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Studies of glycine-activated ionotropic receptors in retina

ProQuest Dissertations and Theses, 2009
Dissertation
Author: Lei Duan
Abstract:
Ionotropic glycine receptors (GlyRs) serve important inhibitory roles throughout the central nervous system, including the retina. They are ligand-gated chloride ion channels that belong to the same cysteine-loop ligand-gated ion channel superfamily as ionotropic GABA receptors. There are four types of ionotropic glycine receptors based on the alpha subunit composition. Unlike its close relative GABA A/C receptors, there are no selective pharmacological agents for different subtypes of glycine receptors. In the first half of my study, I focused on the search of possible antagonists that show selectivity for glycine receptor subtypes. I found that caffeine is a structural analog of strychnine and a competitive antagonist at ionotropic glycine receptors. Docking simulations indicate that caffeine and strychnine may bind to similar sites at the GlyR. The R131A GlyR mutation, which reduces strychnine antagonism without suppressing activation by glycine, also reduces caffeine antagonism. GlyR subtypes show different caffeine sensitivity. Tested against the EC50 of each GlyR subtype, the order of caffeine potency (IC 50 ) is: α2β (248 ± 32 μM) [approximate] α3β (255 ± 16μM) > α4β (517 ± 50 μM) > α1β (837 ± 132 μM). However, considering that the α3β GlyR is the least sensitive to glycine than the other GlyR subtypes, this receptor is most effectively blocked by caffeine. The glycine dose-response curves and the effects of caffeine in retina suggest that amphibian retinal ganglion cells are dominated by the α1β and possibly α4β GlyRs, but are unlikely to utilize the α3β or α4β GlyRs. Comparing the effects of caffeine on glycinergic spontaneous and light-evoked post-synaptic currents indicates that evoked release may be a result of either elevation of glycine concentration at the synapse or recruitment of more synapses. The caffeine IC 50 values for bath applied 100 μM glycine and for the synaptic glycinergic-currents are both 1.7 mM, thus the synaptic glycine concentration may be estimated to be around 100 μM in salamander retinal ganglion cells. As high millimolar concentrations of caffeine can completely inhibit glycinergic synaptic transmission, the use of caffeine to stimulate ryanodine receptors should be under discretion. NMDA receptors (NMDAR), along with AMPA/Kainate receptors, are excitatory ionotropic glutamate receptors possessing integral cation channels. NMDARs are abundantly expressed in the second synaptic layer of the retina; however, previous experiments suggest that the excitatory light responses of retinal ganglion cells are driven almost exclusively by AMPA/Kainate receptors. NMDA receptor activation in retina seems to rely on glutamate spillover to perisynaptic sites where NMDARs are located. In the second part of my study, I found evidence for synaptic NMDA receptor activation in retinal ganglion cells, by changing some of the commonly employed experimental protocols. If the retina was exposed to an adapting light, then there is a progressive enhancement of the NMDA synaptic current. If glycine inhibition was blocked then a large NMDA receptor current can contribute to the ganglion cell light response. Similarly, when magnesium was removed from the extracellular solution then the light response became dominated by the NMDAR receptor current. These experiments indicate the synaptic existence of NMDAR in retina, and that the activation of NMDA receptors is state-dependent.

III    TABLE OF CONTENTS

Acknowledgements …………………………………………………..……………… I List of Figures and Tables ……………………………………………………………VII List of Abbreviations …………………………………………………………………. IX Abstract……………………………………………………………………………. …...XI

Chapter 1 Introduction 1.1. General introduction of retina organization……………………………………. 1 1.2. Synaptic pathways in retina …………………………………………………….5 1.3. Glycine-binding-site-containing receptors in the retina………………………9 1.3.1. Overview of ionotropic glycine receptors……………………………….9 1.3.2. Ionotropic glycine receptor localization in the retina..……………………11 1.3.3. Ionotropic glycine receptor functions in the retina………………………..12 1.3.4. Pharmacology of Ionotropic glycine receptors……………………………13 1.3.5. Pharmacology of caffeine………………………………………………… 14 1.3.6. Metabotropic glycine receptors……………………………………………15

IV    1.3.7. NMDA receptor overview…………………………………………………17 1.3.8. NMDA receptor in retina: disconnection between localization and function…………………………………………………………………………………17

Chapter 2 Materials and Methods 2.1. Retina Slice Preparation…………………………………………………………..20 2.2. Cell culture and cDNA transfection ……………………………………………..21 2.3. Site directed mutagenesis…………………………………………………………22 2.4. Light adaptation and light stimulation……………………………………………22 2.5. Whole cell patch clamp recording……………………………………………….23 2.6. Homology modeling and ligand docking………………………………………24 2.7. Data Analysis…………………………………………………………………..25 2.8. Solutions and pharmacological agents…………………………………………26

