Synaptic inhibition by glycine acting at a metabotropic receptor in tiger salamander retina

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1 J Physiol (2008) pp Synaptic inhibition by glycine acting at a metabotropic receptor in tiger salamander retina Mingli Hou, Lei Duan and Malcolm M. Slaughter Program in Neuroscience and Department of Physiology and Biophysics, State University of New York at Buffalo, 124 Sherman Hall, Buffalo, NY 14214, USA Glycine is the lone fast neurotransmitter for which a metabotropic pathway has not been identified. In retina, we found a strychnine-insensitive glycine response in bipolar and ganglion cells. This glycine response reduced high voltage-activated calcium current. It was G-protein mediated and protein kinase A dependent. The EC 50 of the metabotropic glycine response is 3 μm, an order of magnitude lower than the ionotropic glycine receptor in the same retina. The bipolar cell glutamatergic input to ganglion cells was suppressed by metabotropic glycine action. The synaptic output of about two-thirds of bipolar cells and calcium current in two-thirds of ganglion cells are sensitive to the action of glycine at metabotropic receptors, suggesting this signal regulates specific synaptic pathways in proximal retina. This study resolves the curious absence of a metabotropic glycine pathway in the nervous system and reveals that the major fast inhibitory neurotransmitters, GABA and glycine, both activate G-protein-coupled pathways as well. (Received 4 March 2008; accepted after revision 17 April 2008; first published online 25 April 2008) Corresponding author M. Hou: Department of Physiology and Biophysics, State University of New York at Buffalo, 124 Sherman Hall, 3435 Main Street, Buffalo, NY 14214, USA. mingli hou@hms.harvard.edu Metabotropic receptors transduce extracellular chemical signals to intracellular messengers. There are thousands of metabotropic receptors, dominated in number by those found in the olfactory system. In the nervous system, most neurotransmitters activate metabotropic receptors. A notable exception is glycine, ligand for one member of the cysteine-loop ionotropic receptor family that also includes acetylcholine and GABA (Grenningloh, 1987; Schofield et al. 1987; Moss & Smart, 2001). Within this family, glycine is the only neurotransmitter not associated with a dual activation of ionotropic and metabotropic receptors. The absence of metabotropic glycinergic signals is curious. The goal of this work was to test for the existence of a glycinergic metabotropic signal. In retina, the bipolar to ganglion cell synapse serves as a model system to investigate fast neurotransmitters and their associated metabotropic receptors. Bipolar cells release glutamate, which activates postsynaptic AMPA, kainate and NMDA receptors and also activates presynaptic metabotropic glutamate receptors (Lukasiewicz et al. 1997; Awatramani & Slaughter, 2001; Jacoby & Wu, 2001). The synapse also receives pre- and postsynaptic input mediated by ionotropic GABA and glycine receptors and metabotropic GABA receptors (Wu, 1992; Lukasiewicz et al. 1994; Gao & Wu, 1998; Zhang et al. 1998, 2002). The inhibitory transmitter input at this synapse comes from amacrine cells, of which approximately half release glycine (Marc & Liu, 1985; Marc, 1989). Thus, this is an ideal system to search for the existence of a metabotropic glycine response. Using strychnine to eliminate stimulation of ionotropic glycine receptors, the effects of applied glycine were examined. We found that glycine, through a G-protein pathway, suppresses voltage-gated calcium currents in both bipolar cells and ganglion cells. It also suppresses light signals relayed at the bipolar cell to ganglion cell synapse. Methods Retinal preparation Larval tiger salamanders (Ambystoma tigrinum) were obtained from Kons Direct (Germantown, WI, USA) and Charles Sullivan (Nashville, TN, USA) and were kept in tanks maintained at 4 C on a 12 h dark light cycle. Experiments were performed on isolated neurons and a tissue slice of retina from tiger salamander in accordance with National Institutes of Health and University Animal Care guidelines. The retinal slice preparation was described in detail previously by Wu (Wu, 1987) and modified by Awatramani and Slaughter (Awatramani & Slaughter, 2001). For light response experiments, all operations were performed under infrared illumination to keep the retina fully dark adapted. For studies of isolated DOI: /jphysiol

