NEUROMODULATION BY G-PROTEIN-COUPLED RECEPTORS IN THE AVIAN NUCLEUS ANGULARIS

A thesis submitted

to Kent State University in partial

fulfillment of the requirements for the

degree of Master of Science

By

Wei Shi

Aug, 2011

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Thesis written by

Wei Shi

B.S., Sun Yat-sen University, China, 2007

Approved by

______, Yong Lu, Chair, Master Thesis Committee

______, Alexander V. Galazyuk, Member, Master Thesis Committee

______, Brett R. Schofield, Member, Master Thesis Committee

Accepted by

______, Robert V. Dorman, Director, School of Biomedical Sciences

______, John R. D. Stalvey, Associate Dean, College of Arts and Sciences

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TABLE OF CONTENTS

LIST OF FIGURES...... V-VI

ACKNOWLEDGEMENTS ...... VII

CHAPTER 1. INTRODUCTION...... 1

1.1 Nucleus angularis in sound localization ...... 1

1.1.1 Nucleus angularis: connection, intrinsic and synaptic physiology ...... 1

1.1.2 Acoustic cues for sound localization on the azimuth plane...... 4

1.2 Neuromodulation by GPCRs may improve sound intensity coding in NA...... 11

CHAPTER 2. METHODS ...... 15

2.1 Slice preparation and in vitro whole-cell recordings...... 15

2.2 Synaptic stimulation and recordings of synaptic responses ...... 16

2.3 Data analysis...... 17

2.4 mGluR immunostaining...... 16

2.5 Western blot analysis...... 19

CHAPTER 3. RESULT...... 21

3.1 Electrophysiology results...... 21

3.1.1 Basic intrinsic and synaptic properties of NA neurons...... 21

3.1.2 Activation of groups II and III but not I mGluRs suppressed EPSCs in NA

neurons ...... 22

3.1.3 Activation of GABAbRs suppressed EPSCs in NA neurons...... 25

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3.1.4 Loci for mGluR- and GABAbR-mediated modulation of glutamate

transmission ...... 25

3.2 Immunohistology and Western blot results ...... 26

CHAPTER 4. DISCUSSION AND CONCLUSIONS ...... 52

4.1 Neuromodulation of excitatory transmission in NA...... 52

4.1.1 Involvement of multiple mGluRs in modulation of glutamate release in

NA……… ...... 52

4.1.2 Mechanisms of modulation of glutamate release in NA...... 55

4.1.3 Comparisons of modulation among NM, NL, and NA...... 55

4.1.4 Comparisons of modulation between NA and mammalian cochlear nucleus

……………………………………………………………………………...60

4.2 Future research direction ...... 60

ABRBREVIATIONS ……………………………………………………………….64-65

REFERENCES ...... 66

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LIST OF FIGURES

Figure 1. Time and intensity coding circuits in the avian brainstem ....….……….. 7

Figure 2. Schematic drawings showing the time and intensity coding pathways in the

mammalian and avian auditory brainstem …………………….………... 9

Figure 3. Basic intrinsic and synaptic properties of NA neurons ……...... 23

Figure 4. Activation of group I mGluRs did not affect EPSCs of NA neurons...... 27

Figure 5. Activation of group II mGluRs suppressed EPSCs of NA neurons…..... 29

Figure 6. Activation of group II mGluRs appeared to reduce the excitability of NA

neurons ……….…..………………………………………………………31

Figure 7. Activation of group III mGluRs suppressed EPSCs of NA neurons …... 33

Figure 8. Activation of group III mGluRs might alter the excitability of some NA

neurons ……….……...... 35

Figure 9. Activation of GABAbRs suppressed EPSCs of NA neurons ……………38

Figure 10. Activation of GABAbRs appeared to reduce the excitability of NA

neurons ……...... 40

Figure 11. Activation of group II mGluRs reduced both the frequency and amplitude

of sEPSCs of NA neurons…….……...... 43

Figure 12. Activation of group III mGluRs reduced both the frequency and

amplitude of sEPSCs of NA neurons …………….……………...... 45

Figure 13. Activation of GABAbRs reduced both the frequency and amplitude of

sEPSCs of NA neurons ………...………………...... 47 v

Figure 14. NA neurons seemed to express groups II and III mGluRs …50

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ACKNOWLEDGEMENTS

I thank my committee members Dr. Yong Lu, Dr. Alexander V. Galazyuk, Dr.

Brett R. Schofield for the advice of my thesis. I also want to thank my colleagues Dr.

Zhengquan Tang and William Hamlet for their technical assistance and review of the thesis. I want to express my gratefulness to my parents and my friends who give me a lot of support during my graduate study.

Wei Shi

06/29/2011, Kent, Ohio

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CHAPTER 1

Introduction

1.1 Nucleus angularis in sound localization

1.1.1 Nucleus angularis: connection, intrinsic and synaptic physiology

The avian cochlear nucleus has two components, nucleus magnocellularis (NM) and nucleus angularis (NA) (Fig. 1). After entering the brainstem, the auditory nerve bifurcates, forming two branches. The medial branch innervates the NM, and the lateral one innervates the NA (Boord 1969; Parks and Rubel 1978; Carr and Boudreau 1991).

Neurons in the NM are homologous to bushy cells in the mammalian cochlear nucleus, and they are believed to be homogeneous in both their morphology and physiology. NM neurons have large round cell bodies, and have few or no dendrites. Each NM neuron receives innervation from only 1-3 auditory nerve fibers, forming endbulb terminals which cover up about 60% of the somata surface of NM neurons (review in Ryugo and

Parks 2003). NM neurons have specialized properties suitable for coding timing information of sounds (review in Parks 2000). For example, in response to prolonged positive current injection into the soma, NM neurons fire only a single spike at the onset of the current injection, a characteristic hallmark for timing coding neurons in the central auditory system (Oertel 1999; Trussell 1999). NM neurons send phase-locked excitatory projections to nucleus laminaris (NL) neurons on both sides (Parks and Rubel 1975;

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Rubel and Parks 1975), where processing of bilaterally converging inputs occurs in the time pathway (Overholt et al. 1992; Young and Rubel 1983).

In contrast, NA neurons are heterogeneous in their morphology and physiology, and have no immediate homologous counterpart in the mammalian system (review in

Köppl and Carr 2003). Based on their intrinsic spiking properties in response to prolonged positive current injections into their cell bodies and their morphology, they are classified into three different cell types (one-spike or onset, tonic, and damped) in embryos (Soares et al. 2002). One-spike neurons, like NM neurons, fire a single spike at the onset of the current injection, and they have stubby radiate dendritic trees. Tonic cells respond with trains of action potentials, and they have multipolar cell morphology.

Damped cells produce a few spikes at the onset of the current injection, followed by membrane oscillations with gradual reduction in amplitude. The dendrites of these cells are distributed in an orientation parallel to the iso-frequency axis in NA. In hatchlings, only two cell types (onset and tonic) are observed, with the tonic pattern being dominant

(Fukui and Ohmori 2003). The developmental changes after hatch are relatively subtle, possibly because the majority neural properties of auditory brainstem neurons in the chick are adult-like in late stages of embryos (Gao and Lu 2008).

NA neurons receive both excitatory and inhibitory inputs. The excitatory inputs come from the auditory nerve, using glutamate as the neurotransmitter. Each NA neuron receives innervations from multiple bouton-like terminals of the auditory nerve. Synaptic physiology studies show that excitatory postsynaptic currents (EPSCs) of NA neurons are graded, small in amplitude, and are mediated by both AMPA and NMDA receptors

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(MacLeod and Carr 2005). Unlike NM neurons whose responses are phase-locked

(spiking at only a certain phase of the sinusoidal sound waveforms) to the auditory nerve inputs, responses of most NA neurons are not phase-locked to their auditory nerve inputs above 1 kHz (Warchol and Dallos 1990). These features, along with a slow membrane time constant of NA neurons, lead to substantial temporal and spatial integration of their excitatory inputs, a feature suitable for coding sound intensity. The inhibitory inputs to

NA originate primarily from the superior olivary nucleus (SON), using both GABA and glycine as the neurotransmitter, giving rise to depolarizing inhibition due to a relatively higher intracellular chloride concentration, which persists into maturation (Kuo et al.

2009). This GABA/glycinergic input to the NA may exert potent inhibition via mechanisms found in NM neurons, such as activation of a low-threshold potassium conductance, inactivation of voltage-gated sodium channels, and shunting inhibition

(Monsivais and Rubel 2001).