Chapter 3 Caffeine Inhibition of Ionotropic Glycine Receptors 3.1. Caffeine suppressed glycine-activated currents in retinal ganglion cells………27 3.2. Structural similarity between caffeine and strychnine…………………………30 3.3. Docking of caffeine and strychnine to α3 GlyR……………………………….31

V    3.4. The α1 GlyR R131A mutation and caffeine inhibition………………………. 34 3.5. Caffeine does not act on intracellular sites ……………………………………… 36 3.6. Antagonist properties of caffeine on α1β, α2β, α3β and α4β GlyR on HEK293 cells…………………………………………………………………………………...40 3.7. Caffeine effect on glycinergic synaptic transmission……………………………46 3.8. Caffeine effect on light-evoked and spontaneous synaptic events………………52

Chapter 4 State-Dependent Activation of NMDA Receptors in Retina……………..55 4.1. The state of light adaptation affects the NMDAR-mediated fraction of ganglion cell EPSCs…………………………………………………………………………………...56 4.2. Glycine inhibition can suppress large NMDAR-mediated currents……………….65 4.3. Magnesium unblock reveals large synaptic NMDAR-mediated currents………….68 4.4. AMPA antagonist may enhance NMDAR-mediated currents……………………..68

Chapter 5 Discussion 5.1 General discussion…………………………………………………………………...72 5.2. Caffeine inhibition of ionotropic glycine receptor 5.2.1. Ionotropic glycine receptor pharmacology……………………………………74

VI    5.2.2. Caffeine and strychnine………………………………………………………74 5.2.3. Glycine receptor subtypes in amphibian retina………………………………77 5.2.4. Glycinergic synaptic currents on retinal ganglion cells………………………79 5.2.5. Summary………………………………………………………………………81 5.3. State-Dependent Activation of NMDA Receptors in Retina………………………..82 5.3.1. Summary………………………………………………………………………87

References ……………………………………………………………………………. 89

VII    LIST OF FIGURES AND TABLES Figure 1. Schematic diagram of retinal structure ………………………………………. 3 Figure 2. ON/OFF separation of vertical pathway and synaptic circuitry in the inner plexiform layer………………………………………………………………….7 Figure 3. Structure of members of the methylxanthine family and strychnine ……..….16 Figure 4. Caffeine suppresses exogenous glycine-activated current on retinal ganglion cells………………………………………………………………………………28 Figure 5. Structural similarities between caffeine and strychnine…………………….32 Figure 6. The R131A GlyR mutation decreases caffeine sensitivity of the α1 GlyR...35 Figure 7. Caffeine dose not block GlyRs at intracellular sites…………………………38 Figure 8. Caffeine inhibition of GlyR subtypes………………………………………42 Figure 9. Caffeine inhibits glycine responses in α3β GlyRs. ………………………..45 Figure 10. Caffeine suppression of glycinergic IPSCs in retinal ganglion cel l s………………………….…………………………...…………….48 Figure 11. Effect of caffeine on spontaneous glycinergic IPSCs in retinal ganglion c e l l s …………………………………………………………..…..…50 Figure 12. Effect of caffeine on glycinergic L-IPSCs and L-EPSCs in retinal ganglion cel l s …………………………………………………………….…..…50

VIII    Figure 13. NMDAR component of L-EPSCs differs in the dark and light adapted retinas…………………………………………………………………………….58 Figure 14. NMDAR component of L-EPSC increases with prolonged light exposure…61 Figure15. D-serine, dopamine and melatonin levels may not account for the change of NMDAR currents between the dark/light adapted states……………………....63 Figure 16. Blocking glycinergic inhibition reveals an NMDAR-exclusive current…66 Figure 17. Magnesium unblock reveals prominent NMDAR currents………..…………70 Figure 18. Blocking AMPA/Kainate rreceptors may enhance NMDAR-mediated current…………………………………………………………………………..71 Figure 19. Schematic model of NMDAR synapses from bipolar to ganglion cells….….88 Table 1. Sensitivity of glycine receptor subunits to glycine and caffeine……………44

IX    List of Abbreviations ACh: Acetylcholine AMPA: ± -α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate BAPTA: 1, 2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid CFN: Caffeine cGMP: Cyclic guanosine monophosphate CNQX: 6-cyano-7-nitroquinoxaline-2, 3-Dione D-AP5: D (-)-2-Amino-5-phosphonopentanoic acid DCKA: 5, 7-dichlorokynurenic acid EC 50 : Half maximal effective concentration EPSC: Excitatory postsynaptic current GABA: γ-aminobutyric acid GlyR: glycine receptor G-protein: Guanine nucleotide-binding proteins HEK cells: Human embryonic kidney cells IBMX: Isobutylmethylxanthine IC 50 : Half maximal inhibitory concentration