2 2914 M. Hou and others J Physiol cells, a similar dissection procedure and a standard dissociation procedure were used, as detailed previously (Mitra & Slaughter, 2002). Briefly, the animal was stunned, decapitated and double-pithed. The retina was removed from the eye and placed in a papain-containing Ringer solution (12 U ml 1 papain; Worthington Biochemicals) for 20 min at room temperature (22 C). Then the retina was dissociated in Ringer solution. Cells were placed on coverslips coated with lectin to promote cell adhesion and stored in Ringer solution in a 17 C incubator. Neurons were studied within a few hours of dissociation. Whole-cell patch clamp recording Recordings were made using the whole-cell patch clamp technique. In the retinal slice, recordings were obtained from bipolar cells and ganglion cells. Bipolar cells were identified based on their position in the inner nuclear layer and their light responses. Ganglion cells were identified based on their presence in the ganglion cell layer and their large sodium currents, exceeding 1 na. These criteria did not positively exclude displaced amacrine cells, but based on the number of cells studied and the consistency of the results it is reasonable to conclude that our findings are representative of ganglion cell responses. In the isolated cell preparation, neurons were identified as ganglion cells based on morphology, the presence of a large sodium current, and the absence of an inwardly rectifying current. These criteria were developed by characterizing properties of second- and third-order neurons in the slice preparation. The slices or isolated cells were bathed continuously in control Ringer solution containing the following (mm): 111 NaCl, 2.5 KCl, 1.8 CaCl 2, 1 MgCl 2, 10 dextrose and 5 Hepes, buffered to ph 7.8 with NaOH. Unless stated, the recording pipettes contained the following (mm): 100 potassium gluconate, 5 NaCl, 2 MgCl 2, 5 EGTA and 5 Hepes, and buffered to ph 7.4 with KOH. Data were corrected for the junctional potential. The calculated chloride reversal potential is 66 mv. In addition, the pipette solution contained 4 mm ATP, 20 mm phospho-creatine and 50 U ml 1 creatine phosphokinase to maintain intracellular ATP. To record Ca channel current in bipolar and ganglion cells, the extracellular Ringer solution was changed to one containing 10 mm BaCl 2 and 40 mm tetraethylammonium (TEA)-Cl in equal molar replacement of some of the NaCl and all of the CaCl 2. Tetrodotoxin (TTX, 1 μm) was employed to block sodium currents. Voltage ramps and steps were used to monitor calcium currents. The ramps were appropriate because calcium currents in bipolar and ganglion cells are fast activating and slow inactivating, as shown in Fig. 2. The open tip resistance of the electrodes was 5 7 M. The access resistance, ranging from 10 to 20 M, produced a voltage error usually less than 5 mv and was not corrected. Electrophysiological data were collected with a List EPC-9 amplifier (HEKA Elektronik, Germany), HEKA Pulse software and a Dell Dimension 8300 computer. The analog signals were filtered at 5 khz. Data were analysed with Igor Pro 5.03 software (WaveMetrics Inc, Lake Oswego, OR, USA), Origin 7.0 (Northampton, MA, USA) and Microsoft Excel software. Student s t test was used for statistical comparisons between cells treated with different internal solutions and second-messenger antagonists. The Wilcoxon paired rank test was used to analyse the paired-pulse experiments because this statistical measure is well suited to evaluation of changes observed in each cell in the population under study. In measuring the effects of glycine on calcium currents, dose response curves for glycine were fitted to the Hill equation: I exp I max = 1 ( 1 + EC 50 [glycine] where I exp is the calcium current suppressed by a given concentration of glycine, I max is the maximal calcium