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The effects of the potential glycine receptor antagonist, AMBD, in thalamic ventrobasal nuclei McCarthy, Sarah Monica 2006

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T H E E F F E C T S O F T H E P O T E N T I A L G L Y C I N E R E C E P T O R A N T A G O N I S T , A M B D , I N T H A L A M I C V E N T R O B A S A L N U C L E I by S A R A H M O N I C A M C C A R T H Y B S c , The University of Windsor, 2004 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R O F S C I E N C E in T H E F A C U L T Y O F G R A D U A T E S T U D I E S (Pharmacology and Therapeutics) T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A June 2006 © Sarah Monica McCarthy, 2006 A B S T R A C T This thesis describes the effects of 6-aminomethyl-3-methyl-4H,l,2,4-benzothiadiazine-1,1-dioxide ( A M B D ) on membrane properties and synaptic inhibition in neurons of the ventrobasal ( V B ) nuclei in the thalamus. Although gamma-aminobutyric acid ( G A B A ) has a well-established role as a neurotransmitter in the V B nuclei, recent evidence demonstrates that this area exhibits glycinergic inhibition that is sensitive to blockade by strychnine. A M B D has pharmacological properties that are consistent with glycine receptor antagonism, but its actions in the thalamus are unknown. The major objective was to determine the effects of A M B D on inhibitory postsynaptic currents (IPSCs) in the V B nuclei evoked by electrical stimulation o f the medial lemniscus ( M L ) , the major sensory input. A M B D significantly reduced the peak amplitude o f glycinergic and G A B A A e r g i c mixed IPSCs, pharmacologically isolated glycinergic and GABAAerg ic IPSCs, and purely glycinergic IPSCs. A M B D had no effects on most of the purely G A B A A e r g i c IPSCs. A M B D eliminated the slow and intermediate, not the fast, decay components of mixed glycinergic and GABAAerg ic IPSCs. A M B D decreased the apparent frequency but not the amplitude of spontaneous IPSCs (sIPSCs), implicating a possible presynaptic action. We propose that A M B D has both presynaptic and postsynaptic sites o f action. According to this proposal, A M B D antagonized the effects of glycine-like amino acids at the postsynaptic fast and slow glycine receptors, as wel l as at a presynaptic site that attenuates the effects of G A B A . Blockade of the presynaptic site resulted in reduced G A B A release by nerve terminals. In summary, A M B D has actions expected from a specific antagonist of glycine-like amino acids at thalamic receptors. i i i T A B L E O F C O N T E N T S Abstract i i Table o f Contents i i i List o f Tables v List o f Figures v i Acknowledgements vi i i Chapter I. Introduction 1 1.1 Scope o f the thesis 1 1.2 Background 2 1.2.1 The ventrobasal nuclei of the thalamus 2 1.2.2 Inhibitory neurotransmission in the V B nuclei 4 1.2.2.1 G A B A A e r g i c inhibition 5 1.2.2.2 G A B A A receptor antagonists 6 1.2.2.3 Glycinergic inhibition 6 1.2.2.4 Glycine and p-amino acid transporters 9 1.2.2.5 Glycine receptor antagonists 10 1.2.3 A M B D : a potential p-amino acid antagonist 11 1.2.3.1 The actions of A M B D on non-thalamic neurons 12 1.3 Experimental rationale, objectives and hypothesis 14 Chapter II. Materials and Methods 16 2.1 Whole-cell patch clamp recording 16 2.1.1 Slice preparation 16 2.1.2 Electrophysiological recording 16 2.2 Drugs 19 2.3 Data analysis 19 2.3.1 TPSC analysis 20 2.3.2 sIPSC detection and analysis 20 2.3.3 Concentration-response analysis 21 2.3.4 Statistical analysis 21 Chapter in. Results 22 3.1 Chemical similarity of A M B D obtained from two sources 22 3.2 Effects of A M B D on membrane properties 22 3.2.1 Input resistance and membrane time constant 24 3.2.2 Act ion potential firing 24 3.2.3 Voltage-current relationships 26 iv 3.2.4 Comparison o f effects on baseline currents with bicuculline 28 3.2.5 Summary of effects on membrane properties 28 3.3 Effects o f A M B D on IPSCs 30 3.3.1 Properties o f evoked IPSCs 1 30 3.3.2 Concentration-dependence of IPSC reduction 31 3.3.3 Reversal potentials of IPSCs 33 3.3.4 IPSC latency 33 3.3.5 Peak amplitudes of mixed IPSCs 33 .3.3.6 Time course o f antagonism and recovery 36 3.3.7 Pharmacologically isolated IPSCs 36 3.3.8 Comparison of antagonism with strychnine 39 3.3.9 Purely glycinergic and G A B A A e r g i c IPSCs 41 3.3.10 Rise time and charge transfer 41 3.4 Effects of A M B D on IPSC decay 46 3.4.1 Fast, intermediate and slow IPSC decay 46 3.4.2 Intermediate and slow IPSC components 46 3.4.3 Pharmacologically isolated decays 47 3.4.4 Purely glycinergic and GABAAerg ic decays 51 3.5 Spontaneous IPSCs 56 Chapter IV. Discussion 60 4.1 Summary of the results 60 4.2 Postsynaptic receptor antagonism by A M B D 61 4.2.1 M i x e d IPSCs 61 4.2.2 Isolated and purely glycinergic currents 61 4.2.3 Isolated and purely G A B A A e r g i c currents 62 4.2.4 The potential (5-amino acid receptor 63 4.3 Effects o f A M B D on decay of mixed IPSCs 64 4.3.1 Slow decay time constant 64 4.3.2 Intermediate decay time constant 65 4.4 Presynaptic actions of A M B D 65 4.4.1 Spontaneous IPSCs 65 4.4.2 Modulation o f G A B A release by the glycine receptor 66 4.4.3 Co-release versus co-transmission 67 4.5 Proposed mechanism of A M B D antagonism 67 4.6 Future directions 68 4.7 Conclusions 70 References 71 V LIST O F T A B L E S Table 3.1 Lack of effects of A M B D on Rj, capacitance and x m 25 Table 3.2 Effects of A M B D on IPSC latency 35 Table 3.3 Summary of the effects of A M B D on rise time and charge transfer of mixed and pure IPSCs, and isolated components 45 Table 3.4 Effects of A M B D on decay parameters of mixed IPSCs 49 Table 3.5 Effects of A M B D on decay time constants and amplitudes o f exponentially fitted isolated glycinergic and G A B A A e r g i c IPSCs 53 Table 3.6 Effects of A M B D on decay time constants and amplitudes of exponentially fitted glycinergic and G A B A A e r g i c IPSCs 55 Table 3.7 Summary of A M B D effects on spontaneous IPSCs 59 vi LIST O F FIGURES Figure 1.1 The chemical structures of the glycine receptor agonists, glycine, taurine and P-alanine 8 Figure 1.2 The chemical structure of the glycine receptor antagonist strychnine 10 Figure 1.3 The chemical structure of the potential p-amino acid antagonist A M B D . . . . 11 Figure 3.1 Three spectroscopic techniques confirmed the similar nature o f A M B D obtained from two sources 23 Figure 3.2 A M B D had no effects on action potential firing o f a previously untreated neuron 27 Figure 3.3 Effects o f A M B D on voltage-current relationships 29 Figure 3.4 Concentration-response relationship for A M B D reduction o f IPSCs 32 Figure 3.5 A M B D had no effects on mixed IPSC reversal potential 34 Figure 3.6 A M B D decreased the peak amplitude of representative mixed IPSC 37 Figure 3.7 Time course for A M B D reduction of the mixed IPSC and partial recovery .. 38 Figure 3.8 A M B D reduced the peak amplitude of isolated glycinergic and GABAAerg ic IPSCs 40 Figure 3.9 Comparison of the effects of A M B D with strychnine and bicuculline on mixed and isolated IPSCs 42 Figure 3.10 A M B D exhibited some selectivity in antagonism 43 Figure 3.11 Effects of A M B D on the decay time constants of mixed IPSCs 48 Figure 3.12 Histograms of the decay time constants of mixed IPSCs 50 Figure 3.13 Lack of effect o f A M B D on the decay time constants o f isolated glycinergic or G A B A A e r g i c IPSCs 52 Figure 3.14 A M B D effects on the decay time constants o f purely glycinergic or G A B A A e r g i c IPSCs 54 Figure 3.15 Lack of correlation between amplitude, rise time and decay time constants of spontaneous IPSCs 57 Figure 3.16 A M B D decreased the frequency but not the amplitude of sIPSCs Figure 4.1 Proposed mechanism for A M B D antagonism A C K N O W L E D G E M E N T S M y supervisor Dr. Ernie Pui l , has made this M S c . such a wonderful experience. I have learned so much from Dr. Pui l , both academically and personally. Thank you. I would also like to thank Dr. David Mathers for his suggestions and counsel throughout my studies. Y o u have been so helpful. Thank you to my committee members, Dr. Bernie MacLeod and Dr. Craig Ries for their support and suggestions. Thank you to Viktoriya Dobrovinska and M r . Christian Caritey for their excellent technical assistance with my research. I would also like to thank M i t i Isbasescu for his computer assistance. Thank you to Amer Ghavanini for his continual direction and patience everyday. A special thank you to Stephanie Lee, who has made this experience unforgettable. I am so appreciative of her friendship, both in research and outside. I came into this degree looking for an education. I am leaving with a best friend for life. Thank you. Thank you to C I H R and N S E R C for financially supporting this work. Thank you to Chris Bauer for his support everyday. Y o u make my life so much better. Finally, a huge thanks to my family. I have been able to study because o f your continual support, throughout all o f my lifetime. I dedicate this work to my parents, who have provided me with the confidence to pursue my dreams, whatever they may be. Chapter I. Introduction 1.1 Scope of the thesis This thesis describes the effects of a potential glycine antagonist, 6-aminomethyl-3-methyl-4H,l,2,4-benzothiadiazine-1,1 -dioxide ( A M B D , T A G ) , on neurons in the ventrobasal ( V B ) nuclei of the thalamus. In this thesis, we w i l l refer to this substance as A M B D because its name in much literature, ' T A G ' , infers that it is a specific taurine antagonist. Although gamma-aminobutyric acid ( G A B A ) has a well-established role as a neurotransmitter in the V B nuclei, recent evidence demonstrates that this area exhibits synaptic inhibition sensitive to blockade by strychnine (Ghavanini et al., 2005). Because strychnine antagonizes receptors for glycine-like amino acids, glycine, taurine or 0-alanine, individually or in combination, may mediate this inhibition. Although A M B D has pharmacological properties that are consistent with glycine receptor antagonism, its actions in the thalamus have not been investigated. The major objective o f this thesis was to investigate the synaptic effects of A M B D on inhibitory postsynaptic currents (IPSCs) in the V B nuclei. Based on previous literature, the general hypothesis was that A M B D interacted with synaptic glycine receptors and not G A B A receptors, suppressing the inhibitory effects of glycine and P-amino acids. Min imal effects on G A B A A e r g i c responses might be interpreted as selectivity in A M B D action at the glycine receptor or a hypothetical receptor for p-amino acids. 