X    IPL: Inner plexiform layer IPSC: Inhibitory postsynaptic current LED: Light-emitting diode nAChR: Nicotinic acetylcholine receptor NMDA: N-methyl-D-aspartic acid NMDAR: N-methyl-D-aspartic acid receptor OPL: Outer plexiform layer PTX: Picrotoxin RGC: Retinal ganglion cells SR95531: 2-(3'-carboxy-2'-propyl)-3-amino-6-p-methoxyphenylpyridazinium bromide STR: Strychnine TPMPA: (1, 2, 5, 6-Tetrahydropyridin-4-yl) methylphosphinic acid

XI    Abstract Ionotropic glycine receptors (GlyRs) serve important inhibitory roles throughout the central nervous system, including the retina. They are ligand-gated chloride ion channels that belong to the same cysteine-loop ligand-gated ion channel superfamily as ionotropic GABA receptors. There are four types of ionotropic glycine receptors based on the alpha subunit composition. Unlike its close relative GABA A/C receptors, there are no selective pharmacological agents for different subtypes of glycine receptors. In the first half of my study, I focused on the search of possible antagonists that show selectivity for glycine receptor subtypes. I found that caffeine is a structural analog of strychnine and a competitive antagonist at ionotropic glycine receptors. Docking simulations indicate that caffeine and strychnine may bind to similar sites at the GlyR. The R131A GlyR mutation, which reduces strychnine antagonism without suppressing activation by glycine, also reduces caffeine antagonism. GlyR subtypes show different caffeine sensitivity. Tested against the EC 50 of each GlyR subtype, the order of caffeine potency (IC 50 ) is: α2β (248 ± 32 µM) ≈ α3β (255 ± 16µM) > α4β (517 ± 50 µM) > α1β (837 ± 132 µM). However, considering that the α3β GlyR is the least sensitive to glycine than the other GlyR subtypes, this receptor is most effectively blocked by caffeine. The glycine dose-response curves and the effects of caffeine in retina suggest that amphibian retinal ganglion cells are dominated by the α1β and possibly α4β GlyRs, but are unlikely to utilize the α3β or α4β GlyRs. Comparing the effects of caffeine on glycinergic spontaneous and light- evoked post-synaptic currents indicates that evoked release may be a result of either

XII    elevation of glycine concentration at the synapse or recruitment of more synapses. The caffeine IC 50 values for bath applied 100 µM glycine and for the synaptic glycinergic- currents are both 1.7 mM, thus the synaptic glycine concentration may be estimated to be around 100 µM in salamander retinal ganglion cells. As high millimolar concentrations of caffeine can completely inhibit glycinergic synaptic transmission, the use of caffeine to stimulate ryanodine receptors should be under discretion.

NMDA receptors (NMDAR), along with AMPA/Kainate receptors, are excitatory ionotropic glutamate receptors possessing integral cation channels. NMDARs are abundantly expressed in the second synaptic layer of the retina; however, previous experiments suggest that the excitatory light responses of retinal ganglion cells are driven almost exclusively by AMPA/Kainate receptors. NMDA receptor activation in retina seems to rely on glutamate spillover to perisynaptic sites where NMDARs are located. In the second part of my study, I found evidence for synaptic NMDA receptor activation in retinal ganglion cells, by changing some of the commonly employed experimental protocols. If the retina was exposed to an adapting light, then there is a progressive enhancement of the NMDA synaptic current. If glycine inhibition was blocked then a large NMDA receptor current can contribute to the ganglion cell light response. Similarly, when magnesium was removed from the extracellular solution then the light response became dominated by the NMDAR receptor current. These experiments indicate the synaptic existence of NMDAR in retina, and that the activation of NMDA receptors is state-dependent.

1    Chapter 1 Introduction 1.1. General introduction of retina organization The retina is the first step in the formation of vision. It is embryonically an extension from the forebrain (diencephalon) and maintains connection with the higher vision centers through the optic nerve. The structure of retina is similar across most species. Similar with the brain, the retina consists of neurons and synapses that are highly efficient, using only a small number of neurotransmitters and receptor types to encode large amounts of information. Aside from its paramount importance in vision, the retina serves as an excellent model system to study neurophysiology; its neurological circuits are densely packed into distinct layers and are easily accessible. It possesses only a few types of neurons yet contain almost all of the neurotransmitters and neurotransmitter receptors found in the central nervous system. The retina lines the back two thirds of the inner eye. Light entering the eye travels through a series of refractory structures including cornea, aqueous humor, lens and vitreous humor, and focuses on the retina in the normal eye. The retina transduces light signals into neuronal impulses which are then sent to higher vision centers for further processing. The retina is organized in five intercalating layers of cell bodies and synapses (Figure 1). The vertebrate retina is call the ‘inverted’ retina, due to the fact that the light-sensing photoreceptors are located further away from the light, and light has to travel through the entire retina layers before activating the photoreceptors. While the inverted retina organization may seem to compromise visual accuracy, some argue that