current suppressed, EC 50 is the glycine concentration at which the half-maximal effect was obtained, [glycine] is the glycine concentration and n is the Hill coefficient. Data are expressed as means ± s.e.m.; the error bars in figures represent s.e.m. values. Light stimulation In light response experiments, a full-field red light-emitting diode (LED, 660 nm) and green LED (550 nm) were used to stimulate retinal neurons. The light duration was usually 2 s. For paired-pulse light responses, two 500 ms duration red light stimuli were paired with a 3 s interval. All light-response experiments were performed on a dark background in a light-tight Faraday cage. Drugs Glycine, strychnine, picrotoxin, tetraethylammonium (TEA)-Cl, tetrodotoxin (TTX), 5,7-dichloro-4-hydroxyquinoline-2-carboxylic acid (DCKA), PKA inhibitory fragment 14 22, GF109203X, cyclosporin A, genistein, sarcosine, amoxapine and forskolin were obtained from Sigma Chemical (St Louis, MO, USA). CGP55845, 8,8 -(carbonylbis(imino-3,1-phenylene carbonylimino)bis(1,3,5-naphthalenetrisulphonic acid) (NF023), 8-bromo-cAMP and camp-rp were obtained from Tocris Cookson (Ballwin, MO, USA). GDPβS (1 mm) was placed in pipette internal solution. All secondmessenger antagonists are cell membrane permeable. All other drugs were applied extracellularly, unless specified. A DAD12 air-driven superfusion system (ALA Scientific Instrument, NY, USA) was used in experiments on isolated ) n

3 J Physiol Metabotropic glycine signal 2915 neurons. This provided fast exchange ( 100 ms) over an area of about 300 μm. A gravity-driven superfusion system was used in retinal slice and whole-mount retina experiments. This employed a large-diameter pipette directed at the tissue which required several seconds for drug exchange. Control Ringer solution was applied to the cell or tissue, then exchanged with drugs dissolved in the control Ringer solution, and after an effect was observed the drug was removed by exchange with the control Ringer solution. Results Evidence of a metabotropic glycine action To explore for the possible presence of glycine-activated metabotropic receptors we investigated bipolar and ganglion cells. Both of these cell types express functional GABA and glutamate metabotropic receptors and both cell types receive glycinergic input from amacrine cells (Hartveit et al. 1995; Brandstatter et al. 1996; Koulen et al. 1998; Awatramani & Slaughter, 2001; Shen & Slaughter, 2001). Since many neuronal metabotropic receptors regulate voltage-gated channels (Maguire et al. 1989; Rothe et al. 1994; Pan & Lipton, 1995; Akopian & Witkovsky, 1996; Zhang et al. 1997b; Shen & Slaughter, 1999; Tachibana, 1999; Shen & Jiang, 2007), we tested the effect of glycine on voltage-gated calcium channel currents as shown in Fig. 1. Experiments were performed in the retinal slice preparation and isolated ganglion cells. A voltage ramp ( 100 to +50 mv) was used to activate calcium channel currents, which were isolated by suppressing potassium current with 40 mm TEA and blocking sodium current with 1 μm TTX. In the presence of 10 μm strychnine to block ionotropic glycine receptors, 10 μm glycine (referred to as 10 μm glycine strychnine) suppressed high voltage-activated calcium channel current. This effect was observed in ganglion cells (Fig. 1A) and bipolar cells (Fig. 1D) in Figure 1. Glycine, in the presence of strychnine, suppressed a voltage-dependent inward current in bipolar and ganglion cells in the retinal slice preparation External solution contained 10 mm barium, 40 mm TEA and 1 μm TTX. Inward current was elicited by 2 s voltage ramp from 100 mv to +50 mv. A, a ramp elicited a control voltage-gated inward current in a ganglion cell. Glycine with strychnine (both 10 μm) suppressed the inward current. B, compared to the control trace, 100 μm glycine alone produced an outward current reversing near 70 mv and suppressed the voltage-sensitive inward current in a ganglion cell. C, strychnine (STR, 10 μm) alone (grey trace) had no effect on the inward current (control trace in black). D, in a bipolar cell, 10 μm glycine suppressed the control voltage-dependent inward current in the presence of 10 μm strychnine.