The majority of NA neurons are believed to be multi-functional including encoding intensity of sounds (Köppl and Carr 2003). While some NA neurons possess monotonic rate-intensity functions (the relationships between spike discharge and sound levels), others have non-monotonic functions (Warchol and Dallos 1990; Sato et al. 2010).

This is in great contrast with NM neurons, all of which have only monotonic rate- intensity functions (Warchol and Dallos 1990). NA neurons also have larger dynamic range (the range of sound intensity within which a neuron fires spikes between 5% and

95% of its maximal spike rate) than NM neurons, suggesting of high sensitivity of NA neurons to changes in sound levels (Warchol and Dallos 1990; but also see Köppl and

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Carr 2003). Although both NA and NM neurons receive inhibitory inputs, the distinction in the rate-intensity functions between these two cochlear nuclei may not be simply attributed to synaptic inhibition. Neuromodulation may play a role in shaping the patterns of the rate-intensity functions in the NA. In addition, it is proposed that some NA neurons, especially the one-spike firing cells, may also play roles in encoding timing information of sounds (Soares et al. 2002). Interestingly, the intensity coding process in NA neurons is affected by interaural phase difference of sound stimuli, suggesting interactions between the time and intensity pathways (Sato et al. 2010). Apparently, the time and intensity coding pathways in the avian auditory system are separated at the cochlear nucleus, forming distinct circuits encoding different properties. These distinct pathways, with possible interactions, serve to compute information for sound localization using different acoustic cues.

1.1.2 Acoustic cues for sound localization on the azimuth plane

Sound localization is inherently important for communication and survival of animals and human beings. The auditory peripheral (cochlea), however, does not code locations of sound sources. Such information has to be computed in the central auditory system. A number of acoustic cues are used in this process. Sound localization on the azimuthal plane depends on two major cues, interaural time difference (ITD) and interaural level/intensity difference (ILD), which are encoded by distinct neural circuits in the central auditory system (review in Grothe 2003; Kandler et al. 2009). The ITD coding circuits formed among lower auditory brainstem nuclei in mammals and birds are

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similar. Bushy cells in anterior ventral cochlear nucleus (AVCN) or NM receive phase- locked excitation from the auditory nerve. Bushy cells send phase-locked excitation to coincidence detector neurons in medial superior olive (MSO) or NL. Coincidence detection in MSO or NL may follow the Jeffress model, which assumes two essential elements: a delay line and an array of neurons acting as coincidence detectors (Jeffress

1948). Evidence supporting this model has been demonstrated in birds (Young and Rubel

1983; Carr and Konishi 1990; Overholt et al. 1992; Hyson 2005; MacLeod et al. 2006), and to a less extent in mammals (Yin and Chan 1990; Smith et al. 1993; Grothe 2003;

Joris and Yin 2007). Timing-coding neurons in the auditory brainstem also receive inhibitory inputs. AVCN bushy cells receive inhibitory inputs mediated by glycine

(primarily) and GABA from multiple incompletely understood sources (Ferragamo and

Oertel 2001). MSO neurons receive glycinergic inhibitory inputs from the medial and lateral nucleus of trapezoid body (MNTB and LNTB) (Cant and Hyson 1992; Kuwabara and Zook 1992; Smith et al. 1998, 2000). In birds, both NM and NL receive GABAergic inhibitory inputs from the ipsilateral SON (Burger et al. 2005; Lachica et al. 1994; Yang et al. 1999).

The ILD coding circuits formed among brainstem nuclei in mammals and birds are also similar (Figs. 1 & 2). In mammals, the first station encoding ILD is the lateral superior olive (LSO), which receives inhibitory and excitatory inputs from the contralateral (relayed through inhibitory neurons in the MNTB) and the ipsilateral

AVCN, respectively. Therefore, LSO neurons are inhibition-excitation (IE) cells. In the avian system, NA neurons send their glutamatergic inputs to dorsal nucleus of the lateral

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lemniscus (LLD, formerly VLVp), which is the first station responsible for ILD coding in the birds (Manley et al. 1988; Mogdans and Knudsen 1994). Neurons in the LLD receive excitatory input from the contralateral NA (Sullivan and Konishi 1984; Takahashi and

Konishi 1988). The two LLDs have reciprocal inhibitory innervations, transferring the input to the LLD from the ipsilateral NA to an inhibitory one. Therefore, LLD neurons are EI cells, and they encode ILD with similar mechanisms used at LSO except the signs of the inputs from the two ears are exactly the opposite (IE in LSO vs. EI in LLD). The intensity and timing pathways converge in the external nucleus of the inferior colliculus.

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Fig.1

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Fig. 1. Time and intensity coding circuits in the avian brainstem

A (adapted from Rubel and Parks 1975). A schematic diagram showing the time and intensity coding circuits in the avian brainstem. Upon entering the brainstem, the auditory nerve bifurcates, forming two branches to innervate the cochlear nucleus magnocellularis

(NM) and cochlear nucleus angularis (NA), respectively. Axons of NM neurons also bifurcate. One branch innervates the dorsal dendrites of the ipsilateral NL, and the other branch crosses the midline and innervates the ventral dendrites of the contralateral NL.

Neurons in NA and NL send excitatory inputs to the superior olivary nucleus (SON).

SON neurons in turn send feedback inhibitory inputs to NM, NA, and NL. B (adapted from MacLeod and Carr 2007). The interaural level difference (ILD) coding circuit in the bird. NA projects contralaterally to the dorsal nucleus of lateral lemniscus (LLD, formerly VLVp), and this projection is excitatory. The two VLVp send inhibitory inputs to each other. Therefore, neurons in the VLVp are EI neurons sensitive to ILD, excited by contralateral stimuli and inhibited by ipsilateral stimuli.

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Fig. 2

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Fig. 2. Schematic drawings showing the time and intensity coding pathways in the mammalian and avian auditory brainstem.

A (adapted from Kandler and Gillespie 2005). Left: In mammals, neurons in the lateral superior olive (LSO) encode interaural intensity differences by intergrating excitatory glutamatergic inputs from the ipsilateral cochlea nucleus (CN) with inhibitory glycinergic inputs from the medial nucleus of the trapezoid body (MNTB), which in turn is activated by the contralateral CN. Right: In mammals, neurons in the medial superior olive (MSO) encode interaural time differences by integrating bilateral excitatory inputs from both cochlear nuclei and bilateral inhibitory inputs from the lateral nucleus of the trapezoid body (LNTB) and the MNTB. All of these auditory nuclei are tonotopically organized, as indicated by color gradients. Additional abbreviations: Hf, high frequency; Lf, low frequency. B (adapted from Kubke et al. 1999). In birds, separation into time and sound level pathways (dark lines and dotted lines, respectively) begins with the cochlear nuclei.

VIIIth nerve afferents divide to innervate both the level-coding NA and the time-coding

NM. NM projects bilaterally to NL, which in turn projects to the ipsilateral SON (labeled

SO in the drawing), LLDa, and to the core region of central nucleus of the IC (ICCs).

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1.2 Neuromodulation by GPCRs may improve sound intensity coding in NA

Glutamate and GABA are the most abundant excitatory and inhibitory neurotransmitters in the vertebrate central nervous system (CNS), respectively. These two neurotransmitters act on multiple types of receptors. There are two main classes of glutamate receptors, ionotropic and metabotropic receptors (iGluRs and mGluRs, respectively). iGluRs are themselves ion channels and they mediate fast excitatory synaptic transmission through three different types of receptors: AMPA, NMDA, and kainate receptors. mGluRs are G-protein-coupled receptors (GPCRs) and they mediate slow synaptic transmission through intracellular second messenger systems. There are eight mGluR members (review in Kew and Kemp 2005). They are divided into three groups depending on their amino acid sequences, pharmacology, and signal transduction pathways. Group I includes mGluR1 and mGluR5. They are selectively activated by 3,5- dihydroxyphenylglycine (3,5-DHPG), and are coupled to phosphoinositide hydrolysis with the subsequent production of inositol 1,4,5-triphosphate (IP3), which can cause intracellular calcium release via activation of IP3 receptors located on endoplasmic reticulum (Frenguelli et al. 1993; Yuzaki and Mikoshiba 1992; Takechi et al. 1998).