2 1.2 Background 1.2.1 The ventrobasal nuclei of the thalamus The V B nuclei complex consists of the ventral posterior medial ( V P M ) and ventral posterior lateral ( V P L ) nuclei (Jones, 1991). These nuclei relay information from somatic sensory receptors to cortical layers 1 to 6 of somatosensory neocortex (Brodman areas 1, 2, 3a and 3b; Feldman and Kruger, 1980). V B nuclei neurons are organized into subpopulations with axons that project to only one cortical layer, or multiple layers by collaterals (Manson, 1969; Jones, 1991). Corticothalamic neurons in layer 6 project back to the V B nuclei, producing excitatory and inhibitory inputs, potentially for receptive field adjustment (Alitto and Usrey, 2003). The M L provides the major sensory pathway to the brain and is a major input to the V B nuclei (Mountcastle et al., 1963). The two major types o f neurons identified in the thalamus are numerous thalamocortical (TC) relay neurons and a small population of local circuit interneurons (Yen and Jones, 1983). T C relay neurons can be distinguished from interneurons by somatic size and dendritic pattern. T C relay neurons have large somata and short radiating dendrites, whereas interneurons have small, round somata and extensive dendritic arborization (Yen et al., 1985; Turner et al., 1997). In addition, interneurons have more depolarized resting membrane potentials and lack the ability to fire at high frequencies above 150 H z (Turner etal., 1997). T C relay neurons are further subdivided into two major classes called Type I and U. Type I T C neurons are common, whereas Type II T C neurons are rare. Type I T C neurons are morphologically characterized by large somata, large tufted dendrites, no appendages and thick axons. These neurons are further characterized by low input resistance (R), small afterhyperpolarizations and transient responses to current injection. Type I T C neurons project to cortical layers 4 and 5. Type II T C neurons are morphologically characterized by small somata, thin branching dendrites, thin appendages and thin axons. They are also characterized by high R, large afterhyperpolarizations and non-adapting responses to current injection. Type II T C neurons project to cortical layers 1 and 2 (Yen et al., 1985; Hirai and Jones, 1988; Jones, 1991; Turner et al., 1997). Investigations in the V B nuclei predominantly involve Type I T C neurons due to the small number of interneurons and Type II T C neurons in the rat (Harris and Hendrickson, 1987). T C neurons that receive input from cortical layer 5 are further classified as higher-order thalamic relays, whereas those that receive input from other cortical layers are termed first-order relays (Guillery and Sherman, 2002). Higher-order T C neurons are involved in complex functions such as attention and are sensitive to cortical inactivation. First-order T C relay neurons are not affected by cortical inactivation and are involved in information transfer only (Diamond et al., 1992; Ward et al., 2002; Bokor et al., 2005). The nuclei o f the V B thalamus are the major thalamic relay centres for somatosensory information. V B nuclei are also involved in sleep and wakefulness cycles (Steriade, 2005), or pathophysiological^ in absence epilepsy (Huguenard and Prince, 1994). Most inputs to the V B nuclei are contralateral and somatotopically organized (Waite, 1973; 4 Welker, 1973). V B neurons receive inputs from nociceptive and thermal spinal cord afferents by way of the spinothalamic pathway. They also receive information about touch, pressure and vibration peripheral afferents by way o f the M L tract (Welker, 1973; Akers and Killackey, 1979; Feldman and Kruger, 1980; Jones, 1991). The M L is an excitatory and inhibitory input (Mountcastle et al., 1963; Hirai and Jones, 1988), whereas inputs from the nucleus reticularis (nRt) and zona incerta are only inhibitory (Peschanski et al., 1983; Bartho et al., 2002). In addition, the V B nuclei receive cholinergic and noradrenergic neuromodulatory inputs from the brainstem and basal forebrain (Castro-Alamancos and Calcagnotto, 2001). 1.2.2 Inhibitory neurotransmission in the VB nuclei In the V B nuclei, inhibitory neurotransmission is mediated by G A B A through predominantly postsynaptic G A B A A and G A B A B receptors and glycine-like amino acids through glycine receptors. G A B A c receptors are not apparent in pharmacological studies o f some thalamic nuclei (Wan and Pui l , 2002). G A B A A and glycine receptors are members o f the pentameric ligand-gated ion channel superfamily. G A B A A and glycine receptor activation results in C l " influx, causing postsynaptic inhibition due to hyperpolarization o f the neuron away from the firing threshold and shunting o f excitatory synaptic inputs (Ries and Pui l , 1999; Lynch, 2004). Recent studies have shown that the co-release and co-transmission of glycine and G A B A by glycinergic and G A B A A e r g i c pathways are more common than previously assumed. In the spinal cord, G A B A and glycine are co-released from the same nerve terminal in vesicles containing both neurotransmitters (Chaudhry et al., 1998; Jonas et al., 1998). Furthermore, G A B A and 5 glycine may have a common presynaptic vesicular transporter (Dumoulin et al., 1999). Postsynaptically, the G A B A A and glycine receptors are co-localized, poised for co-release or co-transmission (Bohlhalter et al., 1994). Activation of postsynaptic glycine receptors inhibits G A B A A receptors through a phosphorylation mechanism (L i et al., 2003). 1.2.2.1 GABAAergic inhibition The G A B A A receptor is comprised of five subunits forming a CI" channel (Johnston, 1996). There are currently 19 known G A B A A receptor subunit isoforms: cti-6, P1-3, yi-3, 5], 8, 0 ,7 i and pi-3 (Nayeem et al., 1994; Simon et al., 2004). G A B A A receptors are highly heterogenous because of the large number of subunit isoforms that combine to form functional receptors (Macdonald and Olsen, 1994). The major endogenous agonist of the G A B A A receptor is G A B A , but imidazole-4-acetic acid, taurine, P-alanine and gamma-amino beta-hydroxybutyric acid ( G A B O B ) can also bind to a non-functional form of the receptor (Johnston, 1996). Binding of G A B A to the G A B A A receptor results in opening o f the CI" channel and movement of the membrane potential towards the CI" equilibrium potential (.Eci), causing hyperpolarization or depolarization. G A B A B receptor activation contributes to slow inhibitory transmission and is metabotropic (Bowery, 1989). The G A B A A - m e d i a t e d process contributes to most fast inhibitory transmission in the thalamus (Kaila, 1994). 1.2.2.2 GABAA receptor antagonists There are several known antagonists o f the G A B A A receptor, including picrotoxin, bicuculline and gabazine. Picrotoxin, a mixture of picrotoxinin and picrotin, blocks the C l " channel, and hence antagonizes glycine receptors at concentrations higher than 10 u M (Lynch et al., 1995). Bicuculline is a potent competitive antagonist at G A B A A receptors. This antagonist binds to low affinity G A B A binding sites on the a subunits (Maksay and Ticku, 1984; Sigel et al., 1992). Bicuculline is specific for G A B A A receptors up until a maximum concentration of 50 u.M. Beyond 50 u M , bicuculline antagonizes glycine receptors in hippocampal neurons (Shirasaki et al., 1991). Gabazine (SR-95531) is a competitive antagonist o f G A B A A receptors, and is approximately equipotent with bicuculline (Michaud et al., 1986). In contrast to bicuculline, however, gabazine preferentially binds to high affinity G A B A binding sites, potentially indicating higher specificity (Heaulme et al., 1986). Gabazine is specific to G A B A A receptors up to a maximum concentration of 10 u M (Mori et al., 2002). In V B neurons, bicuculline and gabazine show approximately the same degree of specificity for the G A B A A receptor (Ghavanini et al., 2005). 1.2.2.3 Glycinergic inhibition The glycine receptor has a well-established action in inhibitory neurotransmission in the spinal cord and brainstem (Werman et al., 1968; Grillner et al., 1998). The evidence for glycinergic transmission in the thalamus is based on immunocytochemistry and pharmacological effects. Glycine (Rampon et al., 1996), glycine receptors (Araki et al., 1988), glycine receptor subunits (Ghavanini et al., 2005), glycine transporter 2 (Jursky and Nelson, 1995) and strychnine binding sites (Zarbin et al., 1981; Frostholm and Rotter, 1985) have been demonstrated in the thalamus. Furthermore, stimulation o f the M L activates IPSCs that are sensitive to blockade by strychnine in neurons o f the V B nuclei (Ghavanini et al., 2005). The glycine receptor is a pentameric CI" channel composed o f a and p subunits forming an ion-conducting pore (Lynch, 2004). The a subunit has four isoforms ( a n ) and is required for ligand-binding (Ruiz-Gomez et al., 1990). The p subunit interacts with gephyrin to anchor the synaptic receptor to the cell cytoskeleton (Kirsch and Betz, 1995). In the spinal cord, glycine receptors are predominantly ct2 homomers during development and become ctiP heteromers during adulthood (Malosio et al., 1991). Four extracellular domains (A-D) located on the glycine receptor amino terminal have been proposed to form an agonist-binding pocket (Rajendra et al., 1995; Vafa et al., 1999; Corringer et al., 2000). Activation of the glycine receptor results in the opening of a CI" channel and movement o f the membrane potential towards EQ\- The glycine receptor is inhibitory during adulthood when Ea is more negative than membrane potential (Lynch, 2004). During development, neurons can have a high intracellular CI" concentration, resulting in glycine-mediated depolarizing excitation (Ito and Cherubini, 1991). The increase in CI" channel conductance produces most of the inhibition. A shunt o f excitatory synaptic voltage can occur, depending on its location relative to the regions o f spike generation (Ries andPui l , 1999). 8 In the spinal cord, glycine receptors are activated by glycine, taurine, p-alanine and D - or L-serine (Tokutomi et al., 1989) in the following order of potency: glycine > P-alanine > taurine » serine (Figure 1.1) (Curtis et a l , 1968). D - and L-serine have little or no effect on V B neurons (Ghavanini et al., 2005). Glycine binds to loop A , Ile93, A l a l O l and A s n l 0 2 in the glycine receptor agonist-binding pocket (Vafa et al., 1999). The P -amino acids bind to loop A , in the A l a l O l - T h r l 12 region of the glycine receptor agonist-binding pocket (Han et al., 2001). The p-amino acid binding site is thus structurally close to, but distinct from, the glycine binding site. In V B nuclei, application of P-amino acids causes an increase in conductance (Ghavanini et al., 2005). Glycine Taurine p-alanine Figure 1.1 The chemical structures of the glycine receptor agonists, glycine, taurine and P-alanine. In the developing nervous system, taurine is abundant, particularly in the cerebellum, thalamus and cerebral cortex (Curtis et al., 1971; Huxtable, 1989). It is implicated in cortical plasticity, neuroprotection and inhibition of hyperexcitable states (Kaczmarek, 1976; Huxtable, 1989; Zhao et al., 1999). Taurine is proposed as a potential inhibitory neurotransmitter due in part to its strychnine-sensitive actions on spinal neurons (Curtis et al., 1968; Padjen et a l , 1989). Taurine as a neurotransmitter is controversial because Na+-independent binding has not been demonstrated. Na+-independent binding 9 demonstrates binding to the receptor rather than binding to membrane transporters (Huxtable, 1989). Although there are suggestions o f a taurine-specific receptor, it has not been characterized (reviewed by Huxtable, 1989; Frosini et al., 2003). 