Dopaminergic Control of Hippocampal Neural Circuitry Zev B. Rosen

Dopaminergic control of hippocampal neural circuitry

Zev B. Rosen

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy under the Executive Committee of the Graduate School of Arts and Sciences

COLUMBIA UNIVERSITY

2013

Abstract

Dopaminergic control of hippocampal circuitry

Zev B. Rosen

Memory is a limited resource. Therefore, the circuitry that encodes memory must filter incoming information in accordance with its perceived value. The hippocampus, the hub of the declarative memory system, may achieve memory valuation using its rich variety of neuromodulatory afferent systems.

The dopamine (DA) neurons in the ventral tegmental area (VTA) and susbtantia nigra pars compacta (SNpC) are in a particularly strategic position to aid the hippocampus in gating long-term memory. Their firing rates encode the salience of external cues in the environment and they send axons to the output node of the hippocampus, area CA1. In CA1, exogenous receptor stimulation with DA receptor agonists and antagonists suggests an important role for VTA/SNpC DA in learning and memory as the DA receptors powerfully modulate synaptic transmission, permit LTP induction, and enhance different forms of spatial memory.

However, it remains unknown whether the VTA/SNpC DAergic axons are capable of activating those receptors and triggering the effects on

hippocampal physiology. The VTA/SNpC innervation density in the hippocampus is modest and, in many cases, the axons are distant from the neurons exhibiting the effects. Other sources of DA could couple to those receptors, such as the locus coeruleus, which also releases DA in the CA1 area.

To investigate the VTA/SNpC’s DAergic influence, I took a circuit- based approach and selectively evoked DA release from the VTA/SNpC

DAergic afferents in CA1 in vitro with different patterns of optogenetically guided stimulation. I found that DA release directly modulates the CA3

Schaffer collateral (SC) synaptic excitation of CA1 in a bidirectional manner.

A single light-burst (three 5-ms-long pulses at 66 Hz) suppresses the SC- evoked PSP in CA1 pyramidal neurons (PNs) through a D2-receptor dependent enhancement of parvalbumin-positive interneuron mediated feedforward inhibition. More prolonged DA release using 25 light-bursts (at 1

Hz) increases the SC PSP through a D1-type receptor dependent direct presynaptic effect on excitatory transmission.

Thus, I propose the following model for how VTA/SNpC DAergic afferents effect oppositional synaptic states to influence learning in the hippocampus in accordance with motivational demands. During tonic DA release, the D4 receptors become activated, globally weaken the SC synaptic input to CA1 PNs, and increase plasticity thresholds. In contrast, phasic DA

release activates D1-type receptors, and transitions the SC synapse to a more efficacious state, during which weaker inputs can drive potentiation.

Contents

List of Figures ...... iii

List of Abbreviations ...... v

Acknowledgements ...... vii

Chapter 1

Two orthogonal networks ...... 1

Memory systems ...... 4

Functional neuroanatomy of the hippocampus ...... 7

Memory as a limited resource ...... 13

VTA/SNpC DA signals in striatum ...... 15

VTA/SNpC DA signals in hippocampus ...... 19

DA in the hippocampus: insight from anatomy ...... 21

VTA/SNpC DAergic input to CA1: function ...... 25

VTA/SNpC DAergic input to CA1: synaptic transmission ...... 26

VTA/SNpC DAergic input to CA1: synaptic plasticity ...... 32

VTA/SNpC DAergic input to CA1: memory ...... 35

Aims of this thesis ...... 38

Chapter 2

Introduction ...... 46

Results ...... 54

Discussion ...... 71

Chapter 3

Introduction ...... 79

Results ...... 86

Discussion ...... 111

Chapter 4

Introduction ...... 116

Results ...... 118

Discussion ...... 133

Chapter 5

Conclusion ...... 140

Chapter 6

Methods ...... 147

References ...... 154

List of Figures

1.1 Memory taxonomy...... 9

1.2 Trisynaptic loop ...... 12

1.3 VTA DAergic innervation of the hippocampus...... 27

2.1 Effect of DA on synaptic transmission in the hippocampus . . 49

2.2 ChR2 guided spikes...... 54

2.3 DA bath application effects ...... 56

2.4 ChR2 expresses in DA neurons ...... 60

2.5 VTA DA neurons project to CA1...... 61

2.6 Effects of VTA photostimulation on membrane properties. . . . 63

2.7 Effects of VTA photostimulation on synaptic excitability. . . . . 65

2.8 Pharmacological analysis of photostimulation effects ...... 69

3.1 Photostimulation effect requirea inhibition...... 87

3.2 Photostimulation enhances feedforward inhibition...... 92

3.3 DAMGO abolishes the photostimulation effect...... 95

3.4 Strategy for silencing PV+ interneuron population...... 99

3.5 PSEM expresses and silences the PV+ interneuron population. 100

3.6 PV+ interneuron silencing reverses the photostimulation effect 102

3.7 Strategy for targeted recordings from the PV+ interneurons . . . 105

3.8 TdTomato expressing neurons are PV+ interneurons ...... 106

3.9 Photostimulation enhances SC input to PV+ interneurons...... 109

4.1 Prolonged photostimulation enhances the SC PSP ...... 121

4.2 Enhancement effect requires D1 receptors...... 123

4.3 Pharmacological analysis of enhancement effect...... 128

iii

4.4 Enhancement effect occurs presynaptically...... 130

4.5 DA bath application increases SC PSP with inhibition blocked. . .132

List of abbreviations

Dopamine (DA)

Midbrain dopamine neuron (MDN)

Dentate gyrus (DG)

Cornu ammonis 3 (CA3)

Cornu ammonis 2 (CA2)

Cornu ammonis 1 (CA1)

Entorhinal cortex (EC)

Schaffer collaterals (SC)

Perforant path (PP)

Long-term potentiation (LTP)

Early long-term potentiation (E-LTP)

Late long-term potentiation (L-LTP)

Long-term depression (LTD)

Norepinephrine (NE)

Ventral tegmental area (VTA)

Substantia Nigra pars Compacta (SNpC)

Retrorubral field (RRF)

Resting membrane potential (Vm)

Tyrosine hydroxylase (TH)

Pyramidal neuron (PN)

Interneuron (IN)

Paired pulse facilitation (PPF)

N-Methyl-D-aspartate (NMDA)

Acknowledgements

Science is not a solitary endeavor. I want to give thanks to everyone that supported this process. The incredibly supportive Siegelbaum lab members both past and present were instrumental. John Riley and Eric

Odell, the lab managers— without your support there would be no science. I want to thank Rebecca Piskorowski, Vivien Chevaleyre, Kevin Franks, and

Hu Lei for being cruel to my traces and making me a better physiologist.

Frederick Hitti, Marco Russo, Justine Kupferman for the help with imaging.

Pablo Ariel, Qian Sun, Bina Santoro, Yating Lei, Kalyan Vadurri: thank you for your technical advice. Jayeeta Basu, you took me in, you sparked my amplifier, and guided my manipulator, I owe you everything.

My committee, Gyorgy Buzsaki, Eric Kandel, Attila Losonczy, and

Dave Sulzer: thank you for believing in my research and making me a more thoughtful and rigorous scientist. I also want to acknowledge the animals whose sacrifice to science I hope both increases knowledge and reduces suffering in this world.

I want to thank my cousins: the Singers, Ellen, Scott, Josh, Ayelet, and Shira. Yes, I could have done this without you, but I would have been miserable. To my siblings Kayla, Avi, Ezra, Jessica: you have been an

vii

undying source of joy, encouragement and fortitude. You’re the best.

Mom, Abba, what can I say? Thank you for instilling in me the passion for knowledge that drove those eternal nights at the rig and for bringing the delicious food that sustained them. My mentor Steve, I can only hope that some day I can cultivate as fertile a ground for scientific exploration as you have created here. And God, to whom I owe all.

viii 1

Chapter 1

Introduction

Two orthogonal networks

The prophetic and prolific anatomist Santiago Ramon y Cajal postulated from histological analysis of neural tissue that the brain consists of discrete elements called neurons and that these neurons communicate with one another across their encapsulating membranes (Cajal, 1911). Sherrington eventually named these communicative junctions between two neural membranes “synapses” from the Greek “syn-”, meaning “together”, and

“haptein” meaning “to cling” (Sherrington, 1906). Cajal posited that information flows unidirectionally from dendrite to axon and, at the axon, transmits the signal across the synapse to the dendrite of the downstream neuron. And so it goes from sensation to action.

This principle of dynamic polarization of the neuron doctrine paved the road for the contemporary understanding of the function of the brain. Cajal’s

2 neuron doctrine and the later electrophysiological studies of synaptic transmission by Katz and colleagues pertains most directly to the brain’s

“wired network”. This sort of fixed network could direct discrete input output relations for a wide range of sensory-motor patterns. This wired signaling system in the brain consists of a matrix of highly efficient, fast, synapses characterized by tightly coupled presynaptic and postsynaptic structures containing neurotransmitter exocytosis machinery at the axon bouton active zone and ionotropic receptors in the post-synaptic density.

In addition to this fast, wired network, there is a “slow” signaling network that acts through the release of neurotransmitters which diffuse through the neuropil (Agnati et al., 2006) and activate metabotropic G- protein coupled receptors (GPCRs). The slow system is suited for implementing motivational states—brain states in which an inordinate propensity to seek or avoid a class of objects develops. Hunger, for example refers to the brain state in which the organism seeks food to provide caloric content to the organism, and is implemented as attentional preferences to food related stimuli, negative affect, stomach discomfort, and eating behavior.

The totality of these experiences is referred to as hunger.

The slow neuromodulatory systems are well adapted to implement motivational states as they project divergently and act at an appropriate time scale. These two systems act in concert with one another. The same neurotransmitter can act on both networks and multiple neurotransmitters

3 effecting both types of signaling can even be released from the same bouton

(Sulzer et al., 1998). The wired network is much more precise in both time and space as it directly supports millisecond-scale computation of perception and action. The slow system is spatially and temporally more coarse. This dissociation is reflected by the kinetics of their effectors; ionotropic receptors have activation and deactivation time constants in the millisecond range, whereas GPCRs vary in their activation time constants from 100s of milliseconds for direct protein-protein interactions to hours when they trigger protein synthesis via second messenger cascades (Post and Brown, 1996;

Hamm, 1998). Ultimately, it is the fast system that generates the motor output so the significance of the slow system depends on the degree to which it alters the functioning of the fast system.

Neuromodulatory nuclei that release neurotransmitters such as dopamine, serotonin, and acetylcholine are tuned to fire in response to stimuli in the world that elicit motivational states and they facilitate the consummation of the associated motor programs. Their direct actions on behavior, networks, neurons, synapses, and biochemical signaling pathways, are multifarious. If we consider the neurons in a given neuromodulatory nucleus to be homogeneous, their function will be differentiated by the recipient structures. The brain is thus endowed with two levels of architecture to effectively navigate the environment: perceptual, mnemonic, and motor systems for interpreting, storing, and acting on the current

4 environment and a motivational system that alters the “wired” systems in accordance with the changing demands of the organism and environment.

This thesis aims to elucidate one of the avenues through which motivational systems alter a central and critical component of the memory network, the hippocampus.

Memory systems

In changing environments, it is beneficial for the brain to accrue information about experience so as to optimize future behavior. To do this, the nervous systems evolved an ability to undergo activity dependent plasticity (Sherrington, 1906; Hubel et al., 1977). This extraordinary adaptation, in neurobiological terms, refers to changing the brain’s functional architecture to dynamically alter the input-output mappings between sensory and motor structures in response to new experiences.

On the macroscopic level, the brain subdivides memory into multiple categories and devotes interdependent but distinct structures (figure 1.1) to accomplish each of them. This idea had some historical antecedents in psychology and philosophy (Squire, 2004), but became situated as a biological concept with lesion studies of patients like HM (Scoville and Milner, 1957), whose medial temporal lobectomy, a successful epilepsy treatment, left him with a severe anterograde amnesia. He, and similar patients, could no longer

5 form new memories of conscious facts and events, while motor skills remained intact. These studies led researchers to functionally and anatomically dissociate declarative memory and non-declarative memory

(Milner, 1962).

Declarative memory is subserved by the hippocampus and related structures in the medial temporal lobe, nondeclarative memory by striatum and other structures. Declarative memory is further subdivided into semantic and episodic memory (Tulving, 1972). Semantic memory refers to the

Figure 1.1: Memory taxonomy. Adapted from Squire, L. R. & Knowlton, B.

J., 1994).

6 knowledge of facts (like the organization of the subdivisions of memory) that have been removed from their experienced context. Episodic memory refers to the spatiotemporal flow of experience, the movie that we can play back in our mind.

The hippocampus is involved in encoding all declarative memory, but likely only in the consolidation and retrieval of episodic memory 1. Studies of

HM and other similar patients confirmed the entrenchment of declarative memory in the medial temporal lobe. The hippocampus is the most extensively studied structure anatomically and functionally within the medial temporal lobe and its analysis has provided the most detailed insights into the mechanisms of memory.

The structure and function of the hippocampus and associated structures in the medial temporal lobe are largely conserved across mammalian evolution (Manns and Eichenbaum, 2006). In rodent and non- human primate, the general macroscopic architecture of the medial temporal lobe resembles that in humans and hippocampal lesions impair spatial

(O’Keefe & Nadel, 1978) and general episodic memory (Babb and Crystal,

2005) across mammals. Therefore, the rodent hippocampus is a relevant model for the human and has served as the focal point for research into the neurobiology of memory.

1 The role of the hippocampus in semantic memory retrieval is a matter of some debate. Semantic memories emerge from episodes in our life. It seems like during the consolidation period, semantic memories become reliant on neocortical circuitry.

Functional neuroanatomy of the hippocampus

The structure of the hippocampus implies its function. Therefore gaining a deeper, higher resolution picture of the anatomy provides a basis for insight into the way in which memories are stored and retrieved. The hippocampal formation is a bilateral C-shaped structure in three dimensions that curves from the dorsal-medial to ventral-lateral regions of the rodent brain (Amaral, 1999). It consists of the dentate gyrus (DG), the hippocampus proper (CA3, CA2, and CA1 subfields), the subicular complex (subiculum, presubiculum, and parasubiculum), and the entorhinal cortex (EC) (Amaral and Witter, 1989). Transverse sections of the structure reveal a distinct layered organization. Each layer is called a stratum and maps onto functional subdivisions in the anatomy.

CA1 is the subregion on which this thesis will focus. The cell body layer of the hippocampus proper is confined to one long continuous layer known as stratum pyramidale (SP, figure 1.2, top). The CA1 principal neuron 2 extends its primary apical dendrite out of the soma up to the border of the DG (Amaral, and Lavenex, 2006). The proximal half of the apical dendrite stretches through stratum radiatum (SR). The distal apical tuft dendrite arrays in a

2 The CA1 pyramidal neuron has also served neuroscience as the model cortical neuron.

SO SP SR SLM m

Figure 1.2. Top, the hippocampal trisynaptic loop is revealed in transverse sections. Bottom, CA1 inputs are segregated along the dendritic tree and align with multiple types of inhibition.

9 visibly darker layer known as stratum laconosum moleculare (SLM, figure

1.2, top). The basal dendrites of CA1 neurons emerge from the soma directly into stratum oriens (SO). SLM, SR, and SO are for the most part free of excitatory principal cells but contain an assortment of GABAergic interneurons. The input space conforms to the observed individual layers in most cases, a feature that has made the hippocampus one of the most fruitful models for synaptic transmission and plasticity. Additional interest in the hippocampus exists beyond memory research because many of the cellular and circuit properties apply to neocortex and other brain regions 3.

The anatomy and lesion studies suggest that the hippocampus acts as a processor of all perceptual information, hence its function in episodic memory, which involves the entire spatiotemporal context of experience. It has been a matter of some debate whether the rodent hippocampus performs general episodic memory formation or only spatial forms of memory.

Regardless, in both humans and rodents, it performs some set of operations on universal sensory representations generated in the cortex, transforming them such that they can be permanently stored. Polysensory perceptual representations enter the hippocampus and are processed therein by a series of nested convergent excitatory loops. The outermost, the canonical trisynaptic loop (Andersen et al., 1971), begins with information traveling from

EC layer 2 across long-range, primarily glutamatergic, axons to the dentate

3 Also, its synaptic circuitry and cornucopia of cell types resemble the neocortex.

10 gyrus, where it is conveyed across the perforant path synapses to the dentate gyrus granule cells (figure 1.2, top). The granule cells, then, pass the information along their axons via the mossy fiber synapses onto the thorny excrescences on the dendrites of cornu ammonis area 3 pyramidal neurons

(CA3). CA3 neurons have many recurrent excitatory connections where the autoassociative pattern completion of recognition memory recall is thought to occur (Nakazawa et al., 2002). The CA3 axons then pass the output along a massive axon plexus to CA1 where they form Schaffer collateral (SC) synapses on the proximal and basal dendrites of the CA1 pyramidal neurons in SR and SO (figure 1.2). This completes the tri-synaptic pathway.

CA1 neurons are the principal output of the hippocampal circuit. They

“close the loop” by projecting back to deeper layers of EC and other brain regions. Additionally, the CA1 region receives a direct glutamatergic input from EC layer 3 onto its distal dendrites in the SLM (figure 1.2). CA2 and

CA3 also receive a direct glutamatergic input from EC. These are also known as the perforant path as the axons perforate the subiculum on their trajectory before terminating in synaptic contacts along the apical tuft dendrite of CA1.

It has been theorized that the synaptic matrix of the hippocampus is analogous to a random graph (Buzsaki, 2006) and well suited for storing a representation of space over time.

At each node in this circuit, a wide diversity of interneurons abound, participating in feedforward, feedback, and long range GABAergic inhibition

11 onto the soma, dendrites (Miles et al., 1996), and axons (Kosaka, 1983) of both excitatory and inhibitory neurons (Freund and Buzsaki, 1996). These interneurons will be analyzed in more detail later, but their function is critical for overall network stability (Ben-Ari et al., 1979), the spatiotemporal flow of activity (Pouille and Scanziani, 2001; Royer et al., 2012), and defining and synchronizing cellular ensembles (Buzsaki and Chrobak, 1995; Cobb et al., 1995; Somogyi and Klausberger, 2008).

The synaptic matrix within the hippocampus is the substrate within which statistical regularities within the environment are encoded and retrieved. What changes occur in the hippocampal network when learning occurs? At a cellular level, the mechanism for memory storage is thought to be through Hebbian-style modifications in synaptic strength, whereby temporally correlated presynaptic and postsynaptic activity enhances synaptic strength (Hebb, 1949). Or, more succinctly, “cells that fire together wire together”. In the hippocampus of an anesthetized rabbit a cellular process that fulfilled the requirements of Hebbian plasticity was discovered

(Bliss and Lomo, 1973) and is now known as long-term potentiation (LTP).

These processes at excitatory synapses fit remarkably well with Hebb’s postulate and are thought to be the primary substrates through which learning occurs, although changes in cellular excitability, inhibitory interneuron plasticity, or other ongoing dynamics may also play a role.

Specific pharmacological blockade and congruent knockdown of key molecules

12 in the hippocampus involved in LTP, such as the NMDA receptor (Morris, et al., 1986; Tsien et al., 1996), impairs episodic memory, as measured in spatial navigation tasks in rodents. Complementary studies show that hippocampal dependent learning tasks progressively increase synaptic strength in the hippocampus as learning develops and these changes use the same cellular mechanisms as classical in-vitro LTP (Whitlock et al., 2006), suggesting that synaptic LTP is indeed the substrate of learning. LTP can be produced at all of the major excitatory synaptic junctions in the hippocampus: PP synapses to DG, CA3, CA2, and CA1, mossy fiber synapses onto CA3, and SC synapses onto CA1. Later, many activity-dependent mechanisms for synaptic depression were discovered as well (Levy and Steward, 1979; Bear and

Abraham, 1996).