Group I mGluRs can also inhibit voltage-gated calcium channels (VGCC) and suppress transmitter release (Lester and Jahr 1990; Swartz and Bean 1992). Group II mGluRs consist of mGluR2 and mGluR3. Group III consists of mGluR4, mGluR6, mGluR7 and mGluR8. Both group II and III mGluRs are negatively coupled to adenylyl cyclase, and thus activation of these receptors inhibits the production of protein kinase A. These

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receptors also target to VGCCs, inhibiting calcium influx via either membrane-delimited pathway or second messenger systems (Swartz and Bean 1992; Sahara and Westbrook

1993; Chavis et al. 1994). Consequently, inhibition of calcium channels leads to inhibition of transmitter release (Baskys and Malenka 1991; Desai and Conn 1991;

Knöpfel and Uusisiaari 2008; Nicoletti et al. 2010).

GABA also acts through both ionotropic GABAa receptors (GABAaRs) and metabotropic GABAb receptors (GABAbRs). GABAaRs are ion channels and they mediate fast inhibitory synaptic transmission. GABAbRs are GPCRs and they mediate slow synaptic transmission usually via activation of potassium channels. GABAbR- mediated inhibition of VGCCs and thus inhibition of neurotransmission has been nearly universally found in many brain areas in the CNS (Ulrich and Bettler 2007). Both mGluRs and GABAbRs belong to family 3 GPCRs. Because mGluRs and GABAbRs are activated by the two most abundant naturally released transmitters in the CNS, their modulatory roles in various brain systems have been extensively studied (review in

Cartmell and Schoepp 2000; Schoepp 2001; Ulrich and Bettler 2007).

Most neurons receive both excitatory and inhibitory synaptic inputs. Balanced activation of inhibitory and excitatory pathways to the same neuron is not only important for normal neuronal function but also for cell survival and maintenance. For example, excessive glutamate accumulation is closely associated with excitotoxicity via overload of intracellular calcium through NMDARs in a variety of neurologic diseases (e.g.,

Zorumski et al. 1993; Mattson et al. 2003). On the other hand, synapses are highly plastic, rendering maintenance of homeostasis of neuronal excitability via neuromodulation of

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both glutamate and GABA synapses possible. Revealing roles of neuromodulation under physiological conditions may help us find preventive means and/or treatments for pathological conditions.

In the avian auditory brainstem, neuromodulation mediated by mGluRs and

GABAbRs has been shown to improve temporal coding in the NM and NL neurons.

Briefly, glutamatergic transmission in NM is modulated by presynaptic GABAbRs but not by mGluRs (Brenowitz et al. 1998; Brenowitz and Trussell 2001; Otis and Trussell

1996), whereas GABAergic transmission in NM is subject to a dual modulation by both

GABAbRs (Lu et al. 2005) and mGluRs (Lu 2007). In the NL, glutamatergic transmission is not modulated by GABAbRs or mGluRs, whereas GABAergic transmission is modulated by both types of GPCRs (Tang et al. 2009) (see Discussion for detail). One striking aspect about neuromodulation in NM and NL is that the glutamatergic transmission in both nuclei is not subject to modulation mediated by the autoreceptors, namely mGluRs. In contrast to the relatively extensively studied cases in

NM and NL, the roles of neuromodulation in the intensity-coding NA neurons remain completely unknown. Our general hypothesis is that activation of mGluRs or GABAbRs modulates glutamate transmission in NA neurons, and the neuromodulation may improve sound intensity encoding in these neurons. Using in vitro whole-cell patch recordings from brain slices, we show that activation of either mGluRs or GABAbRs by exogenous agonists suppresses glutamate transmission in NA neurons. Surprisingly, activation of theses receptors also modulated intrinsic firing properties via suppressing cellular excitability. Contrary to our expectation, mGluR- and GABAbR-mediated

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neuromodulation in NA neurons was not cell type dependent, suggesting a universal modulatory role of these receptors in the sound intensity-coding pathway in birds. These actions of GPCRs may dynamically regulate the input-output functions of intensity- coding NA neurons, feeding proper intensity information of sounds to the LLD for ILD encoding.

CHAPTER 2

Methods

2.1 Slice preparation and in vitro whole-cell recordings

Fertilized chicken eggs were incubated using an RX2 Auto Turner (Lyon Electric

Co., Chula Vista, CA) from Embryo day 1(E1) to E18, and a Clearview Brooder (Lyon

Electric Co., Chula Vista, CA) from E19 to E21. Brainstem slices (250-300 µm in thickness) were prepared from chick embryos (E17-19), as described previously (Tang et al., 2009), with modification of the components of the artificial cerebrospinal fluid

(ACSF) used for dissecting and cutting the brain tissue. The ACSF used for dissecting

and slicing the brain tissue contained (in mM): 250 glycerol, 3 KCl, 1.2 KH2PO4, 20

NaHCO3, 3 HEPES, 1.2 CaCl2, 5 MgCl2, and 10 dextrose, pH 7.4 when gassed with 95%

O2 and 5% CO2. The procedures have been approved by the Institutional Animal Care and Use Committee (IACUC) at Northeastern Ohio Universities Colleges of Medicine and Pharmacy, and are in accordance to NIH policies on animal use. Slices were incubated at 34-36˚C for >1 h in normal ACSF containing (in mM): 130 NaCl, 26

NaHCO3, 3 KCl, 3 CaCl2, 1 MgCl2, 1.25 NaH2PO4 and 10 glucose (pH 7.4). For recording, slices were transferred to a 0.5 mL chamber mounted on a Zeiss Axioskop 2

FS Plus microscope (Zeiss, Germany) with a 40X water-immersion objective and infrared, differential interference contrast optics. The chamber was continuously superfused with ACSF (1-2 mL/min) by gravity. The microscope was positioned on the top center of an Isolator CleanTop II and housed inside a Type II Faraday cage

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(Technical Manufacturing Corporation, Peabody, MA). Recordings were performed at

34-36 ˚C, controlled by a Single Channel Temperature Controller TC324B (Warner

Instruments, Hamden, CT).

Patch pipettes were drawn on an Electrode Puller PP-830 (Narishige, Japan) to 1-2

µm tip diameter using borosilicate glass micropipettes (inner diameter of 0.86 mm, outer diameter of 1.60 mm) (VWR Scientific, Seattle, WA). The electrodes had resistances between 3 and 7 MΩ when filled with a solution containing (in mM): 105 K-gluconate,

35 KCl, 5 EGTA, 10 HEPES, 1 MgCl2, 4 ATP-Mg, and 0.3 GTP-Na, with pH of 7.2

(adjusted with KOH) and osmolarity between 280 and 290 mOsm/L. The Cl- concentration (37 mM) in the internal solution approximated the physiological Cl- concentration in NA neurons. Placement of recording electrodes was controlled by a motorized micromanipulator MP-225 (Sutter Instrument, Novato, CA). The liquid junction potential was 10 mV, and data were corrected accordingly. Voltage and current clamp experiments were performed with an AxoPatch 200B (Molecular Devices, Union

City, CA). Voltage-clamp recordings were obtained at a holding potential of -60 mV, unless indicated otherwise. Data were low-pass filtered at 3-10 kHz, and digitized with a

Data Acquisition Interface ITC-18 (Instrutech, Great Neck, NY) at 20 kHz. Recording protocols were written and run using the acquisition and analysis software AxoGraph X

(AxoGraph Scientific, Sydney, Australia).

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2.2 Synaptic stimulation and recordings of synaptic responses

Extracellular stimulation was performed using concentric bipolar electrodes with a tip core diameter of 127 µm (World Precision Instruments, Sarasota, FL). The stimulating electrodes were placed using a Micromanipulator NMN-25 (Narishige, Japan). Square electric pulses of 200 µs duration were delivered through a Stimulator A320RC (World

Precision Instruments, Sarasota, FL). Optimal stimulation parameters were selected for each cell to give postsynaptic currents of maximal amplitude.

All chemicals and drugs were obtained from Sigma (St. Louis, MO) except for

3,5-Dihydroxyphenylglycine(3,5-DHPG), (2S,2’R,3’R)-2-(2’,3’Dicarboxycyclopropyl) glycine (DCG-IV), L-(+)-2-Amino-4-phosphonobutyric acid (L-AP4),6-Imino-3-(4- methoxyphenyl)-1(6H)-pyridazinebutanoic acid (SR95531), and baclofen, which were obtained from Tocris (Ballwin, MO). Drugs were bath-applied using a gravity-driven perfusion system.

Evoked excitatory postsynaptic currents (EPSCs) were elicited with electrical shocks delivered to fibers originating from the eighth nerve and innervating the NA.