2    direct light exposure of photosensitive ganglion cells (Berson et al., 2002) best serves the generation of circadian rhythms found in many vertebrates. The photoreceptor layer consists of cone and rod photoreceptors, with the number of cone photo receptors significantly less than rod photoreceptors, but enriched mostly in and around the fovea for accuracy in central vision. The photoreceptor axon terminals contact horizontal and bipolar cell dendrites in the outer plexiform layer. The inner nuclear layer consists of the cell bodies of bipolar cells, horizontal cells and amacrine cells. The ganglion cell layer contains mostly ganglion cells and some displaced amacrine cells, and it is separated from the inner nuclear layer by synapses of the inner plexiform layer.

3   

(Adapted from Eggers et al, 2006)

Light Figure 1

4    Figure 1. Schematic diagram of retinal structure Photoreceptors (PR) are activated by light and synapse onto bipolar cells (BC) and horizontal cells (HC) in the outer plexiform layer (OPL). Bipolar cells then synapse onto ganglion cells (GC) and amacrine cells (AC) in the inner plexiform layer (IPL). The vertical, excitatory pathway (yellow) consisting of photoreceptors, bipolar cells and ganglion cells is modulated by two lateral, inhibitory pathways (red) consisting of horizontal (outer retina) and amacrine (inner retina) cells.

5    1.2. Synaptic pathways in retina The most direct route of visual signaling in the retina is a disynaptic vertical circuit; the retina uses glutamate (Wu & Dowling, 1978; Slaughter & Miller, 1983a) to transmit signals from photoreceptor to bipolar cells to ganglion cells. Horizontal cells acting at the outer plexiform layer and amacrine cells acting at the inner plexiform layer are involved in the lateral pathway and provide feedback and feedforward inhibition to cells in the vertical pathway. Glycine and GABA are the two major inhibitory neurotransmitters in the lateral pathway. The vertical information flow begins with the transduction of light signals into electrical signals in the photoreceptors. There are photosensitive pigments named rhodopsin located on the outer segments of photoreceptors. Upon light stimulation, through a series of signal cascades, activated rhodopsin activates cGMP phosphodiesterase. The decrease in cellular cGMP levels finally leads to closure of a cGMP gated cation channel, hyperpolarizing the photoreceptor, thus decreasing glutamate release from the photoreceptors. The flow of information is separated into two pathways at the bipolar cells level (Figure 2), due to different glutamate receptor types located on the bipolar cells dendrites. The photoreceptors release glutamate in the dark, which activates ionotropic glutamate receptors located on the OFF bipolar cells, and metabotropic glutamate receptors located on the ON bipolar cells. While activation of ionotropic glutamate receptors depolarizes and excites glutamate release from the OFF bipolar cell, activation of metabotropic glutamate receptors hyperpolarizes and inhibits glutamate release from the ON bipolar cell. Upon light stimulation, glutamate release from photoreceptors is reduced, resulting in activation of ON bipolar cells and

6    suppression of OFF bipolar cells. The ON and OFF bipolar cells synapses onto ganglion cells at the distal (on sublamina) and proximal (off sublamina) half of inner plexiform layer, respectively. Most ganglion cells have synaptic connections with both ON and OFF bipolar cells and are therefore called ON/OFF ganglion cells. The ON/OFF parallel pathways in the retina help to enhance the contrast between dark and light, as ON bipolar cells detects brightness and OFF bipolar cells detects darkness. The lateral pathway in the retina is mediated by horizontal cells and amacrine cells, and it’s activated by photoreceptors and bipolar cells in the vertical pathway (Figure 1). The horizontal cells provide inhibitory feedback to photoreceptors in the first layer of synapses (outer plexiform layer), while amacrine cells regulate the second layer of synapses (inner plexiform layer). Upon excitation by bipolar cells, amacrine cells can provide feedback inhibition to bipolar cells and feed forward inhibition to ganglion cells and other amacrine cells (Figure 2). While other neurotransmitters such as dopamine and acetylcholine are also released by certain groups of amacrine cells, each amacrine cell contains at least either GABA or glycine as the inhibitory neurotransmitter. In the inner plexiform layer, ganglion cell dendrites are postsynaptic to excitatory bipolar cell synapses and inhibitory amacrine cell synapses. Ganglion cells and amacrine cells are the only types of cells in retina that contains voltage-dependent sodium channels. However, the sodium currents generated in ganglion cells are much larger than that of amacrine cells, thus allowing the generation of action potential in ganglion cells. The spiking activities of retinal ganglion cells encode all visual signals

7    from the retina, and are carried through ganglion cell axons in the form of optic nerve to higher vision centers in the brain for further integration.