At around the same time as the discovery of LTP, a major breakthrough for memory science emerged with the discovery of neurons tuned to spatial location. When a rat was placed in a new environment, after a few minutes of exploration, its hippocampal neurons began to fire in particular locations (O'Keefe and Dostrovsky, 1971) in space. These cells were called “place cells” because the neurons seem to encode a given location of a known environment. The place cell map represents an environment in neural space and serves to guide spatial recall. As such, when the environment changes completely and when cues are shifted systematically, the place cells remap to new locations or corresponding locations relative to the cue,

13 suggesting that the place cells encode the animal’s concept of its own position within a fixed environment (Wilson and McNaughton, 1993).

A simplified current model for memory and its retrieval, then, is that the hippocampal synaptic matrix encodes new perceptual representations by arraying the evolving spatiotemporal context into a unique set of synaptic weight changes (the engram), through synaptic potentiation and, possibly, depression, among a defined set of neurons (the neural assembly). Thus, when the animal re-experiences a cue (a percept consisting of a partial representation of the environment) this will activate the same neuronal ensemble and in the same pattern as the one that fired at the time of the initial exposure to that environment. The partial cue reinvigorates the whole of the context by driving the initially encoded cell assembly. In this manner, a past trajectory through a known space will be reawakened when the learned polysensory contextual representation is projected into the hippocampus and activates the same set of place cells, as long as the synaptic plasticity is stable.

Memory as a limited resource

However, this model presents a problem. Since synaptic territory in the hippocampus is limited, just as important as it is to rewire mappings in the network using LTP and LTD, useful structure must be maintained. This

14 tension is described by William James in his discussion of habit in Principles of Psychology:

”Plasticity , then, in the wide sense of the word, means the possession of a structure weak enough to yield to an influence, but strong enough not to yield all at once. Each relatively stable phase of equilibrium in such a structure is marked by what we may call a new set of habits. Organic matter, especially nervous tissue, seems endowed with a very extraordinary degree of plasticity of this sort; so that we may without hesitation lay down as our first proposition the following, that the phenomena of habit in living beings are due to the plasticity of the organic materials of which their bodies are composed .”

How does the brain achieve this proper balance of rigidity and plasticity? Or, stated differently, how does the brain know what to remember and what to forget? This resembles the same sort of optimization problem posed to an organism making decisions under uncertainty. Economic theories of decision-making define an action selected from many alternatives as the one with maximum utility. Perceptions with high memory utility, relevant memories, can be thought of as the perceptions “chosen” to be strongly encoded and, therefore, to have a high probability of being recalled in the future. The key variable that determines memory strength would be the value assigned to ongoing perceptions. The brain then needs a way to calculate the value of a perception (its relevance) and then project that

15 information to memory circuitry to give it preferential access to long-term memory stores. The most relevant perception for future action will shape the brain’s network and irrelevant perceptions will be ignored by the brain’s structure and forgotten 4.

What this would require in the terms stated above, then, would be a memory system that incorporates value and motivational states to adjust plasticity processes accordingly. Motivation related signals are not computed in the hippocampus. Therefore, other brain regions must project this information to the hippocampus. This kind of framework was termed “neo-

Hebbian” (Lisman et al., 2011) because it requires a heterosynaptic signal to guide memory related homosynaptic cellular processes like LTP, LTD.

Hebbian learning models involve correlated firing of cells within a circuit. The activity within that circuit drives the plasticity that supports learning. The neo-hebbian model adds an additional layer, asserting that plasticity processes can be biased by or even gated by extrinsic inputs to the network. These additional inputs will provide further constraints to optimize memory formation in accordance with task demands.

VTA/SNpC DA signals in striatum

4 This problem can also be posed across phylogenetic time for the evolution of the sensory systems that extracts the most relevant perceptions for behavior. Across phylogenetic time the brain has evolved a hard wiring (memory in genes) in response to the statistical regularities in the external world.

There could be numerous ways for a value signal to be projected to the hippocampus and aid in its forming of memory relevance decisions. In economic decision-making theory “rewards” drive learning. A reward is an object or event in the world that naturally elicits approach behavior, reinforces those approach behaviors, and is the outcome of decision-making

(Schultz, 2007). Midbrain DA neurons (MDNs) are at the core of the reward system as they encode the predicted rewarding quality or “value” of a stimulus (Schultz, 1998). DA neurons shift from a tonic firing state (1-3 Hz) to phasic bursting at (10-30 Hz) in response to stimuli that elicit approach behavior, such as unexpected appetitive stimuli like food, water, sex, and novelty (Schultz, 1998; Wise, 2004) 5. They reduce their firing when that reward is absent (Tobler et al., 2003). Moreover, they track associative learning during classical conditioning, firing at first to a surprising and motivating unconditioned stimulus. However, after the animal learns to associate the unconditioned stimulus with a predictive conditioned stimulus,

DA neuron firing shifts in time to the presentation of the conditioned stimulus (Tobler et al., 2005). These data led to the hypothesis that dopamine neuron firing acts as the value signal that comes to reinforce associations

5 It is know also know that stimuli that elicit escape behavior (Brischoux et al., 2009) drive phasic responses in DA neurons as well. Escaping punishment is rewarding.

17 between neutral stimuli (and actions) and rewarding ones (Sutton and Barto,

1998; Schultz, 2007).

The MDN reinforcement hypothesis is well established for striatal- based procedural learning and has been borne out in Pavlovian and operant conditioning paradigms. Rodents choose to lever-press to receive DA neuron stimulation (Philips and Fibiger, 1978), indicating that DA firing is sufficient to reinforce the behavior. Animals find this even more rewarding then life- giving calories and will sustain pain in order to receive it (Routtenberg and

Lindy, 1965). It is indeed the striatal input driving this behavior as specific lesioning of the nigrostriatal DA projection with 6-OHDA impairs habit formation (Faure, 2005). Moreover it is the phasic firing signal of the

VTA/SNpC (10-30 Hz) that drives the learning as optogenetically induced phasic firing of DA neurons of the VTA induces place preference (Tsai et al.,

2009), while tonic firing does not. Thus, rewards drive phasic firing of DA neurons in the VTA/SNpC and that signal instructs learning by some reinforcing action in striatal circuits.

How would DA neuron efferent phasic signals enact this reinforcement? To answer this question we must consider the effectors of DA, the DA receptors, as well as their signaling properties. The DA receptor family belongs to the superfamily of 7-transmembrane spanning G-protein coupled receptors (GPCRs). There are 5 types expressed in the mammalian brain (Missale et al., 1998). The D1-type receptors include the D1 and D5

receptors and are G s coupled so they activate adenylyl cyclase to increase cAMP. The D2 type receptors include D2, D3, and D4 receptors, and are mostly G i/o GPCRs, which inhibit adenylyl cyclase and thus decrease cAMP levels.

If the VTA/SNpC DAergic signal is indeed the reinforcing signal that drives striatal learning then DA receptor blockers should impair learning.

Indeed, antagonists of DA receptors prevent the acquisition and maintenance of lever pressing for DA neuron axon self-stimulation reward or food reward.

DA neurons and receptors are also necessary for classical conditioning

(Spyraki et al., 1982). Rats will ordinarily develop a preference for spatial locations where they are rewarded with food. Under DA receptor blockade, this preference disappears. These strong effects of the DA system on the ability to learn particularly affect striatal dependent learning tasks.

What is the cellular basis for the actions of DA in reward learning?

Both LTP (Centonze, 2001) and LTD (Calabresi, 1992) in the striatum have been found to require DA in the striatum. The theory, then, is that DA neuron phasic firing releases DA onto the striatum and activates D1 and D2 receptors, which drives plasticity by gating LTP and LTD. The reward signal, indicative of the value of a stimulus, is carried by the DA neurons to its terminal field in the striatum, where it permits plasticity to reinforce the association of a behavior with a reward.

VTA/SNpC DA signals in hippocampus

Might VTA/SNpC DA neurons also act to project value signals to the hippocampus to aid decision-making about episodic memory encoding?

Lisman and Grace, 2005 proposed that a hippocampal-VTA-hippocampal loop acts to detect novelty, and then grants the novel stimulus preferential access to hippocampal-dependent memory processes. I propose that this idea be slightly extended and suggest that the currency of memory’s economy is based in DA and other neuromodulators that are sensitive to salience. In other words, relevance decisions are driven in part by neuromodulatory inputs into the hippocampus. These inputs shape learning in accordance with the salience of the ongoing environment and an animal’s internal state by altering synaptic signaling.

Specifically, the theory of Lisman and Grace (2005) states that the hippocampus and DA neurons couple to one another in a recurrent loop to give preference to salient information in its access to memory. The idea is that the hippocampus detects novelty by comparing the ongoing sensory representation with a mnemonic representation. The hippocampal output disinhibits the VTA via an indirect pathway involving three intervening structures: subiculum, nucleus accumbens, and ventral pallidum. The excited

VTA, in turn, releases DA in the hippocampus, which enhances plasticity processes in CA1 to favor encoding of the information that led up to the

20 salience (Lisman and Grace, 2005; Otmakhova et al., 2013). In such a way,

DA facilitates learning and memory. There is considerable evidence for the downward arm of the loop, the novelty detection in the hippocampus (Fyhn et al., 2002) and VTA upregulation as a result (Legault and Wise, 2001).

However, the VTA impact on the hippocampus requires further analysis.

The mesolimbic DA projection from VTA and SNpC, of which the hippocampus is one terminal field, is a suitable candidate to carry mnemonic value information into the hippocampus to enhance the formation of episodic memory. Other neuromodulatory nuclei, such as the serotoninergic raphe nucleus (Li et al., 2013), noradrenergic locus ceruleus (Poschel, and

Ninteman FW, 1963), and different cholinergic nuclei (Blokland, 1996), are also sensitive to the valence of external cues and may play a role in gating memory encoding. This thesis will focus on the upward arm of the DA loop. I will review the relevant literature that supports the existence of the upward arm of the loop; that is, the evidence that VTA/SNpC DAergic projection to the hippocampus can influence CA1 in a way that would regulate learning.

The dopaminergic influence on the hippocampus has been tested from the molecular to behavioral level in a wide variety of preparations ranging from rodent to human. The literature reviewed below will stay focused on rodent studies as they are most relevant to the experiments discussed in the

21 thesis. However, it should be noted that mounting evidence points to the importance of DA signaling in healthy and impaired human episodic memory

(Chowdhury, 2012).

DA in the hippocampus: insight from anatomy

The influence of DA on the hippocampus has been met with some skepticism and many still doubt its ability to have a significant impact on hippocampal neurons and learning and memory. The controversy arises from a major discrepancy between physiology and anatomy, namely that the

DAergic axon pool from VTA is very sparse, despite the potent effects of exogenous receptor stimulation on cellular properties and behavior. However,

I will argue in the next section that there are at least two distinct sources of

DA in the hippocampus and a constellation of potent receptors. How the

DAergic sources can modulate the cellular physiology and behavior via this receptor system is not known, but the preponderance of indirect evidence suggest that the VTA/SNpC source of DA is an effective modulator of hippocampal memory and physiology. Moreover, it seems that acute DA release exerts its own effects, in addition to the basal action of DAergic tone.

Initially, the DA found in the hippocampus was thought to be a precursor along the biosynthetic pathway to norepinephrine (NE) in axons

22 from the locus coeruleus, which provides a strong input to hippocampus.

Since the conversion of DA to NE by the enzyme dopamine beta hydroxylase occurs in the synaptic vesicle, distinguishing a true DA releasing neuron from a noradrenergic one is quite difficult. Later, Scatton et al., 1980 confirmed that DA itself was released independently in the hippocampus. They lesioned known DA pools, the substantia nigra pars compacta (SNpC) and ventral tegmental area (VTA), and observed a significant reduction in DA levels in the hippocampus. Analogous noradrenergic locus coeruleus neuron lesions decreased NE proportionally more than DA, suggesting that there are independent sources of DA and NE.

Later, tracing studies combined with immunohistochemistry verified that the midbrain DA neurons in the VTA (A10), substantia nigra pars SNpC

(A9), and retrorubral field (A8) project axons to the hippocampus (Swanson et al., 1982, Gasbarri et al., 1994, Verney et al., 1985), of which the bulk originate in VTA and terminate in the CA1 subfield and subiculum. These detailed tracing studies found that CA1 is innervated by DA inputs along the entire septo-temporal axis of the hippocampus and subiculum, although there was a significant bias for the ventral side. Within CA1, the axons primarily ramify throughout the alveus and SO with a smattering in SP. In the more dorsal regions the fibers become even sparser and are confined to SO. In the most ventral pole, there are some fibers in SR and SLM.

Immunohistochemistry later replicated these results (Kwon et al., 2008).

sub CA1

Figure 1.3. VTA axons course through the alveus SO border in CA1. They become more diffuse near subiculum. Verney et al., 1985. al = alveus, SO = stratum oriens, Sub = subiculum

The anatomical studies verify that true DA synthesizing axons originating from the midbrain DA neuron pool in VTA/SNpC are present in the hippocampus, but when compared to the striatum, the innervation pattern is still very modest. However, evidence from both cortex (Devoto et al., 2001; Devoto et al., 2005) and hippocampus (Smith and Greene, 2012) suggests that locus coeruleus axons, classically thought of as releasing NE exclusively, can co-release DA. In CA1 the locus coeruleus fiber tract primarily innervates the deeper layers of the hippocampus, SR and SLM, with a slight bias for SLM (Oleskevich et al, 1989). It seems then, that there

24 are two sources of DA in the hippocampus, one in SO originating from

VTA/SNpC and another in SLM originating from LC.

To understand the function of the DA modulatory system, it is therefore necessary to consider each axon source and the target receptors as a signaling unity. What is the function of the VTA/SNpC DAergic input to the hippocampus? What is the function of the LC NE/DAergic input? DA from the

VTA and SNpC axons in SO and SP likely has a different function than the

DA releasing LC axons in SR and SLM, considering that they are anatomically segregated and driven by distinct, although partially overlapping, behavioral conditions.

What is the specific function of the VTA/SNpC DAergic axon pool? Can

VTA/SNPC axons release sufficient DA to alter the cellular and circuit physiology of the hippocampus and influence learning and memory? This question has not been asked directly. However, to get an idea of how the DA receptor system might be recruited by the VTA/SNpC DAergic source we must survey the range of possible actions of DA in isolation.

All of the DA receptor types are expressed throughout the hippocampus. In CA1, the more prevalent D1-type receptor is the D5 receptor

(Ciliax, 2002; Gangarossa et al., 201, Khan et al., 1994), which is localized to cell bodies, dendritic shafts, and dendritic spines. Of the D2-types, D4 is most prevalent (Defagot et al., 1997) and present in cell bodies (Brouwer et al.,

1992; Goldsmith and Joyce, 1994). Cell type distribution of the different

25 receptors is equivocal. However, the literature from prefrontal cortex, whose

DA system is analogous to the hippocampus, suggests that both interneurons and pyramidal cells express D1- and D2-type receptors.

There is a wide diversity of DA receptors present in multiple cell types in the hippocampus. The DA receptor system is additionally complex because the same DA receptor could be coupled to different G-proteins depending on the cell type. Also, the D1- and D2-type receptors can form heteromers

(Verma et al., 2010) and signal synergistically.

VTA/SNpC DAergic input to CA1: function

The VTA/SNpC DAergic axons in the hippocampus are required for spatial memory performance. Selective VTA/SNPC DA axon degradation in the hippocampus induced by a focal infusion of 6-hydroxydopamine 6-OHDA, a toxin that is taken up by the DA transporters, impaired performance on the hippocampal dependent task, the Morris water maze (Gasbarri et al., 1996).

The Morris water maze tests spatial long-term reference memory by querying the animal’s ability to find a hidden platform in a small pool with a few salient visual cues (Morris, 1981). Since the noradrenergic fibers were protected by a norepinephrine transporter antagonist desipramine, only the

VTA/SNpC DA source was lesioned. Importantly, VTA/SNpC DA axon lesions neither impaired performance on a cued-version of the water maze, where the

26 animal had to learn to swim to a visibly cued platform, nor on a one-trial inhibitory avoidance task, indicating that spatial reference memory was impaired, specifically. Complementary experiments using VTA silencing with the GABA A agonist muscimol induced amnesia on the Barnes maze, a land- based version of the water maze (Nazari-Serenjeh et al., 2011). Therefore, intact VTA/SNpC DAergic axons are critical for hippocampal learning.

What is the function of these VTA SNpC DAergic axons? If they regulate memory in the hippocampus, then one would expect them to either modulate synaptic transmission, synaptic plasticity, or both, as these processes underlie the formation of new memories. Synaptic transmission produces the postsynaptic depolarization that is required to unblock the Mg 2+ from the NMDA receptor and trigger LTP. Thus, by globally enhancing or suppressing postsynaptic depolarization the threshold for inducing plasticity can be altered. LTP itself strengthens the synaptic weights that store the memory. Therefore, modulation of either of these processes would provide a pathway with which DA axons could plausibly affect memory processes.

VTA/SNpC DAergic input to CA1: synaptic transmission

Clues to how the VTA/SNpC DAergic input might modulate basal synaptic transmission come from studies using exogenous receptor

27 pharmacology either in-vivo or in the in-vitro slice preparation, or using in- vivo electrical stimulation of VTA neurons. The totality of in-vivo and in-vitro studies on the potential effects of DA on synaptic transmission does indicate that DA is a potent modulator of synaptic transmission in CA1. However, many of the studies contradict one another regarding the nature of the observed effects, likely because the receptor system in place is complex, potent, and perhaps suited for physiological engagement from two separate

DA sources.

Early VTA/SNpC stimulation studies in vivo raise the possibility that

DA may alter the efficacy of the synaptic drive from the SC (CA3 axon) pathway to CA1. In-vivo VTA and SNpC electrical stimulation decrease the

SC-evoked pop spike but leave the SC-evoked field PSP unchanged (Spencer and Wheal, 1990). The fact that there was only an effect on the pop spike and not the PSP suggests that the DA stimulation is affecting either CA1 cellular excitability through modulation of an ion channel in the CA1 neuron or on SC driven inhibitory influences that synapse on CA1. However, electrical stimulation neither can discriminate cell type nor determine whether the stimulation activated neurons within a given nucleus or fibers of passage. In a later study Spencer and Wheal, (1990) created an ibotenic acid lesion in the

VTA/SNpC, a toxin specific to somata, prior to the experiment. The SNpC effect was abolished, but the VTA effect remained, suggesting that perhaps the VTA effect was in fact due to stimulation of fibers of passage. However,

28 there was no pharmacology performed to identify the receptor involved.

Because VTA and SNpC contain mixed populations of neurons, it is not clear that DA mediates the effect of stimulation. Nonetheless, these findings do raise the possibility that the release of DA particularly from the VTA/SNpC originating axons can directly affect the input-output function of CA1 neurons.

Studies using the direct bath application of DA provide information as to how activation of distinct DA receptors regulates fast excitatory and inhibitory synaptic transmission in CA1 pyramidal neurons. In these experiments, DA is typically infused onto the slice via the perfusion medium, setting some unknown concentration (due to oxidation), relatively uniformly throughout the neuropil containing the intact circuit. The dominant result emerging from such studies is that DA acts primarily to suppress the PP

(cortico-CA1) input to SLM by reducing feedforward excitation through a

D1/D2 joint mechanism. In most of these studies, DA does not directly affect the SC input (Lisman et al., 1999; Ito and Schuman, 2papers; Marciani, 1984,

Gribkoff and Ashe, 1984), although Hsu, 1996 reported a depression of the SC response with a low DA concentration. These contradictory data suggest that

DA receptor systems are capable of having effects on both the PP and SC inputs to CA1. However, it is not clear whether PP, SC, neither, or both is targeted by VTA/SNpC mediated DA release.