EPSCs were recorded in the presence of GABAA receptor blocker SR-95531 (Gabazine,

10 µM), and glycine receptor blocker strychnine (1 µM). The morphology of some recorded cells was revealed by adapting a method by Hamam and Kenedy (2003).

Biocytin (0.1-0.5%) was added to the internal solution used for whole-cell recordings.

After physiological recordings, brain slices were fixed overnight in 4% paraformaldehyde. Biocytin was visualized with avidin-biotin peroxidase and diaminobenzidine.

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2.3 Data analysis

The resting membrane potential was read from the amplifiers immediately after the whole-cell configuration was established. Detection of spontaneous EPSCs (sEPSCs) was performed using protocols published previously (Lu, 2007). Briefly, a template with defined rise time and decay time constant was used to detect sEPSCs. The values of the parameters for the template were determined based on the average of real events. The detection threshold was 3 times the noise standard deviation, which allowed the detection of most of the events with the least number of false-positives.

Statistical analyses were performed using Excel (Microsoft, Redmond, WA) and

Statview (Abacus Concepts, Berkeley, CA), and graphs were made in Igor (Wavemetrics,

Lake Oswego, OR). Means and standard errors of the mean (SEM) are reported in the text (n in parenthesis indicates number of cells). Statistical differences were determined by ANOVA post hoc Fisher’s test, unless otherwise indicated.

2.4 mGluR immunostaining

Chick hatchlings were deeply anesthetized (Fatal-Plus, Vortech Pharmaceuticals

Ltd., Dearborn, MI) and transcardially perfused with 4% paraformaldehyde (PFA, pH

7.4) in phosphate buffer. The brains were post-fixed in PFA for 2 h at room temperature and then overnight at 4 °C, rinsed thoroughly in phosphate-buffered saline (PBS), and vibratome-sliced (50 µm in thickness). Free-floating sections were processed for

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immunohistochemistry with standard avidin-biotin-peroxidase methods (Vectastain Elite

ABC; Vector Laboratories, Burlingame, CA). Sections were incubated in 10% normal goat serum in Tris-buffered saline (TBS) containing 0.3% Triton X-100 and avidin (200

µL/mL) for 3–4 hours. After a brief rinse in TBS, the sections were incubated in the mGluR II and III-specific antibodies (rabbit, Abcam Inc; 1:200 for both group II and III mGluRs, respectively) in TBS containing 2% normal goat serum and biotin (200 µL/mL) overnight at RT. After rinsing in TBS, the sections were incubated in biotinylated goat anti-rabbit IgG (1:1,000) at RT for 1 hour, rinsed in TBS, and incubated in ABC Elite solution (5 µL/mL) for 1 hour. After rinsing in phosphate-buffered saline (PBS; 0.075 M, pH 7.3), the sections were processed with 0.06% 3,3-diaminobenzidene HCl and 0.006%

H2O2 diluted in PBS for 15 minutes. The immunolabeling was enhanced by incubation in

0.003% osmium tetroxide in PBS for 30 seconds. After thorough rinsing, the sections were mounted on gelatin-coated slides, dehydrated, and coverslipped.

2.5 Western blot analysis

Chicks (P3) were deeply anaesthetized by isoflurane inhalation (Aerrane, Baxter

International Inc, Deerfield, IL) and rapidly decapitated. The brains were immediately dissected out in ice-cold ACSF, as described for electrophysiological experiments.

Tissues of interest were collected into vials filled with lysis buffer (10 mM Tris HCl, PH

6.8, 1% SDS, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA) and homogenized. Total proteins were extracted by centrifugation and their concentration was determined by

BCA assay (Thermo scientific, Rockford, IL, USA). Samples were then processed

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immediately for western blotting or stored at -80°C. Brain proteins were loaded in 10% gels (50 µg/lane), separated by SDS-PAGE, and wet-transferred onto PVDF membranes

(100 V, 1 h). Membranes were then blocked (1 h, RT) in 5% skim milk prepared in Tris- buffered saline-Tween (TBS-T: 10 mM Tris-HCl, 0.5 M NaCl, 0.5% Tween-20, pH 7.5) prior to incubation with the anti-mGluR II and III primary antibody, diluted at 1:1000 in

TBS-T with 5% skim milk (overnight, 4°C). After three TBS-T rinses, 10 min each, the blot was incubated with horseradish peroxidase-conjugated goat anti-rabbit-IgG (1:3000;

Biorad inc, Hercules, CA, USA) in TBS-T containing 5% skim milk for 1 h. The membrane was washed three times in TBS-T for 10 min each, and then processed for chemiluminescent reaction (ECL kit, Thermo scientific, Rockford, IL, USA) for 5 min and developed on film. Experiments were repeated 8 times.

CHAPTER 3

Result

3.1 Electrophysiology results

3.1.1 Basic intrinsic and synaptic properties of NA neurons

We first confirmed the basic intrinsic and synaptic properties of NA neurons, using protocols similar to previous studies (Soares et al. 2002; MacLeod and Carr 2005).

Based on the firing patterns in response to current injections into their cell bodies, NA neurons were classified into three types. One-spike (Onset) cells fired a single spike at the onset of the depolarizing suprathreshold current injections, followed by a sutbthreshold voltage plateau (Fig. 3A), a characteristic hallmark of time coding neurons in the auditory system (Oertel 1999). Damped cells produced a few spikes at the onset of positive current injection, followed by spikes with reduced amplitude and subthreshold membrane oscillations (Fig. 3B). This type of firing pattern is observed in late embryos

(Soares et al. 2002; this study), but not in hatchlings (Fukui and Ohmori 2003). Tonic cells responded with trains of action potentials without obvious reduction in spike amplitude to a depolarizing current injection. These classification methods were used in all physiologically recorded cells in this thesis.

Synaptic responses of NA neurons were elicited by delivering electrical stimulation to the afferent fibers innervating the NA. A single electrical shock to the medial and ventral area to the NA elicited a postsynaptic current (PSC), which was in

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most cases a combination of EPSC and IPSC, because the auditory nerve fibers and SON fibers innervating the NA travel together in this area. The EPSC and the IPSC were both inward currents under our recording conditions (high and physiological intracellular chloride concentration, 37 mM), and could not be distinguished from each other. We used pharmacological agents to isolate the EPSC. It is known that glutamate mediates the

EPSC, whereas GABA and/or glycine mediate the IPSC in NA neurons (Kuo et al. 2009).

Therefore, we applied GABAaR antagonist SR95531 (10 µM) and glycine receptor antagonist strychnine (1 µM) to block the IPSC, and isolate the EPSC. The isolated EPSC appeared to have a smaller peak current with faster decay (Fig. 3D), suggesting that the

IPSC had a slower decay than the EPSC. The stimulation paradigm was used in the following experiments where effects of various GPCR agonists on the EPSCs of NA neurons were examined.

3.1.2 Activation of groups II and III but not I mGluRs suppressed EPSCs in NA

neurons

Neurons in the NA receive glutamatergic inputs from the auditory nerve.

Glutamate can activate a variety of ionotropic receptors as well as mGluRs. Eight mGluRs have been identified, which are further classified into 3 classes (group I: mGluR

1 and 5; II: mGluR2 and 3; and III: mGluR 4, 6, 7, and 8) based on their pharmacology, homology, and signal transduction pathways (review in Kew and Kemp 2005). Non- specific and specific agonists for each group have been developed. For example, the

agonist 3,5-DHPG can activate group I mGluRs at an EC50 of 6-60 µM. DCG-IV

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Fig. 3

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Fig. 3. Basic intrinsic and synaptic properties of NA neurons

Based on the firing patterns in response to current injections into their cell bodies, NA neurons were classified into three types. A. One-spike (Onset cells) fired a single spike at the onset of the depolarizing suprathreshold current injections. B. Damped cells produced a few spikes at the onset of positive current injection, followed by spikes with reduced amplitude and subthreshold membrane oscillations. C. Tonic cells responded with trains of action potentials to a depolarizing current injection. D. Electrical shocks to the medial and ventral area to the NA elicited postsynaptic current (PSC), which was in most cases a combination of EPSC and IPSC. Application of GABAaR antagonist SR95531 and glycine receptor antagonist strychnine blocked the IPSC, and the EPSC was isolated. The scale bars in panel A also apply to panels B and C.

25

selectively activates group II mGluRs at an EC50 of 0.1-0.3 µM. Meanwhile, L-AP4 activates mGluR 4, 6, and 8 at an EC50 less than 1 µM, but a much higher concentration

(>100 µM) is required to activate mGluR7 (Cartmell and Schoepp 2000). Synaptic transmission can be modulated by each of the three groups of mGluRs, as either autoreceptors or heteroreceptors (Cartmell and Schoepp 2000).