1.2.2.4 Glycine and P-amino acid transporters Glycine is transported from the extracellular space into the cytosol through two N a + / C l " dependent glycine transporters ( G l y T l and GlyT2) (Eulenberg et al., 2005). G l y T l transports 2Na +/C17glycine per cycle in glial cells. GlyT2 transports 3Na +/C17glycine per cycle on the presynaptic terminals of glycinergic neurons (Jursky and Nelson, 1995; Roux and Supplisson, 2000), and hence implicates glycinergic synapses. Immunocytochemistry for GlyT2 has demonstrated glycinergic synapses in the V B nuclei (Zeilhofer et al., 2005). The actions of G l y T l and GlyT2 are antagonized by sarcosine and amoxapine, respectively (Nunez et al., 2000; Harsing et al., 2003). There are two distinct transporters ( T A U T 1 and T A U T 2 ) for the uptake of P -amino acids from the extracellular space (Liu et al., 1992; Smith et al., 1992). The T A U T s transport 2Na +/C17p-amino acid per cycle and are antagonized by guanidinoethane sulfonate (GES) (Huxtable et al., 1979; Nelson, 1998; Barakat et al., 2002). The 12 transmembrane segments of the T A U T s share significant homology with the glycine transporters (Liu et al., 1992). Although not localized to the thalamus, immunocytochemical staining for the T A U T s has been demonstrated throughout the brain, particularly in the cerebellum, cortex and hippocampus (Pow et al., 2002). 1.2.2.5 Glycine receptor antagonists The two principal antagonists of the glycine receptor are strychnine and picrotoxinin. Strychnine is the only established selective antagonist of the glycine receptor (Figure 1.2) (Legendre, 2001). Other substances, such as brucine alkaloids, block glycine actions but have not received extensive investigation presumably because they are much less potent than strychnine (Curtis et al., 1968). In the hippocampus, strychnine is specific at concentrations up to 2 u M , beyond which it also blocks G A B A A receptors (Shirasaki et al., 1991). Glycine and strychnine do not act at the same binding site, but there may be some site overlap on the N terminal region of the glycine receptor a subunit (Graham et al., 1983). Although picrotoxinin is a glycine receptor antagonist, it is less specific and less potent than strychnine and also antagonizes G A B A A receptors (Lynch et al., 1995). The p subunit of the glycine receptor is.involved in the effects o f picrotoxin (Bormann et al., 1993). 11 1.2.3 AMBD: A potential fi-amino acid antagonist In 1982, Girard et al. discovered that 6-aminomethyl-3-methyl-4H, 1,2,4-benzothiadiazine-1,1-dioxide ( A M B D ) (Figure 1.3) acted as a taurine antagonist, but the results of subsequent studies have been ambiguous. Although most studies have indicated that A M B D is specific for the effects of P-amino acids, some investigations have concluded that it also antagonizes the actions of glycine and G A B A (Yarbrough et al., 1981; Okamoto et al., 1983; Mathers, 1993). A M B D is a 1,2,4-benzothiadiazine compound with an acidic centre and basic nitrogens in a rigid and planar conformation. The distance between the acidic centre and basic nitrogens closely resembles G A B A , glycine, taurine and P-alanine (Girard et al., 1982). Although the distance between the acidic and basic groups of taurine is less than A M B D , rotation of the aminomethyl group toward C-5 may produce a closer structural match, resulting in a taurine-specific effect (Girard et al., 1982; Huxtable et al., 1987). Comparisons between A M B D and taurine indicate structural similarity but the exact A M B D binding site remains unresolved. Girard et al. (1982) proposed that receptors recognize the sulfonamide group on A M B D due to its structural similarity to taurine. Figure 1.3 The chemical structure of the potential p-amino acid antagonist A M B D . O O AMBD 12 1.2.3.1 The actions of AMBD on non-thalamic neurons Studies employing ionophoretic application techniques indicate that A M B D specifically antagonizes the inhibitory effects of P-amino acids, with minimal action on either glycine or GABA- induced effects. Ionophoretic application of A M B D on rat and pig cerebellar and spinal neurons in-vivo antagonized taurine- and p-alanine-induced depolarization depression with minimal effects on G A B A - or glycine-induced responses (Yarbrough et al., 1981; Girard et al., 1982; Okamoto et al., 1983; Bi l lard and Batini, 1991). In terms o f receptor specificity, these results are difficult to interpret because the concentration of ionophoretically applied A M B D is unknown. Studies employing non-ionophoretic experimental techniques also indicate A M B D specificity for p-amino acids. In isolated frog spinal cord, bath application o f 0.1-0.25 m M A M B D to dorsal root terminals selectively blocked taurine-induced responses with no effects on G A B A - or glycine- induced responses (Padjen et al., 1989). In the rat substantia nigra, microinjection of A M B D antagonized contraversive turning evoked by taurine injection but had no effects on the binding of G A B A to anxiolytic binding sites (Martin et al., 1981). In whole rabbit brain homogenate, 500 u M A M B D displaced [3H]taurine with no effects on the G A B A A agonists [ 3H]muscimol or [ 3 H ] G A B A (Frosini et al., 2003). In whole rat brain homogenate, 250 u M A M B D reduced uptake o f taurine and [ 3H]taurine, with no effects on G A B A or [ 3 H ] G A B A uptake (Lewin et al., 1994). Most research on A M B D indicates specificity for p-amino acids but a few studies suggest otherwise. Application o f 250 u M A M B D to dissociated salamander retinal cells 13 caused blockade of both taurine- and glycine-induced currents (Pan and Slaughter, 1995). These results are not necessarily applicable to the V B nuclei because the retina has more specialized neurotransmission compared with other C N S areas (Bormann, 2000). In dissociated mouse spinal cord neurons A M B D exhibited a narrow concentration range for taurine specificity (Mathers, 1993). In homogenized mouse cerebral cortex slices, application of 1 m M A M B D attenuated stimulated taurine release (Kontro and Oja, 1987), suggesting a presynaptic action of A M B D . In dissociated ventromedial hypothalamic neurons, bath application o f 10"5 M A M B D had no effects on glycine or taurine-induced CI" currents (Tokutomi et al., 1989). The lack o f effect suggests this concentration was too low for this particular system. Past research on the action of A M B D has demonstrated specific antagonism o f the effects of P-amino acids within a narrow concentration range which varies depending on the system under investigation. This specificity has been demonstrated, using ionophoretic and other techniques, in the cerebellum, spinal cord and substantia nigra of the rat, rabbit, mouse, frog and pig. Differing experimental techniques may explain the contradictory results. For example, high concentrations may have been responsible for some effects observed after ionophoretic application where the drug concentration is unknown. Further research using quantitative drug delivery techniques is required to resolve these issues. 14 1.3 Experimental rationale, objectives and hypothesis The recent demonstration of strychnine-sensitive transmission in the V B nuclei is surprising because of the well-established role of G A B A in this area. The inhibitory action of P-amino acids in the V B nuclei suggests that transmission mediated by the glycine receptor may involve more neurotransmitters than glycine alone (Ghavanini et al., 2005). Glycinergic inhibition may result from co-transmission or co-release, o f combinations of glycine, taurine and p-alanine. The controversy around the action of A M B D could be resolved by demonstration of different glycine receptor binding sites or different receptors for the P-amino acids and glycine. Hence, demonstration that A M B D specifically blocks p-amino acids would be consistent with the hypothesis of differing binding sites or receptors. Therefore, we were interested in determining the effects of A M B D on the multi-transmitter system in the V B nuclei and whether the action of A M B D resembled strychnine which antagonizes glycine-like amino acids. Resolution of these issues may allow further identification of the neurotransmitters involved in V B nuclei IPSCs antagonized by strychnine, but not bicuculline. The objective was to assess with patch clamp the concentration-dependent effects of A M B D on intrinsic, membrane and synaptic properties of individual thalamic neurons. For determining the specificity of A M B D , IPSCs were evoked by stimulation in the M L and pharmacologically identified as G A B A A e r g i c , glycinergic or mixed GABAAerg ic and glycinergic. Glycinergic IPSCs may have resulted from the receptor actions o f glycine, taurine or P-alanine. The effects of A M B D on IPSCs were assessed from changes in peak IPSC amplitude, rise time, decay time and decay time constants. The effects of 15 A M B D on neuronal membrane properties were also investigated. This thesis examined the possibility that A M B D acted on synaptic receptors to specifically antagonize the action of glycine-like amino acids, with minimal effects on GABA-mediated transmission. 16 Chapter II. Materials and Methods 2.1 Whole-cell patch clamp recording 2.1.1 Slice preparation . A l l experiments were approved by the University of British Columbia Committee on Animal Care. Sprague-Dawley rats (12-14 days old) were placed under a gas-tight inverted glass funnel and anesthetized with halothane. Rats were decapitated after approximately 1 minute of deep anesthesia. The brain was rapidly removed and quickly submerged in ice-cold (4 °C), oxygenated (95% O 2 : 5% CO2) artificial cerebrospinal fluid (aCSF) containing (in m M ) : 124 N a C l , 26 N a H C 0 3 , 1.25 N a H 2 P 0 4 , 2.5 KC1, 2 M g C h , 2 C a C b , and 10 dextrose, at p H 7.3-7.4. The brain was sectioned along the interhemispheric fissure into two identical tissue blocks. The medial surface of the block was glued to the Teflon stage of a Vibroslicer (Campden Instruments Ltd. , London, England). Parasagittal slices (200-250 pm thick), containing the V B nuclei and M L were cut. Slices were maintained for 1.5 hours on a polypropylene mesh in a holding chamber containing oxygenated aCSF (23-25 °C) at p H 7.3-7.4. The average osmolality was 315 ± L O m O s m . 