in both insect and rodent olfactory learning and memory is provided in Chapter 2. The mammalian olfactory bulb is enervated with all classes of major neuromodulators, though variations may exist between species (Gomez, Brinon et al. 2005, Matsutani and Yamamoto 2008). In rodents, ACh from the horizontal diagonal band of brocha (Kasa, Hlavati et al.
1995), NE from the locus coeruleus and 5-HT from the dorsal and median raphe preferentially enervate different layers of the bulb where they act through multiple receptor subtypes (Moore, Halaris et al. 1978, Pazos, Cortes et al. 1985,
Pompeiano, Palacios et al. 1992, Pompeiano, Palacios et al. 1994).
ACh has been found to be imperative to learning involving spontaneous discrimination between two similar mixtures of odors. When both muscarinic and nicotinic cholinergic receptors (mAChRs and nAChRs) are blocked, rodents can no longer make fine odor discriminations because their mitral cell receptive fields are widened. In contrast, enhancing cholinergic signaling sharpens the receptive field of mitral cells and improves discrimination abilities (Mandairon, Ferretti et al.
2006, Chaudhury, Escanilla et al. 2009). Broad activation of the OB with odor enrichment or NMDAR agonists can achieve the same effect as ACh for increasing odor discrimination abilities (Mandairon, Stack et al. 2006).
NE has received the most attention in the mammalian olfaction literature since it has been found to be involved in obligatorily in neonate imprinting. NE acts on granule-mitral cell synapses to increase the ratio of inhibitory versus excitatory inputs on mitral cells, the main relay cells of the olfactory bulb. NE acts
12 on β1 receptors on granule cells to increase their inhibition of mitral cells via dendro-dendritic synapses (Wilson, Guthrie et al. 1990). NE from the locus coeruleus (LC) is necessary and sufficient as the arousal signal of the unconditioned stimulus for neonate imprinting (Wilson and Leon 1988, Sullivan,
Wilson et al. 1989, Sullivan, Zyzak et al. 1992, Sullivan, Wilson et al. 1994).
Intracellular mechanisms for this plasticity have been found to involve PKA, cAMP and pCREB, typical of other learning and memory models (McLean,
Harley et al. 1999, Yuan, Harley et al. 2003). NE’s role in neonate imprinting and the resultant plasticity is restricted to the sensitive period until PD10, after which the Locus LC circuit changes dramatically. An increase in α1 inhibitory autoreceptors and decrease in in α2 excitatory autoreceptors in the LC stops the surge of NE from LC to OB which had previously encoded the arousal of maternal stroking on pups. Mostly recently, NE has been found to potentiate the
OSN-Mitral synapse in the OB, moving the site of plasticity further to the periphery (Yuan 2009).
The role of serotonin (5-HT) role in the OB has been least studied. Initial evidence showed that 5-HT2 receptors were found to be a co-facilitate neonate imprinting along with β1 adrenergic receptors (McLean, Darby-King et al. 1993,
McLean, Darby-King et al. 1996, Price, Darby-King et al. 1998). Not only did 5-
HT2ARs co-vocalize with β1 receptors on mitral cells, 5-HTagonists had an additive property when applied with NE B1 agonists as the US as well as contributed to activation of the same cAMP signaling cascade (Yuan, Harley et al. 2000, Yuan, Harley et al. 2003). However, 5-HT was neither sufficient nor necessary for odor imprinting as long as enough NE was present. Given 5-HT’s strong enervation the glomerular layer (McLean and Shipley 1987, McLean and
13 Shipley 1987, McLean, Darby-King et al. 1995) and the differential distribution of
5-HT2A versus 5-HT2C subtypes (Pompeiano, Palacios et al. 1992, Pompeiano,
Palacios et al. 1994), I suspect that 5HT might play a stronger or more specific role in the glomerular layer than previously described in deeper mitral-granule layers.
Dopamine (DA) is a centrifugal modulator in sheep but not rodents (Levy,
Meurisse et al. 1999) and but not have been verified in other species. In rodents, actions of dopamine are purely local from dopaminergic periglomerular cells (PG) that provide presynaptic inhibition via D2 receptors that reside on OSNs and possibly post-synaptically as well (Berkowicz and Trombley 2000, Ennis, Zhou et al. 2001). Since it is not centrifugal, DA most likely cannot confer arousal but rather play a role similar to other intrinsic GABAergic PG cells: sharpening odors during discrimination and decreasing cross excitation in adjacent glomeruli or decreasing discrimination (Wilson and Sullivan 1995, Wei, Linster et al. 2006).
Experience-dependent plasticity at the periphery
The olfactory bulb (OB) might be a unique system where early processing synapses are equipped with the circuitry for context-dependent modulation.
Previous studies have suggested that deeper OB synapses between mitral cells and granule cells are altered by experience as observed during neonate imprinting (Wilson, Guthrie et al. 1990, Yokoi, Mori et al. 1995, Aungst, Heyward et al. 2003, Araneda and Firestein 2006, Lagier, Panzanelli et al. 2007).
However, emerging studies of the glomerulus—the OB’s first relay station, show that this first synapse is also plastic in a experience-dependent manner as well as responsive to neuromodulators. This topic is at the heart of this thesis.
14 The current literature on experience-dependent glomerular plasticity can be divided into studies that focus on functional consequences or those that focus on structural consequences in the glomerulus after changes in experience
(Figure 2). A majority of this literature, however, is limited to findings of plasticity in neonates or juvenile rodents. Experience-dependent plasticity in the developing nervous system is not surprising, even at such a periphery station.
Activity has been deemed a necessary for the refinement of developing primary sensory circuits (Hubel, Wiesel et al. 1977).
In terms of structural plasticity, seminal studies have shown that odor evoked activity during the first weeks of life in necessary for proper development of glomeruli. Odor deprivation prevents the targeting of OSNs into one single glomerulus that expresses one OR (Zou, Feinstein et al. 2004). Conversely, chronic exposure to odor during the neonate period induces the development of extra glomeruli for that odor (Valle-Leija, Blanco-Hernandez et al. 2012).
However, during imprinting, a more meaningful kind of odor experience, glomerular refinement accelerates and reaches unity with less chance for extra glomeruli (Belluscio, Lodovichi et al. 2002). In terms of functional plasticity, neonate rodents show multiple types of plasticity in the glomerulus. Odor exposure causes compensatory decreases in metabolic activity response profiles to that odor in OB (Wilson, Sullivan et al. 1985). Odor deprivation induces an compensatory increases glomeruli’s sensitivity odors by increasing presynaptic release and decreasing spontaneous activity in mitral cells and favors odor- evoked response of mitral cell, an overall increase in signal to noise ratio
(Guthrie, Wilson et al. 1990) as well as increases presynaptic release. This homeostatic plasticity depends on postsynaptic strengthening via
15
Figure 2. Summary of studies on experience-dependent plasticity in the glomerulus. Most of the literature about experience-dependent plasticity at the first relay station has focused on neonate age groups, using manipulations such as imprinting or global manipulations in odor experience such as deprivation or enrichment.. For those studies using adult animals, the findings have been mainly limited to experience or learning dependent structural plasticity glomerulus such as changes glomerulus size or number of OSNs. My study attempts to fill a gap in the literature by focusing on the functional consequences of associative learning-dependent plasticity in adults
16 AMPA and NMDA receptor insertion as well as presynaptic mechanisms such as increase in probability of release (Tyler, Petzold et al. 2007). Neonate imprinting, on the other hand causes an increased excitation in the glomerulus and by increasing AMPAR components but decreasing NMDAR components in glomeruli field potentials (Yuan and Harley 2012).
In neonate rats, classical forms of learning-dependent plasticity such as
LTP and LTD can be electrically induced in the first synapse between OSN and postsynaptic mitral cell, by low or high frequency stimulation(Mutoh, Yuan et al.
2005, Yuan 2009). In these studies, while LTD was shown to be achieved by presynaptic mechanisms while LTP by post-synaptic mechanisms involving
NMDARs, increased calcium signaling and cAMP and pCREB signaling.
However to date, no studies have induced LTP by actual behavioral experiences in neonates. There have been not studies yet on the existence of LTP/LTD in the adult olfactory glomerulus: induced artificially or by natural experience such as learning.
There had been limited research on experience-dependent plasticity in the olfactory glomeruli of adult rodents. Existing studies only focus only on structural plasticity due to experience. For example, odor deprivation or enrichment decreases the number of OSNs in the olfactory epithelium responding to that odorant (Cavallin, Powell et al. 2010). On the other hand, a more meaningful experience such as odor-cue associative learning has the opposite effect. Both reward and fear conditioning increases the number of OSNs responding to that learned odor as well as the size of the glomerulus responding to that odor (Jones, Choi et al. 2008). These studies show that even in adulthood, the glomerulus is plastic to experience and learning. However, they
17 are limited in scope because they focus only on structural change and do not address the functional changes in the circuitry. The studies presented in this thesis will aim to fill a gap in the literature by investigating learning-dependent functional plasticity in the adult glomerulus.
Hypothesis: Learning-dependent plasticity informs a sensory gate
My brief review of the literature on sensory gating, neuromodulators, olfactory bulb learning and memory, and glomerular plasticity points to a very interesting question at intersection of these topics. . How can learning and memory at the first glomerular synapse induce a circuit plasticity that also informs sensory gating? I hypothesize that serotonin, plays dual role of inducing the plasticity needed for learning and memory as well as sensory gating.
Specifically, I propose that during associative learning, centrifugal serotonin arrives in the glomerulus coincidentally with odor input from OSNs. Neurons within the glomerular circuit receiving both inputs have a chance to integrate these two signals to produce learning-dependent synaptic plasticity. Meanwhile,
5-HT’s acute modulation of the same circuit alters the balance of excitation versus inhibition in the glomerulus to produce more efficient sensory gating. It achieves this by acting on multiple receptor subtypes that are differentially expresses on principal relay cells, and GABAergic and glutamatergic interneurons.
Overview of chapters
In chapter 2, in a review of the literature, I will compare the important role of neuromodulators in olfactory learning and memory across two diverse model
18 systems: insects versus mammals. I focus particularly on evidence of learning induced plasticity involving neuromodulators in the Olfactory bulb of rodents and
Antennal lobe of Drosophila, Apis Mellifera and Manduca Sexta, the most common model systems. I hope to elucidate common mechanisms and principles that govern how neuromodulation contributes to experience and learning-dependent plasticity across species.
In chapter 3, I will establish an experimental paradigm that will allow me to assay the role of serotonin and circuit plasticity in olfactory associative learning in rodents. First I develop both positive and negative contexts of associative conditioning to test my hypothesis. Next I define a genetically modified circuit in the M72-GFP mouse that allows me stimulate the circuit using a specific known odor and then perform GFP guided interrogation of functional consequences of learning. Utilizing these two paradigms, I design experiments to studying associative learning-dependent functional plasticity in the adult olfactory system with more precision and accuracy.
In chapter 4, I investigate the role of serotonin as a neuromodulator that may affect peripheral olfactory learning and memory. I first map serotonergic elements within the olfactory bulb, discerning the location of 5-HT innervation and receptor subtype distribution in the glomerular layer. Second, I test the hypothesis that 5-
HT participates in the acquisition of an olfactory memory in adult animals via actions in the OB. I use pharmacology in vivo to block acquisition of odor associative learning and to determine the 5-HT receptor subtypes involved.
Finally, I test the hypothesis that 5-HT signaling plays a long term role in the maintenance of an olfactory memory trace the glomerulus. After associative learning paradigms I used to in instill fear vs. reward memories, I used
19 immunohistochemistry to assay for changes in serotonergic elements in the bulb that would indicate a change in 5-HT signaling strength at the glomerulus processing the learned odor.
In chapter 5, I will link 5-HT’s role in learning and memory with sensory gating. I use whole-cell electrophysiology techniques to investigate the role of serotonin in the modulation of synaptic properties of primary glomerular synapses. I record the activity from juxtaglomerular cells (both excitatory ET cells and inhibitory PG cells) that participate in the ON-OFF cycle of excitation and inhibition at the first relay station. I use 5-HT2AR receptor agonists to assay for the effects of acute serotonin influx into the glomerular circuit. Additionally, I collect data on the effect of fear associative learning on the strength of primary synapses.
Finally in Chapter 6, I conclude with a synthesis of my results in context of the existing literature, an evaluation of my hypothesis, and future directions to elucidate this highly interesting topic of learning-dependent sensory gating at peripheral synapses.
20 REFERENCES
Abdolmaleky, H. M., S. V. Faraone, S. J. Glatt and M. T. Tsuang (2004). "Meta- analysis of association between the T102C polymorphism of the 5HT2a receptor gene and schizophrenia." Schizophr Res 67(1): 53-62.
Abel, T., P. V. Nguyen, M. Barad, T. A. Deuel, E. R. Kandel and R. Bourtchouladze (1997). "Genetic demonstration of a role for PKA in the late phase of LTP and in hippocampus-based long-term memory." Cell 88(5): 615-626.
Adler, L. E., E. M. Cawthra, K. A. Donovan, J. G. Harris, H. T. Nagamoto, A. Olincy and M. C. Waldo (2005). "Improved p50 auditory gating with ondansetron in medicated schizophrenia patients." Am J Psychiatry 162(2): 386-388.
Akhondzadeh, S., M. Malek-Hosseini, A. Ghoreishi, M. Raznahan and S. A. Rezazadeh (2008). "Effect of ritanserin, a 5HT2A/2C antagonist, on negative symptoms of schizophrenia: a double-blind randomized placebo- controlled study." Prog Neuropsychopharmacol Biol Psychiatry 32(8): 1879-1883.
Ambroggi, F., A. Ishikawa, H. L. Fields and S. M. Nicola (2008). "Basolateral amygdala neurons facilitate reward-seeking behavior by exciting nucleus accumbens neurons." Neuron 59(4): 648-661.
Araneda, R. C. and S. Firestein (2006). "Adrenergic enhancement of inhibitory transmission in the accessory olfactory bulb." J Neurosci 26(12): 3292- 3298.
Aungst, J. L., P. M. Heyward, A. C. Puche, S. V. Karnup, A. Hayar, G. Szabo and M. T. Shipley (2003). "Centre-surround inhibition among olfactory bulb glomeruli." Nature 426(6967): 623-629.
Bailey, C. H. and M. Chen (1988). "Long-term memory in Aplysia modulates the total number of varicosities of single identified sensory neurons." Proc Natl Acad Sci U S A 85(7): 2373-2377.
Belluscio, L., C. Lodovichi, P. Feinstein, P. Mombaerts and L. C. Katz (2002). "Odorant receptors instruct functional circuitry in the mouse olfactory bulb." Nature 419(6904): 296-300.
Berkowicz, D. A. and P. Q. Trombley (2000). "Dopaminergic modulation at the olfactory nerve synapse." Brain Res 855(1): 90-99.
Bliss, T. V. and G. L. Collingridge (1993). "A synaptic model of memory: long- term potentiation in the hippocampus." Nature 361(6407): 31-39.
21 Bliss, T. V. and T. Lomo (1973). "Long-lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path." J Physiol 232(2): 331-356.
Bokil, H., N. Laaris, K. Blinder, M. Ennis and A. Keller (2001). "Ephaptic interactions in the mammalian olfactory system." J Neurosci 21(20): RC173.
Bourtchuladze, R., B. Frenguelli, J. Blendy, D. Cioffi, G. Schutz and A. J. Silva (1994). "Deficient long-term memory in mice with a targeted mutation of the cAMP-responsive element-binding protein." Cell 79(1): 59-68.
Brauer, D., A. Strobel, T. Hensch, K. Diers, K. P. Lesch and B. Brocke (2009). "Genetic variation of serotonin receptor function affects prepulse inhibition of the startle." J Neural Transm 116(5): 607-613.
Brunelli, M., V. Castellucci and E. R. Kandel (1976). "Synaptic facilitation and behavioral sensitization in Aplysia: possible role of serotonin and cyclic AMP." Science 194(4270): 1178-1181.
Brunjes, P. C., K. R. Illig and E. A. Meyer (2005). "A field guide to the anterior olfactory nucleus (cortex)." Brain Res Brain Res Rev 50(2): 305-335.
Buchsbaum, M. S., B. T. Christian, D. S. Lehrer, T. K. Narayanan, B. Shi, J. Mantil, E. Kemether, T. R. Oakes and J. Mukherjee (2006). "D2/D3 dopamine receptor binding with [F-18]fallypride in thalamus and cortex of patients with schizophrenia." Schizophr Res 85(1-3): 232-244.
Cavallin, M. A., K. Powell, K. C. Biju and D. A. Fadool (2010). "State-dependent sculpting of olfactory sensory neurons is attributed to sensory enrichment, odor deprivation, and aging." Neurosci Lett 483(2): 90-95.
Chaudhury, D., O. Escanilla and C. Linster (2009). "Bulbar acetylcholine enhances neural and perceptual odor discrimination." J Neurosci 29(1): 52-60.
Christian, B. T., D. S. Lehrer, B. Shi, T. K. Narayanan, P. S. Strohmeyer, M. S. Buchsbaum and J. C. Mantil (2006). "Measuring dopamine neuromodulation in the thalamus: using [F-18]fallypride PET to study dopamine release during a spatial attention task." Neuroimage 31(1): 139-152.
Ennis, M., F. M. Zhou, K. J. Ciombor, V. Aroniadou-Anderjaska, A. Hayar, E. Borrelli, L. A. Zimmer, F. Margolis and M. T. Shipley (2001). "Dopamine D2 receptor-mediated presynaptic inhibition of olfactory nerve terminals." J Neurophysiol 86(6): 2986-2997.
Fitzpatrick, D., I. T. Diamond and D. Raczkowski (1989). "Cholinergic and monoaminergic innervation of the cat's thalamus: comparison of the
22 lateral geniculate nucleus with other principal sensory nuclei." J Comp Neurol 288(4): 647-675.
Galici, R., J. D. Boggs, K. L. Miller, P. Bonaventure and J. R. Atack (2008). "Effects of SB-269970, a 5-HT7 receptor antagonist, in mouse models predictive of antipsychotic-like activity." Behav Pharmacol 19(2): 153-159.
Garcia-Cabezas, M. A., P. Martinez-Sanchez, M. A. Sanchez-Gonzalez, M. Garzon and C. Cavada (2009). "Dopamine innervation in the thalamus: monkey versus rat." Cereb Cortex 19(2): 424-434.
Garcia-Cabezas, M. A., B. Rico, M. A. Sanchez-Gonzalez and C. Cavada (2007). "Distribution of the dopamine innervation in the macaque and human thalamus." Neuroimage 34(3): 965-984.
Geyer, M. A. and D. L. Braff (1982). "Habituation of the Blink reflex in normals and schizophrenic patients." Psychophysiology 19(1): 1-6.
Geyer, M. A. and D. L. Braff (1987). "Startle habituation and sensorimotor gating in schizophrenia and related animal models." Schizophr Bull 13(4): 643- 668.
Glanzman, D. L., S. L. Mackey, R. D. Hawkins, A. M. Dyke, P. E. Lloyd and E. R. Kandel (1989). "Depletion of serotonin in the nervous system of Aplysia reduces the behavioral enhancement of gill withdrawal as well as the heterosynaptic facilitation produced by tail shock." J Neurosci 9(12): 4200-4213.
Gomez, C., J. G. Brinon, M. V. Barbado, E. Weruaga, J. Valero and J. R. Alonso (2005). "Heterogeneous targeting of centrifugal inputs to the glomerular layer of the main olfactory bulb." J Chem Neuroanat 29(4): 238-254.
Govindaiah, G., T. Wang, M. U. Gillette, S. R. Crandall and C. L. Cox (2010). "Regulation of inhibitory synapses by presynaptic D(4) dopamine receptors in thalamus." J Neurophysiol 104(5): 2757-2765.
Guthrie, K. M., D. A. Wilson and M. Leon (1990). "Early unilateral deprivation modifies olfactory bulb function." J Neurosci 10(10): 3402-3412.
Ha, H. I., M. Hendricks, Y. Shen, C. V. Gabel, C. Fang-Yen, Y. Qin, D. Colon- Ramos, K. Shen, A. D. Samuel and Y. Zhang (2010). "Functional organization of a neural network for aversive olfactory learning in Caenorhabditis elegans." Neuron 68(6): 1173-1186.
Haberly, L. B. (2001). "Parallel-distributed processing in olfactory cortex: new insights from morphological and physiological analysis of neuronal circuitry." Chem Senses 26(5): 551-576.
23 Hayar, A., S. Karnup, M. Ennis and M. T. Shipley (2004). "External tufted cells: a major excitatory element that coordinates glomerular activity." J Neurosci 24(30): 6676-6685.
Hayar, A., S. Karnup, M. T. Shipley and M. Ennis (2004). "Olfactory bulb glomeruli: external tufted cells intrinsically burst at theta frequency and are entrained by patterned olfactory input." J Neurosci 24(5): 1190-1199.
Hubel, D. H., T. N. Wiesel and S. LeVay (1977). "Plasticity of ocular dominance columns in monkey striate cortex." Philos Trans R Soc Lond B Biol Sci 278(961): 377-409.
Hurley, L. M., D. M. Devilbiss and B. D. Waterhouse (2004). "A matter of focus: monoaminergic modulation of stimulus coding in mammalian sensory networks." Curr Opin Neurobiol 14(4): 488-495.
Johansen, J. P., C. K. Cain, L. E. Ostroff and J. E. LeDoux (2011). "Molecular mechanisms of fear learning and memory." Cell 147(3): 509-524.
Jones, S. V., D. C. Choi, M. Davis and K. J. Ressler (2008). "Learning-dependent structural plasticity in the adult olfactory pathway." J Neurosci 28(49): 13106-13111.
Kasa, P., I. Hlavati, E. Dobo, A. Wolff, F. Joo and J. R. Wolff (1995). "Synaptic and non-synaptic cholinergic innervation of the various types of neurons in the main olfactory bulb of adult rat: immunocytochemistry of choline acetyltransferase." Neuroscience 67(3): 667-677.
Kauer, J. A., R. C. Malenka and R. A. Nicoll (1988). "A persistent postsynaptic modification mediates long-term potentiation in the hippocampus." Neuron 1(10): 911-917.
Kay, L. M. and S. M. Sherman (2007). "An argument for an olfactory thalamus." Trends Neurosci 30(2): 47-53.
Kiselycznyk, C. L., S. Zhang and C. Linster (2006). "Role of centrifugal projections to the olfactory bulb in olfactory processing." Learn Mem 13(5): 575-579.
Lagier, S., P. Panzanelli, R. E. Russo, A. Nissant, B. Bathellier, M. Sassoe- Pognetto, J. M. Fritschy and P. M. Lledo (2007). "GABAergic inhibition at dendrodendritic synapses tunes gamma oscillations in the olfactory bulb." Proc Natl Acad Sci U S A 104(17): 7259-7264.
LeDoux, J. E. (2000). "Emotion circuits in the brain." Annu Rev Neurosci 23: 155- 184.
Levy, F., M. Meurisse, G. Ferreira, J. Thibault and Y. Tillet (1999). "Afferents to the rostral olfactory bulb in sheep with special emphasis on the
24 cholinergic, noradrenergic and serotonergic connections." J Chem Neuroanat 16(4): 245-263.
Lynch, G., J. Larson, S. Kelso, G. Barrionuevo and F. Schottler (1983). "Intracellular injections of EGTA block induction of hippocampal long-term potentiation." Nature 305(5936): 719-721.
Mandairon, N., C. J. Ferretti, C. M. Stack, D. B. Rubin, T. A. Cleland and C. Linster (2006). "Cholinergic modulation in the olfactory bulb influences spontaneous olfactory discrimination in adult rats." Eur J Neurosci 24(11): 3234-3244.
Mandairon, N., C. Stack, C. Kiselycznyk and C. Linster (2006). "Broad activation of the olfactory bulb produces long-lasting changes in odor perception." Proc Natl Acad Sci U S A 103(36): 13543-13548.
Matsutani, S. and N. Yamamoto (2008). "Centrifugal innervation of the mammalian olfactory bulb." Anat Sci Int 83(4): 218-227.
McCormick, D. A. and T. Bal (1994). "Sensory gating mechanisms of the thalamus." Curr Opin Neurobiol 4(4): 550-556.
McCormick, D. A. and H. C. Pape (1988). "Acetylcholine inhibits identified interneurons in the cat lateral geniculate nucleus." Nature 334(6179): 246-248.
McCormick, D. A. and H. C. Pape (1990). "Noradrenergic and serotonergic modulation of a hyperpolarization-activated cation current in thalamic relay neurones." J Physiol 431: 319-342.
McCormick, D. A., H. C. Pape and A. Williamson (1991). "Actions of norepinephrine in the cerebral cortex and thalamus: implications for function of the central noradrenergic system." Prog Brain Res 88: 293- 305.
McCormick, D. A. and D. A. Prince (1986). "Acetylcholine induces burst firing in thalamic reticular neurones by activating a potassium conductance." Nature 319(6052): 402-405.
McCormick, D. A. and Z. Wang (1991). "Serotonin and noradrenaline excite GABAergic neurones of the guinea-pig and cat nucleus reticularis thalami." J Physiol 442: 235-255.
McLean, J. H., A. Darby-King and E. Hodge (1996). "5-HT2 receptor involvement in conditioned olfactory learning in the neonate rat pup." Behav Neurosci 110(6): 1426-1434.
McLean, J. H., A. Darby-King and G. D. Paterno (1995). "Localization of 5-HT2A receptor mRNA by in situ hybridization in the olfactory bulb of the postnatal rat." J Comp Neurol 353(3): 371-378.
25 McLean, J. H., A. Darby-King, R. M. Sullivan and S. R. King (1993). "Serotonergic influence on olfactory learning in the neonate rat." Behav Neural Biol 60(2): 152-162.
McLean, J. H., C. W. Harley, A. Darby-King and Q. Yuan (1999). "pCREB in the neonate rat olfactory bulb is selectively and transiently increased by odor preference-conditioned training." Learn Mem 6(6): 608-618.
McLean, J. H. and M. T. Shipley (1987). "Serotonergic afferents to the rat olfactory bulb: I. Origins and laminar specificity of serotonergic inputs in the adult rat." J Neurosci 7(10): 3016-3028.
McLean, J. H. and M. T. Shipley (1987). "Serotonergic afferents to the rat olfactory bulb: II. Changes in fiber distribution during development." J Neurosci 7(10): 3029-3039.
Mitchell, E. S. and J. F. Neumaier (2008). "5-HT6 receptor antagonist reversal of emotional learning and prepulse inhibition deficits induced by apomorphine or scopolamine." Pharmacol Biochem Behav 88(3): 291- 298.
Moore, R. Y., A. E. Halaris and B. E. Jones (1978). "Serotonin neurons of the midbrain raphe: ascending projections." J Comp Neurol 180(3): 417-438.
Murphy, G. J., D. P. Darcy and J. S. Isaacson (2005). "Intraglomerular inhibition: signaling mechanisms of an olfactory microcircuit." Nat Neurosci 8(3): 354-364.
Mutoh, H., Q. Yuan and T. Knopfel (2005). "Long-term depression at olfactory nerve synapses." J Neurosci 25(17): 4252-4259.
Neville, K. R. and L. B. Haberly (2003). "Beta and gamma oscillations in the olfactory system of the urethane-anesthetized rat." J Neurophysiol 90(6): 3921-3930.
Nicoll, R. A., J. A. Kauer and R. C. Malenka (1988). "The current excitement in long-term potentiation." Neuron 1(2): 97-103.
Nicoll, R. A., S. Tomita and D. S. Bredt (2006). "Auxiliary subunits assist AMPA- type glutamate receptors." Science 311(5765): 1253-1256.
Padich, R. A., T. C. McCloskey and J. H. Kehne (1996). "5-HT modulation of auditory and visual sensorimotor gating: II. Effects of the 5-HT2A antagonist MDL 100,907 on disruption of sound and light prepulse inhibition produced by 5-HT agonists in Wistar rats." Psychopharmacology (Berl) 124(1-2): 107-116.
Pape, H. C. and D. A. McCormick (1989). "Noradrenaline and serotonin selectively modulate thalamic burst firing by enhancing a hyperpolarization-activated cation current." Nature 340(6236): 715-718.
26 Pavlov, I. P. and G. V. Anrep (1927). Conditioned reflexes; an investigation of the physiological activity of the cerebral cortex. London, Oxford University Press: Humphrey Milford.
Pazos, A., R. Cortes and J. M. Palacios (1985). "Quantitative autoradiographic mapping of serotonin receptors in the rat brain. II. Serotonin-2 receptors." Brain Res 346(2): 231-249.
Pinault, D. (2004). "The thalamic reticular nucleus: structure, function and concept." Brain Res Brain Res Rev 46(1): 1-31.
Pompeiano, M., J. M. Palacios and G. Mengod (1992). "Distribution and cellular localization of mRNA coding for 5-HT1A receptor in the rat brain: correlation with receptor binding." J Neurosci 12(2): 440-453.
Pompeiano, M., J. M. Palacios and G. Mengod (1994). "Distribution of the serotonin 5-HT2 receptor family mRNAs: comparison between 5-HT2A and 5-HT2C receptors." Brain Res Mol Brain Res 23(1-2): 163-178.
Price, T. L., A. Darby-King, C. W. Harley and J. H. McLean (1998). "Serotonin plays a permissive role in conditioned olfactory learning induced by norepinephrine in the neonate rat." Behav Neurosci 112(6): 1430-1437.
Quednow, B. B., K. U. Kuhn, R. Mossner, S. G. Schwab, A. Schuhmacher, W. Maier and M. Wagner (2008). "Sensorimotor gating of schizophrenia patients is influenced by 5-HT2A receptor polymorphisms." Biol Psychiatry 64(5): 434-437.
Saiz, P. A., M. P. Garcia-Portilla, C. Arango, B. Morales, V. Alvarez, E. Coto, J. M. Fernandez, M. T. Bascaran, M. Bousono and J. Bobes (2007). "Association study of serotonin 2A receptor (5-HT2A) and serotonin transporter (5-HTT) gene polymorphisms with schizophrenia." Prog Neuropsychopharmacol Biol Psychiatry 31(3): 741-745.
Sanchez-Gonzalez, M. A., M. A. Garcia-Cabezas, B. Rico and C. Cavada (2005). "The primate thalamus is a key target for brain dopamine." J Neurosci 25(26): 6076-6083.
Sawin, E. R., R. Ranganathan and H. R. Horvitz (2000). "C. elegans locomotory rate is modulated by the environment through a dopaminergic pathway and by experience through a serotonergic pathway." Neuron 26(3): 619- 631.
Schwaerzel, M., M. Monastirioti, H. Scholz, F. Friggi-Grelin, S. Birman and M. Heisenberg (2003). "Dopamine and octopamine differentiate between aversive and appetitive olfactory memories in Drosophila." J Neurosci 23(33): 10495-10502.
Shepherd, G. M. (1972). "Synaptic organization of the mammalian olfactory bulb." Physiol Rev 52(4): 864-917.
27 Shepherd, G. M. (2005). "Perception without a thalamus how does olfaction do it?" Neuron 46(2): 166-168.
Shepherd, G. M., W. R. Chen and C. A. Greer (2004). The Olfactory Bulb. The synaptic organization of the brain. G. M. Shepherd. Oxford, Oxford University Press: 165-216.
Sherman, M. S. and R. W. Guillery (2004). The Thalamus. The synaptic organization of the brain (5th Ed.). G. M. Shepherd. Oxford, Oxford University Press: 331-360.
Sherman, S. M. and C. Koch (1986). "The control of retinogeniculate transmission in the mammalian lateral geniculate nucleus." Exp Brain Res 63(1): 1-20.
Shi, S. H., Y. Hayashi, R. S. Petralia, S. H. Zaman, R. J. Wenthold, K. Svoboda and R. Malinow (1999). "Rapid spine delivery and redistribution of AMPA receptors after synaptic NMDA receptor activation." Science 284(5421): 1811-1816.
Silva, A. J., R. Paylor, J. M. Wehner and S. Tonegawa (1992). "Impaired spatial learning in alpha-calcium-calmodulin kinase II mutant mice." Science 257(5067): 206-211.
Stuber, G. D., D. R. Sparta, A. M. Stamatakis, W. A. van Leeuwen, J. E. Hardjoprajitno, S. Cho, K. M. Tye, K. A. Kempadoo, F. Zhang, K. Deisseroth and A. Bonci (2011). "Excitatory transmission from the amygdala to nucleus accumbens facilitates reward seeking." Nature 475(7356): 377-380.
Sullivan, R. M., D. A. Wilson, C. Lemon and G. A. Gerhardt (1994). "Bilateral 6- OHDA lesions of the locus coeruleus impair associative olfactory learning in newborn rats." Brain Res 643(1-2): 306-309.
Sullivan, R. M., D. A. Wilson and M. Leon (1989). "Norepinephrine and learning- induced plasticity in infant rat olfactory system." J Neurosci 9(11): 3998- 4006.
Sullivan, R. M., D. R. Zyzak, P. Skierkowski and D. A. Wilson (1992). "The role of olfactory bulb norepinephrine in early olfactory learning." Brain Res Dev Brain Res 70(2): 279-282.
Tsai, H. C., F. Zhang, A. Adamantidis, G. D. Stuber, A. Bonci, L. de Lecea and K. Deisseroth (2009). "Phasic firing in dopaminergic neurons is sufficient for behavioral conditioning." Science 324(5930): 1080-1084.
Tye, K. M., G. D. Stuber, B. de Ridder, A. Bonci and P. H. Janak (2008). "Rapid strengthening of thalamo-amygdala synapses mediates cue-reward learning." Nature 453(7199): 1253-1257.
28 Tyler, W. J., G. C. Petzold, S. K. Pal and V. N. Murthy (2007). "Experience- dependent modification of primary sensory synapses in the mammalian olfactory bulb." J Neurosci 27(35): 9427-9438.
Valle-Leija, P., E. Blanco-Hernandez, R. Drucker-Colin, G. Gutierrez-Ospina and R. Vidaltamayo (2012). "Supernumerary formation of olfactory glomeruli induced by chronic odorant exposure: a constructivist expression of neural plasticity." PLoS One 7(4): e35358.
Valverde, F. and L. Lopez-Mascaraque (1991). "Neuroglial arrangements in the olfactory glomeruli of the hedgehog." J Comp Neurol 307(4): 658-674.
Varty, G. B., V. P. Bakshi and M. A. Geyer (1999). "M100907, a serotonin 5- HT2A receptor antagonist and putative antipsychotic, blocks dizocilpine- induced prepulse inhibition deficits in Sprague-Dawley and Wistar rats." Neuropsychopharmacology 20(4): 311-321. von Krosigk, M., T. Bal and D. A. McCormick (1993). "Cellular mechanisms of a synchronized oscillation in the thalamus." Science 261(5119): 361-364.
Wei, C. J., C. Linster and T. A. Cleland (2006). "Dopamine D(2) receptor activation modulates perceived odor intensity." Behav Neurosci 120(2): 393-400.
Wilson, D. A., K. M. Guthrie and M. Leon (1990). "Modification of olfactory bulb synaptic inhibition by early unilateral olfactory deprivation." Neurosci Lett 116(3): 250-256.
Wilson, D. A. and M. Leon (1988). "Spatial patterns of olfactory bulb single-unit responses to learned olfactory cues in young rats." J Neurophysiol 59(6): 1770-1782.
Wilson, D. A. and R. M. Sullivan (1995). "The D2 antagonist spiperone mimics the effects of olfactory deprivation on mitral/tufted cell odor response patterns." J Neurosci 15(8): 5574-5581.
Wilson, D. A., R. M. Sullivan and M. Leon (1985). "Odor familiarity alters mitral cell response in the olfactory bulb of neonatal rats." Brain Res 354(2): 314-317.
Yokoi, M., K. Mori and S. Nakanishi (1995). "Refinement of odor molecule tuning by dendrodendritic synaptic inhibition in the olfactory bulb." Proc Natl Acad Sci U S A 92(8): 3371-3375.
Yuan, Q. (2009). "Theta bursts in the olfactory nerve paired with beta- adrenoceptor activation induce calcium elevation in mitral cells: a mechanism for odor preference learning in the neonate rat." Learn Mem 16(11): 676-681.
29 Yuan, Q. and C. W. Harley (2012). "What a nostril knows: olfactory nerve-evoked AMPA responses increase while NMDA responses decrease at 24-h post-training for lateralized odor preference memory in neonate rat." Learn Mem 19(2): 50-53.
Yuan, Q., C. W. Harley, J. C. Bruce, A. Darby-King and J. H. McLean (2000). "Isoproterenol increases CREB phosphorylation and olfactory nerve- evoked potentials in normal and 5-HT-depleted olfactory bulbs in rat pups only at doses that produce odor preference learning." Learn Mem 7(6): 413-421.
Yuan, Q., C. W. Harley, A. Darby-King, R. L. Neve and J. H. McLean (2003). "Early odor preference learning in the rat: bidirectional effects of cAMP response element-binding protein (CREB) and mutant CREB support a causal role for phosphorylated CREB." J Neurosci 23(11): 4760-4765.
Yuan, Q., C. W. Harley and J. H. McLean (2003). "Mitral cell beta1 and 5-HT2A receptor colocalization and cAMP coregulation: a new model of norepinephrine-induced learning in the olfactory bulb." Learn Mem 10(1): 5-15.
Zhang, Y., H. Lu and C. I. Bargmann (2005). "Pathogenic bacteria induce aversive olfactory learning in Caenorhabditis elegans." Nature 438(7065): 179-184.
Zou, D. J., P. Feinstein, A. L. Rivers, G. A. Mathews, A. Kim, C. A. Greer, P. Mombaerts and S. Firestein (2004). "Postnatal refinement of peripheral olfactory projections." Science 304(5679): 1976-1979.
30 Chapter 2
ROLE OF NEUROMODULATORS ON LEARNING-DEPENDDENT PLASTICITY
IN GLOMERULI OF RODENTS VERSUS INSECTS
Introduction: why study experience plasticity in the olfactory periphery?
The olfactory system is an optimal model to study experience-mediated sensory plasticity. Olfaction is the primary sensory modality of rodents as well as insects. It provides cues for food acquisition, reproduction, and predator avoidance (Doty 1986, Brennan and Kendrick 2006). Logically, environmental, cue-mediated learning must occur in the olfactory system for an organism to optimally respond to is environment and survive.
Research in insects and rodents often proceed in parallel, but rarely do they build upon information from the other’s fields. Recently, however, literature reviews have emerged allowing intersection of the two bodies work. Olfactory processing in rodents and insects have many common themes, sharing not only similar organization and functional principals for odor processing (Bargmann
2006, Wilson 2008)but also, as I propose here, similar types of circuit plasticity that underlies learning and memory. This review compares the peripheral olfactory learning and memory circuits in the olfactory bulb (OB) of rodents versus the antennal lobe (AL) of insects.
The olfactory bulb is a convenient model system to study experience dependent plasticity for both technical and biological reasons. First, studying learning and memory in peripheral circuits has an experimental advantage over studying central circuits. The researcher has direct access of peripheral circuits to manipulate experience, while central circuits such as hippocampal, amygdala,
31 or cortical circuits, for example, involves analysis of synapses several integrations removed from the original stimulus (LeDoux 2000, Sigurdsson,
Doyere et al. 2007, Johansen, Cain et al. 2011).
Moreover, the OB and AL’s first synapses are functionally organized into glomeruli, each consisting of OSNs expressing one odorant receptor (OR), responding to one structurally similar set of odorants.(Buck and Axel 1991,
Mombaerts, Wang et al. 1996, Davis 2005, Masse, Turner et al. 2009). In the recent years, a functional map of glomeruli on the OB has been sketched using intrinsic imaging(Rubin and Katz 1999) and genetically encoded fluorescent probes. (Meister and Bonhoeffer 2001, Wachowiak and Cohen 2001, Belluscio,
Lodovichi et al. 2002, Soucy, Albeanu et al. 2009). OR genes have been identified for specific glomeruli (M72/1, P2, I2, MOR23), making possible transgenic mice that processes one reporter-tagged glomeruli selectively responding known pure odorants (Mombaerts, Wang et al. 1996). In parallel, in insect olfaction research, Drosophila, Apis Mellifera (Honeybee), and Manduca
Sexta (Sphinx Moth) are most popular models and their ALs have also been carefully mapped (Galizia and Menzel 2000, Vosshall, Wong et al. 2000).
Genetically labeled circuits and encoded functional probes have been well developed in Drosophila even more than its mouse counterpart (Wang, Wong et al. 2003, Larsson, Domingos et al. 2004, Fishilevich and Vosshall 2005). These tools allow for precise targeting of stimuli as well as circuit interrogation.
Since the olfactory systems first input synapses as well as modulatory synapses are located in very surface of the olfactory bulb (OB) or antennal lobe
(AL), these tools are additionally useful because I can access the brain easily for imaging of activity in vivo, in real time. Techniques such as intrinsic imaging,
32 activity dependent probes such as synaptopHluorin (SpH), Ca2+ probes and voltage sensitive dyes should be used. In addition it is easy to access with application of pharmacological agents, and easy access for recording and stimulation.
The main biological advantage studying learning and memory in the olfactory modality is because it is the only modality with rich sources of centrifugal feedback at the first relay station. This has been reviewed in detail in
Chapters 1 and 2. Shaping of information by cortical and neuromodulator feedback exists as early as the OSN synapse, where as other modalities must pass through the thalamus as its first station for central feedback and gating
(Sherman and Guillery 2004, Kay and Sherman 2007). For learning to occur, the circuit must have both feed forward (CS) information as well feedback information that carries the arousal conferred by the positive or negative unconditioned stimulus (US). The olfactory system is unique in that higher order computations occur at the accessible periphery.
Insects versus rodents: anatomy and function of the olfactory periphery
In both the olfactory bulb (OB) as well antennal lobe (AL), odorants first bind to the apical dendrites of the olfactory sensory neurons (OSNS) within the nasal epithelium of rodents or sensilla of antennae of insects. Olfactory receptor neurons in mammals and insects are structurally different and are believed to be from divergent ancestry (Mombaerts 1999, Bargmann 2006). Vertebrate OSNs use classical GPCR Golf which uses cAMP to open a cyclic nucleotide gated cation channel as well as a chloride channel (Kurahashi and Yau 1994) to depolarize the cell. Among invertebrates, some worms such as C. Elegans uses
33 GPCR just like mammals. Meanwhile, insects use a special class of receptors that are structurally different than mammalian ones and most likely ionotropic.
Insect ORs are gated via an unknown mechanism: either by directly gating ions or transferring its activity to a ubiquitously expressed ion channel that is expressed in every OSN cell.
OSN axons with the same OR identity converge into structurally and functionally distinct glomeruli in the olfactory bulb or antenna lobe of the insect or mammalian bulb (Couto, Alenius et al. 2005). Here, OSNs of vertebrates release glutamate and insects release acetylcholine as the main excitatory transmitter to relay information on the principal output cells: the mitral or projection neuron PN) respectively. In both systems, there are only a few (1-5) mitral cells sampling each glomerulus. Some believe that this type of organization helps ensure higher signal to noise ratio. Electrophysiological observation show that while OSN responses are slow and less specific to a particular odor in both models, PN and mitral cell responses are much more temporally specific as well as odor specific
(Wilson, Turner et al. 2004, Bhandawat, Olsen et al. 2007, Masse, Turner et al.
2009). To accomplish, the glomerular processing stations of both model organisms provide extensive refinement of the odor-derived signal before output form the AL or OB (Mori, Nagao et al. 1999, Cleland 2010).
The complex shaping and gating of odors information in the OB or AL is accomplished at the glomerulus by juxtaglomerular interneurons: mostly
GABAergic inhibitory interneurons and some excitatory neurons that act in concert to process odors at this first station. In vertebrates, they have been well categorized as periglomerular (PG) cells (GABAergic), External Tufted (ET) cells
(glutamatergic) and short axon (SA) cells (glutamatergic). While the ET cell
34 ramifies only one glomerulus and the PG cell mainly one glomeruli and sparsely its neighbors, short axon cells span long distances to excite the GABAergic interneurons of a faraway glomerulus. Excitation by one mitral cell is enough cause a symphony of GABAergic inhibition from a large population of GABAergic interneurons. OSNs will synapse directly onto ET cells who are the glutamatergic intermediary between the OSN input and Mitral Cell or GABAergic PG cell. ET cells make dendro-dendritic synapses onto mitral cells to influence their spike pattern. ET dendro-dendritic excitation of GABAergic PG cells cause them to provide strong feed forward inhibition onto relay o mitral cells as well as feedback inhibition upon the OSN terminals. Moreover, ET cells can synchronize the activity of an entire glomerulus via intrinsic bursting. (Hayar, Karnup et al. 2004,
Hayar, Karnup et al. 2004, Hayar, Shipley et al. 2005, Hayar and Ennis 2007).
Insect juxtaglomerular local interneurons are similar to mammalian ones in several ways. They are also mostly GABAergic but with a small percentage excitatory (Cholinergic) (Hildebrand, Christensen et al. 1992, Chou, Spletter et al.
2010) in parallel with PG and ET cell types in mammals. Both principal relay cells: mitral cells and projection neurons of insects do not always receive direct excitation from OSNs and sometimes depend on excitatory interneurons for excitation. GABAergic interneurons may also depend on glutamatergic juxtaglomerular neurons for excitation. In insects, these glutamatergic interneurons excitatory burst spontaneously just like their ET counterparts
(Hildebrand, Christensen et al. 1992, Masse, Turner et al. 2009, Seki, Rybak et al. 2010).
These common elements allow the glomerular relay station to piriform complex processing of odor information at the first synapse. The two classes of
35 juxtaglomerular cells provide both an amplification of excitation as well as a strong, concerted GABAergic inhibition of the first synapse between OSN and mitral or PN relay neuron. Such processing improves the signal to nose ratio of the odor signal as well as improves the temporal fidelity of mitral or PN cell firing as well as temporal fidelity of information in the relay neurons. The glomerular station via its two classes of juxtaglomerular cells can amplify intimal excitation of mitral cells followed by strong GABAergic inhibition which represent a rapid ON-
OFF response to a single OSN stimulation (Wilson and Laurent 2005, Gire and
Schoppa 2009). The responses sharpened responses of mitral cells are bundled into the lateral olfactory tract or antennocerebral tract in the mammal and insect respectively. The olfactory tubercle and Piriform cortex receive information from these first order relays in mammals while the lateral horn and mushroom bodies receive insect PN inputs (Shepherd 1972, Masse, Turner et al. 2009).
Besides a difference in the type of odorant receptors and use of different excitatory neurotransmitters, there are two main differences between the OB and
AL. First, the olfactory bulb has two processing stations: the peripheral glomerular station that surrounds the mitral cell apical dendrite and the deeper granule-mitral cell station at the mitral cell lateral dendrites. The deeper mitral- granule cell synapses are thought to provide additional output control before information leaves the bulb (Price and Powell 1970, Wilson, Guthrie et al. 1990,
Yokoi, Mori et al. 1995). Insects lack this second relay station and depend exclusively on glomerular inhibition. Second difference is that, while the rodent glomerular layer receives extensive feedback from the olfactory cortex
(Strowbridge 2009, Franks, Russo et al. 2011). The insect AL does not appear to
36 receive feedback the higher processing centers such as the MB and LH (Masse,
Turner et al. 2009).