As discussed above, tracing studies show that the VTA and SNpC

DAergic axons, primarily send their axons to SO of CA1 (Gasbarri et al.,

1994), which is several hundred m away from the stratum laconosum moleculare (SLM), the layer where the PP inputs form synapses onto CA1 pyramidal neurons. Thus, it is unlikely that any effect of DA on PP inputs to

CA1 could be driven by the VTA/SNpC DA input. Rather, it is more likely that any physiological action of DA receptor activation on the PP results from the influence of the locus coeruleus DA input to the hippocampus.

Specific receptor agonist experiments do show that both D1-type and

D2-type receptors can efficaciously regulate SC to CA1 synaptic transmission in some conditions. There are, as yet, no reports of experiments to test the actions of specific DA receptor agonists on PP to CA1 synaptic transmission.

Two studies found that agonist stimulation of the D1-type receptor potentiated the SC basal synaptic transmission in an NMDA receptor dependent manner via D1/D5 receptors (Huang and Kandel, 1995;

Navakkode, 2007), but a later study failed to observe this effect (Mockett et al., 2004). These inconsistencies likely speak to the complexity of examining the neuromodulatory system using exogenous agonists in in-vitro slice preparations. The slice preparation methods and concentrations of agonists used differ between the studies, which may have influenced signaling through the D1-type receptors.

D2-type agonists have been reported to exert a variety of effects on SC synaptic transmission. The pan D2 agonist has no direct impact on SC evoked synaptic transmission (Huang and Kandel, 1995). The D3 specific agonist, however, inhibits the SC-evoked feedforward IPSC (Hammad and Wagner,

2006; Swant and Wagner, 2008); this consequently enhances the depolarization of CA1 pyramidal neurons in response to SC stimulation. The

D4 agonist was reported to depotentiate SC synapses that have previously undergone LTP, but this study did not investigate changes in basal transmission (Kwon et al., 2008). In other brain areas, such as prefrontal cortex (Wang et al., 2002), thalamus, and globus pallidus (Shin et al., 2003), the D4 agonist downregulates inhibition by interacting indirectly with

GABA-A receptors. Immunohistochemistry and in-situ hybridization show that the D4 receptor is expressed in parvalbumin-expressing (PV+) interneurons in the hippocampus. These receptors may, then, regulate feedforward inhibition (Kwon et al., 2008).

To summarize, the studies using exogenous DA show that the PP input to CA1 neurons can be suppressed by DA bath application whereas the SC input can be suppressed by DA bath application or potentiated by D1/D5 agonist application. The D3 and D4 agonists may indirectly enhance or suppress the SC-mediated depolarization of CA1 PNs, respectively, through regulating synaptic inhibition. Since the locus coeruleus monaminergic axonal pool is aligned with the PP input in SLM, an area with very sparse

31 input from the VTA/SNpC, it is likely that the DA receptors in that layer are activated under physiological conditions by DA that is released from the LC terminals.

The SC excitatory axons form synapses in SR and in SO and the experiments discussed above focused largely on synaptic transmission in response to stimulation of the SC axons present in the SR. However, the vast majority of VTA/SNpC DAergic axons are found in SO, quite distant from SR.

Therefore, it is unclear whether the various effects of exogenous DA application on the SC synapses that have been reported in the literature are more relevant to the actions of LC-dependent versus VTA/SNpC-dependent

DA release. Support for a role for LC-mediated DA release in the hippocampus comes from the finding that an amphetamine-induced potentiation of the SC PSP (evoked by stimulation of SC axons in SR) via

D1/D5 receptors was not altered following depletion of DA in the VTA/SNpC, suggesting that the SC enhancements may result from the functional impact of DA released from LC axons (Smith and Greene, 2012). Moreover, direct

VTA/SNpC stimulation in-vivo (Spencer and Wheal, 1990) or bath application of low concentrations of DA in vitro (Hsu et al., 1996) result in suppressive effects on the SC-evoked excitation of CA1 neurons, suggesting that the primary role of VTA/SNpC mediated DA release may be to inhibit activation of CA1 by its SC inputs. These conflicting results clearly indicate the limitations of experiments using bath application of DA, and motivate my

32 experiments using an optogenetic approach to selectively trigger DA release from VTA/SNpC axons.

VTA/SNpC DAergic input to CA1: synaptic plasticity

In addition to directly affecting SC and PP synaptic transmission,

VTA/SNpC DA release could regulate memory by modulating ongoing synaptic plasticity processes. The data on the D1/D5 receptor show a consistent permissive and facilitative effect on SC LTP and learning and memory, though whether DA affects early (E) LTP versus late (L) LTP has been a matter of debate. Early LTP is activated by one or two rounds of brief tetanic stimulation of the SC inputs, lasts for 1-2 hours and does not require the synthesis of new proteins. Late LTP requires multiple rounds of SC tetanic stimulation (3-4), lasts for > 3 hours and depends on transcription and translation. Pharmacological and genetic analyses established that the D1- type receptor is necessary for LTP at the SC (CA3-CA1) synapse. Thus, the

D1-type antagonist SCH23390 abolishes tetanic stimulation induced L-LTP

(Huang and Kandel, 1995) and reduces E-LTP (Granado, 2008). D1-type agonists enhance only E-LTP (Otmakhova and Lisman, 1996).

The effect of D1/D5 receptor antagonists on SC LTP have been corroborated by genetic studies, where the DRD1 (the D1 gene) knockout blocks the induction of both E- and L-LTP (Granado, 2008) in-vitro and in

33 vivo (Ortiz et al., 2010). In the latter study, shRNA knockdown of the D1 receptor in CA1 impaired E-LTP and flattened the learning curve in both a spatial memory task and a hippocampal-dependent version of trace eye-blink conditioning. Ortiz et al., 2010 also measured field postsynaptic potentials during the trace conditioning evoked by SC stimulation. They observed that

D1 knockdown in the hippocampus diminished the progressive increase in synaptic strength that accompanies the development of the conditioning, linking the effect on synaptic plasticity directly to learning.

Additionally, spike timing dependent plasticity (STDP) at SC (CA3-

CA1) synapses, a form of plasticity related to E-LTP, does not develop in DA depleted slices, an effect that can be rescued with bath application of 20 M

DA and blocked with the D1-type antagonist (Edelmann and Lessman, 2013).

In hippocampal neuron cultures, DA extended the time window and reduced the threshold for STDP induction (Zhang et al., 2009). It has been suggested from these studies that DA signaling, acting primarily through the D1-type receptor, is a requirement for the induction of LTP at the SC synapse.

It is not completely understood as to how D1 receptors permit SC E- or

L-LTP. The current hypothesis for L-LTP is that the D1 receptor activates

PKA via the Gs signaling pathway, which leads to the phosphorylation of

CREB (Huang and Kandel, 1995), and subsequent synthesis and insertion of proteins that maintain plasticity, such as AMPA receptors (Smith et al.,

2005). Also, D1-type receptors can directly modulate the NMDA receptor

34 current (Varela, et al., 2009) and may control LTP induction through its action therein.

These studies of SC LTP reveal a strong dependence of plasticity on

DA signaling. However, it is still not clear if the effects observed on LTP described above using direct manipulations of the D1/D5 receptors captures an effect of VTA/SNpC DA signaling. As mentioned above, the DA axons primarily course through SO in the basal dendrites and the effects reported above largely focus on synaptic transmission and plasticity at the SC inputs onto the proximal apical dendrites of CA1 neurons in stratum radiatum. It is possible that DA, when released from axons in SO, diffuses to SR. Or, perhaps, these receptors may be activated under physiological conditions by

DA release from locus coeruleus axons.

Evidence that the VTA may be important for SC LTP comes from the finding that, in an anesthetized rat, silencing of the VTA with local perfusion of lidocaine right after induction of LTP through tetanic stimulation of the SC pathway transiently suppressed the expression of SC LTP (Ghanbarian and

Motamedi, 2012). This implies that some aspect of LTP expression requires activity in the VTA/SNpC, although whether this is mediated by DA release or release of some other transmitter present in this mid-brain region requires further investigation. Finally, it is not clear if DA signaling is merely permissive through ongoing DA tone due to tonic firing of DA neurons, or

35 whether acute signaling through DA release mediated by phasic firing of

DAergic neurons actively gates induction of LTP during learning.

VTA/SNpC DAergic input to CA1: memory

Is the VTA/SNpC DA system truly required for hippocampal- dependent memory storage? There is now abundant evidence that D1-type receptors are, indeed, required for spatial memory encoding and its maintenance. D1-type receptor antagonists infused into CA1 during the exposure of rats to an associative tastant-location learning paradigm weakened one trial taste-location associations (Bethus et al., 2010). Recall 24 hours later was severely impaired, whereas recall 30 minutes following the encoding of the memory was intact. Paired associates learned 24 hours prior to antagonist infusion were also still intact. This experiment suggests that

D1-type receptors throughout CA1 are specifically required during exposure to the to-be-remembered stimulus for the persistence of the engram for extended periods, but not for initial short-term memory. This time-dependent effect is reminiscent of the LTP results reported by Huang and Kandel, 1995, where D1-type antagonists blocked L-LTP but not E-LTP. However, since this experiment solely manipulated D1-type receptors in the hippocampus, the source of the DA for memory facilitation is ambiguous.

In an inhibitory avoidance task, a hippocampal-dependent contextual fear learning paradigm, D1-type receptors were necessary during maintenance 12 hours after the learning phase but were not required during or immediately after encoding (Rossato et al., 2009). In this same study, local tetrodotoxin (TTX) lesion of the VTA also impaired memory maintenance.

Together, the VTA/SNpC DAergic axon lesion (Gasbarri et al., 1996) and D1- type antagonist study on the inhibitory avoidance task (Rossato et al., 2009) suggest that VTA/SNpC DA is necessary for hippocampal spatial memory

(Gasbarri, et al., 1996) and consolidation of contextual associations (Rossato et al., 2009). However, it is remains unclear which axon pool activates the

D1-type receptor required for encoding (Bethus, 2010) the paired associate as in that experiment only D1/D5 antagonist infusion into the hippocampus was used to assess the function of DA.

The possible recruitment of D2 receptors by DA released from VTA

SNpC in the service of learning has not been less extensively studied, although the D2 receptors in the hippocampus are potent regulators of learning. The wide array of effects observed makes it difficult to predict how these receptors might function in physiological conditions with DA release from VTA/SNpC axons. D2-type receptor stimulation, depending on conditions, has been reported to either facilitate or impair learning. D2-type antagonist administration to the hippocampus impaired (Wilkerson and

Levin, 1999) spatial working memory, suggesting a facilitative role for D2-

37 type receptors. However, in the passive avoidance task neither a D2-type pan antagonist nor a D1-type antagonist affected memory (Nazari-Serenjeh et al.,

2011). In contrast, amnesia could be induced by infusing muscimol into the

VTA in that same task (Nazari-Serenjeh et al., 2011). In this situation, the

D2-type antagonist applied to the hippocampus restored memory (Nazari-

Serenjeh et al., 2011). These latter results suggest that in the absence of

VTA/SNpC signaling D2-type receptors are actively suppressing memory.

One possibility is that it is DA released from LC axons that activates the D2 receptors to constrain memory performance. Alternatively, since VTA/SNpC

DA tone in the hippocampus was likely reduced by muscimol infusion, it may be that a residual low level of DA release from VTA/SNpC axons is particularly effective in activating those D2-type receptors that are coupled to the suppression of memory encoding.

In more specific receptor activation experiments, a D2 agonist applied to the hippocampus enhanced spatial working memory performance

(Wilkerson and Levin, 1999). Interestingly, in this experiment the D1-type antagonist SCH23390 had no effect on learning. Another study found that the D2 agonist could also ameliorate spatial memory impairment induced by the muscarinic antagonist, scopolamine (Fujishiro et al., 2005). In summary, the data is reasonably consistent with D2 having a facilitative role, at least in control conditions. In most studies there is little data as to whether these receptors may be recruited by VTA/SNpC or LC monaminergic fibers.

However, the finding that the D2 receptor signaling profile changes following blockade of VTA/SNpC signaling (Nazari-Serenjeh et al., 2011) is consistent with distinct roles of D2 receptor activation in response to DA release from its two neural sources within the hippocampus.

Aims of this thesis

To summarize briefly, DA has three well-established effects on hippocampal physiology. First, it suppresses the basal synaptic transmission from PP axons (originating in EC layer 3) to CA1 pyramidal neurons via D1- type and D2 receptors (Otmakhova and Lisman, 1999). Second, DA is permissive for the induction of different forms of LTP through its action on

D1-type receptors. Third, DA acts as a gate on long-term memory storage through activation of D1-type and possibly D2-type receptors in CA1 in several different types of memory tasks. From these data it has been hypothesized that salient stimuli drive phasic firing of DA neurons, which then facilitates LTP in the SC mnemonic input to CA1 while simultaneously blocking the direct sensory representation carried by PP input (Grace and

Lisman, 2005). Consistent with this view the D1/D5 antagonist also impairs the stabilization of place fields in CA1 over time (Kentros et al., 2004). In this way, DA neurons in the VTA/SNpC that fire in response to saliency can aid in

39 facilitating encoding of the information that preceded the saliency signal and prevent interference from the ongoing sensory representation.

However, this model considers the entire DA receptor landscape to be a target of the VTA/SNpC. In fact, there are likely two separate pools of

DAergic axons projecting to the hippocampus, one originating in the

VTA/SNpC, the other in the LC, so that two separate modulatory neural pathways will likely activate the DA receptor system in distinct layers of the hippocampus. It is clear that the VTA/SNpC DA axons in the hippocampus are critical for hippocampal learning processes (Gasbarri, 2006). Yet, that does not preclude the LC DA signal from having a strong physiological role.

DA and specific receptor agonist bath application studies can only infer the underlying mechanisms for the function of those axons in learning processes under the assumption that all of the receptors expressed are ordinarily activated by a single axonal pool of modulatory inputs.

It seems from the evidence provided above that this is not the case.

Rather, it is probable that in distinct behavioral conditions, DA will be released from LC and VTA/SNpC and each will activate its own coupled receptor profile to exert a unique range of effects. The agonist and antagonist studies do not distinguish between receptors that would be activated by one modulatory pathway versus the other. And, since the VTA/SNpC and LC fire in different conditions, the receptor profile must be linked to its afferent partner to construct a model for neuromodulatory control of memory.

Therefore, a circuit-based approach is needed for understanding how and when separate saliency signals, despite their potential use of a common transmitter, are projected to the hippocampus to regulate memory storage.

In this thesis I use such a circuit-based approach to analyze selectively the effects of DA release from VTA/SNpC axons located within the hippocampus on synaptic transmission from both PP and SC inputs onto CA1 pyramidal neurons and interneurons. To account for the constraints set by the VTA/SNpC axonal anatomy in time and space, I turned to optogenetic tools. These allow for the controlled release of endogenous DA from the impinging genetically defined axons originating from the VTA and SNpC.

First, I confirmed that bath application of DA reduced the PP-evoked

PSPs and left the SC PSPs intact in CA1 pyramidal cells, replicating previous findings (Otmakhova and Lisman, 1999; Ito and Schuman, 2007). Second, I utilized optogenetic tools (Boyden et al., 2005) to selectively stimulate

VTA/SNpC DAergic axons in CA1 to define how this particular modulatory input affects both excitatory and inhibitory synaptic transmission. I found that VTA/SNpC stimulation with a single brief light train (3 pulses at 66 Hz) activates D2-type receptors to produce a long-lasting suppression of the SC depolarizing postsynaptic potential (PSP) in the CA1 pyramidal neurons, with no change in the PP PSP, the opposite pattern of the results with bath application of DA. This suppression results from an increase in the amount of feedforward inhibition activated by the SC afferents. I further found that this

41 increased inhibition was selectively mediated by an increased excitation of the PV+ population of interneuron by their SC inputs. Finally, more prolonged light burst protocols consisting of 25 bursts applied at 1 Hz, a protocol that approximates the phasic firing of DA neurons during an animal’s foraging for rewards (Wang et al., 2011), potentiates the SC PSP through a direct presynaptic modulation of the SC input onto the CA1 pyramidal neurons. Thus, distinct patterns of activity of VTA/SNpC DAergic neurons will produce a bidirectional modulation of CA1 synaptic excitation that can alter synaptic plasticity rules in accordance with task demands and thereby aid in economical memory formation.

Chapter 2

Introduction

The DA system is one of the most powerful modulatory systems in the brain.

In its projection regions, it can alter cellular, circuit, and network properties, as well as behavior. In the standard model of neuromodulatory function, the central signaling apparatus is the total overall receptor output, given some concentration of agonist. The anatomy of the DA system in the hippocampus does not conform to this model. DA is likely released onto the CA1 subfield of the hippocampus from two segregated populations of axons. One emerges from VTA/SNpC, the other from LC. How might DAergic signals from the

VTA/SNpC neurons that are known to encode salience influence synaptic transmission in the hippocampus?

CA1 pyramidal neurons are the only major glutamatergic output of the hippocampus. CA1 receives excitatory glutamatergic input from two major sources, the PP axons that originate in entorhinal cortex and the SC axons that originate in CA3. The entorhinal cortex neurons in layer 3 are tuned to sensory features of the environment (Vinagrodova, 1984) and provide contextual information into CA1 directly, in addition to a “spatial metric”

(Buzsaki and Moser, 2013). CA3, on the other hand, carries the mnemonic

43 representation that that same context has generated via the tri-synaptic pathway. When information arrives to CA1 from the PP or SC axons, it can only leave the hippocampus and go on to drive entorhinal cortex if the depolarization resulting from synaptic transmission causes the CA1 membrane to reach action potential threshold. Also LTP, the best-established substrate for learning, is triggered by a depolarization of the post-synaptic membrane that is sufficient to relieve the magnesium block of the NMDA receptor and conduct Ca 2+ . Therefore, the relative synaptic strengths of the

PP and SC pathways determine which type of information will dominate hippocampal output, and consequently, which ones will induce LTP.

Anatomically, it seems like the DAergic input from VTA/SNpC is more aligned with the SC input from CA3 as its axons course through SO and SP of

CA1. The LC axons run throughout the hippocampus, but are most dense in

SLM, where the PP axons form synapses on the CA1 apical tuft dendrite and onto GABAergic interneurons. The influence of the VTA/SNpC DAergic neurons on synaptic transmission in CA1 has been studied either by applying exogenous agonists or by electrically stimulating VTA and SNpC directly, a mixed pool of neurons and fibers of passage. But, since DA is likely released from two distinct pools, exogenous application artificially activates receptors and likely conflates physiologically independent signaling channels. The effect of VTA/SNpC stimulation itself could reflect the activation of a mixed population of DAergic, glutamatergic, and GABAergic projection neurons.

These studies reveal the range of possible actions of DA. However, some of these are anatomically improbable, arguing for the necessity of taking an afferent-based approach to modulatory signaling. Whether VTA/SNpC DA release can effect one or several of these actions is still a matter of speculation.

DA bath application has an input specific effect. It reduces the PP- evoked synaptic depolarization of CA1 and leaves the SC-evoked depolarization intact (Otmakhova and Lisman, 1999; Ito and Schuman, 2007;

2012). Though DAergic terminals can appose GABAergic interneurons (Carr and Sesack, 1996), this effect did not require inhibition. Rather, exogenously applied DA directly suppressed the PP’s glutamatergic depolarization of CA1 in the apical dendrite (Figure X, C) and did so in a dose dependent manner, as the effect increased as the concentrations of DA increased from .5 uM up to

100 M (Figure X, B), where it saturated. This effect requires both D1 and

D2 receptors (Otmakhova and Lisman, 1999). From these data the authors hypothesize that the input specific suppression of the PP by DA might aid in

CA1’s ability to select the mnemonic output of CA3 over the online contextual representation from the entorhinal cortex. The exposed context that preceded the DA release would be favorably stored within the CA3-CA1 network, perhaps due to less interference from the PP synaptic input.