To examine whether the glutamatergic transmission in NA neurons was modulated by one or more groups of mGluRs, we activated mGluRs using bath application of agonists specific for each group while recording EPSCs from NA neurons.

Two general observations are worth noting. First, none of the drugs applied in the experiments of this thesis changed the firing patterns of NA neurons (data not shown).

Second, contrary to our prediction, the drugs effects on EPSCs were not cell type dependent (data not shown). Therefore, we lumped the data together based on drugs applied in our statistical analyses. Group I mGluR agonist 3,5-DHPG (200 µM) did not have significant effects on the EPSC amplitude (Fig. 4, normalized EPSC amplitude during 3,5-DHPG application: 0.74±0.25, n=4, P > 0.05). In contrast, at a concentration

at least 3 times higher than their EC50, either group II mGluR agonist DCG-IV (2 µM, n=10: 3 tonic cells, 2 damped cells, 5 onset cells) or L-AP4 (10 µM, n=12: 4 tonic cells, 2 damped cells, 6 onset cells) significantly reduced the amplitude of EPSCs (Figs. 5, 7; normalized EPSC amplitude during DCG-IV application: 0.54±0.10, P < 0.01; normalized EPSC amplitude during L-AP4 application: 0.37±0.06, P < 0.01).

Although activation of mGluRs did not change the firing patterns of NA neurons, the cellular excitability and thus the spike counts in response to prolonged suprathreshold

26

current injections appeared to be changed by mGluR activation. Figure 6 shows the effects of DCG-IV (2 µM), a group II mGluR agonist, on the membrane voltage responses of NA neurons to somatic current injections. Selected membrane voltage recordings of one tonic firing NA neuron in response to a negative current injection (-0.4 nA) and two suprathreshold (0.2 and 0.75 nA) current injections showed reduced number of spikes during DCG-IV application, and the responses recovered after DCG-IV washout (Fig. 6A, B). Because NA neurons have heterogeneous firing patterns, we adopted two processes to achieve accurate analyses of pooled data. First, we excluded one-spike firing cells because these cells fired only one spike in response to prolonged suprathreshold current injections under all conditions tested here, and thus including them would bias the analyses resulting in underestimated total number of spikes and diminished the effects, if any, of the drugs on cells with other firing patterns. Second, because each cell had different threshold current (the minimum current required to make the cell fire action potentials) and fired different number of spikes in response to the same current injection, we normalized the number of spikes of the non one-spike firing cells to the maximal spike count of the NA neuron that fired the most spikes under control conditions, and plotted against the amplitude of the injected currents relative to the neurons’ threshold currents. Results showed a tendency of reduced excitability of NA neurons, displayed by higher threshold currents (Fig. 6B) and less spike counts, when group II mGluRs were activated (Fig. 6C, n=6).

The effects of L-AP4 (10 µM), a group III mGluR agonist, on the membrane voltage responses of NA neurons to somatic current injections were variable among

27

Fig. 4

28

Fig. 4. Activation of group I mGluRs did not affect EPSCs of NA neurons

Effects of 3,5-DHPG (200 µM), a group I mGluR agonist, on the amplitude of EPSCs in

NA neurons. A. The EPSC amplitude of one NA neuron was plotted against time. B.

Averaged EPSCs obtained under conditions of control (B1), 3,5-DHPG (B2), and washout of the agonist (B3). C. The sampled neuron had a one-spike firing pattern. D.

Pooled data showed that 3,5-DHPG did not significantly affect EPSCs (n=4), although the data need to be substantiated because of the limited number of cells studied. In this and subsequent figures, *, **, and *** indicate P < 0.05, P < 0.01, and P < 0.001, respectively (ANOVA post hoc Fisher’s test unless indicated otherwise). NS: not significant. Means ± SE are shown. Cells were held at -60 mV for voltage-clamp recordings unless indicated otherwise. Current-clamp recordings were performed without any somatic current injections.

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Fig. 5

30

Fig. 5. Activation of group II mGluRs suppressed EPSCs of NA neurons

Effects of DCG-IV (2 µM), a group II mGluR agonist, on the amplitude of EPSCs in NA neurons. A. The EPSC amplitude of one NA neuron was plotted against time. B.

Averaged EPSCs obtained under conditions of control (B1), DCG-IV (B2), and washout of the agonist (B3). C. The sampled neuron had a one-spike firing pattern. D. Pooled data showed that DCG-IV significantly reduced EPSCs (n=10: 5 one-spike, 2 damped, and 3 tonic cells).

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Fig. 6.

32

Fig. 6. Activation of group II mGluRs appeared to reduce the excitability of NA neurons

Effects of DCG-IV (2 µM), a group II mGluR agonist, on the membrane voltage responses of NA neurons to somatic current injections. A. Selected membrane voltage recordings of one tonic firing NA neuron in response to a negative current injection (-0.4 nA) and two suprathreshold (0.2 and 0.75 nA) current injections. B. Number of spikes of the sampled neuron plotted against the amplitude of the injected currents. C. Number of spikes of non one-spike firing cells was normalized to the maximal spike count of the NA neuron that fired the most spikes under control conditions, and plotted against the amplitude of the injected currents relative to the neurons’ threshold currents. A tendency of reduced excitability when group II mGluRs were activated was observed (n=6).

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Fig. 7

34

Fig. 7. Activation of group III mGluRs suppressed EPSCs of NA neurons

Effects of L-AP4 (10 µM), a group III mGluR agonist, on the amplitude of EPSCs in NA neurons. A. The EPSC amplitude of one NA neuron was plotted against time. B.

Averaged EPSCs obtained under conditions of control (B1), L-AP4 (B2), and washout of the agonist (B3). C. The sampled neuron had a damped firing pattern. D. Pooled data showed that L-AP4 significantly reduced EPSCs (n=12: 6 one-spike, 2 damped, and 4 tonic cells).

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Fig. 8

36

Fig. 8. Activation of group III mGluRs might alter the excitability of some NA neurons

Effects of L-AP4 (10 µM), a group III mGluR agonist, on the membrane voltage responses of NA neurons to somatic current injections. A. Selected membrane voltage recordings of one tonic firing NA neuron in response to a negative current injection (-0.4 nA) and two suprathreshold (0.2 and 0.75 nA) current injections. B. Number of spikes of the sampled neuron plotted against the amplitude of the injected currents. The drug application did not seem to affect the firing of this neuron. C. Number of spikes of non one-spike firing cells was normalized to the maximal spike count of the NA neuron that fired the most spikes under control conditions, and plotted against the amplitude of the injected currents relative to the neurons’ threshold currents. A tendency of reduced excitability when group III mGluRs were activated was observed (n=5). The discrepancy between the sampled neuron and the population and the large standard errors of the pooled data suggest that group III mGluRs might alter the excitability of some NA neurons but not others.

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different neurons. In the sampled neuron, the drug application did not seem to affect the firing of this neuron (Fig. 8A, B). However, a tendency of reduced excitability when group III mGluRs were activated was observed (Fig. 8C, n=5). The discrepancy between the sampled neuron and the population and the large standard errors of the pooled data suggest that group III mGluRs might alter the excitability of some NA neurons but not others.

3.1.3 Activation of GABAbRs suppressed EPSCs in NA neurons

Neurons in the NA receive inhibitory inputs primarily from the superior olive and also from locally distributed inhibitory neurons (Takhashi and Konishi 1988; Carr et al.

1989; Yang et al. 1999; Monsivais et al. 2000). GABA is believed to be the major neurotransmitter mediating the inhibition in avian auditory brainstem, although glycinergic input is also present in NA neurons (Kuo et al. 2009). GABA can activate both ionotropic GABAaRs as well as metabotropic GABAbRs. GABAbRs are highly expressed in the chicken auditory brainstem neurons (Burger et al. 2005). To test whether GABAbRs modulate glutamatergic transmission in the NA, we examined the effects of a specific GABAbR agonist baclofen on the EPSCs. Baclofen, at a presumably saturating concentration (100 µM), significantly reduced EPSCs to 30±8% of the control in NA neurons (Fig. 9, normalized EPSC amplitude during baclofen application:

0.31±0.08, p < 0.01; n=11: 1 tonic cell, 4 damped cells, 6 onset cells). Activation of

GABAbRs also appeared to reduce the excitability of NA neurons; baclofen application increased the threshold current and reduced the excitability of NA neurons (Fig. 10, n=5), although the responses did not recover after washout of baclofen.