Both the OB and the AL, however, receive extensive centrifugal neuromodulator inputs that convey important information about the arousal state of the animal (Shepherd 2005, Kay and Sherman 2007). Some of these centrifugal fibers terminate extensively or preferentially in the glomerular layer.
Neuromodulators for vertebrates include acetylcholine (ACh), norepinephrine
(NE), and serotonin (5-HT) in rodents, while Dopamine (DA) is mostly local (Price and Powell 1970, Gray and Skinner 1988, Kiselycznyk, Zhang et al. 2006,
Matsutani and Yamamoto 2008). Meanwhile insects receive heavy DA, octopamine (OA) and 5-HT (Nassel 2002, Masse, Turner et al. 2009, Carlsson,
Diesner et al. 2010). These neuromodulators play essential functions to induce experience dependent plasticity and are crucial for acquiring and maintaining early olfactory memories across species.
Global changes in experience plasticity causes functional and structural plasticity in the antenna lobe versus olfactory bulb glomerulus
Experience dependent plasticity in the olfactory periphery of both insects and rodents.
Global manipulations of odor experience by deprivation or enrichment usually cause compensatory homeostatic changes. These changes may not dependent on centrifugal feedback. In the rodent olfactory bulb, olfactory deprivation by uninaris occlusion in neonates and adolescents causes compensatory changes glomerular activity in the occluded bulb. Deprivation
37 recruits more glomeruli to respond to the same odor, widens receptive fields of mitral cells, but increases signal to noise ratio by decreasing spontaneous activity while favoring odor-evoked activity (Guthrie, Wilson et al. 1990, Guthrie, Pullara et al. 1991, Wilson and Sullivan 1995), . Mitral cells experience less feed-forward inhibition from both deeper layer granule (Wilson, Guthrie et al. 1990) as well as less feedback inhibition from dopaminergic periglomerular cells (Wilson and
Sullivan 1995). The mechanisms of this kind of homeostatic plasticity at the glomerulus has been attributed to both pre and postsynaptic changes including increase in probability of release, insertion of new glutamatergic synapses and as well strengthening synapses by insertion of GLuR1 and NR1 AMPA and NMDA receptors (Tyler, Petzold et al. 2007). Moreover, deprivation during the neonate period also causes structural changes in the developing glomerulus. Without odor-evoked activity, OSNs fail to converge into unitary glomeruli and target to their correct positions within the olfactory bulb, often resulting in multiple, smaller glomeruli sharing the same OR (Zheng, Feinstein et al. 2000, Zou, Feinstein et al. 2004). Only one study to date has assayed the effects odor deprivation in adult rodents. This study found that adult mice may lack this compensatory plasticity: odor deprivation causes atrophy instead: a decrease in OSNs as well as decrease in glomerular size (Cavallin, Powell et al. 2010).
Global odor enrichment increases discrimination abilities in both neonates and adult rodents (Mandairon, Stack et al. 2006, Mandairon, Stack et al. 2006,
Mandairon, Stack et al. 2006, Woo, Hingco et al. 2007, Mandairon, Didier et al.
2008). In adults, this has been attributed to increased GABAergic inhibition by periglomerular cells. Structural changes also occur with deprivation. Odor enrichment causes number of OSNs as well as glomerular size to decrease in
38 adult animals (Cavallin, Powell et al. 2010) and leads to formation of supranumery glomeruli in neonates (Valle-Leija, Blanco-Hernandez et al. 2012).
In the insect AL, deprivation and enrichment also induces plasticity.
Severing OSN projections in Drosophila causes increased excitation within the glomerulus, due to a cell death/injury signal (Kazama, Yaksi et al. 2011).
Inactivity of OSNs causes compensatory up regulation of excitatory signaling via nAChRs followed by shaker potassium channels (Ping and Tsunoda 2012).
Following an period of deprivation, re-exposure to odors causes increases sensitivity to lower thresholds of these odors (Iyengar, Chakraborty et al. 2010).
Enrichment, the opposite manipulation, also induces plasticity. For example, honey bees experience dramatic functional and structural plasticity at the glomerulus when shifting roles from nursing bee to forging bee, an odor enrichment experience. Precocious induction of forging (and thus experience) in young bees increases glomerular volume and synapse density (Brown, Napper et al. 2002, Brown, Napper et al. 2004). Precocious foraging also has been temporally linked to increased performance in olfactory rewards associative
(sucrose-proboscis extension) tasks as well as increase in glomerular volume
(Sigg, Thompson et al. 1997). Recently studies have immerged that show that the refinement of glomerular circuits can be affected by experience (Tessier and
Broadie 2009).
Neuromodulators play key roles in learning and memory or sensory gating at central olfactory circuits: piriform cortex versus Mushroom Body
The literature about mechanisms of olfactory learning and memory in the higher processing centers such as the piriform cortex (PC) and mushroom body 39 (MB) is quite extensive, but more so for insects. Mechanisms for role of modulators are well elucidated at that stage of processing compared to the relatively, new frontier of the olfactory periphery. Though not the focus of this review, it is valuable to review this information since the mechanisms by which neuromodulator facilitate associative learning in the PF and MB can provide insights their roles they play in the OB and AL.
In rodent models, neuromodulators serotonin (5-HT), acetylcholine (ACh) and norepinephrine (NE) heavily innerve PC and affect both lateral olfactory tract
(LOT) afferents from the olfactory bulb as well as inter-cortical interactions between interneurons and pyramidal neurons. Meanwhile, octopamine (OA), DA and 5-HT from various distinct neuropil that resemble nuclei innervate the MB.
Thorough reviews in both rodent and insect systems (Davis 2005, Fletcher and
Chen 2010) suggests that all neuromodulators attempt to achieve the common goal enhancing sensory gating for odors that have meaning for the animal.
Acetylcholine permits formation of new memories and improves odor discrimination in the piriform cortex, but is just a excitatory transmitter in insects
Acetylcholine modulates piriform cortex (PC) circuits in several distinct ways.
First ACh from the (Horizontal Limb of the Diagonal Band of Broca) HDBL binds to mAChRs in the PC to facilitate the formation of new memories. Ach reduces pyramidal cell habituation to repeated stimulus, suppresses transmission between intrinsic neurons but not afferent synapses and facilitates LTP
(Hasselmo and Barkai 1995, Linster, Wyble et al. 1999, Barkai and Saar 2001)
The selectivity of Ach inhibition on intrinsic but not afferent fibers facilitates feed
40 forward information versus associative connections, and may be a mechanism to acquire new memories to replace old ones (interference) (De Rosa and
Hasselmo 2000). Ach also participates in odor discrimination of learned odors.
Lesioning the HDLB, disrupts odor discrimination and causes generalized odors in response to odorants that are meaningful to the animal (CS+ during associative learning (Linster, Garcia et al. 2001, Linster and Hasselmo 2001).
ACh may facilitate odor discrimination by prolonging the window for coincidence detection and thus spike timing dependent plasticity by decreased after- hyperpolarization in pyramidal neurons following reward learning (Saar,
Grossman et al. 1998, Saar, Grossman et al. 2001). Finally, it is interesting to note that Acetylcholine release into the piriform cortex is tightly controlled rate by the rate of information flow from the OB to the PC. Increased flow of information from the bulb via the LOT activates over 20% of cholinergic neurons in the HDLB
(Linster and Hasselmo 2000). Thus the degree of cholinergic modulation of the olfactory bulb and olfactory cortex may be controlled in real time by the incoming sensory inputs.
For insects, Acetylcholine is the major excitatory transmitter in insect central nervous systems via nAChRs similar to glutamate’s role in the vertebrate system.
Thus, ACh is not considered a neuromodulator in this context.
Norepinephrine increases signal to noise for learning in piriform cortex and octopamine is sufficient to convey the signal of reward in associative learning.
Norepinephrine (NE) fibers from the locus coeruleus widely innervate the piriform cortex, with heavy presence in the afferent fiber layer (layer I) as well as pyramidal layer (layer III). Over 45% of pyramidal cells respond to NE, either directly via β1adrenergic receptors or by the NE excitation of GABAergic 41 interneurons via α1 receptors (Gellman and Aghajanian 1993, Marek and
Aghajanian 1996). At low concentrations, NE excites synapses of from Mitral cell axons onto pyramidal neurons, while high concentrations induce inhibition to quiet the system (Collins, Probett et al. 1984). Stimulation of the locus coeruleus
(LC) during odor delivery improves sensory gating in the PC: over 60% of cells experience improved temporal resolutions and lower thresholds for firing (Bouret and Sara 2002). Overall the role of NE is similar to ACh signaling to increase signal to noise ratio of incoming information by favoring feed forward information at the expense of associations between pyramidal neurons (Hasselmo, Linster et al. 1997)
Octopamine enervates the MB from tightly defined clusters of cells called
DUM/VUM neurons. The role of octopamine in the MB is mainly to facilitate reward learning and memory. In Apis Mellifera, a subset of VUM neurons in the mushroom body is sufficient for appetitive (odor-sucrose paired0 associative learning and produces when stimulated during odor exposure, causes learned response of proboscis extension (Hammer, 1993). The octopaminergic reward signal has been shown to be prevalent in the MBs of several species (see below).
Dopamine in is a punishment signal in the mushroom body and but role in the piriform cortex is unknown.
In the piriform cortex, DA’s roles is described to be much like the other neuromodulators NE and 5-HT. DA increases firing rates of GABAergic interneurons that provide inhibition to pyramidal neurons (Gellman and
Aghajanian 1993).
42 In insects, DA as neuromodulator is usually presented in contrast to OA, since they seem to mediate opposite valences of associative learning in the MB.
In Drosophila, a specific set of dopaminergic neurons projecting to the MB confers the arousal of a noxious shock. Similar to the VUM neuron in honey bees, optogenetically stimulating these neurons is sufficient to produce aversive association with an odor (Claridge-Chang, Roorda et al. 2009) Conversely, drosophila that lack dopamine (due to a tyrosine dehydroxylase mutation) cannot learn to associate and odor with an aversive cue, but instead are masochists, and prefer odors that have been paired with shock as well as have a diminished preference for sucrose in general. Such learning deficits and perversions are rescued by administration of L-Dopa, a precursor to DA (Riemensperger, Isabel et al. 2011). In the honey bee, activation of D1 receptors are necessary for aversive olfactory learning (Kim, Lee et al. 2007). While both DA and OA participate in aversive and appetitive learning, respectively but both converge on the same cAMP intracellular signaling cascades(Schwaerzel, Monastirioti et al.
2003). In another Drosophila model, dopamine confers the arousal associated with hunger and appetite. DA neurons that modulate the MB receive inputs from
Neuropeptide (NPF) F neurons (mammalian homolog to Neuropeptide Y (NPY), which supply the cue for hunger and feeding. Manipulating the activity of these
NPF neurons adjusts the strength of the unconditioned stimulus (Krashes,
DasGupta et al. 2009).
Serotonin may be involved in regulating signal to noise in the piriform cortex and play a role in aversive associative learning in the mushroom body.
Serotonin’s projection patterns from the Dorsal Raphe Nucleus (DRN) to the
PC are similar to NE, with preference for layer I and layer II (Datiche, Luppi et al. 43 1995). Receptor subtypes 5-HT1/2 are heavily expressed in the piriform cortex
(Pompeiano, Palacios et al. 1992, Pompeiano, Palacios et al. 1994). In the PC, serotonin acts on 5-HT1 family receptors to enhance pyramidal cell activity while acting on 5HT2 receptors located on GABAergic interneurons to provide inhibitory tone (Aghajanian, Sprouse et al. 1990). At this time, no specific studies have been done on 5-HT’s role in olfactory learning and memory in the PC and thus this field would benefit from further investigation.
Serotonin innervates the mushroom body extensively from clusters of serotonergic neurons (Blenau and Thamm 2011) as well as from the DCG
(deuterocerebrum giant cell). Serotonin receptors (Apis AM-5-HT receptors and
Drosophila DM-5-HT receptors) are closely related to mammalian homologs (5-
HTβ=5HT2 and 5-HTα=5-HT1). Few studies have been done in this area except for a recent study that in drosophila, a serotonergic tone may be involved in the acquisition of amnesia resistant aversive associative learning (odor-foot shock)
(Lee, Lin et al. 2011).
Neuromodulators may be involved in learning and memory at the glomerulus
Although the role of neuromodulators in olfactory and learning and have been well established in higher association centers like the Mushroom Body (MB) in the insect and piriform cortex (PC) in the rodent, their role at the periphery is only emerging. For insects, biogenic amines dopamine, serotonin and octopamine innervate the AL and are dramatically higher in forgers than nursing bees regardless of age, suggesting that these modulators may not only be developmental regulated but also correspond to the experience of the animal 44 (Schulz and Robinson 1999). For mammals, differences across species exist, but in the rodent model, mapping studies show centrifugal fibers from the Locus
Coeruleus and Basal Forebrain most heavily innervate the granule cell layer to modulate mitral-granule synapses and lightly innervate the glomerular layer. On the other hand, both ACh and Dorsal Raphe 5-HT preferentially innervate the glomerular layer to modulate the OSN to Mitral, PG or ET synapses (Shepherd
1972, Halasz and Shepherd 1983, Gomez, Brinon et al. 2005, Matsutani and
Yamamoto 2008).
Dopamine‘s role in olfactory learning in memory in the antennal lobe is unknown, while rodents only have local but not centrifugal dopamine
Dopaminergic fibers innervate the ALs of honey bee ALs from the lateral deutocerebral spinal rind. Innervation corresponds to development of glomeruli, which was accelerated by DA (Kirchhof, Homberg et al. 1999).
Electrophysiological studies have shown that DA application to primary cultures of AL neurons reduces the outward current of less than half the neurons which is hypothesized to be a calcium activated potassium current (Perk and Mercer
2006). Honey bee dopamine receptors Amdrop1, 2, 3 have localized to the AL as well as drosophila dopamine receptors Dmdrop1, 2. While Amdrop1, 2 encodes for D1 like receptors that increase cAMP, Amdrop3 encode for D2 like receptors that decrease cAMP via Gi. (Blenau, Erber et al. 1998, Humphries, Mustard et al.
2003, Beggs, Hamilton et al. 2005). In culture, D2 like receptors develop in concert with glomerular neutrophil (Kirchhof and Mercer 1997). Histology shows that DA and GABA may be released from a subset of the same juxtaglomerular cells that receive synaptic inputs from antennal nerve neurons (Distler 1990).
This cooperation between GABA and DA may parallel vertebrate model. 45 Dopamine’s role for mediating punishment for fear associative learning in the MB has not been extended to the AL. To date no studies have been published studying the effects of DA on learning and memory at the AL level of peripheral processing.
Dopamine in the rodent OB is not centrifugal but rather intrinsic. A small population of periglomerular cells expresses tyrosine hydroxylase. They release dopamine which binds to presynaptic D2 receptors on OSNs for to provide feedback inhibition (Berkowicz and Trombley 2000, Ennis, Zhou et al. 2001). In this sense they act much like the other GABAergic population of periglomerular cells which act on GABAB receptors on OSNS to achieve the same effect. This local dopamine also serves to modulate the power of GABAergic inhibition via D1 receptors on granule cells and D2 receptors on mitral and ET cells. Both
GABAergic and dopaminergic populations of intrinsic neurons are born in the sub ventricular zone and replaced regularly (Betarbet, Zigova et al. 1996).
Modulation via D2 receptors alters odor discrimination as well as odor intensity in the glomerulus (Wilson and Sullivan 1995, Wei, Linster et al. 2006). In other species such as sheep and primates, there exists centrifugal dopaminergic enervation from dopaminergic nuclei such as the VTA (Levy, Meurisse et al.
1999).
Octopamine is involved in associative reward learning in the antennal lobe
Both Octopamine and Tyromine are the invertebrate versions of
Norepinephrine and Epinephrine respectively in the vertebrate system.
Octopamine is known to modulate every sensory modality of the insect. The source of octopamine are concentrated sets of neurons DUM and VUM(dorsal and ventral unpaired median) neurons (Roeder 2005). Though VUM is famous 46 for serving as the unconditioned stimulus signal during odor-reward associative learning in the MB (Hammer 1993), the role of OA in the AL is much less known.
OA innervates the AL and its receptor OAR1 resides in GABAergic interneurons of honeybee glomerulus as well as higher up in the calyces and mushroom body
(Sinakevitch, Mustard et al. 2011). In honey bees, OA seems to play a role in peripheral associative learning. Eliminating octopaminergic action in the AL via receptor antagonists or SiRNA of AmOARs disturb both acquisition and recall phases of sucrose-proboscis extension reward associative learning(Farooqui,
Robinson et al. 2003). Reward associative learning also causes a transient increase in PKA in the AL (Hildebrandt and Muller 1995) as does OA, which may be an early component of learning and memory.
Norepinephrine serves as the unconditioned stimulus in neonate olfactory imprinting at mitral-granule synapses of the olfactory bulb
Norepinephrine is the vertebrate counterpart of Octopamine. Norepinephrine
(NE)’s role in neonate odor imprinting and circuit plasticity of the deeper mitral- granule synapse of the OB has been dissected in detail. Neonate pups before
P10 can be reliably imprinted to an odor when that odor is forward paired with
(predicts) tactile stimulation that mimics mother’s stroking. NE arriving from the
LC is responsible for encoding the arousal or unconditioned stimulus conferred by the tactile stimulation (Sullivan, Wilson et al. 1994, Sullivan, Stackenwalt et al.
2000). NE is both necessary and sufficient to induce imprinting behavior by acting on β1 adrenergic receptors (Sullivan, Wilson et al. 1989, Sullivan, Zyzak et al. 1992, Sullivan, Wilson et al. 1994, Wilson, Pham et al. 1994, Sullivan,
47 Stackenwalt et al. 2000). Imprinted animals experience changes in mitral cell activity: greater ratio of inhibition versus excitation (Wilson, Sullivan et al. 1987,
Wilson and Leon 1988).
This olfactory imprinting uses classical learning and memory second cellular mechanism by increasing cAMP, PKA (decreasing phosphodiesterase) and pCREB which induces differential transcription (McLean, Harley et al. 1999,
Yuan, Harley et al. 2000, Yuan, Harley et al. 2003, McLean, Darby-King et al.
2005). This critical period for imprinting ends as locus coeruleus circuitry changes at weaning: adrenergic receptors turn over from α1 auto excitatory to α2 auto inhibitory, decreasing the NE surge to the olfactory bulb and thus the arousal valence of stroking (Moriceau and Sullivan 2004). However several adrenergic receptors are active in modulation in the bulb even after P14, and continue to modulate mitral-granule synapses, possibly playing a role in adult learning and memory.
Recently neural plasticity associated with imprinting has been suggested to exist in glomerular synapses as well. Norepinephrine and its receptors β1 have been found in in the glomerular layer and receptors increase expression in development (Woo and Leon 1995). Although β1 adrenergic receptors are known to be located mitral cells from the previous imprinting studies, and recently, this has plasticity has been also found in the glomerulus (Yuan, Harley et al. 2003, Yuan 2009, Yuan and Harley 2012)
48 Serotonin modulates gating in the antennal lobe glomeruli, and play a facilitator role in the olfactory for neonate learning and memory at deep mitral-granule synapse
The role of serotonin on olfactory processing behavior and processing insects has been well reviewed (Kloppenburg and Mercer 2008). In well the studied Manduca Sexta, a unique serotonin neuron (5-HT_IRN) resides in each antenna lobe project to the contralateral lobe as well as higher processing centers such as the protocerebrum including the mushroom bodies which then extends an axon back into the contralateral lobe. Serotonin fibers sparsely innervates each glomeruli from early in development to adulthood (Kent, Hoskins et al. 1987). 5-HT fibers appear to innervate cell bodies in the glomerular region but spare the incoming OSNs, a pattern that is similar in rodents. In contralateral lobe, the 5-HT fibers make many output synapses as well as a few input synapses with the interneurons within each glomerulus. This suggests that 5-HT controls intraglomerular dynamics and receive feedback from them (Sun, Tolbert et al. 1993).
Overall 5-HT increases the responsiveness of the AL glomerulus to odors. In culture, AL neurons show acute effects of 5-HT on AL neurons: a reduction the
K+ currents, both rapidly and slowly activating subtypes (Mercer, Hayashi et al.
1995), which resulted in macroglomerular neurons being more easily depolarizable by weak odors (Kloppenburg, Ferns et al. 1999). 5-HT also increased the frequencies of action potentials, broadened action potentials and increased input resistance of AL cultured neurons, changing the excitability is attributed to regulation of 3 types of potassium currents (Mercer, Kloppenburg et al. 1996)
49 In vivo, electrophysiology and in vivo imaging studies confirm this phenomenon. Field recordings from M. Sexta glomeruli indicate that 5-HT increased the magnitude and duration of glomerular Local Field Potentials
(LFP)s (Kloppenburg and Heinbockel 2000). Voltage sensitive dyes report that
5H-T increase the response of the post-synaptic cells of the glomerulus during
OSN stimulation (Hill, Okada et al. 2003). In drosophila, 5-HT increase firing rates, firing duration and magnitude of these cells of the AL glomerulus as well as enhances cross-correlation between units. Serotonin shifts the threshold of response to a lower concentrations for some units, while for others increasing gain by shifting the dose-response curve for other units(Dacks, Christensen et al.
2008). Calcium imaging showed that 5-HT works similarly in rodents and insects.
It increases activity of some PN cells as well as GABAergic interneurons to reduce OSN release via GABAB receptors (Dacks, Christensen et al. 2008,
Dacks, Green et al. 2009). Despite the knowledge of the acute effects of 5-HT in the AL, no studies to date have implicated 5-HT to play a role in learning and memory at the glomerulus of the AL.
In the vertebrate olfactory bulb, 5-HT fibers preferentially innervate the glomerular layer more than any other layer of the bulb, and represents one of the heaviest recipients of 5-HT in the entire brain (Gomez, Brinon et al. 2005). Both
5-HT1 as well as 5-HT2 receptors all expressed in the OB. The 5-HT1 family resides mostly on the granule cell layer (McLean, Darby-King et al. 1995).
Meanwhile, expression of the 5-HT2AR receptor is heaviest in the glomerular layer, with receptors preferentially located on mitral cells and juxtaglomerular cells as well as the middle tufted cells while receptors are present in the
50 glomerular layer, absent in the EPL and abundant in the granule cell layer
(Pompeiano, Palacios et al. 1994).
Functionally, serotonin modulates all elements of glomerular microcircuits.
Serotonin acts on both mitral cells directly as well as indirectly via its actions on both GABAergic (PG) and glutamatergic (ET) interneurons. Serotonin depolarizes a third of PG cells via 5-HT2C receptors, which in turn in inhibit mitral cells via GABAAR. Serotonin also depolarizes and increases spiking of ET glutamatergic interneurons via 5-HT2A receptors (Liu, Aungst et al. 2012), which in turn drives both PG and Mitral cells and synchronizes excitation and inhibition in the circuit. Finally, 5-HT2A receptors are also located on mitral cells where they depolarize mitral cells more directly (Hardy, Palouzier-Paulignan et al.
2005). An influx of serotonin from the DRN increases the activity of juxtaglomerular cells at least partially via 5-HT2C receptors. The increase in
GABAergic inhibition by interneurons suppresses OSN release via GABAB receptors, closely resembling the Drosophila model (Petzold, Hagiwara et al.
2009).
Serotonin’s role in olfactory learning and memory has only been studied with respect at deeper mitral-granule synapses during imprinting. Though NE is the main player by providing the necessary and sufficient signal for unconditioned stimulus, imprinting behavior, 5-HT may play a facilitator but not necessary role
(Price, Darby-King et al. 1998). When 5-HT is depleted, β1adrenergic agonists or downstream signaling molecules such as pCREB can rescue the behavior.
Serotonin seems to work via 5-HT2A receptors which co-localize with βadrenergic receptors at mitral cells to potentiate the NE response (McLean, Darby-King et al.
51 1993, McLean, Darby-King et al. 1996, Price, Darby-King et al. 1998, Yuan,
Harley et al. 2000, Yuan, Harley et al. 2003).
Serotonin’s preferential innervation of glomerular layer makes it a prime candidate to facilitate associative learning at the earliest stages of information processing as well. Recall that, 5-HT, as reviewed, is a classical transmitter used to convey arousal in other simpler organisms during associative learning and memory. Few studies have been about the role of 5-HT, or any other neuromodulator, for that matter on learning and memory at glomerular synapses of OB. In fact, few studies studied learning and memory at the glomerulus, especially in adults.
Associative learning induced plasticity in the antennal lobe versus olfactory bulb
Most of the literature concerning neuromodulators in the AL is limited describing to its functions modulating glomerular responses. Only a few studies have linked neuromodulators to learning and memory the glomeruli in the AL or to the Mitral-Granule processing station in the OB. Evidence on the presence of learning-dependent plasticity in the glomeruli of the AL and OB is fairly extensive, though I barely know the roles that neuromodulators play in the process. In the insect AL, both reward and fear associative learning in insects have been shown to induce glomerular plasticity. For reward learning, honeybees who learn to associate odor with sucrose show greater glomerular responses to reward- associated odors, next largest responses to novel odors and the smallest responses to familiar odors (Faber, Joerges et al. 1999). This is good example of how sensory gating may be altered in the first synapse based on the meaning of
52 the odor. Moreover, reward odor glomeruli decor related their activity to non- reward glomeruli to make their responses more distinct (Faber, Joerges et al.
1999). Similarly, in M. Sexta, in vivo electrophysiology of glomerular units show recruitment of additional glomerular units in response to the learned odor and decreased number of units responding to non-meaningful familiar odors as well as changes in temporal profiles of learned odors(Daly, Christensen et al. 2004).
In the opposite valence, Odor-shock learning in Drosophila results in rapid recruitment of additional projection neurons to responding to that odor, broadening the responsive glomerular field of the learned odor, increase in OSNs transmitter release (Yu, Ponomarev et al. 2004). Classical NMDAR mechanisms are suspected to be a mechanism in the process since disruption of genes that reduce NR1 NMDAR subunits specifically in the AL attenuates fear associative learning (Tamura, Horiuchi et al. 2010).
In the rodent olfactory glomerulus, learning dependent plasticity has not been in the adult animal. Since the glomerulus encoding a learned odor shows structural plasticity after associative learning in adults(Jones, Choi et al. 2008), this gives hope that functional plasticity may also exist here at the first synapse. I suggest that some kind of activity dependent signal arising from post-synaptic cells within the glomerulus is facilitating the structural plasticity of OSNs specifically at the odor-learning glomerulus. In neonates, LTP and LTD can be induced by classical modes of theta stimulation paired with NE but have not been induced by natural experience such as learning and memory (Mutoh, Yuan et al.
2005, Yuan 2009). In this case, the mitral cell may serve as a site of integration for OSNs carrying the Cs+ signal arriving simultaneous as the US conferred by a centrifugal modulator. In adults, no evidence of functional, learning-dependent
53 plasticity or even global experience deponent plasticity has been shown. The studies presented in this thesis address this problem of functional plasticity in the adult olfactory glomerulus following associative learning.
Conclusion: common themes and translation
Common themes between rodent and insect models
My analysis of the role of neuromodulators in experience and learning- dependent plasticity in the antennal love of insects and the olfactory bulb of rodents reveals many common themes. , Although I did find divergences in of circuit organization, signal transduction as well as neuromodulators specific to each group, the principles and rules governing odor processing as well as the role of neuromodulators are surprisingly similar. It is still difficult to believe that the insect and mammalian systems did not develop from common ancestry, but instead convergent evolution: thus independently arriving at the same efficient solution for the same problems of sensory processing (Bargmann 2006).
At higher olfactory synapses in the Mushroom body of insects use centrifugal neuromodulators to confer the US of associative learning for both positive and negative contexts using dopamine and octopamine via multiple receptors subtypes. Similarly, in the deep mitral-granule synapse of the OB, the
OA’s vertebrate counterpart Norepinephrine plays similar rules.
Second, both these types of plasticity involve the cAMP, PKA, pCREB classical cascade to store learning-dependent associative olfactory memories.
These are shared mechanisms first found in studied in Aplysia (McLean, Harley et al. 1999, Yuan, Harley et al. 2000, Yuan, Harley et al. 2003, Honjo and
54 Furukubo-Tokunaga 2009) and later further developed in rodent hippocampal models.
Third, very few studies have investigated the role of centrifugal modulators in glomerulus of the AL and OB. Thus at this point in time, I cannot decisively conclude that neuromodulators play a key role at this most peripheral synapse like in slightly more central synapses. However, large amounts of supporting evidence are accumulating. Centrifugal modulators not only heavily innervate the glomerular layer, but play important roles in modulating the synapses of all cell types within the glomerulus both at the pre and post synaptic side. Both in insects and in rodents, neuromodulators may act simultaneously on relay neurons as well as interneurons to produce the desired sensory gating effect. In parallel to this body of literature, exciting new findings show that associative learning dependent plasticity may exist at the glomerular synapses of both the insect AL and rodent. This plasticity still needs to be verified across age groups not just in development, but also in adulthood. Although the mechanisms of this plasticity have yet to be investigated, neuromodulators present themselves as the most obvious candidate.
Future studies should focus on this exciting new possibility of neuromodulator-dependent learning and memory the olfactory periphery, especially since much of the groundwork has already been done. To achieve the most interpretable results, one may wish to use a genetically identified circuit that allows for precise activation with odors and precise interrogation guided by a reporter. Alternately, one can take advantage of the many activity dependent probes for stimulation or imaging activity both in vitro and in vivo in awake, behaving animals.
55
Evolutionarily shared principals and translation?
Even subjectively, one often knows that olfactory memories are strongly affective. What was first described as the Proustian effect, memories, often very old ones, can be initiated by gustatory or olfactory cues and affect strong emotional states. Empirical evidence for this phenomenon has been thoroughly reviewed (Chu and Downes 2000). Reports are that these strong memories brought on by olfactory cues almost never neutral (almost always has positive or negative valence) and are skewed towards memories obtained during childhood rather adulthood.
Can the principles and rules I learned from peripheral olfactory learning and memory be applied to primates and humans? Even between insects and rodent olfactory systems, with their evolutionary different origins, I see abundant similarities in principles and rules outline above. The translation between rodents to primates and humans should not be a larger leap. Non-human primate and human olfactory systems have been shown to have similar anatomical structure, centrifugal neuromodulator innervation, including in the olfactory bulb (Smith,
Baker et al. 1993). The human piriform cortex show experience mediated plasticity: such as habituation and increased ability for discrimination in response to odor exposure (Li, Luxenberg et al. 2006). Unimodal regions of olfactory processing such as piriform cortex contain traces of associative memories between olfactory input and visual input (Gottfried, Smith et al. 2004). Thus, the mechanisms for learning-dependent sensory input gain that I find in insect and rodent models may be well conserved in primates and primates.
56 Learning-dependent sensory plasticity at the first synapse may be essential for this task of assigning meanings to cues, without which the organism cannot make sense of the external world. Further studies in insects and rodents will provide insights into disease states that suffer from improper sensory gating, specifically schizophrenic disorders. Without the plasticity to optimize sensory gating by learning, one would face a fragmented world with hallucinations, anxiety, paranoia, and possibly delusional cognition. Clarifying the mechanisms of abnormal sensory gating in both models and a through comparison will provide insight how to therapeutically attack this problem.
57 REFERENCES
Aghajanian, G. K., J. S. Sprouse, P. Sheldon and K. Rasmussen (1990). "Electrophysiology of the central serotonin system: receptor subtypes and transducer mechanisms." Ann N Y Acad Sci 600: 93-103; discussion 103.
Bargmann, C. I. (2006). "Comparative chemosensation from receptors to ecology." Nature 444(7117): 295-301.
Barkai, E. and D. Saar (2001). "Cellular correlates of olfactory learning in the rat piriform cortex." Rev Neurosci 12(2): 111-120.
Beggs, K. T., I. S. Hamilton, P. T. Kurshan, J. A. Mustard and A. R. Mercer (2005). "Characterization of a D2-like dopamine receptor (AmDOP3) in honey bee, Apis mellifera." Insect Biochem Mol Biol 35(8): 873-882.
Belluscio, L., C. Lodovichi, P. Feinstein, P. Mombaerts and L. C. Katz (2002). "Odorant receptors instruct functional circuitry in the mouse olfactory bulb." Nature 419: 296-300.
Berkowicz, D. A. and P. Q. Trombley (2000). "Dopaminergic modulation at the olfactory nerve synapse." Brain Res 855(1): 90-99.
Betarbet, R., T. Zigova, R. A. Bakay and M. B. Luskin (1996). "Dopaminergic and GABAergic interneurons of the olfactory bulb are derived from the neonatal subventricular zone." Int J Dev Neurosci 14(7-8): 921-930.
Bhandawat, V., S. R. Olsen, N. W. Gouwens, M. L. Schlief and R. I. Wilson (2007). "Sensory processing in the Drosophila antennal lobe increases reliability and separability of ensemble odor representations." Nat Neurosci 10(11): 1474-1482.
Blenau, W., J. Erber and A. Baumann (1998). "Characterization of a dopamine D1 receptor from Apis mellifera: cloning, functional expression, pharmacology, and mRNA localization in the brain." J Neurochem 70(1): 15-23.
Blenau, W. and M. Thamm (2011). "Distribution of serotonin (5-HT) and its receptors in the insect brain with focus on the mushroom bodies: lessons from Drosophila melanogaster and Apis mellifera." Arthropod Struct Dev 40(5): 381-394.
Bouret, S. and S. J. Sara (2002). "Locus coeruleus activation modulates firing rate and temporal organization of odour-induced single-cell responses in rat piriform cortex." Eur J Neurosci 16(12): 2371-2382.
Brennan, P. A. and K. M. Kendrick (2006). "Mammalian social odours: attraction and individual recognition." Philos Trans R Soc Lond B Biol Sci 361(1476): 2061-2078.
58 Brown, S. M., R. M. Napper and A. R. Mercer (2004). "Foraging experience, glomerulus volume, and synapse number: A stereological study of the honey bee antennal lobe." J Neurobiol 60(1): 40-50.
Brown, S. M., R. M. Napper, C. M. Thompson and A. R. Mercer (2002). "Stereological analysis reveals striking differences in the structural plasticity of two readily identifiable glomeruli in the antennal lobes of the adult worker honeybee." J Neurosci 22(19): 8514-8522.
Buck, L. B. and R. Axel (1991). "A novel multigene family may encode odorant receptors: a molecular basis for odor recognition." Cell 65: 175-187.
Carlsson, M. A., M. Diesner, J. Schachtner and D. R. Nassel (2010). "Multiple neuropeptides in the Drosophila antennal lobe suggest complex modulatory circuits." J Comp Neurol 518(16): 3359-3380.
Cavallin, M. A., K. Powell, K. C. Biju and D. A. Fadool (2010). "State-dependent sculpting of olfactory sensory neurons is attributed to sensory enrichment, odor deprivation, and aging." Neurosci Lett 483(2): 90-95.
Chou, Y. H., M. L. Spletter, E. Yaksi, J. C. Leong, R. I. Wilson and L. Luo (2010). "Diversity and wiring variability of olfactory local interneurons in the Drosophila antennal lobe." Nat Neurosci 13(4): 439-449.
Chu, S. and J. J. Downes (2000). "Odour-evoked autobiographical memories: psychological investigations of proustian phenomena." Chem Senses 25(1): 111-116.
Claridge-Chang, A., R. D. Roorda, E. Vrontou, L. Sjulson, H. Li, J. Hirsh and G. Miesenbock (2009). "Writing memories with light-addressable reinforcement circuitry." Cell 139(2): 405-415.
Cleland, T. A. (2010). "Early transformations in odor representation." Trends Neurosci 33(3): 130-139.
Collins, G. G., G. A. Probett, J. Anson and N. J. McLaughlin (1984). "Excitatory and inhibitory effects of noradrenaline on synaptic transmission in the rat olfactory cortex slice." Brain Res 294(2): 211-223.
Couto, A., M. Alenius and B. J. Dickson (2005). "Molecular, anatomical, and functional organization of the Drosophila olfactory system." Curr Biol 15(17): 1535-1547.
Dacks, A. M., T. A. Christensen and J. G. Hildebrand (2008). "Modulation of olfactory information processing in the antennal lobe of Manduca sexta by serotonin." J Neurophysiol 99(5): 2077-2085.
Dacks, A. M., D. S. Green, C. M. Root, A. J. Nighorn and J. W. Wang (2009). "Serotonin modulates olfactory processing in the antennal lobe of Drosophila." J Neurogenet 23(4): 366-377.
59 Daly, K. C., T. A. Christensen, H. Lei, B. H. Smith and J. G. Hildebrand (2004). "Learning modulates the ensemble representations for odors in primary olfactory networks." Proc Natl Acad Sci U S A 101(28): 10476-10481.
Datiche, F., P. H. Luppi and M. Cattarelli (1995). "Serotonergic and non- serotonergic projections from the raphe nuclei to the piriform cortex in the rat: a cholera toxin B subunit (CTb) and 5-HT immunohistochemical study." Brain Res 671(1): 27-37.
Davis, R. L. (2005). "Olfactory memory formation in Drosophila: from molecular to systems neuroscience." Annu Rev Neurosci 28: 275-302.
De Rosa, E. and M. E. Hasselmo (2000). "Muscarinic cholinergic neuromodulation reduces proactive interference between stored odor memories during associative learning in rats." Behav Neurosci 114(1): 32- 41.
Distler, P. (1990). "Synaptic connections of dopamine-immunoreactive neurons in the antennal lobes of Periplaneta americana. Colocalization with GABA- like immunoreactivity." Histochemistry 93(4): 401-408.
Doty, R. L. (1986). "Odor-guided behavior in mammals." Experientia 42(3): 257- 271.
Ennis, M., F. M. Zhou, K. J. Ciombor, V. Aroniadou-Anderjaska, A. Hayar, E. Borrelli, L. A. Zimmer, F. Margolis and M. T. Shipley (2001). "Dopamine D2 receptor-mediated presynaptic inhibition of olfactory nerve terminals." J Neurophysiol 86(6): 2986-2997.
Faber, T., J. Joerges and R. Menzel (1999). "Associative learning modifies neural representations of odors in the insect brain." Nat Neurosci 2(1): 74-78.
Farooqui, T., K. Robinson, H. Vaessin and B. H. Smith (2003). "Modulation of early olfactory processing by an octopaminergic reinforcement pathway in the honeybee." J Neurosci 23(12): 5370-5380.
Fishilevich, E. and L. B. Vosshall (2005). "Genetic and functional subdivision of the Drosophila antennal lobe." Curr Biol 15(17): 1548-1553.
Fletcher, M. L. and W. R. Chen (2010). "Neural correlates of olfactory learning: Critical role of centrifugal neuromodulation." Learn Mem 17(11): 561-570.
Franks, K. M., M. J. Russo, D. L. Sosulski, A. A. Mulligan, S. A. Siegelbaum and R. Axel (2011). "Recurrent circuitry dynamically shapes the activation of piriform cortex." Neuron 72(1): 49-56.
Galizia, C. G. and R. Menzel (2000). "Odour perception in honeybees: coding information in glomerular patterns." Curr Opin Neurobiol 10(4): 504-510.
60 Gellman, R. L. and G. K. Aghajanian (1993). "Pyramidal cells in piriform cortex receive a convergence of inputs from monoamine activated GABAergic interneurons." Brain Res 600(1): 63-73.
Gire, D. H. and N. E. Schoppa (2009). "Control of on/off glomerular signaling by a local GABAergic microcircuit in the olfactory bulb." J Neurosci 29(43): 13454-13464.
Gomez, C., J. G. Brinon, M. V. Barbado, E. Weruaga, J. Valero and J. R. Alonso (2005). "Heterogeneous targeting of centrifugal inputs to the glomerular layer of the main olfactory bulb." J Chem Neuroanat 29(4): 238-254.
Gottfried, J. A., A. P. Smith, M. D. Rugg and R. J. Dolan (2004). "Remembrance of odors past: human olfactory cortex in cross-modal recognition memory." Neuron 42(4): 687-695.
Gray, C. M. and J. E. Skinner (1988). "Centrifugal regulation of neuronal activity in the olfactory bulb of the waking rabbit as revealed by reversible cryogenic blockade." Exp Brain Res 69(2): 378-386.
Guthrie, K. M., J. M. Pullara, J. F. Marshall and M. Leon (1991). "Olfactory deprivation increases dopamine D2 receptor density in the rat olfactory bulb." Synapse 8(1): 61-70.
Guthrie, K. M., D. A. Wilson and M. Leon (1990). "Early unilateral deprivation modifies olfactory bulb function." J Neurosci 10(10): 3402-3412.
Halasz, N. and G. M. Shepherd (1983). "Neurochemistry of the vertebrate olfactory bulb." Neuroscience 10(3): 579-619.
Hammer, M. (1993). "An Identified Neuron Mediates the Unconditioned Stimulus in Associative Olfactory Learning in Honeybees." Nature 366(6450): 59- 63.
Hardy, A., B. Palouzier-Paulignan, A. Duchamp, J. P. Royet and P. Duchamp- Viret (2005). "5-Hydroxytryptamine action in the rat olfactory bulb: in vitro electrophysiological patch-clamp recordings of juxtaglomerular and mitral cells." Neuroscience 131(3): 717-731.
Hasselmo, M. E. and E. Barkai (1995). "Cholinergic modulation of activity- dependent synaptic plasticity in the piriform cortex and associative memory function in a network biophysical simulation." J Neurosci 15(10): 6592-6604.
Hasselmo, M. E., C. Linster, M. Patil, D. Ma and M. Cekic (1997). "Noradrenergic suppression of synaptic transmission may influence cortical signal-to- noise ratio." J Neurophysiol 77(6): 3326-3339.
61 Hayar, A. and M. Ennis (2007). "Endogenous GABA and glutamate finely tune the bursting of olfactory bulb external tufted cells." J Neurophysiol 98(2): 1052-1056.
Hayar, A., S. Karnup, M. Ennis and M. T. Shipley (2004). "External tufted cells: a major excitatory element that coordinates glomerular activity." J Neurosci 24(30): 6676-6685.
Hayar, A., S. Karnup, M. T. Shipley and M. Ennis (2004). "Olfactory bulb glomeruli: external tufted cells intrinsically burst at theta frequency and are entrained by patterned olfactory input." J Neurosci 24(5): 1190-1199.
Hayar, A., M. T. Shipley and M. Ennis (2005). "Olfactory bulb external tufted cells are synchronized by multiple intraglomerular mechanisms." J Neurosci 25(36): 8197-8208.
Hildebrand, J. G., T. A. Christensen, I. D. Harrow, U. Homberg, S. G. Matsumoto and B. R. Waldrop (1992). "The roles of local interneurons in the processing of olfactory information in the antennal lobes of the moth Manduca sexta." Acta Biol Hung 43(1-4): 167-174.
Hildebrandt, H. and U. Muller (1995). "Octopamine mediates rapid stimulation of protein kinase A in the antennal lobe of honeybees." J Neurobiol 27(1): 44-50.
Hill, E. S., K. Okada and R. Kanzaki (2003). "Visualization of modulatory effects of serotonin in the silkmoth antennal lobe." J Exp Biol 206(Pt 2): 345-352.
Honjo, K. and K. Furukubo-Tokunaga (2009). "Distinctive neuronal networks and biochemical pathways for appetitive and aversive memory in Drosophila larvae." J Neurosci 29(3): 852-862.
Humphries, M. A., J. A. Mustard, S. J. Hunter, A. Mercer, V. Ward and P. R. Ebert (2003). "Invertebrate D2 type dopamine receptor exhibits age- based plasticity of expression in the mushroom bodies of the honeybee brain." J Neurobiol 55(3): 315-330.
Iyengar, A., T. S. Chakraborty, S. P. Goswami, C. F. Wu and O. Siddiqi (2010). "Post-eclosion odor experience modifies olfactory receptor neuron coding in Drosophila." Proc Natl Acad Sci U S A 107(21): 9855-9860.
Johansen, J. P., C. K. Cain, L. E. Ostroff and J. E. LeDoux (2011). "Molecular mechanisms of fear learning and memory." Cell 147(3): 509-524.
Jones, S. V., D. C. Choi, M. Davis and K. J. Ressler (2008). "Learning-dependent structural plasticity in the adult olfactory pathway." J Neurosci 28(49): 13106-13111.
Kay, L. M. and S. M. Sherman (2007). "An argument for an olfactory thalamus." Trends Neurosci 30(2): 47-53.
62 Kazama, H., E. Yaksi and R. I. Wilson (2011). "Cell death triggers olfactory circuit plasticity via glial signaling in Drosophila." J Neurosci 31(21): 7619-7630.
Kent, K. S., S. G. Hoskins and J. G. Hildebrand (1987). "A novel serotonin- immunoreactive neuron in the antennal lobe of the sphinx moth Manduca sexta persists throughout postembryonic life." J Neurobiol 18(5): 451-465.
Kim, Y. C., H. G. Lee and K. A. Han (2007). "D1 dopamine receptor dDA1 is required in the mushroom body neurons for aversive and appetitive learning in Drosophila." J Neurosci 27(29): 7640-7647.
Kirchhof, B. S., U. Homberg and A. R. Mercer (1999). "Development of dopamine-immunoreactive neurons associated with the antennal lobes of the honey bee, Apis mellifera." J Comp Neurol 411(4): 643-653.
Kirchhof, B. S. and A. R. Mercer (1997). "Antennal lobe neurons of the honey bee, Apis mellifera, express a D2-like dopamine receptor in vitro." J Comp Neurol 383(2): 189-198.
Kiselycznyk, C. L., S. Zhang and C. Linster (2006). "Role of centrifugal projections to the olfactory bulb in olfactory processing." Learn Mem 13(5): 575-579.
Kloppenburg, P., D. Ferns and A. R. Mercer (1999). "Serotonin enhances central olfactory neuron responses to female sex pheromone in the male sphinx moth manduca sexta." J Neurosci 19(19): 8172-8181.
Kloppenburg, P. and T. Heinbockel (2000). "5-Hydroxy-tryptamine modulates pheromone-evoked local field potentials in the macroglomerular complex of the sphinx moth Manduca sexta." J Exp Biol 203(Pt 11): 1701-1709.
Kloppenburg, P. and A. R. Mercer (2008). "Serotonin modulation of moth central olfactory neurons." Annu Rev Entomol 53: 179-190.
Krashes, M. J., S. DasGupta, A. Vreede, B. White, J. D. Armstrong and S. Waddell (2009). "A neural circuit mechanism integrating motivational state with memory expression in Drosophila." Cell 139(2): 416-427.
Kurahashi, T. and K. W. Yau (1994). "Olfactory transduction. Tale of an unusual chloride current." Curr Biol 4(3): 256-258.
Larsson, M. C., A. I. Domingos, W. D. Jones, M. E. Chiappe, H. Amrein and L. B. Vosshall (2004). "Or83b encodes a broadly expressed odorant receptor essential for Drosophila olfaction." Neuron 43(5): 703-714.
LeDoux, J. E. (2000). "Emotion circuits in the brain." Annu Rev Neurosci 23: 155- 184.