Figure 2.1: The effect of DA on synaptic transmission in the hippocampus is unclear.

Figure 2.1: The effect of DA on synaptic transmission in the hippocampus is unclear. A. CA1 neurons receive glutamatergic inputs from the Schaffer Collateral (SC) axons in stratum radiatum (SR) and from the Perforant Path (PP) axons in stratum laconosum moleculare (SLM). Both of these inputs are shaped by powerful inhibitory interneurons (shown in red). Stimulating electrodes Ire placed in both layers to evoke action potentials and subsequent synaptic transmission. B. DA suppresses the PP evoked field PSP by 50%, whereas the SC evoked field PSP is reduced by

10%. Adapted from Otmakhova and Lisman, 1999. C. The effect of DA is on feedforward excitation as it does not change in the presence of blockers of inhibition. D. SC PSP is reduced by DA. Adapted from Hsu, 1996.

However, Hsu (1996) reports that DA suppresses the SC input, not the

PP (Figure X.) and Gribkoff and Ashe (1984) found a minimal effect on the

SC PSP, but found that millimolar concentrations produced a long lasting potentiation of the SC evoked population spike in extracellular recordings from the CA1 pyramidal layer. Gribkoff and Ashe (1984) used higher concentrations, into the milliMolar range, while the others used nano and microMolar range concentrations. Hsu (1996), who observed that DA depress the SC-evoked depolarization measured the synaptic input using whole cell recordings, whereas the others measured field potentials from the dendritic region within SR and SLM, where the SC and PP synapses are formed, respectively.

Nevertheless, in these exogenous DA application experiments, the volume and concentration of neurotransmitter are set arbitrarily, which results in the impartial activation of DA receptors. In the physiological scenario, the DA receptors are linked to a particular afferent pool with its own firing properties. It remains unclear, therefore, how the VTA/SNpC axons innervating the hippocampus stimulate receptor signaling and alter neuronal output and learning and memory.

Therefore, in order to respect the constraints set by the axonal anatomy and enzymatic machinery, I optogenetically manipulated viable

DAergic VTA/SNpC inputs to CA1, in-vitro. The main advantage of optogenetics is that it provides cell-type and temporal specificity to neuron

48 control (Boyden et al., 2005). The light activated protein channelrhodopsin2

(ChR2) can be expressed in a genetically identified neuron type of choice.

And then, in that neuron, light will gate the channel and produce an inward mixed cation current. Optogenetically driven axons in the slice can release neurotransmitter with high temporal precision. A brief pulse of 470nm light can reliably gate a ChR2 conductance, produce a rapid depolarization of the membrane to threshold, and generate action potential triggered exocytosis

(sensitive to TTX) in severed axons present in slices (Cruikshank et al.,

2010). This method has been used to drive action potential firing in DAergic neurons in the VTA at the soma; the kinetics of the channel are fast enough to capture tonic and phasic (Figure 2.2) modes of firing (Tsai et al., 2009).

Optogenetic control of DA neurons allows us to ask how the VTA/SNpC DA signal alters SC and PP synaptic transmission to CA1 in an in-vitro slice preparation with greater clarity.

Figure 2.2. ChR2 guided spikes in a VTA DA neuron.

Adapted from Tsai et al., 2009

Results

The goal of my experiments is to compare the effects of DA when applied through the bath solution with the effects of DA released by optogenetic activation of VTA/SNpC axons in the hippocampal slice preparation. First, I repeated the bath application experiments to insure that results in mouse brain slices are analogous to those in rat slices used in the aforementioned synaptic transmission experiments. I prepared acute transverse sections of the hippocampus using a horizontal slice angle (shifted

12 degrees from the true horizontal axis) in order to preserve the intrinsic circuitry of the hippocampus: the PP inputs and trisynaptic loop. I then used whole cell current clamp recordings from CA1 pyramidal neurons to measure evoked synaptic potentials in response to electrical stimulation of the SC and

PP input pathways using bipolar stimulating electrodes placed in SR and

SLM, respectively (Figure 2.3).

Identified pyramidal neurons exhibited PP and SC synaptic potential waveforms consistent with the literature. SLM stimulation of the PP axon bundle evoked a depolarizing postsynaptic potential (PSP) followed by a strongly hyperpolarizing response, due to powerful feedforward inhibition. SR stimulation of the SC axon bundle evoked a larger depolarization and smaller inhibition. The PP and SC stimulating pulses were each applied at a 900 ms

51 interval to prevent cross-input contamination (Remondes and Schuman,

2002).

After establishing a baseline measure of the PSP, I bath-applied 20 M

DA. As has been shown in previous experiments in rat (Otmakhova and

Lisman, 1999; Ito and Schuman, 2007; Ito and Schuman, 2009), 20 M DA applied in the perfusion medium reduced the peak PP-evoked synaptic depolarization of CA1 PNs to 45% +-11% of baseline (n = 4, p = .01, paired t- test; Figure 2.3). In contrast, DA had no effect on mean SC-evoked depolarization (96% +-12% of baseline, n = 9, p = .59, paired t-test; Figure

2.3). Note that there was a greater degree of variability in the effect of DA on the SC-evoked response in individual experiments compared to the consistent depression of the PP-evoked PSP despite there being no net change in the mean SC PSP (Figure 2.3). For more detailed methods, see Chapter 5.

PP 2.5 SC e

d 20 M DA 2.0 u

t i

l 2.0 p m

a 1.5

P 1.5 S P

d 1.0

e 1.0 z i l a m 0.5 r 0.5 o n DA+ 0.0

0.0 ) .

-5 0 5 10 15 20 n

DA- i m

Time (minutes) 0 1 (

C S PP PP (10min.)

Figure 2.3. A. 20 µM DA suppresses the PP peak PSP and leaves the

SC peak PSP unchanged.

Figure 2.3. A. 20 µM DA suppresses the PP peak PSP and leaves the

SC peak PSP unchanged. Top panel,electrode configuration. Bottom panel, left, circles represent the PSP amplitude normalized to the average of the baseline 5 minutes prior to drug application and averaged across 3 time points. B, Top, dark trace shows SC-evoked PSP before DA application. Light trace shows SC PSP 10 minutes after DA application. Bottom, dark trace shows PP PSP prior to DA application. Light trace shows PP PSP 10 minutes after DA application. Representative traces are averages taken from the time period prior to drug application and 10 minutes post application from an experiment characteristic of the population mean. Right, population data for all cells showing fold change in peak SC and PP PSP amplitude following DA application (normalized to PSP prior to DA).

Bath application of DA in the perfusion medium creates a uniform concentration of DA in the slice and activates whatever constellation of DA receptors is present. To ask how the VTA/SNpC DAergic input alters hippocampal synaptic transmission, I utilized optogenetic tools to drive action potentials in the VTA/SNpC DA neurons’ axons present in CA1, and thereby recapitulate the in-vivo response to activity in the VTA/SNpC-CA1 circuit under in-vitro conditions.

To express ChR2 in the DA neurons alone, I used a viral strategy combined with mouse genetics to target the ChR2 protein to the DA neurons of VTA and SNpC. I used the TH-Cre driver line (Jackson labs, 008601,

Savitt et al., 2005), which expresses the Cre recombinase enzyme under the control of the tyrosine hydroxylase (TH) promoter. Tyrosine hydroxylase is an enzyme in the biosynthetic pathway for DA, NE, and epinephrine and so all neurons in the brain that synthesize these molecules will express Cre. I gained functional control over the VTA/SnPC DA neurons by stereotactically delivering into the midbrain (figure 2.4 A) a Cre recombinase inducible adeno-associated viral vector (AAV) carrying the gene encoding the ChR2-

EYFP fusion protein within a double-floxed inverted open reading frame

(Atasoy et al., 2008), wherein the ChR2-EYFP gene construct is present in the anti-sense orientation. Cre recombinase flips the orientation of ChR2-

EYFP , thereby enabling its continuous expression under the strong and ubiquitous EF-1α promoter.

Since the Cre is under transcriptional control of the tyrosine hydroxylase promoter it is restricted to monoaminergic neurons. The restricted spread of the stereotactically injected virus keeps the noradrenergic neurons from becoming transduced. Neurons across the extent of VTA and SNpC were infected as indicated by YFP fluorescence and positive co-staining for the monoamine neuron marker, the enzyme TH

(Figure 2.4 B and C). These neurons projected to the hippocampus (Figure

2.5).

The majority of VTA/SNpC axons primarily course through SO of the

CA1 region and stained positively for tyrosine hydroxylase (Figure 2.5A and

B), as has been reported by previous anatomical tracing and immunohistochemistry experiments (Verney et al., 1985; Kwon et al., 2008;

Gasbarri et al. 1994). There are also a few axons in SP in some areas, but this was inconsistent (figure 2.5 A and B). I saw few if any axons that reached SR and none in SLM.

In contrast to results with bath application of DA, stimulation of the

VTA/SNpC axons with a brief light flash suppressed the SC-evoked PSP.

Similar to the bath application experiments,I recorded from CA1 PNs in acute hippocampal transverse slices using the whole cell current clamp configuration and evoked postsynaptic potentials (PSPs) by electrically stimulating PP and SC input pathways. After recording PSPs for xx min to establish a baseline level response, the slice was exposed to full field 470 nm

Figure 2.4. YFP fluorescence and TH staining in the midbrain. A.

Midbrain VTA and SNpC dopamine areas of a Th -Cre transgenic mice were infected with ChR2-EYFP with an AAV packaged with a flexed EF1 α::ChR2-

YFP construct. B. Confocal 5x image of ChR2 -EYFP fluoresc ence signal

(green) in the ventral tegmental area (VTA) and substantia nigra (SNpC) of the midbrain of TH-Cre mice injected with flexed EF1 α::ChR2-YFP AAV. C.

20x magnification image of the VTA neurons infected with ChR2 -EYFP

(green) and stained for TH (re d).

Figure 2.5. YFP fluorescence and TH staining in the CA1 field of the hippocampus. A. An example image taken from a slice of a TH -Cre mouse that had been injected in its midbrain with the rAAV (EF1a::DIO - hChR2(H134R)-EYFP-WPRE -hGH) . Hippocampal slices were obtained from the brains of these mice for electrophysiological recordings. 20X confocal images were taken from slices After recordings slices were post -fixed in PFA labeled with an anti-GFP antibody, and then stained with a secon dary ab.

Green- anti-GFP ab fluorescence. Blue -DAPI counterstain. B. Another example.

illumination using a single, brief burst of light flashes (3 pulses each with a 5 ms duration delivered at a 15 ms interpulse interval) (figure 2.6 A).

The light burst did not have any immediate effect on the resting membrane potential (Vm) (figure 2.6 A), suggesting that these axons did not release neurotransmitters that caused significant activation of ionotropic glutamate or GABA receptors on the CA1 neuron, although glutamate and

GABA have been found to be co-released with DA in other brain areas (Sulzer et al., 1998; Tritsch et al., 2012). The light pulse protocol also failed to produce any slow change in input resistance over the course of 20 minutes

(figure 2.6 B), suggesting that DA did not modulate ion channels that contribute to the resting properties of the CA1 pyramidal neuron soma.

Figure 2.6. Unitary light burst protocol did not affect input resistance A. Schematic of the unitary burst light pro tocol, which consisted of 3 pulses (5 ms each) given at 66 Hz. 470 nm flashes of light were delivered using an LED focused through a 40x objective to achieve “full field illumination”. The resting Vm did not exhibit any changes during the light stimulati on. B. Input resistance stayed constant over the course of the experiment. Pre = five minutes prior to the light burst protocol administration. Post = 15 minutes following light protocol administration.

The single light burst produced a remarkably long-lived suppression of the SC evoked depolarizing component of the PSP in CA1 pyramidal neurons to 42 +- 9% of baseline (n = 11, p =.0001, paired t-test; figure 2.7) after 10 to

15 minutes. The effect took five minutes to set in and then continued to increase until the PSP reached a stable level 15 minutes following light stimulation. The PSP did not return to initial baseline level for as long as the recordings lasted (up to xxx min). In contrast, the PP evoked PSP was unaffected by the light pulses (111 +-13% of baseline, n = 10, p = .42, paired t- test; Figure 2.7), indicating that the suppression did not result from a degradation of slice health or recording quality. The effect size scaled with light intensity (Figure X) and lasted for the entire 20-minute recording period. This inhibitory effect on the depolarization of the soma is input specific, like the effect of bath application, but it shows the opposite input profile.

B C D

Figure 2.7. Photostimulation of the VTA/SNpC DAergic afferents suppresses the SC synaptic depolarization of CA1 and spares the PP .

A. Electrode configuration for measuring evoked synaptic potentials along the

PP and SC pathways. B. Blue arrow indicates the time point at which the unitary burst protocol was administered. Each circle is an average of 3 trial

PSP amplitudes. Blue circles represent PP PSP amplitudes normalized to average of the baseline (N = 7). Black circles represent normalized SC PSP amplitudes (N = 12). Error bars indicate the standard error of the mean. Only neurons in which PP stimulation elicited an early net depolarizing PSP were included. C. SC evoked PSP waveforms before (black trace) and after (gray trace) photostimulation. Minus symbol demarcates the baseline PSP waveform. Plus symbol demarcates the PSP waveform following photostimulation. PP synaptic potentials before (dark blue trace) and after

(aqua trace) photostimulation. D. Distribution of normalized PSPs 10 minutes following photostimulation for both SC and PP PSPs.

VTA/SNpC stimulation releases DA and activates the D4

receptor to suppress the the SC excitation of CA1

How does the stimulation of TH+ VTA/SNpC axons alter synaptic transmission along the SC pathway to CA1 pyramidal neurons? What

63 neurotransmitter system and receptor accounts for the suppression of the SC- evoked depolarization? I applied a battery of pharmacological agents to determine the receptor responsible for the suppressive effect. When the selective D2-type and D1-type antagonists, haloperidol (2 M) and SCH23390

(20 M), respectively, were included in the perfusion medium, the effect of photostimulation on the SC PSP was abolished (116 +- 15% of baseline, n = 5, p = .456, paired t-test; Fig. 2 B 3), suggesting that VTA/SNpC stimulation via brief optogenetic depolarization releases DA to activate D1-type or D2-type receptors.

Blockade of only D1-type receptors with SCH23390 (20 M) failed to block the ability of photostimulation of VTA/SNpC axons to suppress the SC- evoked PSP, which was reduced (by or to) 58 +-5% of its baseline level (n = 5, p = .005, paired t-test, Fig. 2, B 1), similar to the photostimulation response in the absence of the D1 antagonist. In contrast, application of the D2-type receptor antagonist haloperidol (2 M) fully blocked the effect of photostimulation to reduce the SC-evoked PSP, which was equal to 114 +-

26% of its initial baseline level (n = 6, p = .365, paired t-test; Fig. 2, B 3). This suggests that photostimulation of TH+ fibers suppresses the SC depolarization of CA1 neurons by releasing sufficient neurotransmitter to activate D2 type receptors.

Haloperidol has high affinity (Ki < 10 nM) for all three D2 receptor subtypes (Seeman and Von Tol, 1994). Therefore, the above experiment

64 cannot distinguish between D2 subtypes. Since the D4 receptor is the most ubiquitously expressed D2-type receptor in the hippocampus (Defagot, 1994),

I examined its importance using the specific D4 receptor subtype antagonist

L745,870 (200 nM). Selective D4 receptor blockade completely abolished the effect of photostimulation (Figure 2.8D), suggesting that DA released by

VTA/SNpC axons activates the D4 receptor to suppress the SC depolarization of CA1. However, I cannot completely rule out the possibility that photostimulation of VTA/SNpC axons release a neurotransmitter other than

DA that acts as an agonist for the D4 receptor.

Figure 2.8 . Optogenetic stimulation of VTA fibers reduces SC depolarization of CA1 by activating D2 -type receptors.

Figure 2.8 . Optogenetic stimulation of VTA fibers reduces SC depolarization of CA1 by activating D2-type receptors.

A) Electrode configuration and relevant circuit diagram.

B) SC synaptic potentials waveforms before (black traces) and after (gray traces) light protocol. B 1) Representative traces of photorelease effects in the presence of D1/D5 antagonist SCH23390 (20 M; vertical scale bar, 4mV; horizontal scale bar 20ms), B 2), D2/D3/D4 antagonist haloperidol (20 M), vertical scale bar, 1mV; horizontal scale bar, 20ms, and B 3) both drugs

(vertical scale bar, 4mV; horizontal scale bar, 20 ms. C) PSP amplitudes measured over time. D) D1+ D2 antagonists (N = 5, paired t-test, P = .45).

Haloperidol (N = 6, paired t-test, P = .36). SCH23390 (N = 5; paired t-test, P =

.005). NE is a noradrenergic receptor antagonist cocktail consisting of 5 M proporanolol, 10 M prazosin, and 10 M yohimbine (N = 4; paired t-test, P =

.03).

Discussion

My experiments demonstrate that brief stimulation of the VTA/SNpC pool of

DAergic axons suppresses the SC depolarization of CA1 PNs through activation of the D4 receptor. This runs counter to the standard model, which posits, based on bath application of DA, that the VTA/SNpC DAergic input suppresses the SC-evoked depolarization of CA1 and leaves the SC-evoked- depolarization intact. Other effects have been observed, weakening this model, such as Hsu (1996), who reported that DA suppresses the PP-evoked synaptic depolarization, but the most consistent effect, and the one that I replicated in my own experiments, is that bath-applied DA does indeed suppress the PP-evoked depolarizing PSP recorded in CA1 pyramidal neurons (Otmakhova and Lisman, 1999; Ito and Schuman, 2007; 2011).

Hsu (1996) measured the SC synaptic input with whole cell recording, while Ito and Schuman (2007) and Otmakhova and Lisman (1999) measured the field EPSP. PSPs measured in whole cell recordings are more sensitive to changes in somatic inhibition compared to field potentials, which are dominated by local synaptic currents. If the effect of DA on the SC PSP is driven by alterations in inhibition onto the CA1 pyramidal cell soma, then only the whole-cell measurements would capture it. Also, the whole-cell recording procedure involves dialyzing the cell with an artificial cytosolic pipette solution. This could have altered key signaling molecules and

68 disrupted the DA receptor function. However, my experiments using whole- cell recording match the results obtained from field potentials (Otmakhova and Lisman, 1999).

The standard model attempts to fit the suppressive effect of DA on the

PP PSP to the facilitative role that the VTA/SNpC axons and the DA receptors have on memory (Grace and Lisman, 2005). It is argued that the saliency signal from VTA/SNpC neurons suppresses the contextual sensory

PP input to CA1, resulting in the boosting of the CA3 mnemonic input to

CA1. Thus, when salient stimuli emerge from the environment, they can induce more efficient plasticity at the SC CA3-CA1 synapses and thereby aid in memory encoding. However, this view operates under the assumption that effects with bath-applied DA provide a reasonable approximation to the effect of the physiological engagement of the VTA/SNpC DAergic input.

I find, in contrast, that optogenetically guided VTA/SNpC stimulation, using just three brief pulses of light, releases DA in a way that leads to a long-lasting suppression of the SC synaptic input to CA1 and leaves the PP synaptic input unchanged. This very modest stimulation protocol is nonetheless able to activate the relatively high affinity D4 receptor (Seeman and Van Tol, 1994), which I find underlies the suppressive effect on the SC

PSP. The D1-type receptor seems not to be involved. Thus, at low levels of activity, the VTA/SNpC axon suppresses the strength of the overall synaptic excitation from CA3 to CA1 via the SC pathway.

