Wei Thesis-Final

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 i 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 ii 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 iii 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 iv 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 vi 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 vii 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; 1 2 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 3 (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

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