4 2916 M. Hou and others J Physiol the slice preparation. Glycine (10 μm), in the presence of 10 μm strychnine, suppressed 35 ± 2% (n = 63) of calcium channel current in ganglion cells. In an additional 31 ganglion cells glycine plus strychnine had no effect. Strychnine alone did not affect the calcium current (Fig. 1C). In bipolar cells, calcium channel current was suppressed in 7 of 11 cells by 10 μm glycine strychnine. For comparison, the effect of glycine in the absence of strychnine is shown in Fig. 1B. Under these conditions, glycine reduced the voltage-gated calcium current but also produced an outward current that reversed near 70 mv, characteristic of an ionotropic glycine receptor-mediated chloride current. Similarly, in isolated retinal ganglion cells, 10 μm glycine strychnine produced a mean suppression of 24 ± 6% of the voltage-gated calcium current (n = 5, Fig. 2A). Isolated cell experiments indicate that glycine s action on calcium channel current is not due to polysynaptic effects. Collectively, these results demonstrate that glycine can suppress voltage-activated calcium channel currents when ionotropic receptors are fully blocked. To confirm that glycine regulated a calcium current, 50 μm cadmium was applied to block voltage-gated calcium channels. As showed in Fig. 2B, cadmium totally suppressed the inward current in a ganglion cell, evoked by a voltage ramp from 100 to +50 mv (n = 4). The current blocked by cadmium was used as a measure of the full calcium current when determining the fraction of the calcium current suppressed by the putative metabotropic glycine action (Fig. 2B). After pre-treatment with cadmium, glycine was applied with cadmium and only an outward chloride current was observed (compare Figs 1B and 2C). In the presence Figure 2. Glycine inhibition of ganglion cell calcium channel current in isolated neurons and retinal slice preparation in the presence of strychnine External solution contained 10 mm barium, 40 mm TEA and 1 μm TTX. Inward current was elicited by a 2 s voltage ramp from 100 to +50 mv. A, in an isolated third-order neuron, 10 μm glycine strychnine suppressed the control inward current. B, in a ganglion cell in the retinal slice, 10 μm glycine with 10 μm strychnine reduced the control voltage-activated inward current while 50 μm cadmium totally suppressed the inward current elicited by a voltage ramp. C, in the presence of 50 μm cadmium, 10 μm glycine without strychnine produced an outward current that reversed near 70 mv. Addition of 10 μm strychnine blocked this outward current. D, inward calcium currents observed in a ganglion cell that was held at 80 mv then stepped in 10 mv increments from 30 mv to 0 mv.

5 J Physiol Metabotropic glycine signal 2917 of strychnine and cadmium, glycine produced no current (Fig. 2C), indicating that glycine responses were due to a strychnine-sensitive chloride current and a strychnine-insensitive regulation of a calcium current. In most experiments a voltage ramp was used to elicit the calcium current. This is appropriate because the calcium currents investigated were fast activating and slow inactivating (Fig. 2D). Glycine in the presence of 10 μm strychnine suppressed high-voltage activated calcium currents in retinal ganglion cells in a dose-dependent manner, with an EC 50 of 3.3 μm (Fig. 3A). This compares with an EC 50 of 39 μm for the ionotropic glycine receptor in retinal ganglion cells (Wang & Slaughter, 2005). It is commonly observed that metabotropic receptors have a lower EC 50 than ionotropic receptors for the same ligand, suggesting that glycine s suppression of calcium current is mediated through activation of metabotropic receptors. The time course of glycine s action on calcium channel current was measured by using a 50 ms voltage step from 80 mv to 0 mv, repeated every 10s (Fig.3B). Plotting the calcium current with time of glycine exposure in nine cells indicated that 10 μm glycine strychnine suppressed the calcium current with an average time constant of 26.2 ± 3.3 s (Fig. 3C). Glycine-induced chloride currents are much faster, peaking within 1 or 2 s (which represents the delay of the perfusion system). While eliminating the possibility that glycine s action was due to activation of ionotropic glycine-gated chloride channels, there was also the possibility that glycine activates metabotropic GABA receptors, since both GABA and glycine share similar structures containing single carboxyl and amine groups. To test this possibility, we used CGP55845, a potent GABA B receptor (GABA B R) antagonist (Davies et al. 1993) After pre-treatment with 5 μm CGP55845, 10 μm glycine strychnine still reduced the calcium current (Fig. 4A). Overall, in 12 cells tested, the effect of glycine on calcium channel current was unchanged by the presence of this blocker of metabotropic GABA receptors (Fig. 4B). Figure 3. The time course and potency of glycine s action A, the dose-dependent suppression of calcium current by glycine in the presence of 10 μm strychnine was plotted and fitted to Hill equation: I exp = I max /(1 + (EC 50 /[glycine]) n ) where EC 50 is the glycine concentration at which half-maximal suppression by glycine was obtained and n is Hill coefficient. The glycine EC 50 was 3.3 ± 0.3 μm. The fit was based on normalized data from ganglion cells: 1 μm glycine (n = 5), 2 μm glycine (n = 11), 3 μm glycine (n = 10), 5 μm glycine (n = 8), 10 μm glycine (n = 63), 50 μm glycine (n = 19), 100 μm glycine (n = 27), 500 μm glycine (n = 10). B, the calcium current was recorded at intervals of 10 s, during the continuous application of 10 μm glycine with 10 μm strychnine. The currents were evoked by voltage steps from 80 to 0 mv. C, using the protocol in B but measuring the calcium channel current every 5 s, the normalized steady-state calcium currents from 9 ganglion cells were plotted, showing the decline with time. The continous line is a fit to a single exponential curve, yielding a time constant of 26.2 ± 3.3 s for the effect of glycine.