Noradrenergic neurons also express TH and the D4 receptor does have some affinity for NE, albeit 50 fold lower than for DA (Newman-Tancredi et al., 1997). It remains possible, then, that the light evoked effects are due to release of NE from LC axons. This was not the case as the suppression of the

SC-evoked synaptic depolarization persisted in the presence of a cocktail of alpha and beta antagonists: prazosin, yohimbine, and propranolol (49 +-6% of baseline, n = 3, p = .03, paired t-test; Figure 2.8D). The most parsimonious explanation for these data is that a few action potentials in the pool of

VTA/SNpC DAergic axons in SO of CA1 releases sufficient DA to activate D4 receptors in proximity and these D4 receptors suppresses the SC synaptic depolarization of CA1. VTA/SNpC axons, while sparse in number, exert a powerful input specific inhibitory effect on the transfer of information from

CA3 to CA1, while leaving the PP synaptic input intact.

The PP and SC axons have different functions. Their inputs are segregated along the dendritic tree of CA1 PNs, where the PP axons synapse on the apical tuft dendrites in SLM and the SC axons synapse on the oblique proximal dendrites in SR. Corresponding to the input segregation, the dendritic structure compartmentalizes into molecularly segmented regions with distinct electrical properties (Lorincz et al., 2002; Tsay, 2007) in SR and

SLM. The fact that these inputs are modulated differentially by DA release from VTA/SNpC axons gives these pathways an additional layer of independent modulation.

Fittingly, the PP and SC subserve distinct aspects of learning and memory. PP axon lesions cause profound memory consolidation deficits and altered place cell tuning (Remondes and Schuman, 2004; Brun et al., 2008.)

Genetic lesion of the medial PP impairs trace fear conditioning (Suh et al.,

2011). On the other hand, novelty based memory effects are more reliant on the SC input (Nakashiba, 2008). Genetic lesion of the SC pathway through tetanus toxin expression in CA3 neurons did not impair spatial memory on the water maze, but did significantly diminish one trial fear-conditioning in a novel context (Nakashiba et al., 2008). Place fields in a novel context were much more diffuse in the CA3-lesioned mice and the network was generally overly excitable. Interestingly, the SC lesion did not impair one-trial fear conditioning in a familiar context. CA3, then, is critical for forming memories in novel environments. The VTA/SNpC neurons fire phasically to novel stimuli and therefore may produce an “iceberg effect”, setting the threshold for memory storage, whereby global network suppression allows only those

CA1 neurons that receive the strongest presynaptic input to reach threshold and participate in memory encoding.

What accounts for the difference between exogenous DA application and VTA/SNpC release of DA? Our bath application experiments, which also show the PP suppression, confirm that this is not due to species differences between rat and mouse. The most likely explanation for the discrepancy is anatomical. The PP inputs in SLM are hundreds of microns away from where

71 the VTA/SNpC DAergic axons ramify in stratum oriens (SO). Although this indicates that the receptors predicted to be present in SLM are capable of influencing the circuit, because of their distance from the axons, it is unlikely that these receptors are activated by VTA/SNpC released DA within a physiological range of activity. Indeed the distance of SO from SLM is generally considered to be outside the bounds of the DA diffusion radius, which has been estimated to be between 10 m (Cragg et al., 2001) and 50 m

(Fuxe, 2010). It is much more likely for VTA/SNpC DAergic axons to modulate targets within this diffusional range, including the commissural excitatory SC inputs to CA1, located in SO, and local inhibitory interneurons within SO and SP that participate in feedforward and feedback inhibition.

Whereas the lack of effect of optogenetic release of DA from VTA/SNpC terminals on PP-evoked PSPs can be accounted for by the underlying anatomy, it is less clear why bath applied DA fails to recapitulate the effect I observe with optogenetic release of DA to suppress the SC PSP. In fact, Hsu

(1996) did report a DA-induced suppression of the SC PSP that was mediated by D2-type receptors. Hsu (1996) used a lower DA concentration, 0.1 – 1 M, compared to the usual concentration of 20 M. However, Otmakhova and

Lisman (1999) did assess the action of DA concentrations as low as 1 M, and failed to observe any effect on the SC-evoked field PSP. It seems then, that if the suppressive effect on the SC-evoked synaptic depolarization by

VTA/SNpC DA in our experiments is similar to the effect observed by Hsu

(1996), it may require a low concentration of DA and may only be observed in whole-cell recording conditions. At the standard concentration of 20 M, the

D4 receptor signaling may desensitize or trigger parallel facilitative effects on the PSP.

What are the DA receptors doing in SLM if the DA released from

VTA/SNpC axons cannot activate them? These receptors are clearly functional as they strongly suppress the PP-evoked PSP. If they are not normally activated by input from VTA/SNpC neurons, what is the source of transmitter that engages these receptors in vivo? There are several possibilities. First, the DA receptors in SLM may show a low but measurable degree of spontaneous activity in the absence of ligand, resulting in weak activation of downstream G protein signaling pathways that would serve to maintain a slightly depressed basal synaptic state. Second, the receptors in

SLM may be activated under physiological conditions by NE released from locus coeruleus axons that course through SLM. Third, based on evidence from amphetamine application experiments, the DA in SLM may be supplied by the locus coeruleus neuron terminals, that are known to release DA, especially at high firing rates (Smith and Greene, 2012). Finally, the DA receptors in SLM may not be engaged under physiological conditions but are only activated through exogenous agonist application.

What could be the function of the depression elicited by VTA/SNpC DA release? The DA neurons shift from tonic (1-4 Hz) to phasic firing mode (10-

30 Hz) upon encountering salient stimuli. From a series of studies in the ventral striatum, it has been hypothesized that the tonic mode of DA firing maintains a basal DA tone in the nM range that activates the higher affinity

D2 type receptors (Goto and Grace, 2005). Phasic DA firing events induce a fast, transient, and compartmentalized rise in DA concentration to the low

M to mM range that enables the activation of the lower affinity D1 type receptors.

If this holds true in the hippocampus then the network-wide synaptic suppression observed here may reflect the basal state of the CA1 synaptic input space under DAergic tone. Such a persistent suppression of the SC input during extended periods in familiar contexts could prevent synaptic plasticity events from occurring in response to non-novel stimuli. However, since the hippocampus has very low levels of the dopamine transporter DAT

(Kwon, 2008), the striatal system may not be comparable. We cannot argue conclusively from our data that the effect of a single burst of photostimulation represents the effects of tonic or phasic DA neuron firing, but it is widely thought that D2 receptors are tonic mode detectors, given their higher affinity for DA (Goto and Grace, 2005; Palmiter, 2008).

A final possibility is that the suppressive effect of DA on the SC- mediated excitation of CA1 pyramidal neurons contributes to a general stabilization of a highly recurrent hippocampal network prone to epileptiform activity.

D4 is the least well understood of the DA receptors and the most ubiquitously expressed D2 type receptor in the hippocampus (Defagot, 1997).

D4 garnered significant interest in schizophrenia research when it was discovered that it is clozapine’s principal target (cite). The DA hypothesis of schizophrenia itself, that the brain’s DA systems are overactive (Seeman and

Van Tol, 1993), stems from the fact that neuroleptic drugs block D2-type receptors. The potential importance of the D4 receptor is underscored by the finding that its density in brain tissue of human patients with schizophrenia is 600% higher than healthy controls (Seeman, 1992).

D4 receptor activation had previously been shown to depotentiate the

SC evoked synaptic depolarization of CA1, after prior induction of LTP (Kwon et al., 2008). This effect occurred when D4 was activated by endogenous DA release triggered by neuregulin1 application. But the D4 agonist could also produce the effect in the absence of neuregulin1 signaling. Our result is somewhat reminiscent of this effect in that it represents a decrease in SC- mediated excitation, but the effect I have described does not require an evident potentiated synaptic state. To get an idea of how this novel

VTA/SNpC DAergic effect relates to the psychopathology of schizophrenia will require further investigation, perhaps using some of the various mouse models of this disease.

Chapter 3

Introduction

How does D4 receptor activation induced by DA release from

VTA/SNpC neurons reduce the SC synaptic depolarization of CA1? The CA3-

CA1 SC synapse is a canonical example of a feedforward excitatory circuit under tight regulation by inhibition (Buzsáki, 1984). The CA3 principal neurons’ glutamatergic SC axons terminate in SR and SO of CA1 where they form synapses onto excitatory CA1 pyramidal neurons’ apical and basal dendrites and onto a host of inhibitory interneurons.

Upon firing action potentials, CA3 releases glutamate onto the CA1 principal cells, gates ligand gated glutamate receptors, primarily AMPA and

NMDA, in the dendritic spine membrane, to produce the inward current and depolarization of the membrane measured as the PSP in current clamp recordings. The interneurons that also receive SC glutamatergic input will also get depolarized, fire action potentials, and release GABA onto the CA1 pyramidal neurons. GABA A and GABA B receptor activation generates an

IPSP in the CA1 neuron that sufficiently overlaps in time in the CA1 soma with the glutamatergic EPSP to reduce the amplitude and sharpen the peak

76 of the PSP waveform (Pouille and Scanziani, 2001). Thus, the SC evoked PSP waveform in the CA1 neuron is a composite of a monosynaptic ally generated glutamatergic EPSP and a disynaptically generated GABAergic IPSP.

Therefore, VTA/SNpC DAergic stimulation can reduce the PSP in CA1 by either directly suppressing SC excitatory transmission or by increasing inhibitory transmission via feedforward inhibitory circuits.

Anatomically, VTA/SNpC axons are positioned to modulate both excitatory and inhibitory circuits. SO and SP, where these DAergic axons terminate, contain multiple types of inhibitory interneurons’ axons, dendrites, and somata, as well as the CA3 commissural axons and the basal dendrites of the CA1 principal neurons. Both excitatory and inhibitory microcircuits can be modulated by DA in the cortex (Zhou and Hablitz, 2002;

Yang, 2002) and DA receptors are present on both excitatory and inhibitory neurons in the hippocampus (Gangarossa et al., 2012) and in the cortex

(Mrzljak, 1996).

The depression of the PP-evoked synaptic depolarization induced by

DA bath application does not require inhibition, as has been reported in the literature (Otmakhova and Lisman, 1998). However, the effect observed by

Hsu (1996), the suppression of the SC-evoked PSP was reduced in the presence of picrotoxin a GABA A antagonist. Moreover, one possible explanation for why this suppression of the SC-evoked PSP (Hsu, 1996) was not observed in field recordings is that it results from somatic inhibition.

In this Chapter I explore the synaptic mechanism whereby photoactivation of VTA/SNpC axons in acute hippocampal slices with a single brief burst of light (3 ms-long pulses) results in a long-lasting suppression of the SC-evoked depolarization of CA1 pyramidal neurons. My results demonstrate that this effect is caused by an enhancement of feed-forward inhibition from PV+ basket cells onto CA1 PNs. Moreover I find that this effect is mediated by an increased synaptic excitation of the PV interneurons by their Schaffer collateral inputs.

Interneuron diversity

Inhibition in the hippocampal network has many different functions at various levels of resolution. Excitatory principal cells, in the vast majority of cases 6, direct the downstream areas’ activity as they are responsible for the continuity of information flow through the brain. However, the range of possible transformations that can be made on that information locally is expanded widely by inhibition.

GABAergic conductances can lead to inhibition in two ways. First they shunt the target cells’ postsynaptic membrane to the reversal potential of the

GABA receptor, which is close to -75mV in adult CA1 pyramidal neuron.

Thus, this conductance is normally inhibitory with respect to the action

6 Long range interneurons are rare, but likely very important for synchronizing distantly situated networks .

78 potential threshold. In addition, activation of GABAergic conductance can alter the resting Vm, which may be excitatory or inhibitory, depending on the difference between the Vm and the GABA receptor reversal potential.

However, in adult hippocampal pyramidal neurons the reversal potential is normally at or negative to the resting potential. As a result, such conductances usually generate hyperpolarizing currents (Glickfield et al.,

2009) across the entire somatodendritic axis.

Inhibition is carried in the hippocampus by an incredibly diverse array of interneurons. These neurons primarily release GABA as their neurotransmitter, but can also co-release different neuropeptides. The importance of interneurons is underscored by the extraordinarily wide array of GABAergic interneurons types in the hippocampus, classified by a combination of molecular, neurochemical, morphological, electrophysiological, functional, and connectivity characteristics. At least 21 types of interneurons in the hippocampus have been identified (Freund and Buzsaki, 1996;

Somogyi and Klausberger, 2008).

In mature pyramidal neurons GABA receptor conductances constrain the amplitude and kinetics of coincident subthreshold EPSPs (Pouille and

Scanziani, 2001), which has powerful effects on the rate and timing of spike output (Losonczy et al., 2010; Lovett-Barron, 2012; Royer et al., 2012). For the network as a whole, such actions are critical for maintaining its overall stability (Schuler et al., 2001), gain control of inputs (Mitchell and Silver,

2003), gating of inputs, and entraining networks to synchronize their outputs

(Cobb et al., 1995; Cardin et al., 2009; Bartos et al., 2007). The different interneurons divide the labor amongst them, providing the circuit with a wide range of possible inhibitory operations (Lovett-Barron, 2012;

Klausberger, 2003; Wilson et al., 2012; Klausberger and Somogyi, 2008). It is therefore, critical that the type of interneuron targeted by VTA/SNpC DA is identified as it will allow application of the results to the function of

VTA/SNpC DA in-vivo, during learning.

Many of the interneuron types that receive inputs from the SC pathway, such as the PV+ soma-targeting basket cells and axo-axonic cells, somatostatin+ and PV+ dendrite targeting bistratified cells, cholecystokinin+ basket cells, SC-associated interneurons, and ivy cells are well positioned to modulate the SC-evoked PSP as they send their own axons to the CA1 soma, dendrite, or axon initial segment. Moreover, some of these interneurons can be distinguished by the expression of particular GPCRs, indicating their ability to be dynamically controlled by motivationally sensitive extrinsic modulatory systems (Freund). Inhibition, then, likely does not provide a fixed input/output modulation of the circuit. Rather, its modulatory range may be altered by a supervisory layer of control from neurotransmitters released during various behavioral states. Moreover, it is known that neuromodulators exert powerful control over particular interneuron classes.

Muscarinic receptors are expressed in somatostatin+ and PV+ interneurons.

The CCK+ basket cell population expresses 5-HT3, estrogen, and cannabinoid receptors, while the Ivy cells and PV+ interneurons seem to uniquely express the -opioid receptor on the axon terminal, which silences their output.

DA receptor expression patterns are hard to determine based on immunohistochemistry and the subcellular and cell-type expression patterns are controversial. Yet, there seems to be a preponderance of evidence favoring a role for D2-type receptors in modulating PV+ interneurons. In the prefrontal cortex, GABAergic interneurons showing the characteristic fast- spiking behavior of the parvalbumin positive (PV+) population express D2 type receptors (Le Moine and Gaspar, 1998) and D2-type receptors have been shown to modulate their excitability (Tseng and O’Donnell, 2007) and release probability (Zhou and Hablitz, 1999). The majority of studies indicate that the D4 receptor protein and transcript are restricted to superficial SP and SO of CA1 (Defagot et al., 1994; Mrzljak et al., 1996), where PV+ cell somas are located. In both the CA1 and the neocortex, most D4 expressing cells were found to be interneurons (Andersson et al., 2012; Mrzljak et al., 1996) and

75% of cells displaying D4 immunoreactivity are PV+ (Andersson et al.,

2012). Therefore, it appears that the D4 receptor is poised to modulate PV+ inhibition. Additionally, the D4 agonist increases kainate-induced gamma oscillatory power in CA3 (Andersson et al., 2012), a process thought to be

81 controlled by the PV+ interneuron population (Cardin et al., 2009; Penttonen et al., 1998).

Results

To distinguish whether the suppression of the SC PSP in CA1 PNs results from a suppression of the EPSP or an enhancement of the IPSP, I performed a series of experiments in which inhibition was blocked using antagonists of GABA A and GABA B receptors, SR95531 (2 M) and CGP 55845

(1 M), respectively.

Using the same TH-Cre mouse from the previous set of experiments, I virally expressed ChR2 in the DA neurons of VTA and SNpC, and repeated the previously described (in Chapter 2) optogenetic electrophysiology experiments, using photostimulation with a unitary burst of light to activate the ChR2+ DAergic axons from VTA/SNpC DAergic axons present in the acute hippocampal slice. I found that inclusion of GABA receptor antagonists in the slice perfusion medium eliminated the suppressive effect of optogenetic

VTA/SNpC stimulation on the SC-evoked PSP in the CA1 pyramidal neuron.

Following the light protocol, the PSP was unchanged (119 +-19% of baseline, n = 7, p = .24, paired t-test; Figure 3.1), suggesting that VTA/SNpC release of

DA suppresses the SC-evoked PSP by enhancing rapid feed-forward inhibition . As I previously found that the suppression of the SC PSP requires

D4 receptor activation, the enhanced inhibition should also require this receptor.

Figure 3.1: VTA/SNpC stimulation suppresses the SC -evoked depolarization of CA1 pyramidal neurons by enhancing inhibition.

A) Electrode configuration and relevant circuit diagram. B) Under GABA A and GABA B blockade using SR-95531 and CGP-35348 optogenetic stimulation has no effect on the SC synaptic potential. Error bars represent standard errors of the mean. Black circles are averages of 3 points and therefore represent the PSP amplitude over the course of 1 minute. The blue arrow demarcates the time point at which the single burst optogenetic stimulation was administered. C) Representative traces taken from before (black) and 10 minutes following (gray) VTA/SNpC optogenetic stimulation. Vertical scale bar 2mV; horizontal scale bar, 20 ms. (N = 9).

If VTA/SNpC suppresses the SC PSP by increasing inhibitory conductances then the IPSC evoked by the SC synaptic drive of feedforward inhibitory circuits ought to increase. In the same preparation as above, I patch clamped CA1 pyramidal neurons in the whole cell voltage clamp configuration, and directly measured the SC evoked IPSC by setting the command potential of the CA1 pyramidal neuron near the reversal potential for excitation (at +10 mV). At this membrane potential there is little or no electrochemical driving force on the cations carrying the EPSC. With my recording solutions, the GABA A receptor IPSC has a predicted reversal potential near -76 mV so GABA A activation should produce a strong outward current at + 10 mV. We substituted Cs + for K + in our internal solution to improve voltage control. The Cs + blocks several of the voltage gated K + channels, enhances the resistivity of the membrane, and thus allows for voltage control of membrane in more distal dendritic processes (Spruston et al., 1993), although the control will still not be ideal (Williams and Wozny,

2011).

Under these conditions, activation of the SC axons with a single current pulse from an extracellular stimulating electrode placed in SR evoked a large fast outward current in the CA1 PNs. This current was fully blocked by GABA A and GABA B antagonists, indicating that the currents represent a

GABAergic IPSC. The stimulating electrode is placed in SR in these

86 experiments, far away from the pyramidal cell to avoid activating directly any local interneurons near the recorded PN.

As in the measurements of resting Vm, light pulses evoked no observable fast current response under voltage clamp (Figure 3.2), suggesting that any GABA co-release from the DA terminals has little ability to activate

GABAA receptors in the CA1 PNs, in contrast to what is reported in other systems (Tritsch et al., 2012). As predicted from the current clamp experiments, VTA/SNpC stimulation produced a nearly 2-fold increase in the

SC evoked IPSC to (187 +-37% of the baseline, n = 12, p = .02, paired t-test;

Figure 3.2). On several occasions, the IPSC waveform converted from a single peaked event to a multipeaked event waveform. These results provide direct support for my hypothesis that DA release from VTA/SNpC neurons enhances SC-activated feed-forward inhibition of CA1 PNs.

We applied NBQX to verify that the IPSC was indeed evoked by SC action potentials releasing glutamate onto interneurons that then integrate the depolarization and drive a GABAergic IPSC onto the CA1 pyramidal cell.