2.1.2 Electrophysiological recording Recording pipettes were drawn from borosilicate glass tubing with internal filament (World Precision Instruments, Sarasota, U S A ) using a vertical electrode puller (Narishige Instruments, Tokyo, Japan). For recording of IPSCs, pipettes were filled with an intracellular solution containing (in m M ) : 140 K-gluconate, 5 KC1 , 4 N a C l , 3 M g C l 2 , 1 C a C l 2 , 1 0 E G T A , 1 H E P E S , 3 M g A T P , 0.3 N a 2 G T P . Under these conditions, EC\ was 17 -53 m V and was -84 m V , when intracellular [Ca 2 + ] was approximately 5 n M (calculated using M a x Chelator software). The p H was adjusted to 7.3-7.4 using 50% gluconic acid and K O H . The average osmolality was 252 ± 2.0 mOsm. For recording of spontaneous EPSCs (sJPSCs) the intracellular solution contained (in m M ) : 16.5 Cs-gluconate, 128.5 C s C l , 4 N a C l , 3 M g C l 2 , 1 C a C l 2 , 10 E G T A , 10 H E P E S , 3 M g A T P , 0.3 N a 2 G T P , 3 QX-314. Under these conditions, EC\ was 0 m V . The p H was adjusted to 7.3-7.4 using 50% gluconic acid and C s O H . Adenosine triphosphate (ATP) and guanosine triphosphate (GTP) were added to the pipette solution immediately prior to recording. Electrode resistances ranged between 5 and 10 M Q . After 1-2 hours incubation in aCSF, slices were placed in a Perspex chamber with volume of 1.5-2 ml . Slices were immobilized using a polypropylene mesh and perfused with bubbled (95% 0 2 and 5% C 0 2 ) aCSF with 1 m M kynurenate (23-25 °C) at a rate of 2 ml/minute. Kynurenate was used to block ionotropic glutamatergic transmission and isolate IPSCs (cf. Ghavanini et al., 2005). The V B nuclei and M L were visually identified using differential interference contrast (DIC) microscopy at 400x magnification (Axioscope, Carl Zeiss, Germany). Whole cell patch clamp recording was performed using a List EPC-7 ( H E K A , Lambrecht, Germany) in the voltage-clamp or current-clamp mode. Signals were filtered at 3 kHz , digitized at 10 k H z with a 16-bit data acquisition system (Axon Instruments) and stored for later analysis using pClamp software (Axon Instruments) on a Pentium computer. Neurons were accepted for further study i f they had stable membrane 18 potentials and responded to depolarizing current pulse injections with overshooting action potentials. IPSCs were evoked from a bipolar tungsten electrode (World Precision Instruments) connected to an isolated stimulator (Digitimer, Hertfordshire, U K ) . The electrode was placed in the M L , approximately 3 mm from the recording electrode. Stimuli at < 0.5 H z with single pulses of duration 0.05 - 1 ms were employed and adjusted for maximal IPSC responses. 10 IPSCs were evoked for each treatment. Stimuli remained constant for single neurons throughout the procedure. Neurons were held at -80 m V for all IPSC experiments. To identify the acting inhibitory neurotransmitter, IPSCs were characterized using 2 u.M strychnine and 20 u M bicuculline to isolate the glycinergic and GABAAerg ic components, respectively. More than 95% inhibition of the IPSC by strychnine or bicuculline indicated purely glycinergic or purely GABAAerg ic responses. Partial blockade of an IPSC by either antagonists indicated mixed glycinergic or G A B A A e r g i c responses. Percentage inhibition of IPSC amplitude was calculated to quantify inhibition by A M B D , strychnine and bicuculline. Percentage inhibition was measured by calculating the percentage of original TPSC amplitude that was inhibited after application of an antagonist. 19 2.2 Drugs Stock solutions were prepared in distilled water or dimethyl sulfoxide ( D M S O ) and diluted in aCSF. Drugs for IPSC experiments were applied by bath perfusion for ~ 8 minutes at 2 ml/min. A l l drugs applied to the slice were previously oxygenated. Bicuculline methiodide, strychnine and kynurenate were purchased from Sigma Chemical Company (St. Louis, U S A ) . The first batch of A M B D was a kind gift o f Merck Frosst Company (Montreal, Quebec, Canada) and the second batch was synthesized by BioFine International (Vancouver, B C , Canada). A l l drugs were washed out after application. Complete recovery was defined as 25% IPSC inhibition or less. 2.3 Data analysis Electrophysiological data analysis was conducted using pClamp (Clafnpfit, A x o n 1 Instruments), Microsoft Excel and CorelDraw (Ottawa, Canada) software. Membrane potential was adjusted for a junction potential o f -11 m V . Rj and membrane time constant (xm) were calculated from < 5 m V voltage responses to hyperpolarizing injections of current. Voltage-current relationships were determined from voltage responses to depolarizing and hyperpolarizing intracellular injections o f current from -150 p A to 150 p A in neurons held at -60 m V . Tetrodotoxin (TTX) was not used in voltage-current experiments, so the recorded reversals may include presynaptic and In-dependent contributions. Reversal potentials were obtained from the intersection of the control and drug curves. 2.3.1 IPSC analysis After aligning current peaks in time, traces of 10 successive IPSCs from each treatment protocol were averaged for analysis. Rise time of the IPSC was determined by measuring the time between the initial deflection of the baseline current and peak IPSC amplitude. Charge transfer was determined by measuring the area under the IPSC. The decay phases of the averaged IPSCs were fitted with exponential functions to determine decay time constants. The single exponential function was, y = Ae* where A was the peak amplitude and x was the decay time constant. The double exponential function was, y = A,e^+ A2e^-where A i and A2 were the peak amplitudes of the terms with fast and slow time constants T i and i2. 2.3.2 sIPSC detection and analysis pClamp software template search was used to detect sIPSCs using a sliding template procedure. The sIPSCs recorded in the V B thalamus have 3 distinct time courses; fast sIPSCs decay within 100 ms, intermediate sIPSCs decay within 100-200 ms and slow sIPSCs decay within 500-1000 ms (cf. Ghavanini et al., 2006). Based on this finding, short, intermediate and slow sIPSC templates were produced using averaged sIPSCs that were visually detected. Detection threshold was set at 5 p A and all accepted events were visually monitored. 21 2.3.3 Concentration-response analysis A concentration-response relationship for A M B D antagonism o f the IPSC was established using cumulative drug application in a step-wise manner. Sigmoid curves were fitted to the data using Prism GraphPad software (San Diego, U S A ) . The fitting equation for the single sigmoid relationship was, _ Max Response y ~ (l+IC5 0-[drug])n where max response was the plateau response, IC50 was the concentration at half-maximal response and n was the slope of the sigmoid curve. 2.3.4 Statistical analysis A l l data were expressed as mean ± S E M and n denoted the number o f neurons tested. Data were statistically analyzed using the N C S S Statistical Analysis System (Kaysville, U S A ) . The Student's Mest was used for comparing two groups and the analysis of variance ( A N O V A ) was used for multiple comparisons. Significance was defined as P < 0.05. 22 Chapter III. Results 3.1 Chemical similarity of A M B D obtained from two sources We used three spectroscopic methods for analysis of 6-aminomethyl-3-methyl-4H, 1,2,4-benzothiadiazine-1,1-dioxide ( A M B D or T A G ) obtained from Merck and Biofine (cf. Methods). Ultraviolet ( U V ) spectrum absorbance peaks, high-pressure liquid chromatography ( H P L C ) absorbance peaks and proton nuclear magnetic resonance ( N M R ) spikes were identified and compared to determine i f A M B D manufactured by the two sources were identical. N o substantial differences were noted between the sources (Figure 3.1). Minor differences were likely attributable to salt preparation (cf. Kirkpatrick and Sandberg, 1973). The results of analysis with U V , H P L C and N M R techniques provided confirmation of the identity of A M B D from the two sources. 3.2 Effects of A M B D on membrane properties Before observing the effects o f A M B D on synaptic inhibition, it was necessary to determine i f A M B D (250 u M ) had effects on membrane properties that might account for its proposed antagonism. Initially, the 250 u M concentration of A M B D was chosen based on previous research that determined that this concentration specifically antagonized taurine when bath applied to neurons (Mathers, 1993). We assessed the effects of A M B D on membrane potential ( V m ) , R , x m , action potential firing, voltage-current relationships and baseline current. A M B D was applied to 19 previously untreated neurons, 19 bicuculline (20 uM) pre-treated neurons and 19 strychnine (2 u,M) pre-treated neurons. Strychnine and bicuculline were co-applied after A M B D treatment. The average V m for all neurons was -52 ± 1.5 m V (n = 39). 23 < a> o c co € o CO XI < =3 < E. <u o c to .Q I o CO < NMR B. BIOFINE 3 < E. cu o c nj i o CO - O < 12 10 8 6 4 8 (ppm) MERCK 3 < E, cu o c CO .Q < 14 12 10 8 6 4 2 5 (ppm) HPLC 3.575 Time (min) 537 Time (min) UV Spectroscopy CO o CO < "O cu N O z 2250 2000 1750 1500 1250 1000 750 500 250 o! ^Merck Biofine 200 250 300 350 400 450 500 550 Wavelength (nm) Figure 3.1 Three spectroscopic techniques confirmed the similar nature of A M B D obtained from two sources. (A) N M R absorbances of the two substances were a close match. Note slightly differing 8 axes. (B) H P L C peaks for the two sources of A M B D closely matched eachother. (C) Overlapping U V absorbances also closely matched. 24 3.2.1 Input resistance and membrane time constant We assessed the effects of A M B D (250 u M ) on R; and x m . Input capacitance was calculated using Rj and x m measurements. Average R o f neurons in control solutions was 323 ± 44 M Q and the average x m was 41 ± 6 ms (n =19). The average input capacitance of neurons was 130 ± 10 pF (n = 19). Application of A M B D had no significant effects on R o f previously untreated (n = 19), bicuculline (20 u M ) pre-treated (n = 19) or strychnine (2 u M ) pre-treated (n = 19) neurons (paired Mest, P > 0.05). A M B D also had no significant effects on input capacitance of previously untreated (n = 19), bicuculline (20 u M ) pre-treated (n = 19) or strychnine (2 uM) pre-treated (n = 19) neurons (paired Mest, P > 0.05) (Table 3.1). Hence, A M B D had no significant effects on R, x m or capacitance that accounted for its proposed antagonism. 