Lee, P. T., H. W. Lin, Y. H. Chang, T. F. Fu, J. Dubnau, J. Hirsh, T. Lee and A. S. Chiang (2011). "Serotonin-mushroom body circuit modulating the
63 formation of anesthesia-resistant memory in Drosophila." Proc Natl Acad Sci U S A 108(33): 13794-13799.
Levy, F., M. Meurisse, G. Ferreira, J. Thibault and Y. Tillet (1999). "Afferents to the rostral olfactory bulb in sheep with special emphasis on the cholinergic, noradrenergic and serotonergic connections." J Chem Neuroanat 16(4): 245-263.
Li, W., E. Luxenberg, T. Parrish and J. A. Gottfried (2006). "Learning to smell the roses: experience-dependent neural plasticity in human piriform and orbitofrontal cortices." Neuron 52(6): 1097-1108.
Linster, C., P. A. Garcia, M. E. Hasselmo and M. G. Baxter (2001). "Selective loss of cholinergic neurons projecting to the olfactory system increases perceptual generalization between similar, but not dissimilar, odorants." Behav Neurosci 115(4): 826-833.
Linster, C. and M. E. Hasselmo (2000). "Neural activity in the horizontal limb of the diagonal band of broca can be modulated by electrical stimulation of the olfactory bulb and cortex in rats." Neurosci Lett 282(3): 157-160.
Linster, C. and M. E. Hasselmo (2001). "Neuromodulation and the functional dynamics of piriform cortex." Chem Senses 26(5): 585-594.
Linster, C., B. P. Wyble and M. E. Hasselmo (1999). "Electrical stimulation of the horizontal limb of the diagonal band of broca modulates population EPSPs in piriform cortex." J Neurophysiol 81(6): 2737-2742.
Liu, S., J. L. Aungst, A. C. Puche and M. T. Shipley (2012). "Serotonin modulates the population activity profile of olfactory bulb external tufted cells." J Neurophysiol 107(1): 473-483.
Mandairon, N., A. Didier and C. Linster (2008). "Odor enrichment increases interneurons responsiveness in spatially defined regions of the olfactory bulb correlated with perception." Neurobiol Learn Mem 90(1): 178-184.
Mandairon, N., C. Stack, C. Kiselycznyk and C. Linster (2006). "Broad activation of the olfactory bulb produces long-lasting changes in odor perception." Proc Natl Acad Sci U S A 103(36): 13543-13548.
Mandairon, N., C. Stack, C. Kiselycznyk and C. Linster (2006). "Enrichment to odors improves olfactory discrimination in adult rats." Behav Neurosci 120(1): 173-179.
Mandairon, N., C. Stack and C. Linster (2006). "Olfactory enrichment improves the recognition of individual components in mixtures." Physiol Behav 89(3): 379-384.
64 Marek, G. J. and G. K. Aghajanian (1996). "Alpha 1B-adrenoceptor-mediated excitation of piriform cortical interneurons." Eur J Pharmacol 305(1-3): 95- 100.
Masse, N. Y., G. C. Turner and G. S. Jefferis (2009). "Olfactory information processing in Drosophila." Curr Biol 19(16): R700-713.
Matsutani, S. and N. Yamamoto (2008). "Centrifugal innervation of the mammalian olfactory bulb." Anat Sci Int 83(4): 218-227.
McLean, J. H., A. Darby-King and C. W. Harley (2005). "Potentiation and prolongation of long-term odor memory in neonate rats using a phosphodiesterase inhibitor." Neuroscience 135(2): 329-334.
McLean, J. H., A. Darby-King and E. Hodge (1996). "5-HT2 receptor involvement in conditioned olfactory learning in the neonate rat pup." Behav Neurosci 110(6): 1426-1434.
McLean, J. H., A. Darby-King and G. D. Paterno (1995). "Localization of 5-HT2A receptor mRNA by in situ hybridization in the olfactory bulb of the postnatal rat." J Comp Neurol 353(3): 371-378.
McLean, J. H., A. Darby-King, R. M. Sullivan and S. R. King (1993). "Serotonergic influence on olfactory learning in the neonate rat." Behav Neural Biol 60(2): 152-162.
McLean, J. H., C. W. Harley, A. Darby-King and Q. Yuan (1999). "pCREB in the neonate rat olfactory bulb is selectively and transiently increased by odor preference-conditioned training." Learn Mem 6(6): 608-618.
Meister, M. and T. Bonhoeffer (2001). "Tuning and topography in an odor map on the rat olfactory bulb." J Neurosci 21(4): 1351-1360.
Mercer, A. R., J. H. Hayashi and J. G. Hildebrand (1995). "Modulatory effects of 5-hydroxytryptamine on voltage-activated currents in cultured antennal lobe neurones of the sphinx moth Manduca sexta." J Exp Biol 198(Pt 3): 613-627.
Mercer, A. R., P. Kloppenburg and J. G. Hildebrand (1996). "Serotonin-induced changes in the excitability of cultured antennal-lobe neurons of the sphinx moth Manduca sexta." J Comp Physiol A 178(1): 21-31.
Mombaerts, P. (1999). "Seven-transmembrane proteins as odorant and chemosensory receptors." Science 286(5440): 707-711.
Mombaerts, P., F. Wang, C. Dulac, S. K. Chao, A. Nemes, M. Mendelsohn, J. Edmondson and R. Axel (1996). "Visualizing an olfactory sensory map." Cell 87(4): 675-686.
65 Mori, K., H. Nagao and Y. Yoshihara (1999). "The olfactory bulb: coding and processing of odor molecule information." Science 286(5440): 711-715.
Moriceau, S. and R. M. Sullivan (2004). "Unique neural circuitry for neonatal olfactory learning." J Neurosci 24(5): 1182-1189.
Mutoh, H., Q. Yuan and T. Knopfel (2005). "Long-term depression at olfactory nerve synapses." J Neurosci 25(17): 4252-4259.
Nassel, D. R. (2002). "Neuropeptides in the nervous system of Drosophila and other insects: multiple roles as neuromodulators and neurohormones." Prog Neurobiol 68(1): 1-84.
Perk, C. G. and A. R. Mercer (2006). "Dopamine modulation of honey bee (Apis mellifera) antennal-lobe neurons." J Neurophysiol 95(2): 1147-1157.
Petzold, G. C., A. Hagiwara and V. N. Murthy (2009). "Serotonergic modulation of odor input to the mammalian olfactory bulb." Nat Neurosci.
Ping, Y. and S. Tsunoda (2012). "Inactivity-induced increase in nAChRs upregulates Shal K(+) channels to stabilize synaptic potentials." Nat Neurosci 15(1): 90-97.
Pompeiano, M., J. M. Palacios and G. Mengod (1992). "Distribution and cellular localization of mRNA coding for 5-HT1A receptor in the rat brain: correlation with receptor binding." J Neurosci 12(2): 440-453.
Pompeiano, M., J. M. Palacios and G. Mengod (1994). "Distribution of the serotonin 5-HT2 receptor family mRNAs: comparison between 5-HT2A and 5-HT2C receptors." Brain Res Mol Brain Res 23(1-2): 163-178.
Price, J. L. and T. P. Powell (1970). "An experimental study of the origin and the course of the centrifugal fibres to the olfactory bulb in the rat." J Anat 107(Pt 2): 215-237.
Price, J. L. and T. P. Powell (1970). "The synaptology of the granule cells of the olfactory bulb." J Cell Sci 7(1): 125-155.
Price, T. L., A. Darby-King, C. W. Harley and J. H. McLean (1998). "Serotonin plays a permissive role in conditioned olfactory learning induced by norepinephrine in the neonate rat." Behav Neurosci 112(6): 1430-1437.
Riemensperger, T., G. Isabel, H. Coulom, K. Neuser, L. Seugnet, K. Kume, M. Iche-Torres, M. Cassar, R. Strauss, T. Preat, J. Hirsh and S. Birman (2011). "Behavioral consequences of dopamine deficiency in the Drosophila central nervous system." Proc Natl Acad Sci U S A 108(2): 834-839.
Roeder, T. (2005). "Tyramine and octopamine: ruling behavior and metabolism." Annu Rev Entomol 50: 447-477.
66 Rubin, B. D. and L. C. Katz (1999). "Optical imaging of odorant representations in the mammalian olfactory bulb." Neuron 23(3): 499-511.
Saar, D., Y. Grossman and E. Barkai (1998). "Reduced after-hyperpolarization in rat piriform cortex pyramidal neurons is associated with increased learning capability during operant conditioning." Eur J Neurosci 10(4): 1518-1523.
Saar, D., Y. Grossman and E. Barkai (2001). "Long-lasting cholinergic modulation underlies rule learning in rats." J Neurosci 21(4): 1385-1392.
Schulz, D. J. and G. E. Robinson (1999). "Biogenic amines and division of labor in honey bee colonies: behaviorally related changes in the antennal lobes and age-related changes in the mushroom bodies." J Comp Physiol A 184(5): 481-488.
Schwaerzel, M., M. Monastirioti, H. Scholz, F. Friggi-Grelin, S. Birman and M. Heisenberg (2003). "Dopamine and octopamine differentiate between aversive and appetitive olfactory memories in Drosophila." J Neurosci 23(33): 10495-10502.
Seki, Y., J. Rybak, D. Wicher, S. Sachse and B. S. Hansson (2010). "Physiological and morphological characterization of local interneurons in the Drosophila antennal lobe." J Neurophysiol 104(2): 1007-1019.
Shepherd, G. M. (1972). "Synaptic organization of the mammalian olfactory bulb." Physiol Rev 52(4): 864-917.
Shepherd, G. M. (2005). "Perception without a thalamus how does olfaction do it?" Neuron 46(2): 166-168.
Sherman, M. S. and R. W. Guillery (2004). The Thalamus. The synaptic organization of the brain (5th Ed.). G. M. Shepherd. Oxford, Oxford University Press: 331-360.
Sigg, D., C. M. Thompson and A. R. Mercer (1997). "Activity-dependent changes to the brain and behavior of the honey bee, Apis mellifera (L.)." J Neurosci 17(18): 7148-7156.
Sigurdsson, T., V. Doyere, C. K. Cain and J. E. LeDoux (2007). "Long-term potentiation in the amygdala: a cellular mechanism of fear learning and memory." Neuropharmacology 52(1): 215-227.
Sinakevitch, I., J. A. Mustard and B. H. Smith (2011). "Distribution of the octopamine receptor AmOA1 in the honey bee brain." PLoS One 6(1): e14536.
Smith, R. L., H. Baker and C. A. Greer (1993). "Immunohistochemical analyses of the human olfactory bulb." J Comp Neurol 333(4): 519-530.
67 Soucy, E. R., D. F. Albeanu, A. L. Fantana, V. N. Murthy and M. Meister (2009). "Precision and diversity in an odor map on the olfactory bulb." Nat Neurosci 12(2): 210-220.
Strowbridge, B. W. (2009). "Role of cortical feedback in regulating inhibitory microcircuits." Ann N Y Acad Sci 1170: 270-274.
Sullivan, R. M., G. Stackenwalt, F. Nasr, C. Lemon and D. A. Wilson (2000). "Association of an odor with activation of olfactory bulb noradrenergic beta-receptors or locus coeruleus stimulation is sufficient to produce learned approach responses to that odor in neonatal rats." Behav Neurosci 114(5): 957-962.
Sullivan, R. M., D. A. Wilson, C. Lemon and G. A. Gerhardt (1994). "Bilateral 6- OHDA lesions of the locus coeruleus impair associative olfactory learning in newborn rats." Brain Res 643(1-2): 306-309.
Sullivan, R. M., D. A. Wilson and M. Leon (1989). "Norepinephrine and learning- induced plasticity in infant rat olfactory system." J Neurosci 9(11): 3998- 4006.
Sullivan, R. M., D. R. Zyzak, P. Skierkowski and D. A. Wilson (1992). "The role of olfactory bulb norepinephrine in early olfactory learning." Brain Res Dev Brain Res 70(2): 279-282.
Sun, X. J., L. P. Tolbert and J. G. Hildebrand (1993). "Ramification pattern and ultrastructural characteristics of the serotonin-immunoreactive neuron in the antennal lobe of the moth Manduca sexta: a laser scanning confocal and electron microscopic study." J Comp Neurol 338(1): 5-16.
Tamura, T., D. Horiuchi, Y. C. Chen, M. Sone, T. Miyashita, M. Saitoe, N. Yoshimura, A. S. Chiang and H. Okazawa (2010). "Drosophila PQBP1 regulates learning acquisition at projection neurons in aversive olfactory conditioning." J Neurosci 30(42): 14091-14101.
Tessier, C. R. and K. Broadie (2009). "Activity-dependent modulation of neural circuit synaptic connectivity." Front Mol Neurosci 2: 8.
Tyler, W. J., G. C. Petzold, S. K. Pal and V. N. Murthy (2007). "Experience- dependent modification of primary sensory synapses in the mammalian olfactory bulb." J Neurosci 27(35): 9427-9438.
Valle-Leija, P., E. Blanco-Hernandez, R. Drucker-Colin, G. Gutierrez-Ospina and R. Vidaltamayo (2012). "Supernumerary formation of olfactory glomeruli induced by chronic odorant exposure: a constructivist expression of neural plasticity." PLoS One 7(4): e35358.
Vosshall, L. B., A. M. Wong and R. Axel (2000). "An olfactory sensory map in the fly brain." Cell 102(2): 147-159.
68 Wachowiak, M. and L. B. Cohen (2001). "Representation of odorants by receptor neuron input to the mouse olfactory bulb." Neuron 32(4): 723-735.
Wang, J. W., A. M. Wong, J. Flores, L. B. Vosshall and R. Axel (2003). "Two- photon calcium imaging reveals an odor-evoked map of activity in the fly brain." Cell 112(2): 271-282.
Wei, C. J., C. Linster and T. A. Cleland (2006). "Dopamine D(2) receptor activation modulates perceived odor intensity." Behav Neurosci 120(2): 393-400.
Wilson, D. A., K. M. Guthrie and M. Leon (1990). "Modification of olfactory bulb synaptic inhibition by early unilateral olfactory deprivation." Neurosci Lett 116(3): 250-256.
Wilson, D. A. and M. Leon (1988). "Spatial patterns of olfactory bulb single-unit responses to learned olfactory cues in young rats." J Neurophysiol 59(6): 1770-1782.
Wilson, D. A., T. C. Pham and R. M. Sullivan (1994). "Norepinephrine and posttraining memory consolidation in neonatal rats." Behav Neurosci 108(6): 1053-1058.
Wilson, D. A. and R. M. Sullivan (1995). "The D2 antagonist spiperone mimics the effects of olfactory deprivation on mitral/tufted cell odor response patterns." J Neurosci 15(8): 5574-5581.
Wilson, D. A., R. M. Sullivan and M. Leon (1987). "Single-unit analysis of postnatal olfactory learning: modified olfactory bulb output response patterns to learned attractive odors." J Neurosci 7(10): 3154-3162.
Wilson, R. I. (2008). "Neural and behavioral mechanisms of olfactory perception." Curr Opin Neurobiol 18(4): 408-412.
Wilson, R. I. and G. Laurent (2005). "Role of GABAergic inhibition in shaping odor-evoked spatiotemporal patterns in the Drosophila antennal lobe." J Neurosci 25(40): 9069-9079.
Wilson, R. I., G. C. Turner and G. Laurent (2004). "Transformation of olfactory representations in the Drosophila antennal lobe." Science 303(5656): 366-370.
Woo, C. C., E. E. Hingco, B. A. Johnson and M. Leon (2007). "Broad activation of the glomerular layer enhances subsequent olfactory responses." Chem Senses 32(1): 51-55.
Woo, C. C. and M. Leon (1995). "Distribution and development of beta- adrenergic receptors in the rat olfactory bulb." J Comp Neurol 352(1): 1- 10.
69 Yokoi, M., K. Mori and S. Nakanishi (1995). "Refinement of odor molecule tuning by dendrodendritic synaptic inhibition in the olfactory bulb." Proc Natl Acad Sci U S A 92(8): 3371-3375.
Yu, D., A. Ponomarev and R. L. Davis (2004). "Altered representation of the spatial code for odors after olfactory classical conditioning; memory trace formation by synaptic recruitment." Neuron 42(3): 437-449.
Yuan, Q. (2009). "Theta bursts in the olfactory nerve paired with beta- adrenoceptor activation induce calcium elevation in mitral cells: a mechanism for odor preference learning in the neonate rat." Learn Mem 16(11): 676-681.
Yuan, Q. and C. W. Harley (2012). "What a nostril knows: olfactory nerve-evoked AMPA responses increase while NMDA responses decrease at 24-h post-training for lateralized odor preference memory in neonate rat." Learn Mem 19(2): 50-53.
Yuan, Q., C. W. Harley, J. C. Bruce, A. Darby-King and J. H. McLean (2000). "Isoproterenol increases CREB phosphorylation and olfactory nerve- evoked potentials in normal and 5-HT-depleted olfactory bulbs in rat pups only at doses that produce odor preference learning." Learn Mem 7(6): 413-421.
Yuan, Q., C. W. Harley, A. Darby-King, R. L. Neve and J. H. McLean (2003). "Early odor preference learning in the rat: bidirectional effects of cAMP response element-binding protein (CREB) and mutant CREB support a causal role for phosphorylated CREB." J Neurosci 23(11): 4760-4765.
Yuan, Q., C. W. Harley and J. H. McLean (2003). "Mitral cell beta1 and 5-HT2A receptor colocalization and cAMP coregulation: a new model of norepinephrine-induced learning in the olfactory bulb." Learn Mem 10(1): 5-15.
Zheng, C., P. Feinstein, T. Bozza, I. Rodriguez and P. Mombaerts (2000). "Peripheral olfactory projections are differentially affected in mice deficient in a cyclic nucleotide-gated channel subunit." Neuron 26(1): 81-91.
Zou, D. J., P. Feinstein, A. L. Rivers, G. A. Mathews, A. Kim, C. A. Greer, P. Mombaerts and S. Firestein (2004). "Postnatal refinement of peripheral olfactory projections." Science 304(5679): 1976-1979.
70 Chapter 3
A MODEL TO STUDY LEARNING-MEDIATED PLATICITY AND SENSORY
GATING IN THE MAMMILIAN OLFACTORY SYSTEM
Introduction
Experience-dependent plasticity has been abundantly studied in the developing nervous system and is a necessity during critical periods for the establishment of refined, normally functioning circuits (Hubel, Wiesel et al. 1977,
Katz and Shatz 1996, Cohen-Cory 2002). In adult animals, a similar type plasticity is possible during very limited time frames either 1) when an animal acquires new information during learning (Frey, Huang et al. 1993, Turrigiano and Nelson 2000, Dan and Poo 2006, Kandel 2009) or 2) in a homeostatic manner to global changes in activity patterns (Turrigiano and Nelson 2000,
Burrone and Murthy 2003).
Using the olfactory bulb (OB) as a model to study experience dependent plasticity has many benefits due its shared principals across species as a archaic sensory modality (Strausfeld and Hildebrand 1999, Kaupp 2010). In fact, for rodents, it is ethologically one of the most important sensory modalities (Doty
1986, Brennan and Kendrick 2006). Moreover its location near the periphery allows for direct manipulation of stimuli. Meanwhile olfaction is a unique modality where feedback modulation that may be important for context also can be found at the periphery (Kiselycznyk, Zhang et al. 2006, Kay and Sherman 2007).
The OB is functionally organized into glomeruli, each of which is composed of the convergence of olfactory sensory neurons expressing the same odorant receptor (OR) that bind the same set of structurally similar odorants (Bozza,
71 Feinstein et al. 2002, Treloar, Feinstein et al. 2002). Taking advantage of this organization, geneticists have developed several tools that could be useful to the study of functional and structural plasticity at the glomerular relay station. These include mouse strains that express a structural reporter (such as GFP/Lac-Z) or a functional reporter/probe (synaptopHluorin, channelrhodopsin) in one or more olfactory sensory neurons (Mombaerts, Wang et al. 1996, Belluscio, Lodovichi et al. 2002, Bozza, McGann et al. 2004, Feinstein and Mombaerts 2004, Bozza,
Vassalli et al. 2009, Soucy, Albeanu et al. 2009, Smear, Shusterman et al. 2011).
OMP (olfactory marker protein) promoters are used to express fluorescence or activity dependent probes in all OSNs for mapping. In contrast, using a single OR promoter, results in expression of that reporter limited to only one glomerulus
(M72, P2, MOR23). Missing from the repertoire of genetically modified reporter mice are those with calcium indicators such as GCaMP expressed in the OSNs.
As one can imagine, these set of tools are powerful for studying not only glomerular targeting and structure, but also experience dependent functional plasticity.
The M72-iresTau GFP mouse, created in Peter Mombaerts’ lab, is the genetically modified strain I chose for my studies. For the M7-GFP mouse, GFP is under the M72 OR’s promoter and is fused with axonal transport protein Tau for maximal structural labeling of OSN axons. The GFP labeled OSNs have been found to converge to unity by post natal day 30 in 17% of cases (Potter, Zheng et al. 2001, Zou, Feinstein et al. 2004) and can to be activated by narrow class of structurally similar odorants including acetophenone, benzaldehyde (Bozza,
Feinstein et al. 2002, Zou, Feinstein et al. 2004) and possibly ethyl tailgate
(Soucy, Albeanu et al. 2009). In the literature, the M72-iresTau GFP mouse or
72 the M72-tauLacz mouse (a non fluorescent reporter) has been used to study structural plasticity of glomeruli following deprivation, learning, imprinting, associative learning in neonates as well as adults, using acetophenone (Zou,
Feinstein et al. 2004, Kerr and Belluscio 2006, Jones, Choi et al. 2008, Cavallin,
Powell et al. 2010, Valle-Leija, Blanco-Hernandez et al. 2012). There have been no studies using the M72-GFP mouse study experience or learning dependent functional plasticity. The present study is the first to utilize the M72-GFP glomerulus to study experience or learning dependent functional plasticity. More specifically, the M72-GFP mouse is used in order to achieve, targeted manipulation of odor experience by specific activation of an identifiable peripheral circuit, followed by GFP targeted interrogation of the functional effects resulting from behavioral manipulation.
Studies of learning and memory in the rodent olfactory bulb have used a plethora of paradigms to alter experience in both neonates and adult animals.
The simplest type of experience manipulation is global manipulation of experience. Sensory deprivation is achieved via either a knocking out function of
OSN signaling (Lin, Wang et al. 2000, Zheng, Feinstein et al. 2000) or unilateral naris occlusion by fusing closed one nostril. Meanwhile, sensory enrichment
(either many odors or odor-specific) is achieved by brief or chronic exposure to a single or broad set of pure or natural odorants (Mandairon, Stack et al. 2006,
Cavallin, Powell et al. 2010, Valle-Leija, Blanco-Hernandez et al. 2012). In neonates, both odor deprivation and enrichment disrupts normal glomerular refinement and results in extra glomeruli (Zou, Feinstein et al. 2004, Valle-Leija,
Blanco-Hernandez et al. 2012). Functional consequences include sensory deprivation induced compensatory increases in activity and synaptic strength
73 (Guthrie, Wilson et al. 1990, Wilson, Guthrie et al. 1990, Guthrie, Pullara et al.
1991). Adult animals also show structural changes in glomeruli after deprivation and enrichment: both causes decrease in glomerulus size and OSN number
(Cavallin, Powell et al. 2010). Meanwhile, odor enrichment to a wide ensemble of odors increases ability to discriminate between structurally similar odors
(Mandairon, Didier et al. 2008).
Olfactory learning is a more complex type of experience manipulation beyond just increasing or decreasing overall activity. Associative learning requires associations between odor inputs and information from other modalities.
In neonates, associative learning is limited to imprinting: a bonding between mother to pup that is limited to a sensitive period between PD1-PD10. An imprinting paradigm pairing maternal presence or stroking of the pup with a neutral odor will illicit imprinting approach behavior to that odor (Wilson, Sullivan et al. 1987, Sullivan, Wilson et al. 1989, Moriceau and Sullivan 2004). The imprinting process accelerates the refinement OSNs into a unitary glomerulus for that specific odor compared glomeruli on control odors (Kerr and Belluscio 2006).
Imprinting is also mediated by functional plasticity: granule cell to mitral inhibitory cell synapses are strengthened by β1 norepinephrine (NE) signaling (Wilson,
Sullivan et al. 1987, Sullivan, Wilson et al. 1989, Sullivan, Zyzak et al. 1992,
Sullivan, Stackenwalt et al. 2000, Wilson and Linster 2008). Recently, studies have proposed that the imprinting related functional plasticity can be found in the neonate glomerulus (Yuan, Mutoh et al. 2004, Mutoh, Yuan et al. 2005).
In adults, associative learning requires the pairing of two temporally specific cues: the conditioned stimulus (CS) which initially processes neutral meaning followed shortly by the unconditioned stimulus (US) that carries inherent
74 emotional value for the animal. The US can be either positive and negative valences, and the training paradigm to establish these relationships can either be classical or operant (Pavlov and Anrep 1927, Skinner 1938). For the most part, olfactory associative learning paradigms for rodents are very etiologically relevant. Reward paradigms in the olfaction literature are mostly operant and involve an initially neutral odor (CS) paired and thus predicting the availability of sucrose, water, food (US) which requires some operant task (digging or nose poke). Occasionally odor-reward conditioning is performed using a classical sucrose or cocaine (place preference) paradigm. In sum, these reward paradigms have been used to show that acetylcholine is not involved in studies odor discrimination (Mandairon, Ferretti et al. 2006, Chaudhury, Escanilla et al.
2009) or the integration of new neurons (Mandairon, Sultan et al. 2011). In contrast, olfactory fear associative learning paradigms have been less tested and involve odor paired with a foot shock (Jones, Heldt et al. 2005). A recent study incorporated both reward and fear paradigms to establish a relationship between associative learning and glomerular structural plasticity (increase in size, and number OSNs of the learned odor) (Jones, Choi et al. 2008). My study aims use the same behavioral manipulations: fear and reward associative learning to instill implicit learning experiences with different valences.
In this chapter, I will establish an experimental paradigm to study learning- induced functional plasticity to optimize a peripheral sensory gate at the glomerulus. I will aim to 1) to identify a glomerular circuit in the M72-GFP mouse by which I can manipulate experience in an odor-specific manner followed by guided interrogation for learning-dependent changes to 2) develop a behavioral paradigm for olfactory associative learning in both fear and reward contexts.
75 Methods
Animals
M72-iresTau-GFP mice were used for C-Fos activation, behavioral paradigms, in vivo pharmacological experiments, and post-learning immunohistochemistry assays. GAD64::GFP mice were used for 5-HT and 5-HT receptors circuit mapping. All animals were ages 2-5 months. For circuit identification, both male and female mice were used. For study involving associative learning paradigms, only male mice were used to reduce variation based on sex, potential fluctuations in odor perception due fluctuating reproductive hormones and potential odor activation or behavioral influences caused intermixing sexes within the same training/testing area.
Mice were reared in 12h/12h light/dark schedule in standard cage housing with ad libidum diets as per the standard protocol for mice by animal care facilities. All animals were reared and scarified according to IAUCUC institutional guidelines at Arizona State University. All mice that were subjected to behavioral training were food deprived and maintained at 80-85% of ad libidum feed weight for the duration of the behavioral conditioning. Food deprivation was necessary to facilitate sucrose reward training but also was implemented before fear conditioning training to keep the same nutritional conditions across all training groups.
Odor exposure for C-Fos activation
Single housed mice were exposed to one of three odors: acetophenone, benzaldehyde or ethyl tailgate (Sigma) diluted 1:100 in mineral oil; 100uL was placed in an open 1mL Eppendorf tube at 4 corners of their home cages. Only
76 one odor was tested at a time and mice cages were placed in a ventilated hood to prevent odor contamination for other odors in the room. Mice were exposed to the odor for 30 minutes before the odor was removed and the mice remained in their cage for an additional 20 minutes (for C-Fos early genes to be turned on) before sacrificed, perfused and prepared for histology
Immunohistochemistry for C-Fos activation,
Animals were anesthetized using a Ketamine (70 mg/kg) and Xylazine (7 mg/kg) cocktail, transcardially perfused with 4% paraformaldehyde in phosphate buffered saline, and their brains were harvested according to IACUC approved protocols. The forebrain and olfactory bulbs of post-fixed brains were then removed and washed 3 x 30 min in fresh phosphate buffered saline (PBS) at 4°
C before being prepared for slicing. Parasagittal sections (70 µm) of the olfactory bulbs and forebrain were made using a vibratome. Slices were then prepared for staining, mounting, and confocal microscopy. All slices were mounted using
Vectashield (H-1000, Vector Laboratories, Burlingame, CA) to minimize photobleaching before being cover slipped. Different combinations of antibodies were used depending on the assay.
C-Fos immunofluorescence for response to known odors
Following transcardial perfusion, the brains of mice were post-fixed in 4% paraformaldehyde overnight at 4° C. The forebrain and olfactory bulbs of post- fixed brains were then removed and washed 3 x 30 min in fresh phosphate buffered saline (PBS) at 4° C before being prepared for slicing. Parasagittal sections (70 µm) of the olfactory bulbs and forebrain were made using a
77 vibratome. Slices were then prepared for staining, mounting, and confocal microscopy. All slices were mounted using Vectashield (H-1000, Vector
Laboratories, Burlingame, CA) to minimize photobleaching before being cover slipped. Different combinations of antibodies were used depending on the assay.
Prior to staining, all slices were permeabilized using Triton X-100 (0.1% in
PBS) for 15 minutes at room temperature. To label cell bodies in some experiments, slices were exposed to TO-PRO-3 (1:1000; Life Technologies,
Carlsbad, CA) in PBS for 15 minutes at room temperature prior to incubation in primary and secondary antibodies. Labeling with primary antibodies was conducted overnight at 4° C in a blocking buffer consisting of 10% bovine serum albumin in PBS.
To assay cellular activation following odor exposure, slices were labeled with a rabbit anti-c-Fos antibody (1:500; SC-253, Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA). Following the overnight incubation, slices were washed 3 x 10 minutes in fresh PBS before being incubated in an Alexa-568 anti-rabbit secondary antibody (1:1000; Life Technologies) overnight at 4° C to confer fluorescence.
Confocal imaging of immunohistochemistry
All imaging was performed on an upright Olympus Fluoview FV-300 laser- scanning confocal microscope (Olympus America, Inc., Center Valley, PA, USA).
The 488 nm laser line of an Argon laser was used to excite GFP originating from immunolabeled slices prepared from GAD65::GFP or M72-GFP mice. TO-PRO-3 was excited using a 633 nm HeNe laser. Images were acquired using a 10X air
(0.3 NA), 20X oil (1.2 NA), or 60X oil (1.45 NA) objective lens. Individual optical
78 sections (2 µm apart) were taken from regions of interest through the entire extent of the tissue and stored on a computer for laser offline analysis.
Two photon imaging of M72 glomerulus structure
For 2-Photon imaging, brains of M72-GFP mice were excised rapidly after
CO2 anesthesia followed by rapid decapitation according to IAUCUC protocols.
Brains were submerged in ice cold, high sucrose aCSF and secured to a petri dish for ex-vivo imaging. A Prarrie-2P with Tsunami sapphire laser was used to obtain Z stacks at 10µM steps of the glomerulus from the OB surface to about
100µM into the tissue or just past the depth of glomerular layer.
Intrinsic Imaging for response to known odors
M72-GFP mice were anesthetized with Ketamine/Zylazine until they reached a surgical plane of anesthesia and body temperature was maintained with a heating blanket. Animals were head fixed into a stereotax and the cranium over the olfactory bulbs was carefully thinned with a dental drill and removed with forceps. The exposed brain then washed with cold aCSF and 2% agarose was added in addition to a glass coverslip to minimize movement of the brain.
Animals were imaged on custom build intrinsic imaging rig with CCD camera.
Short wavelength 500nM light was used to focus on the glomerular layer using blood vessels as reference and long wavelength 780nM light was used to obtain intrinsic images at 25 frame/sec. A custom build olfactometer was programed deliver odors: acetophenone, benzaldehyde or ethyl tailgate (1:100 in mineral oil) at a constant rate while clean air was delivered as controls. Delta F/F was
79 calculated by using applying normalized frame by frame across pre-odor fresh air versus during odor presentation frames.
Olfactory conditioning apparatus
I used a custom built operant conditioning chamber and software (Med
Associates, Inc., St. Albans, Vermont) for fear conditioning or reward conditioning. The chamber had a two-channel olfactometer for odor delivery, and fans for odor evacuation. The conditioning chamber was housed inside a sound dampening box, which had another fan for evacuating odors as well as vacuum.
For fear conditioning the chamber had a metal grid capable of delivery foot shock. For reward conditioning, the chamber also had a sucrose storage unit that dispensed sucrose pellets upon lever press, a mouse-sized lever, and a port next to the leveler in which sucrose was dispensed, A high-definition webcam was placed inside the sound dampening box, such that the activity of mice inside the conditioning chamber could be remotely observed and recorded. All animals were acclimated to the chamber for 30 minutes on each of two days prior to the commencement of a training paradigm.
Fear conditioning: classical conditioning using odor CS and foot shock US.
For fear conditioning, the conditioned stimulus was acetophenone (1:100) in mineral odor mixed with fresh air. The unconditioned stimulus was a 1mA foot shock (1sec, 1mA) delivered via the metal grid floor. Training was performed for
2 training days, 5 trials per day. Each day, after 3 minutes of acclimation, each trial consisted of 20 sec odor, the last second coinciding with a 1 sec foot shock, followed by a 1 min 30 sec ITI before the entire trial was repeated 5 times.
80 Mice undergoing behavioral conditioning were randomly assigned to one of five experimental groups: a fear group (N = 10 ; CS+/US+), an odor only group
(N = 7 ; CS+/US-), a shock only group (N = 6 ; CS-/US+), a reverse paired group
(N = 6 ; US+/CS+). For the fear group, the foot shock was delivered 20 sec after the onset of the 20 sec odor delivery. For the odor only group, no foot shock was delivered following 20 sec odor while the shock only group was not exposed to the odor of acetophenone (but rather fresh air) that predicted shock during training. The reverse paired group received a foot shock prior to odor presentation at the beginning of the 20 sec odor period. The naïve group was only exposed to the conditioning chamber and acclimated for the same days.
After two training days animals were tested for their behavioral response to the CS odor. The interior of the chamber was modified so context did not resemble that during training days. Black and white stripes were affixed to the chamber walls, a small red light-emitting diode was introduced to the chamber, and a piece of sandpaper was used to cover the metal grid floor. Mice were acclimated to the chamber for five minutes prior to testing. Testing consisted of 2 trials separated by 3 minutes. Mice were video recorded during these testing trials and their movement trajectory and freezing behaviors during each of the trial periods were later analyzed offline. Movement traces were created using
Image J by tracking the mouse’s movement across each frame of the recording.
Reward conditioning: olfactory cue mediate sucrose operant conditioning
For reward odor conditioning, the conditioned stimulus (CS) was also acetophenone (1:100) in mineral odor mixed with fresh air. The operant paradigm required mice to lever press at a 1:1 fixed ratio for a reinforcer: a sucrose pellet
81 reward (I will call this reinforcer US for parallel). Only during a 30 sec odor presentation period was the lever active to dispense sucrose when pressed. The conditioned response was measured (# time bar is pressed during both odor presentation versus during non-odor ITI) was recorded and time stamped by the
Med Associates software.
After 2 days of food deprivation to achieve proper motivation for learning, target weight was achieved. Mice were acclimated to the chamber for 2 days.
Then, shaping consisted of 3 additional days when the mice learned to press the bar for sucrose during 30 minute sessions when the bar was always active to dispense sucrose. Upon successful shaping, associative learning training days were 14 days total, consisted of 5 trials per day consisting, each trial of 3 minutes of chamber acclimation followed by 5 training trials. Each trial consists of 30 sec of odor presentation in which the lever may or may not be active (depending on group) followed by 1 min, 30 sec of inter-trial interval.
Mice undergoing behavioral conditioning were randomly assigned to one of five experimental groups: a reward group (N = 6; CS+/US+), an odor only group
(N = 7; CS+/US-), or a sucrose only group (N = 7; CS-/US+). For the reward group, the level was active to dispense sucrose during the 30 sec of odor presentation. For the odor only group, no foot shock was given, but acetophenone was dispensed at the same intervals. The sucrose only group was exposed to fresh air instead of acetophenone as the predictive cue for an active, sucrose dispensing lever. The naïve group were exposed to the conditioning chamber and acclimated, but were not primed or trained. The 14th day was also a test day, when animal’s bar press responses are recorded for analysis for the robustness of the correct conditioned response. The percentage of correct lever
82 presses (ie. Lever presses that occurred during the odor presentation period/ lever presses in the entire trial) was calculated.
Results
Verification of location and morphology of the M72-GFP glomerulus within the olfactory bulb
To identify the M72 glomerulus location within the olfactory bulbs, I first used wide field fluorescence imaging at low magnification. I found that the M72 glomeruli in 3-5 month old M72-GFP mice are well defined, distinct entities with full axonal innervation from the olfactory epithelium. The two dorsal pair of M72
GFP glomeruli was reliably located in the dorsal, lateral, and caudal quadrant of the olfactory bulb as viewed from a dorsal perspective (Figure 3A). Confocal microscopy at 10x and 20x and 60x confirms this unique and replicable location of the M72 glomerulus (Figure 3B). Two photon microscopy of the M72 glomerulus showed that despite its reliable location and unitary existence, the
M72 glomerulus varies in size (ranging from ~100-200uM in diameter), shape, as well as variety of patterns of OSN axonal innervation (Figure 3C). Identifying the
M72-GFP glomerulus provides information about efficient ways to access the glomerulus during in vivo manipulations or for slice electrophysiology.
M72-GFP glomerulus is activated by a set of known odors including acetophenone
After confirmation of location and morphology of the M72-GFP glomerulus, my next step was to test its functionality. Previous studies mapping odorants topographically onto the olfactory bulb or studies using M72-GFP
83
Figure 3. The M72-GFP glomerulus is located on the dorsal lateral surface of the olfactory bulb and varies in morphology across individuals. A. Confocal images (dorsal view) of a pair of ex-vivo, olfactory bulbs showing dorsal-lateral position of M72 glomerulus. Another pair resides ventrally and is more difficult to access. B. M72-GFP glomerulus is situated within TO-PRO-3(red) stained cell body layers of the olfactory bulb (GL = glomerular layer, EPL = external plexiform layer, M=mitral cell layer, IPL = internal plexiform layer, GR = granule layer and a 60x Z-stack of the same M72-GFP glomerulus C. 2-Photon Z-series of 4 different M72 -GFP glomeruli showing difference in morphology across individuals, but unitary convergence by postnatal day 60.
84 glomerulus reported that three structurally similar odorants might activate this glomerulus: acetophenone, benzaldehyde and ethyl tiglate.
When choosing an odorant to use as my experimental, unconditioned stimulus odor, I must confirm that the odorant 1) does indeed activate the M72 glomerulus and 2) it does not activate the other non M72 glomeruli. One cannot expect that M72 glomerulus is activated exclusively by one odorant since structurally similar odorants will have a gradation of affinities to each type of odor receptor protein. Nor can one expect that any one pure odor exclusively activates the M72 glomerulus without a activating any adjacent glomeruli, since the topographically organized olfactory bulb has several glomeruli responding to structurally similar odorants within spatial proximity. Moreover, I expect higher concentrations of odorants (like the one I used) to cause some unspecific activation. I hoped to set a zone about 5 glomeruli away from the M72 glomerulus beyond which activation was sparse so I can set a region as "control" or non-M72 for comparison.
First I used c-Fos immunohistochemistry to assay for the turning on of immediate early genes, an assay of neuronal activity. I specifically looked for C-
Fos immune-positive staining in juxtaglomerular cells of the M72 versus control glomeruli (more than 5 away) as an indication of neuronal activity in response to odor. I found significant c-Fos activation of the cells surrounding the M72-GFP glomerulus after a 30 minute exposure to all three of the above odorants, although ethyl tiglate appeared to have the weakest activation with least # of c-
Fos positive cells c-Fos. Meanwhile, although immediately adjacent glomeruli had some c-Fos positive staining, glomeruli more than 5 away from the M72-GFP
85 glomerulus showed few or no c-Fos positive cells and thus this distance was deemed to be the safe zone outside of which were control glomeruli (Figure 4A).
I used intrinsic imaging of dorsal surface of the olfactory bulb to further confirm this observation that the M72-GFP glomerulus was indeed activated by these three odorants. During a brief exposure to these odorants, intrinsic signals were obtained by a CCD camera with 900 nM illumination. Since intrinsic imaging captures reflectance of hemoglobin deoxygenation within blood vessels to estimate neuronal activity, I measured ΔR/R or in other words, the change in reflectance due to onset of odor from a baseline reflectance during fresh air exposure normalized. Representative intrinsic data (ΔR/R) shows that a small set of glomeruli are activated by ethyl tiglate, benzaldehyde and acetophenone
(Figure 4B). Cross correlation analysis reveals that acetophenone and benzaldehyde share are common small subset of 3-4 glomeruli at the dorsal, lateral, caudal edge of the olfactory bulb corresponding to the previously verified location of the M72-GFP glomerulus (Figure 4C). Meanwhile ethyl tiglate did not share an activation map with the two more established odorants, so was eliminated from my choices of CS odor. Overall, both immunohistochemistry and real time intrinsic imaging showed that acetophenone and benzaldehyde reliably and robustly activated the M72 glomerulus. I finally chose acetophenone due to the overwhelming preference for its use in the literature to activate the M72 glomerulus, so that I could compare my results to previous studies.
Reward and fear learning: M72 mice learn to associate acetophenone with either foot shock or sucrose reward
86
Figure 4. The M72-GFP glomerulus is activated by acetophenone, benzaldehyde and ethyl tiglate. A. Representative confocal z-stacks of C- Fos immunohistochemistry of the olfactory bulb following 30 minute exposure to the one of the 3 candidate odorants: acetophenone, benzaldehyde or ethyl tiglate in the home cage. Immediate early gene expression is indicated by bright red juxtaglomerular cells surrounding the M72 glomerulus versus control glomeruli (more than 5 glomeruli away) of the same animal. B. Representative images obtained from intrinsic imaging during exposure to odorants (1:100) in mineral oil: acetophenone, benzaldehyde or ethyl tiglate compared to clean air baseline. Intrinsic signals shown are ΔR/R C. Cross- correlation maps showing similarities in responses to the 3 odorants. Highlighted boxes indicate position of the dorsal lateral M72 glomerulus. acetophenone and benzaldehyde both activated the M72-glomerulus (left).
87 I sought to establish that M72-GFP mice could learn to associate an odor stimulus (CS) acetophenone with either a positive or negative valence unconditioned stimulus. For fear learning, 2 days of acclimation was followed by
2 days of training and cumulated on test day. For reward learning, following acclimation and shaping, animals took 14 days to acquire the conditioned response in the operant reward task (Figure 5A).
On test day, I measured percentage of time the animal spent freezing (no movement) during three time periods: 20 sec immediately preceding odor delivery (pre-odor), during the 20 sec of odor delivery period (odor) and 20 sec immediately following odor delivery (post-odor). Successful associative learning was defined by increased freezing only during odor delivery period but low freezing times. I found that only animals in the fear learning group (CS+/US+ forward paired) demonstrated successful learning on test day as shown in the inverted U-curve trend from pre-odor to odor to post-odor periods. Percentage time spent freezing during odor presentation was significantly higher compared to pre-odor period or when compared to post odor period (Pre-odor: 6.33 ± 2.91%,
Odor: 75.33 ± 2.56%; Post odor: 6.23 ±3 .044%, Repeated Measures ANOVA: F
= 203.235, p < 0.001, Pairwise LSD: Pre odor versus odor P < 0.001, Post odor versus odor p < 0.001, n = 10) (Figure 5B).
None of control learning groups show this inverted U shaped curve indicating selective freezing only during odor presentation. The odor only group did not show freezing in any period (Repeated measures ANOVA, F = 0.143, n = 7). The shock only group did show significant freezing during the pre-odor period as well as odor period only to be relieved from freezing after the shock. This was to be expected since without any odor cues to predict, foot shock, it became context
88
Figure 5. M72 -GFP mice successfully learn to associate M72 activating odorant acetophenone with aversive or appetitive unconditioned stimuli. A. Behavioral paradigm for Pavlonian fear conditioning included fear learning group (CS+/US+) where odor predicts a 1s,1mA foot shock. B. Mean ± SEM % of time animals displayed freezing behavior on test day during the 20 sec preceding odor delivery (pre-odor), duration of odor delivery (odor) following odor delivery (post- odor). Only animals in fear group (CS+/US+) showed successful odor-cue fear learning indicated by selective freezing during odor delivery period. C. Distance traveled on test day by animals trained under fear or control paradigm. Only (CS+/US+) animals show lack of movement during odor presentation period (blue). D. Behavioral paradigm for odor reward operant conditioning includes reward learning group (CS+/US+). Odor delivery cues 30sec of active sucrose dispensing lever. E. Learning curve shows Mean ± SEM % of total lever presses that are correct (ie. pressed during odor presentation when bar is actively dispenses sucrose rather that during ITI when bar is inactive for sucrose). Only animals in reward (CS+/US+) group showed a positive learning curve indicated by increased % of correct responses after two weeks of training.
89 fear-conditioned to the chamber (Pre-odor: 55.71 ± 16.13%, Odor: 63.57 ±
14.95%, Post-odor: 17.14 ± 8.79%, F = 5.97, p < 0.05, Repeated Measures
ANOVA: F = 5.97, Pairwise LSD: odor versus post-odor p < 0.05, n=6).
Conversely, the backward-paired animals showed the opposite trend with freezing only in the post-odor period (Pre-odor: 24.17 ± 11.86%, Odor: 24.17 ±
16.35%, Post odor: 70.00 ± 7.30%, F = 7.44, p < 0.05, Pairwise LSD: pre-odor versus post-odor p < 0.01, n = 6) (Figure 5B). Further, more the selective odor- mediated freezing during odor delivery period by fear learning animals
(CS+/US+), but no other control groups can be visualized in the movement traces showing distance traveled over time (Figure 5C).
Reward learning
For the reward learning, I paired odor exposure with the availability of sucrose reward upon performing an operant lever press at fixed ratio of 1:1. After a 2 days acclimation, the animals were shaped or trained to press a lever for sucrose that was initially active 100% of the time for 3 days. By the fourth day, animals that had successfully learned the operant task went on to a 14 day operant associative training period. For the reward learning group, a 30 sec odor delivery coincided with an active lever that would dispense sucrose. For the sucrose only group, sucrose was availability upon bar press was cued by fresh air abut no odor cue. Lastly, for the odor only group, 30 seconds acetophenone was dispensed during each trial in parallel to the other groups, but no sucrose reward was given (Figure 5D).