NBQX flattened the IPSC waveform (Figure 3.2). Therefore, the effect depends on the excitatory transmission from the SC and is not due to direct inhibition: the unphysiological charging of inhibitory axons that synapse on

CA1 pyramidal neurons, but do not ordinarily receive any SC input. The

VTA/SNpC DAergic axons produce an inhibition dependent reduction of the

SC PSP and enhance the feedforward inhibitory drive. Together, this

87 suggests that stimulation of the VTA/SNpC DAergic axons reduces the SC- evoked PSP because SC driven disynaptic feedforward inhibition becomes enhanced and reduces the peak of the excitatory potential through shunting or hyperpolarizing inhibition in the CA1 soma or dendrite.

3 Pre Post NBQX 2

200 pA

1 0.02 s

Normalized IPSC amplitude IPSC Normalized 0 0 5 10 15 Time (minutes)

Figure 3.2: VTA SNpC stimulation enhances SC -evoked inhibitory currents in CA1 PNs.

Figure 3.2: VTA SNpC stimulation enhances SC-evoked inhibitory currents in CA1 PNs. A. Electrode configuration and circuit schematic for recording SC-evoked inhibitory currents in CA1 pyramidal neurons and driving VTA/SNpC DAergic axons. B. The single burst of light produced no fast currents in CA1 pyramidal neurons at a holding potential of +10mV. C.

Black circles represent amplitude measurements of SC evoked IPSCs. CA1 whole cell recording in voltage clamp mode at a holding potential of +10mV.

Intracellular solution included cesium. The blue arrow indicates the point at which the photostimulation protocol was given. The error bars represent standard errors of the mean. D. The minus symbol is above the IPSC trace taken from the baseline period (black trace); the plus symbol is above the

IPSC trace taken 10 minutes after photostimulation (gray trace). Vertical scale bar, 200pA; horizontal scale bar, 20 ms.

DA release suppresses the SC drive onto CA1 by enhancing a

PV+ interneuron mediated feedforward inhibition circuit

Which of the interneuron subtypes that participate in feedforward inhibition does VTA/SNpC DA release modulate? Based on the immunohistochemistry, in-situ hybridization, and physiology described above, the PV+ basket cell population is a likely candidate. As an initial strategy I utilized a pharmacological approach to probe the role of the PV+ fast spiking IN population. The -opioid receptor is strongly expressed on the axons of two distinct interneuron subtypes that receive input from the SC axons: the PV+ fast-spiking basket cells (Drake and Milner, 2002) and NPY+ ivy cells (Krook-

Magnuson, 2011). DAMGO, the -opioid agonist, inhibits release from these neurons (Glickfield and Scanziani, 2008). I therefore perfused DAMGO onto the slice while recording SC-evoked PSPs in current clamp. DAMGO application caused little change in the SC PSP, suggesting that the PV+ basket cells and NPY+ ivy cells may not be important contributors to feedforward inhibition under basal conditions (Basu et al., 2013). However,

DAMGO (0.2 – 1 M) caused a striking change in the effect of photostimulated DA release from VTA/SNpC axons. The presence of DAMGO completely blocked the normal effect of photostimulation to suppress the

PSP. In fact, there was now a trend for DA release t o increase the PSP (151

+- 28% of baseline n = 7, p =.28, paired t -test; figure 3.3). These results suggest that the activity of either PV+ basket cells or ivy cells is required for the VTA/SNpC-driven suppressive effect on the SC depolarization.

Figure 3.3: DAMGO reverses the suppressive effect of VTA/SNpC stimulation on the SC -evoked PSP in the CA1 pyramidal neuron.

Figure 3.3: DAMGO reverses the suppressive effect of VTA/SNpC stimulation on the SC-evoked PSP in the CA1 pyramidal neuron.

A. Electrode configuration for measuring the effect of VTA/SNpC stimulation on the SC-evoked synaptic potential in the CA1 pyramidal neuron. B. Blue arrow indicates the time point at which the light protocol was administered in control external solution. Red circles represent the mean normalized peak

PSP amplitude over time (N = 7) measured in the CA1 neuron with DAMGO present in the external solution. Blue circles are the averages in interleaved control slices (N = 6). Error bars depict the standard error of the mean. C.

Top, control conditions. Dark trace depicts the SC-evoked PSP before photostimulation. Light trace depicts the SC PSP 10 minutes after photostimulation. Bottom, DAMGO present. Dark trace depicts SC-evoked

PSP before photostimulation. Light trace depicts SC PSP 10 minutes after photostimulation.

PV+ basket cells are a particularly attractive candidate as they receive an ample glutamatergic synaptic input onto their dendrites, which extend from SO through SR, from SC axons (Gulyas, 1999), PV+ basket cells project their own widely divergent GABAergic axons onto the CA1 somata and proximal dendrites. To test more definitively if it is indeed the PV+ basket cell inhibition that is enhanced by VTA/SNpC driven DA release, we silenced the PV+ basket cell population using a pharmacogenetic approach recently shown to be an effective strategy for analyzing the function of genetically defined classes of interneurons (Lovett-Barron et al., 2012).

The ligand gated inhibitory channel, the pharmacologically selective actuator module PSAM L141F,Y115F -GlyR (PSAM), is a chimeric protein consisting of the intracellular Cl --selective pore domain of a glycine receptor and the mutated ligand-binding domain of the nicotinic alpha7 acetylcholine receptor (Magnus et al., 2011). This ligand-gated chloride channel is opened by the synthetic pharmacologically selective effector molecule (PSEM 0308 ), a pharmacoligcal agent that does not activate the wild-type nicotinic receptor.

When PSAM is expressed in a select neuron population, application of the

PSEM 0308 ligand gates the inhibitory conductance in these neurons, which hyperpolarizes the membrane and powerfully shunts excitatory currents.

Synaptic inputs and spiking events in genetically identified interneurons have been silenced with this tool (Magnus, et al., 2011; Lovett-Barron et al.,

2012) as long as the ligand was present. Using this method, I can functionally remove the PV+ interneuron population from the circuit while minimally perturbing other cells.

To examine the importance of PV+ INs for the suppressive effect of photostimulated DA release, I crossed a PV-IRES-Cre line (Jackson labs,

008069, Hippenmeyer et al., 2005), which expressed Cre selectively in PV+ neurons, to the TH-Cre driver line. Two AAVs were delivered stereotactically into distinct brain regions in the double transgenic mouse: a flexed PSAM-

2A-ChR2 construct (plasmid generously provided by the Sternson lab) was injected into the CA1 cell body layer (Figure 3.4) and a flexed ChR2-YFP was injected into the midbrain VTA/SNpC. It has been shown that in the presence of PSEM 0308 , the PSAM conductance quenches any spiking activity in the neuron evoked by direct current injection or synaptic input (Lovett-Barron et al., 2011; Magnus et al., 2011). The ChR2 will express in all cells expressing

PSAM and enable verification of the silencing efficacy of the system.

Figure 3. 4: Strategy to test whether the PV+ interneuron are necessary for the VTA/SNpC modulation of CA1 excitation.

Since ChR2 was expressed in the same neurons, we confirmed the expression

of the receptor and the efficacy of the silencing system by evoking IPSCs in

CA1 pyramidal neurons through photostimulation of PV+ interneurons

expressing ChR2-YFP and PSAM. In the a bsence of PSEM, A brief light pulse

elicited a fast IPSC in the CA1 PN voltage -clamped to +10 mV (Figure, 3.4).

Application of PSEM 0308 (3 M) powerfully reduced the IPSC to only 8 + -1.5%

of its initial level after 30 minutes (Figure 3.5).

Figure 3. 5: The PSAM system effectively blocks PV+ interneuron output. A. CA1 neurons were recorded in current clamp in slices containing

PV+ interneurons expressing ChR2 and PSAM. B. PV+ interneurons were photostimulated with 0.5 ms full field light flashes, which evoked a monophasic IPSC recorded in the CA1 neuron soma at a +10mV holding potential. Top, PV+ photo -IPSC before (black trace) and 30 minutes after

(grey trace) PSEM 0308 application. Bottom, mean peak IPSC measurements normalized to the first 3 minutes p rior to PSEM 0308 application (N = 3).

Does DA release from VTA/SNpC axons suppress the SC-evoked depolarization in CA1 PNs by enhancing inhibition mediated by the PV+ interneurons? The SC-evoked depolarization was measured by electrically stimulating the SC inputs in the SR region. Application of PSEM 0308 (3 M) fully blocked the normal action of photostimulated DA release to suppress the

SC-evoked PSP. In the presence of PSEM, photostimulation actually increased the SC PSP to 127 +-11% of its initial value (n = 8, p = .04, paired t- test; Figure 3.6). In alternate slices from the same brains in the absence of

PSEM, VTA/SNpC photostimulation produced a normal suppression of the

SC PSP 71 +-6% of baseline ( n = 9, p = .002, paired t-test; Figure 3.6). The ligand PSEM 0308 did not produce any off-target effects as PSEM 0308 alone did not block the suppressive effect of DA in double transgenic animals injected only in the midbrain with AAV expressing ChR2 (Figure 3.6). thusm selective silencing of PV+ cells not only blocked the suppressive effect of DA but unveiled its facilitatory effect on the SC PSP in CA1 pyramidal neurons.

Figure 3.6: PV+ interneurons mediate the suppressive effect of

VTA/SNpC stimulation on the SC excitation of CA1.

Figure 3.6: PV+ interneurons mediate the suppressive effect of

VTA/SNpC stimulation on the SC excitation of CA1. A. Experimental schematic and circuit. PSAM and ChR2 were expressed in the PV+ basket cell population. ChR2 was expressed in the VTA/SNpC DAergic afferents. B 1.

Representative traces of the SC PSP before (dark blue trace) and 10 minutes after (aqua trace) optogenetic stimulation of VTA/SNpC inputs recorded from slices with PSAM expressed in the PV+ interneuron population superfused with control solutions. B 2. The SC-evoked PSP before photostimulation (black trace) and 10 minutes after photostimulation (gray trace) recorded from slices with PSAM expressed in the PV+ interneuron population and superfused with PSEM 0308 . C. Black circles represent normalized PSP peak amplitudes in control ACSF; blue circles represent normalized PSP peak amplitudes in the presence of PSEM 0308 . D. Mean fold change in PSP amplitude from baseline in: 1. Control slices expressing PSAM (PSAM+, N = 9; paired t-test,

P = .003), 2. An experimental group of slices expressing PSAM and exposed to

3 M PSEM 0308 (PSAM+PSEM+, N = 8; paired t-test, P = .05), and 3. A second control group exposed to 3 M PSEM 0308 without PSAM (N = 6; paired t-test, P < .05).

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PV+ GABAergic interneurons are thus necessary for the reduction of the SC-evoked depolarization of CA1 in response to photostimulation of DA release from VTA/SNpC DAergic afferents. The enhancement in response to

DA release unveiled by the PV+ IN silencing suggests that the DA release may also have an additional facilitative effect, which I explore directly in the next chapter.

VTA/SNpC DAergic afferent stimulation enhances the SC

pathways’ synaptic excitation of PV+ interneurons in CA1

The experiments in the previous section provide strong evidence in favor of the hypothesis that PV+ interneurons mediate the suppressive effect on the

SC PSP in CA1 PNs following photostimulation of VTA/SNpC DAergic afferents. Moreover my results suggest that this suppression results from an enhancement in inhibition from PV+ interneurons to the CA1 PNs. In principle, DA release from VTA/SNpC afferents may facilitate inhibition mediated by PV+ INs either by enhancing the efficacy with which the SC afferents excite the PV+ cells or by increasing the efficacy of the inhibitory synapse between the PV+ INs and CA1 PNs.

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As D4 receptors are expressed, in part, on PV+ interneurons, DA may regulate act on the PV+ interneuron itself. However, such a D4 receptor - mediated action could target either the SC-PV excitatory synapse or the PV -

IN-CA1 inhibitory synapse. To approach this question I examined the effect of VTA/SNpC photstimulation on the SC -evoked synaptic depolarization of the PV+ interneuron itself. To aid in targeting the PV+ interneuron population for recording, an AAV containing a flexed TD -tomato plasmid was stereotactically delivered to the CA1 region of the PV -Cre X TH-Cre double transgenic mouse. In addition, the flexed ChR2 virus was delivered into the midbrain (Figure 3.7).

Figure 3.7: Strategy for targeting PV+ interneurons for whole cell recording and neuromodulation.

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Figure 3.8: Whole cell recordings from PV+ interneurons . A. Electrode configuration and circuit schematic. PV+ interneuron expressing tdTomato depicted in red. B 1. Epifluorescence image of PV+ interneurons transduced with the tdTomato fluorescent protein for visualization and targeted patch clamp. B 2. Dodt contrast image of the whole cell recording achieved from the fluorescing neuron. C 1 Characteristic s piking pattern and hyperpolarization response to +300 pA and -300 pA DC current injections in patched fluorescent cells. C 2. Characteristic afterhyperpolarization of patched fluorescent cells.

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Under epifluorescence, I visualized the TDtomato+ cells to target them for whole-cell patch clamp recording. The PV+ cell bodies were located in stratum oriens and stratum pyramidale. 100% of fluorescing cells exhibited the classical electrophysiological signatures of PV+ fast spiking interneurons

(Figure 3.8C): no accommodation, resting potential around -60 mV, sharp afterhyperpolarization, and a minimal hyperpolarization-induced depolarization sag.

VTA/SNpC stimulation provided a marked enhancement in the SC- evoked PSP in the PV+ IN (to 235 +-46% of baseline, n = 8, p = .02, paired t- test; Figure 3.9). The D2 antagonist, haloperidol, abolished this effect, as the photostimulation caused no significant change in the PSP in its presence

(91% of baseline, n = 4, p = .16, paired t-test; Figure 3.9). VTA/SNpC DAergic axon stimulation, then, produces a D2-type receptor-dependent enhancement of the SC-evoked PSP onto the PV+ IN. This effect had roughly the same time course as the enhancement of the feedforward IPSC and reduction of the

SC-evoked PSP in the CA1 neuron in response to photostimulated DA release.

In addition to participating in disynaptic circuits between the SC and

CA1 neuron, interneurons in CA1 and cortex are known to target other interneurons and some of these receive glutamatergic input from the SC as well. The PV+ interneuron population receives inhibitory input from both other PV+ interneurons and CCK+ interneurons (Armstrong and Soltesz,

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2012). Therefore, it is possible for this enhancement to result from an upstream effect, namely a depression of inhibitory drive onto the PV+ population. However, GABA receptor blockade (using the same concentrations used above) had no effect on the ability of DA release to enhance the synaptic depolarization in the PV+ INs in response to electrical stimulation of the SC inputs. Under such conditions, DA release increased the size of the pure SC EPSP (isolated due to the blockade of inhibition) to

268 +-76% of its initial level (n = 9, p = .05, Figure 3.9), suggesting that the

D2 receptor directly enhances excitatory synaptic transmission between the

SC terminals and the PV+ interneurons.

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* *

Figure 3.9. Photostimulation of the VTA/SNpC afferents to CA1 enhances the SC-evoked depolarization of PV+ interneurons.

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Figure 3.9. Photostimulation of the VTA/SNpC afferents to CA1 enhances the SC-evoked depolarization of PV+ interneurons. A.

VTA/SNpC axons were photostimulated at the time point indicated by the blue arrow. PSP amplitude measurements are normalized to the mean of the baseline and then averaged. The normalized mean peak PSP data from experiments in control ACSF are indicated by the blue circles. Interleaved experiments in haloperidol (2 M) are indicated by the black circles. Error bars represent the standard errors of the mean. B. Characteristic example traces of SC evoked PSPs in each drug condition. Dark traces are taken from the baseline period, lighter traces from 10 minutes following photostimulation. Minus and plus symbols indicate whether the trace was taken from the baseline (-) or following the photostimulation (+). Vertical scale bars, 2 mV; horizontal scale bars, 20 ms. G) Summary data for all cells showing the mean fold change of the SC-evoked PSP from baseline following

VTA/SNpC photostimulation under control conditions, ACSF (N = 8, paired t- test, P = .02), with GABA receptors blocked, SR+CGP (N = 8, paired t-test, P

= .05), or with D2-type receptors blocked, Haloperidol (N = 4, paired t-test, P

= .63).

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Discussion

The working model for the influence of the VTA/SNpC DAergic projection to CA1 is the following. Brief VTA/SNpC DAergic axon stimulation suppresses the SC synaptic drive onto the CA1 neuron by enhancing PV+ mediated feedforward inhibition. DA activates D4 receptors that enhance the SC axons’ direct excitatory synaptic input onto the PV+ interneuron population. The consequently amplified PV+ interneuron-driven

IPSC in CA1 pyramidal neurons reduces the net PSP in response to SC stimulation. Though we do not have direct evidence that it is the enhancement of inhibitory drive onto CA1 that suppresses the excitation, this is the simplest interpretation of the totality of my results.

Firstly, the silencing of the PV+ interneuron population blocks the suppressive effect of VTA/SNpC stimulation on the SC PSP in the CA1 pyramidal neuron. Therefore, PV+ interneurons are required for the production of the suppression. Secondly, The pan-D2 antagonist blocks both the VTA/SNpC-driven suppression of the SC-evoked PSP in the CA1 neuron as well as the enhancement of the SC-evoked PSP in the PV+ interneuron, suggesting that both effects emerge from a common mechanism. The alternative possibility would be that a second D2-type receptor action controls another process that results in the suppression of the SC-evoked

PSP.

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The enhancement of the SC-evoked PSP onto the PV+ interneuron was quite large. This effect should result in a higher spike rate in an already discharging PV+ interneuron and a greater spike probability across the population. PV+ interneurons are extraordinarily efficient carriers of activity

(Jonas et al., 2004) as they rest only a few mV away from spike threshold, can fire at > 200 Hz, and have a high release probability (p=0.9). Even small shifts in depolarization of the PV+ cell would have a large impact on the

IPSC in CA1.

There may be alternative effects of VTA/SNpC DA release as well. It is possible that the increase in the SC-evoked PSP onto the CA1 pyramidal neuron revealed after PV+ interneuron silencing with PSEM occurs because of an enhancement in the direct feedforward excitatory input or from disinhibition of another interneuron that receives SC input. The latter is more likely as the full pharmacological blockade of the GABA receptors abolished all effects. Nevertheless, the enhancement of the SC synaptic drive onto the PV+ interneuron population supersedes the other effects of DA release in the normal conditions of my experiments.

My data suggest that VTA/SNpC DAergic afferents suppress synaptic excitation of CA1 PNs by their SC inputs via amplification of SC-driven feedforward inhibition mediated by PV+ interneurons. Yet the acute silencing of PV+ interneurons seems to only very weakly shift the CA1 input-output curve (Lovett-Barron et al., 2012). One possible explanation would be that

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Lovett-Barron (2012) analyzed the SC-evoked bursting output and did not focus on subthreshold events. Perhaps once the neuron spikes, it becomes dominated by feedback dendritic inhibition that controls the subsequent plateau potentials in the dendrite that contribute to the bursting output; whereas the initial climb to spike threshold is under greater control of somatic inhibition. Alternatively, PV+ interneurons may normally make only a very small contribution to FFI evoked by the SC inputs. Therefore, silencing such weak inhibitory drive would have little impact. Increasing the

PV+ IPSC, however, as observed in my experiments, could recruit these INs as efficacious inhibitors of feedforward synaptic excitation of CA1 PNs.

Implications

On a cellular level, PV+ basket cells are thought to primarily control spike generation (Cobb et al., 1995) and timing as they shift the firing of CA1 pyramidal neurons relative to the phase of theta oscillations in vivo (Royer et al., 2012), and in vitro (Losonczy, 2011). These INs also synchronize cortical networks (Cardin et al., 2009; Jonas review, 2003; Klausberger and Somogyi,

2003) in the gamma oscillatory band (30-100 Hz) in vivo (Cardin et al., 2009), in vitro (Fuchs et al., 2007), and in silico (Whittington et al., 1995).