3.2.2 Action potential firing We examined Na +-dependent action potentials evoked by current pulse injection to determine whether the proposed antagonistic effects of A M B D resulted from alterations in N a + and K + channels. Repetitive firing of action potentials was evoked by intracellular current pulse injection. Application of A M B D (250 u M ) had no effects on action potential firing at 25-40 H z in previously untreated (n = 19), bicuculline (20 uM) pre-treated (n = 19) or strychnine (2 uM) pre-treated (n = 19) neurons (cf. Figure 3.2). In addition, there were no significant 25 Table 3.1 Lack of effects of A M B D on R j , capacitance and x m . Control A M B D (n = 19) (250 uM) Ri ( M Q ) 323 ± 4 4 222 ± 38 Capacitance (pF) 130 ± 10 120 ± 3 0 x (ms) 41 ± 6 26 ± 7 Bicuculline A M B D Strychnine A M B D pre-treated (250 uM) pre-treated (250 uM) (n=19) (n=19) Ri ( M Q ) 314 ± 8 0 294 ± 54 233 ± 35 257 ± 5 1 Capacitance (pF) 90 ± 1 0 9 0 ± 2 0 110 ± 20 9 0 ± 4 0 x m (ms) 27 ± 6 25 ± 4 25 ± 7 28 ± 14 Values are mean ± S E M . 26 effects on action potential amplitude (paired Mest, P < 0.05) or configuration. Ha l f width of action potentials from previously untreated neurons was 1.0 ± 0.1 ms before A M B D application and 1.0 ± 0.2 ms after A M B D application (n = 5) (P < 0.05, paired t-test). These results suggest that the antagonistic effects of A M B D did not likely result from alteration o f voltage-dependent N a + or K + channels. 3.2.3 Voltage-current relationships We examined the effects of A M B D (250 uM) on voltage-current relationships to assess its action on rectifying properties. Furthermore, we examined these before and after application of bicuculline (20 uM) or strychnine (2 uM) to determine whether A M B D had actions comparable to these antagonists or whether bicuculline or strychnine altered A M B D effects. Figure 3.3 depicts the voltage-current relationships for currents from control (A), bicuculline pre-treated (B) and strychnine pre-treated (C) neurons and the effects of A M B D on these currents. After application of A M B D to previously untreated neurons, the control and drug curves had an intercept of -51 m V (n = 19). The voltage-current relationship for previously untreated neurons indicated a slight AMBD-media ted blockade o f rectification during large hyperpolarizing pulses. Application of bicuculline or strychnine alone had no effects on the current intercept. After application of A M B D to 27 Control B . AMBD 50 ms 100 pA 20 mV 50 pA 25 ms 10 mV Figure 3.2 A M B D (250 uM) had no effects on action potential firing of a previously untreated neuron. (A) action potential firing of a previously untreated neuron. (B) the same neuron after application of A M B D . (C) control (grey) and A M B D (black) traces were superimposed. Upper traces show injected current pulses and lower traces depict evoked action potentials. 28 neurons pre-treated with bicuculline, the control and drug curves intercepted at -59 m V (n = 19). After application of A M B D to neurons pre-treated with strychnine, the intercept was -58 m V (n = 19). These intercepts were within 6 m V s of EC\ (-53 m V ) , suggesting that A M B D had no effects on CT-mediated currents. 3.2.4 Comparison of effects on baseline currents with bicuculline Recent studies indicate that gabazine has reversible effects on the baseline G A B A -mediated tonic current in V B neurons (Cope et al., 2005). To determine whether A M B D (250 uM) acted in a way comparable to gabazine, baseline currents were determined before and after A M B D application to previously untreated neurons. Alterations in baseline currents (Al) were measured by subtracting baseline current after drug application from control baseline current adjusted to zero. After application o f bicuculline, A l was 46 ± 54 p A . After application o f strychnine, A l was 38 ± 33 p A . A l after A M B D application was 3 ± 31 p A . Bicuculline, strychnine and A M B D had no significant effects on baseline current (paired /-tests, P > 0.05). 3.2.5 Summary of effects on membrane properties In summary, A M B D had no significant effects on R , x m , or input capacitance. A M B D did not alter action potential firing frequency, amplitude, configuration or half-width, hence it l ikely had no effects on voltage-dependent N a + or K + channels. Voltage-current relationships, over a physiological range, showed no significant alteration. The V - I relationship for previously untreated neurons indicated a slight AMBD-media ted 29 Current (pA) i i i -150 -100 -50 50 100 150 B. Control AMBD Strych and Bic Current (pA) 100 Control Bicuculline AMBD + Bicuculline Current (pA) -100 -50 5" E a at 3 I -20' -40H -80 -100--120 50 100 Control Strychnine AMBD + Strychnine Figure 3.3 Effects of A M B D (250 uM) on voltage-current relationships are shown for currents from control (A), bicuculline (20 uM) pre-treated (B) and strychnine (2 uJVI) pre-treated (C) neurons. Arrows depict current intercepts obtained from the intersection of control and antagonist curves. Intercepts for control and drug curves were -51 m V (n = 19) for previously untreated, -59 m V (n = 19) for bicuculline pre-treated and -58 m V (n = 19) for strychnine pre-treated neurons. 30 blockade o f rectification during large hyperpolarizing pulses. Although this effect did not alter our results (see below), it may have effects in future current-clamp or in-vivo studies. Sulfonamide drugs, including A M B D , have been shown to alter ATP-sensitive K + (KATP) channels, and thereby may alter rectification in this way (Ashcroft and Gribble, 2000). A M B D did not affect the mixed IPSC reversal potential and hence, C l " transport processes that maintain [Cl"] gradients. 3.3 Effects of A M B D on IPSCs 3.3.1 Properties of evoked IPSCs IPSCs were evoked by electrical stimulation o f the M L to determine the effects o f A M B D on synaptic inhibition. IPSCs were evoked in 100% of neurons tested (n — 39). The mixed IPSCs (cf. Methods) were further characterized using bicuculline (20 u M ) or strychnine (2 u M ) to isolate the glycinergic and GABAAerg ic components, respectively. The mixed IPSC amplitude was 59 ± 4.0% G A B A A e r g i c and 41 ± 4.0% glycinergic (cf. similar values of Ghavanini et al., 2005). Only one ot the recorded IPSCs was totally blocked by strychnine, indicating purely glycinergic transmission. 20% of the IPSCs were completely blocked by bicuculline, indicating purely G A B A A e r g i c transmission (n = 5) and 76% (n = 19) had mixed G A B A A e r g i c and glycinergic components. The average reversal potential of mixed IPSCs was -54 ± 1 . 0 m V (n = 19) (Figure 3.5 A ) . The average reversal potential of isolated glycinergic IPSCs was -52 ± 1 . 0 m V and -56 ± 3.0 m V for isolated G A B A A e r g i c IPSCs. These reversal potentials were not significantly 31 different from Ea o f -53 m V (One sample /-test, P > 0.05), indicating that the IPSCs were mediated by Cl" . The latency to IPSCs was also determined. Latency was defined as the time from the beginning of the stimulus artifact to the onset of the IPSC. The average latency for mixed IPSCs was 2.0 ± 0.4 ms (n = 19). The average latency for isolated glycinergic and G A B A A e r g i c IPSCs was 2.0 ± 0.5 ms (n = 19). The latency of the one purely glycinergic IPSC was 2.3 ms and purely G A B A A e r g i c IPSCs had an average latency o f 3.8 ± 1.0 ms (n = 5). These latencies were not significantly different (paired t-tests, P > 0.05). 3.3.2 Concentration-dependence of IPSC reduction We anticipated that the proposed antagonistic properties o f A M B D would be concentration-dependent (cf. Mathers, 1993). We determined the concentration-response relationship for the effects of A M B D on the peak amplitude of mixed IPSCs. Five concentrations of A M B D were bath applied in a step-wise manner to 7 previously untreated neurons. The antagonism of IPSCs by A M B D was concentration-dependent (Repeated measures A N O V A , P < 0.05). Figure 3.4 depicts the relationship between A M B D concentration and percentage antagonism of the IPSC. The curve was well-fitted by a single H i l l function with an IC50 o f 77 u M . The H i l l slope was 1.3 ± 0.8, implying that a single A M B D molecule was required for antagonism (Shirasaki et al, 1991). Based on the 32 Figure 3.4 Concentration-response relationship for A M B D reduction of IPSCs. A M B D was applied cumulatively in a step-wise manner to neurons held at -80 m V (n = 7 for all points). The dotted line indicates the IC50 o f 77 u M . H i l l slope was 1.3 ± 0.8. 33 concentration-response curve, the 250 u M A M B D concentration was used for all further experiments. 3.3.3 Reversal potentials of IPSCs We determined whether A M B D (250 uM) had effects on IPSC reversal potential to determine whether it altered the CI" gradient. IPSC reversal potential was obtained by changing the holding potential in 10 m V steps between -40 m V and -70 m V . Application of A M B D to mixed IPSCs had no effects on the reversal potential. The average reversal potential of mixed IPSCs after application o f A M B D was -53 ± 2.0 m V (n = 19) (Figure 3.5 B) . Neither the control nor A M B D reversal potentials differed significantly from Ec\ (One sample Mest, P > 0.05). Therefore, A M B D had no effects on the CI" gradient. 3.3.4 IPSC latency IPSC latency was measured to determine the effects of A M B D (250 u M ) on the time between applied stimulus and TPSC onset. Table 3.2 shows the effects o f A M B D on TPSC latency. In summary, A M B D had no significant effects on latency o f mixed, isolated or pure IPSCs (paired Mests, P > 0.05). 3.3.5 Peak amplitudes of mixed IPSCs The effect o f A M B D (250 u M ) on mixed IPSCs was determined to identify potential antagonism o f the glycinergic and G A B A A e r g i c components before isolation. Application of A M B D resulted in partial block of mixed IPSCs in every neuron tested (n = 19). The 34 Figure 3.5 A M B D (250 uM) had no effects on mixed IPSC reversal potential. IPSCs (left) and current-voltage relationship (right) of a mixed IPSC before (A) and after (B) A M B D (n = 19). Arrows depict average reversal potential of the mixed IPSC before (-54 ± 1.0 m V ) and after A M B D (-53 ± 2.0 m V ) . 35 Table 3.2 Effects of A M B D on IPSC latency. Mixed A M B D (n=19) (250 uM) Latency (ms) 2.0 ± 0.4 2.1 ± 0 . 5 Isolated glycinergic (n=19) A M B D (250 uM) Isolated GABAAergic (n=19) A M B D (250 uM) Latency (ms) 2.0 ± 0.5 2.0 ± 0 . 7 2.0 ± 0.5 2.2 ± 0.2 Purely GABAAergic (n = 5) A M B D (250 uM) Purely glycinergic (n=l) A M B D (250 uM) Latency (ms) 3.8 ± 1.0 3.5 ± 0 . 