(Adapted from http://webvision.med.utah.edu )

Cone photoreceptor Bipolar cell Amacrine cell Ganglion cell Inner Plexiform Layer ON OFF Figure 2

8    Figure 2. ON/OFF separation of vertical pathway and synaptic circuitry in the inner plexiform layer. The diagram shows that signal from a cone phororeceptor is transmited through both an ON bipolar – ON ganglion cell pathway and an OFF bipolar – OFF ganglion cell pathway. In the inner plexiform layer, ganglion cell dendrites are postsynaptic to excitatory bipolar cell synapses and inhibitory amacrine cell synapses. Bipolar cells also excite amacrine cells. Often amacrine cells make reciprocal synapses to the bipolar cell axons from which they receive excitation, and form synapses with other amacrine cells.

9    1.3. Glycine-binding-site-containing receptors in the retina Being the simplest form of all amino acid neurotransmitters, glycine plays important inhibitory roles in the central nervous system. The notion of glycine as a neurotransmitter emerged in the sixties, when glycine was found in high concentrations in the spinal cord (Aprison & Werman, 1965), and suppresses action potentials in spinal neurons (Curtis & Watkins, 1960; Werman et al., 1967). Later on glycine was found to be synthesized (Shank & Aprison, 1970) and released (Hopkin & Neal, 1970) by neurons. We now know that glycine is the ligand for ionotropic glycine receptors, metabotropic glycine receptors and the co-activator site on NMDA receptors.

1.3.1. Overview of ionotropic glycine receptors The ionotropic glycine receptor (GlyR) was first cloned in 1987 (Grenningloh et al., 1987) and is a member of the cysteine-loop ligand-gated ion channel superfamily, of which nicotinic acetylcholine receptor (nAChR) is the prototypic member. Other members of the receptor superfamily include 5-hydroxytryptamine (5-HT) 3 and GABA A/C . All these receptor proteins have a large N-terminal extracellular domain, four transmembrane segments (M1–M4), a long intracellular loop connecting M3 and M4, and a short extracellular C-terminus. High sequence homologies are seen within the transmembrane segments and an extracellular cysteine-bonded motif (Cysteine- loop). Homology modeling of GlyR protein with the crystal structure of acetylcholine binding protein (Brejc et al., 2001) and three-dimensional micrograph of the nAchR (Unwin, 2005) provided insights for the understanding of the structure and ligand

10    binding pockets of the GlyR. The GlyR is a pentameric structure with an integral chloride ion-selective channel. The ionotropic glycine receptor is generally considered inhibitory since the chloride ion equilibrium potential is usually around the cell membrane resting potential. However, during embryonic development, the intracellular chloride ion concentrations are elevated due to the presence of a Na + - K + -2Cl - co- transporter, NKCC1, and the lack of a K + -Cl - cotransporter, KCC2 (Price et al., 2005); this results in GlyR mediated neuronal excitation. Molecular cloning studies revealed that there are four types of GlyR alpha subunit (α1-α4) (Smiley & Yazulla, 1990; Matzenbach et al., 1994; Laube et al., 2002; Haverkamp et al., 2003) and one type of beta subunit. The native GlyR is formed from 2 alpha and 3 beta subunits (Grudzinska et al., 2005). Ligand-binding sites are located at the (+) and (–) interfaces between subunits. In heterologous expression, the alpha subunits are necessary and sufficient to form functional channels. However, in native tissues, the beta subunits are essential for receptor clustering at the postsynaptic density by interacting with gephyrin, an anchoring protein (Meyer et al., 1995; Kneussel & Betz, 2000). The synaptic GlyRs are mainly located in the spinal cord, brain stem, cerebellum and retina, whereas in higher brain regions GABA receptors are the main inhibitory receptors. The GlyRs are most well known for regulation of motor reflexes and nociceptive sensory pathways.