6 2918 M. Hou and others J Physiol The second messenger cascade Many metabotropic neurotransmitter receptors act through a G-protein cascade and are therefore susceptible to block by internal GDPβS. Experiments in both the retinal slice preparation (Fig. 5A) and in isolated ganglion cells (Fig. 5B) indicate that GDPβS suppresses glycine regulation of the calcium current. As summarized in Fig. 5C, inclusion of 1 mm GDPβS in the pipette (n = 12) almost completely eliminated the suppression of voltage-gated calcium current by 10 μm glycine strychnine (Student s two-tail t test: P < 0.01). The suramin analogue NF023, 8,8 -(carbonylbis(imino- 3,1-phenylene carbonylimino) bis(1,3,5-naphthalenetrisulphonic acid), blocks G-protein activation by inhibiting the dissociation of GDP from the G-protein complex (Beindl et al. 1996). Inclusion of 100 μm NF023 in the recording pipette also blocked 10 μm glycine strycnine inhibition of calcium current (n = 6, example shown in Fig. 5D). Thus, agents that disrupt the G-protein transduction pathway suppressed glycine s action. A number of pharmacological agents were tested to explore second messenger cascades associated with glycine s action. Agents that activated the PKA pathway occluded the effects of glycine strychnine while agents that inhibited the PKA pathway mimicked the effects of glycine strychnine. In the presence of 100 μm forskolin, which activates adenylyl cyclase, 10 μm glycine strychnine did not suppress the calcium channel current (grey trace superimposed on control calcium current in black, Fig. 6A). The effect of 10 μm glycine strychnine without forskolin is shown for comparision (n = 9). If the PKA inhibitory fragment (PKI, 20 μm) was included in the pipette (n = 4), then the effect of 10 μm glycine strychnine was blocked (Fig. 6B). Bath application of the PKI fragment (20 μm), which is cell permeable, suppressed the calcium current (labelled black trace) and the suppression exceeded that of 10 μm glycine strychnine (Fig. 6C). PKI also occluded the effect of glycine (grey trace superimposed on black PKI trace in Fig. 6C). In contrast, agents that blocked calcium calmodulin or tyrosine kinase pathways did not significantly affect the action of glycine on calcium channel current (Fig. 6D). To examine the connection between cyclic nucleotide levels and the action of glycine, the effects of 8-bromocAMP and camp-rp were compared. Application of 8-bromo-cAMP reversed the effects of glycine. For example, as shown in Fig. 6E, 10 μm glycine strychnine reduced the calcium current in a ganglion cell, but when 100 μm 8-bromo-cAMP was also added the calcium current was restored. When applied alone, 8-bromo-cAMP produced a small enhancement of the calcium channel current. This suggests that camp augments the calcium channel current and glycine reduces the current by reducing camp. In support of this model, application of the PKA antagonist camp-rp strongly suppressed the calcium current. Its effect on the calcium current was greater than that of glycine (black labelled trace in Fig. 6F) and it occluded the action of glycine (grey trace superimposed on black camp-rp trace). In 10 cells, 50 μm camp-rp reduced the current by 40 ± 3%, compared to the mean glycine suppression of 35 ± 2%. Overall, the results are consistent with a model in which glycine leads to activation of an inhibitory G-protein which suppresses Figure 4. A GABA B R antagonist did not block the inhibitory effect of glycine A, the retinal slice was pretreated with 5 μm CGP55845, a potent GABA B R antagonist. In the presence of 10 μm strychnine and 5 μm CGP55845, 10 μm glycine suppressed the calcium channel current of a ganglion cell in the slice preparation. External solution contained 10 mm barium, 40 mm TEA and 1 μm TTX. Inward current was elicited bya2svoltage ramp from 100 to +50 mv. B, the histogram indicates that 10 μm glycine and 10 μm strychnine suppressed a similar percentage of calcium current with (n = 12) or without (n = 63) 5 μm CGP55845.