7 2.3 3.1 Values are mean ± SEM. 36 average reduction in peak amplitude of the mixed IPSC was 67 ± 5.0%. Figure 3.6 depicts the effects of A M B D on a representative mixed IPSC. 3.3.6 Time course of antagonism and recovery We determined the time course of action and recovery for A M B D (250 uM) antagonism. Percentage inhibition of the peak amplitude of a mixed IPSC was measured every minute during the application o f A M B D and every 15 minutes after terminating application. Peak amplitude reduction began within 2 to 4 minutes after initiating application and showed no further reduction after 10 minutes (n = 5). Fu l l recovery was not recorded despite stable recording for periods up to 1.5 hours. Partial recovery was observed in 6 neurons. Partial recovery from A M B D began within 30 to 45 minutes and was complete within 60 minutes (n = 6) (Figure 3.7). Past studies using an identical bath perfusion technique showed that complete recovery from bicuculline required 30 minutes or less. Hence, the rate-limiting step in A M B D recovery was not perfusion, but likely, tissue uptake and binding characteristics. 3.3.7 Pharmacologically isolated IPSCs The effects of A M B D on isolated glycinergic and GABAAerg ic IPSCs were examined to determine whether the observed mixed IPSC antagonism resulted from reduction of one or both components. Glycinergic and G A B A A e r g i c components were isolated from the mixed IPSC using bicuculline (20 uM) and strychnine (2 uM) , respectively. Application of A M B D (250 uM) to 19 neurons with the isolated glycinergic component resulted in -80 mV Strychnine & Bicuculline 10 ms Mixed Figure 3.6 A M B D (250 uM) decreased the peak amplitude of a representative mixed IPSC by 24 pA (25%). 38 AMBD 100! O W 9= 75 E .2 50 c o Dl | 25 < I I I J * . . . . i i 1 i 1 i 10 20 30 40 50 60 Time (min) Figure 3.7 Time course for A M B D (250 uM) reduction of the mixed IPSC and recovery. Percentage antagonism of peak amplitude recorded every minute for 8 minutes during A M B D application (n = 5) and every 15 minutes during A M B D washout (n = 6). reduction of this current in 12 neurons (63%) and no effect in 7 neurons (37%). A M B D reduced the peak amplitude o f the isolated glycinergic IPSC by an average of 41 ± 11% (n = 19). A M B D reduced the isolated G A B A A e r g i c IPSC in 79% (n = 15), and had no effect in 21% (n = 4). A M B D reduced the isolated G A B A A e r g i c IPSC peak amplitudes by an average of 70 ± 18.0% (n = 19). Figure 3.8 depicts the pharmacological isolation of glycinergic and G A B A A e r g i c components from a mixed IPSC and the effects o f A M B D on these components. Our observations indicated that mixed IPSC antagonism resulted from A M B D actions on both the isolated glycinergic and the G A B A A e r g i c components. The total antagonism approximated the amount of reduction observed in mixed IPSCs. 3.3.8 Comparison of antagonism with strychnine We determined whether A M B D antagonized the glycine receptor like strychnine. To assess whether A M B D reduced the mixed and isolated IPSCs in a manner similar to strychnine, we compared average percentage inhibitions of peak EPSC amplitude induced by A M B D (250 uM) , strychnine (2 uM) and bicuculline (20 uM) . The mixed IPSC peak amplitude was reduced by A M B D (67 ± 5%), bicuculline (59 ± 4%) and strychnine (41 ± 4%) (n = 19). Percentage inhibitions of the mixed IPSC by A M B D and bicuculline were not significantly different (paired Mest, P > 0.05) but percentage inhibitions by A M B D and strychnine were significantly different (paired t- test, P < 0.05). A M B D produced less antagonism o f the isolated glycinergic IPSC (41 ± 11%) than strychnine (96 + 1%) (n = 19) (paired Mest, P < 0.05). The isolated G A B A A e r g i c IPSC was reduced by A M B D 40 A. Bicuculline & Strychnine Figure 3.8 A M B D (250 uM) reduced the peak amplitude of isolated glycinergic and GABAAergic IPSCs in 2 neurons (A, B, C). (A) bicuculline (20 uM) and strychnine (2 uM) revealed distinct glycinergic and GABAAergic components. (B) A M B D reduced the isolated glycinergic component. (C) A M B D reduced the isolated GABAAergic component. 41 (70 + 18%) and bicuculline (98 ± 1%) (n - 19). Percentage inhibitions of the isolated GABAAergic IPSC by A M B D were significantly less than percentage inhibitions by bicuculline (paired t-test, P < 0.05). A M B D reduced the mixed IPSC to a greater extent than strychnine and decreased the glycinergic and GABAAergic IPSCs after their pharmacological isolations (Figure 3.9). In summary, A M B D did not antagonize the glycine receptor to the same extent as strychnine, and additionally exhibited properties of a G A B A A antagonist. 3.3.9 Purely glycinergic and GABAAergic IPSCs The effects of A M B D (250 uM) were also examined on one purely glycinergic and 5 purely GABAAergic IPSCs. In one neuron, A M B D reduced the peak amplitude of the glycinergic IPSC by 81%. Strychnine eliminated the purely glycinergic IPSC. Interestingly, A M B D had no effects on 4 GABAAergic IPSCs and partially antagonized 1 GABAAergic IPSC by 17% (Figure 3.10). A M B D reduced the purely glycinergic IPSC in one neuron but had no effect on the majority of purely GABAAergic IPSCs, suggesting potential specificity for the glycine receptor. 3.3.10 Rise time and charge transfer Before A M B D antagonism, strychnine (2 uM) and bicuculline (20 uM) had no significant effects on the rise time of mixed, pure and isolated glycinergic and GABAAergic IPSCs (paired /-tests, P > 0.05). Hence, we were able to attribute any potential effects exclusively to AMBD. 42 O </> ro 100 (n = 19) (n = 19) • A M B D (250 uM) • Bicuculline (20 uM) • Strychnine (2 uM) Mixed Isolated Isolated Glycinergic GABA A ergic IPSC Component Figure 3.9 Comparison of the effects of A M B D with strychnine and bicuculline on mixed and isolated IPSCs. Values are mean ± S E M . Asterisks indicate that percentage antagonisms of the mixed and isolated glycinergic IPSCs by A M B D and strychnine, and of the isolated G A B A A e r g i c IPSCs by A M B D and bicuculline, were statistically different from each other (P < 0.05, paired Mests). 43 Figure 3.10 A M B D (250 uM) exhibited selectivity in antagonism. A M B D reduced a purely glycinergic IPSC (A) but had no effects on purely GABAAergic IPSCs (B). 44 A M B D had no significant effects on the rise time of mixed, pure, and isolated glycinergic and G A B A A e r g i c IPSCs (paired /-tests, P > 0.05). Table 3.3 summarizes the rise times of IPSCs before and after A M B D . Rise time was not associated with IPSC antagonism by A M B D . We calculated percentage inhibition of charge transfer for mixed, pure and isolated glycinergic and G A B A A e r g i c IPSCs. A M B D (250 uM) had no significant effects on charge transfer of mixed IPSCs, with an average reduction o f 43 ± 11% (n = 19) (P > 0.05, paired /-test). A M B D also had no significant effects on charge transfer o f isolated G A B A A e r g i c IPSCs, with an average reduction of 59 ± 17% (n = 19) (P > 0.05, paired /-test). A M B D had no significant effects on the isolated glycinergic components charge transfer, with an average reduction of 49 ± 8 % (n = 19) (P > 0.05, paired /-test). A M B D (250 u M ) reduced charge transfer of the purely glycinergic IPSC by 85% (n = 1). Lastly, A M B D had no significant effects on the charge transfer of purely G A B A A e r g i c IPSCs (n = 4), with an average reduction o f 2 ± 2% (Table 3.3) (P > 0.05, paired /-test). In contrast to the A M B D reduction in IPSC peak amplitude, measurements of charge transfer were associated with large variations in S E M , contributing to an inability to demonstrate significance. 45 Table 3.3 Summary of the effects of A M B D on rise time and charge transfer of mixed and pure IPSCs, and isolated components. Control A M B D (0=19) (250 uM) Rise Time (ms) 1.0 ± 0 . 5 1.0 ± 0 . 5 Area (pC) -6.7+1.7 -4.4 ± 2.2 Isolated glycinergic (o=19) A M B D (250 uM) Isolated G A B A A e r g i c (n=19) A M B D (250 uM) Rise Time (ms) Area (pC) 1.0 ± 0 . 5 -0.7 ± 0.3 2.0 ± 1 . 0 -0.9 ± 0.5 1.0 ± 0 . 5 -5.9 ± 2 . 1 1.0+1.0 -4.7 ± 4.0 Purely G A B A A e r g i c (n = 4) A M B D (250 uM) Purely glycinergic (11=1) A M B D (250 uM) Rise Time (ms) Area (pC) 1.0 ± 0 . 5 -0.4 ± 0.2 2.0 ± 1 . 0 -0.5 ± 1.1 1.0 -5.1 1.0 -0.8 Values are mean ± S E M . 46 3.4 Effects of A M B D on IPSC decay 3.4.1 Fast, intermediate and slow IPSC decay Previous studies suggested that mixed IPSCs in the V B nuclei had slow and fast decay time constants attributable to glycine receptor activation, whereas GABAAerg ic IPSCs had intermediate decay time constants (Ghavanini et al., 2006). We determined whether A M B D had effects on the decay of mixed IPSCs or isolated components and whether these effects were alike to the observed IPSC reduction. IPSC decay phases were fitted with exponential terms to determine the time constants. The decay time constants differed slightly from Ghavanini et al. (2006), presumably because of recordings from a differing neuronal sample. 3.4.2 Intermediate and slow IPSC components The mixed IPSCs decayed with a biexponential time course and were separatable into two groups based on differential strychnine- and bicuculline-sensitivity: (1) IPSCs with bicuculline-sensitive intermediate (21 ± 0.5 ms) and strychnine-sensitive fast (8 ± 1.0 ms) time constants (n = 14), and (2) IPSCs with bicuculline-sensitive intermediate (19 ± 2.0 ms) and strychnine-sensitive slow (101 ± 12.0 ms) time constants (n = 10). The fast, intermediate and slow time constants were significantly different from each other (One way A N O V A , P < 0.05). Application of A M B D (250 p M ) to group (1) neurons eliminated the intermediate IPSC component. The remaining component had a time constant of 6 ± 1.0 ms, which was not significantly different from the control fast component (paired /-test, P > 0.05). 47 Application of A M B D to group (2) neurons eliminated the slow IPSC component, leaving a current with a time constant of 13 ± 3.0 ms, which was not significantly different from the control intermediate component (paired /-test, P > 0-05) (Figure 3.11, Table 3.4). Histograms of decay time constants are shown in Figure 3.12. A M B D abolished the intermediate and slow components in mixed IPSCs, and had no effects on the fast components. 3.4.3 Pharmacologically isolated decays The observed AMBD-media ted reduction in isolated glycinergic IPSC amplitude (see above) was not associated with alterations in decay. Isolation of the glycinergic component from the mixed IPSC containing fast and intermediate components revealed a monoexponential decay time constant of 8 ± 1.0 ms (n = 4). The time constant was not significantly different from the mixed IPSC fast constant (paired /-test, P > 0.05). The glycinergic component isolated from a mixed IPSC containing intermediate and slow time constants had a single decay time constant of 83 ms (n = 1). The observed AMBD-media ted reduction in isolated G A B A A e r g i c IPSC amplitude (see above) was also not associated with alterations in decay. Isolation of the GABAAerg ic component from mixed IPSCs with fast and intermediate components revealed a single decay time constant o f 15 ± 3.0 ms (n = 8). The time constant was not significantly different from the mixed IPSC intermediate constant (paired /-test, P > 0.05). The 48 Figure 3.11 Effects of A M B D (250 uM) on the decay time constants of mixed IPSCs with fast and intermediate time constants (A) and mixed IPSCs with intermediate and slow time constants (B). IPSCs were fitted with mono- or bi-exponentials (black). 