The success of associative learning was accessed on each day during training by the percentage of correct lever presses for sucrose. For the reward
90 group, this means calculating the number of lever presses during odor presentation period when the bar actively dispense sucrose divided by the number of total lever presses during non-odor time during that trial (scaled by 3, since the non-odor ITI was 3x the odor presentation time). The learning progress of each group was plotted over 13 days of training and 1 final day of testing for
14 days total. Comparing performance on Dayv1 of Training to Day 14, test day: only the reward (CS+/US+) group experienced positive learning curve from Day1 to Day 14 on test day. (Day 1: 29.83 ± 3.03%, Test day 14: 74.20 ± 6.17%,
Paired Test: p < 0.005, n = 6). Meanwhile control groups did not improve at all from day 1 to day 14: Sucrose only (Day1 34.9 ± 4.21%, Test Day 14: 37.30 ±
4.58%, Paired T-test p = 0.41, n = 7) Odor only (Day1: 42.24 ± 11.58%, Test
Day14: 44.24 ± 8.80%, Paired T-test: p = 0.74, n = 7) (Figure 5E).
Discussion
In this study I developed an experimental paradigm that can be used to study learning-dependent plasticity at the first relay station of the adult rodent olfactory nervous system. My paradigm allows a very selective manipulation of experience of the first relay station by activation of the M72-GFP glomerulus with a known, pure odorant acetophenone. It also allows a guided post-hoc analysis of the consequences of such learning experiences since GFP marks the exact location in the circuit one should interrogate for structural or functional changes.
The existing studies of plasticity at glomerular relay station have been focused on structural plasticity while ignoring functional plasticity, while other studies are limited to glomerular refinement in neonates (Kerr and Belluscio
2006, Jones, Choi et al. 2008, Cavallin, Powell et al. 2010, Valle-Leija, Blanco-
91 Hernandez et al. 2012), while ignoring functional plasticity after learning.
Moreover, a majority of the literature has focused on experience dependent plasticity at deeper OB granule-mitral cell synapses (Wilson, Sullivan et al. 1985,
Wilson, Sullivan et al. 1987, Guthrie, Wilson et al. 1990, Wilson, Guthrie et al.
1990, Sullivan and Wilson 1995). Not enough due attention has been given to the first synapses in the glomerulus, which are capable of just as complicated signal processing and receives just as rich centrifugal modulation as the deeper synapses in the bulb.
Second, implementing an associative learning olfactory learning paradigm with positive and negative contexts in adult mice allows me to study the more etiologically relevant question of learning rather than global increases in experience on plasticity such as deprivation (Meisami and Safari 1981, Guthrie,
Wilson et al. 1990, Wilson, Guthrie et al. 1990, Guthrie, Pullara et al. 1991,
Wilson and Wood 1992, Tyler, Petzold et al. 2007) or enrichment (Mandairon,
Stack et al. 2006, Mandairon, Stack et al. 2006, Escanilla, Mandairon et al. 2008,
Mandairon, Didier et al. 2008, Cavallin, Powell et al. 2010). Unlike other studies, I focus on both contexts of learning in the mature adult nervous system rather than the limited phenomena of imprinting in neonates (Sullivan, Wilson et al. 1989,
Sullivan, Zyzak et al. 1992, Wilson, Best et al. 2004). Such studies have a broader implication about learning-dependent changes over the entire lifetime of the animal. Moreover, by using both rewarding and fear contexts, I not only pose the question in terms of both arousal level (associative learning versus non- learning) and but also the emotional valence of the arousal signal or US.
My first set of results confirmed the reliable location and unitary presence of the M72-GFP glomerulus during the age period I wished to study: adults above
92 60 days of age, as visualized via wide-field, confocal, as well as 2photon imaging. Olfactory glomeruli, under normal developmental conditions often form in pairs that are at opposite corners of the bulb. I confirmed at low magnification, that each bulb has 2 pairs of M72-glomerulus: one at the dorsal lateral edge and one at the ventral medial edge. For future experiments, I decided to use the most dorsal lateral pair, since they more accessible for pharmacology, imaging or when cutting a sagittal slice of the OB. Using confocal imaging and concurrent labeling with TO-PRO-3 DNA stain for cell bodies, I determined that the M72- glomerulus was located in the position at about the 5th glomerulus from the most dorsal caudal glomerulus in in the parasagittal slice it was found. I determined the size of the glomerulus to range from 100-200µM range and thus using 70µM sections; I were very likely to find a plane of the glomerulus near its midpoint.
Finally, with 2photon imaging, I concluded that the M72-GFP glomerulus has large variance in structure and OSN innervation pattern (however, most were from the rostral to caudal direction. My findings did concur with the literature
(Potter, Zheng et al. 2001) that reported M72 glomerulus converged to unity by 2 to 3 months of age: the ages at which I studied learning induce plasticity in mice.
This characterization of the M72-GFP in my strain of mice was necessarily to confirm the usability of these mice in my next set of investigation.
My goal was to selectively manipulate odor input in the glomerulus and be able to find the site of odor activation after the manipulation for post-hoc examination of plasticity. Before using a particular odor in an associative learning paradigm, I wished to verify that the “known odor”’ indeed activates the M72 glomerulus reliably at the concentrations I planned to use for behavioral conditioning. In the literature, methods such as electrophysiology, calcium
93 imaging and intrinsic imaging have provided evidence that acetophenone is the strongest activator of the M72 glomerulus followed by benzaldehyde (Bozza,
Feinstein et al. 2002, Feinstein, Bozza et al. 2004, Nguyen, Zhou et al. 2007) and also to some extent ethyl tailgate (Soucy, Albeanu et al. 2009). My results from the C-Fos experiments show that all three proposed odors activate my glomerulus of choice at concentrations of 1:00 in mineral oil, with Ethyl tailgate being slightly less robust. C-Fos is the protein product of an immediate early gene that is activated by neuronal activity and has been used as a reliable indicator of neuronal activation in the neuroscience literature (Dragunow and
Faull 1989). Finally, I chose acetophenone as my odorant for future learning paradigms since 1) it was reliable activator in all my experiments using both C-
Fos and intrinsic imaging, 2) it has been shown to be the highest affinity odor for the M72 OR and finally, 3)numerous studies using the M72 glomerulus to assay structural plasticity have all used acetophenone (Jones, Choi et al. 2008,
Cavallin, Powell et al. 2010, Valle-Leija, Blanco-Hernandez et al. 2012), and thus
I can better make direct comparisons with the literature.
Moreover, I confirmed that there a certain amount of specificity in the activation by the three known odors since glomeruli that were 5 positions away from the M72 glomerulus were not activated or very sparsely activated using my concentration of 1:100 dilution. My intrinsic imaging also confirmed a narrow field of activation of a few glomeruli around the M72-glomerulus that was shared by benzaldehyde and acetophenone, but not ethyl tailgate My studies agree with the literature, that chemically similar odors activate glomeruli adjacent to each other but pure odorants at non saturating concentrations activate a field of glomeruli a range of over 5 glomeruli in each direction (Soucy, Albeanu et al. 2009) as
94 assayed by synaptopHluroin and intrinsic imaging. My odor activation during associative learning used odor concentrations well above threshold, and thus several non-m72 glomeruli were activated. For this set of studies, the reliability of activation in each learning experience is much more important than the specificity. I must be certain that during each trial of learning that the M72 glomerulus is activated. However, I can always use glomeruli more than 5 positions away as internal control glomeruli with which to compare my results.
The results from associative learning experiments in positive and negative context verify that I have established two reliable conditioning paradigms with opposite valence. Although well-established rodent paradigms exist for auditory or contextual fear learning using a foot-shock as the reinforcing
US (Gewirtz and Davis 2000, LeDoux 2000, Carcaud, Roussel et al. 2009,
Johansen, Cain et al. 2011), odor-shock fear conditioning for olfactory learning has been rarely used in rodents (Jones, Heldt et al. 2005). In other species, fear conditioning can be easily achieved by pairing the odor with a noxious chemical
(C. Elegans) or by pairing odor to foot shock (insects) (Quinn, Harris et al. 1974,
Carcaud, Roussel et al. 2009). In the few rodent studies using odor-shock, it was found that neonate rodents could not learn fear associations when the mother is present (Moriceau, Wilson et al. 2006). The second showed that glomeruli are structurally plastic after reward and fear learning in adults using acetophenone activation the M72-glomerulus (Jones, Choi et al. 2008). My study represents the first study to use the odor-shock classical condition paradigm to study functional plasticity in the olfactory bulb, especially in the glomerulus. My paradigm can achieve fear associative learning following 7 days total of learning including acclimation and testing. The results show a robust freezing behavior during odor
95 presentation specifically in the learning group (CS+/US+) forward paired odor- shocks. Several control groups show non-odor cue specific freezing behavior or no freezing at all, demonstrating the specificity of this learning.
For reward associative learning, the most often used olfactory behavior paradigm studied in the olfactory literature is the imprinting paradigm. However this paradigm is time limited to sensitive period between PD1-PD10 (Moriceau and Sullivan 2004) and thus represents a very specific type of reward learning and memory available only to neonates. More generalized odor-reward learning paradigms in adult rodents have been developed in the operant (nose poke, digging for food) and non-operant/classical forms (place preference). However, these paradigms have been used exclusively to study odor discrimination or the effect of learning on olfactory neurogenesis (Bodyak and Slotnick 1999,
Mandairon, Ferretti et al. 2006, Escanilla, Mandairon et al. 2008, Mandairon,
Peace et al. 2008, Mandairon, Sultan et al. 2011). I developed a more generalized olfactory reward behavioral paradigm that is clearly operant and is comparable to other operant paradigms that require lever press under the contingency of a cue in other modalities (sound, sight, context). My paradigm is the first documented in the literature pairs and odor cue with the with a sucrose reward contingent upon an operant lever press task. My paradigm is easy to implement, requiring only 17 days total from shaping (learning the tasks for lever press, which is harder in mice) to learning to testing. This paradigm produced reliable results after 14 days of association learning, when adult mice achieve correct lever responses over 70% accuracy. In my study, I exclusively used a
1:1 fixe ratio because my aim was simply to establish the associative relationship between odor and reward, not test breakpoint or the degree of pleasure provided
96 by the US. Just like with fear conditioning, I used several control groups given odor only or sucrose only provide evidence of the specificity of learning.
I faced several technical or practical challenges in developing olfactory learning paradigms for adult mice. First I attempted to use a purely olfactory US that was not aided by visual or sound cues. Such a restriction may have retarded learning to some degree, but also helped control for the number of inputs from other modalities that served as CS. Although I could not control the contextual arousal that may have been caused by the conditioning chamber (Curzon,
Rustay et al. 2009), I tried to isolate the cue’s salience as the US by decreasing contextual variables. For example, on test day, I remodeled the chamber to eliminate contextual familiarity so I only assayed for the effect of the odor cue
(CS) mediated freezing behavior. Another problem I faced was the precise temporal control of the CS or odor stimulus. Unlike CS of other modalities such as sound, light, or even touch, odors cannot be rapidly injected and evacuated within a training space with such a temporal precision. I attempted to achieve clean odor-on and odor-off states the by precise control of odor air stream input, and use of a double fans (in the internal chamber as well as the external sound proof chamber), coupled with a strong vacuum that was working during the entire experiment to eliminate odor pollution. This issue of temporal precision may only be problematic for quantification of behavior, however, since slow onset and offset is also the case for the encountering of odors in a natural environment.
A third problem I faced was establishing motivation for reward learning.
Animals that are food or water deprived learn much quicker on an associative task with food/water as the US reward. In fact, in insects, studies have found that the hunger state of the organism can influence the strength neural signal for US
97 reward (Krashes, DasGupta et al. 2009). In early attempts to establish the reward paradigm, I found that, without food deprivation, mice could not learn to associate the lever pressing task with sucrose dispense during shaping (even before odor was even introduced). Compared to rats, which easily learn a lever press task for sucrose without food deprivation, mice may be less adept at operant tasks and need higher motivation to learn. I solved this problem by food depriving the animals for 2 days before the first shaping/learning session on a diet between
1.8-2.8g food/day as to maintain 80-85% of ad libidum body weight. To parallel the same type of hunger/motivational/nutritional state in the fear conditioned group, this deprivation paradigm was applied universally regardless of context.
In conclusion, the experiments in this chapter achieved the goal of the identification of a functional circuit and the establishment of reliable behavioral paradigms for associative fear and reward learning in adult M72-GFP mice. This provides a foundation to now study experience dependent plasticity and sensory gating by manipulating experience in a ethologically meaningful way via associative learning of both reward and fear paradigms. Also, I could activate and assay my GFP labeled circuit with a known odorant that specifically binds to a narrow range of receptors and thus activates a small subset of glomeruli.
These preliminary experiments set the way for me to probe topics that are sparsely addressed by the literature: the question of associative learning- dependent functional plasticity and sensory gating in the adult glomerular relay station.
98 REFERENCES
Belluscio, L., C. Lodovichi, P. Feinstein, P. Mombaerts and L. C. Katz (2002). "Odorant receptors instruct functional circuitry in the mouse olfactory bulb." Nature 419: 296-300.
Bodyak, N. and B. Slotnick (1999). "Performance of mice in an automated olfactometer: odor detection, discrimination and odor memory." Chem Senses 24(6): 637-645.
Bozza, T., P. Feinstein, C. Zheng and P. Mombaerts (2002). "Odorant receptor expression defines functional units in the mouse olfactory system." J Neurosci 22(8): 3033-3043.
Bozza, T., J. P. McGann, P. Mombaerts and M. Wachowiak (2004). "In vivo imaging of neuronal activity by targeted expression of a genetically encoded probe in the mouse." Neuron 42(1): 9-21.
Bozza, T., A. Vassalli, S. Fuss, J. J. Zhang, B. Weiland, R. Pacifico, P. Feinstein and P. Mombaerts (2009). "Mapping of class I and class II odorant receptors to glomerular domains by two distinct types of olfactory sensory neurons in the mouse." Neuron 61(2): 220-233.
Brennan, P. A. and K. M. Kendrick (2006). "Mammalian social odours: attraction and individual recognition." Philos Trans R Soc Lond B Biol Sci 361(1476): 2061-2078.
Burrone, J. and V. N. Murthy (2003). "Synaptic gain control and homeostasis." Curr Opin Neurobiol 13(5): 560-567.
Carcaud, J., E. Roussel, M. Giurfa and J. C. Sandoz (2009). "Odour aversion after olfactory conditioning of the sting extension reflex in honeybees." J Exp Biol 212(Pt 5): 620-626.
Cavallin, M. A., K. Powell, K. C. Biju and D. A. Fadool (2010). "State-dependent sculpting of olfactory sensory neurons is attributed to sensory enrichment, odor deprivation, and aging." Neurosci Lett 483(2): 90-95.
Chaudhury, D., O. Escanilla and C. Linster (2009). "Bulbar acetylcholine enhances neural and perceptual odor discrimination." J Neurosci 29(1): 52-60.
Cohen-Cory, S. (2002). "The developing synapse: construction and modulation of synaptic structures and circuits." Science 298(5594): 770-776.
Curzon, P., N. R. Rustay and K. E. Browman (2009). "Cued and Contextual Fear Conditioning for Rodents."
99 Dan, Y. and M. M. Poo (2006). "Spike timing-dependent plasticity: from synapse to perception." Physiol Rev 86(3): 1033-1048.
Doty, R. L. (1986). "Odor-guided behavior in mammals." Experientia 42(3): 257- 271.
Dragunow, M. and R. Faull (1989). "The Use of C-Fos as a Metabolic Marker in Neuronal Pathway Tracing." Journal of Neuroscience Methods 29(3): 261-265.
Escanilla, O., N. Mandairon and C. Linster (2008). "Odor-reward learning and enrichment have similar effects on odor perception." Physiol Behav 94(4): 621-626.
Feinstein, P., T. Bozza, I. Rodriguez, A. Vassalli and P. Mombaerts (2004). "Axon guidance of mouse olfactory sensory neurons by odorant receptors and the beta2 adrenergic receptor." Cell 117(6): 833-846.
Feinstein, P. and P. Mombaerts (2004). "A contextual model for axonal sorting into glomeruli in the mouse olfactory system." Cell 117(6): 817-831.
Frey, U., Y. Y. Huang and E. R. Kandel (1993). "Effects of cAMP simulate a late stage of LTP in hippocampal CA1 neurons." Science 260(5114): 1661- 1664.
Gewirtz, J. C. and M. Davis (2000). "Using pavlovian higher-order conditioning paradigms to investigate the neural substrates of emotional learning and memory." Learn Mem 7(5): 257-266.
Guthrie, K. M., J. M. Pullara, J. F. Marshall and M. Leon (1991). "Olfactory deprivation increases dopamine D2 receptor density in the rat olfactory bulb." Synapse 8(1): 61-70.
Guthrie, K. M., D. A. Wilson and M. Leon (1990). "Early unilateral deprivation modifies olfactory bulb function." J Neurosci 10(10): 3402-3412.
Hubel, D. H., T. N. Wiesel and S. LeVay (1977). "Plasticity of ocular dominance columns in monkey striate cortex." Philos Trans R Soc Lond B Biol Sci 278(961): 377-409.
Johansen, J. P., C. K. Cain, L. E. Ostroff and J. E. LeDoux (2011). "Molecular mechanisms of fear learning and memory." Cell 147(3): 509-524.
Jones, S. V., D. C. Choi, M. Davis and K. J. Ressler (2008). "Learning-dependent structural plasticity in the adult olfactory pathway." J Neurosci 28(49): 13106-13111.
Jones, S. V., S. A. Heldt, M. Davis and K. J. Ressler (2005). "Olfactory-mediated fear conditioning in mice: simultaneous measurements of fear-potentiated startle and freezing." Behav Neurosci 119(1): 329-335.
100 Kandel, E. R. (2009). "The biology of memory: a forty-year perspective." J Neurosci 29(41): 12748-12756.
Katz, L. C. and C. J. Shatz (1996). "Synaptic activity and the construction of cortical circuits." Science 274(5290): 1133-1138.
Kaupp, U. B. (2010). "Olfactory signalling in vertebrates and insects: differences and commonalities." Nat Rev Neurosci 11(3): 188-200.
Kay, L. M. and S. M. Sherman (2007). "An argument for an olfactory thalamus." Trends Neurosci 30(2): 47-53.
Kerr, M. A. and L. Belluscio (2006). "Olfactory experience accelerates glomerular refinement in the mammalian olfactory bulb." Nat Neurosci 9(4): 484-486.
Kiselycznyk, C. L., S. Zhang and C. Linster (2006). "Role of centrifugal projections to the olfactory bulb in olfactory processing." Learn Mem 13(5): 575-579.
Krashes, M. J., S. DasGupta, A. Vreede, B. White, J. D. Armstrong and S. Waddell (2009). "A neural circuit mechanism integrating motivational state with memory expression in Drosophila." Cell 139(2): 416-427.
LeDoux, J. E. (2000). "Emotion circuits in the brain." Annu Rev Neurosci 23: 155- 184.
Lin, D. M., F. Wang, G. Lowe, G. H. Gold, R. Axel, J. Ngai and L. Brunet (2000). "Formation of precise connections in the olfactory bulb occurs in the absence of odorant-evoked neuronal activity." Neuron 26(1): 69-80.
Mandairon, N., A. Didier and C. Linster (2008). "Odor enrichment increases interneurons responsiveness in spatially defined regions of the olfactory bulb correlated with perception." Neurobiol Learn Mem 90(1): 178-184.
Mandairon, N., C. J. Ferretti, C. M. Stack, D. B. Rubin, T. A. Cleland and C. Linster (2006). "Cholinergic modulation in the olfactory bulb influences spontaneous olfactory discrimination in adult rats." Eur J Neurosci 24(11): 3234-3244.
Mandairon, N., S. Peace, A. Karnow, J. Kim, M. Ennis and C. Linster (2008). "Noradrenergic modulation in the olfactory bulb influences spontaneous and reward-motivated discrimination, but not the formation of habituation memory." Eur J Neurosci 27(5): 1210-1219.
Mandairon, N., C. Stack, C. Kiselycznyk and C. Linster (2006). "Enrichment to odors improves olfactory discrimination in adult rats." Behav Neurosci 120(1): 173-179.
101 Mandairon, N., C. Stack and C. Linster (2006). "Olfactory enrichment improves the recognition of individual components in mixtures." Physiol Behav 89(3): 379-384.
Mandairon, N., S. Sultan, M. Nouvian, J. Sacquet and A. Didier (2011). "Involvement of newborn neurons in olfactory associative learning? The operant or non-operant component of the task makes all the difference." J Neurosci 31(35): 12455-12460.
Meisami, E. and L. Safari (1981). "A quantitative study of the effects of early unilateral olfactory deprivation on the number and distribution of mitral and tufted cells and of glomeruli in the rat olfactory bulb." Brain Res 221(1): 81-107.
Mombaerts, P., F. Wang, C. Dulac, S. K. Chao, A. Nemes, M. Mendelsohn, J. Edmondson and R. Axel (1996). "Visualizing an olfactory sensory map." Cell 87(4): 675-686.
Moriceau, S. and R. M. Sullivan (2004). "Unique neural circuitry for neonatal olfactory learning." J Neurosci 24(5): 1182-1189.
Moriceau, S., D. A. Wilson, S. Levine and R. M. Sullivan (2006). "Dual circuitry for odor-shock conditioning during infancy: corticosterone switches between fear and attraction via amygdala." J Neurosci 26(25): 6737-6748.
Mutoh, H., Q. Yuan and T. Knopfel (2005). "Long-term depression at olfactory nerve synapses." J Neurosci 25(17): 4252-4259.
Nguyen, M. Q., Z. Zhou, C. A. Marks, N. J. Ryba and L. Belluscio (2007). "Prominent roles for odorant receptor coding sequences in allelic exclusion." Cell 131(5): 1009-1017.
Pavlov, I. P. and G. V. Anrep (1927). Conditioned reflexes; an investigation of the physiological activity of the cerebral cortex. London, Oxford University Press: Humphrey Milford.
Potter, S. M., C. Zheng, D. S. Koos, P. Feinstein, S. E. Fraser and P. Mombaerts (2001). "Structure and emergence of specific olfactory glomeruli in the mouse." J Neurosci 21(24): 9713-9723.
Quinn, W. G., W. A. Harris and S. Benzer (1974). "Conditioned behavior in Drosophila melanogaster." Proc Natl Acad Sci U S A 71(3): 708-712.
Skinner, B. F. (1938). The behavior of organisms; an experimental analysis. New York, London,, D. Appleton-Century Company.
Smear, M., R. Shusterman, R. O'Connor, T. Bozza and D. Rinberg (2011). "Perception of sniff phase in mouse olfaction." Nature 479(7373): 397- 400.
102 Soucy, E. R., D. F. Albeanu, A. L. Fantana, V. N. Murthy and M. Meister (2009). "Precision and diversity in an odor map on the olfactory bulb." Nat Neurosci 12(2): 210-220.
Strausfeld, N. J. and J. G. Hildebrand (1999). "Olfactory systems: common design, uncommon origins?" Curr Opin Neurobiol 9(5): 634-639.
Sullivan, R. M., G. Stackenwalt, F. Nasr, C. Lemon and D. A. Wilson (2000). "Association of an odor with activation of olfactory bulb noradrenergic beta-receptors or locus coeruleus stimulation is sufficient to produce learned approach responses to that odor in neonatal rats." Behav Neurosci 114(5): 957-962.
Sullivan, R. M. and D. A. Wilson (1995). "Dissociation of behavioral and neural correlates of early associative learning." Dev Psychobiol 28(4): 213-219.
Sullivan, R. M., D. A. Wilson and M. Leon (1989). "Norepinephrine and learning- induced plasticity in infant rat olfactory system." J Neurosci 9(11): 3998- 4006.
Sullivan, R. M., D. R. Zyzak, P. Skierkowski and D. A. Wilson (1992). "The role of olfactory bulb norepinephrine in early olfactory learning." Brain Res Dev Brain Res 70(2): 279-282.
Treloar, H. B., P. Feinstein, P. Mombaerts and C. A. Greer (2002). "Specificity of glomerular targeting by olfactory sensory axons." J Neurosci 22(7): 2469- 2477.
Turrigiano, G. G. and S. B. Nelson (2000). "Hebb and homeostasis in neuronal plasticity." Curr Opin Neurobiol 10(3): 358-364.
Tyler, W. J., G. C. Petzold, S. K. Pal and V. N. Murthy (2007). "Experience- dependent modification of primary sensory synapses in the mammalian olfactory bulb." J Neurosci 27(35): 9427-9438.
Valle-Leija, P., E. Blanco-Hernandez, R. Drucker-Colin, G. Gutierrez-Ospina and R. Vidaltamayo (2012). "Supernumerary formation of olfactory glomeruli induced by chronic odorant exposure: a constructivist expression of neural plasticity." PLoS One 7(4): e35358.
Wilson, D. A., A. R. Best and R. M. Sullivan (2004). "Plasticity in the olfactory system: lessons for the neurobiology of memory." Neuroscientist 10(6): 513-524.
Wilson, D. A., K. M. Guthrie and M. Leon (1990). "Modification of olfactory bulb synaptic inhibition by early unilateral olfactory deprivation." Neurosci Lett 116(3): 250-256.
Wilson, D. A. and C. Linster (2008). "Neurobiology of a simple memory." J Neurophysiol 100(1): 2-7.
103 Wilson, D. A., R. M. Sullivan and M. Leon (1985). "Odor familiarity alters mitral cell response in the olfactory bulb of neonatal rats." Brain Res 354(2): 314-317.
Wilson, D. A., R. M. Sullivan and M. Leon (1987). "Single-unit analysis of postnatal olfactory learning: modified olfactory bulb output response patterns to learned attractive odors." J Neurosci 7(10): 3154-3162.
Wilson, D. A. and J. G. Wood (1992). "Functional consequences of unilateral olfactory deprivation: time-course and age sensitivity." Neuroscience 49(1): 183-192.
Yuan, Q., H. Mutoh, F. Debarbieux and T. Knopfel (2004). "Calcium signaling in mitral cell dendrites of olfactory bulbs of neonatal rats and mice during olfactory nerve Stimulation and beta-adrenoceptor activation." Learn Mem 11(4): 406-411.
Zheng, C., P. Feinstein, T. Bozza, I. Rodriguez and P. Mombaerts (2000). "Peripheral olfactory projections are differentially affected in mice deficient in a cyclic nucleotide-gated channel subunit." Neuron 26(1): 81-91.
Zou, D. J., P. Feinstein, A. L. Rivers, G. A. Mathews, A. Kim, C. A. Greer, P. Mombaerts and S. Firestein (2004). "Postnatal refinement of peripheral olfactory projections." Science 304(5679): 1976-1979.
104 Chapter 4
SEROTONIN MEDIATES OLFACTORY LEARNING PARTIALLY BY
MODULATING EXPRESSION OF 5HT2A RECEPTORS IN GLOMERULI
Introduction
Serotonin’s role in learning and memory in the olfactory bulb
Centrifugal modulators have been found to play a key role in olfactory learning and memory. Acetylcholine (ACh) plays crucial roles in spontaneous odor discrimination in adult animals (Fletcher and Wilson 2002, Wilson, Fletcher et al. 2004, Mandairon, Ferretti et al. 2006, Chaudhury, Escanilla et al. 2009). Its mechanisms are thought to be via M1 and M2 receptors to increase lateral inhibition between glomeruli. Norepinephrine (NE) has been extensively studied in the context of neonate olfactory imprinting: a special form of associative learning available only during the first 10 days of life. In fact NE can play the role of the unconditioned stimulus when it alone is paired with an odor cue during the imprinting sensitive period. NE uses β1 adrenergic receptors to increase the strength of GABAergic synapses from granule cells to mitral cells, and potentially sharpen the output response of the mitral cell (Wilson and Leon 1988, Sullivan,
Wilson et al. 1989, Sullivan, Zyzak et al. 1992, Wilson and Sullivan 1994, Wilson,
Best et al. 2004).
Meanwhile serotonin’s role in olfactory bulb learning and memory has been the least studied. Initial evidence suggested that 5-HT may facilitate imprinting via 5-HT2 family receptors, since both 5-HT depletion and 5-HT2 antagonist attenuated imprinting behavior (McLean, Darby-King et al. 1993, McLean, Darby-
King et al. 1996, Price, Darby-King et al. 1998). However 5-HT only serves as
105 compliment to NE, which is the stronger, necessary and sufficient for neonate imprinting. Since both β1 adrenergic receptors and 5HT2A receptors are co- localized on Mitral cells (Yuan, Harley et al. 2003), the two neuromodulators are thought to converge on the same intracellular signaling mechanisms causing an increase in cAMP, PKA and pCREB, a classical learning and memory cascade with potential for LTP-like changes in synaptic strength (Yuan, Harley et al. 2003,
Yuan, Mutoh et al. 2004, Mutoh, Yuan et al. 2005).
Serotonin innervation and receptor distribution in the olfactory bulb
Studies have shown that serotonin’s innervation of the olfactory bulb, and specifically in the glomerular layer, is one of the densest compared to any other region in the brain. Given such a rich presence, few studies have elucidated functional and behavioral roles of 5-HT in olfactory bulb, especially not in the glomerular layer. The dorsal raphe nucleus projects to the OB with innervation of the outer granule and mitral layers, glomerular layer (Dahlstrom, Fuxe et al.
1965, Fuxe 1965, Moore, Halaris et al. 1978), but 5-HT preferentially innervates the glomerular layer with a dense plexus (Price and Powell 1970, Halaris, Jones et al. 1976, Moore, Halaris et al. 1978, Bobillier, Seguin et al. 1979, Steinbusch
1981, McLean and Shipley 1987). Cross species analysis reveals that while rodents show heaviest serotonin innervation in the glomerular layer, cat and monkey olfactory bulbs show a majority of serotonin fibers in the granule cell layer (Takeuchi, Kimura et al. 1982). In primates and humans 5-HT enervation of the OB is significantly less than rodents, rabbits or cats (Jacobs and Azmitia
1992), perhaps indicating an evolutionary shift from dependence on olfaction to dependence on vision being as main sensory modality.
106 Serotonin receptor mRNA situ hybridization and immunohistochemistry show that the OB expresses 5-HT2A,C receptors (Pompeiano, Palacios et al.
1994, Hamada, Senzaki et al. 1998), 5-HT1A,B receptors (Pazos and Palacios
1985, Pompeiano, Palacios et al. 1992) and 5-HT3 receptors (Tecott, Maricq et al. 1993, Gehlert, Shekhar et al. 2005). The role of 5HT1 family receptors and
5HT3 receptors in the OB are relatively unknown. The 5-HT1 family receptors preferentially reside in the granule and external plexiform layers. Studies using
5HT1 specific antagonists in the OB, however, have not found any acute effects of pharmacology on functional properties of the circuit (Hardy, Palouzier-
Paulignan et al. 2005, Petzold, Hagiwara et al. 2009). The novel, ionotropic 5-
HT3 receptor has not yet been investigated in the OB. However, in other primary sensory circuits such as the retina, 5-HT3 receptors work in concert with receptors to excite ON-center and inhibit OFF-center neurons to facilitate bipolar cell conduction (Brunken and Jin 1993).
Serotonin 2A family receptors are prevalent in the OB and have modulatory effects on many circuits in the OB. Serotonin 2A are preferentially located in the glomerular layer and external tufted layer. In fact ET and Mitral and OB show stronger levels of 5-HT2AR mRNA than any other brain region (Hamada, Senzaki et al. 1998). The 5-HT2CRs, however, are found with low to medium levels of expression in the glomerular and granule cell layers (Pompeiano, Palacios et al.
1994, McLean, Darby-King et al. 1995). 5-HT2A receptors are expressed in both dendrites and axons of the cells (Cornea-Hebert, Riad et al. 1999). More recently, immunohistochemistry has shown that 5-HT2ARs receptors are expressed on mitral cells along with β1 adrenergic receptors, possibly acting
107 synergistically to produce plasticity in the mitral- granule circuit (Yuan, Harley et al. 2003).
Serotonin actions in the OB
Actions of serotonin the olfactory bulb have been assessed in several key studies using electrophysiology and functional imaging. More details about how serotonin modulates the glomerular circuit will be discussed in Chapter 5. The overall functional role that 5-HT plays at glomerular level may be to sharpen mitral cell outputs from the bulb. Serotonin’s direct actions are to excite glutamatergic mitral cells and external tufted (ET) cells are via 5-HT2A receptors and also excite GABAergic PG cells via 5-HT2C receptors. In ET cells, acute activation of 5-HT2A receptors increase spiking output and bursting output by depolarizing the cell via a TRP channel (Hurley and Pollak 1999, Hardy,
Palouzier-Paulignan et al. 2005, Liu, Aungst et al. 2012). Despite exciting both glutamatergic ET cells and GABAergic PG cells, the overall effect of an influx of
5-HT from the DRN causes the relay station to be inhibited. This seems to be achieved by increase GABAergic release by PG cells causing presynaptic inhibition of OSNs transmitter release by GABAB Rs (Petzold, Hagiwara et al.
2009).
Informed by the literature about the function of each glomerular circuit element, I can make a more comprehensive hypothesis of the role for 5-HT in the circuit and how it integrates both sensory gating and learning and memory. Given that 5-HT and 5-HT2A receptors are heavily present in the glomerular layer and have strongly modulate circuit interactions within the glomerulus, I propose that it has a functional role in olfactory guided behavior. Since it already plays a
108 significant role at deeper synapses in the OB for young neonate animals, I suggest the 5-HT may also be involved in the plasticity underlying learning and memory in the glomerulus in the adult animal.
In this study, I ask the question: how does serotonin play a role in olfactory associative learning memory in the olfactory bulb of adult olfactory bulb, especially in the glomerulus? I hypothesize that serotonin is involved in both the acquisition stage of learning (perhaps to convey an unconditioned stimulus) and maintenance stage of an olfactory memory trace (to solidify a long change in sensory gating properties). To test this hypothesis, first I map the olfactory bulb for 5-HT fibers and its family of 2A and 2C receptors. Second, I test if serotonin is involved in learning acquisition by in vivo behavioral pharmacology. Finally, I test if serotonin signaling is permanently altered as part of a long term memory trace in in the maintenance stage of learning memory
Methods
Animals
Animals used were M72-iresTauGFP for in vivo pharmacology experiments and post-learning assays of 5-HT elements and GAD::65 GFP for 5-HT circuit mapping. Animals were reared in a 12/12 light dark cycle with ad libidum access to food according to standard animal care protocols. All animals were cared for and raised according to IAUCUC institutional guidelines. All mice were ages 2-5 months of age. For circuit mapping, both male and female mice were used. For all experiments involving behavioral conditioning, only male mice were used.
109 Behavioral Training
All behavioral conditioning for in vivo pharmacology or post-learning assays of 5-HT circuitry was performed according to the protocol described in the methods section of Chapter 3. For in vivo pharmacology experiments, I used 3 groups of animals that each underwent fear conditioning: odor-shock pairing
(US+/CS+) conditioning as described previously, but received drug or vehicle injections/infusions on training days prior to training sessions.
For immunoassays post-learning, I used both fear conditioning and reward conditioning. For the post-learning immunohistochemistry assays, I used both fear and reward learning (CS+/US+) animals as well as all the control groups
(CS+/US-) (CS-/US+) described in chapter 3 and also added a naïve group (CS-
US-). For all experiments, on test day, appropriate output behavior indicating successful associative learning was assayed as described previously. Freezing behavior (for fear conditioning) and correct % of lever presses were recorded.
Animals were sacrificed 48 hours after the last learning session for perfusion/brain fixation according to the IAUCUC approved protocol.
Immunohistochemistry
Transcardial perfusion, paraformaldehyde fixation, slicing and histology of olfactory bulbs were performed by standard protocol according to the methods described by in chapter 3. A standard immunofluorescence protocol was used as described in the methods in chapter 3. To label serotonin fibers and puncta, slices were incubated in rabbit anti-serotonin antibody (1:1000; Abcam-8882,
Abcam, Cambridge, MA). To assay for 5-HT2A receptors, slices were incubated in rabbit anti-5HT2A antibody (1:1000; Abcam-16028). To assay for 5HT2C receptors,
110 slices were incubated in rabbit anti-5HT2C antibody (1:1000; Abcam-32172).
Second Antibody Alexa-568 anti-rabbit secondary antibody (1:1000; Life
Technologies) overnight at 4° C to confer fluorescence. To label cell bodies experiments, slices were exposed to TO-PRO-3 (1:1000; Life Technologies,
Carlsbad, CA) in PBS for 15 minutes at room temperature following incubation in primary and secondary antibodies.
Confocal microscopy
All imaging was performed on an upright Olympus Fluoview FV-300 laser- scanning confocal microscope (Olympus America, Inc., Center Valley, PA, USA).
I used the 488 nm laser line of an Argon laser to excite GFP originating from immunolabeled slices prepared from GAD65::GFP or M72-GFP mice. I excited
TO-PRO-3 using a 633 nm HeNe laser and the Alexa-568 antibodies were excited using a 543 nm HeNe laser. Images were acquired using a 10X air (0.3
NA), 20X oil (1.2 NA), or 60X oil (1.45 NA) objective lens. Individual optical sections (2 µm) were taken from regions of interest through the entire extent of the tissue and stored on a computer for later offline analysis.
Behavioral pharmacology
In different groups of M72-GFP mice, I used pharmacological blockers of 5-
HT2A and 5-HT2C receptors to determine their effects on odor-mediated fear conditioned behaviors. To block 5-HT2A receptors, M72-GFP mice (N =9 ) received an intraperitoneal (i.p.) injection of the 5-HT2A receptor antagonist MDL-
100907 (0.1 mg/kg, Tocris Bioscience, Bristol, United Kingdom) in 0.2mL 0.9% saline. To block 5-HT2C receptors, M72-GFP mice (n = 6 ) received an i.p
111 injection of the 5-HT2C receptor antagonist SB-242,084 (0.1 mg/kg, Tocris
Bioscience) in 0.2mL 0.9% saline. A control group of M72-GFP mice (N =6 ) received an i.p. injection of 0.2mL 0.9% saline vehicle. On each training day following injections, mice were allowed 20 minutes of recovery time to allow the drug to begin taking action before fear conditioning training sessions began. On the test day, all animals received 0.2mL 0.9% saline injections 20 minutes before testing.
Another group of M72-GFP mice received intra-bulbar infusions of the N- methyl-D-aspartate (NMDA) receptor antagonist amino-5-phosphonovaleric acid
(APV) and the 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid (AMPA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 5-HT2A receptor antagonist MDL-100907, or vehicle to their determine effects on odor- mediated fear conditioned behaviors.
These animals were surgically affixed with cannulas overlying their olfactory bulbs to facilitate daily infusions of the drug or vehicle. Briefly, mice were anesthetized using an i.p. injection of a Ketamine (70 mg/kg) and Xylazine (7 mg/kg) cocktail. Once mice reached a surgical plane of anesthesia, a small hole over the dorsal surface of each olfactory bulb and the dura covering the bulb was perforated using a 30-gauge needle. A 21-gauge stainless steel cannula was then inserted into the craniotomy and secured in place using a small amount of dental acrylic cement. Cannulas were then capped using a blunted 30-gauge section of stainless steel tubing. Mice were recovered from surgery and monitored for 24 hours post-surgery. Mice were then allowed to recover an additional two days before chamber acclimation began.
112 On each of the two training days, 15 minutes prior to being placed in the chamber, one group of mice (N = 9) received a 5 µL infusion of MDL-100907
(100mM) in normal artificial cerebrospinal fluid (aCSF; see electrophysiology section below) through the cannula over each bulb, another group of mice (N =3) received a 5 µL infusion of a CNQX (10µM) and APV (50µM) mixture into each bulb, and a final vehicle control group (N =6 ) received 5 µL infusions of aCSF into each bulb. On the testing day, all animals received a 5 µL infusion of aCSF into each bulb 15 minutes prior to testing.
Analysis of immunohistochemistry
For analysis of immunofluorescence, the region of M72 glomerulus area was defined using TO-PRO-3 to outline cells of the region corresponding to the
GFP labeled M72 glomerulus. For control glomeruli from each animal, five random glomeruli were defined in a similar manner using TO-PRO-3 from the same slice of olfactory bulb as the M72 glomerulus. The control glomeruli were spaced at least five glomeruli away from the M72 glomerulus to avoid cross activation. I determined that glomeruli spaced this distance away from the M72 away were not co-activated as determined by c-Fos labeling since c-Fos signals were not only observed in M72 glomerulus but also in adjacent glomeruli following odor activation. The densities of 5-HT puncta and 5-HT2A receptors were calculated for the M72 glomerulus, as well as control glomeruli using
ImageJ. Serotonin fibers entering the olfactory bulb could be visualized as puncta in the glomerular layer. I suspected the first possibility since, in my parasagittal slice, 5-HT fibers coming into the olfactory bulb slice from the brain (near the rostral migratory stream) was in fiber form, but by the time it reached glomeruli, it
113 was is discontinuous puncta form (probably due to fibers turning in direction to enervate the glomerulus).
Results
I found that serotonin innervation was highest in the glomerular layer and much lower in the external plexiform layer and granule cell layer. When I over- layed 5-HT with GAD65-GFP, I found that a proportion of GABAergic periglomerular cell bodies co-labeled with 5-HT. This may indicate that 5-HT fibers synapse onto juxtaglomerular neurons (both PG and ET). Moreover, 5-HT fibers were not found in the OSN layer (Figure 6A).
Serotonin 2A receptors were also missing from the OSN layer, but expressed most also densely in the glomerular layer, external tufted cell layer, middle tufted cell layer and mitral cell layer. Noticeably 5-HT2ARs were located in the apical dendrites of Mitral cells and dendrites of ET that strongly arborize extensively in the glomerulus to provide 5-HT immuno-reactivity in this a cellular region. The 5-HT2ARs positive cell bodies did no co- localize with GAD65-GFP cell bodies (Figure 6B). This may indicate that these receptors reside exclusively on glutamatergic external tufted cells while sparing GABAergic periglomerular cells. However 5-HT2AR positive dendrites from ET cells did overlay with GAD65-
GFP cell bodies and presumably GAD65-GFP cell dendrites. ET cells may synapse onto neighboring GABAergic periglomerular cells in the same glomerulus to alter their activity. The positioning of 5-HT2ARs on the dendrites as well as cell bodies may indicate that these receptors modifies activity locally at post synaptic sites of the glutatmergic cells on which it resides, in addition to the electrical properties and intrinsic excitability of that cell at large (Figure 6B).
114
Figure 6. Serotonin (5-HT), 5-HT2A and 5-HT2C receptors are differentially located across OB layers and differentially co-localize with GABAergic interneurons. Immunofluorescence for 5-HT, 5-HT2AR or 5-HT2CR co-labeled with GABAergic marker GAD65-GFP and cell body stain TOPRO-3. A.5-HT innervation of the glomerular layer is heavy, but of mitral and granule cell layers is weak. High-magnification shows 5-HT innervation of both GABAergic and non-GABAergic juxtaglomrular (JG) cells. B. 5-HT2ARs are heavily expressed in mitral and glomerular layers, but not in granule layer. High-mag shows lack of co-localization with non-GABAergic ET cells (white arrows) C. 5-HT2CRs are heavily expressed in the glomerular and granule cell layers but not mitral cell layer. High-mag shows co-localization with GABAergic PG cells (white arrows).
115 Immunostaining for 5-HT2C receptors yielded fewer positive cells in the glomerular layer, only a very small proportion of the total number of TOPRO-3 stained cells. In contrast to 5HT2A receptors, 5-HT2CR positive cells were found to be lightly expressed in the mitral cell layer and most abundantly expressed in the granule cell layer. Not only was the pattern of expression different, 5HT2C receptors are mainly located on the cell body of the cells that express it. This may indicate that its function is less for modulation at synaptic sites but rather altering overall neuronal excitability. Overlay with GAD65-GFP indicates that 5-
HT2CR positive cells co-localize GABAergic interneurons (both in the PG cells of the glomerular layer as well as in the deeper granule cells). The co-localization is far from exclusive, however, as only a minority of GABAergic PG cells are 5-
HT2CR positive. OSNs did not express 5HT2C receptors (Figure 6C).
Disruption in 5-HT2AR signaling but not 5-HT2cR signaling attenuates associative learning
To investigate the role of serotonin signaling via 5-HT2 family receptors in the acquisition of associative fear learning, I used 5-HT2AR (MDL100907) and 5-
HT2cR (SB242084) specific antagonists to disrupt signaling during the learning acquisition stage of fear conditioning. In my first set of experiments, intraperitoneal injection of these specific, blood brain barrier permeable drugs and were used at minimum physiological relevant dosages to prevent cross activation of the other receptor of the same family. The drugs were administered
116
Figure 7. Pharmacological blockade of 5-HT2A receptors but not 5-HT2C receptors attenuates fear conditioned freezing behavior. A. Training and drug treatment paradigms for intra-peritoneal pharmacological blockade of 5- HT learning system. Injections occurred on training days prior to odor-shock associative training sessions. No drugs were given on test day. B. Mean ± SEM percentage of time animals spent freezing on test day when given saline vehicle, selective 5-HT2AR antagonist MDL100907 or selective 5-HT2CR antagonist SB242084 during training. Animals receiving TCB-2 showed attenuation of associative learning as showing by lower freezing times during odor presentation (P < 0.05 n = 6) C. Training and drug treatment paradigm for intra-bulb pharmacological blockade of receptors (proposed US+) or odor input (proposed CS+) in the olfactory bulb only. Intra-bulb infusions were given on both training days prior to associative odor-shock training. No drugs were given on test day. D. Mean ± SEM of percentage of time animals spent freezing on test day. Treatment groups include infusions of aCSF, MDL100907 or CNQX+APV to block glutamatergic synaptic transmission of odor inputs. Animals receiving intra-bulbar TCB-2 experience decreased %time freezing indicative of attenuation of learning (P < 0.05).
117 precisely 20 minutes previous to behavioral conditioning during the 2 days of training (Figure 7A). I found that mice receiving 5-HT2AR but not 5-HT2C antagonist showed significant attenuation in fear behavior compared vehicle treated mice. When I compared percentage of time freezing during odor presentation period across groups, freezing was attenuated for the MDL100907 treated group (Saline: 78.57 ± 3.32, MDL100907: 50 ± 15.25, SB242084: 74.00 ±
5.78) (ANOVA F = 4.164 (2, 19), p < 0.05, n =12 (saline), n = 6 (2A antagonist), n
= 6 (2C antagonist), Post hoc Tukey's: MDL100907: versus saline p < 0.05;
SB242084 versus saline p = 0.885). (Figure 7B).
However the effect caused by 5HT2AR antagonist only attenuated freezing behavior, but did not completely obliterate it. The data show there was still a shallow inverted-U curve for learning with MDL100907 treatment (Repeated measures ANOVA F =11.25 (2, 8), p < 0.05 n = 6, Odor versus Pre-odor: p <
0.05, Odor versus post odor: p < 0.05, Pre-odor versus post: odor p = 0.99)
(Figure 7B).
In the second set of experiments I verified the above result using intracranial of drugs directly into the olfactory bulb cavity. Again, I administered the antagonist 20 minutes before behavioral conditioning on training days (Figure
7C). I focused on the 5-HT2A receptors since they were the only group with significant effects in the previous experiment. Additionally, I introduced another group of mice who received CNQX+APV infusion, thus blocking glutamatergic excitation from olfactory sensory neurons. I predicted that either blocking either the CS (via CNQX+ APV) or blocking the US (using 5-HT2AR) should negatively affect the animal’s ability to acquire associative fear learning.