7 J Physiol Metabotropic glycine signal 2919 camp levels and consequently PKA activity. The resulting reduction in phosphorylation suppresses calcium channel current. Metabotropic glycine action suppresses bipolar cell synaptic signals The previous results indicate that retinal bipolar cells and ganglion cells possess glycine-activated metabotropic receptors that suppress voltage-gated calcium currents. Therefore, one prediction is that these metabotropic receptors could reduce the excitatory glutamatergic signals from bipolar cells to ganglion cells. To test this, ganglion cells in the retinal slice preparation were voltage clamped to 80 mv. Ionotropic GABA and glycine receptors were blocked with a combination of 50 μm picrotoxin and 10 μm strychnine. The retina was stimulated with red or green light. Under these conditions, the excitatory glutamatergic input from bipolar cells could be observed. Glycine suppressed the light-evoked EPSCs in ganglion cells. The mean suppression of ON EPSCs in response to green light was 43 ± 3% and to red light was 42 ± 5% (n = 36, Fig. 7A). Glycine did not suppress the EPSCs in another 18 ganglion cells. The reduced EPSC is probably due to glycine-mediated suppression of presynaptic calcium channels at the bipolar cell axon terminal. However, it is also possible that glycine suppressed the overall light response of bipolar cells or that it reduced the responsiveness of postsynaptic, ganglion cell glutamate receptors. To examine the first possibility, whole-cell recordings were made from bipolar cells (n = 10). The bipolar cell light responses were not significantly altered by 10 μm glycine strychnine, as exemplified by the recording Figure 5. The glycine response is G-protein coupled Retinal ganglion cells were dialysed with GDPβS in the retinal slice preparation (A) or in isolated cell preparation (B). 1 mm GDPβS was applied through the recording pipette solution. When the cells were treated with GDPβS, 10 μm glycine with 10 μm strychnine (grey traces) did not suppress the control calcium current (black traces). C, a histogram summary of the experiments in the retinal slice shows that GDPβS in the recording pipette (n = 12) significantly blocked the effect of glycine on calcium current. Student s two-tail t test: P < D, another G-protein blocker, NF023 (100 μm), in the pipette blocked the action of glycine (grey trace). External solution contained 10 mm barium, 40 mm TEA and 1 μm TTX. Inward current was elicited by a 2 s voltage ramp from 100 to +50 mv.