Gamma oscillations, (25-120hz), emerge from phased inhibitory inputs from a network of PV+ interneurons (Cardin et al., 2009 ) and have been

110 hypothesized to coordinate local cell assemblies and distant brain regions in the service of cognitive operations, including declarative memory

(Montgomery and Buzsaki, 2007). Therefore, as SC drive onto the PV+ interneuron population is enhanced by DA release, in an intact network oscillatory power in the gamma band should increase, which could profoundly enhance the efficiency of signal propagation from CA1 to its downstream targets. Our data predict, then, that contextual and state-dependent information carried by VTA/SNpC DAergic afferents modulates memory processes by activating inhibitory transmission though PV+ interneurons.

This may result in an enhancement in overall gamma power or, perhaps, an increase in the threshold for information transfer and plasticity, as discussed in Chapter 2.

The PV+ interneuron population is crucial for the regulation of CA1 activity during spatial working memory (SWM) performance (Murray et al.,2011). Selective silencing of the population impaired SWM but not spatial reference memory performance on the radial arm maze (Murray et al., 2011).

As discussed in the introduction, D1 receptors in the hippocampus are dispensable for SWM performance (Wilkerson and Levin, 1999). However, focal infusion of D2 agonists and antagonists into the hippocampus facilitate and impair SWM, respectively (Wilkerson and Levin, 1999), supporting the idea that D2 receptors and PV+ interneurons could be linked to a common

111 process in the hippocampus. Further analysis bridging the circuit and network effects of DA release on PV+ interneurons will be required to test whether DA action on CA1 interneurons modulates memory performance.

These experiments predict that PV+ basket cells may operate in multiple different regimes, depending on the behavioral state and context, as their excitation is strongly enhanced by subcortical VTA/SNpC DAergic projection neurons that relay motivational information to the hippocampus.

Fittingly, PV+ interneurons are sensitive to the behavioral state of the animal, increasing their firing rate dramatically when they transition from sleep to quiet wakefulness to movement, fitting with the hypothesis that these interneurons are modulated by arousal-related neurotransmitter systems such as DA (Lapray et al., 2012). DAergic and other subcortical inputs must be accounted for to gain a complete understanding of the function of inhibitory circuits in learning and memory,

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Chapter 4

Introduction

Prolonged VTA/SNPC stimulation enhances the SC

depolarization of CA1

The DA neuron system is bimodal at many levels. The midbrain DA neurons in VTA/SNpC vary their firing pattern between two distinct activity modes. The neurons maintain a persistent 1-4 Hz firing rate, known as the

“tonic mode”, which sets the basal DA level in the neuropil. In response to salient events in the environment the population shifts to phasic mode, a brief burst of action potentials at 10-30 Hz lasting for an average of 150 ms.

Tonically released DA is likely what is measured by microdialysis. Based on studies in the most densely innervated terminal region of the VTA/SNpC, the striatum (Goto and Grace, 2005; Grace et al., 2007), and computational modeling (Dreyer et al., 2010), it has been hypothesized that tonic firing acts via stimulation of the higher affinity D2-type receptors. In the striatum, D2 receptors are required for LTD in medium spiny neurons (Shen et al., 2008).

Conversely, phasic firing is thought to induce a brief spike in DA concentration in a more confined area and act via stimulation of the lower affinity D1-type receptors (Goto and Grace, 2005). In medium spiny neurons, the D1 receptors are required for the induction of LTP (Shen et al., 2008).

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Therefore, in the striatum, the bimodal firing behavior maps onto two independent effector channels with opposite effects on LTP.

In the previous chapter I reported that, endogenous release of DA from the VTA/SNpC DAergic axonal afferents suppresses the SC depolarization of

CA1 PNs. This effect resulted from a single burst of 3 5-millisecond-long light pulses in a high frequency burst. This required activation of the D4 receptor, a member of the higher affinity D2-type family, and was independent of D1- type receptors. Yet, in the literature, the primary observed effects of bath- applied DA are to reduce PP synaptic input to CA1 PNs (Otmakhova and

Lisman, 1999; Ito and Schuman, 2007), facilitate LTP in the SC input to CA1

PNs via D1-type receptors (Huang and Kandel, 1995; Li et al., 2003), and permit learning and memory by acting through D1-type receptors (Jay, 2003;

Ortiz et al, 2010). These effects have been observed in multiple species, behavioral tasks, in culture, in vitro, and in vivo, using exogenous receptor stimulation and blockade with agonists and antagonists.

The lack of an effect of optogenetic release of DA from VTA/SNpC terminals on the PP synaptic input to CA1 PNs in SLM and on D1-type receptor action in SR can be explained anatomically as the DA terminals are most prominent in SO and SP CA1 layers. Yet it is also possible that with stronger or more prolonged stimulation, analogous to phasic firing of the

VTA/SNpC, greater DA concentrations will be generated that may activate

114 the lower affinity D1-type receptors and/or receptors at a further distance from the axons. Considering the preponderance of evidence supporting a strong role for D1-type receptors in LTP and spatial reference memory and the known bimodal frequency structure of DA neuron activity, I set out to test whether the D1/D5 receptor population was under the control of the

VTA/SNpC DAergic axon pool using a stronger pattern of axon stimulation resembling the phasic firing of the neurons.

I demonstrate, in the following chapter, how more prolonged periods of photostimulation produce an opposite effect to that seen with a single light burst. There is a pronounced increase in the size of the SC EPSP in the CA1

PN due to a direct presynaptic enhancement of excitatory synaptic transmission mediated by D1/D5 receptors.

Results

A single burst of VTA/SNPC stimulation, a total of 15 ms of light, reduces the

SC-evoked depolarization of CA1 PNs by enhancing feedforward inhibitory drive mediated by PV+ interneurons through activation of D4 DA receptors.

However, when mice are foraging for rewards DA neurons fire high frequency bursts (“phasic firing”) at approximately 1 Hz (Wang et al., 2011). We

115 attempted to model this in our optogenetic stimulation of the VTA/SNPC inputs through a minimal change in our previous light protocol. Prolonged

VTA/SNPC DAergic axon stimulation, consisting of 25 single bursts of light given at 1 Hz, enhanced the SC-evoked depolarization of CA1 PNs, the opposite effect seen with a single light burst. In current clamp recordings from CA1 PNs, the light protocol enhanced the peak amplitude of the SC- evoked PSP (to 148 +- 16% of baseline, n = 21, p = .008, paired t-test; Figure

4.1). The PSP continued to increase until it reached a maximum of a 1.7 fold potentiation about 15 minutes after photostimulation.

Given that the suppressive effect of a single light burst on the SC PSP was mediated by the D4 receptor, the bimodal behavior of DA in the striatum, and the previously observed facilitative effects of exogenous DA receptor stimulation on LTP of the SC pathway to CA1, I predicted that the

D1-type receptor would mediate this synaptic enhancement. Indeed, the D1- type antagonist SCH23390 (20 M) blocked the ability of the prolonged

VTA/SNpC stimulation protocol to enhance the SC-evoked PSP, which remained at 107 +-16% of baseline (n = 7, p = .65, paired t-test; figure 4.2) 15 minutes after light delivery. In contrast, the D2-type receptor antagonist, haloperidol (2 M), did not alter the enhancement in the SC PSP with prolonged VTA/SNPC stimulation (PSP increased to 157 +-18% of baseline, n

= 15, p = .005, paired t-test; figure 4.2). The cocktail of the two antagonists together also blocked the effect of prolonged photostimulation, with the PSP

116 remaining at 110 +-18% of baseline (n = 4, p = .49, paired t-test; figure 4.3).

These experiments indicate that the prolonged release of DA induced by 25 bursts of VTA/SNPC photostimulations at 1 Hz enhances the SC PSP by releasing DA, which activates D1-type receptors. As in the striatum, distinct

DA release patterns map onto two distinct and opposing synaptic effects, governed by the 2 classes of DA receptors

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Figure 4.1: Prolonged VTA/SNpC stimulation enhances the SC synaptic depolarization of CA1.

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Figure 4.1: Prolonged VTA/SNpC stimulation enhances the SC synaptic depolarization of CA1. A. Electrode configuration and relevant circuit diagram for the experiment. SC PSPs were measured in CA1 PNs using whole cell recordings, as previously described. B. The 25-burst stimulation protocol consisted of the single burst of light used in the previous experiments (3 pulses, 5 ms each, 66 Hz), given 25 times at 1 Hz. The blue arrow indicates the time point at which the protocol was given. Black circles indicate the normalized peak PSP amplitude means across cells (N = 21) for the prolonged burst stimulation experiments and the blue circles the normalized mean peak PSP amplitude across cells for the single burst experiment. Error bars represent the standard error of the mean. C.

Representative traces of the SC evoked PSP in the CA1 PN before (dark trace with minus symbol) and 15 minutes following (light trace with plus symbol) photostimulation.

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Figure 4.2: Prolonged VTA/SNpC stimulation induces an enhanceme nt of the SC -evoked PSP in CA1 by activating D1 -type receptors.

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Figure 4.2: Prolonged VTA/SNpC stimulation induces an enhancement of the SC-evoked PSP in CA1 by activating D1-type receptors. A. Each black circle is the mean peak PSP amplitude across cells

(N = 7) normalized to the baseline with the D1 antagonist SCH23390 present throughout. Each blue circle is the mean peak PSP amplitude across cells (N

= 15) normalized to the baseline over the course of the experiments with haloperidol present. The blue arrow points out the time point of photostimulation. B. Top, haloperidol. PSP waveforms recorded from representative experiments. The dark trace taken from the baseline period; the light trace taken 15 minutes following the light stimulation protocol.

Bottom, SCH23390. The dark trace is taken from the baseline; the light trace

15 minutes after light stimulation.

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Prolonged VTA/SNPC stimulation enhances the SC evoked EPSP in

CA1 via presynaptic mechanisms

Since the suppressive effect of single burst stimulation of the

VTA/SNPC axons was mediated by an enhancement of feedforward inhibitory circuitry, I reasoned that, perhaps, conversely, the D1-type receptor reduced feedforward inhibition to enhance the PSP in CA1 PNs. However, VTA/SNpC photostimulation, with inhibition blocked with the GABA A and GABA B antagonists SR95531 (2 M) and CGP 55845 (1 M), increased the SC PSP to

200 +-38% of baseline (n = 8, p = .03, paired t-test; figure 4.3), equal to (or slightly greater than the enhancement with inhibition intact. This suggests that, unlike the D4 mediated effect, the D1-type receptor population activated by DA release, acts to facilitate the feedforward excitatory SC-CA1 synapse.

It has been demonstrated that the D1/D5 receptor is capable of modulating the SC-evoked PSP in CA1 PNs via NMDA receptor modulation, either directly or indirectly. Exogenous activation of the D1/D5 receptors in

CA1 directly increases the SC-evoked PSP (Huang and Kandel, 1995) on CA1

PNs in an NMDA receptor-dependent manner and also modulates the NMDA receptor EPSC evoked by both SC and PP stimulation (Varela et al., 2009).

D1/D5 receptor activation can also enhance 100 Hz LTP via the NR2B

122 subunit of the NMDA receptor (Stramiello and Wagner, 2008). The D1/D5 receptors can even interface directly with the NMDA receptor (Lee et al.,

2002), and, perhaps, alter its subunit composition (Varela et al., 2009).

The question then arises—can endogenous stimulation of DA release recapitulate the effects of exogenous application of D1/D5 agonists? Is the

VTA/SNpC stimulation induced enhancement of the SC-evoked PSP via D1- type receptors that I observed analogous to the D1/D5 agonist effects. If so, then the effect of photostimulation should be NMDA receptor dependent.

However, I found that NMDA receptors were not involved in the potentiation induced by prolonged VTA/SNpC stimulation. In the presence of the NMDA receptor antagonist D-APV, the SC-evoked PSP in CA1 PNs increased to

151% of baseline (n = 6, p = .02, paired t-test, figure 4.3), suggesting that

NMDA receptors are dispensable for producing this long-term synaptic enhancement.

On which neuron does DA act to enhance the SC EPSP through D1/D5 receptor stimulation? The SC PSP could increase as a result of a postsynaptic modulation of AMPA receptors in the CA1 PN. Alternatively,

DA may act presynpatically, to enhance glutamate release from the SC terminals. I examined paired-pulse facilitation (PPF), a form of plasticity of presynaptic origin (Katz and Miledi, 1968), to determine if the D1-type dependent enhancement occurs pre or postsynaptically. Increases in relerase

123 probability are generally associated with a decrease in PPR as the release probability during the first pulse moves closer to 1. The SC inputs were stimulated with 5 current pulses at 20 Hz before and after photostimulation of DA release to assess the short-term plasticity properties of the synapse.

There was a significant decrease in paired pulse facilitation (Figure 4.4) when the impact of D1-type receptors on the feedforward excitatory synapse was isolated by the presence of haloperidol. Paired pulse ratio 15 minutes after VTA/SNPC stimulation was significantly reduced from 1.91 +-.25 to

1.24 +-.08 (n = 8, p = .03, paired t-test, Figure 4.4). This suggests that persistent stimulation of the VTA/SNPCs enhances the SC synapse through a

D1-type receptor mediated increase in release probability.

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Figure 4.3: Pharmacological analysis of the prolonged VTA/SNpC stimulation effect.

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Figure 4.3: Pharmacological analysis of the prolonged VTA/SNpC stimulation effect. Bars signify the normalized mean PSP amplitudes across cells 10-15 minutes following the extended burst stimulations. Error bars signify the standard errors of the mean. Stars indicate statistical significance of at least p < .05. Numbers in parentheses are the number of cells in the data set. SC = Schaffer collaterals, Halo = haloperidol, SCH =

SCH23390, PP = perforant path. To maximize signal to noise ratio in the experiments, further pharmacological analysis of the effect was performed in the presence of D2-type antagonist haloperidol, as in these conditions the

PSP increase was larger, likely because the concurrent D2-dependent suppression was blocked.

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0.5mV 3mV 100ms 50ms

Figure 4.4. PPR decreases after prolonged VTA/SNpC photostimulation. A. Paired pulse ratios before (black bars) and 15 minutes after (blue bars the prolonged photostimulation protocol of 25 bursts at 1 Hz in control solutions (ACSF), haloperidol, and haloperidol + inhibition blockers. In parentheses are the number of cells in each condition. For paired pulse ratio analysis method see methods. Asterisk signifies statistical significance. B. 20 Hz electrical stimulation of the SC pathway. Black trace recorded during the baseline period; blue trace recorded 15 minutes post

VTA/SNpC photostimulation. C. Traces in B normalized to the peak amplitude of the first local maximum.

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In chapter 2, the question was raised as to why bath application of 20

M DA does not recapitulate the suppression of the SC-evoked synaptic depolarization of CA1 by single burst stimulation of the VTA/SNpC axons.

The D1-type mediated enhancement could help explain the absence of an effect. The single burst VTA/SNpC stimulation produces a D2 receptor effect, a reduction in synaptic strength, because it releases only sufficient DA to activate the D4 receptors, which have a higher affinity for DA compared to the D1-type receptors. Bath application of 20 M DA may be driving the D4- mediated suppressive effect concurrently with the D1-type mediated enhancement such that the two cancel each other out. We reasoned, then, that under conditions of inhibition blockade, which completely prevented the reduction in the PSP following the single burst photostimulation, a potentiation effect should be unveiled. Indeed, with inhibition blocked, bath application of 20 M DA produced a trend towards an increase in the SC PSP in CA1 PNs to 163 +- 36% (n = 5, p = .15) of baseline (Figure 4.5) after 15 minutes.

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SR 2M; CGP 1M

20M DA e d 2.5 u t

i l

p 2.0 m a

P 1.5 S P

E 1.0 d e z i

a 0.5 m r

o n 0.0 -5 0 5 10 15 20 Time (minutes)

Figure 4.5: Under inhibition blockade DA bath application shows a trend to enhance the SC depolarization of CA1. Each black circle demarcates the mean peak PSP amplitude across cells (n = 5) normalized to the baseline for each one-minute period over the course of the experiments.

SR and CGP are present throughout the experiment. DA applied at time = 0 and maintained in the perfusion medium for the course of the experiment.

Discussion

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In a manner reminiscent of the bimodal nature of DA function in the striatum, different levels of VTA/SNpC DAergic axon activity push and pull the efficiency of the SC synapse onto CA1 PNs, resulting in synaptic enhancement or suppression. Single bursts of light suppress the SC PSP by enhancing SC-driven PV+ interneuron mediated feedforward inhibition.

Photostimulation of the VTA/SNpC axons with 25 bursts of light at 1 Hz activates D1-type receptors to produce a long-lasting synaptic enhancement of the direct excitatory SC input to CA1 PNs via a presynaptic mechanism.

Thus, a single neuromodulatory circuit can drive oppositional alterations in synaptic strength.

The enhancing effect is probably presynaptic because paired pulse facilitation decreased after the effect set in. These experiments were done with both inhibition and D2 receptors blocked. With D2 receptors intact, inferences cannot be made regarding the synaptic locus of the D1-type effect as the D2 effect is likely co-occurring and may also cause changes in short term plasticity. Similarly, with inhibition intact, the EPSP is contaminated by the IPSP so changes in PPF could result from effects in the inhibitory circuit.

The D1-type receptors have been shown to be present in somata, dendritic shafts, as well as dendritic spines in the CA1 region. In CA3, they are present as well, but less prevalent. Therefore, it is more likely that the

130 activated D1-type receptors are on the dendrites of the CA1 neuron, which would raise the intriguing possibility that the D1-type receptor triggers the release of modulatory retrograde messengers, as they can do in ventral striatum (Harvey and Lacey, 1997).

Although a plethora of receptor agonist studies link D1-type receptors to SC-CA1 PN synaptic function via NMDA receptor modulation, the long- term enhancement induced by VTA/SNpC photostimulation that we observed was independent of this receptor. This highlights the distinction between the mechanisms of the VTA/SNpC stimulation induced and the D1-D5 agonist induced long-term enhancements. Additionally, the D1/D5 agonist induced direct enhancement of the SC PSP has much slower kinetics, only emerging several hours after bath application (Huang and Kandel, 1995; Navakkode, et al., 2007). The VTA/SNpC stimulation of DA release, on the other hand, starts increasing the PSP within a few minutes. It is possible that the prolonged stimulation would have produced an NMDA receptor-dependent enhancement after several hours, but cells were not held long enough to test this. But, nevertheless, it is clear that the D1/D5 agonist driven effect does not resemble the effect of VTA/SNpC stimulation. This leaves open the possibility that the D1/D5, NMDA receptor dependent slow effect is not actually derived from VTA/SNpC activity.

Amphetamine enhances the SC EPSP with a similar time course as the effect of prolonged stimulation of VTA SNpC (Smith and Greene, 2012) and

131 does so by releasing DA and activating D1-type receptors. However, the amphetamine enhancement has been demonstrated to release DA from the locus coeruleus axon pool, suggesting that the CA3-CA1 SC synapse has two sources of facilitative modulation. It remains to be established whether DA release from locus coeruleus neurons is only an amphetamine-related phenomenon, or can be engaged by physiological activity.

Optogenetically guided VTA/SNpC stimulation in the slice attempts to capture the specific influence of these neurons on CA1 and provides a more physiological circuit-based approach to studying neuromodulatory control then exogenous receptor stimulation. However, ChR2 artificially drives axons and we do not know if the release is analogous to release in-vivo. Nor can we be sure if the light pulses administered to the axons, having been designed based on the light-firing relationaship at the soma, will actually have the same effect as axon excitation in vivo by its upstream partners. It is possible that the severed axons are hyperexcitable and our effects are artificially strong, as a result. Further in-vivo experiments will be necessary to confirm the veracity of these results.