118 I found that blocking either 5-HT2AR with (MDL100907) or blocking glutamatergic signaling(CNQX/APV) attenuated fear associative learning(Figure
7C). Blockade of 5-HT2AR signaling or glutamatergic signaling specifically in the bulb resulted in reduced percentage freezing times during in the MDL100907 treated groups and the CNQX+APV group when compared to the vehicle treated group. (aCSF: 73.33±3.58, MDL100907: 40.63±10.15, CNQX+APV:
26.67±13.02) (ANOVA F (2,14)=5.47, P<0.05, n=6 (aCSF), n=9 (MDL100907), n=3(CNQX+APV), Post-hoc Tukey’s: MDL100907 versus aCSF p<0.05,
CNQX+APV versus aCSF P<0.05, CNQX+APV versus MDL100907 antagonist
P=0.643). (Figure 11D).
Neither of the two intracranial treatments was enough to completely obliterate learning, only attenuate it. Mice treated with MDL100907 still showed more freezing during odor presentation compared to pre-odor periods) (Pre-odor:
12.50±4.82, Odor, 40.63±10.15, Post-odor 41.25±10.72) (ANOVA F (2,14) =4.77,
P<0.05, n=6) (Post-hoc comparisons: Pre-odor versus Odor: p<0.01, Odor versus Post-odor: p=0.96). The animals were could still weakly acquire an association between odor cue and foot shock, but could not stop freezing in the post-odor period. Mice receiving CNQX+APV treatment, freezing behavior was fairly low in all three periods with no odor-specific freezing (Pre-odor: 1.67±1.67,
Odor: 26.67±13.02, Post-odor: 32.67±13.02) (Repeated measures ANOVA
F=3.94 P=0.113, n=3). These mice may have become anosmic due to blockade of glutamatergic transmission from OSNs (Figure 7D).
119
Changes 5-HT2AR expression and 5-HT fiber density persist 48 hours after learning as part of a memory trace
I established that serotonin via 5-HT2AR signaling plays a role in the acquisition of odor associative learning. Next, I questioned if changes in the 5-
HT2AR signaling system also participates in the maintenance of that olfactory memory. I used both fear and reward learning paradigms that were established in
Chapter 3 to instill olfactory associations between acetophenone and shock or sucrose in adult M72-GFP mouse. I also included the full panel of control groups described in Chapter 3. Fourty-eight hours after learning, I used the M72-GFP circuit to guide my glomerulus specific interrogation for learning-dependent changes in serotonergic signaling using immunohistochemistry.
By overlaying the either confocal Z-stacks of 5-HT or 5-HT2A immunofluorescence with Z-stacks of the M72-GFP glomerulus, I quantified the density of 5-HT puncta as well as the density of 5-HT2AR positive cells in the glomerular and juxtaglomerular region. Then, I compared this to the density of 5-
HT and 5-HT2AR in non-M72 glomeruli of the same bulb that are at least 5 glomeruli away (Figures 8B, D). Area was defined by either the M72-glomerulus outline along with TO-PRO-3, which clearly outlines glomeruli by staining all juxtaglomerular cells. My data showed that even at 48 hours after the last learning session, both pre and post synaptic elements of serotonergic circuit were denser in the M72 glomerulus vs. control glomeruli of fear learning animals.
Specifically, I found that density of 5-HT innervation of M72 glomeruli glomerulus was also higher than control glomeruli in the same bulb, but only in fear learned animals. (Fear learned M72 glomerulus: 2.93x10-2 ± 3.99x10-3
120
Figure 8. Fear associative learning increases density of serotonin puncta and 5HT2AR in the M72 versus control glomerulus. A. Mean ± SEM serotonin puncta density in the M72-GFP glomerulus compared control glomeruli of the same animal that learned to associative fear learning with acetophenone. Control groups include odor only, shock only and naive animals. M72 glomerulus had higher 5-HT puncta density compared to control glomeruli only in the fear learned animals (p < 0.05). B. Example of immunohistochemistry for 5-HT puncta M72 (LEFT) versus control glomerulus (RIGHT) of a fear learned mouse C. Mean ± SEM 5-HT2AR immuno-positive cell density in M72-GFP versus control glomeruli in the same mice after fear associative learning and control paradigms. M72 glomerulus had a higher 5- TH2AR positive cell count than control glomeruli in the same animal (p < 0.05). D. Example of immunohistochemistry for 5-HT2AR in M72 (LEFT) versus control glomerulus (RIGHT) of fear learned mice.
121
Figure 9. Reward associative learning increases density of 5HT2AR in the M72 versus control glomerulus. A. Mean ± SEM serotonin puncta density in the M72-GFP glomerulus compared control glomeruli of the same animals after reward associative learning paradigms. Control groups include odor only, sucrose only and naive animals. There were no significant differences in 5-HT puncta in any groups. B. Example of immunohistochemistry for serotonin puncta M72 (LEFT) versus control glomerulus (RIGHT) of mice who were fear conditioned. C. Mean ±SEM density of 5HT2AR immuno-positive cells in M72-GFP versus control glomeruli in the same mice after reward associative learning paradigms. Control groups include, odor only, sucrose only and naïve mice. M72 glomerulus had a higher 5-HT2AR positive cell count than control glomeruli in the same animal (p<0.05). D. Example of immunohistochemistry of 5HT2AR positive cells in M72 (LEFT) versus control (RIGHT) glomerulus in mice who were reward conditioned.
122 puncta/ µm2, Control glomeruli: 1.83x10-3 ± 2.03x10-3 puncta/ µm2) (Paired T
Test, p < 0.05, n = 5). Moreover, I found that 5-HT2AR density was also higher in
M72 glomeruli and control glomeruli, but again, only in the fear learning group.
-3 -4 2 (M72 glomerulus: 2.33x10 ± 2.26x10 5HT2AR positive cells/µm , Control
-3 -4 2 glomerulus 1.48x10 ± 1.39x10 5-HT2AR positive cells/µm , Paired T-test, p <
0.05, n = 11) (Figure 8C). I preformed the same experiment in reward conditioned animals with quantification of 5-HT and 5-HT2ARs of mice in the reward learning group as well as control groups, comparing M72 glomeruli with control glomeruli in the same bulb (Figure 9B,D). I did not find a difference between M72 and control glomeruli with respect to 5-HT puncta density in the reward learning group nor any other control group (Reward M72 glomeruli:
1.68x10-3 ± 1.77 puncta/ µm2 and reward control glomeruli:1.95x10-2 ± 2.83x10-3
2 puncta/ µm ) (Paired T test, p = 0.78) (Figure 8A). With respect to 5-HT2AR, however, I found higher levels of 5-HT2A receptors in the M72 glomerulus over control glomerulus, again, only in animals of the reward learning group (M72
-3 -4 2 glomerulus: 2.47x10 ± 3.04x10 5-HT2AR positive cells/µm , Control glomeruli
-3 -4 2, 1.44x10 ± 1.13x10 5-HT2AR positive cells/µm Paired T-test, p < 0.05, n = 5)
(Figure 12C). None of the control groups showed any significant differences in 5-
HT2AR density between M72- versus control glomeruli (Figure 8C).
Discussion
In the studies in of this chapter, I tested the hypothesis that serotonin signaling via 5-HT2AR receptors plays an important role in both the acquisition and maintenance of associative olfactory memory at the glomerular relay station.
First, there were several lines of logic that lead me to believe 5HT2A receptors
123 may participate in the initial acquisition of fear memories. For example, neuromodulators are essential for either carrying the signal for the unconditioned stimulus for olfactory associative learning in many species and circuits (including
Drosophila, Apis Mellifera, C. Elegans, Aplysia, and rodents) by carrying information about the arousal state of the animal (Brunelli, Castellucci et al. 1976,
Glanzman, Mackey et al. 1989, Sullivan, Wilson et al. 1989, Sullivan, Zyzak et al.
1992, Hammer 1993, Nuttley, Atkinson-Leadbeater et al. 2002, Schwaerzel,
Monastirioti et al. 2003, Kim, Lee et al. 2007). Yet a second reason is that neuromodulator serotonin and its receptor 5-HT2AR specifically are densely present in the glomerular layer of the OB (Price and Powell 1970, Moore, Halaris et al. 1978, McLean and Shipley 1987, McLean and Shipley 1987, Pompeiano,
Palacios et al. 1994, McLean, Darby-King et al. 1995).
Additionally, I had reasons to suspect that plasticity of serotonergic circuits in the glomerulus may participate to maintain the memory trace of the olfactory memory. Foremost, if sensory gates were to exist at the periphery
(Shepherd 2005, Kay and Sherman 2007) and function optimally, then it must be informed learning and memory. Thus some traces of learning and memory must exist at the glomerular relay station as they do in the close by mitral-granule station (Wilson, Sullivan et al. 1987, Sullivan, Wilson et al. 1989, Wilson and
Sullivan 1994, Yuan, Harley et al. 2000, Yuan, Harley et al. 2003, Yuan, Harley et al. 2003). The glomerular station is already capable of harboring experience dependent plasticity due global enrichment or deprivation. This plasticity exists functionally (Guthrie, Wilson et al. 1990, Wilson and Wood 1992, Mandairon,
Stack et al. 2006, Mandairon, Stack et al. 2006, Mandairon, Stack et al. 2006,
Tyler, Petzold et al. 2007) and structurally (Kerr and Belluscio 2006, Cavallin,
124 Powell et al. 2010, Valle-Leija, Blanco-Hernandez et al. 2012). Most recently, artificial induction of LTP and LTD like plasticity has also been found at the glomerulus, at least in neonates (Yuan, Mutoh et al. 2004, Mutoh, Yuan et al.
2005, Yuan 2009). I propose that the long term synaptic plasticity endowed by formation of associative memories in adults is the same long term plasticity that is retained as part of an optimized sensory gate. Moreover, sensory gates in central synapses heavily modulated by centrifugal neuromodulators, among them serotonin (McCormick and Pape 1988, Pape and McCormick 1989, McCormick and Pape 1990, Lee and McCormick 1995, Monckton and McCormick 2002).
Overall, my results support my hypothesis. I demonstrated that serotonin signaling participates in the acquisition and maintenance of olfactory fear associative learning and memory. I also showed that this was mediated by 5-
HT2A receptors, but not 5HT2C receptors in the olfactory bulb, with a potential memory trace at the glomerulus maintained in part by elevated 5-HT2AR signaling.
5-HT and 5HT2 receptors circuit mapping
The results from my mapping study of serotonergic elements in the circuits confirmed previous studies while providing new insights on the specific function of 5-HT in the olfactory bulb. First I showed that serotonin preferentially innervates the glomerular layer over all other lamina of the OB which agrees with older studies using electron microscopy, autoradiography, retrograde tracing or immunohistochemistry (Halaris, Jones et al. 1976, Halasz and Shepherd 1983,
McLean and Shipley 1987, McLean and Shipley 1987, Gomez, Brinon et al.
2005). Second I showed that complimentary to the presynaptic 5-HT fibers,
5HT2A receptors are also preferentially expressed in the glomerular layer in
125 accordance with previous mapping studies. Then, I found that 5-HT2CR receptors were faintly and sparsely found the glomerular layer, but were expressed moderately granule cell layer as found in previous studies (Pompeiano, Palacios et al. 1992, Pompeiano, Palacios et al. 1994, McLean, Darby-King et al. 1995,
McLean, Darby-King et al. 1996), except one recent study(Petzold, Hagiwara et al. 2009). Finally, I found that neither 5-HT fibers nor any 5-HT2 receptors are found in the OSN nerve layer.
Confocal microscopy allowed me to see in in detail that receptors were heavily expressed not only in the cell body but also the dendrites of cells in the glomerular, external tufted later and mitral cell layer—many of these dendrites terminated in the glomerulus which resulted in dense 5-HT2AR presence in the first relay station. Meanwhile, 5-HT2CRs were located in some juxtaglomerular cell bodies and granule cell bodies, but did not seem to be expressed in dendrites. This observation can provide clues on the potentially different roles of each receptor either influence synaptic gain in dendrites or to adjust the intrinsic excitability and firing rate of a neuron.
My study’s use of triple channel confocal microscopy and immunofluorescence allowed me to contribute some new details about the positioning of serotonergic elements. Using GAD65-GFP mice allowed me to overlay 5-HT or 5-HT2AR with GAD containing GABAergic cells in the olfactory bulb, namely periglomerular cells (PG) and granule cells (GC). I found that 5-HT fibers terminated on both GABAergic and non GABAergic cells in the glomerular layer. More interestingly, 5-HT2ARs were only found on non-GABAergic
(presumably glutamatergic) cells while 5-HT2ARs were found on GABAergic PG cells. This leads me to believe that 5-HT2AR is expressed on glutamatergic Mitral
126 and ET cells while 5-HT2CR is expressed, faintly on GABAergic PG cells. My results were the first to show demonstrate the differential location of the two receptor subtypes on GABAergic versus glutamatergic elements of the circuit using histology. This data is in agreement with functional studies (Hardy,
Palouzier-Paulignan et al. 2005, Petzold, Hagiwara et al. 2009, Liu, Aungst et al.
2012) using electrophysiology and calcium imaging that have located 5-HT2ARs to Mitral cells and ET cells while locating 5-HT2CRs to GABAergic PG cells.
My study is limited in that I only assayed for 5-HT2 family receptors in the bulb, while future assays can include mapping for 5-HT1A/B receptors
(Pompeiano, Palacios et al. 1992) as well as for the newly found 5-HT3 ionotropic receptor which has been implicated in sensory processing (Brunken and Jin
1993). It would be interesting to know if these two families of receptors also show laminar specification, or be expressed on certain cell types over others. For example, 5-HT1A family receptors play key role in sensory gating in the thalamus
(Monckton and McCormick 2002), and may also be involved in similar processes in the OB. Moreover, it will be especially interesting to see the locations of the 5-
HT3 ionotropic receptors, since they have the possibility of facilitating fast synaptic actions (Maricq, Peterson et al. 1991).
Mapping serotonin and its receptors in the bulb was an important first step in this investigation since it allowed me to identify the potential receptors subtypes that may be involved in learning and memory at the glomerulus. This informed my decisions on the choice of pharmacological agents to use in the next set of behavioral pharmacology experiments as well as which receptor subtype to concentrate on in my post-learning interrogation for longer term plasticity.
127 Given my knowledge of synaptic interactions between each element of the circuit as well as their role in information processing in the glomerulus (Hayar,
Karnup et al. 2004, Hayar, Karnup et al. 2004, Hayar, Shipley et al. 2005, Hayar and Ennis 2007, Gire and Schoppa 2009), their response to 5-HT via 2A and 2C receptor subtypes (Hardy, Palouzier-Paulignan et al. 2005, Petzold, Hagiwara et al. 2009, Liu, Aungst et al. 2012), and now the exact location of serotonin elements within olfactory bulb circuit, I can make a generalized hypothesis on 5-
HT actions in the olfactory bulb to promote sensory gating. For example, I might propose that 5-HT2AR receptors depolarize ET Cells and Mitral cells, the glutamatergic elements of the circuits so that these cells will fire more easily leading to increase mitral cell output to cortex. Meanwhile, I might propose that 5-
HT2CR receptors, located on GABAergic PG cells, also depolarize these cells, increasing PG firing rates to release copious amounts of GABA to provide 1) feedback inhibition to OSNs and 2)feed-forward inhibition to ET and Mitral cells.
5-HT2A receptor signaling participates in acquisition of olfactory fear memories
In the second part of this study, I tested the hypothesis that 5-HT confers the arousal signal for the unconditional stimulus of olfactory associative learning at the olfactory glomerulus. My hypothesis was based in the literature where many studies have found that neuromodulators either completely sufficient as the unconditioned stimulus or at least play a partial role as the unconditional stimulus. In the insect, literature, for example dopamine and octopamine have been found to be sufficient, by their actions in the mushroom bodies to induce associative fear or reward learning in drosophila and honey bees (Hammer 1993,
Schwaerzel, Monastirioti et al. 2003, Kim, Lee et al. 2007). In other invertebrate
128 models such as Aplysia or C. Elegans (Brunelli, Castellucci et al. 1976, Bailey,
Giustetto et al. 2000, Zhang, Lu et al. 2005, Lee, Choi et al. 2009), serotonin is a signal for the aversive unconditioned stimulus. In the rodent literature, neonate odor imprinting can be artificially induced by pairing norepinephrine OB infusions with an odor (Sullivan, Wilson et al. 1989, Sullivan, Zyzak et al. 1992). Several other studies suggest that in the neonate rat, serotonin works in concert with NE to achieve imprinting (Price, Darby-King et al. 1998, Yuan, Harley et al. 2003).
I found that indeed, disrupting serotonergic signaling either systemically or directly in the olfactory bulb during training attenuated olfactory fear associative learning. Serotonin’s effects in the bulb seem to be mediate by 5-
HT2A receptors not 5-HT2C receptors. Moreover, I found that blockade of either 5-
HT2A R or odor input signals via CNQX/APV also attenuated learning. My study is the first to show the role of serotonin in the acquisition of associative learning in the adult rodent olfactory bulb and then isolate the receptor subtype that is responsible.
The design of my experiment ensured that I were testing for serotonin’s role during acquisition, since pharmacological blockade was only given during previous to training periods, but not on test day. This type of pharmacological blockade has been use previously to assay for the necessity of NE for neonate imprinting (Sullivan, Zyzak et al. 1992), ACh for adult odor discrimination
(Mandairon, Ferretti et al. 2006) and 5-HT for neonate imprinting (McLean,
Darby-King et al. 1996). In my experiments, the specificity of 5HT2A versus 5HT2C antagonist was ensured by using the most specific drugs available at fairly low dosages. Low concentrations have the advantage of not cross activating other receptor subtypes, but on the other hand may not demonstrate the full effect. For
129 example I saw only attenuation, not complete obliteration of olfactory fear learning with 5-HT2AR blockade. Low drug concentrations as well as practical barriers such as drug metabolism, efficient crossing of blood brain barrier, or tissue penetration in the bulb could be the cause. I believe, however, that it is mostly due to the low .01kg/mg dose I used for both antagonists (lowest possible to induce physiological effects) (O'Neill, Heron-Maxwell et al. 1999, Fletcher,
Grottick et al. 2002) so that I may avoid other side effects that may affect behavior as well as cross binding to other receptors. In the literature, blockade of olfactory learning/discrimination is dose dependent and is attenuated to some degree rather than completely blocked pharmacology (Wilson, Pham et al. 1994,
Yuan, Harley et al. 2003). Outside of technical issues, it is certainly likely that other neuromodulators beside serotonin provide the other part of the unconditioned stimulus. Both in the olfactory bulb, piriform cortex or thalamus, neuromodulators often work in concert on the same cells to achieve the desire effect (McCormick 1992, McCormick and Bal 1994, Yuan, Harley et al. 2000,
Yuan, Harley et al. 2003, Yuan, Harley et al. 2003). The strong presence of ACh in the glomerulus, for example, might suggest it as a potential co-candidate providing the arousal tone for fear conditioning (Gomez, Brinon et al. 2005).
My finding that the 5-HT2ARs specifically mediate serotonin’s role in olfactory learning was unsurprising given my immunohistochemistry data in experiment one, which demonstrated high concentrations of 5-HT2AR in the bulb, especially the glomerulus compared to 5-HT2CR. Examples of 5HT2AR’s essential role in other types of procedural learning has been documented (Farber, Hanslick et al. 1998, Ellingrod, Perry et al. 2002, Gouzoulis-Mayfrank, Heekeren et al.
2006, Akhondzadeh, Malek-Hosseini et al. 2008, Lee, Choi et al. 2009).
130 This study is limited in several key ways. One main limitation is that we were unable to show that the acquisition of learning, and thus 5-HT2AR’s actions in bulb occurred specifically at the glomerulus, but only in the OB. I did ensure that the volume of drug used only penetrated the olfactory bulb and did not seep into other regions based on previous diffusion studies and verification with dye staining. From laminar distribution of both 5-HT and 5-HT2AR receptors in the bulb, I can be fairly confident that an influx of centrifugal serotonin, for the most part will arrive in the glomerular layer more than any other layer. However, to be exact, I would need to design experiments with focal 5-HT2AR blockade just in the glomerular level. Another weakness to the study is that I did not assay for the effects of 5HT2CR with intra-OB infusions. A comprehensive approach would have an entire range of concentrations for both 5-HT2A and 5-HT2C antagonists to compare at optimum dosages for blocking fear learning. Finally, I look forward to future studies examining the role of 5-HT2 family receptors in a reward learning paradigm. This will clarify if 5-HT signaling simply mediates arousal of the US, or if it can also convey the specific valence of the US.
Serotonin 2A receptor signaling participates in maintenance of an olfactory memory
In final part of this study, I utilize my genetically identified circuit in the
M72-GFP mice to study how serotonin signaling (via 5-HT2AR) participates in the maintenance of a memory trace at the glomerular level of information processing.
I found that 48 hours after fear associative learning, the serotonin elements of the glomerular circuit (5-HT fibers and 5-HT2ARs) increased in density only in the glomerulus processing the learned odor. After reward associative learning, I also
131 found a specific increase in 5-HT2ARs in the M72-glomerulus over control glomeruli.
My use of the acetophenone to specifically activate the M72-GFP glomerulus and then assay long term plasticity at his particular glomerulus in comparison to control glomeruli in the same animal gives these results additional validity and specificity. Previous studies of functional plasticity in the bulb have mostly used either 1) global deprivation or enrichment with a mixture of odors or
2) specific activation of 1 odor but post-hoc analysis from random glomeruli which may or may not have responded to the odor or an estimation of the location of the glomerulus processing the learned odor. 3) Targeted activation and targeted post-hoc analysis limited to quantifying OSN number or structural changes in M72 glomerular size. This use of the glomerular circuit allows me to precisely identify the synapses which received a coincidence of CS odor and US arousal during associative learning and compare them to internal controls in the same bulb of the same animals.
My findings showed an increase in both presynaptic 5-HT innervations as well as up regulation of postsynaptic 5-HT2AR receptors only in the learning M72 glomerular circuit. What exactly occurred during repeated learning trials thatmay promote an increase in both density serotonin fibers/amount of serotonin enervating the learned glomerulus as well as 5HT2A receptors? The increase serotonin puncta density could be interpreted as either 1) a change in morphology of the presynaptic fibers of 5-HT to provide more axon collaterals or release sites to the M72 glomerulus or 2) changes in amount of serotonin synthesized and released per fiber. The increases in receptors could be interpreted as either 1) increase expression of 5-HT2AR per mitral or ET cell
132 (such that cells that once were below threshold of detection now expressed enough 5-HT2ARs to be counted). These are possible ways in which the both pre and post synaptic serotonergic elements make a concerted effort to strengthen the efficacy of 5-HT2AR signaling only at the glomerulus carrying the learned odor that integrated CS and US.
There are several limitations to this study that may affect the interpretation of my data. First, quantification using immunohistochemistry assayed by confocal imaging has its problems. I avoided densitometry methods to quantify fluorescence, which could be biased by imaging techniques (laser intensities, PMT gains, auto fluorescence, image manipulation) and instead chose to quantify 5-HT puncta or 5-HT2AR positive cells. This allowed me to apply an all-or none standard of detection that was equal for each slice imaged. If a cell contained enough receptors to be detected, then it was a 5-HT2AR positive cell. Increases in receptor expression might move a cell from non-detectable to detectable and thus increase the # of 5-HT2AR positive cells. Second, it is well known that the most common synaptic modification following learning induced synaptic plasticity such as LTP in the post synaptic cell is an increase in glutamatergic elements, including the classical insertion of AMPA receptors. I did not assay changes in glutamate receptor number of subunit composition, which should be pursued in a future study. Recent studies demonstrating the possibility of electrically stimulated LTP between OSNs and Mitral cells show changes intracellular calcium, AMPAR versus NMDAR mediated current. Moreover the insertion of AMPARs is known to be mechanism to for homeostatic increase in synaptic strength following odor deprivation (Tyler, Petzold et al. 2007). In this study I only focused only on changes in serotonergic elements. Yet another
133 limitation is that I do not know at what time point the glomerular circuit increased its serotonergic elements as part of forming a memory trace. From other models of memory consolidation, however, I suspect that it takes place between 24 hours after learning. I also do not know how long the changes in serotonergic signaling persist: a week, month, or lifetime of the organism.
Traditional models of learning and memory have been succinctly reviewed (Kandel 2009) and cam be simplified into several steps in 1) an acquisition phase in which initial instances of coincidence of CS and US result in elevated activity between the neurons carrying CS and US information.
2)coincidental synaptic activity allow for NMDAR/Calcium mediated mechanisms that converge on intracellular cascades that strengthen the synapse by increasing glutamatergic transmission 3) repeated trials of learning establishes increasingly stronger synapses carrying the CS and US information 4)During consolidation phase, when short term plasticity is converted into a long term one, the above cascades cause changes in gene expression, translation and protein synthesis resulting in such as: changes in morphology,(pre or post synaptic), changes in vesicle refilling, release machinery or release pool (presynaptic), or presynaptic receptor expression, secretion of secretion of growth factors and change in any surface proteins (channels or receptors of the post synaptic cell) or that allows the neuron to keep this plasticity for the long range.
The changes in serotonin circuits in my glomerulus occurred after at least
2X5 trials of fear conditioning over 2 days or 5x14trials of reward conditioning over 2 weeks. I assayed for plasticity 48 hours after learning session. This means that the plasticity I found likely belongs to the long term plasticity (Frey, Huang et al. 1993, Abel, Nguyen et al. 1997) that may involve genomic changes, and
134 protein synthesis, as well as structural changes and formation of new synapses.
The increases in 5-HT2AR as well as 5-HT fiber innervation could be the consequences of these types of late long term plasticity.
What are the functional and behavioral implications of increasing serotonin signaling at the glomerulus that processes the learned odor? Once serotonergic circuit elements are increased in the M72-GFP learned glomerulus, this glomerulus becomes is perpetually aroused and ultrasensitive to any serotonergic tone. Even at basal levels of serotonin (not to mention slightly aroused or anxious), the M72 glomerulus already in a state of heighted sensory gating. (This state of heighted gating is similar to the effect of having and acute influx of serotonin, which may increase Mitral cell output while strengthening feedback inhibition for sharper detection of odors). Next time, the animal encounters the odor acetophenone; the M72 glomerulus will be poised to process it with optimal efficiency even without the US arousal of foot shock. In this manner, acetophenone has been canonized as a meaningful odor that deserves high attention at the periphery.
The findings of this last set of studies are unique because they are the first to show a longer term memory trace as early as the first synapse of the sensory periphery. I can also logically link the role of serotonin learning and memory to its role in the optimization of sensory gates. Previously, learning and memory are thought to be stored only in higher order synapses: for example contextual/place associations in the hippocampus, fear associations in the amygdala, or procedural association in the cerebellum (Bliss and Lomo 1973,
Morris, Garrud et al. 1982, Schindler, Gormezano et al. 1983, Dobson, Sederati et al. 1989, Davis, Butcher et al. 1992, Bliss and Collingridge 1993, Sigurdsson,
135 Doyere et al. 2007, Johansen, Cain et al. 2011). Although the neonate olfactory imprinting story described above was the first evidence of plasticity in the bulb, it was found at a second order synapse, and only in neonates during sensitive period of development. Due to the glomerular relay station’s unique circuitry in the middle of a feed-forward, feedback loop, it has all elements needed for learning and memory. Using specific activation followed by specific circuit interrogation, I were able to evaluate the plasticity of the serotonergic system in exact glomerulus processing that input. This study is the first to show associative learning-dependent functional plasticity exist in the glomerulus of adult animals.
In conclusion, I have established that serotonin, especially via 5-HT2AR signaling plays key roles in olfactory learning and memory at the OB and specifically in the glomerulus. Serotonin and its receptor 5-HT2AR dominate the glomerular layer and 5-HT2AR signaling takes place exclusively on glutamatergic mitral and ET cells. Serotonin, by activating 5-HT2ARs provides a major part of the unconditional stimulus signal during odor-shock olfactory fear learning by acting on circuits within the olfactory bulb. Longer term plasticity of serotonergic elements is part of a memory trace within the glomerular micro-circuit that encodes a meaningful, learned odor. These results point to serotonin as play dual roles of learning acquisition and maintaining memory traces that may be necessary for plastic sensory gates that can stabilized by important learning experiences.
136 REFERENCES
Abel, T., P. V. Nguyen, M. Barad, T. A. Deuel, E. R. Kandel and R. Bourtchouladze (1997). "Genetic demonstration of a role for PKA in the late phase of LTP and in hippocampus-based long-term memory." Cell 88(5): 615-626.
Akhondzadeh, S., M. Malek-Hosseini, A. Ghoreishi, M. Raznahan and S. A. Rezazadeh (2008). "Effect of ritanserin, a 5HT2A/2C antagonist, on negative symptoms of schizophrenia: a double-blind randomized placebo- controlled study." Prog Neuropsychopharmacol Biol Psychiatry 32(8): 1879-1883.
Bailey, C. H., M. Giustetto, H. Zhu, M. Chen and E. R. Kandel (2000). "A novel function for serotonin-mediated short-term facilitation in aplysia: conversion of a transient, cell-wide homosynaptic hebbian plasticity into a persistent, protein synthesis-independent synapse-specific enhancement." Proc Natl Acad Sci U S A 97(21): 11581-11586.
Bliss, T. V. and G. L. Collingridge (1993). "A synaptic model of memory: long- term potentiation in the hippocampus." Nature 361(6407): 31-39.
Bliss, T. V. and T. Lomo (1973). "Long-lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path." J Physiol 232(2): 331-356.
Bobillier, P., S. Seguin, A. Degueurce, B. D. Lewis and J. F. Pujol (1979). "The efferent connections of the nucleus raphe centralis superior in the rat as revealed by radioautography." Brain Res 166(1): 1-8.
Brunelli, M., V. Castellucci and E. R. Kandel (1976). "Synaptic facilitation and behavioral sensitization in Aplysia: possible role of serotonin and cyclic AMP." Science 194(4270): 1178-1181.
Brunken, W. J. and X. T. Jin (1993). "A role for 5HT3 receptors in visual processing in the mammalian retina." Vis Neurosci 10(3): 511-522.
Cavallin, M. A., K. Powell, K. C. Biju and D. A. Fadool (2010). "State-dependent sculpting of olfactory sensory neurons is attributed to sensory enrichment, odor deprivation, and aging." Neurosci Lett 483(2): 90-95.
Chaudhury, D., O. Escanilla and C. Linster (2009). "Bulbar acetylcholine enhances neural and perceptual odor discrimination." J Neurosci 29(1): 52-60.
Cornea-Hebert, V., M. Riad, C. Wu, S. K. Singh and L. Descarries (1999). "Cellular and subcellular distribution of the serotonin 5-HT2A receptor in the central nervous system of adult rat." J Comp Neurol 409(2): 187-209.
137 Dahlstrom, A., K. Fuxe, L. Olson and U. Ungerstedt (1965). "On the distribution and possible function of monamine nerve terminals in the olfactory bulb of the rabbit." Life Sci 4(21): 2071-2074.
Davis, S., S. P. Butcher and R. G. Morris (1992). "The NMDA receptor antagonist D-2-amino-5-phosphonopentanoate (D-AP5) impairs spatial learning and LTP in vivo at intracerebral concentrations comparable to those that block LTP in vitro." J Neurosci 12(1): 21-34.
Dobson, A. T., F. Sederati, G. Devi-Rao, W. M. Flanagan, M. J. Farrell, J. G. Stevens, E. K. Wagner and L. T. Feldman (1989). "Identification of the latency-associated transcript promoter by expression of rabbit beta-globin mRNA in mouse sensory nerve ganglia latently infected with a recombinant herpes simplex virus." J Virol 63(9): 3844-3851.
Ellingrod, V. L., P. J. Perry, B. C. Lund, K. Bever-Stille, F. Fleming, T. L. Holman and D. Miller (2002). "5HT2A and 5HT2C receptor polymorphisms and predicting clinical response to olanzapine in schizophrenia." J Clin Psychopharmacol 22(6): 622-624.
Farber, N. B., J. Hanslick, C. Kirby, L. McWilliams and J. W. Olney (1998). "Serotonergic agents that activate 5HT2A receptors prevent NMDA antagonist neurotoxicity." Neuropsychopharmacology 18(1): 57-62.
Fletcher, M. L. and D. A. Wilson (2002). "Experience modifies olfactory acuity: acetylcholine-dependent learning decreases behavioral generalization between similar odorants." J Neurosci 22(2): RC201.
Fletcher, P. J., A. J. Grottick and G. A. Higgins (2002). "Differential effects of the 5-HT(2A) receptor antagonist M100907 and the 5-HT(2C) receptor antagonist SB242084 on cocaine-induced locomotor activity, cocaine self- administration and cocaine-induced reinstatement of responding." Neuropsychopharmacology 27(4): 576-586.
Frey, U., Y. Y. Huang and E. R. Kandel (1993). "Effects of cAMP simulate a late stage of LTP in hippocampal CA1 neurons." Science 260(5114): 1661- 1664.
Fuxe, K. (1965). "Evidence for the Existence of Monoamine Neurons in the Central Nervous System. 3. The Monoamine Nerve Terminal." Z Zellforsch Mikrosk Anat 65: 573-596.
Gehlert, D. R., A. Shekhar, S. M. Morin, P. A. Hipskind, C. Zink, S. L. Gackenheimer, J. Shaw, S. D. Fitz and T. J. Sajdyk (2005). "Stress and central Urocortin increase anxiety-like behavior in the social interaction test via the CRF1 receptor." Eur J Pharmacol 509(2-3): 145-153.
Gire, D. H. and N. E. Schoppa (2009). "Control of on/off glomerular signaling by a local GABAergic microcircuit in the olfactory bulb." J Neurosci 29(43): 13454-13464.
138 Glanzman, D. L., S. L. Mackey, R. D. Hawkins, A. M. Dyke, P. E. Lloyd and E. R. Kandel (1989). "Depletion of serotonin in the nervous system of Aplysia reduces the behavioral enhancement of gill withdrawal as well as the heterosynaptic facilitation produced by tail shock." J Neurosci 9(12): 4200-4213.
Gomez, C., J. G. Brinon, M. V. Barbado, E. Weruaga, J. Valero and J. R. Alonso (2005). "Heterogeneous targeting of centrifugal inputs to the glomerular layer of the main olfactory bulb." J Chem Neuroanat 29(4): 238-254.
Gouzoulis-Mayfrank, E., K. Heekeren, A. Neukirch, M. Stoll, C. Stock, J. Daumann, M. Obradovic and K. A. Kovar (2006). "Inhibition of return in the human 5HT2A agonist and NMDA antagonist model of psychosis." Neuropsychopharmacology 31(2): 431-441.
Guthrie, K. M., D. A. Wilson and M. Leon (1990). "Early unilateral deprivation modifies olfactory bulb function." J Neurosci 10(10): 3402-3412.
Halaris, A. E., B. E. Jones and R. Y. Moore (1976). "Axonal transport in serotonin neurons of the midbrain raphe." Brain Res 107(3): 555-574.
Halasz, N. and G. M. Shepherd (1983). "Neurochemistry of the vertebrate olfactory bulb." Neuroscience 10(3): 579-619.
Hamada, S., K. Senzaki, K. Hamaguchi-Hamada, K. Tabuchi, H. Yamamoto, T. Yamamoto, S. Yoshikawa, H. Okano and N. Okado (1998). "Localization of 5-HT2A receptor in rat cerebral cortex and olfactory system revealed by immunohistochemistry using two antibodies raised in rabbit and chicken." Brain Res Mol Brain Res 54(2): 199-211.
Hammer, M. (1993). "An Identified Neuron Mediates the Unconditioned Stimulus in Associative Olfactory Learning in Honeybees." Nature 366(6450): 59- 63.
Hardy, A., B. Palouzier-Paulignan, A. Duchamp, J. P. Royet and P. Duchamp- Viret (2005). "5-Hydroxytryptamine action in the rat olfactory bulb: in vitro electrophysiological patch-clamp recordings of juxtaglomerular and mitral cells." Neuroscience 131(3): 717-731.
Hayar, A. and M. Ennis (2007). "Endogenous GABA and glutamate finely tune the bursting of olfactory bulb external tufted cells." J Neurophysiol 98(2): 1052-1056.
Hayar, A., S. Karnup, M. Ennis and M. T. Shipley (2004). "External tufted cells: a major excitatory element that coordinates glomerular activity." J Neurosci 24(30): 6676-6685.
Hayar, A., S. Karnup, M. T. Shipley and M. Ennis (2004). "Olfactory bulb glomeruli: external tufted cells intrinsically burst at theta frequency and are entrained by patterned olfactory input." J Neurosci 24(5): 1190-1199.
139 Hayar, A., M. T. Shipley and M. Ennis (2005). "Olfactory bulb external tufted cells are synchronized by multiple intraglomerular mechanisms." J Neurosci 25(36): 8197-8208.
Hurley, L. M. and G. D. Pollak (1999). "Serotonin differentially modulates responses to tones and frequency-modulated sweeps in the inferior colliculus." J Neurosci 19(18): 8071-8082.
Jacobs, B. L. and E. C. Azmitia (1992). "Structure and function of the brain serotonin system." Physiol Rev 72(1): 165-229.
Johansen, J. P., C. K. Cain, L. E. Ostroff and J. E. LeDoux (2011). "Molecular mechanisms of fear learning and memory." Cell 147(3): 509-524.
Kandel, E. R. (2009). "The biology of memory: a forty-year perspective." J Neurosci 29(41): 12748-12756.
Kay, L. M. and S. M. Sherman (2007). "An argument for an olfactory thalamus." Trends Neurosci 30(2): 47-53.
Kerr, M. A. and L. Belluscio (2006). "Olfactory experience accelerates glomerular refinement in the mammalian olfactory bulb." Nat Neurosci 9(4): 484-486.
Kim, Y. C., H. G. Lee and K. A. Han (2007). "D1 dopamine receptor dDA1 is required in the mushroom body neurons for aversive and appetitive learning in Drosophila." J Neurosci 27(29): 7640-7647.
Lee, K. H. and D. A. McCormick (1995). "Acetylcholine excites GABAergic neurons of the ferret perigeniculate nucleus through nicotinic receptors." J Neurophysiol 73(5): 2123-2128.
Lee, Y. S., S. L. Choi, S. H. Lee, H. Kim, H. Park, N. Lee, Y. S. Chae, D. J. Jang, E. R. Kandel and B. K. Kaang (2009). "Identification of a serotonin receptor coupled to adenylyl cyclase involved in learning-related heterosynaptic facilitation in Aplysia." Proc Natl Acad Sci U S A 106(34): 14634-14639.
Liu, S., J. L. Aungst, A. C. Puche and M. T. Shipley (2012). "Serotonin modulates the population activity profile of olfactory bulb external tufted cells." J Neurophysiol 107(1): 473-483.
Mandairon, N., C. J. Ferretti, C. M. Stack, D. B. Rubin, T. A. Cleland and C. Linster (2006). "Cholinergic modulation in the olfactory bulb influences spontaneous olfactory discrimination in adult rats." Eur J Neurosci 24(11): 3234-3244.
Mandairon, N., C. Stack, C. Kiselycznyk and C. Linster (2006). "Broad activation of the olfactory bulb produces long-lasting changes in odor perception." Proc Natl Acad Sci U S A 103(36): 13543-13548.
140 Mandairon, N., C. Stack, C. Kiselycznyk and C. Linster (2006). "Enrichment to odors improves olfactory discrimination in adult rats." Behav Neurosci 120(1): 173-179.
Mandairon, N., C. Stack and C. Linster (2006). "Olfactory enrichment improves the recognition of individual components in mixtures." Physiol Behav 89(3): 379-384.
Maricq, A. V., A. S. Peterson, A. J. Brake, R. M. Myers and D. Julius (1991). "Primary structure and functional expression of the 5HT3 receptor, a serotonin-gated ion channel." Science 254(5030): 432-437.
McCormick, D. A. (1992). "Neurotransmitter actions in the thalamus and cerebral cortex." J Clin Neurophysiol 9(2): 212-223.
McCormick, D. A. and T. Bal (1994). "Sensory gating mechanisms of the thalamus." Curr Opin Neurobiol 4(4): 550-556.
McCormick, D. A. and H. C. Pape (1988). "Acetylcholine inhibits identified interneurons in the cat lateral geniculate nucleus." Nature 334(6179): 246-248.
McCormick, D. A. and H. C. Pape (1990). "Noradrenergic and serotonergic modulation of a hyperpolarization-activated cation current in thalamic relay neurones." J Physiol 431: 319-342.
McLean, J. H., A. Darby-King and E. Hodge (1996). "5-HT2 receptor involvement in conditioned olfactory learning in the neonate rat pup." Behav Neurosci 110(6): 1426-1434.
McLean, J. H., A. Darby-King and G. D. Paterno (1995). "Localization of 5-HT2A receptor mRNA by in situ hybridization in the olfactory bulb of the postnatal rat." J Comp Neurol 353(3): 371-378.
McLean, J. H., A. Darby-King, R. M. Sullivan and S. R. King (1993). "Serotonergic influence on olfactory learning in the neonate rat." Behav Neural Biol 60(2): 152-162.
McLean, J. H. and M. T. Shipley (1987). "Serotonergic afferents to the rat olfactory bulb: I. Origins and laminar specificity of serotonergic inputs in the adult rat." J Neurosci 7(10): 3016-3028.
McLean, J. H. and M. T. Shipley (1987). "Serotonergic afferents to the rat olfactory bulb: II. Changes in fiber distribution during development." J Neurosci 7(10): 3029-3039.
Monckton, J. E. and D. A. McCormick (2002). "Neuromodulatory role of serotonin in the ferret thalamus." J Neurophysiol 87(4): 2124-2136.
141 Moore, R. Y., A. E. Halaris and B. E. Jones (1978). "Serotonin neurons of the midbrain raphe: ascending projections." J Comp Neurol 180(3): 417-438.
Morris, R. G., P. Garrud, J. N. Rawlins and J. O'Keefe (1982). "Place navigation impaired in rats with hippocampal lesions." Nature 297(5868): 681-683.
Mutoh, H., Q. Yuan and T. Knopfel (2005). "Long-term depression at olfactory nerve synapses." J Neurosci 25(17): 4252-4259.
Nuttley, W. M., K. P. Atkinson-Leadbeater and D. Van Der Kooy (2002). "Serotonin mediates food-odor associative learning in the nematode Caenorhabditiselegans." Proc Natl Acad Sci U S A 99(19): 12449-12454.
O'Neill, M. F., C. L. Heron-Maxwell and G. Shaw (1999). "5-HT2 receptor antagonism reduces hyperactivity induced by amphetamine, cocaine, and MK-801 but not D1 agonist C-APB." Pharmacol Biochem Behav 63(2): 237-243.
Pape, H. C. and D. A. McCormick (1989). "Noradrenaline and serotonin selectively modulate thalamic burst firing by enhancing a hyperpolarization-activated cation current." Nature 340(6236): 715-718.
Pazos, A. and J. M. Palacios (1985). "Quantitative autoradiographic mapping of serotonin receptors in the rat brain. I. Serotonin-1 receptors." Brain Res 346(2): 205-230.
Petzold, G. C., A. Hagiwara and V. N. Murthy (2009). "Serotonergic modulation of odor input to the mammalian olfactory bulb." Nat Neurosci.
Pompeiano, M., J. M. Palacios and G. Mengod (1992). "Distribution and cellular localization of mRNA coding for 5-HT1A receptor in the rat brain: correlation with receptor binding." J Neurosci 12(2): 440-453.
Pompeiano, M., J. M. Palacios and G. Mengod (1994). "Distribution of the serotonin 5-HT2 receptor family mRNAs: comparison between 5-HT2A and 5-HT2C receptors." Brain Res Mol Brain Res 23(1-2): 163-178.
Price, J. L. and T. P. Powell (1970). "An electron-microscopic study of the termination of the afferent fibres to the olfactory bulb from the cerebral hemisphere." J Cell Sci 7(1): 157-187.
Price, J. L. and T. P. Powell (1970). "An experimental study of the origin and the course of the centrifugal fibres to the olfactory bulb in the rat." J Anat 107(Pt 2): 215-237.
Price, T. L., A. Darby-King, C. W. Harley and J. H. McLean (1998). "Serotonin plays a permissive role in conditioned olfactory learning induced by norepinephrine in the neonate rat." Behav Neurosci 112(6): 1430-1437.
142 Schindler, C. W., I. Gormezano and J. A. Harvey (1983). "Effect of morphine on acquisition of the classically conditioned nictitating membrane response of the rabbit." J Pharmacol Exp Ther 227(3): 639-643.
Schwaerzel, M., M. Monastirioti, H. Scholz, F. Friggi-Grelin, S. Birman and M. Heisenberg (2003). "Dopamine and octopamine differentiate between aversive and appetitive olfactory memories in Drosophila." J Neurosci 23(33): 10495-10502.
Shepherd, G. M. (2005). "Perception without a thalamus how does olfaction do it?" Neuron 46(2): 166-168.
Sigurdsson, T., V. Doyere, C. K. Cain and J. E. LeDoux (2007). "Long-term potentiation in the amygdala: a cellular mechanism of fear learning and memory." Neuropharmacology 52(1): 215-227.
Steinbusch, H. W. (1981). "Distribution of serotonin-immunoreactivity in the central nervous system of the rat-cell bodies and terminals." Neuroscience 6(4): 557-618.
Sullivan, R. M., D. A. Wilson and M. Leon (1989). "Norepinephrine and learning- induced plasticity in infant rat olfactory system." J Neurosci 9(11): 3998- 4006.
Sullivan, R. M., D. R. Zyzak, P. Skierkowski and D. A. Wilson (1992). "The role of olfactory bulb norepinephrine in early olfactory learning." Brain Res Dev Brain Res 70(2): 279-282.
Takeuchi, Y., H. Kimura and Y. Sano (1982). "Immunohistochemical demonstration of serotonin nerve fibers in the olfactory bulb of the rat, cat and monkey." Histochemistry 75(4): 461-471.
Tecott, L. H., A. V. Maricq and D. Julius (1993). "Nervous system distribution of the serotonin 5-HT3 receptor mRNA." Proc Natl Acad Sci U S A 90(4): 1430-1434.
Tyler, W. J., G. C. Petzold, S. K. Pal and V. N. Murthy (2007). "Experience- dependent modification of primary sensory synapses in the mammalian olfactory bulb." J Neurosci 27(35): 9427-9438.
Valle-Leija, P., E. Blanco-Hernandez, R. Drucker-Colin, G. Gutierrez-Ospina and R. Vidaltamayo (2012). "Supernumerary formation of olfactory glomeruli induced by chronic odorant exposure: a constructivist expression of neural plasticity." PLoS One 7(4): e35358.
Wilson, D. A., A. R. Best and R. M. Sullivan (2004). "Plasticity in the olfactory system: lessons for the neurobiology of memory." Neuroscientist 10(6): 513-524.
143 Wilson, D. A., M. L. Fletcher and R. M. Sullivan (2004). "Acetylcholine and olfactory perceptual learning." Learn Mem 11(1): 28-34.