49 Table 3.4 Effects of A M B D on decay parameters of mixed IPSCs. Mixed IPSCs with fast and intermediate components Component Parameter Control A M B D (n = 14) (250 uM) Fast Time constant (ms) 8+1.0 6 ± 1.0 Amplitude (pA) -375 ± 124 - 2 1 5 ± 1 0 1 * Intermediate Time constant (ms) 21 ± 0 . 5 0 Amplitude (pA) -123 ± 4 3 0 Mixed IPSCs with intermediate and slow components Component Parameter Control A M B D (a =10) (250 uM) Intermediate Time constant (ms) 19 ± 2 . 0 13 ± 3 . 0 Amplitude (pA) -205 ± 96 -136 ± 2 2 * Slow Time constant (ms) 101 ± 13 0 Amplitude (pA) -22 ± 4 0 Values are mean ± SEM. * P < 0.05 (paired Mes t ) . 50 B. 8-7-6-c 5-§ 4-j ° 3-2 -1 0 0 40 60 80 100 120 Decay Time Constant (ms) 140 160 u 1411 J l 20 40 60 80 100 120 Decay Time Constant (ms) 140 160 Figure 3.12 Histograms o f the decay time constants of mixed IPSCs. M i x e d IPSCs that decayed with a biexponential time course were separated into 2 groups. (A) the first group had a bicuculline-sensitive intermediate (21 ± 0.5 ms) and a strychnine-sensitive fast (8 ± 1.0 ms) component (n = 14), arrows. (B) the second group had a bicuculline-sensitive intermediate (19 ± 2.0 ms) and a strychnine-sensitive slow (101 ± 12.0 ms) component (n = 10), arrows. 51 G A B A A e r g i c component from mixed IPSCs with intermediate and slow time constants had an intermediate decay time constant o f 19 ± 3.0 ms (n = 7). The time constant was not significantly different from the mixed IPSC intermediate component (paired /-test, P > 0.05). These results are consistent with previously published decay time constants for isolated glycinergic and G A B A A e r g i c IPSCs (Ghavanini et al., 2006). A s summarized in Table 3.5, A M B D (250 u,M) had no significant effects on the isolated glycinergic or G A B A A e r g i c IPSC decay time constants (paired Mest, P > 0.05) (Figure 3.13). 3.4.4 Purely glycinergic and GABAAergic decays The decay time constants of purely glycinergic and G A B A A e r g i c IPSCs were determined to establish whether the observed IPSC antagonism was associated with alterations in decay. The decay of the purely glycinergic IPSC had a single decay time constant o f 6.0 ms (n = 1). The decay of the purely G A B A A e r g i c IPSCs had a single intermediate decay time constant of 19 ± 1.0 ms (n = 4). A M B D (250 uM) had no effects on decay time constants of purely glycinergic and G A B A A e r g i c IPSCs. The respective time constants were 6.0 ms (n = 1) and 17 ± 1.0 ms (n = 4) (paired Mest, P > 0.05 for G A B A A e r g i c ) (Figure 3.14, Table 3.6). Glycinergic IPSC antagonism by A M B D and its failure to antagonize G A B A A e r g i c IPSCs were not associated with changes in decay. Figure 3.13 Lack of effect of A M B D (250 uM) on the decay time constants o f isolated glycinergic (A) or G A B A A e r g i c (B) IPSCs. IPSCs were fitted with single exponentials (black). 53 Table 3.5 Effects of A M B D on decay time constants and amplitudes of exponentially fitted isolated glycinergic and GABAAergic IPSCs. Isolated from IPSCs with fast and intermediate components Parameter Glycinergic A M B D GABA A e rg i c A M B D (n = 4) (250 uM) (n = 8) (250 uM) (n = 4) ( n = D Time constant (ms) 8 + 1.0 9 + 1.0 15 ± 3 . 0 19 Amplitude (pA) -317190 -288 ± 85 -164 + 95 -520 Isolated from IPSCs with intermediate and slow components Parameter Glycinergic A M B D GABA A e rg i c A M B D ( n = l ) (250 uM) (n = 7) (250 uM) ( n = D ( n = l ) Time constant (ms) 83 15 19 + 3.0 30 Amplitude (pA) -58 -9 -121+47 -44 Values are mean ± SEM. 54 200 pA 10 ms Figure 3.14 A M B D (250 uM) effects on the decay time constants of purely glycinergic (A) or G A B A A e r g i c (B) IPSCs. The IPSCs were fitted with single exponentials (black). Table 3.6 Effects of A M B D on decay time constants and amplitudes of exponentially fitted glycinergic and GABAAerg ic IPSCs. Parameter Purely A M B D Purely A M B D GABA A ergic (250 uM) glycinergic (250 uM) ( a =4) (n= l ) Time constant (ms) 19 ± 1.0 17 ± 1.0 6 6 Amplitude (pA) -26 + 9 -25 ±11 -1030 -124 Values are mean ± S E M . 56 3.5 Spontaneous IPSCs We determined the effects o f A M B D on sIPSCs to assess possible presynaptic actions. Without A M B D , we recorded an average sIPSC frequency of 1.0 ± 0.2 H z (n = 5). The average amplitude of sIPSCs was -19 + 2 p A (n = 5). The average rise time o f sIPSCs was 1.0 ± 0.3 ms (n = 5). There was no correlation between the amplitude, rise time and decay time constants o f the detected events (R 2 < 0.05) (Figure 3.15). Hence, the detected events were sIPSCs and not random noise. A M B D (250 uM) significantly decreased the frequency of sIPSCs to 0.2 ± 0.03 H z (P < 0.05, paired /-test) (Figure 3.16). After A M B D application, the amplitude o f sIPSCs was -19 ± 3 p A , which was not different from control (P > 0.05, paired /-test). The rise time of sIPSCs after A M B D application was 1.0 ± 0.4 ms, which was not different from control (P > 0.05, paired t-test). Table 3.7 summarizes the effects o f A M B D on sIPSCs. 58 Control 250 uM AMBD j-IOOpA 100 ms Figure 3.16 A M B D decreased the frequency but not the amplitude o f sIPSCs. Arrows indicate visually accepted sIPSCs. Table 3.7 Summary of A M B D effects on spontaneous IPSCs. Control A M B D (n = 5) (250 u M ) Frequency (Hz) 1.0 ± 0 . 2 0.2 ± 0.03 * Amplitude (pA) 19 + 2 19 + 3 Rise time (ms) 1.0 + 0.3 1.0 + 0.4 Values are mean ± S E M . * P <0.05, paired /-test. 60 Chapter IV. Discussion 4.1 Summary of the results This thesis studied the effects of a potential glycine receptor antagonist, A M B D , on neurons in the V B nuclei of the thalamus. The aim was to assess the ability of A M B D to selectively antagonize IPSCs and sIPSCs in V B neurons. The hypothesis was that A M B D interacts with synaptic glycine receptors and not G A B A A receptors, to specifically suppress the inhibitory effects of glycine-like amino acids. A M B D significantly reduced the peak amplitude of electrically stimulated mixed IPSCs, pharmacologically isolated IPSCs, and purely glycinergic IPSCs. The reduction of peak amplitude of mixed IPSCs by A M B D was concentration-dependent, with an IC50 of 77 uM and a Hill slope of ~ 1.3. Like strychnine, A M B D had no effects on four purely GABAAergic IPSCs, or their time constants. A M B D did not antagonize IPSCs in a manner identical to strychnine. A M B D abolished the intermediate and slow components of mixed glycinergic and GABAAergic IPSCs, whereas strychnine eliminated the fast, as well as the slow, components of mixed IPSCs. A M B D had no effects on the decay time constants of pharmacologically isolated IPSCs. A M B D decreased the apparent frequency but not the amplitude of spontaneous IPSCs (sIPSCs). A M B D had little or no effects on the passive and active membrane properties that could account for the observed antagonism. It therefore remains to succinctly discuss the findings that are most relevant to an assessment of A M B D as a selective antagonist of receptors for amino acids. 61 4.2 Postsynaptic receptor antagonism by A M B D 4.2.1 Mixed IPSCs We have shown for the first time, that A M B D significantly reduced the peak amplitude by ~ 67% of mixed glycinergic and G A B A A e r g i c EPSCs in 19 thalamocortical neurons. These studies also confirmed that glycinergic and GABAAerg ic receptors mediate the mixed IPSCs evoked by electrical stimulation of the medial lemniscus ( M L ) (cf. Ghavanini et al. 2005). The mixed IPSC likely results from release of several glycine-like amino acids and G A B A from co-transmitting glycinergic and G A B A A e r g i c pathways (cf. Ghavanini et al., 2005). Although purely glycinergic and G A B A A e r g i c IPSCs were observed in several neurons, it was necessary to isolate the glycinergic and G A B A A e r g i c components from the mixed IPSCs with strychnine or bicuculline for a detailed analysis of A M B D actions. 4.2.2 Isolated and purely glycinergic currents We showed that bicuculline antagonized the G A B A A e r g i c component of the mixed IPSC, evident as a decrease in peak amplitude. Concomitant application o f strychnine abolished the remaining component. A M B D antagonized the peak amplitude of the latter, glycinergic component by ~ 41% in 19 neurons. A M B D also reduced the peak amplitude of purely glycinergic IPSCs by 81% in one neuron. B y comparison, strychnine produced ~ 96% antagonism o f the glycinergic component in the same 19 neurons, and eliminated the purely glycinergic IPSCs in one neuron. Although less potent than strychnine, A M B D was an effective antagonist of glycinergic inhibition in V B thalamus. 62 4.2.3 Isolated and purely GABAAergic currents Unexpectedly, the actions of A M B D were not entirely specific for glycinergic receptors, and blocked G A B A A e r g i c IPSCs after isolation with strychnine. A M B D reduced the peak amplitude o f isolated G A B A A e r g i c IPSCs by ~ 70% in 19 neurons. A M B D did not alter purely G A B A A e r g i c IPSCs in four out of five neurons. The purely G A B A A e r g i c IPSCs altered by A M B D in one neuron may have been mixed, with a small glycinergic component, therefore these were excluded from further analysis (cf. Results). A n explanation for the additional antagonism at the G A B A A receptor is that, the G A B A A e r g i c current associated with the mixed IPSC may differ in pharmacological sensitivity from purely G A B A A e r g i c IPSCs. The specificity of released glycine-like amino acids for the synaptic glycine receptor may not be uniformly absolute, since 0-alanine can additionally affect the G A B A A receptor (cf. W u et al., 1993). Thus, A M B D antagonism may critically depend on the type of agonist interacting with the G A B A A receptor or equivocally, access of antagonists to the mixed and isolated receptor sites. This explanation implies that purely G A B A A e r g i c currents may be mediated by a different G A B A A receptor than the isolated G A B A A e r g i c currents. A M B D could block a new postsynaptic receptor with an unknown subunit composition or act at a distinct binding site on the G A B A A receptor that is sensitive to A M B D (cf. Kuhse et al., 1990). The mixed IPSC presumably results from glycine, taurine, and p-alanine, with unknown release-stoichiometry, as well as G A B A . Although the exact stoichiometry is unknown, 63 specificity of A M B D for the P -amino acids, and less for glycine, could explain the reduced potency of A M B D on mixed IPSCs, in comparison to strychnine. Numerous studies in the C N S have suggested the existence of a p -amino acid receptor that is relatively insensitive to glycine but activated by taurine and P-alanine (reviewed by Huxtable, 1989). 