11    1.3.2. Ionotropic glycine receptor localization in the retina In the retina, glycine localizes in approximately half of the amacrine cells (Pourcho & Goebel, 1985; Marc, 1989). These glycinergic amacrine cells typically have small, diffuse dendritic trees(Pourcho & Owczarzak, 1991). GlyRs are found in all cells postsynaptic to glycinergic amacrine cells, including bipolar cells, amacrine cells and ganglion cells (Sassoe-Pognetto & Wassle, 1997; Jusuf et al., 2005). Glycinergic amacrine cells have a high-affinity uptake system for glycine (Pourcho & Goebel, 1985; Wassle et al., 1986; Marc, 1989) by expressing the glycine transporter GlyT1 (Zafra et al., 1995; Menger et al., 1998; Pow & Hendrickson, 1999). In situ hybridization and immunohistochemical staining revealed that all four alpha subunits and the beta subunit are expressed in the retina (Greferath et al., 1994; Haverkamp et al., 2003; Heinze et al., 2007). GlyR α1 subunit was found in the off- sublamina of the inner plexiform layer, on the AII amacrine cell to OFF-cone bipolar cell synapses that are part of the rod pathway (Grunert & Wassle, 1996; Sassoe- Pognetto & Wassle, 1997; Haverkamp et al., 2003). Some GlyR α1 subunit immunoreactivity was observed in the outer plexiform layer(Smiley & Yazulla, 1990), possibly postsynaptic to glycinergic interplexiform cells. Consistent with this finding, electrophysiological studies showed that bipolar cells receive glycinergic input in the outer plexiform layer (Maple & Wu, 1998). GlyR α2 subunits were found on bipolar cell axon terminals, amacrine and ganglion cell dendrites (Haverkamp et al., 2004; Jusuf et al., 2005). GlyR α3 subunits exist in four bands within the inner plexiform layer where they were associated primarily with neurons of the cone pathways (Haverkamp et al., 2003). GlyR α4 subunits immunoreactivity was also found in the

12    inner plexiform layer in a more evenly distributed manner. Processes of displaced (ON-) cholinergic amacrine cells coincides with the α4 subunit puncta (Heinze et al., 2007). GlyR were also found on Muller cells in bullfrog retina (Lee et al., 2005). The α2 GlyR has slower kinetics compared to α1 and α3 GlyRs (De Saint Jan et al., 2001). Physiological studies on expressed GlyRs from perch retina showed α1 GlyR desensitize at a slower rate compared to α3 GlyR (Gisselmann et al., 2002). In tiger salamander retinal ganglion cells, glycine responses can be separated into a 500 nM strychnine insensitive fast component and a 5,7- dichlorokynurenic acid (DCKA) sensitive slow component (Han et al., 1997), possibly due to different GlyR subunit compositions. These differences in expression patterns and response kinetics suggest specific physiological functions of GlyR subtypes.

1.3.3. Ionotropic glycine receptor functions in the retina Ionotropic glycine receptors play critical roles in shaping the light signal pathways in the retina (overview in Wassle, 2004). The retina does not have a discrete class of rod OFF bipolar cells. The unique rod pathway from rod ON bipolar cell to OFF cone bipolar cell is critically dependent on glycinergic AII amacrine cells (Zhou & Dacheux, 2005). Activation of GlyR suppresses glutamate release from bipolar cells (Maple & Wu, 1998) and reduces light responses in ganglion cells at both presynaptic and postsynaptic levels (Roska et al., 1998). Both glycinergic and GABAergic amacrine cell activity can be inhibited by glycine (Zhang et al., 1997; Roska et al.,

13    1998). Glycine inhibition at the inner plexiform layer was shown to contribute to the center-surround receptive field of ganglion cells (Stone & Pinto, 1992). Glycinergic synapses were also involved in the information exchange between ON and OFF bipolar cells in rabbit retina (Volgyi et al., 2004; Eggers & Lukasiewicz, 2006).

1.3.4. Pharmacology of Ionotropic glycine receptors Glycine binds between the interfaces of two adjacent GlyR subunits (Rajendra et al., 1995). Strychnine, a plant alkaloid highly selective for GlyRs (Figure 3B), is a potent competitive antagonist on all subtypes of GlyR at nanomolar concentration (Young & Snyder, 1973). Several mutational studies have shown that strychnine and glycine share certain binding sites, as disruption of key amino acid residues can dramatically reduce receptor affinity to both ligands (Marvizon et al., 1986; Ruiz- Gomez et al., 1990; Vandenberg et al., 1992a; Vandenberg et al., 1992b; Rajendra et al., 1995). At present, the agonist and antagonist profiles of GlyR remain rudimentary compared to that of the other members of the superfamily, such as acetylcholine, GABA, or serotonin receptors. There are no subtype specific antagonists for GlyR, which makes the functional study of different GlyRs difficult. A few antagonists were reported to show modest selective inhibition between α2 and α1 GlyRs. Cyanotriphenylborate, a use dependent blocker, has higher affinity for α1 than α2 (Enz & Bormann, 1995). Picrotoxin, a commonly used non-competitive antagonist for GABA A/C , can inhibit α2 subunit more than α1 subunit (Wang & Slaughter, 2005). 5,7- dichlorokynurenic acid (DCKA), a potent blocker of the glycine site on the NMDA receptor, inhibits recombinant α2 homomeric GlyRs, but not α1 homomeric GlyR (Han

14    et al., 2004). Emergence of such antagonists provides insights for future subtype selective GlyR antagonist design.