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Fig. 9

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Fig. 9. Activation of GABAbRs suppressed EPSCs of NA neurons

Effects of baclofen (100 µM), a specific GABAbR agonist, on the amplitude of EPSCs in

NA neurons. A. The EPSC amplitude of one NA neuron was plotted against time. Some natural rundown of the EPSCs existed for this cell. B. Averaged EPSCs obtained under conditions of control (B1), baclofen (B2), and washout of the agonist (B3). C. The sampled neuron had a tonic firing pattern. D. Pooled data showed that baclofen significantly reduced EPSCs (n=11: 6 one-spike, 4 damped, and 1 tonic cell).

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Fig. 10.

41

Fig. 10. Activation of GABAbRs appeared to reduce the excitability of NA neurons

Effects of baclofen (100 µM), a specific GABAbR agonist, on the membrane voltage responses of NA neurons to somatic current injections. A. Selected membrane voltage recordings of one tonic firing NA neuron in response to a negative current injection (-0.4 nA) and two suprathreshold (0.25 and 0.6 nA) current injections. B. Number of spikes of the sampled neuron plotted against the amplitude of the injected currents. Baclofen increased the threshold current of this neuron substantially. C. Number of spikes of non one-spike firing cells was normalized to the maximal spike count of the NA neuron that fired the most spikes under control conditions, and plotted against the amplitude of the injected currents relative to the neurons’ threshold currents. A tendency of reduced excitability when GABAbRs were activated was observed (n=5), although the responses did not recover after washout of baclofen.

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3.1.4 Loci for mGluR- and GABAbR-mediated modulation of glutamate

transmission

To determine the loci of mGluR- and GABAbR-mediated modulation of glutamate transmission in the NA, we examined the effects of different mGluR agonists on spontaneous EPSCs (sEPSCs). In the NA, sEPSCs recorded in the slice preparations represent the miniature EPSCs (mEPSCs) (MacLeod and Carr 2005). Therefore, the effects of drugs on sEPSCs can indirectly indicate whether the modulation of glutamate transmission in NA arises from a presynaptic, postsynaptic, or dual mechanism.

Modulation of the frequency but not the amplitude of sEPSCs would imply a presynaptic mechanism, modulation of the amplitude but not of the frequency of sEPSCs would imply a postsynaptic mechanism, and modulation of both the frequency and amplitude of sEPSCs would imply a mechanism involving both pre- and postsynaptic elements of the synapse (e.g., Chu and Moenter 2005; Piet et al. 2003; Valenti et al. 2003).

Somewhat surprisingly, activation of group II mGluRs, group III mGluRs, or

GABAbRs each reduced both the frequency and amplitude of sEPSCs of NA neurons

(Figs. 11-13). As shown in Figure 11, group II agonist DCG-IV (2 µM) significantly reduced the frequency of sEPSCs (Fig. 11B, n=8 cells: 5 onset cells, and 3 tonic cells, P <

0.01), and the amplitude of sEPSCs (Fig. 11C, n=8, P < 0.05). Group III mGluR agonist

L-AP4 (10 µM) produced similar effects (Fig. 12, n=7 cells: 2 damped cells, 4 tonic cells, and 1 onset cell), and so did GABAbR agonist baclofen (100 µM) (Fig. 13, n=8 cells: 3 onset cells, and 5 tonic cells). We remain cautious about these results because of the

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Fig. 11

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Fig. 11. Activation of group II mGluRs reduced both the frequency and amplitude of sEPSCs of NA neurons

A. Representative sEPSCs recorded under the conditions of control, DCG-IV (2 µM), and washout. Shown on the right are individual detected event of the sample cell, at an expanded time scale. Note different scale bars for the original and the individual trace. B

& C. DCG-IV (2 µM) significantly reduced both the frequency and the amplitude of sEPSCs (n=8: 5 one-spike, and 3 tonic cells).

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Fig. 12

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Fig. 12. Activation of group III mGluRs reduced both the frequency and amplitude of sEPSCs of NA neurons

A. Representative sEPSCs recorded under the conditions of control, L-AP4 (10 µM), and washout. Shown on the right are the averaged traces of all detected events of the sample cell. Note different scale bars for the original and the averaged traces. B & C. L-AP4 (10

µM) significantly reduced both the frequency and the amplitude of sEPSCs (n=7: 1 one- spike, 2 damped, and 4 tonic cells).

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Fig. 13.

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Fig. 13. Activation of GABAbRs reduced both the frequency and amplitude of sEPSCs of NA neurons

A. Representative sEPSCs recorded under the conditions of control, baclofen (100 µM), and washout. Shown on the right are individual detected event of the sample cell, at an expanded time scale. Note different scale bars for the original and the individual trace. B

& C. Baclofen (100 µM) significantly reduced both the frequency and the amplitude of sEPSCs (n=8: 3 one-spike, and 5 tonic cells).

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relatively high noise level in these recordings of sEPSCs, which may mask some of the small sEPSCs.

3.2 Immunohistology and Western blot results

The presence of group II and III mGluRs in the NA was studied using immunostaining and Western blot techniques (Fig. 14). Preliminary data showed that antibodies raised in the rat against group II and group III mGluRs seemed to be able to detect the expression of their corresponding receptors in NA neurons (Fig. 14A, B), and omitting the primary antibodies resulted in relatively negative staining (Fig. 14C). We also tested the specificity of the antibodies with Western blot. The mouse cerebellum (for group II mGluRs) the rat cerebellum (for group III mGluRs) were used as positive controls. In protein extracts from multiple chicken tissues (cerebellum, cerebrum, ventral part of the brainstem, and dorsal part of the brainstem containing NM, NL, and NA), a band (about 100 kD) corresponding to group II mGluRs or group III mGluRs was detected at the expected molecular weight (Fig. 14D).

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Fig. 14

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Fig. 14. NA neurons seemed to express groups II and III mGluRs

A. Antibodies against group II mGluRs seemed to stain some NA neurons. B. Antibodies against group III mGluRs also seemed to stain some NA neurons. C. The staining was negative when the primary antibody was omitted. D. Western blot appeared to confirm the specificity of the antibodies. Expression in the mouse or rat cerebellum (CBL) was used as a positive control. The red lines in panels A and B define approximately the boundaries of the NA. Scale bar in A (100 µm) also applies to B and C.

CHAPTER 4

Discussion and Conclusions

4.1 Neuromodulation of excitatory transmission in NA

4.1.1 Involvement of multiple mGluRs in modulation of glutamate release in NA

GPCR-mediated modulation of synaptic transmission has been found in numerous central neuronal systems. Although such modulation almost universally exists in the

CNS, the types of GPCRs involved, and the mechanisms underlying the regulation dramatically differ among different systems. In the NA, multiple mGluRs agonists

(DCG-IV and L-AP4 for groups II and III, respectively) significantly reduced EPSCs, suggesting involvement of multiple mGluR members in modulation of glutamate release in NA neurons. Modulation of the synaptic transmission by multiple mGluRs at the same synapses is not uncommon (e.g. review in Knöpfel and Uusisaari 2008; Drew and

Vaughan 2004; Zheng and Johnson 2003). Such seemingly redundant modulation may be accounted for by the fact that mGluRs of different groups are clustered at different membrane loci, and they may exert modulation of glutamate release via different signaling pathways at different locations. Furthermore, the density of each group mGluRs on the presynaptic membrane may be low so that multiple members from different groups need to be activated simultaneously in order to generate synergistic regulation of glutamate release. In some cases, interactions between receptors activated by different transmitters also exist (Hirono et al. 2001).

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Group I agonist 3,5-DHPG did not affect EPSCs significantly. This is unlikely due to the concentration of the agonist. We chose the concentrations of 200, 2, and 10 µM for

3,5-DHPG, DCG-IV, and L-AP4, respectively. These concentrations are at least three

times their EC50 (Cartmell and Schoepp 2000; Schoepp et al. 1999), so activation of corresponding receptors, if present, is expected to be nearly saturating. In addition, the specificity of these agonists has been tested in previous studies, which showed selectivity of these agonists on avian mGluRs (Sampaio and Paes-de-Carvalho 1998; Tasca et al.