Wilson, D. A. and M. Leon (1988). "Spatial patterns of olfactory bulb single-unit responses to learned olfactory cues in young rats." J Neurophysiol 59(6): 1770-1782.
Wilson, D. A., T. C. Pham and R. M. Sullivan (1994). "Norepinephrine and posttraining memory consolidation in neonatal rats." Behav Neurosci 108(6): 1053-1058.
Wilson, D. A. and R. M. Sullivan (1994). "Neurobiology of associative learning in the neonate: early olfactory learning." Behav Neural Biol 61(1): 1-18.
Wilson, D. A., R. M. Sullivan and M. Leon (1987). "Single-unit analysis of postnatal olfactory learning: modified olfactory bulb output response patterns to learned attractive odors." J Neurosci 7(10): 3154-3162.
Wilson, D. A. and J. G. Wood (1992). "Functional consequences of unilateral olfactory deprivation: time-course and age sensitivity." Neuroscience 49(1): 183-192.
Yuan, Q. (2009). "Theta bursts in the olfactory nerve paired with beta- adrenoceptor activation induce calcium elevation in mitral cells: a mechanism for odor preference learning in the neonate rat." Learn Mem 16(11): 676-681.
Yuan, Q., C. W. Harley, J. C. Bruce, A. Darby-King and J. H. McLean (2000). "Isoproterenol increases CREB phosphorylation and olfactory nerve- evoked potentials in normal and 5-HT-depleted olfactory bulbs in rat pups only at doses that produce odor preference learning." Learn Mem 7(6): 413-421.
Yuan, Q., C. W. Harley, A. Darby-King, R. L. Neve and J. H. McLean (2003). "Early odor preference learning in the rat: bidirectional effects of cAMP response element-binding protein (CREB) and mutant CREB support a causal role for phosphorylated CREB." J Neurosci 23(11): 4760-4765.
Yuan, Q., C. W. Harley and J. H. McLean (2003). "Mitral cell beta1 and 5-HT2A receptor colocalization and cAMP coregulation: a new model of norepinephrine-induced learning in the olfactory bulb." Learn Mem 10(1): 5-15.
Yuan, Q., H. Mutoh, F. Debarbieux and T. Knopfel (2004). "Calcium signaling in mitral cell dendrites of olfactory bulbs of neonatal rats and mice during olfactory nerve Stimulation and beta-adrenoceptor activation." Learn Mem 11(4): 406-411.
144 Zhang, Y., H. Lu and C. I. Bargmann (2005). "Pathogenic bacteria induce aversive olfactory learning in Caenorhabditis elegans." Nature 438(7065): 179-184.
145 Chapter 5
5HT2A RECEPTOR ACTIVATION MODULATES EXCTIATORY GLOMERULAR
SYNAPSES
Introduction
Current theories of learning and memory at the circuit and synapse level inform me that learning and memory depend on the plasticity of synapses
(Kauer, Malenka et al. 1988, Abel, Nguyen et al. 1997, Dan and Poo 2006,
Sigurdsson, Doyere et al. 2007). I proposed in earlier chapters, that 5-HT2AR receptors play an integral role in both the initial learning and as well memory maintenance at olfactory bulb glomeruli. How exactly does 5-HT modulate glomerular circuits to achieve these behavioral consequences? In this chapter, I attempt to begin to answer this question by assessing exactly how 5-HT2AR acutely modulates excitatory glomerular synapses by using 5-HT2AR specific antagonists coupled with slice-electrophysiological techniques. I hope to gain more information about how 5-HT modifies glomerular processing. The results of this study will provide insight about mechanisms by which 5-HT can play dual roles in both learning and memory as well as optimal sensory gating at the olfactory periphery.
Serotonergic modulation of the olfactory bulb circuitry has only been investigated in a few studies (Hardy, Palouzier-Paulignan et al. 2005, Petzold,
Albeanu et al. 2008, Liu, Aungst et al. 2012). From these studies, I observe that
5-HT modulatory great influence on almost all the circuit elements of the glomerulus via 5HT2 family receptors. Serotonin directly depolarizes glutamatergic Mitral cells and External tufted (ET) cells exclusively via 5-HT2A receptors but depolarizes GABAergic periglomerular (PG cells) via 5-HT2C
146 receptors. However many mitral cells also experience net inhibition, most likely due to these strong influence of this GABAergic inhibition via a large symphony of PG cells (Hardy, Palouzier-Paulignan et al. 2005, Liu, Aungst et al. 2012).
Both 5-HT2A and 5-HT2C receptors are couple to Gq and activate similar intracellular cascades involving PKC, PLC and release of intracellular stores of calcium (Hoyer, Hannon et al. 2002, Liu, Aungst et al. 2012). Although the exact intermediaries are unknown, these mechanisms ultimately close K+ channels, while opening a non-selective cation channel, which, in ET cells have been identified as a TRP channel (Aghajanian, Sprouse et al. 1990). Application of 5-
HT2A agonists causes a slow, long lasting depolarization on the order of minutes in mitral cells and ET cells, causing both cell types to increase in spike rate as well as increases intrinsic bursting in ET cells (Liu, Aungst et al. 2012). The mechanism by which 5-HT2C causes increased activity in GABAergic PG cells is still not clear but suspected to be similar.to the shared intracellular signaling cascade.
Given 5-HT’s action on each element of the glomerulus, how does this neuromodulator alter normal glomerular processing? Recent in vivo study provides some clues: electric stimulation of the DRN(to mimic natural release) caused both 1)juxtaglomerular neurons to increase in calcium response and
2)the OSNs to decrease in transmitter release (Petzold, Hagiwara et al. 2009). In light of the previous studies, these findings suggest that 5-HT’s functional role is to increase the activity of GABAergic PG cells and thus provide greater inhibition to both the presynaptic OSN and post synaptic mitral cell. This process might allow the glomerulus to more efficiently gate information into the olfactory bulb.
147 Informed by these studies, I expand previous hypotheses to provide a role for ET cells as a key player the mediating 5-HT’s role in sensory gating the glomerulus. ET cells hold a powerful position in the glomerulus. They are the only cell making guaranteed monosynaptic, connections with OSNs, and thus often serve as an intermediary—receiving OSN excitation and in turn driving the activity of both Mitral cells and PG cells. ET cells, can, via strong dendro- dendritic synapses as well as powerful intrinsic bursting that can synchronize the entire glomerulus (Hayar, Karnup et al. 2004, Hayar, Karnup et al. 2004, Gire and Schoppa 2009). Because ET cells provide excitation to both the feed forward component (Mitral cells) and inhibitory component (PG cells), they serve as amplitude modulators for the ON-OFF cycle of glomerular excitation followed by inhibition. I hypothesize that 5-HT2AR signaling heavily influence the activity of the ET cell, and thus amplitude of the ON-OFF within the glomerulus. Through this mechanism, I hypothesize that centrifugal serotonin can modulate the sensory gating based on the arousal state of the animal and learning experiences.
Methods
Animals
Adult M72-GFP mice ages 2 months to 5 months were used for electrophysiology experiments and behavioral conditioning preceding them. I tended to use slightly younger animals than in previous parts of this study because age greatly deteriorates the health of acute slices. Animals were housed, cared for and sacrificed according to approved IACUC protocols as described in Chapter 3.
148
Behavioral Training
All behavioral conditioning was performed according to the associative learning protocols described in the methods section of Chapter 3. An odor-shock paired fear paradigm (CS+/US+) was used to instill associative learning . The success of learning was accessed on test day by analysis of freezing behavior specific to odor presentation. All animals were sacrificed for electrophysiology experiments 48 hours after the last learning session or 24 hours after testing.
Acute Slice Preparation
Parasagittal acute slice were obtained from adult, male, M72-GFP mice age
2-5 months. Mice were anesthetized in a CO2 chamber and rapidly decapitated.
The brain and olfactory bulbs were removed quickly and submerged in ice cold high sucrose aCSF. Slices were also cut in this aCSF containing in (mM): 83
NaCl, 2.5 KCl, 3.3 MgSO4, 1 NaH2PO4, 26.2 NaHCO3, 22 glucose, 72 sucrose, and 0.5 CaCl2, and equilibrated with 95% O2/5% CO2 at pH 7.35-7.4. 300uM parasagittal slices were cut with a Vibratome 1000 while cooled in an ice bath chamber. Slices recovered for 20 minutes at 37degrees C and then left at room temperature until they were ready for electrophysiology. All recordings were done within 6 hours of slice preparation
Electrophysiology
Male M72-GFP mice were asphyxiated in a CO2 chamber and rapidly decapitated according to IAUCUC protocols. Their brains were removed and placed in an ice cold aCSF containing a high sucrose concentration comprised of
149 (in mM): 83 NaCl, 2.5 KCl, 3.3 MgSO4, 1 NaH2PO4, 26.2 NaHCO3, 22 glucose,
72 sucrose, and 0.5 CaCl2, and equilibrated with 95% O2/5% CO2 at pH 7.35-
7.40. The brain was incubated in this ice cold aCSF for 5 minutes before being sliced. Acute parasagittal slices (300 µm) of the olfactory bulbs and anterior forebrain were prepared using a vibratome while submerged in the oxygenated high sucrose aCSF. Slices were then allowed to recover for 20 minutes at 37° C and then left at room temperature in normal aCSF until they were used for electrophysiology. All recordings were performed within 6 hours of slice preparation. Normal aCSF contained (in mM): 119 NaCl, 2.5 KCl, 1.3 MgSO4, 1
NaH2PO4, 26.2 NaHCO3, 22 glucose, and 2.5 CaCl2 equilibrated with 95% O2/5%
CO2 at pH 7.35-7.40.
Prior to use slices were transferred into a recording chamber containing normal aCSF on the Olympus FV-300 confocal microscope equipped with a patch-clamp setup. Slices were visualized using a 20X (0.8 NA) water immersion lens. Wide-field fluorescence and differential interference contrast illumination were used to visualize M72 glomeruli, juxtaglomerular cells projecting to the M72 glomerulus, and incoming olfactory sensory (OSN) neurons. Whole-cell voltage- clamp recordings were made from visually identified juxtaglomerular (JG) cells including external tufted (ET) and periglomerular (PG) neurons using an intracellular recording solution consisting of (in mM): 0.05 Alexa-564, 120 Cs- gluconate, 17.5 CsCl, 10 NaCl, 10 Na-HEPES, 0.2 EGTA, 2 Mg-ATP, 0.2 Na-
GTP, 2 ascorbate, and 0.2 QX-314 (N-(2,6-dimethylphenyl)acetamide-2- tetraethylammonium bromide). Patch electrodes had resistances ranging from 5 -
9 MΩ depending on cell type targeted and whole-cell access was maintained <
25 MΩ in all experiments. Cells were voltage-clamped and maintained at a
150 holding potential of -70mV. Membrane currents were acquired using the
MultiClamp 700B and digitized with a Digidata 1440A (Molecular Devices, Foster
City, CA). Data was stored on a computer for later offline analysis.
I examined the influence of 5-HT2A receptors on excitatory synaptic events in juxtaglomerular neurons by bath applying specific 5-HT2AR antagonist TCB-2 (1
µM) during whole-cell recordings of OSN-evoked excitatory postsynaptic currents
(EPSCs), spontaneous excitatory postsynaptic currents (sEPSCs), and miniature excitatory postsynaptic currents (mEPSCs).
To record OSN-evoked EPSCs I blocked presynaptic inhibition of OSN fibers by adding the GABAB receptor antagonist CGP-55845 and the D2 dopamine receptor antagonist sulpiride to the aCSF for a final concentration of 5
µM and 100 µM respectively. Stimulating electrodes were (1 - 2 MΩ) placed on visually identified OSN afferents and stimulated using a Grass SD9 stimulator and an AMPI Isoflex 9001 stimulus isolator with 200 µsec, 50 µA pulses. I recorded OSN-evoked EPSCs in response to single stimuli and paired stimuli separated by 50, 100, 500, and 1000 msec. Monosynaptic inputs cells were defined as cells exhibiting < 3msec response latencies.
To record sEPSCs only the GABAA receptor antagonist picrotoxin (50 µM) was added to the aCSF and to record mEPSCs both picrotoxin and tetrodotoxin
(TTX 0.5 µM) was included in the aCSF. Both sEPSCs and mEPSCs were recorded during three epochs (60 sec each) with and without the addition of
TCB-2.
Results
151 Activation of 5-HT2A receptors decreases ET and PG cell excitatory currents to an evoked OSN stimulus
What are the effects of 5-HT2AR activation on the effective drive of OSN inputs? To investigate this, I stimulated a bundle of OSNs belonging to a single glomerulus (200µS, 50µA) and recorded from a juxtaglomerular cell (either PG or
ET) of that glomerulus that had a response to OSN stimulation (Figure 10A).
Then I added 1uM TCB-2 to the bath and conducted the same recording son the same cell. Representative eEPSCs from a ET versus a PG cells show that PG cell eEPSCs are much smaller in amplitude compared to ET cell eEPSCs. For example the representative cell shows a 5 fold difference between eEPSCs of
PG cells versus eEPSCs of ET cells. Furthermore, ET cell eEPSCs tend to have a short latency time between stimulation and the induction of inward current, which defines a monosynaptic synapse. I categorized monosynaptic currents as those with latency less than 3 msec. PG cells have a longer latency since they are mostly not monosynaptic and use the OSNàETàPG route for excitation
(Figure 10B). Results indicated that 5HT2AR agonist TCB-2(1µm) decreased the amplitude of evoked EPSCs in ET cells (baseline: 4604.94 ± 1112.06pA, TCB-2:
4100.33 ± 1019.71 Paired T test, p < 0.05, n = 6). In PG cells, TCB-2 did not significantly decrease eEPSC amplitude but there was strong trend towards a decrease (baseline: 2183.58 ± 1065.10, TCB-2: 1558.84 ± 697.93, Paired T-test p = 0.059 n = 3) (Figure 10C). Although the excitatory drive between OSNs and its post synaptic targets seem to have weakened, I could not tell from this experiment whether the changes were pre or post synaptic.
Activation of 5-HT2A receptors decreases probability of presynaptic release at olfactory sensory neurons.
152
Figure 10. 5-HT2AR agonist decreases the amplitude of OSN-evoked EPSCs in both ET cells. A. Diagram of stimulation and recording sites in the glomerular layer of the olfactory bulb. B. Representative ET and PG cell OSN-evoked EPSCs (average of 10 trials) in response to 1uM TCB-2. C. Mean+/- SEM of eEPSC amplitudes are decreased in response to TCB-2 only in PG cells, paired T-test: *p < 0.05. (ET cells = 6, PG cells = 3).
153 The next set of experiments was aimed to test if presynaptic probability of release (PPR) of neurotransmitters. I used a paired pulse stimulation paradigm since paired-pulse ratios provide information about this PPR. My protocol consisted of same stimulation used to evoke eEPSCs above of (200µsec, 50µA) with an inter-stimulus interval (ITI) of 50msec, 100msec, 500msec and
1000msec. This is shown in representative examples of an ET and one of the few PG cells that received monosynaptic input from OSNs (Figure 11A,B). OSN synapses are high probability releases that normally have a low P2/P1 ratio to short it ITI that recover with longer ITI (Murphy, Darcy et al. 2005).
In ET cells, I found that for 50msec ITI, the P2/P1 ratio was significantly affected by TCB-2 application (baseline: 0.22 ± 0.0061, TCB-2: 0.88 ± 0.049,
Paired test: p < 0.05, n = 3) (Figure 11C,D). At 100msec inter-stimulus intervals, the effect of the treatment was no longer significant (baseline: 0.55 ± 0.28, TCB-
2: 0.93 ± 0.027, Paired test: P = 0.38, n = 3). At 500ms inter-stimulus intervals, there were no significant differences between baseline and treatment: 500ms
(baseline: 0.96 ± 0.010, TCB-2: 0.98 ± 0.0091, Paired test: p < 0.074, n = 6). At
1000 msec, no PPD was apparent and thus there was no significant effect of
TCB-2 (baseline: 0.98 ± 0.0046, TCB-2: 0.99 ± 0.0042, Paired test: p = 0.40, n =
6). These results indicate that at short inter-stimulus intervals, activating 5HT2A receptors decreases probability of neurotransmitter release at OSN terminals.
I also recorded from PG cells while performing paired pulse stimulations.
Almost all the PG cells I recorded were polysynaptic to OSNs as evidenced by a longer latency period that is above 3 msec. The eEPSCs recorded in these cells, thus, are not direct reflections of probability of release of OSNs, but rather the second hand effects and rather reflect ET cell PPRs onto PG cells. I see
154
Fig ure 11. 5HT2AR activation decreases probability of OSN neurotransmitter release. A. Representative pair-pulsed OSN evoked EPSCs in a ET cell at baseline (black) and after the application of 1uM 5-HT2AR specific agonist TCB-2 (red). Inter-stimulus intervals of paired pulses are of 50, 100, 500 and 1000 msec. TCB-2 reduced the first pulse as well as the depression of the second pulse for all stimulus intervals B. Representative evoked EPSCs trace from the minority of PG that are monosynaptic with OSNs by the same pair pulsed stimulation. TCB-2 has not noticeable effect on this PG cells. C. Mean ± SEM of paired pulse ratio (P1/P2) at baseline and after TCB-2 application in ET cells and PG cells over the series of inter-stimulus intervals. Paired pulse ratios increase d after TCB-2 application in ET cells at all inter-stimulus intervals but significant only at 50 msec interval, *p < 0.05. No significant effect of TCB-2 on PG cells. D. Mean ± SEM of percentage change in paired pulse ratio (P2/P1) within analyzed by each cell before and after TCB-2 application over the series of inter -stimulus intervals. ET cells experience significant within cell percentage changes in PPR at 50ms interval after TCB-2, *p < 0.05. PG cells do not show significant changes in PPR.
155
Figure 12. 5HT2AR agonist TCB-2 decreases mEPSC amplitude in ET cells and decreases frequency in PG and ET cells A. Representative mEPSC traces from an ET and PG cell in response to application of TCB-2 (1µM). B. Representative average trace of one mEPSC event from an ET versus PG cell in response to TCB-2. C. Cumulative distribution and mean ± SEM of mEPSC amplitudes from ET and PG cells in response to TCB-2. TCB- 2 decreased amplitudes in a population comparison (KS test, *p < 0.05) and within cell (Paired T-test, *p < 0.05). No change in PG cell amplitudes. D. Cumulative probabilities of mEPSC inter-event intervals and mean± SEM of mESPC frequencies in PG cells, and ET cells in response to TCB-2 application. Population mESPC inter-event intervals increase (frequency decr eases) for both ET and PG cells (KS test: *p < 0.05 for both). Mean mEPSC frequency does not significantly change for either cell type, ET cells, pa ired T-test, p = 0.083. (ET cells=5, PG cells=6 for all analysis).
156 no statistically significant changes in paired pulse ratio after TCB-2 application in
PG cells at any of my inter-stimulus intervals (Paired T-tests: 50 msec: p = 0.30,
100 msec, baseline: p = 0.35, 500 msec: p = 0.91, 1000 msec: p = 0.33, n=3)
(Figure 11C,D). These data suggest that TCB-2 increase paired-pulse ratio, which may be indicative that 5-HT2AR activation in the glomerulus decreases
OSN presynaptic release.
Activation of 5-HT2A receptors decreases quantal synaptic transmission at OSN synapses
Next, I investigated the effect of 5-HT2AR signaling on the postsynaptic properties of ET and PG juxtaglomerular cells. I recorded miniature EPSCs at baseline and applied TCB-2. Cells were held at -70mV in the presence of TTX
(0.5nM) to block action potentials from OSNs as GABAA antagonist picrotoxin
(50µM) isolate excitatory currents.
Representative traces of an ET and PG cell mEPSCs before and after TCB2 treatment as well as the average mEPSCs event are shown(Figure 12A, B). I performed both population comparisons between baseline and TCB-2 treatment groups across all cells as well as within cell comparisons of before and after treatment. I found that 5HT2AR agonist TCB-2 significantly reduced ET cell mEPSCs amplitude in a distribution comparison (KS test: p < 0.05) or within cell pairwise comparison (baseline: 20.577 ± 0.98 pA, TCB-2: 16.98 ± 0.54 pA, ,
Paired T-test, p < 0.05, n = 6). For PG cells, TCB-2, not significantly affect mEPSCs amplitude in PG cells by either kind of comparison. (KS test = NS, baseline: 17.84 ± 3.37 pA, TCB-2: 17.36 ± 4.29pA, Paired T-test: p = 0.67, n=5)
(Figure 12C). 157 TCB-2 also increased ET cell mEPSC inter-event intervals and decreased frequencies as shown in a population comparison of means (baseline ITI: 14.36 ±
1.64 msec, TCB-2 ITI: 10.27 ± 1.86 msec, KS test, p < 0.05) but not in the with cell comparison (Paired T-test: p = 0.11, n = 6). For PG cells, mESPC frequencies decreased in population comparisons only (baseline ITI: 3.79 ± 1.19 msec, TCB-2 ITI: 3.87 ± 1.25 msec, KS test: p < 0.05) but not cell comparisons
(Paired T-test p < 0.84, n = 5; Figure 12D).
Taken together, the amplitude results may indicate that TCB-2 causes an decrease quantal transmission, which may be due to insertion of new glutamate receptors. Meanwhile the frequency results show that acute TCB-2 does not appear to affect the number of glutamatergic synapse between OSNs and PG cells, but may affect that number between OSNs and ET cells.
Activation of Receptors decreases spontaneous excitatory transmission from olfactory sensory neurons onto ET cells.
In my next set of experiments, I set out to measure the effects of 5HT2AR activation on the functional excitatory drive of the circuit by measuring spontaneous excitatory currents (sEPSCs) in ET and PG cells. I still used picrotoxin to block out GABAergic IPSCs with picrotoxin, but eliminated TTX to assay spontaneous activity of the circuit. Representative sEPSC traces from ET cells and PG cells before and after TCB-2 are shown (Figure 13A). The average event from the same representative cell shows a decrease in ET amplitude but no change in PG amplitude from baseline (Figure 13B). Comparing the population of baseline or vs. TCB-2 treated cells, I observed a decreased the amplitude sEPSCs in ET cells (KS test, p < 0.05), comparing means, I found the
158
Figure 13. 5-HT2AR activation decreases sEPSC amplitude and frequency in ET cells but does not affect PG cells. A. Representative sEPSC trace from ET and PG cell in response to TCB-2 (1µM) treatment. B. Representative average sEPSC event from ET and PG cell in response to TCB-2. C. Cumulative probability plot and mean+ SEM for sEPSC amplitude in response to TCB-2. Population of sEPSC amplitudes decrease in ET cells (KS test: *p < 0.05). No change in PG cells. Mean sEPSC amplitudes decreases in response to TCB-2 in ET cells (Paired T-test: *p < 0.05. No change in PG cells. D. Cumulative probability plot for sEPSC inter-event interval and mean ± SEM for sEPSC frequencies in ET and PG cells in response to TCB-2. Population sEPSC inter- event intervals increase (frequency decreases) in ET cells ( KS test: *p < 0.05). No change in PG cells. Mean sEPSC frequency decreases in response to TCB-2 treatment in ET cells (Paired T-test: *p < 0.05). No change in PG cells (ET cells n = 8, PG cells n = 13 for all analysis).
159 same decrease (baseline: 20.27 ± 1.31 pA, TCB-2: 18.88 ± 1.23 pA, Paired T- test: p < 0.05, n = 8) (Figure 15C). TCB-2 does not cause any significant change in PG cell sEPSC amplitude as assayed by population comparisons and no in the mean comparisons (baseline: 14.18 ± 0.97 pA, TCB-2 13.87 ± 0.99 pA, p=0.51, n
= 13) (Figure 13C). TCB-2 also decreased the frequency of sEPSCs (increased inter-event-interval) of ET cells as assayed by population comparisons (KS test P
< 0.05) or comparison of means (baseline frequency:12.75 ± 3.11 pA, TCB-2:
10.46 ± 3.09 pA, Paired T-test: p < 0.05). TCB-2 did not affect the sEPSC frequency of PG cells (baseline: 4.67 ± 0.64 pA, TCB-2: 4.67 ± 0.69 pA, Paired
T-test p=0.99) (Figure 13D).
Activation of receptors decreases the effects of ET cell busting on other glomerular elements
ET cells intrinsically burst even without OSN input and in presence of glutamatergic and GABAergic blockers. This bursting is dependent on sodium channel that slow depolarization’s causing membrane oscillations (Hayar, Karnup et al. 2004). The effects of these bursts can be seen in ET, Mitral or PG cells within the same glomerulus as bursts in postsynaptic events. Busting set by ET cells may infect the entire glomerulus via dendro-dendritic actions in both cell types or in ET cells via electrical coupling with other ET cells. I tested if 5-HT2AR activation affected the effects of ET cell bursting on PG and ET post synaptic cells.
Spontaneous EPSC recordings were obtained from PG and ET cells
(Figure 14A). Representative trace of ET and PG cells that burst (40% of ET
160
Figure 14. 5-HT2AR decreases the effects of bursting on juxtaglomerular cells. A. Example of confocal Z-stack of an acute slice containing the M72- GFP glomerulus overlayed with patch-clamped juxtaglomerular cell filled with Alexa Fluor Hydrazide 568 in the internal solution. Juxtaglomerular (JG) cells include glutamatergic ET cell (left) and GABAergic PG cell(right) B. Representative sEPSC traces from an ET cell(right) and PG Cell (left) that show bursts of sEPSCs. Recordings were performed at baseline and two minutes after application of agonist TCB-2. C. TCB-2 increases the duration of sEPSC bursts in a pooled analysis of JG (ET+PG) cells, (Paired T-test: *p < 0.05). D. TCB-2 has no effect on bursting frequency of juxtaglomerular cells. E. TCB-2 has no effect on number of events per sEPSC burst in juxtaglomerular cells.
161 cells, 67% of PG cells) are shown (Figure 14B). I found that as a whole, this group of juxtaglomerular experienced a slight, but statistically significant decreased duration of bursting per sEPSC burst (baseline burst duration: 421.08
± 33.90 msec, TCB-burst duration: 378.67 ± 33.30 msec, Paired T test: p < 0.05, n = 10) (Figure 14C). I saw no significant changes after TCB-2 in other measures of busting such as frequency of busting (baseline 0.49± 0.076Hz, TCB-2: 0.45 ±
0.07 Hz, Paired T-test: p = 0.30 (Figure 14D), events per burst (baseline: 6.97 ±
0.64 events, TCB-2: 6.62 ± 0.65 events, p = 0.27) (Figure 14E). These data may suggest that 5-HT2AR activation may decrease the effects of bursting on PG and
ET cells.
Fear associative learning increase excitatory drive of olfactory sensory neurons at glomerulus
In my final experiment, I aimed to find the effect of associative learning on excitatory glomerular synapses 48 hours after learning. I subjected animals to a standard fear condition protocol (acetophenone paired with foot-shock) as described in Chapters 3 and I recorded spontaneous EPSCs from the juxtaglomerular (ET+PG cells) of the M72-glomerulus and compared them to those of controlled glomeruli of fear trained mice. I found that 48 hours after learning, there was a persistent synaptic plasticity, in the form of an increased excitatory drive in the M72 glomerulus compared to control (non-M72)glomeruli of fear trained animals. . The M72 glomerulus that processes the learned odor showed a larger amplitude of sEPSCs (Control glomerulus amplitude: 15.38 ±
1.00 pA, n=3, M72 glomerulus amplitude: 21.77 ± 1.76 pA, n = 3, T-test: p <
0.05). The M72 glomerulus also had a trend for having larger frequency of
162
Figure 15. Fear associative learning increases excitatory drive in M72-GFP glomerulus versus control glomeruli (JG) cells. A. Example of confocal Z-stack of an acute slice containing the M72-GFP glomerulus over-layed with patch clamped ET cell filled with Alexa Fluor Hydrazide 568. B. Representative sEPSC traces from an ET cell postsynaptic to the M72-GFP glomerulus and ET cell post synaptic to a control glomerulus. C. Fear associative learning increases the sEPSC amplitude in M72-glomerulus compared to control glomeruli (Paired t test: p<.05, n=3) D. Fear learning does not increase the frequency of sEPSCs in the M72-GFP glomerulus compared to controls in a statistically significant manner in this limited sample size.
163 sEPSCs. However this was not significant due to the small sample size (Control glomerulus frequency: 2.73 ± 1.14 Hz, M72 glomerulus frequency: 34.42 ± 18.68
Hz, T-test, p = 0.10, n= 3 respectively). I conclude from these data, that fear learning experience induces a persistent plasticity in the glomerular synapses that respond to the learned odor.
Discussion
The experiments performed in this study were motivated by the question: how does serotonin modulate glomerular synapses? Understanding 5-HT mediated plasticity at the glomerulus will allow me to hypothesize how this plasticity may play role in functional computations that affect the behaving animal. It will bring me closer to understanding how 5-HT signaling at the glomerulus provides mechanisms for both learning and memory as well as sensory gating.
Overall, my results showed that activation of 5-HT2AR receptors decrease the strength of excitatory synapses at the glomerular relay station. This decrease was mediated via by both presynaptic mechanisms such as a decrease in probability of OSN neurotransmitter release as well as post synaptic mechanisms such as a decrease in quantal currents (which may indicate insertion of glutamate receptors) or perhaps even the addition of new glutamatergic synapses in the postsynaptic cell and dampen the effects of bursting excitation on juxtaglomerular neurons. Finally, I also found that associative fear learning, can strengthen glutamatergic synapses of the glomerulus, at least by increasing overall excitatory drive. These findings are significant since it is the first time
164 learning-dependent plasticity has been found in the adult olfactory bulb, much less the glomerular station. These finding are especially interesting in that they fit well with my hypothesis that 5-HT neuromodulation plays dual roles of learning and memory integrator and sensory gate modulator.
In the literature, there have been two previous electrophysiological studies that investigate the effects of acute serotonin in the olfactory. Collectively, they inform me that 5-HT uses 5HT2ARs depolarize excitatory elements of the circuit (Mitral and ET cells) and simultaneously acts on 5-HT2C receptors located on periglomerular cells to increase GABAergic inhibition mitral cells(Hardy,
Palouzier-Paulignan et al. 2005, Liu, Aungst et al. 2012). The results of these studies agree with mapping studies for serotonin receptors(McLean and Shipley
1987, Pompeiano, Palacios et al. 1994), including the immunofluorescence mapping of 5-HT2A and 5-HT2C I present in chapter 4 of this study. The above studies(Hayar, Karnup et al. 2004, Hardy, Palouzier-Paulignan et al. 2005) use current clamp to visualize the 5-HT mediated small, but long lasting depolarization of the target cell to verify its direct action on that cell type and to identify the ionic channel(found to be TRP channel) responsible for its conduction. Then they heavily uses current clamp to measure 5-HT signaling mediated changes in spiking and bursting, to make statements about how 5-HT affected the output of that cell. These two types of assays are very informative since I can gain knowledge of the output of the mitral cell (final output from the
OB) as well as 5-HT’s effect on many individual elements of the circuit.
My electrophysiological studies were more functional interrogation than circuit dissection and thus differ from previous studies in my approach. I want to measure the effects 5-HT2ARs signaling on glutamatergic synapses in the
165 glomerulus. Thus, I use the voltage clamp not to visualize the effects of 5-HT2AR activation the on the ET cell itself, but rather use the ET or PG cell as readout for how a steady state of 5-HT2AR signaling affects the glomerular circuit as a whole.
ET cells are especially useful to use as detectors of OSN synaptic strength since they are the only glomerular cell type that always monosynaptic with OSNS.
Mitral cells and PG cells often do not even make direct connections with OSNs but instead rely on ET cells for excitatory drive (Hayar, Karnup et al. 2004, Gire and Schoppa 2009). In my experiments, the acute application of agonist TCB-2 on the scale of 2 to 5 minutes is designed to mimic the effect of endogenous serotonin released from DRN centrifugal fibers during an arousing even which results in influx of 5-HT into the glomerular layer binding to abundantly expressed
5-HT2Areceptors on ET and mitral cells. This is the same influx of serotonin that I prose arrives at the OB, and most heavily the glomerular layer during associative learning in Chapter 4. With this approach I hope to measure the net effects of 5-
HT2AR activation on excitatory synapses in the circuit as an assay of functional drive of incoming odor signals.
My first experiments with OSN evoked EPSCs are attempts to make rough estimates of how excitatory glomerular synapses respond to an odor stimulus under the influence of 5-HT2AR activation. I electrically stimulated the OSN and recorded juxtaglomerular ET and PG cells that are postsynaptic to it. In agreement with previous literature (Hayar, Karnup et al. 2004, Tyler, Petzold et al. 2007), I found large monosynaptic evoked currents in ET cells and small polysynaptic evoked currents in PG cells. Activation of 5HT2A receptors reduced the amplitude of evoked ET cell currents, but PG cell amplitudes were little affected. What is mediating this decrease in amplitude? Previous studies have
166 reported 5-HT causing inhibition to the glomerulus by activation of PG cells which in turn inhibit OSN presynaptic neurotransmitter release (Petzold, Hagiwara et al.
2009). However in this particular experiment in my attempt to isolate glutamatergic synapses, pharmacology was used such at both sources of extrinsic inhibition (pre and post synaptic inhibition) were blocked. My next set of experiments then set out to dissect the decrease in synaptic strength has presynaptic or postsynaptic origins.
One of the presynaptic mechanisms by which 5-HT2AR may decrease the strength of glutamatergic synapses is by decreasing the presynaptic probability of release. I found that 5-HT2AR activation increased he paired pulse ratio. In other words, it decreased the paired pulse depression that usually occurs at this high probability synapse (Murphy, Darcy et al. 2005). In the natural circuit,
GABAergic PG cells of the glomerulus provide a majority of glomerular inhibition via GABAB receptors located on OSNs (feed-back inhibition) (Aroniadou-
Anderjaska, Zhou et al. 2000, Wachowiak, McGann et al. 2005) as well as
GABAA receptors on all post synaptic elements (ET, PG, Mitral cells) (feed forward inhibition) (Murphy, Darcy et al. 2005, Shao, Puche et al. 2009).
However, by using GABAA and GABAB receptor antagonist in my bath solution, I essentially filtered out all sources of pre and postsynaptic GABAergic inhibition. I also had blocked any possible dopaminergic presynaptic inhibition via D2 receptors from the small population of dopaminergic PG cells (Berkowicz and
Trombley 2000, Ennis, Zhou et al. 2001).
The only remaining source of presynaptic inhibition that I did not block was glutamatergic presynaptic inhibition of OSNs via mGLURs (Schoppa and
Westbrook 1997) which has been reported to be a mechanism for LTD at the first
167 synapse (Mutoh, Yuan et al. 2005). Serotonin 2A’s actions in ET cells and Mitral cells can increase their excitability and firing rates (Hardy, Palouzier-Paulignan et al. 2005, Liu, Aungst et al. 2012), especially with the lack of GABAergic inhibition.
Thus 5-HT2AR’s may increase dendritic release by these glutamatergic cells and thus cause mGLuR (mostly likely Group III mGLURs) presynaptic inhibition to decrease neurotransmitter release. Finally, however, the ultimate reason for decrease in probability of release could be intrinsic in nature—that 5-HT2AR activation may have changed the availability of available vesicles from the ready releasable pool of the OSN terminal. Metabotropic glutamate receptors have been shown to use such a mechanism to control presynaptic release (Billups,
Graham et al. 2005).
I further suspected post synaptic mechanisms are heavily involved in the decrease of glutamatergic synaptic strength. 5-HT2AR activation decreased miniature eEPSC events, indicating a decrease in the effect of each quanta of neurotransmitter on the post synaptic cell. This is usually mediated by postsynaptic glutamatergic receptor insertion into synapses. I also observed weaker, evidence for an increase in mEPSC frequency, which can be interpreted as an increase in the number of glutamatergic synapses.
I know that 5HT2ARs are heavily expressed in ET cells (Chapter 3,
(Pompeiano, Palacios et al. 1992, McLean, Darby-King et al. 1995), I suspect that 5-HT2AR signaling in the post synaptic cell may facilitate strengthening of glutamatergic post synaptic elements that lead to these phenomena. For example, 5-HT2AR receptors might up-regulate intracellular mechanisms that promote glutamatergic synapse formation or strengthening of existing synapses by insertion of AMPA or NMDAR receptors. Rapid modification of glutamatergic
168 synapses by receptor insertion/removal have been documented in the literature in hippocampus (Carroll, Lissin et al. 1999) and in mitral cell (change of
AMPA/NMDAR current ratios (Yuan 2009, Yuan and Harley 2012) following only
5 minutes of high frequency stimulation. This makes it feasible that the changes I saw in my mEPSC experiments are due to fast removal of glutamatergic receptors from the synapse. Moreover, 5-HT2AR receptors have been implicated in inducing plasticity of dendritic spines (Peddie, Davies et al. 2008, Jones,
Srivastava et al. 2009), which are often remodeled during the formation of new synapses or changing synaptic strength by accommodating more receptors.
Thus 5-HT2ARs are well poised to play a role for controlling post synaptic glutamatergic activity. Future studies should address this issue more directly with electrophysiological studies comparing NMDA/AMPA currents or imaging of the trafficking of these receptors under the influence of serotonin.
Which intracellular mechanisms might 5-HT2AR be using to alter glutamatergic synapse number as strength? 5HT2A receptors are coupled to Go receptors which use the PLC/IP3 or DAG/PKC pathway to manipulate intracellular levels of calcium (Aghajanian, Sprouse et al. 1990, Hoyer, Hannon et al. 2002). Other evidence suggests that although 5-HT2AR in concert with β1 ultimately promotes an increase in cAMP, one of the key regulators of long term plasticity (Yuan, Harley et al. 2003). Although intermediate mechanisms are unknown, 5-HT2ARs, within about 2-3 minutes activation, causes the closing of potassium channels as well as opens a non-selective ion channel which in ET cell has been identified as a TRP channel (Aghajanian, Sprouse et al. 1990, Liu,
Aungst et al. 2012). In this way, 5-HT2ARs depolarize mitral cells and increase their spiking rates. 5-HT2ARs receptors have been localized to not only cell
169 bodies but the dendrites of Mitral and ET cells that arborize extensively in the glomerulus (Chapter 4). In fact 5-HT2ARs have even been co-localized with NR1 subunits at the same dendritic spines with electron microscopy. Thus, I can imagine that 5-HT2AR may use similar intracellular mechanisms to modulate the strength of synaptic inputs of the cell either locally or via some diffusible signal.
There is second, but less likely interpretation that the decrease in mEPSC amplitude, or quantal transmission may actually be partially presynaptic in origin.
Since I did not block D2 or GABAB receptors in mEPSC recordings, I cannot eliminate the possibility that presynaptic inhibition may be causing a decrease in quantal transmission by decreasing the amount of neurotransmitter per vesicle.
This can be tested in future studies using partial agonists of GABAB receptors
(Tyler, Petzold et al. 2007)
Finally, I found that 5-HT2AR activation decreased frequency and amplitude was decreased ET cell sEPSCs. Among my recordings, spontaneous excitatory currents (sEPSCs) are the most accurate representation of the glomerular circuit at its natural state. Only GABAA was blocked to isolate glutamatergic signals.
With D2, GABAB, presynaptic inhibition intact, some of the decrease in excitatory drive may be attributed to 5-HT2ARs activation of ET cells which in turn drive
GABAergic inhibition of the OSN presynaptically (Petzold, Albeanu et al. 2008).
The lack of TTX permits. synaptic transmission from OSNs onto other juxtaglomerular cells as well as action potentials to take place in ET, mitral and
PG cells. Since all these cell types participate in dendro-dendritic signaling, this would be engaging circuit mechanisms that were not present in the other types of the recordings. Thus I can interpret these results as 5-HT2AR having the effect of
170 decreasing excitatory drive a the first synapse. Moreover, my results from bursting analysis also show decreases in excitatory drive.
Last, but perhaps most interestingly, I performed one final experiment to assay the effects of fear learning on excitatory drive the glomerular circuit. I found that fear learning animals possessed an increase in excitatory drive at the glomerulus that was persistent 48 hours after learning. At first this result seemed contrary my previous results and my expectations. I had reasoned that fear conditioned animals would experience the same type of dampening of excitation in the M72-glomerulus as with 5-HT2AR agonist application, since fear learned animals showed a higher level of 5-HT2ARs 48 hours post-learning (Chapter 4).
However, upon careful review, there is no contradiction between an increase in
5-HT2ARs 48 hours post learning and an increase excitatory drive. In the intact circuit, increased expression of 5-HT2ARs would most likely cause an increase in the amplitude of the ON-OFF response in the glomerulus, which results in a net increase GABAergic inhibition at the glomerulus to shape mitral cell activity. This may result in in sharper mitral cell responsible that have more temporal fidelity to the odor signal, a greater signal to noise, as well shifting thresholds to avoid saturation (Gire and Schoppa 2009, Petzold, Hagiwara et al. 2009, Shao, Puche et al. 2009, Liu, Aungst et al. 2012). The recordings in the first part of this study only capture a portion of this inhibition. Meanwhile, an increase in excitatory drive caused by fear learning would increase excitatory signal from the OSN to the mitral output cell and the ET cell, further amplifying the ON-OFF responses. In the M72 glomerulus of the fear learned animal---this might mean a stronger as well as more optimally gated signal for the meaningful odor.
171 The most obvious next set of studies should use serotonin pharmacology to assay if the increases in excitatory drive in fear conditioned animals are due to 5-
HT signaling. Further studies should be conducted to dissect out if this plasticity is due to strengthening is via pre or post synaptic mechanisms (such as increase in probability of release or insertion of glutamatergic receptors. Another set of studies could more comprehensively assay how glomerular elements work together to produce this dramatic increase in excitation after learning. Yet another interesting study could assay the length for which this kind of learning induced plasticity persists, marking its onset and offset (if ever, in the life time of the animal). Finally, it would be very interesting to investigate how the strength of the glomerular synapse is altered by reward learning.
In conclusion, these findings provide evidence that 5-HT2AR can induce plasticity for optimizing sensory gating. My first set of results indicates that 5-
HT2ARs mediate decrease in glomerular excitatory drive even when extrinsic sources of inhibition are removed. In a short time frame, 5-HT2AR signaling appears to be weakening the OSN synapse either by mediating increased circuit inhibition or intrinsically by down regulating glutamatergic transmission in ET and possibly mitral cells. In the long term however, associative learning may cause increases in excitatory drive but still maintains elevated levels of serotonin signaling for gating this strong signal. Functionally speaking, this is description of a learning informed sensory gate. For the emotionally salient learned odor, glomerular synapses will be optimized to attend to it with excitatory drive but simultaneously also with a high amplitude of the ON-OFF cycle for optimal gating
In such a away learning and memory is the means by which this a meaningful odor is prioritized for admission to higher centers.
172 REFERENCES
Abel, T., P. V. Nguyen, M. Barad, T. A. Deuel, E. R. Kandel and R. Bourtchouladze (1997). "Genetic demonstration of a role for PKA in the late phase of LTP and in hippocampus-based long-term memory." Cell 88(5): 615-626.
Aghajanian, G. K., J. S. Sprouse, P. Sheldon and K. Rasmussen (1990). "Electrophysiology of the central serotonin system: receptor subtypes and transducer mechanisms." Ann N Y Acad Sci 600: 93-103; discussion 103.
Aroniadou-Anderjaska, V., F. M. Zhou, C. A. Priest, M. Ennis and M. T. Shipley (2000). "Tonic and synaptically evoked presynaptic inhibition of sensory input to the rat olfactory bulb via GABA(B) heteroreceptors." J Neurophysiol 84(3): 1194-1203.
Berkowicz, D. A. and P. Q. Trombley (2000). "Dopaminergic modulation at the olfactory nerve synapse." Brain Res 855(1): 90-99.
Billups, B., B. P. Graham, A. Y. Wong and I. D. Forsythe (2005). "Unmasking group III metabotropic glutamate autoreceptor function at excitatory synapses in the rat CNS." J Physiol 565(Pt 3): 885-896.
Carroll, R. C., D. V. Lissin, M. von Zastrow, R. A. Nicoll and R. C. Malenka (1999). "Rapid redistribution of glutamate receptors contributes to long- term depression in hippocampal cultures." Nat Neurosci 2(5): 454-460.
Dan, Y. and M. M. Poo (2006). "Spike timing-dependent plasticity: from synapse to perception." Physiol Rev 86(3): 1033-1048.
Ennis, M., F. M. Zhou, K. J. Ciombor, V. Aroniadou-Anderjaska, A. Hayar, E. Borrelli, L. A. Zimmer, F. Margolis and M. T. Shipley (2001). "Dopamine D2 receptor-mediated presynaptic inhibition of olfactory nerve terminals." J Neurophysiol 86(6): 2986-2997.
Gire, D. H. and N. E. Schoppa (2009). "Control of on/off glomerular signaling by a local GABAergic microcircuit in the olfactory bulb." J Neurosci 29(43): 13454-13464.
Hardy, A., B. Palouzier-Paulignan, A. Duchamp, J. P. Royet and P. Duchamp- Viret (2005). "5-Hydroxytryptamine action in the rat olfactory bulb: in vitro electrophysiological patch-clamp recordings of juxtaglomerular and mitral cells." Neuroscience 131(3): 717-731.
Hayar, A., S. Karnup, M. Ennis and M. T. Shipley (2004). "External tufted cells: a major excitatory element that coordinates glomerular activity." J Neurosci 24(30): 6676-6685.
173 Hayar, A., S. Karnup, M. T. Shipley and M. Ennis (2004). "Olfactory bulb glomeruli: external tufted cells intrinsically burst at theta frequency and are entrained by patterned olfactory input." J Neurosci 24(5): 1190-1199.
Hoyer, D., J. P. Hannon and G. R. Martin (2002). "Molecular, pharmacological and functional diversity of 5-HT receptors." Pharmacol Biochem Behav 71(4): 533-554.
Jones, K. A., D. P. Srivastava, J. A. Allen, R. T. Strachan, B. L. Roth and P. Penzes (2009). "Rapid modulation of spine morphology by the 5-HT2A serotonin receptor through kalirin-7 signaling." Proc Natl Acad Sci U S A 106(46): 19575-19580.
Kauer, J. A., R. C. Malenka and R. A. Nicoll (1988). "A persistent postsynaptic modification mediates long-term potentiation in the hippocampus." Neuron 1(10): 911-917.
Liu, S., J. L. Aungst, A. C. Puche and M. T. Shipley (2012). "Serotonin modulates the population activity profile of olfactory bulb external tufted cells." J Neurophysiol 107(1): 473-483.
McLean, J. H., A. Darby-King and G. D. Paterno (1995). "Localization of 5-HT2A receptor mRNA by in situ hybridization in the olfactory bulb of the postnatal rat." J Comp Neurol 353(3): 371-378.
McLean, J. H. and M. T. Shipley (1987). "Serotonergic afferents to the rat olfactory bulb: II. Changes in fiber distribution during development." J Neurosci 7(10): 3029-3039.
Murphy, G. J., D. P. Darcy and J. S. Isaacson (2005). "Intraglomerular inhibition: signaling mechanisms of an olfactory microcircuit." Nat Neurosci 8(3): 354-364.