1.3.5. Pharmacology of caffeine The history of coffee, one of the most popular beverages worldwide, dates back to the ninth century. It is widely consumed not only for its aroma and distinctive taste, but also for the central nervous system stimulation it brings. Caffeine is the natural pharmacologically active substance behind the stimulatory effect of coffee. Caffeine belongs to the methylxanthine family. Other members of the family include theophyline, theobromine and IBMX (Figure 3A). At physiological concentrations, caffeine exerts most of its CNS stimulant role through blocking adenosine receptors (Nichols et al., 1970; Sattin & Rall, 1970; Phillis et al., 1979). At a few hundred micromolar concentrations, caffeine and other members of methylxanthine family are general phosphodiesterase inhibitors, resulting in increased cyclic nucleotide levels (Vurnikos- Danellis & Harris, 1968). At very high concentrations (~10 mM) caffeine stimulates ryanodine receptors located on the smooth endoplasmic reticulum and causes internal calcium release (reviewed by Zucchi and Ronca-Testoni, 1997). In turtle retinal ganglion cells, caffeine can suppress GABA A R through activation of internal calcium release, as the suppression can be overcome by adding the calcium buffering agent, BAPTA, in the pipette solution (Akopian et al., 1998). In the first part of my thesis research, I report another surprising physiological feature of caffeine on GlyRs and how this affects glycine synaptic transmission in retina.

15   

1.3.6. Metabotropic glycine receptors Glycine receptors are most frequently compared with the GABA receptor family, which consists of ionotropic GABA A /C receptors and a metabotropic GABA B receptor. However, no metabotropic glycine receptor was reported until recently. Studies on salamander retina revealed a strychnine-insensitive glycine response in bipolar and ganglion cells (Hou et al., 2008). This glycine response which reduced high voltage-activated calcium current was G-protein mediated and protein kinase A dependent.

 

Figure 3 A

Figure 3 A . Structure

3 B. Structur e of caffeine e of strychn i and other m i ne, an iono t m embers of t h

t ropic GlyR h e methylx a antagonist. 1 a nthine fami l 1 6  l y.

17    1.3.7. NMDA receptor overview The ionotropic glutamate receptors, which are non-selective cation channels, are divided into two groups; NMDA receptors and non-NMDA receptors. The non-NMDA receptors consist of AMPA and kainate receptors. NMDA receptors are notable for high permeability to Ca 2+ (MacDermott et al., 1986; Cline & Tsien, 1991), voltage- dependent magnesium block (Mayer et al., 1984; Ozawa et al., 1998; Dingledine et al., 1999), slow gating kinetics (Ozawa et al., 1998) and big single channel conductances (Jahr & Stevens, 1987; Rosenmund et al., 1998). The NMDA receptor is also unique in that it needs a co-agonist in order to be activated. Co-agonists, including glycine (Johnson & Ascher, 1987; Kleckner & Dingledine, 1988; Boje et al., 1992) and D- serine (Stevens et al., 2003), are generally thought to bind to NR1 subunits while glutamate binds to NR2 subunits (McBain & Mayer, 1994).

1.3.8. NMDA receptor in retina: disconnection between localization and function The most direct route of visual signaling in the vertebrate retina is a disynaptic circuit in which photoreceptors release glutamate to activate bipolar cells and they in turn use glutamate to stimulate ganglion cells. Although AMPA/Kainate and metabotropic glutamate receptor types are abundantly expressed throughout the retina, NMDA receptors are mostly restricted to the second synaptic layer (IPL). In the outer retina, although a few studies reported NR1C2’ and NR1/2 immunoreactivity on photoreceptors (Fletcher et al., 2000; Kalloniatis et al., 2004), horizontal cells (Grunder