1999; Caramelo et al. 1999; Dutar et al 1999; Gomes et al. 2004; Kreimborg et al. 2001;

Lu and Rubel 2005). Assuming saturated activation of the metabotropic receptors by the agonists at the concentrations used, group III mGluR agonist L-AP4 and GABAbR agonist baclofen produced the strongest reduction in the amplitude of EPSCs (Figs. 7 and

9), suggesting that group III mGluRs, along with GABAbRs, may play a key role in modulating glutamate transmission in NA neurons.

4.1.2 Mechanisms of modulation of glutamate release in NA

The loci for GPCR action on synaptic transmission can be presynaptic, postsynaptic, or both. Direct evidence showing presynaptic modulation loci of GPCRs comes from presynaptic recordings. However, because the size of most presynaptic terminals is too small, such recordings have been successfully achieved in only few large synapses like the glutamatergic terminals at MNTB (Forsythe 1994). The conclusion about loci of GPCR modulation relies on indirect evidence obtained from studies that examine the effects of GPCR activation on miniature transmitter release, pair-pulse

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ratios, and variation coefficients of EPSCs. We used the first method to determine the action loci of mGluRs and GABAbRs on glutamatergic transmission in NA. The rationale for this strategy is that modulation of the frequency but not the amplitude of mIPSCs would imply a presynaptic mechanism, modulation of the amplitude but not the frequency would imply a postsynaptic mechanism, and modulation of both would imply a dual (both pre- and postsynaptic) mechanism (Chen and van den Pol 1998; Jarolimek and

Misgeld 1997; Kabashima et al. 1997).

We recorded sEPSCs, which are highly likely represent mEPSCs in NA neurons, because in slice preparations we did not observe spike activity in NA neurons, and like in

NM or NL, blocking action potential-dependent release does not affect sEPSCs in NA neurons (MacLeod and Carr 2005). Somewhat surprisingly, all the three drugs that reduced EPSC amplitude of NA neurons (group II mGluR agonist DCG-IV, group III agonist L-AP4, and GABAbR agonist baclofen) produced a significant reduction in both the frequency and the amplitude of sIPSCs, suggesting a dual mechanism. The effects on the frequency of sEPSCs were expected, based on previous studies (reviewed in Cartmell and Schoepp 2000; Schoepp 2001; Ulrich and Bettler 2007) and in particular on the studies in NM and NL where exclusively presynaptic modulation of transmission has been observed (Brenowitz et al. 1998; Brenowitz and Trussell 2001; Otis and Trussell

1996; Lu et al. 2005; Lu 2007; Tang et al. 2009). VGCCs are subject to modulation by mGluRs (reviewed in Catterall 2000; Stefani et al. 1996) and GABAbRs (Ulrich and

Bettler 2007). Since evoked transmitter release is dependent on Ca2+ influx through

VGCCs in presynaptic terminals, modulation of VGCCs by mGluRs and GABAbRs is

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highly likely the mechanism underlying the presynaptic regulation of glutamate release at

NA.

The effects on the amplitude of sEPSCs are somewhat surprising. Groups II and III mGluRs are generally expressed on presynaptic terminals (reviewed in Nicoletti et al.

2010), and postsynaptic effects on synaptic transmission are rare. Effects of GABAbRs on postsynaptic neurons are heterogeneous. Activation of GABAbRs hyperpolarizes a variety of neurons in the CNS by activating K+ channels (reviewed in Misgeld et al.

1995), but does not hyperpolarize the membrane potential in many other central neurons

(e.g. Misgeld et al. 1989; Newberry and Nicoll 1984; Stevens et al. 1985, 1999). The postsynaptic effects of these drugs on NA neurons need to be further examined. We remain cautious about the observation that both mGluR and GABAbR agonists reduced the amplitude of sEPSCs in NA neurons, and consider our results and conclusion on this part preliminary because of the limited number of cells collected for each drug, and the moderate quality of chart recordings of sEPSCs.

4.1.3 Comparisons of modulation among NM, NL, and NA

The glutamatergic transmission at NM is modulated by GABAbRs

(heteroreceptors) but not by mGluRs (autoreceptors) (Otis and Trussell 1996; Otis et al.

1996; Brenowitz et al. 1998; Brenowitz and Trussell 2001). Surprisingly, the glutamatergic transmission in NL neurons is not subject to modulation by either group of receptors (Tang et al., 2009), whereas the glutamatergic transmission at NA is modulated

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by both types of GPCRs (this study). The GABAergic transmission at both NM and NL is subject to a dual modulation by both GABAbRs and mGluRs (Lu et al. 2005; Lu 2007;

Tang et al. 2009). Possible functions of modulation in these nuclei are discussed below.

Because neuromodulation of the inhibitory transmission in NA has not been studied, we focus our discussion here primarily on modulation of glutamate release.

For heteroreceptor GABAbR-mediated modulation of glutamatergic transmission to occur, synaptically released GABA needs to “spillover” to activate GABAbRs on the excitatory terminals. It is well known that synaptically released transmitter is actively uptaken into glial cells and neurons through transmitter transporters, so it is normally difficult for transmitter molecules to reach their receptors at distance in order to initiate crosstalk between two distinct types of synapses (Barbour and Häusser 1997). However, some geometric features of some synapses may facilitate “spillover”. NM neurons have large glutamatergic synapses covering more than half of the cell body surface (Ryugo and

Parks 2003). GABAergic terminals innervating NM neurons are presumably located nearby glutamatergic terminals (Lachica et al. 1994). These morphological features may facilitate interactions between the glutamatergic and GABAergic pathways and allow presynaptic GABAbRs on the glutamatergic terminals to be activated by GABA spillover

(Otis and Trussell 1996; Otis et al. 1996; Brenowitz et al. 1998; Brenowitz and Trussell

2001; Lu et al. 2005). In contrast, at NA, because both glutamate and GABA terminals are likely to be small bouton-like structures, activation of GABAbRs on glutamate terminals may require relatively short distance of traveling of GABA molecules. In other words, we speculate that at least some of the glutamate and GABA synapses on NA

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neurons are expected to be spatially close to each other in order for spilled GABA to reach their receptors on the glutamatergic terminals. On the other hand, if modulation of

EPSCs by postsynaptic GABAbRs does exist (waiting to be confirmed), it may provide a relatively more accessible route simply because of the large size of the postsynaptic cell bodies and hence access of GABA molecules to activate the receptors.

In the NM, regulation of synaptic strength of the glutamatergic inputs is critical for

NM neuronal function. GABA modulates glutamate release at a presynaptic locus via metabotropic GABAbRs, reducing synaptic depression (Otis and Trussell 1996;

Brenowitz et al. 1998; Brenowitz and Trussell 2001). This modulation helps increase the synaptic efficacy and ensure high safety factor of glutamate transmission at the endbulb synapses, rendering NM neurons capability of following high frequency inputs. On the other hand, modulation of the GABAergic transmission by GABAbRs (Lu et al. 2005) and mGluRs (Lu 2007), may help preserve temporal information of sounds by preventing excessive release of GABA. Neurons in the SON, driven by excitatory inputs from NL and NA, are probably activated and fire spikes in a sound level dependent manner

(Burger et al. 2005; Dasika et al. 2005). This is critical in engaging the feedback mechanism to balance the excitatory inputs to the timing-coding neurons; stronger excitatory glutamatergic inputs to the avian cochlear nucleus lead to stronger feedback inhibitory inputs. However, too strong activation of the GABAergic pathway could disrupt phase-locking fidelity by generating GABA spikes (Lu and Trussell 2001;

Monsivais et al. 2001). Activation of GPCRs on inhibitory terminals suppresses

GABAergic transmission in NM, preventing excitatory action of GABA while leaving

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their inhibitory actions intact. Hence, even though they have functionally opposite ligands, the two synaptic pathways may achieve balanced excitatory and inhibitory inputs to NM through reciprocal inhibition of transmitter release via their metabotropic receptors. In NA, such reciprocal interactions between the excitatory and the inhibitory inputs are unknown because neuromodulation of the inhibitory transmission has not been studied.