Mutoh, H., Q. Yuan and T. Knopfel (2005). "Long-term depression at olfactory nerve synapses." J Neurosci 25(17): 4252-4259.
Peddie, C. J., H. A. Davies, F. M. Colyer, M. G. Stewart and J. J. Rodriguez (2008). "Colocalisation of serotonin2A receptors with the glutamate receptor subunits NR1 and GluR2 in the dentate gyrus: an ultrastructural study of a modulatory role." Exp Neurol 211(2): 561-573.
Petzold, G. C., D. F. Albeanu, T. F. Sato and V. N. Murthy (2008). "Coupling of neural activity to blood flow in olfactory glomeruli is mediated by astrocytic pathways." Neuron 58(6): 897-910.
Petzold, G. C., A. Hagiwara and V. N. Murthy (2009). "Serotonergic modulation of odor input to the mammalian olfactory bulb." Nat Neurosci.
174 Pompeiano, M., J. M. Palacios and G. Mengod (1992). "Distribution and cellular localization of mRNA coding for 5-HT1A receptor in the rat brain: correlation with receptor binding." J Neurosci 12(2): 440-453.
Pompeiano, M., J. M. Palacios and G. Mengod (1994). "Distribution of the serotonin 5-HT2 receptor family mRNAs: comparison between 5-HT2A and 5-HT2C receptors." Brain Res Mol Brain Res 23(1-2): 163-178.
Schoppa, N. E. and G. L. Westbrook (1997). "Modulation of mEPSCs in olfactory bulb mitral cells by metabotropic glutamate receptors." J Neurophysiol 78(3): 1468-1475.
Shao, Z., A. C. Puche, E. Kiyokage, G. Szabo and M. T. Shipley (2009). "Two GABAergic intraglomerular circuits differentially regulate tonic and phasic presynaptic inhibition of olfactory nerve terminals." J Neurophysiol 101(4): 1988-2001.
Sigurdsson, T., V. Doyere, C. K. Cain and J. E. LeDoux (2007). "Long-term potentiation in the amygdala: a cellular mechanism of fear learning and memory." Neuropharmacology 52(1): 215-227.
Tyler, W. J., G. C. Petzold, S. K. Pal and V. N. Murthy (2007). "Experience- dependent modification of primary sensory synapses in the mammalian olfactory bulb." J Neurosci 27(35): 9427-9438.
Wachowiak, M., J. P. McGann, P. M. Heyward, Z. Shao, A. C. Puche and M. T. Shipley (2005). "Inhibition [corrected] of olfactory receptor neuron input to olfactory bulb glomeruli mediated by suppression of presynaptic calcium influx." J Neurophysiol 94(4): 2700-2712.
Yuan, Q. (2009). "Theta bursts in the olfactory nerve paired with beta- adrenoceptor activation induce calcium elevation in mitral cells: a mechanism for odor preference learning in the neonate rat." Learn Mem 16(11): 676-681.
Yuan, Q. and C. W. Harley (2012). "What a nostril knows: olfactory nerve-evoked AMPA responses increase while NMDA responses decrease at 24-h post-training for lateralized odor preference memory in neonate rat." Learn Mem 19(2): 50-53.
Yuan, Q., C. W. Harley and J. H. McLean (2003). "Mitral cell beta1 and 5-HT2A receptor colocalization and cAMP coregulation: a new model of norepinephrine-induced learning in the olfactory bulb." Learn Mem 10(1): 5-15.
175 Chapter 6
DISCUSSION
This study was motivated by the question of how learning and memory sensory gating can inform and optimized sensory gating. Using the rodent olfactory bulb (OB) as a model system, I hypothesized that centrifugal serotonergic signaling is a key mechanism in this process of learning-dependent gating at olfactory glomeruli. More specifically serotonin (5-HT) provides the arousal for the associative learning and memory, which induces the circuit plasticity necessary to form a better adapted sensory gate, one that preferentially attends to important, meaningful odors. From my experiments, I found evidence to support for 5-HT2A receptor dependent olfactory learning and memory as well as 5-HT2Areceptor modulated sensory gating at the glomerulus of the rodent olfactory system.
In chapter 2, I presented a comparative review of the rodent versus insect olfaction literature showing that centrifugal neuromodulators play a role in learning and memory as well as peripheral sensory plasticity in diverse classes of animals. I found that, despite the differences in circuit organization, signal transduction, or complexity of higher olfactory processing, the role of neuromodulators in learning and memory may be part of a common evolutionary theme and may be translational across species.
In both schools of literature, many studies have shown that neuromodulators are key players in olfactory learning and memory in higher olfactory centers such as the mushroom body or lateral horn (Hammer 1993,
Schwaerzel, Monastirioti et al. 2003, Kim, Lee et al. 2007, Claridge-Chang,
Roorda et al. 2009, Krashes, DasGupta et al. 2009, Lee, Lin et al. 2011) and to a
176 less thorough extent in the rodent piriform cortex (Gellman and Aghajanian 1993,
Hasselmo and Barkai 1995, Hasselmo, Linster et al. 1997, Linster, Garcia et al.
2001, Linster and Hasselmo 2001, Saar, Grossman et al. 2001). However, studies about learning and memory in the more periphery olfactory bulb and antennal lobe (especially in the glomerular relay station) are just emerging based. To date, the strong presence of neuromodulator mediated plasticity has been identified in the antennal lobe (Kirchhof and Mercer 1997, Kirchhof,
Homberg et al. 1999, Perk and Mercer 2006, Sinakevitch, Mustard et al. 2011) as well as olfactory bulb (Phillips, Powell et al. 1963, Linster and Cleland 2002,
Hardy, Palouzier-Paulignan et al. 2005, Chaudhury, Escanilla et al. 2009, Yuan
2009, Liu, Aungst et al. 2012). Similarly, the experience and learning induced plasticity have also been shown in the antennal lobe (Faber, Joerges et al. 1999,
Daly, Christensen et al. 2004, Yu, Ponomarev et al. 2004, Tamura, Horiuchi et al.
2010) and olfactory bulb (Wilson, Sullivan et al. 1987, Tyler, Petzold et al. 2007,
Jones, Choi et al. 2008, Cavallin, Powell et al. 2010, Valle-Leija, Blanco-
Hernandez et al. 2012). Studies that show neuromodulator influence on learning and memory at the glomerulus are very few within this subset of the literature.
They either only show plasticity in neonates synapses deeper than the glomerulus (Wilson, Sullivan et al. 1987, Sullivan, Wilson et al. 1989, Sullivan,
Zyzak et al. 1992, Sullivan, Stackenwalt et al. 2000, Yuan, Harley et al. 2003,
Yuan, Harley et al. 2003, Wilson and Linster 2008) or show very short lived plasticity on the order of minutes (Farooqui, Robinson et al. 2003). To date, no studies have provided definitive evidence that learning as well as memory traces exist in the antennal lobe or olfactory bulb glomeruli due to associative learning in adults.
177 In chapter 3, I developed an experimental paradigm in which I can study learning-dependent sensory gating in adult animal. I coupled associative learning with a genetically identified circuit. The behavioral paradigm allowed me to study adult learning and memory in an ethological relevant positive and negative context while the M72-GFP circuit gave me control over experimental stimulation followed by accurate, post learning interrogation for functional plasticity. Most studies in the literature showing learning induced plasticity in the OB has been focused on one of several paradigms including 1) global deprivation/enrichment experience (Guthrie, Wilson et al. 1990, Wilson, Guthrie et al. 1990, Zou,
Feinstein et al. 2004, Tyler, Petzold et al. 2007) 2) neonate imprinting(Wilson and
Linster 2008) or 3) odor discrimination (Mandairon, Stack et al. 2006, Mandairon,
Didier et al. 2008), The first type of experience manipulation usually causes homeostatic or compensatory changes and does not really qualify as a type of learning. Neonate learning is development specific and disappears after the first
14 days of life—thus I cannot generalize these conclusions to older animals or plasticity in the mature nervous system. There has been only one study to date using associative learning in adult mice, but limited its assays to structural plasticity of the glomerulus (Jones, Choi et al. 2008). This study focuses associative learning’s effects on functional plasticity in the glomerulus of adult mice.
The behavioral paradigms I developed were much more ethologically relevant since odor-cues provide information about important events in a rodent’s life such as feeding, reproduction, predator avoidance. Also more importantly, this paradigm allows me to tackle the problem of adult learning and memory, which is generalizable across the animal’s lifetime, not only during a critical
178 period. I do acknowledge that my reward and fear paradigms are not parallel and may pose a problem when comparing between positive and negative valances. Differences include duration of learning to establish the behavior as well as type of learning task (classical conditioning or operant conditioning).
These factors may affect the type of plasticity induced or circuits activated. In future studies, an effort should be made to balance the paradigms: such as two classical paradigms or two operant paradigms of similar duration.
One problem faced by many other previous studies about experience dependent functional plasticity in the OB is that they used generalized odor activation followed by a general interrogation of glomeruli anywhere in the bulb
(Guthrie, Wilson et al. 1990, Mandairon, Didier et al. 2008, Chaudhury, Escanilla et al. 2009). Or at times, researchers tried to make a best estimate the location of the glomeruli responding to the odor used in learning(Wilson and Leon 1988). I tried to overcome this problem by using the M72-GFP mouse developed by Peter
Mombaerts (Potter, Zheng et al. 2001, Feinstein and Mombaerts 2004) with a verified and restricted activation of the M72 glomerulus with acetophenone and the ability to identify the location in post-hoc analyses via GFP. This follows in the suit of several other studies that have used these M72-GFP mice for manipulating an odor input and assaying glomerular structural plasticity or refinement in the glomerulus (Zou, Feinstein et al. 2004, Kerr and Belluscio 2006,
Jones, Choi et al. 2008, Cavallin, Powell et al. 2010, Valle-Leija, Blanco-
Hernandez et al. 2012). In contrast, this study is the first to use the M72 glomerulus as a marker to investigate learning induced functional plasticity.
Future studies could take advantage of these models further for example, by using OMP-SpH (Bozza, McGann et al. 2004) mice to assay the effects of
179 learning on OSN transmitter release following learning. Alternately, with the advent of the OMP-ChR mouse (Smear, Shusterman et al. 2011) one could artificially provide the conditioned stimulus via light stimulation and unconditioned stimulus via pharmacology or DRN stimulation in an attempt to instill associative learning artificially. In this way, I can achieve even more specific in activation of the circuit.
In chapter 4 I found that serotonin signaling played a key role in olfactory learning by acting on the glomerular relay station. First, serotonergic circuit mapping showed the presence of both presynaptic 5-HT fibers from the dorsal raphe nucleus (DRN) as well as post synaptic receptors especially 5-HT2A receptors and to a lesser extent 5-HT2CR receptors in the glomerular layer. Next,
I showed that 5-HT2AR signaling but not 5-HT2CR involved in the acquisition of fear memories via actions in the olfactory bulb, possibly by providing the arousal tone of the unconditioned stimulus. Finally, evidence suggests that 5-HT2AR signaling is elevated persistently even 2 days after both fear and reward learning, and thus may be part of a term memory trace at the glomerular relay station specifically at the glomerulus processing the learned odor.
Results from mapping, along with recent developments in the literature detailing the about the functional properties of each circuit elements (Mitral, ET,
PG cells)(Hayar, Karnup et al. 2004, Hayar, Karnup et al. 2004, Hayar, Shipley et al. 2005, Gire and Schoppa 2009) helped me formulate a hypothesis about the role serotonin in the circuit. I conjectured that an influx of 5-HT’s directly excite mitral output cells ET cells (which are intermediaries to GABAergic inhibition and mitral cell excitation). I know that 5-HT2AR’s Gq coupled signaling cascade ultimately leads to pathways to cause slow depolarization via cation TRP 180 channels while closing K+ leak channels (Aghajanian, Sprouse et al. 1990, Liu,
Aungst et al. 2012). The net effect of serotonin influx in an intact circuit, is that both excitatory and inhibitory elements of the circuit increase in activity: an a higher amplitude ON is followed by a higher amplitude OFF. Such cascades may alter the strength of glutamatergic signaling during integration of OSN input and
5-HT influx from the DRN during associative learning (Figure 16).
My in vivo pharmacology results supported this hypothesis that 5-HT, specifically via 5-HT2ARs, play a part in the unconditional stimulus (US) signal in the acquisition of fear associative learning in the OB. Similar methods of intrabulbular application of antagonists have been used in the olfactory bulb imprinting literature to show that NE and 5-HT collectively provide the US tone for neonate learning or to show that Ach plays a role in odor discrimination (Sullivan,
Zyzak et al. 1992, McLean, Darby-King et al. 1996, Mandairon, Ferretti et al.
2006). My study is the first to show the serotonin plays a role in adult associative learning by its actions in the olfactory bulb. Unfortunately, I were unable to isolate the learning to 5-HT’s influence specifically in the glomerular layer in this set of studies.
There have been no studies yet on whether learning and memory traces can exist in the glomerulus, especially in adult rodents. I found the glomerulus processing a learned odor after associative learning experienced an elevation of
5-HT levels as well as expression of 5-HT2ARs. Unfortunately, it was outside the scope of this study to investigate the mechanisms that may have translated the coincidence of OSN glutamate and 5-HT2AR activation during learning into a longer term increase in 5-HT circuit elements (fibers and receptors) that I observed M72 glomerulus. I can conjecture, however, that perhaps traditional
181 mechanisms in learning dependent plasticity involving NMDAR mediate calcium signaling and resultant activation of intracellular signaling such as the mechanisms found for hippocampal spatial learning may play a role (Davis,
Butcher et al. 1992, Tsien, Huerta et al. 1996) in causing upregulation of 5-
HT2ARs. This cascade is activated, for example, in the mitral cells following neonate imprinting (McLean, Harley et al. 1999, Yuan, Harley et al. 2000, Yuan,
Harley et al. 2003) and can also be induced with pairing of OSN stimulation coupled with β1 agonist. Although NE is the main activator of this pathway that involves cAMP, PKA and converges on pCREB, 5-HT has also been implicated to converge upon the same pathway(Price, Darby-King et al. 1998, Yuan, Harley et al. 2003) despite the fact that it is a Gq and not Gα coupled receptor. Gq receptors however do converge on PKC which controls CamKII, signaling molecule implicated in hippocampal late LTP and learning and memory
(Malenka, Kauer et al. 1989, Malinow, Schulman et al. 1989). In both fear and reward learning paradigms, learning occurred over a period of either 2 days
(fear) or 14 days(reward), and a memory trace was assayed 48 hours following the last training session. In this time scale, plenty of time has elapsed for intracellular messengers to alter gene expression, and synthesize new proteins
(Frey, Huang et al. 1993, Abel, Nguyen et al. 1997), one of which may include of
5-HT2ARs. The same coincidence may also be responsible for the increase in 5-
HT fiber density or amount of 5-HT synthesized within each fiber. The first would require morphological changes in the presynaptic axon which may include addition of additional buttons, growth of new fibers while the second could be caused by increased 5-HT synthesis and packaging into vesicles. These changes in the presynaptic 5-HT axon could be caused by retrograde signals
182 from the post synapse . For example cAMP was found to be an activity dependent signal that retrograded influence the targeting of OSNs during development and could also affect presynaptic serotoninergic fibers. BDNF also has been implicated as an activity dependent signal in the glomerulus and regulates dendritic growth of PG cells (Berghuis, Agerman et al. 2006). BDNF is known to cause growth of 5-HT fibers in the cortex (Spenger, Hyman et al. 1995,
Mamounas, Altar et al. 2000), and thus it is very likely that signals like BDNF could be generated by postsynaptic cells can be facilitating the increased 5-HT innervation that I observed which is specific to the M72 glomerulus. In short, 5-
HT2AR may translate the coincidence of OSN input and 5-HT2AR activation during learning into long term plasticity involving the synthesis of more 5-HT2AR receptors or an increase 5-HT innervation pattern These two changes guarantee that the M72-glomeruls of learned odor has heightened sensitive to future serotonergic tone.
In chapter 5, I measured the effects of 5-HT2AR signaling on excitatory glomerular synapses as well as the effects of associative learning the same synapse. I found evidence that 5-HT2AR receptors cause an overall decrease in excitatory drive at the first synapse between OSN and postsynaptic elements. In absence of feed-forward and feedback GABAergic inhibition in my recordings, I isolated presynaptic mechanism for this decrease in excitatory drive that may be due partially to presynaptic mGLuRs. Meanwhile, modifications of the post synaptic compartment could involve increase in glutamatergic receptors per synapse or number of glutamatergic synapse. Finally I find evidence that learning dependent plasticity might exist in glomerulus following fear associative learning in adult rodents, which has not been shown in the literature before.
183 The results from this section could be more thoroughly interpreted with the understanding that, my experiments were done to isolate glutamatergic synapses. However, within the intact circuit, intraglomerular interaction is extremely important in mediating the potential gating mechanism. For my overall hypothesis of 5-HT2AR mediate sensory in the behaving animal, it is very important to understand these intraglomerular mechanisms and how they can be affected by acute serotonin signaling. From the first set studies dedicated purely to synaptic interactions in glomerular information processing (Hayar, Karnup et al. 2004, Hayar, Karnup et al. 2004, Hayar, Shipley et al. 2005, Murphy, Darcy et al. 2005, Gire and Schoppa 2009, Najac, De Saint Jan et al. 2011), I learned that
1)ET cells serve as the main driving force of both excitation and inhibition in the glomerulus since they are that are reliably and strongly receive monosynaptic input from OSNs 2) ET cells can drive both GABAergic PG cells as well as Mitral cells via strong dendro-dendritic synapses that in PG cells cause strong waves of recurrent inhibition and in Mitral cells cause a slow long lasting post synaptic potential that determines mitral cell output. Since ET cells intrinsically burst, it can at once synchronize the glomerulus to fire together as well as tune the amplitude of the entire glomerulus by simultaneously affecting ON and OFF responses. 3) GABAergic inhibition from PG cells is step behind Mitral cell excitation, and thus, the mitral cell receives this inhibition mid LLD, thus limits this
LLD as well as spiking. 4) GABAergic inhibition facilitates improved sensory gating by narrowing the duration of LLDs which enhances temporal fidelity, and return to hyperpolarization for the next signal and dampening LLD amplitudes which too prevents saturation of the signal. Since the intensity of the odor is encoded in mitral cell spike rates, achieving temporal specificity and recovering
184
Figure 16. Proposed roles of serotonergic elements within the glomerular circuit during learning. Serotonin from dorsal raphe confers arousal of the unconditioned stimulus arrives coincidentally at glomerulus with odor inputs carrying conditioned stimulus information. Integration of signals occurs at excitatory cells within the glomerulus via 5-HT2AR and glutamatergic signaling. The selective location of 5-HT2ARs and 5-HT2CRs on glutamatergic and GABAergic cells respectively allows for regulation of the ON-OFF response of the glomerulus and thus contributes to learning informed sensory gating.
185 quickly from depolarization will allow the mitral cell to conduct stronger and more accurate signals to the piriform cortex.
Meanwhile, the second set of literature informs me that the acute effects of 5-HT in the bulb are by acting on ET cells and mitral cells to increase excitatory drive. Meanwhile 5-HT also acts on 5-HT2CRs on PG cells to provide enhanced inhibition for better information gating as described above (Hardy,
Palouzier-Paulignan et al. 2005, Petzold, Hagiwara et al. 2009, Liu, Aungst et al.
2012). Collectively, these studies lead me to the hypothesize that 5-HT signaling serves to modulate the amplitude of these ON-OFF circuits to both increase precision and intensity of mitral cell output for a given OSN input, in order to preferential gating of the information from this glomerulus (Figure 16).
Although short term effects of 5-HT2AR is to decrease excitatory drive, after learning occurs, however, 5-HT2AR expression and excitatory drive increases in the glomerulus of the learned odor. Thus, the learning induced sensory gate may initial dampen glutamatergic synapses, but the convergence of
CS and US ultimately translates, over time, into an increased excitatory drive from OSNs but simultaneous increased the power of GABAergic gating via persistent increases in 5HT2AR signaling.
In conclusion, despite the central location traditionally ascribed to fundamental processes such as learning, memory and sensory gating, I have found evidence that these complex processes occur as early as the olfactory glomeruli, the primary synapses of the rodent olfactory system. In fact, these processes may share the same underlying mechanisms that begin with increased 5-HT2AR signaling during acquisition with rapid dampening of excitatory drive within minutes, however translating hours to days into
186 persistently stronger glutamatergic glomerular synapses efficiently gated by high levels of glomerulus specific 5-HT2AR signaling. In other words, the short term plasticity used in learning acquisition may first begin to alter the sensory gate.
Meanwhile, a long-term neural plasticity resulting from memory consolidation in the glomerulus may participates in the stabilization of an optimized sensory gate.
Based on the rich literature I reviewed and the findings of this study, propose a more generalized model for how sensory experience modifies the sensory gate (Figure 17). I proposed that upon first encountering a neutral, novel odor, an organism responds to it with some set amplitude in the first glomerular relay station. In one scenario, the odor is repeatedly presented as a neutral, meaningless odor so the animal becomes habituated to it. As a result, the synapses encoding this odor are weakened by homeostatic mechanisms and the sensory gate is altered so the animal it attends to this stimulus minimally. The second possibility is that the odor is presented coincidentally with an arousing stimulus and associative learning occurs (in either positive or negative contexts).
The sensory gate is altered via neuromodulators and maintained by Hebbian or spike timing time dependent plasticity or plasticity in neuromodulator systems themselves. As a result, the sensory gate increases in amplitude so that it may attend to this stimulus with the utmost priority.
How my knowledge of neuromodulator-mediated learning-dependent amplitude modulation and sensory gating provide insights into the mechanisms sensory gating deficits in psychiatric disease? One can imagine a third scenario, where the normal systems in place for controlling ON-OFF cycles of sensory processing are missing or perturbed either due to neuromodulator imbalance/deficiencies or lack of some step of the intracellular mechanisms
187
Figure 17. Model: Learning-dependent amplitude modulation of excitation and inhibition in an ON-OFF circuit optimizes sensory gating. Serotonergic neuromodulation contributes to amplitude modulation of both excitation and inhibition. Greater amplitudes of ON and OFF increase signal to noise, temporal fidelity and prevents saturation of signal. Meaningful odors are preferentially gated and heightened attention by the nervous system. Meanwhile habituated odors receive less attention. Finally, models of diseased sensory gates may involve in a disturbance in neuromodulator control of control of ON-OFF cycles at the periphery.
188 needed to initiate learning induced plasticity or maintain this plasticity on longer time scales. These disturbances may occur at the periphery, perhaps for another modalities, at its first gating station, the thalamus. In my study, 5-HT especially via 5-HT2AR signaling is a key candidate for modulating the ON-OFF response of the glomerulus to provide learning informed amplitude modulation. In the literature, 5-HT2AR signaling has been implicated in a possible mechanism for sensory gating deficits and the positive symptoms of hallucination in schizophrenia. Evidence shows that 2A family receptor signaling is linked with hallucinatory drugs, positive symptoms of schizophrenia, schizophrenic genotypes and sensory gating deficits in animal as well as human models of disturbed sensory gating (Geyer and Braff 1987, Padich, McCloskey et al. 1996,
Swerdlow and Geyer 1998, Adler, Cawthra et al. 2005, Akhondzadeh, Malek-
Hosseini et al. 2008, Quednow, Kuhn et al. 2008, Brauer, Strobel et al. 2009).
Future studies may wish to use animal or human models of schizophrenia to investigate the punitive role of 5-HT2AR or other signaling pathways that are disturbed in these disease states causing problems in peripheral sensory gating.
From here, I can find ways to rescue disturbed learning-mediated sensory gates
(perhaps those lacking plasticity or those that are too unstable) to restore a flexible but dependable perception of reality.
189 REFERENCES
Abdolmaleky, H. M., S. V. Faraone, S. J. Glatt and M. T. Tsuang (2004). "Meta- analysis of association between the T102C polymorphism of the 5HT2a receptor gene and schizophrenia." Schizophr Res 67(1): 53-62.
Abel, T., P. V. Nguyen, M. Barad, T. A. Deuel, E. R. Kandel and R. Bourtchouladze (1997). "Genetic demonstration of a role for PKA in the late phase of LTP and in hippocampus-based long-term memory." Cell 88(5): 615- 626.
Adler, L. E., E. M. Cawthra, K. A. Donovan, J. G. Harris, H. T. Nagamoto, A. Olincy and M. C. Waldo (2005). "Improved p50 auditory gating with ondansetron in medicated schizophrenia patients." Am J Psychiatry 162(2): 386-388.
Aghajanian, G. K., J. S. Sprouse, P. Sheldon and K. Rasmussen (1990). "Electrophysiology of the central serotonin system: receptor subtypes and transducer mechanisms." Ann N Y Acad Sci 600: 93-103; discussion 103.
Akhondzadeh, S., M. Malek-Hosseini, A. Ghoreishi, M. Raznahan and S. A. Rezazadeh (2008). "Effect of ritanserin, a 5HT2A/2C antagonist, on negative symptoms of schizophrenia: a double-blind randomized placebo-controlled study." Prog Neuropsychopharmacol Biol Psychiatry 32(8): 1879-1883.
Ambroggi, F., A. Ishikawa, H. L. Fields and S. M. Nicola (2008). "Basolateral amygdala neurons facilitate reward-seeking behavior by exciting nucleus accumbens neurons." Neuron 59(4): 648-661.
Araneda, R. C. and S. Firestein (2006). "Adrenergic enhancement of inhibitory transmission in the accessory olfactory bulb." J Neurosci 26(12): 3292-3298.
Aroniadou-Anderjaska, V., F. M. Zhou, C. A. Priest, M. Ennis and M. T. Shipley (2000). "Tonic and synaptically evoked presynaptic inhibition of sensory input to the rat olfactory bulb via GABA(B) heteroreceptors." J Neurophysiol 84(3): 1194- 1203.
Aungst, J. L., P. M. Heyward, A. C. Puche, S. V. Karnup, A. Hayar, G. Szabo and M. T. Shipley (2003). "Centre-surround inhibition among olfactory bulb glomeruli." Nature 426(6967): 623-629.
Bailey, C. H. and M. Chen (1988). "Long-term memory in Aplysia modulates the total number of varicosities of single identified sensory neurons." Proc Natl Acad Sci U S A 85(7): 2373-2377.
Bailey, C. H., M. Giustetto, H. Zhu, M. Chen and E. R. Kandel (2000). "A novel function for serotonin-mediated short-term facilitation in aplysia: conversion of a transient, cell-wide homosynaptic hebbian plasticity into a persistent, protein synthesis-independent synapse-specific enhancement." Proc Natl Acad Sci U S A 97(21): 11581-11586.
190 Bargmann, C. I. (2006). "Comparative chemosensation from receptors to ecology." Nature 444(7117): 295-301.
Barkai, E. and D. Saar (2001). "Cellular correlates of olfactory learning in the rat piriform cortex." Rev Neurosci 12(2): 111-120.
Beggs, K. T., I. S. Hamilton, P. T. Kurshan, J. A. Mustard and A. R. Mercer (2005). "Characterization of a D2-like dopamine receptor (AmDOP3) in honey bee, Apis mellifera." Insect Biochem Mol Biol 35(8): 873-882.
Belluscio, L., C. Lodovichi, P. Feinstein, P. Mombaerts and L. C. Katz (2002). "Odorant receptors instruct functional circuitry in the mouse olfactory bulb." Nature 419(6904): 296-300.
Belluscio, L., C. Lodovichi, P. Feinstein, P. Mombaerts and L. C. Katz (2002). "Odorant receptors instruct functional circuitry in the mouse olfactory bulb." Nature 419: 296-300.
Berghuis, P., K. Agerman, M. B. Dobszay, L. Minichiello, T. Harkany and P. Ernfors (2006). "Brain-derived neurotrophic factor selectively regulates dendritogenesis of parvalbumin-containing interneurons in the main olfactory bulb through the PLCgamma pathway." J Neurobiol 66(13): 1437-1451.
Berkowicz, D. A. and P. Q. Trombley (2000). "Dopaminergic modulation at the olfactory nerve synapse." Brain Res 855(1): 90-99.
Betarbet, R., T. Zigova, R. A. Bakay and M. B. Luskin (1996). "Dopaminergic and GABAergic interneurons of the olfactory bulb are derived from the neonatal subventricular zone." Int J Dev Neurosci 14(7-8): 921-930.
Bhandawat, V., S. R. Olsen, N. W. Gouwens, M. L. Schlief and R. I. Wilson (2007). "Sensory processing in the Drosophila antennal lobe increases reliability and separability of ensemble odor representations." Nat Neurosci 10(11): 1474- 1482.
Billups, B., B. P. Graham, A. Y. Wong and I. D. Forsythe (2005). "Unmasking group III metabotropic glutamate autoreceptor function at excitatory synapses in the rat CNS." J Physiol 565(Pt 3): 885-896.
Blenau, W., J. Erber and A. Baumann (1998). "Characterization of a dopamine D1 receptor from Apis mellifera: cloning, functional expression, pharmacology, and mRNA localization in the brain." J Neurochem 70(1): 15-23.
Blenau, W. and M. Thamm (2011). "Distribution of serotonin (5-HT) and its receptors in the insect brain with focus on the mushroom bodies: lessons from Drosophila melanogaster and Apis mellifera." Arthropod Struct Dev 40(5): 381- 394.
Bliss, T. V. and G. L. Collingridge (1993). "A synaptic model of memory: long- term potentiation in the hippocampus." Nature 361(6407): 31-39.
191 Bliss, T. V. and T. Lomo (1973). "Long-lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path." J Physiol 232(2): 331-356.
Bobillier, P., S. Seguin, A. Degueurce, B. D. Lewis and J. F. Pujol (1979). "The efferent connections of the nucleus raphe centralis superior in the rat as revealed by radioautography." Brain Res 166(1): 1-8.
Bodyak, N. and B. Slotnick (1999). "Performance of mice in an automated olfactometer: odor detection, discrimination and odor memory." Chem Senses 24(6): 637-645.
Bokil, H., N. Laaris, K. Blinder, M. Ennis and A. Keller (2001). "Ephaptic interactions in the mammalian olfactory system." J Neurosci 21(20): RC173.
Bouret, S. and S. J. Sara (2002). "Locus coeruleus activation modulates firing rate and temporal organization of odour-induced single-cell responses in rat piriform cortex." Eur J Neurosci 16(12): 2371-2382.
Bourtchuladze, R., B. Frenguelli, J. Blendy, D. Cioffi, G. Schutz and A. J. Silva (1994). "Deficient long-term memory in mice with a targeted mutation of the cAMP-responsive element-binding protein." Cell 79(1): 59-68.
Bozza, T., P. Feinstein, C. Zheng and P. Mombaerts (2002). "Odorant receptor expression defines functional units in the mouse olfactory system." J Neurosci 22(8): 3033-3043.
Bozza, T., J. P. McGann, P. Mombaerts and M. Wachowiak (2004). "In vivo imaging of neuronal activity by targeted expression of a genetically encoded probe in the mouse." Neuron 42(1): 9-21.
Bozza, T., A. Vassalli, S. Fuss, J. J. Zhang, B. Weiland, R. Pacifico, P. Feinstein and P. Mombaerts (2009). "Mapping of class I and class II odorant receptors to glomerular domains by two distinct types of olfactory sensory neurons in the mouse." Neuron 61(2): 220-233.
Brauer, D., A. Strobel, T. Hensch, K. Diers, K. P. Lesch and B. Brocke (2009). "Genetic variation of serotonin receptor function affects prepulse inhibition of the startle." J Neural Transm 116(5): 607-613.
Brennan, P. A. and K. M. Kendrick (2006). "Mammalian social odours: attraction and individual recognition." Philos Trans R Soc Lond B Biol Sci 361(1476): 2061- 2078.
Brown, S. M., R. M. Napper and A. R. Mercer (2004). "Foraging experience, glomerulus volume, and synapse number: A stereological study of the honey bee antennal lobe." J Neurobiol 60(1): 40-50.
Brown, S. M., R. M. Napper, C. M. Thompson and A. R. Mercer (2002). "Stereological analysis reveals striking differences in the structural plasticity of
192 two readily identifiable glomeruli in the antennal lobes of the adult worker honeybee." J Neurosci 22(19): 8514-8522.
Brunelli, M., V. Castellucci and E. R. Kandel (1976). "Synaptic facilitation and behavioral sensitization in Aplysia: possible role of serotonin and cyclic AMP." Science 194(4270): 1178-1181.
Brunjes, P. C., K. R. Illig and E. A. Meyer (2005). "A field guide to the anterior olfactory nucleus (cortex)." Brain Res Brain Res Rev 50(2): 305-335.
Brunken, W. J. and X. T. Jin (1993). "A role for 5HT3 receptors in visual processing in the mammalian retina." Vis Neurosci 10(3): 511-522.
Buchsbaum, M. S., B. T. Christian, D. S. Lehrer, T. K. Narayanan, B. Shi, J. Mantil, E. Kemether, T. R. Oakes and J. Mukherjee (2006). "D2/D3 dopamine receptor binding with [F-18]fallypride in thalamus and cortex of patients with schizophrenia." Schizophr Res 85(1-3): 232-244.
Buck, L. B. and R. Axel (1991). "A novel multigene family may encode odorant receptors: a molecular basis for odor recognition." Cell 65: 175-187.
Burrone, J. and V. N. Murthy (2003). "Synaptic gain control and homeostasis." Curr Opin Neurobiol 13(5): 560-567.
Carcaud, J., E. Roussel, M. Giurfa and J. C. Sandoz (2009). "Odour aversion after olfactory conditioning of the sting extension reflex in honeybees." J Exp Biol 212(Pt 5): 620-626.
Carlsson, M. A., M. Diesner, J. Schachtner and D. R. Nassel (2010). "Multiple neuropeptides in the Drosophila antennal lobe suggest complex modulatory circuits." J Comp Neurol 518(16): 3359-3380.
Carroll, R. C., D. V. Lissin, M. von Zastrow, R. A. Nicoll and R. C. Malenka (1999). "Rapid redistribution of glutamate receptors contributes to long-term depression in hippocampal cultures." Nat Neurosci 2(5): 454-460.
Cavallin, M. A., K. Powell, K. C. Biju and D. A. Fadool (2010). "State-dependent sculpting of olfactory sensory neurons is attributed to sensory enrichment, odor deprivation, and aging." Neurosci Lett 483(2): 90-95.
Chaudhury, D., O. Escanilla and C. Linster (2009). "Bulbar acetylcholine enhances neural and perceptual odor discrimination." J Neurosci 29(1): 52-60.
Chou, Y. H., M. L. Spletter, E. Yaksi, J. C. Leong, R. I. Wilson and L. Luo (2010). "Diversity and wiring variability of olfactory local interneurons in the Drosophila antennal lobe." Nat Neurosci 13(4): 439-449.
Christian, B. T., D. S. Lehrer, B. Shi, T. K. Narayanan, P. S. Strohmeyer, M. S. Buchsbaum and J. C. Mantil (2006). "Measuring dopamine neuromodulation in
193 the thalamus: using [F-18]fallypride PET to study dopamine release during a spatial attention task." Neuroimage 31(1): 139-152.
Chu, S. and J. J. Downes (2000). "Odour-evoked autobiographical memories: psychological investigations of proustian phenomena." Chem Senses 25(1): 111- 116.
Claridge-Chang, A., R. D. Roorda, E. Vrontou, L. Sjulson, H. Li, J. Hirsh and G. Miesenbock (2009). "Writing memories with light-addressable reinforcement circuitry." Cell 139(2): 405-415.
Cleland, T. A. (2010). "Early transformations in odor representation." Trends Neurosci 33(3): 130-139.
Cohen-Cory, S. (2002). "The developing synapse: construction and modulation of synaptic structures and circuits." Science 298(5594): 770-776.
Collins, G. G., G. A. Probett, J. Anson and N. J. McLaughlin (1984). "Excitatory and inhibitory effects of noradrenaline on synaptic transmission in the rat olfactory cortex slice." Brain Res 294(2): 211-223.
Cornea-Hebert, V., M. Riad, C. Wu, S. K. Singh and L. Descarries (1999). "Cellular and subcellular distribution of the serotonin 5-HT2A receptor in the central nervous system of adult rat." J Comp Neurol 409(2): 187-209.
Couto, A., M. Alenius and B. J. Dickson (2005). "Molecular, anatomical, and functional organization of the Drosophila olfactory system." Curr Biol 15(17): 1535-1547.
Curzon, P., N. R. Rustay and K. E. Browman (2009). "Cued and Contextual Fear Conditioning for Rodents."
Dacks, A. M., T. A. Christensen and J. G. Hildebrand (2008). "Modulation of olfactory information processing in the antennal lobe of Manduca sexta by serotonin." J Neurophysiol 99(5): 2077-2085.
Dacks, A. M., D. S. Green, C. M. Root, A. J. Nighorn and J. W. Wang (2009). "Serotonin modulates olfactory processing in the antennal lobe of Drosophila." J Neurogenet 23(4): 366-377.
Dahlstrom, A., K. Fuxe, L. Olson and U. Ungerstedt (1965). "On the distribution and possible function of monamine nerve terminals in the olfactory bulb of the rabbit." Life Sci 4(21): 2071-2074.
Daly, K. C., T. A. Christensen, H. Lei, B. H. Smith and J. G. Hildebrand (2004). "Learning modulates the ensemble representations for odors in primary olfactory networks." Proc Natl Acad Sci U S A 101(28): 10476-10481.
Dan, Y. and M. M. Poo (2006). "Spike timing-dependent plasticity: from synapse to perception." Physiol Rev 86(3): 1033-1048.
194 Datiche, F., P. H. Luppi and M. Cattarelli (1995). "Serotonergic and non- serotonergic projections from the raphe nuclei to the piriform cortex in the rat: a cholera toxin B subunit (CTb) and 5-HT immunohistochemical study." Brain Res 671(1): 27-37.
Davis, R. L. (2005). "Olfactory memory formation in Drosophila: from molecular to systems neuroscience." Annu Rev Neurosci 28: 275-302.
Davis, S., S. P. Butcher and R. G. Morris (1992). "The NMDA receptor antagonist D-2-amino-5-phosphonopentanoate (D-AP5) impairs spatial learning and LTP in vivo at intracerebral concentrations comparable to those that block LTP in vitro." J Neurosci 12(1): 21-34.
De Rosa, E. and M. E. Hasselmo (2000). "Muscarinic cholinergic neuromodulation reduces proactive interference between stored odor memories during associative learning in rats." Behav Neurosci 114(1): 32-41.
Distler, P. (1990). "Synaptic connections of dopamine-immunoreactive neurons in the antennal lobes of Periplaneta americana. Colocalization with GABA-like immunoreactivity." Histochemistry 93(4): 401-408.
Dobson, A. T., F. Sederati, G. Devi-Rao, W. M. Flanagan, M. J. Farrell, J. G. Stevens, E. K. Wagner and L. T. Feldman (1989). "Identification of the latency- associated transcript promoter by expression of rabbit beta-globin mRNA in mouse sensory nerve ganglia latently infected with a recombinant herpes simplex virus." J Virol 63(9): 3844-3851.
Doty, R. L. (1986). "Odor-guided behavior in mammals." Experientia 42(3): 257- 271.
Dragunow, M. and R. Faull (1989). "The Use of C-Fos as a Metabolic Marker in Neuronal Pathway Tracing." Journal of Neuroscience Methods 29(3): 261-265.
Ellingrod, V. L., P. J. Perry, B. C. Lund, K. Bever-Stille, F. Fleming, T. L. Holman and D. Miller (2002). "5HT2A and 5HT2C receptor polymorphisms and predicting clinical response to olanzapine in schizophrenia." J Clin Psychopharmacol 22(6): 622-624.
Ennis, M., F. M. Zhou, K. J. Ciombor, V. Aroniadou-Anderjaska, A. Hayar, E. Borrelli, L. A. Zimmer, F. Margolis and M. T. Shipley (2001). "Dopamine D2 receptor-mediated presynaptic inhibition of olfactory nerve terminals." J Neurophysiol 86(6): 2986-2997.
Escanilla, O., N. Mandairon and C. Linster (2008). "Odor-reward learning and enrichment have similar effects on odor perception." Physiol Behav 94(4): 621- 626.
Faber, T., J. Joerges and R. Menzel (1999). "Associative learning modifies neural representations of odors in the insect brain." Nat Neurosci 2(1): 74-78.
195 Farber, N. B., J. Hanslick, C. Kirby, L. McWilliams and J. W. Olney (1998). "Serotonergic agents that activate 5HT2A receptors prevent NMDA antagonist neurotoxicity." Neuropsychopharmacology 18(1): 57-62.
Farooqui, T., K. Robinson, H. Vaessin and B. H. Smith (2003). "Modulation of early olfactory processing by an octopaminergic reinforcement pathway in the honeybee." J Neurosci 23(12): 5370-5380.
Feinstein, P., T. Bozza, I. Rodriguez, A. Vassalli and P. Mombaerts (2004). "Axon guidance of mouse olfactory sensory neurons by odorant receptors and the beta2 adrenergic receptor." Cell 117(6): 833-846.
Feinstein, P. and P. Mombaerts (2004). "A contextual model for axonal sorting into glomeruli in the mouse olfactory system." Cell 117(6): 817-831.
Fishilevich, E. and L. B. Vosshall (2005). "Genetic and functional subdivision of the Drosophila antennal lobe." Curr Biol 15(17): 1548-1553.
Fitzpatrick, D., I. T. Diamond and D. Raczkowski (1989). "Cholinergic and monoaminergic innervation of the cat's thalamus: comparison of the lateral geniculate nucleus with other principal sensory nuclei." J Comp Neurol 288(4): 647-675.
Fletcher, M. L. and W. R. Chen (2010). "Neural correlates of olfactory learning: Critical role of centrifugal neuromodulation." Learn Mem 17(11): 561-570.
Fletcher, M. L. and D. A. Wilson (2002). "Experience modifies olfactory acuity: acetylcholine-dependent learning decreases behavioral generalization between similar odorants." J Neurosci 22(2): RC201.
Fletcher, P. J., A. J. Grottick and G. A. Higgins (2002). "Differential effects of the 5-HT(2A) receptor antagonist M100907 and the 5-HT(2C) receptor antagonist SB242084 on cocaine-induced locomotor activity, cocaine self-administration and cocaine-induced reinstatement of responding." Neuropsychopharmacology 27(4): 576-586.
Franks, K. M., M. J. Russo, D. L. Sosulski, A. A. Mulligan, S. A. Siegelbaum and R. Axel (2011). "Recurrent circuitry dynamically shapes the activation of piriform cortex." Neuron 72(1): 49-56.
Frey, U., Y. Y. Huang and E. R. Kandel (1993). "Effects of cAMP simulate a late stage of LTP in hippocampal CA1 neurons." Science 260(5114): 1661-1664.
Fuxe, K. (1965). "Evidence for the Existence of Monoamine Neurons in the Central Nervous System. 3. The Monoamine Nerve Terminal." Z Zellforsch Mikrosk Anat 65: 573-596.
Galici, R., J. D. Boggs, K. L. Miller, P. Bonaventure and J. R. Atack (2008). "Effects of SB-269970, a 5-HT7 receptor antagonist, in mouse models predictive of antipsychotic-like activity." Behav Pharmacol 19(2): 153-159.
196 Galizia, C. G. and R. Menzel (2000). "Odour perception in honeybees: coding information in glomerular patterns." Curr Opin Neurobiol 10(4): 504-510.
Garcia-Cabezas, M. A., P. Martinez-Sanchez, M. A. Sanchez-Gonzalez, M. Garzon and C. Cavada (2009). "Dopamine innervation in the thalamus: monkey versus rat." Cereb Cortex 19(2): 424-434.
Garcia-Cabezas, M. A., B. Rico, M. A. Sanchez-Gonzalez and C. Cavada (2007). "Distribution of the dopamine innervation in the macaque and human thalamus." Neuroimage 34(3): 965-984.
Gehlert, D. R., A. Shekhar, S. M. Morin, P. A. Hipskind, C. Zink, S. L. Gackenheimer, J. Shaw, S. D. Fitz and T. J. Sajdyk (2005). "Stress and central Urocortin increase anxiety-like behavior in the social interaction test via the CRF1 receptor." Eur J Pharmacol 509(2-3): 145-153.
Gellman, R. L. and G. K. Aghajanian (1993). "Pyramidal cells in piriform cortex receive a convergence of inputs from monoamine activated GABAergic interneurons." Brain Res 600(1): 63-73.
Gewirtz, J. C. and M. Davis (2000). "Using pavlovian higher-order conditioning paradigms to investigate the neural substrates of emotional learning and memory." Learn Mem 7(5): 257-266.
Geyer, M. A. and D. L. Braff (1982). "Habituation of the Blink reflex in normals and schizophrenic patients." Psychophysiology 19(1): 1-6.
Geyer, M. A. and D. L. Braff (1987). "Startle habituation and sensorimotor gating in schizophrenia and related animal models." Schizophr Bull 13(4): 643-668.
Gire, D. H. and N. E. Schoppa (2009). "Control of on/off glomerular signaling by a local GABAergic microcircuit in the olfactory bulb." J Neurosci 29(43): 13454- 13464.
Glanzman, D. L., S. L. Mackey, R. D. Hawkins, A. M. Dyke, P. E. Lloyd and E. R. Kandel (1989). "Depletion of serotonin in the nervous system of Aplysia reduces the behavioral enhancement of gill withdrawal as well as the heterosynaptic facilitation produced by tail shock." J Neurosci 9(12): 4200-4213.
Gomez, C., J. G. Brinon, M. V. Barbado, E. Weruaga, J. Valero and J. R. Alonso (2005). "Heterogeneous targeting of centrifugal inputs to the glomerular layer of the main olfactory bulb." J Chem Neuroanat 29(4): 238-254.
Gottfried, J. A., A. P. Smith, M. D. Rugg and R. J. Dolan (2004). "Remembrance of odors past: human olfactory cortex in cross-modal recognition memory." Neuron 42(4): 687-695.
Gouzoulis-Mayfrank, E., K. Heekeren, A. Neukirch, M. Stoll, C. Stock, J. Daumann, M. Obradovic and K. A. Kovar (2006). "Inhibition of return in the
197 human 5HT2A agonist and NMDA antagonist model of psychosis." Neuropsychopharmacology 31(2): 431-441.
Govindaiah, G., T. Wang, M. U. Gillette, S. R. Crandall and C. L. Cox (2010). "Regulation of inhibitory synapses by presynaptic D(4) dopamine receptors in thalamus." J Neurophysiol 104(5): 2757-2765.
Gray, C. M. and J. E. Skinner (1988). "Centrifugal regulation of neuronal activity in the olfactory bulb of the waking rabbit as revealed by reversible cryogenic blockade." Exp Brain Res 69(2): 378-386.
Guthrie, K. M., J. M. Pullara, J. F. Marshall and M. Leon (1991). "Olfactory deprivation increases dopamine D2 receptor density in the rat olfactory bulb." Synapse 8(1): 61-70.
Guthrie, K. M., D. A. Wilson and M. Leon (1990). "Early unilateral deprivation modifies olfactory bulb function." J Neurosci 10(10): 3402-3412.