4.2.4 The potential P-amino acid receptor Studies on concentration-response relationships for glycine and the P -amino acids have revealed discrepancies in medullary neurons that provide evidence for a distinct p -amino acid receptor. The H i l l slopes for the agonist actions of glycine and p -amino acids were not similar in the medulla (Gatti et al., 1985), suggesting that their effects were mediated by different receptors or by different binding sites on the same receptor. In the V B nuclei, the concentration-response curves for the agonist actions o f P-alanine, glycine and taurine have different slopes (cf. Ghavanini et al., 2005). Furthermore, the latter studies showed that co-application of strychnine and bicuculline did not completely block the effects o f P-alanine, although the reversal potential for its action indicated C l " mediation. These results suggest that p -amino acid receptors or uncharacterized sites on the glycine or G A B A A receptor mediate the effects of p -amino acids. The existence o f a P -amino acid receptor may require evidence for high-affinity, N a + -independent binding of P -amino acids, in contrast to membrane transporters. The specific binding to receptors is Na+-independent, whereas binding o f agonists to transporters is Na+-dependent (Huxtable, 1989). Although AMBD-sens i t ive , N a + -64 independent binding of taurine to brain synaptosomes has been reported for cerebral cortex (Kontro and Oja, 1983; Kontro et al., 1984; Kontro and Oja, 1987), these results are considered inconclusive because o f the use of a homogenized tissue preparation (cf. Huxtable, 1989). 4.3 Effects of A M B D on decay of mixed IPSCs 4.3.1 Slow decay time constant A n outstanding effect o f A M B D was the elimination of the slow component o f mixed IPSCs comprised of slow and intermediate components, in all 10 neurons. The IPSC decay time constant is similar to extrasynaptic channel burst duration during glycine and P -amino acid application in the V B nuclei (Ghavanini et al., 2006). The lifetimes o f channel bursts (cf. Beato et al., 2002) likely mediate the decay time constant of IPSCs. The average lifetimes of short- and long-duration bursts were similar to the decay time constants of fast and slow IPSCs, respectively (Ghavanini et al., 2006). Theoretically, glycine, taurine or p-alanine could mediate inhibition. However, taurine and P-alanine more commonly activate channels with long-duration bursts than glycine at extrasynaptic receptors. These results suggest that taurine and p-alanine may mediate slow synaptic inhibition and could have neurotransmitter roles in the V B nuclei. If the slow component results from the action of p -amino acids and not glycine, as suggested by Ghavanini et al. (2006), it seems likely that A M B D was specific for the synaptic actions o f p -amino acids. 65 4.3.2 Intermediate and fast decay time constants Our results showed that A M B D abolished the intermediate component and reduced the peak amplitude of the fast component of mixed IPSCs with fast and intermediate components. In the V B nuclei, the fast and slow components are attributable to glycine receptor activation, whereas the intermediate components are attributable to G A B A A receptor activation (see also, Ghavanini et al., 2005; 2006). It is plausible that mixed IPSCs with fast and intermediate components were mediated by p-alanine rather than G A B A , because P-alanine binds to both glycine and G A B A A receptors (Wu et al., 1993). Based on this reasoning, our results imply that A M B D eliminated the intermediate component of mixed IPSCs, possibly due to specific antagonism of p-alanine-mediated inhibition, rather than antagonism of inhibition mediated by released G A B A . In addition, our results show that A M B D reduced the peak amplitude of the fast component, suggesting some antagonism of fast glycinergic inhibition. 4.4 Presynaptic actions of A M B D 4.4.1 Spontaneous IPSCs A M B D decreased the apparent frequency of sIPSCs with no effects on amplitude, suggesting a presynaptic effect (cf. Figure 3.16). Past studies on embryonic hippocampal neurons have indicated that sIPSCs largely result from the release of single synaptic vesicles (mono-quantal packets of neurotransmitter). A decrease in the frequency of sIPSCs after application of a pharmacological agent is expected from a presynaptic drug action on nerve terminal release, whereas a concomitant decrease in sIPSC amplitude indicates a postsynaptic action (Fatt and Katz, 1952; Vautrin et al., 1993). Kontro and 66 Oja (1987) found that A M B D attenuated K +-stimulated taurine release in cerebral cortex. Hence, A M B D may have actions on nerve terminals or presynaptic neurons. Although analysis of sIPSCs is powerful for the identification of presynaptic actions, it is not without ambiguities. Postsynaptic blockade of the glycine or G A B A A receptors may produce a reduction in frequency of sIPSCs, without a presynaptic effect (cf. Vautrin et al., 1993). Although we propose that the observed decrease in sTPSC frequency results from a presynaptic effect on transmitter release, the decrease could have resulted from postsynaptic A M B D blockade. 4.4.2 Modulation of GABA release by the glycine receptor Theoretically, AMBD-media ted blockade of a presynaptic site that attenuates G A B A release from nerve terminals could decrease G A B A A e r g i c transmission, leading to reduction o f isolated GABAAerg ic IPSCs. For example, studies on glycine and G A B A co-release in the medial nucleus of the trapezoid body and spinal cord have shown that glycine activates presynaptic glycine receptors that increase the release o f G A B A (Jones, 1991; Turecek and Trussell, 2001). These and other studies (Malminen and Kontro, 1989) may suggest that glycine-like amino acids and taurine released from glia could modulate G A B A release from nerve terminals. In support of this possibility, our results show that A M B D reduced the peak amplitude of mixed IPSCs, as well as isolated glycinergic and G A B A A e r g i c IPSCs (cf. Figure 3.6 and Figure 3.8). Furthermore, A M B D had little effect on postsynaptic G A B A A receptors (cf. 67 Figure 3.10). Given our observations of decreased frequency of sIPSCs, the present data imply that the effects of A M B D on isolated G A B A A e r g i c IPSCs may have resulted from actions on a presynaptic site that mediates G A B A release from nerve terminals. 4.4.3 Co-release versus co-transmission We did not observe a prevalence of bi-phasic sIPSCs expected from co-release o f G A B A and the glycine-like amino acids. Bi-phasic sIPSCs are associated with the release o f two transmitters within single quanta that activate two channels with different kinetics. Neurons showing exclusively glycinergic or G A B A A e r g i c responses suggest co-transmission by independent pathways (cf. Donato and Nistri , 2000; Dumoulin et al., 2001; ^Ghavanini et al., 2006). In the V B nuclei, the provenance o f released G A B A and the glycine-like amino acids is likely from co-localized nerve terminals. 4.5 Proposed mechanism of A M B D antagonism We propose that A M B D has both presynaptic and postsynaptic sites o f action. Our results show that A M B D eliminated the slow component and reduced the peak amplitude of the fast component of mixed IPSCs and antagonized isolated and purely glycinergic IPSCs. We suggest that A M B D antagonized the actions o f glycine-like transmitters at the postsynaptic receptor or receptors mediating fast and slow IPSCs. The glycine receptor mediating slow IPSCs may specifically bind p-amino acids, supporting the role of A M B D as an antagonist of a potential P-amino acid receptor. In addition, our results show that A M B D reduced the peak amplitude o f isolated G A B A A e r g i c IPSCs with no effects on the majority of purely G A B A A e r g i c IPSCs and decreased the apparent 68 frequency of sIPSCs. We suggest that A M B D blocks a presynaptic site that normally increases spatially co-localized G A B A release, thereby decreasing the amplitude o f isolated G A B A A e r g i c IPSCs. Figure 4.1 depicts the proposed mechanism for A M B D antagonism. 4.6 Future directions Studies on differential antagonism of extrasynaptic receptors activated by exogenous application of glycine-like amino acids and G A B A may help to resolve whether A M B D is specific for particular agonist interactions at the G A B A A receptor. Unfortunately, there may be substantial differences between synaptic and extrasynaptic receptors, such as CI" permeability and conductance (Ghavanini et al., 2006). Synthesis and pharmacological studies o f p-alanine analogues represent an important future approach. Although in vivo studies o f A M B D actions were not within the scope of this thesis, these experiments would provide insight into the potential neurotransmitter role o f glycine-like amino acids. Injection of A M B D into live animals would further discriminate between the antagonistic actions of A M B D and strychnine, and potentially clarify the role of the p-amino acid receptor. Classical studies have shown that strychnine reduces the threshold for sensory evoked convulsions and induces allodynia in vivo (Sherrington, 1947), suggesting that glycinergic transmission plays an important role in sensory processing (cf. Ran et al., 2004). Determination of whether A M B D also reduces the threshold for sensory evoked convulsions could potentially provide evidence for the role of p-amino acids in sensory pathophysiology. The involvement o f the thalamus in pain 69 Figure 4.1 Proposed mechanism for A M B D antagonism. 70 transmission (Ohye, 1998), suggests that the characterization o f P-amino acid receptor agonists antagonized by A M B D may help in the development of analgesic drugs (cf. Hornfeldt et al., 1992). 4.7 Conclusions The major finding of these studies was that A M B D specifically antagonized the peak amplitude of mixed, isolated, and purely glycinergic IPSCs, but had little effect on purely GABAAerg ic IPSCs. Furthermore, A M B D abolished the slow and intermediate components of biexponentially fitted mixed IPSCs and decreased the frequency of sIPSCs, with no effects on their amplitude. A M B D antagonism of IPSCs did not result from actions on passive or active membrane properties. Rather, AMBD-media ted IPSC reduction was a likely consequence o f both postsynaptic and presynaptic effects. We propose that A M B D specifically antagonizes transmitter action at the postsynaptic glycine receptors that mediate fast and slow IPSCs. We raise this possibility from observations o f specific AMBD-media ted elimination of the slow component o f mixed IPSCs and reduction in the peak amplitude o f the fast component. 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