In the NL, it is unusual that the glutamatergic transmission is affected by neither

GABAbRs nor by mGluRs (Tang et al. 2009). Given the universal modulatory effects of these receptors on glutamate synapses in the CNS (Cartmell and Schoepp 2000; Ulrich and Bettler 2007), we speculate that this lack of modulation may uniquely fit the NL’s function in coincidence detection. The morphological symmetry of bilateral dendrites of

NL neurons implies physiological symmetry between the two segregated excitatory inputs, which may be optimal for coincidence detection. Neurons of the NL show similar thresholds to tone-burst stimulation presented to either ear (Rubel and Parks 1975; Köppl and Carr 2008), suggesting that they have approximately equal sensitivity to their segregated excitatory inputs. The importance of balanced excitatory inputs to coincidence detectors can also be inferred from computer modeling studies in which the same membrane parameters are assigned to the two excitatory inputs (Agmon-Snir et al. 1998;

Grau-Serrat et al. 2003; Dasika et al. 2007). The lack of modulation of the excitatory inputs may be one key to preserving physiological symmetry, because modulation of one but not the other excitatory input or the differential degree of modulation of the two inputs would lead to imbalanced excitation. A differential driving force may bias the NL

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firing toward the stronger input, reducing the acuity for sound localization. Therefore, maintenance of the overall balance of excitation and inhibition in NL neurons may rely primarily on regulation of the inhibitory inputs to the NL, and the selective regulation of the GABAergic transmission by GABAbRs and mGluRs in the NL may be crucial to the maintenance of fine temporal information in the auditory brainstem.

In the NA, the glutamatergic transmission is subject to a dual modulation by both mGluRs and GABAbRs. The mGluR-mediated modulation of glutamate release in NA is a striking division from the timing-coding NM and NL neurons where such autoreceptor- mediated modulation of glutamatergic transmission is absent. This could be simply because mGluRs are not expressed on the excitatory terminals of NM and NL neurons.

However, this is unlikely because mGluRs are found to be expressed in NM and NL (our unpublished observation), although expression loci (presynaptic or postsynaptic, or both) have not been determined. Alternatively, mGluRs on presynaptic glutamatergic terminals of NM/NL neurons might be saturated or desensitized, and therefore no further activation can be observed when exogenous agonists are applied. Another possible explanation for the difference in mGluR-mediated regulation of glutamate release between NA and

NM/NL neurons may reside in the neurons sensitivity to sound intensity. NA neurons have much steeper rate-intensity functions and have larger dynamic ranges than NM and

NL neurons (Warchol and Dallos 1990). Therefore, NA neurons are expected to respond with increasing amount of glutamate release sound intensity is raised, leading to a high propensity of activation of mGluRs, whereas NM and NL neurons are relatively

60

insensitive to changes in sound intensity, leading to a possible flat amount of glutamate release and hence a low propensity of activation of mGluRs.

The dual modulation of glutamate release in NA may bear functional significance.

We propose that a tonic activation of mGluRs may exist limiting the basal level of glutamate release at NA. In a slice preparation, NA neurons do not fire action potentials spontaneously. However, they are considerably active in vivo, with a spontaneous firing rate of about 25 Hz (Warchol and Dallos 1990). An even higher rate is expected when sound stimuli are present because during acoustic stimulation, the firing rate of cochlear nuclear synapses can be up to several times higher (Sachs and Abbas 1974; Liberman

1978; Manley et al. 1985, 1997). The high spike activity in NA neurons indicates highly active release of glutamate from the auditory nerve. High-rate activity may well delay the clearance of glutamate (Scanziani et al. 1997). It is likely that the neurotransmitter glutamate is present relatively constantly in the synaptic cleft and surrounding areas at a level that is sufficient in activating mGluRs and exerting a tonic regulation on its own release. However, whether such endogenous mGluR activity exists in NA neurons waits to be examined. GABAbR-mediated modulation may be recruited as intensive GABA release occurs. Both modulations can regulate the input-output functions of NA neurons, and the degree of these modulations may determine the proper dynamic range and thus their intensity coding capability. Some NA neurons (about 22%) display non-monotonic input-output functions (Warchol and Dallos 1990), indicating that inhibitory input plays roles in sculpturing their intensity coding properties. Consequently, modulation of the

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inhibitory input will inevitably affect the dynamic range of these neurons. Future work should also investigate modulation of the inhibitory inputs in NA.

4.1.4 Comparisons of modulation between NA and mammalian cochlear nucleus

In general, neuromodulation mediated by mGluRs and GABAbRs in the mammalian cochlear nucleus has not been extensively studied. The most studied is the dorsal cochlear nucleus (DCN), a structurally cerebellum-like nucleus, believed to function in encoding spectral information of sounds (Oertel and Young 2004). A number of studies have suggested expression of mGluRs in the DCN (Shigemoto et al. 1993;

Petralia et al. 1996). In vitro studies have showed that activation of group I mGluRs suppresses glutamatergic transmission of the parallel fiber inputs in fusiform cells of the

DCN (Molitor and Manis 1997), and multiple mGluRs are involved in long-term synaptic plasticity in the same cells (Fujino and Oertel 2003). Modulation of sound level processing by mGluRs in the DCN in vivo has also been explored, and highly variable effects (suppressed, enhanced, and unchanged responses) of mGluRs are observed, regardless of cell type (Sanes et al. 1998). However, the majority of NA neurons do not seem to be similar to DCN neurons in their morphology or physiology (Köppl and Carr

2003). Indeed some NA neurons are considered similar to the multipolar neurons in the mammalian ventral cochlear nucleus (MacLeod and Carr 2007). Expression of

GABAbRs is found in anteroventral and posteroventral (AVCN and PVCN) (Juiz et al.

1994; Lujan et al. 2004). However, modulation of glutamatergic transmission by

62

GABAbRs in the VCN has not been reported. There only exists a report in an abstract form, which showed that group I mGluRs depolarize AVCN bushy cells but have no effects on glutamatergic transmission (Chanda and Xu-Friedman, ARO, 2011).

Therefore, detailed comparison between NA and mammalian cochlear nucleus requires more studies.

4.2 Future research direction

Seven areas are identified for future studies regarding neuromodulation in the NA.

First, more data using current paradigms/protocols used in this thesis are needed in order to substantiate the findings out of this thesis. The effects of mGluRs and GABAbRs on the sEPSCs and the intrinsic firing properties observed in this thesis need to be confirmed with higher quality recordings. Second, although modulation of glutamate release by application of exogenous agonists for GPCRs was found in NA neurons, it is physiologically more significant to show that endogenous activity of these receptors also exert modulation. To test this, effects of synaptically released GABA or glutamate on the glutamatergic transmission at NA should be examined by using antagonists for mGluRs or GABAbRs. Third, experiments investigating protein expression are needed to reveal the distribution pattern, density of various mGluRs in NA neurons. Fourth, given the existence of dual modulation of the inhibitory transmission by mGluRs and GABAbRs in both the NM and NL, we predict that such neuromodulation also exists in the NA, participating in regulation of GABA/glycine release and hence the synaptic strength of inhibition impinging upon NA neurons. Therefore, it is likely that the balance between

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excitation and inhibition in the intensity-coding NA neurons can be achieved via GPCR- mediated regulation of both the excitatory and the inhibitory inputs. Fifth, a mix of short- term plasticity (facilitation and depression) of EPSCs is characteristic of NA neurons while purely synaptic depression is characteristic of timing coding neurons (MacLeod and Carr 2007; MacLeod and Horiuchi 2011), so it is imperative that the modulatory effects of GPCRs on short-term plasticity properties of NA neurons be investigated. We predict that the amplitude of individual EPSCs of NA neurons in response to train stimulations would be affected indistinguishably, and the response profile or short-term plasticity patterns would be maintained. Sixth, we predicted cell type specific neuromodulation before this thesis was initiated. However, we observed universal modulation of EPSCs in NA neurons by mGluRs and GABAbRs. This may be accounted for by the fact that all different morphological types of NA neurons project to the LLD where ILD coding is initiated (Soares and Carr 2001). However, in depth studies are required to further investigate cell type dependent modulation in the NA. Finally, physiological roles of these modulations should be tested under in vivo conditions.

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Abbreviations:

ACSF: artificial cerebrospinal fluid

AMPA: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

AVCN: anterior ventral cochlear nucleus

DCN: dorsal cochlear nucleus

EPSC: excitatory postsynaptic current

GABA: gamma- aminobutyric acid

GPCR: G-protein-coupled receptor

IP3: inositol 1,4,5-triphosphate

IPSC: inhibitory postsynaptic current

ILD: interaural level difference

ITD: interaural time difference

LNTB: lateral nucleus of trapezoid body

LSO: lateral superior olive mGluR: metabotropic glutamate receptor

MNTB: medial nucleus of trapezoid body

MSO: medial superior olive

NA: nucleus angularis

NMDA: N-methyl D-aspartate

NL: nucleus laminaris

NM: nucleus magnocellularis

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SON: superior olivary nucleus

VGCC: voltage-gated calcium channels

VLVp: posterior division of the ventral nucleus of the lateral lemniscus

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