18    et al., 2000) and bipolar cells (Wenzel et al., 1997; Grunert et al., 2003), no functional NMDA currents were recorded on these cells (Ariel et al., 1986; Thoreson & Miller, 1993; Zhou et al., 1993; Euler et al., 1996; Sasaki & Kaneko, 1996), with the lone exceptions of catfish and carp horizontal cells (O'Dell & Christensen, 1989; Shen et al., 2006). In the inner retina, however, NR1/NR2A-D/NR3A NMDA receptor subunits were abundantly expressed on amacrine cells (Vandenbranden et al., 2000; Kalloniatis et al., 2004) and ganglion cells (Hartveit et al., 1994; Brandstatter et al., 1998; Vandenbranden et al., 2000; Sucher et al., 2003). And curiously, despite their abundance in the inner plexiform layer, glutamate synaptic transmission relies mainly on AMPA receptors (Coleman & Miller, 1988; Tran et al., 1999). Yet exogenous NMDA application elicits large responses in most amacrine (Slaughter & Miller, 1983b; Massey & Miller, 1990) and ganglion cells (Slaughter & Miller, 1983b; Lukasiewicz & McReynolds, 1985; Mittman et al., 1990), leading to the suggestion that NMDA receptors are located perisynaptically. Strong evidence supports the current model of NMDA activation in retina: glutamate spillover activates perisynaptic NMDA receptors. It had been shown that evoked EPSCs in ganglion cells consist of both non-NMDA and NMDA components (Mittman et al., 1990; Diamond & Copenhagen, 1993; Matsui et al., 1998; Higgs & Lukasiewicz, 1999; Chen & Diamond, 2002). However, spontaneous EPSCs were mediated only by AMPA/Kainate receptors (Taylor et al., 1995; Matsui et al., 1998; Chen & Diamond, 2002). This suggests that NMDA receptors may only be activated by enhanced glutamate release. Consistent with this finding, using paired pulse recordings, Tachibana and colleges (Matsui et al., 1998) found that NMDA receptors on ganglion cells are responsible for sustained responses

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Abstract: Ionotropic glycine receptors (GlyRs) serve important inhibitory roles throughout the central nervous system, including the retina. They are ligand-gated chloride ion channels that belong to the same cysteine-loop ligand-gated ion channel superfamily as ionotropic GABA receptors. There are four types of ionotropic glycine receptors based on the alpha subunit composition. Unlike its close relative GABA A/C receptors, there are no selective pharmacological agents for different subtypes of glycine receptors. In the first half of my study, I focused on the search of possible antagonists that show selectivity for glycine receptor subtypes. I found that caffeine is a structural analog of strychnine and a competitive antagonist at ionotropic glycine receptors. Docking simulations indicate that caffeine and strychnine may bind to similar sites at the GlyR. The R131A GlyR mutation, which reduces strychnine antagonism without suppressing activation by glycine, also reduces caffeine antagonism. GlyR subtypes show different caffeine sensitivity. Tested against the EC50 of each GlyR subtype, the order of caffeine potency (IC 50 ) is: α2β (248 ± 32 μM) [approximate] α3β (255 ± 16μM) > α4β (517 ± 50 μM) > α1β (837 ± 132 μM). However, considering that the α3β GlyR is the least sensitive to glycine than the other GlyR subtypes, this receptor is most effectively blocked by caffeine. The glycine dose-response curves and the effects of caffeine in retina suggest that amphibian retinal ganglion cells are dominated by the α1β and possibly α4β GlyRs, but are unlikely to utilize the α3β or α4β GlyRs. Comparing the effects of caffeine on glycinergic spontaneous and light-evoked post-synaptic currents indicates that evoked release may be a result of either elevation of glycine concentration at the synapse or recruitment of more synapses. The caffeine IC 50 values for bath applied 100 μM glycine and for the synaptic glycinergic-currents are both 1.7 mM, thus the synaptic glycine concentration may be estimated to be around 100 μM in salamander retinal ganglion cells. As high millimolar concentrations of caffeine can completely inhibit glycinergic synaptic transmission, the use of caffeine to stimulate ryanodine receptors should be under discretion. NMDA receptors (NMDAR), along with AMPA/Kainate receptors, are excitatory ionotropic glutamate receptors possessing integral cation channels. NMDARs are abundantly expressed in the second synaptic layer of the retina; however, previous experiments suggest that the excitatory light responses of retinal ganglion cells are driven almost exclusively by AMPA/Kainate receptors. NMDA receptor activation in retina seems to rely on glutamate spillover to perisynaptic sites where NMDARs are located. In the second part of my study, I found evidence for synaptic NMDA receptor activation in retinal ganglion cells, by changing some of the commonly employed experimental protocols. If the retina was exposed to an adapting light, then there is a progressive enhancement of the NMDA synaptic current. If glycine inhibition was blocked then a large NMDA receptor current can contribute to the ganglion cell light response. Similarly, when magnesium was removed from the extracellular solution then the light response became dominated by the NMDAR receptor current. These experiments indicate the synaptic existence of NMDAR in retina, and that the activation of NMDA receptors is state-dependent.