Implications

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The single burst photostimulation protocol likely recapitulates the tonic DA state as the prolonged burst protocol recapitulates the phasic DA state. My slices are likely depleted of DA, as acute slices prepared in a high sucrose solution have recently been shown to contain a 40% lower DA level than slices prepared in regular artificial cerebral spinal fluid (Edelmann and

Lessmann, 2011). In DA depleted slices, brief photoactivation of the axons, generating a few action potentials, may bring the ambient levels of DA up to the tonic state. Accordingly, it has been shown from pharmacological, modeling, and genetic studies that tonic and phasic activity patterns are transduced by D2-type and D1-type receptor signaling, respectively. Although there are some behaviors controlled by the D1-type receptors that do not require phasic bursting, the double dissociation largely holds. Further evidence that D1 receptor activation requires phasic bursting comes from a study of a VTA/SNpC specific NR1 deletion knockout mouse, which, eliminates bursting in the DA neurons alone, and this is sufficient to recapitulate the phenotype of the D1 knockout mouse (Palmiter, 2008).

Symmetrically, the D2D1 double knockout mostly recapitulates the effects of complete DA depletion through synthesis blockade (Palmiter, 2008), suggesting that the tonic firing related DA functions are largely controlled by the D2-type receptors.

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The two opposing effects observed in my experiments, together, could maintain the proper tension between plasticity and stability in the network.

That is, the phasic firing in response to salience enhances the mean synaptic efficacy between the entire CA3-CA1 population, reducing the input threshold for plasticity globally. Then, after persistent low frequency release events, this enhancement of excitation becomes balanced by enhanced inhibition to prevent interference with the synaptic weights that coded the memory.

Tonic and phasic VTA/SNpC firing have distinct effects on behavior.

Phasic firing in VTA induced by optogenetic stimulation is sufficient to induce place preference in a two-compartment chamber (Tsai et al., 2009), yet tonic firing had no effect on preference, suggesting that phasic release events are critical for assigning a neutral stimulus with motivational salience. In the reverse experiment, the DA neuron restricted NR1 deletion knockout mouse with tonically fixed DA neurons mentioned above, could perform many DA- dependent tasks that are impaired after complete block of DA synthesis

(Palmiter, 2008), such as feeding, working memory, and locomotion (Zweifel et al., 2009). For these tasks the tonic DA firing is likely sufficient. However, deficient VTA/SNpC DA neuron bursting impaired all cue-based learning tasks, whether they were motivated by aversion or reward, including conditioned place preference, Morris water maze, operant conditioning, and the appetitive T-maze (Zweifel et al., 2009).

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Since the Morris water maze requires D1/D5 receptors and VTA

DAergic afferents in the hippocampus, it is possible that the phasic firing of the VTA/DAergic afferents in the hippocampus activate D1/D5 receptors in order to facilitate the learning of the Morris water maze. The mice with impaired DA neuron bursting were delayed in their acquisition of the spatial reference memory required in the water maze.

Based on these results, phasic firing of VTA/SNpC DAergic afferents may facilitate acquisition of the water maze task by broadcasting the saliency signal, a direct synaptic enhancement, to the CA3-CA1 synaptic network following the aversive water immersion US. This action may reduce the threshold for synaptic plasticity between neurons encoding a given trajectory and the ones encoding the contextual cues in the environment. A second possibility, not mutually exclusive, is that the VTA/SNpC neurons fire phasically in response to the platform location and retrospectively stabilize recently activated ensembles.

Working memory, on the other hand, which requires hippocampal PV+ interneurons as well D2 receptors, is intact after genetically suppressing the phasic firing ability of VTA/SNpC DA neurons in the hippocampus. This supports the notion that tonic DA release specifically targets D2-type receptors in the hippocampus. Thus, DA neuron firing may drive synaptic state transitions in the hippocampus to optimized its function for different behavioral tasks.

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Chapter 5

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Conclusion

Cortical networks change their properties with behavioral state.

Neuromodulatory centers, such as the DA system, are thought to implement these slow, adaptive, changes in network structure. 30 years of research points to the potential of the VTA/SNpC DAergic input in affecting cellular, circuit, and network function in the hippocampus as well as hippocampal dependent learning and memory. One influential model asserts that the hippocampus and VTA/SNpC couple to one another in a recurrent loop whereby hippocampally computed novelty signals disinhibit the VTA/SNpC via an indirect pathway involving subiculum, nucleus accumbens, and ventral pallidum. The VTA/SNpC, in turn, releases DA in the hippocampus.

DA release, then, enhances plasticity processes on the SC-CA1 PN synapse and suppresses the contextual input from the PP input to favor encoding of the information that led up to the salience (Lisman and Grace, 2005;

Otmakhova et al., 2013). In such a way, VTA facilitates learning and memory.

Because of the limitations of exogenous application, it has been controversial as to whether the release of DA from a fairly sparse pool of

VTA/SNpC DAergic axons could generate concentrations that produce the wide array of effects that have been observed. And, considering the

137 alternative source of DA, the locus coeruleus in much closer proximity to the primary effect of exogenous application of DA, the PP inputs, skeptics have posited that the main effects of DA on synaptic transmission, plasticity, and learning arise from a source in the locus coeruleus. The range of effects of DA receptors have been well elucidated, however, the receptor profile coupled to the VTA/SNpC DAergic axons remains unknown.

We provide evidence for the VTA-hippocampus loop model by demonstrating that the “upward arm” of the loop, the VTA/SNpC- hippocampal input, is indeed capable of powerfully modulating CA1 circuitry.

I show, in a series of experiments in vitro, that the VTA/SNpC DAergic neurons are a plausible candidate for gating long-term memory storage via its signaling in the CA1 region of the hippocampus. In using a circuit-based approach to modulatory function, the partial extent of the receptor system physiologically accessible to the VTA/SNpC DA source was revealed.

I observed that VTA/SNpC exerts a bidirectional effect that depends on the pattern of its release. The unitary burst stimulation, a very modest amount of light (5ms pulses each at 66 Hz) releases DA and suppresses the

SC-evoked excitation of CA1 PNs by activating the D4 receptor, which enhances convergent PV+ interneuron mediated inhibitory drive. The enhancement in feedforward inhibition suppresses excitation. Strengthening the DA signal by prolonging the duration of light stimulation strengthens the

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SC excitation of CA1 PNs via a D1 receptor dependent increase in glutamate release at the direct SC excitatory synapse.

Therefore, our data support the model in its general concept of providing a likely facilitative effect on memory encoding, yet call for its significant refinement, regarding the implementation. The previous model predicts that DA modulates plasticity in the SC pathway to CA1, we observe, in contrast, that DA does not merely permit LTP, it enacts bidirectional and global network-wide changes in basal synaptic efficacy. Bath application studies suggest that DA only modulates plasticity—permitting plasticity induced by unique patterns glutamate and GABA transmission (Otmakhova and Lisman, 1996; Ortiz, 2010). The D1-type mediated potentiation that we evoke does not require NMDA receptors, whereas the previously reported D1- type mediated plasticity modulations do require them (Huang and Kandel,

1995; Navakkode et al., 2007). Although we can’t rule out from our experiments that ongoing AMPA receptor signaling is necessary, our effects are on basal transmission and do not require classical plasticity triggers like tetanic stimulation (Huang and Kandel, 1995) or spike-timing dependent plasticity pairing protocols (Edelmann and Lessman, 2011). Instead, we observe global modulation of synaptic strength. A recent study in the nucleus accumbens (Ishikawa et al., 2013) also finds that, in contrast to studies using exogenous receptor stimulation, optogenetically guided release of DA induces a heterosynaptic LTD of inhibition, not a modulation of plasticity.

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The two patterns of DA stimulation effect synaptic state transitions in the SC pathway to CA1 PNs, which can act in concert as a salience filter on memory encoding. In vivo, the DA neurons shift from a tonic firing state (1-3

Hz) to phasic bursts of (10-30 Hz) in response to motivationally salient stimuli. The unitary light burst stimulus resembles tonic MDN firing and the phasic burst train, phasic firing. We hypothesize, then, that the tonic mode functions as a suppressor of global excitation of CA1, which could raise the threshold for synaptic plasticity in mundane environments (deprived of stimuli that drive phasic firing of DA neurons). This would help the memory system strategically filter out the familiar stimuli from memory. Upon arrival of salient stimuli, phasic burst activity in MDNs will globally enhance excitation and boost plasticity processes.

Also, the synergistic effect of a general increase in excitatory input from the SC to CA1 PNs coupled with enhancement of PV+interneuron mediated feedforward inhibition could enhance the synchronization of CA1 neurons (Cobb, 1996) in the gamma band, a network oscillation that may have a role in synaptic plasticity (Bi and Poo, 1998), memory encoding

(Sederberg et al., 2006), and selecting particular cell assemblies for memory retrieval (Colgin et al, 2009; Montgomery and Buzsaki, 2007).

The DAergic axon pool is sparse and yet the effects on the SC synapses are considerable. This discrepancy left additional doubt regarding the physiological plausibility of the bath application studies. Here, too, the effect

140 is much more dramatic than the observable structure may imply. There could be two ways that such a dramatic effect occurs. One possibility is that fine

DAergic axons are invisible to immunohistochemistry and GFP tracing.

Second, the effect of minimal DA stimulation is through interneurons whose morphology is uniquely suited to modulate ample sections of the circuit. In cortex and hippocampus many of the neuromodulatory systems, including serotonin (Freund et al., 1990), acetylcholine (Cobb and Davies, 2005), and norepinephrine (Bergles, 1996), have been shown to also preferentially target

INs. It is a relatively efficient way to project motivational state information to cortical circuits as INs are highly influential players in the circuit dynamics. They integrate large numbers of distinct converging neuronal inputs and extend widely divergent axonal arbors. Supervisory control over these interneurons by extrinsic neuromodulatory inputs is an energetically cheap way of controlling the synaptic circuitry

The long lasting depression of the synaptic excitation of CA1 neurons occurs as a result of the activation of D4 receptors. The D4 receptors exert this effect by potentiating the SC input to PV+ interneurons. Although the net result on the circuit output is suppressive, the mechanism of this potentiation warrants further investigation as D2-type receptors, of which D4 is a member, classically depress transmission. This may be a novel signaling pathway of the D4 receptor.

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Future directions

The results described herein have implications for the understanding of the function of neuromodulators in general. Bath application experiments are fundamental, but do not provide a complete picture of their action on synaptic transmission. Further optogenetic analysis of other neuromodulatory circuits will also be necessary to gain a complete picture of their physiological function. In the hippocampus, I wish to extend the research described in this thesis by comparing the locus coeruleus and

VTA/SNpC monoaminergic inputs to the hippocampus in vitro as independent and comodulators of hippocampal function. This analysis will test whether DA can be released from locus coeruleus axons, and if so, which if any of the known receptor pathways they recruit. I hypothesize that at low levels of firing the locus coeruleus releases NE, but at high levels of firing, when the rate limiting enzyme for NE-DA conversion dopamine beta hydroxylase is saturated, DA will begin to be released and modulate the PP basal synaptic input in SLM.

Next, I would like to use in vivo optogenetic and electrophysiological techniques to verify that the in vitro effects of VTA/SNpC and LC occur in the intact network. Then, I can test its relevance in vivo during learning. In particular, from the studies mentioned in the discussions of Chapters 2, 3, and 4, the D1-type receptors are necessary for both the in vitro synaptic

142 enhancement effects on the SC synaptic input to CA1 as well as performance in spatial reference memory. D2-type receptors, in contrast are required for the suppressive SC-CA1 synaptic effects and working memory. I can test if the two patterns of stimulation, known to activate D1-type and D2-type receptors, are governing the learning in these two types of tasks and then use electrophysiological recording or imaging to establish a correlation between the behavioral benefits and the network effects. This will aid in building models for the functioning of the hippocampus in an unperturbed state.

Chapter 6

Methods

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All experiments were conducted in accordance with the principles established by the US National Institute of Health with approval of the Columbia

University Institutional Animal Care and use Committee.

Animals

The initial bath application experiments were performed on C57/BL6 mice

(Jackson labs). Optogenetics experiments were performed on B6.Cg-Tg(Th- cre)1Tmd/J (Jackson labs) maintained as heterozygotes by crossing to

C57/BL6 mice. These heterozygotes were crossed to homozygous B6;129P2-

Pvalb tm1(cre)Arbr /J (Jackson labs) for the PV+ IN targeted manipulation. For information on how the mice were made see the Jackson labs website.

Viral constructs

For control of the dopaminergic axons an adeno-associated virus was injected:

AAV2/5 packaged with the following expression vector EF1a::DIO- hChR2(H134R)-EYFP-WPRE-hGH (Penn Vector). For Td-tomato expression in the hippocampus we injected an AAV2/5 CAG::DIO-tdTomato-WPRE

(Penn Vector). To express PSAM in interneurons we injected AAV2/1

CAG::DIO-ChR2(2xHA) PSAML141FY115F-GlyR virus (the plasmid and

144 ligand were generous gifts from the Sternson lab, the virus custom made by

Penn Vector). The construction and mechanism of action of PSAM and its second-generation ligand PSEM 308 have been described in previous publications (Magnus et al., 2011)

Stereotactic surgery for viral transduction in-vivo

Mice from P35-P90 were anesthetized with ketamine/xylazine or isofluorane and then mounted in a stereotactic frame. All injections into the MDNs and hippocampus were bilateral. Coordinates relative to bregma for midbrain injections were +-.5, -3.4, -4.3) and for hippocampal injections were (+-3.7,-

3.0, -2.7). For all injections, viruses were pressure injected from a thin glass pipette fixed onto the stereotax. 1 l of high titer (10 13 ) particles/ml virus injected/hemisphere for midbrain injections, 200 nl/hemisphere for hippocampal injections. The hippocampal injection site was chosen based on the position of the horizontal sections prepared for electrophysiology.

Following injection, mice were returned to their home cage for 2.5-4 weeks. In this time window all electrophysiological experiments were done. Following recording to avoid inadvertent excitation of CHR2 prior to the experiment, the YFP signal was verified in the ventral most horizontal slice which contained VTA as well as in dorsal slices which contained striatum. If no fluorescence was observed, the recordings for that day were discarded (only

145 two instances). 100% of slices of the Th-CreXPV-Cre flexed tdTomato injected animals displayed mosaic fluorescence throughout the pyramidal cell layer and stratum oriens of CA1.

Tissue preparation for electrophysiology

For all experiments horizontal brain slices were prepared from the hippocampus of P42-P90 mice. Except for the initial experiments where mice were decapitated directly after anesthesia, animals were anesthetized and then perfused intracardially with ice-cold (2°C) sucrose-replaced ACSF (S-

ACSF) containing (in mM): NaCl (10), NaH 2PO 4 (1.2), KCl (2.5), NaHCO 3 (25), glucose (25), CaCl 2 (0.5), MgCl 2 (7), sucrose (190), pyruvate (2), continuously bubbled with 95%/5% O 2/CO 2. There were no differences in results between perfused and unperfused brains so data were pooled. After decapitation and brain removal, brains were submerged in the S-ACSF and hemisected.

Cerebellum and the anterior portion of the brain were cut. The hemisected brain was then cut in the horizontal plane at a 10-12° in the ventromedial direction to optimally achieve a transverse section (perpendicular to the longitudinal axis of the hippocampus) and sliced into 400 m sections on a

Leica VT1200s sectioning system. Slices between bregma distance -2.16 --> -

3.96 were then transferred to an incubation chamber containing a 34° solution consisting of 50% S-ACSF and 50% recording ACSF containing (in

146

mM): NaCl (125), NaH 2PO 4 (1.25), KCl (2.5), NaHCO 3 (25), Glucose (25),

CaCl 2 (2), MgCl 2 (1), pyruvate (2) for 20 minutes. Slices were then transferred to room temperature where they awaited recording. When ready to record slices were placed into a submerged chamber in the rig at 30-32°C, constantly supplied at 4-5ml/minute with 95%/5% O 2/CO 2 R-ACSF.

Electrophysiological Recording and Analysis

Whole cell recordings were obtained from pyramidal neurons and interneurons using patch pipettes (3-5M ) filled with KMeSO 4 (135), KCl (5),

EGTA-Na (0.1), HEPES (10), NaCl (2), ATP (5), GTP (0.4), phosphocreatine

(10) with (pH of 7.2; 280–290 mOsm) for current clamp recordings and the same with CsMeSO 4 (135) replacing KMeSO 4 for voltage clamp recordings.

Series resistance (typically 10–25 M ) was monitored throughout each experiment; cells with more than 15% change in series resistance were excluded from analysis. Focal stimulating electrodes, a patch pipette filled with R-ACSF, were used to apply unipolar shocks of .1ms in duration using a constant voltage stimulator. Pyramidal cell recordings were performed using the both the visual and blind patch method. Interneuron recordings were obtained by selecting fluorescing (tdtomato+) cells and patching them visually. 100% of fluorescing cells patched displayed fast-spiking behavior.

147

Recordings were obttained using a two-channel multiclamp 700B amplifier

(Axon instuments). Data were digitized on a PC using a digidata 1440 under the control of the AxographX acquisition package (Berkeley, CA). Data were acquired at 20 kHz with a 4 kHz low-pass Bessel filter applied using the internal circuitry of the Multiclamp amplifier.

Analysis was completed using AxographX software, Matlab student version (Mathworks, Natick, MA), Excel (Microsoft, Redmond, WA), and

Prism (GraphPad Software, La Jolla, CA). Electrical stimulation was given once every 20s, a frequency that avoids any plasticity induction. In experiments with both SC and PP stimulation, the two stimulation pulses were given 1s apart to avoid cross contamination of the PSPs. The amplitude of synaptic responses was measured as a small windowed average of the peak potential following stimulation. Data were normalized to the average of the baseline 5 minutes before the manipulation and then box-car filtered such that each point represents the average of 3 points (1 minute each). The fold change was, therefore, the average magnitude of the EPSP 10 minutes post- manipulation divided by the average magnitude of the baseline EPSP. For measurements of short term plasticity e.g. paired pulse facilitation, the data was normalized to the amplitude of the first evoked psp peak and then PPF was calculated by dividing 2nd PSP amplitude by the 1st PSP amplitude.

Statistical comparisons were performed using Student’s t test. Results are reported as mean ± SEM.

148

Pharmacology

In most experiments drugs were added to the bath solution by dilution from stock solutions (1000-10000-fold concentrated). All drugs were obtained from either Sigma or Tocris except for PSEM (Sternson lab) and used at the following concentrations ( M): Dopamine(20), Haloperidol (2), SCH23390 (20), prazosin(10), propranolol (5), yohimbine (10), SR-95531(2), CGP-55845 (1),

D-APV (50), NBQX (10), and PSEM (3). For DA experiments a reducing agent sodium metabisulfite (.075 mM) was included in the R-ACSF to prevent immediate oxidation of the compound. PSEM was always applied for 30 minutes prior to optogenetic stimulation. For experiments under inhibition blockade, experiments were only started after 10 minutes of PSP stability.

Light delivery

For optogenetics experiments a 470 nm blue LED (Thorlabs) was mounted on the backport of the microscope and focused through a 40x objective centered around the recorded cell. For the unitary light burst experiments designed to stimulate MDN afferents, the light protocol consisted of a single burst of three five ms pulses of light given every 15 ms. The persistent light stimulation experiments repeated this protocol 25 times at 1Hz. Light-evoked

149

PV-IPSC experiments were done using

Histology

After recording a subset of slices were fixed in PFA overnight for staining.

After washing off the PFA, slices were permeabilized in 1X PBS+.2% Triton, and then blocked in PBS +.2% Triton + 3% goat serum. Primary antibodies were done overnight at 4°. Secondary antibodies were applied for 2 hours.

Slices were mounted with immunogold (Invitrogen), and fluorescence imaging performed on an inverted laser scanning confocal microscope (Zeiss LSM 700).

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