Ha, H. I., M. Hendricks, Y. Shen, C. V. Gabel, C. Fang-Yen, Y. Qin, D. Colon- Ramos, K. Shen, A. D. Samuel and Y. Zhang (2010). "Functional organization of a neural network for aversive olfactory learning in Caenorhabditis elegans." Neuron 68(6): 1173-1186.
Haberly, L. B. (2001). "Parallel-distributed processing in olfactory cortex: new insights from morphological and physiological analysis of neuronal circuitry." Chem Senses 26(5): 551-576.
Halaris, A. E., B. E. Jones and R. Y. Moore (1976). "Axonal transport in serotonin neurons of the midbrain raphe." Brain Res 107(3): 555-574.
Halasz, N. and G. M. Shepherd (1983). "Neurochemistry of the vertebrate olfactory bulb." Neuroscience 10(3): 579-619.
Hamada, S., K. Senzaki, K. Hamaguchi-Hamada, K. Tabuchi, H. Yamamoto, T. Yamamoto, S. Yoshikawa, H. Okano and N. Okado (1998). "Localization of 5- HT2A receptor in rat cerebral cortex and olfactory system revealed by immunohistochemistry using two antibodies raised in rabbit and chicken." Brain Res Mol Brain Res 54(2): 199-211.
Hammer, M. (1993). "An Identified Neuron Mediates the Unconditioned Stimulus in Associative Olfactory Learning in Honeybees." Nature 366(6450): 59-63.
Hardy, A., B. Palouzier-Paulignan, A. Duchamp, J. P. Royet and P. Duchamp- Viret (2005). "5-Hydroxytryptamine action in the rat olfactory bulb: in vitro electrophysiological patch-clamp recordings of juxtaglomerular and mitral cells." Neuroscience 131(3): 717-731.
Hasselmo, M. E. and E. Barkai (1995). "Cholinergic modulation of activity- dependent synaptic plasticity in the piriform cortex and associative memory function in a network biophysical simulation." J Neurosci 15(10): 6592-6604.
198 Hasselmo, M. E., C. Linster, M. Patil, D. Ma and M. Cekic (1997). "Noradrenergic suppression of synaptic transmission may influence cortical signal-to-noise ratio." J Neurophysiol 77(6): 3326-3339.
Hayar, A. and M. Ennis (2007). "Endogenous GABA and glutamate finely tune the bursting of olfactory bulb external tufted cells." J Neurophysiol 98(2): 1052- 1056.
Hayar, A., S. Karnup, M. Ennis and M. T. Shipley (2004). "External tufted cells: a major excitatory element that coordinates glomerular activity." J Neurosci 24(30): 6676-6685.
Hayar, A., S. Karnup, M. T. Shipley and M. Ennis (2004). "Olfactory bulb glomeruli: external tufted cells intrinsically burst at theta frequency and are entrained by patterned olfactory input." J Neurosci 24(5): 1190-1199.
Hayar, A., M. T. Shipley and M. Ennis (2005). "Olfactory bulb external tufted cells are synchronized by multiple intraglomerular mechanisms." J Neurosci 25(36): 8197-8208.
Hildebrand, J. G., T. A. Christensen, I. D. Harrow, U. Homberg, S. G. Matsumoto and B. R. Waldrop (1992). "The roles of local interneurons in the processing of olfactory information in the antennal lobes of the moth Manduca sexta." Acta Biol Hung 43(1-4): 167-174.
Hildebrandt, H. and U. Muller (1995). "Octopamine mediates rapid stimulation of protein kinase A in the antennal lobe of honeybees." J Neurobiol 27(1): 44-50.
Hill, E. S., K. Okada and R. Kanzaki (2003). "Visualization of modulatory effects of serotonin in the silkmoth antennal lobe." J Exp Biol 206(Pt 2): 345-352.
Honjo, K. and K. Furukubo-Tokunaga (2009). "Distinctive neuronal networks and biochemical pathways for appetitive and aversive memory in Drosophila larvae." J Neurosci 29(3): 852-862.
Hoyer, D., J. P. Hannon and G. R. Martin (2002). "Molecular, pharmacological and functional diversity of 5-HT receptors." Pharmacol Biochem Behav 71(4): 533-554.
Hubel, D. H., T. N. Wiesel and S. LeVay (1977). "Plasticity of ocular dominance columns in monkey striate cortex." Philos Trans R Soc Lond B Biol Sci 278(961): 377-409.
Humphries, M. A., J. A. Mustard, S. J. Hunter, A. Mercer, V. Ward and P. R. Ebert (2003). "Invertebrate D2 type dopamine receptor exhibits age-based plasticity of expression in the mushroom bodies of the honeybee brain." J Neurobiol 55(3): 315-330.
199 Hurley, L. M., D. M. Devilbiss and B. D. Waterhouse (2004). "A matter of focus: monoaminergic modulation of stimulus coding in mammalian sensory networks." Curr Opin Neurobiol 14(4): 488-495.
Hurley, L. M. and G. D. Pollak (1999). "Serotonin differentially modulates responses to tones and frequency-modulated sweeps in the inferior colliculus." J Neurosci 19(18): 8071-8082.
Iyengar, A., T. S. Chakraborty, S. P. Goswami, C. F. Wu and O. Siddiqi (2010). "Post-eclosion odor experience modifies olfactory receptor neuron coding in Drosophila." Proc Natl Acad Sci U S A 107(21): 9855-9860.
Jacobs, B. L. and E. C. Azmitia (1992). "Structure and function of the brain serotonin system." Physiol Rev 72(1): 165-229.
Johansen, J. P., C. K. Cain, L. E. Ostroff and J. E. LeDoux (2011). "Molecular mechanisms of fear learning and memory." Cell 147(3): 509-524.
Jones, K. A., D. P. Srivastava, J. A. Allen, R. T. Strachan, B. L. Roth and P. Penzes (2009). "Rapid modulation of spine morphology by the 5-HT2A serotonin receptor through kalirin-7 signaling." Proc Natl Acad Sci U S A 106(46): 19575- 19580.
Jones, S. V., D. C. Choi, M. Davis and K. J. Ressler (2008). "Learning-dependent structural plasticity in the adult olfactory pathway." J Neurosci 28(49): 13106- 13111.
Jones, S. V., S. A. Heldt, M. Davis and K. J. Ressler (2005). "Olfactory-mediated fear conditioning in mice: simultaneous measurements of fear-potentiated startle and freezing." Behav Neurosci 119(1): 329-335.
Kandel, E. R. (2009). "The biology of memory: a forty-year perspective." J Neurosci 29(41): 12748-12756.
Kasa, P., I. Hlavati, E. Dobo, A. Wolff, F. Joo and J. R. Wolff (1995). "Synaptic and non-synaptic cholinergic innervation of the various types of neurons in the main olfactory bulb of adult rat: immunocytochemistry of choline acetyltransferase." Neuroscience 67(3): 667-677.
Katz, L. C. and C. J. Shatz (1996). "Synaptic activity and the construction of cortical circuits." Science 274(5290): 1133-1138.
Kauer, J. A., R. C. Malenka and R. A. Nicoll (1988). "A persistent postsynaptic modification mediates long-term potentiation in the hippocampus." Neuron 1(10): 911-917.
Kaupp, U. B. (2010). "Olfactory signalling in vertebrates and insects: differences and commonalities." Nat Rev Neurosci 11(3): 188-200.
200 Kay, L. M. and S. M. Sherman (2007). "An argument for an olfactory thalamus." Trends Neurosci 30(2): 47-53.
Kazama, H., E. Yaksi and R. I. Wilson (2011). "Cell death triggers olfactory circuit plasticity via glial signaling in Drosophila." J Neurosci 31(21): 7619-7630.
Kent, K. S., S. G. Hoskins and J. G. Hildebrand (1987). "A novel serotonin- immunoreactive neuron in the antennal lobe of the sphinx moth Manduca sexta persists throughout postembryonic life." J Neurobiol 18(5): 451-465.
Kerr, M. A. and L. Belluscio (2006). "Olfactory experience accelerates glomerular refinement in the mammalian olfactory bulb." Nat Neurosci 9(4): 484-486.
Kim, Y. C., H. G. Lee and K. A. Han (2007). "D1 dopamine receptor dDA1 is required in the mushroom body neurons for aversive and appetitive learning in Drosophila." J Neurosci 27(29): 7640-7647.
Kirchhof, B. S., U. Homberg and A. R. Mercer (1999). "Development of dopamine-immunoreactive neurons associated with the antennal lobes of the honey bee, Apis mellifera." J Comp Neurol 411(4): 643-653.
Kirchhof, B. S. and A. R. Mercer (1997). "Antennal lobe neurons of the honey bee, Apis mellifera, express a D2-like dopamine receptor in vitro." J Comp Neurol 383(2): 189-198.
Kiselycznyk, C. L., S. Zhang and C. Linster (2006). "Role of centrifugal projections to the olfactory bulb in olfactory processing." Learn Mem 13(5): 575- 579.
Kloppenburg, P., D. Ferns and A. R. Mercer (1999). "Serotonin enhances central olfactory neuron responses to female sex pheromone in the male sphinx moth manduca sexta." J Neurosci 19(19): 8172-8181.
Kloppenburg, P. and T. Heinbockel (2000). "5-Hydroxy-tryptamine modulates pheromone-evoked local field potentials in the macroglomerular complex of the sphinx moth Manduca sexta." J Exp Biol 203(Pt 11): 1701-1709.
Kloppenburg, P. and A. R. Mercer (2008). "Serotonin modulation of moth central olfactory neurons." Annu Rev Entomol 53: 179-190.
Krashes, M. J., S. DasGupta, A. Vreede, B. White, J. D. Armstrong and S. Waddell (2009). "A neural circuit mechanism integrating motivational state with memory expression in Drosophila." Cell 139(2): 416-427.
Kurahashi, T. and K. W. Yau (1994). "Olfactory transduction. Tale of an unusual chloride current." Curr Biol 4(3): 256-258.
Lagier, S., P. Panzanelli, R. E. Russo, A. Nissant, B. Bathellier, M. Sassoe- Pognetto, J. M. Fritschy and P. M. Lledo (2007). "GABAergic inhibition at
201 dendrodendritic synapses tunes gamma oscillations in the olfactory bulb." Proc Natl Acad Sci U S A 104(17): 7259-7264.
Larsson, M. C., A. I. Domingos, W. D. Jones, M. E. Chiappe, H. Amrein and L. B. Vosshall (2004). "Or83b encodes a broadly expressed odorant receptor essential for Drosophila olfaction." Neuron 43(5): 703-714.
LeDoux, J. E. (2000). "Emotion circuits in the brain." Annu Rev Neurosci 23: 155- 184.
Lee, K. H. and D. A. McCormick (1995). "Acetylcholine excites GABAergic neurons of the ferret perigeniculate nucleus through nicotinic receptors." J Neurophysiol 73(5): 2123-2128.
Lee, P. T., H. W. Lin, Y. H. Chang, T. F. Fu, J. Dubnau, J. Hirsh, T. Lee and A. S. Chiang (2011). "Serotonin-mushroom body circuit modulating the formation of anesthesia-resistant memory in Drosophila." Proc Natl Acad Sci U S A 108(33): 13794-13799.
Lee, Y. S., S. L. Choi, S. H. Lee, H. Kim, H. Park, N. Lee, Y. S. Chae, D. J. Jang, E. R. Kandel and B. K. Kaang (2009). "Identification of a serotonin receptor coupled to adenylyl cyclase involved in learning-related heterosynaptic facilitation in Aplysia." Proc Natl Acad Sci U S A 106(34): 14634-14639.
Levy, F., M. Meurisse, G. Ferreira, J. Thibault and Y. Tillet (1999). "Afferents to the rostral olfactory bulb in sheep with special emphasis on the cholinergic, noradrenergic and serotonergic connections." J Chem Neuroanat 16(4): 245-263.
Li, W., E. Luxenberg, T. Parrish and J. A. Gottfried (2006). "Learning to smell the roses: experience-dependent neural plasticity in human piriform and orbitofrontal cortices." Neuron 52(6): 1097-1108.
Lin, D. M., F. Wang, G. Lowe, G. H. Gold, R. Axel, J. Ngai and L. Brunet (2000). "Formation of precise connections in the olfactory bulb occurs in the absence of odorant-evoked neuronal activity." Neuron 26(1): 69-80.
Linster, C. and T. A. Cleland (2002). "Cholinergic modulation of sensory representations in the olfactory bulb." Neural Netw 15(4-6): 709-717.
Linster, C., P. A. Garcia, M. E. Hasselmo and M. G. Baxter (2001). "Selective loss of cholinergic neurons projecting to the olfactory system increases perceptual generalization between similar, but not dissimilar, odorants." Behav Neurosci 115(4): 826-833.
Linster, C. and M. E. Hasselmo (2000). "Neural activity in the horizontal limb of the diagonal band of broca can be modulated by electrical stimulation of the olfactory bulb and cortex in rats." Neurosci Lett 282(3): 157-160.
Linster, C. and M. E. Hasselmo (2001). "Neuromodulation and the functional dynamics of piriform cortex." Chem Senses 26(5): 585-594.
202 Linster, C., B. P. Wyble and M. E. Hasselmo (1999). "Electrical stimulation of the horizontal limb of the diagonal band of broca modulates population EPSPs in piriform cortex." J Neurophysiol 81(6): 2737-2742.
Liu, S., J. L. Aungst, A. C. Puche and M. T. Shipley (2012). "Serotonin modulates the population activity profile of olfactory bulb external tufted cells." J Neurophysiol 107(1): 473-483.
Lynch, G., J. Larson, S. Kelso, G. Barrionuevo and F. Schottler (1983). "Intracellular injections of EGTA block induction of hippocampal long-term potentiation." Nature 305(5936): 719-721.
Malenka, R. C., J. A. Kauer, D. J. Perkel, M. D. Mauk, P. T. Kelly, R. A. Nicoll and M. N. Waxham (1989). "An essential role for postsynaptic calmodulin and protein kinase activity in long-term potentiation." Nature 340(6234): 554-557.
Malinow, R., H. Schulman and R. W. Tsien (1989). "Inhibition of postsynaptic PKC or CaMKII blocks induction but not expression of LTP." Science 245(4920): 862-866.
Mamounas, L. A., C. A. Altar, M. E. Blue, D. R. Kaplan, L. Tessarollo and W. E. Lyons (2000). "BDNF promotes the regenerative sprouting, but not survival, of injured serotonergic axons in the adult rat brain." J Neurosci 20(2): 771-782.
Mandairon, N., A. Didier and C. Linster (2008). "Odor enrichment increases interneurons responsiveness in spatially defined regions of the olfactory bulb correlated with perception." Neurobiol Learn Mem 90(1): 178-184.
Mandairon, N., C. J. Ferretti, C. M. Stack, D. B. Rubin, T. A. Cleland and C. Linster (2006). "Cholinergic modulation in the olfactory bulb influences spontaneous olfactory discrimination in adult rats." Eur J Neurosci 24(11): 3234- 3244.
Mandairon, N., S. Peace, A. Karnow, J. Kim, M. Ennis and C. Linster (2008). "Noradrenergic modulation in the olfactory bulb influences spontaneous and reward-motivated discrimination, but not the formation of habituation memory." Eur J Neurosci 27(5): 1210-1219.
Mandairon, N., C. Stack, C. Kiselycznyk and C. Linster (2006). "Broad activation of the olfactory bulb produces long-lasting changes in odor perception." Proc Natl Acad Sci U S A 103(36): 13543-13548.
Mandairon, N., C. Stack, C. Kiselycznyk and C. Linster (2006). "Enrichment to odors improves olfactory discrimination in adult rats." Behav Neurosci 120(1): 173-179.
Mandairon, N., C. Stack and C. Linster (2006). "Olfactory enrichment improves the recognition of individual components in mixtures." Physiol Behav 89(3): 379- 384.
203 Mandairon, N., S. Sultan, M. Nouvian, J. Sacquet and A. Didier (2011). "Involvement of newborn neurons in olfactory associative learning? The operant or non-operant component of the task makes all the difference." J Neurosci 31(35): 12455-12460.
Marek, G. J. and G. K. Aghajanian (1996). "Alpha 1B-adrenoceptor-mediated excitation of piriform cortical interneurons." Eur J Pharmacol 305(1-3): 95-100.
Maricq, A. V., A. S. Peterson, A. J. Brake, R. M. Myers and D. Julius (1991). "Primary structure and functional expression of the 5HT3 receptor, a serotonin- gated ion channel." Science 254(5030): 432-437.
Masse, N. Y., G. C. Turner and G. S. Jefferis (2009). "Olfactory information processing in Drosophila." Curr Biol 19(16): R700-713.
Matsutani, S. and N. Yamamoto (2008). "Centrifugal innervation of the mammalian olfactory bulb." Anat Sci Int 83(4): 218-227.
McCormick, D. A. (1992). "Neurotransmitter actions in the thalamus and cerebral cortex." J Clin Neurophysiol 9(2): 212-223.
McCormick, D. A. and T. Bal (1994). "Sensory gating mechanisms of the thalamus." Curr Opin Neurobiol 4(4): 550-556.
McCormick, D. A. and H. C. Pape (1988). "Acetylcholine inhibits identified interneurons in the cat lateral geniculate nucleus." Nature 334(6179): 246-248.
McCormick, D. A. and H. C. Pape (1990). "Noradrenergic and serotonergic modulation of a hyperpolarization-activated cation current in thalamic relay neurones." J Physiol 431: 319-342.
McCormick, D. A., H. C. Pape and A. Williamson (1991). "Actions of norepinephrine in the cerebral cortex and thalamus: implications for function of the central noradrenergic system." Prog Brain Res 88: 293-305.
McCormick, D. A. and D. A. Prince (1986). "Acetylcholine induces burst firing in thalamic reticular neurones by activating a potassium conductance." Nature 319(6052): 402-405.
McCormick, D. A. and Z. Wang (1991). "Serotonin and noradrenaline excite GABAergic neurones of the guinea-pig and cat nucleus reticularis thalami." J Physiol 442: 235-255.
McLean, J. H., A. Darby-King and C. W. Harley (2005). "Potentiation and prolongation of long-term odor memory in neonate rats using a phosphodiesterase inhibitor." Neuroscience 135(2): 329-334.
McLean, J. H., A. Darby-King and E. Hodge (1996). "5-HT2 receptor involvement in conditioned olfactory learning in the neonate rat pup." Behav Neurosci 110(6): 1426-1434.
204 McLean, J. H., A. Darby-King and G. D. Paterno (1995). "Localization of 5-HT2A receptor mRNA by in situ hybridization in the olfactory bulb of the postnatal rat." J Comp Neurol 353(3): 371-378.
McLean, J. H., A. Darby-King, R. M. Sullivan and S. R. King (1993). "Serotonergic influence on olfactory learning in the neonate rat." Behav Neural Biol 60(2): 152-162.
McLean, J. H., C. W. Harley, A. Darby-King and Q. Yuan (1999). "pCREB in the neonate rat olfactory bulb is selectively and transiently increased by odor preference-conditioned training." Learn Mem 6(6): 608-618.
McLean, J. H. and M. T. Shipley (1987). "Serotonergic afferents to the rat olfactory bulb: I. Origins and laminar specificity of serotonergic inputs in the adult rat." J Neurosci 7(10): 3016-3028.
McLean, J. H. and M. T. Shipley (1987). "Serotonergic afferents to the rat olfactory bulb: II. Changes in fiber distribution during development." J Neurosci 7(10): 3029-3039.
Meisami, E. and L. Safari (1981). "A quantitative study of the effects of early unilateral olfactory deprivation on the number and distribution of mitral and tufted cells and of glomeruli in the rat olfactory bulb." Brain Res 221(1): 81-107.
Meister, M. and T. Bonhoeffer (2001). "Tuning and topography in an odor map on the rat olfactory bulb." J Neurosci 21(4): 1351-1360.
Mercer, A. R., J. H. Hayashi and J. G. Hildebrand (1995). "Modulatory effects of 5-hydroxytryptamine on voltage-activated currents in cultured antennal lobe neurones of the sphinx moth Manduca sexta." J Exp Biol 198(Pt 3): 613-627.
Mercer, A. R., P. Kloppenburg and J. G. Hildebrand (1996). "Serotonin-induced changes in the excitability of cultured antennal-lobe neurons of the sphinx moth Manduca sexta." J Comp Physiol A 178(1): 21-31.
Mitchell, E. S. and J. F. Neumaier (2008). "5-HT6 receptor antagonist reversal of emotional learning and prepulse inhibition deficits induced by apomorphine or scopolamine." Pharmacol Biochem Behav 88(3): 291-298.
Mombaerts, P. (1999). "Seven-transmembrane proteins as odorant and chemosensory receptors." Science 286(5440): 707-711.
Mombaerts, P., F. Wang, C. Dulac, S. K. Chao, A. Nemes, M. Mendelsohn, J. Edmondson and R. Axel (1996). "Visualizing an olfactory sensory map." Cell 87(4): 675-686.
Monckton, J. E. and D. A. McCormick (2002). "Neuromodulatory role of serotonin in the ferret thalamus." J Neurophysiol 87(4): 2124-2136.
205 Moore, R. Y., A. E. Halaris and B. E. Jones (1978). "Serotonin neurons of the midbrain raphe: ascending projections." J Comp Neurol 180(3): 417-438.
Mori, K., H. Nagao and Y. Yoshihara (1999). "The olfactory bulb: coding and processing of odor molecule information." Science 286(5440): 711-715.
Moriceau, S. and R. M. Sullivan (2004). "Unique neural circuitry for neonatal olfactory learning." J Neurosci 24(5): 1182-1189.
Moriceau, S., D. A. Wilson, S. Levine and R. M. Sullivan (2006). "Dual circuitry for odor-shock conditioning during infancy: corticosterone switches between fear and attraction via amygdala." J Neurosci 26(25): 6737-6748.
Morris, R. G., P. Garrud, J. N. Rawlins and J. O'Keefe (1982). "Place navigation impaired in rats with hippocampal lesions." Nature 297(5868): 681-683.
Murphy, G. J., D. P. Darcy and J. S. Isaacson (2005). "Intraglomerular inhibition: signaling mechanisms of an olfactory microcircuit." Nat Neurosci 8(3): 354-364.
Mutoh, H., Q. Yuan and T. Knopfel (2005). "Long-term depression at olfactory nerve synapses." J Neurosci 25(17): 4252-4259.
Najac, M., D. De Saint Jan, L. Reguero, P. Grandes and S. Charpak (2011). "Monosynaptic and polysynaptic feed-forward inputs to mitral cells from olfactory sensory neurons." J Neurosci 31(24): 8722-8729.
Nassel, D. R. (2002). "Neuropeptides in the nervous system of Drosophila and other insects: multiple roles as neuromodulators and neurohormones." Prog Neurobiol 68(1): 1-84.
Neville, K. R. and L. B. Haberly (2003). "Beta and gamma oscillations in the olfactory system of the urethane-anesthetized rat." J Neurophysiol 90(6): 3921- 3930.
Nguyen, M. Q., Z. Zhou, C. A. Marks, N. J. Ryba and L. Belluscio (2007). "Prominent roles for odorant receptor coding sequences in allelic exclusion." Cell 131(5): 1009-1017.
Nicoll, R. A., J. A. Kauer and R. C. Malenka (1988). "The current excitement in long-term potentiation." Neuron 1(2): 97-103.
Nicoll, R. A., S. Tomita and D. S. Bredt (2006). "Auxiliary subunits assist AMPA- type glutamate receptors." Science 311(5765): 1253-1256.
Nuttley, W. M., K. P. Atkinson-Leadbeater and D. Van Der Kooy (2002). "Serotonin mediates food-odor associative learning in the nematode Caenorhabditiselegans." Proc Natl Acad Sci U S A 99(19): 12449-12454.
206 O'Neill, M. F., C. L. Heron-Maxwell and G. Shaw (1999). "5-HT2 receptor antagonism reduces hyperactivity induced by amphetamine, cocaine, and MK- 801 but not D1 agonist C-APB." Pharmacol Biochem Behav 63(2): 237-243.
Padich, R. A., T. C. McCloskey and J. H. Kehne (1996). "5-HT modulation of auditory and visual sensorimotor gating: II. Effects of the 5-HT2A antagonist MDL 100,907 on disruption of sound and light prepulse inhibition produced by 5-HT agonists in Wistar rats." Psychopharmacology (Berl) 124(1-2): 107-116.
Pape, H. C. and D. A. McCormick (1989). "Noradrenaline and serotonin selectively modulate thalamic burst firing by enhancing a hyperpolarization- activated cation current." Nature 340(6236): 715-718.
Pavlov, I. P. and G. V. Anrep (1927). Conditioned reflexes; an investigation of the physiological activity of the cerebral cortex. London, Oxford University Press: Humphrey Milford.
Pazos, A., R. Cortes and J. M. Palacios (1985). "Quantitative autoradiographic mapping of serotonin receptors in the rat brain. II. Serotonin-2 receptors." Brain Res 346(2): 231-249.
Pazos, A. and J. M. Palacios (1985). "Quantitative autoradiographic mapping of serotonin receptors in the rat brain. I. Serotonin-1 receptors." Brain Res 346(2): 205-230.
Peddie, C. J., H. A. Davies, F. M. Colyer, M. G. Stewart and J. J. Rodriguez (2008). "Colocalisation of serotonin2A receptors with the glutamate receptor subunits NR1 and GluR2 in the dentate gyrus: an ultrastructural study of a modulatory role." Exp Neurol 211(2): 561-573.
Perk, C. G. and A. R. Mercer (2006). "Dopamine modulation of honey bee (Apis mellifera) antennal-lobe neurons." J Neurophysiol 95(2): 1147-1157.
Petzold, G. C., D. F. Albeanu, T. F. Sato and V. N. Murthy (2008). "Coupling of neural activity to blood flow in olfactory glomeruli is mediated by astrocytic pathways." Neuron 58(6): 897-910.
Petzold, G. C., A. Hagiwara and V. N. Murthy (2009). "Serotonergic modulation of odor input to the mammalian olfactory bulb." Nat Neurosci.
Phillips, C. G., T. P. Powell and G. M. Shepherd (1963). "Responses Of Mitral Cells To Stimulation Of The Lateral Olfactory Tract In The Rabbit." J Physiol 168: 65-88.
Pinault, D. (2004). "The thalamic reticular nucleus: structure, function and concept." Brain Res Brain Res Rev 46(1): 1-31.
Ping, Y. and S. Tsunoda (2012). "Inactivity-induced increase in nAChRs upregulates Shal K(+) channels to stabilize synaptic potentials." Nat Neurosci 15(1): 90-97.
207 Pompeiano, M., J. M. Palacios and G. Mengod (1992). "Distribution and cellular localization of mRNA coding for 5-HT1A receptor in the rat brain: correlation with receptor binding." J Neurosci 12(2): 440-453.
Pompeiano, M., J. M. Palacios and G. Mengod (1994). "Distribution of the serotonin 5-HT2 receptor family mRNAs: comparison between 5-HT2A and 5- HT2C receptors." Brain Res Mol Brain Res 23(1-2): 163-178.
Potter, S. M., C. Zheng, D. S. Koos, P. Feinstein, S. E. Fraser and P. Mombaerts (2001). "Structure and emergence of specific olfactory glomeruli in the mouse." J Neurosci 21(24): 9713-9723.
Price, J. L. and T. P. Powell (1970). "An electron-microscopic study of the termination of the afferent fibres to the olfactory bulb from the cerebral hemisphere." J Cell Sci 7(1): 157-187.
Price, J. L. and T. P. Powell (1970). "An experimental study of the origin and the course of the centrifugal fibres to the olfactory bulb in the rat." J Anat 107(Pt 2): 215-237.
Price, J. L. and T. P. Powell (1970). "The synaptology of the granule cells of the olfactory bulb." J Cell Sci 7(1): 125-155.
Price, T. L., A. Darby-King, C. W. Harley and J. H. McLean (1998). "Serotonin plays a permissive role in conditioned olfactory learning induced by norepinephrine in the neonate rat." Behav Neurosci 112(6): 1430-1437.
Quednow, B. B., K. U. Kuhn, R. Mossner, S. G. Schwab, A. Schuhmacher, W. Maier and M. Wagner (2008). "Sensorimotor gating of schizophrenia patients is influenced by 5-HT2A receptor polymorphisms." Biol Psychiatry 64(5): 434-437.
Quinn, W. G., W. A. Harris and S. Benzer (1974). "Conditioned behavior in Drosophila melanogaster." Proc Natl Acad Sci U S A 71(3): 708-712.
Riemensperger, T., G. Isabel, H. Coulom, K. Neuser, L. Seugnet, K. Kume, M. Iche-Torres, M. Cassar, R. Strauss, T. Preat, J. Hirsh and S. Birman (2011). "Behavioral consequences of dopamine deficiency in the Drosophila central nervous system." Proc Natl Acad Sci U S A 108(2): 834-839.
Roeder, T. (2005). "Tyramine and octopamine: ruling behavior and metabolism." Annu Rev Entomol 50: 447-477.
Rubin, B. D. and L. C. Katz (1999). "Optical imaging of odorant representations in the mammalian olfactory bulb." Neuron 23(3): 499-511.
Saar, D., Y. Grossman and E. Barkai (1998). "Reduced after-hyperpolarization in rat piriform cortex pyramidal neurons is associated with increased learning capability during operant conditioning." Eur J Neurosci 10(4): 1518-1523.
208 Saar, D., Y. Grossman and E. Barkai (2001). "Long-lasting cholinergic modulation underlies rule learning in rats." J Neurosci 21(4): 1385-1392.
Saiz, P. A., M. P. Garcia-Portilla, C. Arango, B. Morales, V. Alvarez, E. Coto, J. M. Fernandez, M. T. Bascaran, M. Bousono and J. Bobes (2007). "Association study of serotonin 2A receptor (5-HT2A) and serotonin transporter (5-HTT) gene polymorphisms with schizophrenia." Prog Neuropsychopharmacol Biol Psychiatry 31(3): 741-745.
Sanchez-Gonzalez, M. A., M. A. Garcia-Cabezas, B. Rico and C. Cavada (2005). "The primate thalamus is a key target for brain dopamine." J Neurosci 25(26): 6076-6083.
Sawin, E. R., R. Ranganathan and H. R. Horvitz (2000). "C. elegans locomotory rate is modulated by the environment through a dopaminergic pathway and by experience through a serotonergic pathway." Neuron 26(3): 619-631.
Schindler, C. W., I. Gormezano and J. A. Harvey (1983). "Effect of morphine on acquisition of the classically conditioned nictitating membrane response of the rabbit." J Pharmacol Exp Ther 227(3): 639-643.
Schoppa, N. E. and G. L. Westbrook (1997). "Modulation of mEPSCs in olfactory bulb mitral cells by metabotropic glutamate receptors." J Neurophysiol 78(3): 1468-1475.
Schulz, D. J. and G. E. Robinson (1999). "Biogenic amines and division of labor in honey bee colonies: behaviorally related changes in the antennal lobes and age-related changes in the mushroom bodies." J Comp Physiol A 184(5): 481- 488.
Schwaerzel, M., M. Monastirioti, H. Scholz, F. Friggi-Grelin, S. Birman and M. Heisenberg (2003). "Dopamine and octopamine differentiate between aversive and appetitive olfactory memories in Drosophila." J Neurosci 23(33): 10495- 10502.
Seki, Y., J. Rybak, D. Wicher, S. Sachse and B. S. Hansson (2010). "Physiological and morphological characterization of local interneurons in the Drosophila antennal lobe." J Neurophysiol 104(2): 1007-1019.
Shao, Z., A. C. Puche, E. Kiyokage, G. Szabo and M. T. Shipley (2009). "Two GABAergic intraglomerular circuits differentially regulate tonic and phasic presynaptic inhibition of olfactory nerve terminals." J Neurophysiol 101(4): 1988- 2001.
Shepherd, G. M. (1972). "Synaptic organization of the mammalian olfactory bulb." Physiol Rev 52(4): 864-917.
Shepherd, G. M. (2005). "Perception without a thalamus how does olfaction do it?" Neuron 46(2): 166-168.
209 Shepherd, G. M., W. R. Chen and C. A. Greer (2004). The Olfactory Bulb. The synaptic organization of the brain. G. M. Shepherd. Oxford, Oxford University Press: 165-216.
Sherman, M. S. and R. W. Guillery (2004). The Thalamus. The synaptic organization of the brain (5th Ed.). G. M. Shepherd. Oxford, Oxford University Press: 331-360.
Sherman, S. M. and C. Koch (1986). "The control of retinogeniculate transmission in the mammalian lateral geniculate nucleus." Exp Brain Res 63(1): 1-20.
Shi, S. H., Y. Hayashi, R. S. Petralia, S. H. Zaman, R. J. Wenthold, K. Svoboda and R. Malinow (1999). "Rapid spine delivery and redistribution of AMPA receptors after synaptic NMDA receptor activation." Science 284(5421): 1811- 1816.
Sigg, D., C. M. Thompson and A. R. Mercer (1997). "Activity-dependent changes to the brain and behavior of the honey bee, Apis mellifera (L.)." J Neurosci 17(18): 7148-7156.
Sigurdsson, T., V. Doyere, C. K. Cain and J. E. LeDoux (2007). "Long-term potentiation in the amygdala: a cellular mechanism of fear learning and memory." Neuropharmacology 52(1): 215-227.
Silva, A. J., R. Paylor, J. M. Wehner and S. Tonegawa (1992). "Impaired spatial learning in alpha-calcium-calmodulin kinase II mutant mice." Science 257(5067): 206-211.
Sinakevitch, I., J. A. Mustard and B. H. Smith (2011). "Distribution of the octopamine receptor AmOA1 in the honey bee brain." PLoS One 6(1): e14536.
Skinner, B. F. (1938). The behavior of organisms; an experimental analysis. New York, London,, D. Appleton-Century Company.
Smear, M., R. Shusterman, R. O'Connor, T. Bozza and D. Rinberg (2011). "Perception of sniff phase in mouse olfaction." Nature 479(7373): 397-400.
Smith, R. L., H. Baker and C. A. Greer (1993). "Immunohistochemical analyses of the human olfactory bulb." J Comp Neurol 333(4): 519-530.
Soucy, E. R., D. F. Albeanu, A. L. Fantana, V. N. Murthy and M. Meister (2009). "Precision and diversity in an odor map on the olfactory bulb." Nat Neurosci 12(2): 210-220.
Spenger, C., C. Hyman, L. Studer, M. Egli, L. Evtouchenko, C. Jackson, A. Dahl- Jorgensen, R. M. Lindsay and R. W. Seiler (1995). "Effects of BDNF on dopaminergic, serotonergic, and GABAergic neurons in cultures of human fetal ventral mesencephalon." Exp Neurol 133(1): 50-63.
210 Steinbusch, H. W. (1981). "Distribution of serotonin-immunoreactivity in the central nervous system of the rat-cell bodies and terminals." Neuroscience 6(4): 557-618.
Strausfeld, N. J. and J. G. Hildebrand (1999). "Olfactory systems: common design, uncommon origins?" Curr Opin Neurobiol 9(5): 634-639.
Strowbridge, B. W. (2009). "Role of cortical feedback in regulating inhibitory microcircuits." Ann N Y Acad Sci 1170: 270-274.
Stuber, G. D., D. R. Sparta, A. M. Stamatakis, W. A. van Leeuwen, J. E. Hardjoprajitno, S. Cho, K. M. Tye, K. A. Kempadoo, F. Zhang, K. Deisseroth and A. Bonci (2011). "Excitatory transmission from the amygdala to nucleus accumbens facilitates reward seeking." Nature 475(7356): 377-380.
Sullivan, R. M., G. Stackenwalt, F. Nasr, C. Lemon and D. A. Wilson (2000). "Association of an odor with activation of olfactory bulb noradrenergic beta- receptors or locus coeruleus stimulation is sufficient to produce learned approach responses to that odor in neonatal rats." Behav Neurosci 114(5): 957-962.
Sullivan, R. M. and D. A. Wilson (1995). "Dissociation of behavioral and neural correlates of early associative learning." Dev Psychobiol 28(4): 213-219.
Sullivan, R. M., D. A. Wilson, C. Lemon and G. A. Gerhardt (1994). "Bilateral 6- OHDA lesions of the locus coeruleus impair associative olfactory learning in newborn rats." Brain Res 643(1-2): 306-309.
Sullivan, R. M., D. A. Wilson and M. Leon (1989). "Norepinephrine and learning- induced plasticity in infant rat olfactory system." J Neurosci 9(11): 3998-4006.
Sullivan, R. M., D. R. Zyzak, P. Skierkowski and D. A. Wilson (1992). "The role of olfactory bulb norepinephrine in early olfactory learning." Brain Res Dev Brain Res 70(2): 279-282.
Sun, X. J., L. P. Tolbert and J. G. Hildebrand (1993). "Ramification pattern and ultrastructural characteristics of the serotonin-immunoreactive neuron in the antennal lobe of the moth Manduca sexta: a laser scanning confocal and electron microscopic study." J Comp Neurol 338(1): 5-16.
Swerdlow, N. R. and M. A. Geyer (1998). "Using an animal model of deficient sensorimotor gating to study the pathophysiology and new treatments of schizophrenia." Schizophr Bull 24(2): 285-301.
Takeuchi, Y., H. Kimura and Y. Sano (1982). "Immunohistochemical demonstration of serotonin nerve fibers in the olfactory bulb of the rat, cat and monkey." Histochemistry 75(4): 461-471.
Tamura, T., D. Horiuchi, Y. C. Chen, M. Sone, T. Miyashita, M. Saitoe, N. Yoshimura, A. S. Chiang and H. Okazawa (2010). "Drosophila PQBP1 regulates
211 learning acquisition at projection neurons in aversive olfactory conditioning." J Neurosci 30(42): 14091-14101.
Tecott, L. H., A. V. Maricq and D. Julius (1993). "Nervous system distribution of the serotonin 5-HT3 receptor mRNA." Proc Natl Acad Sci U S A 90(4): 1430- 1434.
Tessier, C. R. and K. Broadie (2009). "Activity-dependent modulation of neural circuit synaptic connectivity." Front Mol Neurosci 2: 8.
Treloar, H. B., P. Feinstein, P. Mombaerts and C. A. Greer (2002). "Specificity of glomerular targeting by olfactory sensory axons." J Neurosci 22(7): 2469-2477.
Tsai, H. C., F. Zhang, A. Adamantidis, G. D. Stuber, A. Bonci, L. de Lecea and K. Deisseroth (2009). "Phasic firing in dopaminergic neurons is sufficient for behavioral conditioning." Science 324(5930): 1080-1084.
Tsien, J. Z., P. T. Huerta and S. Tonegawa (1996). "The essential role of hippocampal CA1 NMDA receptor-dependent synaptic plasticity in spatial memory." Cell 87(7): 1327-1338.
Turrigiano, G. G. and S. B. Nelson (2000). "Hebb and homeostasis in neuronal plasticity." Curr Opin Neurobiol 10(3): 358-364.
Tye, K. M., G. D. Stuber, B. de Ridder, A. Bonci and P. H. Janak (2008). "Rapid strengthening of thalamo-amygdala synapses mediates cue-reward learning." Nature 453(7199): 1253-1257.
Tyler, W. J., G. C. Petzold, S. K. Pal and V. N. Murthy (2007). "Experience- dependent modification of primary sensory synapses in the mammalian olfactory bulb." J Neurosci 27(35): 9427-9438.
Valle-Leija, P., E. Blanco-Hernandez, R. Drucker-Colin, G. Gutierrez-Ospina and R. Vidaltamayo (2012). "Supernumerary formation of olfactory glomeruli induced by chronic odorant exposure: a constructivist expression of neural plasticity." PLoS One 7(4): e35358.
Valverde, F. and L. Lopez-Mascaraque (1991). "Neuroglial arrangements in the olfactory glomeruli of the hedgehog." J Comp Neurol 307(4): 658-674.
Varty, G. B., V. P. Bakshi and M. A. Geyer (1999). "M100907, a serotonin 5- HT2A receptor antagonist and putative antipsychotic, blocks dizocilpine-induced prepulse inhibition deficits in Sprague-Dawley and Wistar rats." Neuropsychopharmacology 20(4): 311-321. von Krosigk, M., T. Bal and D. A. McCormick (1993). "Cellular mechanisms of a synchronized oscillation in the thalamus." Science 261(5119): 361-364.
Vosshall, L. B., A. M. Wong and R. Axel (2000). "An olfactory sensory map in the fly brain." Cell 102(2): 147-159.
212 Wachowiak, M. and L. B. Cohen (2001). "Representation of odorants by receptor neuron input to the mouse olfactory bulb." Neuron 32(4): 723-735.
Wachowiak, M., J. P. McGann, P. M. Heyward, Z. Shao, A. C. Puche and M. T. Shipley (2005). "Inhibition [corrected] of olfactory receptor neuron input to olfactory bulb glomeruli mediated by suppression of presynaptic calcium influx." J Neurophysiol 94(4): 2700-2712.
Wang, J. W., A. M. Wong, J. Flores, L. B. Vosshall and R. Axel (2003). "Two- photon calcium imaging reveals an odor-evoked map of activity in the fly brain." Cell 112(2): 271-282.
Wei, C. J., C. Linster and T. A. Cleland (2006). "Dopamine D(2) receptor activation modulates perceived odor intensity." Behav Neurosci 120(2): 393-400.
Wilson, D. A., A. R. Best and R. M. Sullivan (2004). "Plasticity in the olfactory system: lessons for the neurobiology of memory." Neuroscientist 10(6): 513-524.
Wilson, D. A., M. L. Fletcher and R. M. Sullivan (2004). "Acetylcholine and olfactory perceptual learning." Learn Mem 11(1): 28-34.
Wilson, D. A., K. M. Guthrie and M. Leon (1990). "Modification of olfactory bulb synaptic inhibition by early unilateral olfactory deprivation." Neurosci Lett 116(3): 250-256.
Wilson, D. A. and M. Leon (1988). "Spatial patterns of olfactory bulb single-unit responses to learned olfactory cues in young rats." J Neurophysiol 59(6): 1770- 1782.
Wilson, D. A. and C. Linster (2008). "Neurobiology of a simple memory." J Neurophysiol 100(1): 2-7.
Wilson, D. A., T. C. Pham and R. M. Sullivan (1994). "Norepinephrine and posttraining memory consolidation in neonatal rats." Behav Neurosci 108(6): 1053-1058.
Wilson, D. A. and R. M. Sullivan (1994). "Neurobiology of associative learning in the neonate: early olfactory learning." Behav Neural Biol 61(1): 1-18.
Wilson, D. A. and R. M. Sullivan (1995). "The D2 antagonist spiperone mimics the effects of olfactory deprivation on mitral/tufted cell odor response patterns." J Neurosci 15(8): 5574-5581.
Wilson, D. A., R. M. Sullivan and M. Leon (1985). "Odor familiarity alters mitral cell response in the olfactory bulb of neonatal rats." Brain Res 354(2): 314-317.
Wilson, D. A., R. M. Sullivan and M. Leon (1987). "Single-unit analysis of postnatal olfactory learning: modified olfactory bulb output response patterns to learned attractive odors." J Neurosci 7(10): 3154-3162.
213 Wilson, D. A. and J. G. Wood (1992). "Functional consequences of unilateral olfactory deprivation: time-course and age sensitivity." Neuroscience 49(1): 183- 192.
Wilson, R. I. (2008). "Neural and behavioral mechanisms of olfactory perception." Curr Opin Neurobiol 18(4): 408-412.
Wilson, R. I. and G. Laurent (2005). "Role of GABAergic inhibition in shaping odor-evoked spatiotemporal patterns in the Drosophila antennal lobe." J Neurosci 25(40): 9069-9079.
Wilson, R. I., G. C. Turner and G. Laurent (2004). "Transformation of olfactory representations in the Drosophila antennal lobe." Science 303(5656): 366-370.
Woo, C. C., E. E. Hingco, B. A. Johnson and M. Leon (2007). "Broad activation of the glomerular layer enhances subsequent olfactory responses." Chem Senses 32(1): 51-55.
Woo, C. C. and M. Leon (1995). "Distribution and development of beta- adrenergic receptors in the rat olfactory bulb." J Comp Neurol 352(1): 1-10.
Yokoi, M., K. Mori and S. Nakanishi (1995). "Refinement of odor molecule tuning by dendrodendritic synaptic inhibition in the olfactory bulb." Proc Natl Acad Sci U S A 92(8): 3371-3375.
Yu, D., A. Ponomarev and R. L. Davis (2004). "Altered representation of the spatial code for odors after olfactory classical conditioning; memory trace formation by synaptic recruitment." Neuron 42(3): 437-449.
Yuan, Q. (2009). "Theta bursts in the olfactory nerve paired with beta- adrenoceptor activation induce calcium elevation in mitral cells: a mechanism for odor preference learning in the neonate rat." Learn Mem 16(11): 676-681.
Yuan, Q. and C. W. Harley (2012). "What a nostril knows: olfactory nerve-evoked AMPA responses increase while NMDA responses decrease at 24-h post- training for lateralized odor preference memory in neonate rat." Learn Mem 19(2): 50-53.
Yuan, Q., C. W. Harley, J. C. Bruce, A. Darby-King and J. H. McLean (2000). "Isoproterenol increases CREB phosphorylation and olfactory nerve-evoked potentials in normal and 5-HT-depleted olfactory bulbs in rat pups only at doses that produce odor preference learning." Learn Mem 7(6): 413-421.
Yuan, Q., C. W. Harley, A. Darby-King, R. L. Neve and J. H. McLean (2003). "Early odor preference learning in the rat: bidirectional effects of cAMP response element-binding protein (CREB) and mutant CREB support a causal role for phosphorylated CREB." J Neurosci 23(11): 4760-4765.
214 Yuan, Q., C. W. Harley and J. H. McLean (2003). "Mitral cell beta1 and 5-HT2A receptor colocalization and cAMP coregulation: a new model of norepinephrine- induced learning in the olfactory bulb." Learn Mem 10(1): 5-15.
Yuan, Q., H. Mutoh, F. Debarbieux and T. Knopfel (2004). "Calcium signaling in mitral cell dendrites of olfactory bulbs of neonatal rats and mice during olfactory nerve Stimulation and beta-adrenoceptor activation." Learn Mem 11(4): 406-411.
Zhang, Y., H. Lu and C. I. Bargmann (2005). "Pathogenic bacteria induce aversive olfactory learning in Caenorhabditis elegans." Nature 438(7065): 179- 184.
Zheng, C., P. Feinstein, T. Bozza, I. Rodriguez and P. Mombaerts (2000). "Peripheral olfactory projections are differentially affected in mice deficient in a cyclic nucleotide-gated channel subunit." Neuron 26(1): 81-91.
Zou, D. J., P. Feinstein, A. L. Rivers, G. A. Mathews, A. Kim, C. A. Greer, P. Mombaerts and S. Firestein (2004). "Postnatal refinement of peripheral olfactory projections." Science 304(5679): 1976-1979.
215 APPENDIX A
INSTITUTIONAL APPROVAL FOR THE HUMANE USE OF ANIMALS
216
217