8 2920 M. Hou and others J Physiol Figure 6. The second messenger cascade associated with glycine s action Recordings were made from ganglion cells in the retinal slice preparation with an external solution containing 10 mm barium, 40 mm TEA and 1 μm TTX. Inward current was elicited by a 2 s voltage ramp from 100 to +50 mv. A, glycine plus strychnine (both 10 μm) suppressed the control calcium current (black trace), and this effect was reversed by 100 μm forskolin (grey trace that is superimposed on control trace). B, PKA inhibitor (PKI, 20 μm) in the recording pipette solution blocked the action of 10 μm glycine strychnine on calcium current. Black trace is calcium current produced under control conditions, grey trace shows the effect of glycine strychnine. C, glycine plus strychnine (each 10 μm) reduced the control calcium channel current. After washout and return to the control current, application of 20 μm PKI in the bath solution reduced the calcium current (labelled black trace) and occluded the action of 10 μm glycine strychnine (grey trace that superimposes with PKI trace). D, a summary of the effect of second messenger antagonists on action of glycine. Forskolin (n = 9) and PKI (n = 4) blocked the action of glycine (Student s two-tail t test: P < 0.01). Other second messenger antagonists did not significantly affect the

9 J Physiol Metabotropic glycine signal 2921 of an ON bipolar cell shown in Fig. 7B. To test the second possibility, exogenous 100 μm glutamate was applied to ganglion cells before and during treatment with glycine. There was no apparent effect of 10 μm glycine strychnine on the glutamate response in any of the seven cells tested (Fig. 7A, inset). In addition to inhibitory glycine receptors, glycine is also required for the activation of NMDA receptors. In retina, amacrine and ganglion cells (but not bipolar cells) possess NMDA receptors and their glycine recognition sites are not saturated under normal physiological conditions (Taylor et al. 1995; Tian et al. 1998; Stevens et al. 2003). Thus, glycine might stimulate NMDA receptors on amacrine cells leading to a polysynaptic suppression of bipolar cell transmitter release. To evaluate this possibility, glycine s action was tested in the presence of DCKA, a potent inhibitor of the NMDA receptor glycine binding site. Glycine (10 μm), in combination with 10 μm strychnine and 1 μm DCKA, suppressed the calcium current elicited by a voltage ramp (Fig. 7C). Also, 10 μm glycine in the presence of 1 μm DCKA and 10 μm strychnine suppressed ganglion cell light responses (Fig. 7D). We do not have an antagonist with which to evaluate the action of endogenous glycine at the metabotropic receptor. However, glycinergic neurons depend upon extracellular uptake to fill their vesicular stores (Pow, 1998). Therefore, we examined the effect of endogenous glycine in the retinal slice preparation by blocking its uptake with sarcosine and amoxapine. To determine the time course of the action of these inhibitors we measured their effect on the ionotropic glycinergic IPSC in ganglion cells. GABAergic IPSCs were blocked with 50 μm picrotoxin and the ganglion cell was clamped at 10 mv, thus isolating an outward glycinergic chloride current. Under these conditions 100 μm sarcosine and 100 μm amoxapine were applied. At first the IPSC was enhanced, but within 2 min it was strongly suppressed (Fig. 7E). These results could signify that initially synaptic glycine levels were elevated because the uptake systems were blocked, but after a couple of minutes the presynaptic glycine pools became depleted and the glycinergic IPSC diminished. Using this timeframe, we examined the effect of uptake inhibitors on activation of glycine-sensitive metabotropic receptors. This was done by eliciting light-evoked EPSCs while blocking IPSCs with strychnine and picrotoxin (Fig. 7F). At first the EPSC was suppressed (data not shown), but within 2 min the EPSC was enhanced (n = 10). This is opposite to the effect on glycinergic IPSCs. It can be interpreted to indicate that as presynaptic glycine levels decline in amacrine cells, there is a loss of glycinergic metabotropic feedback inhibition to the bipolar cell terminal. Therefore, bipolar cells release more glutamate and the EPSCs in ganglion cells are augmented. To determine if a similar second messenger cascade is operative in regulating bipolar cell transmitter release, the effect of forskolin was tested. Forskolin (100 μm) did not alter the ON or OFF EPSCs in response to red or green light stimuli, but it did eliminate the inhibitory effect of glycine (Fig. 8A). In measurements of the ON response to red or green light in 10 cells, 10 μm glycine strychnine reduced the mean EPSC by 44% and 28%, respectively. In contrast, 100 μm forskolin increased the mean EPSC by 7% and 3% in response to red and green light, respectively (P < 0.005). Furthermore, after the light response was suppressed by 10 μm glycine strychnine, the light-evoked EPSCs were restored by applying forskolin in addition to glycine strychnine (Fig. 8B). An alternative method of evaluating the action of glycine at the bipolar to ganglion cell synapse is to use paired-pulse light stimulation. If the retina is stimulated twice in rapid succession, the second response (P 2 ) of a ganglion cell is smaller than the first (P 1 ). At least part of the reason for the reduction is vesicle depletion at the bipolar to ganglion cell synapse. This can be monitored by comparing the ratio of responses (P 2 /P 1 ). Application of 10 μm glycine strychnine reduced the response to the first stimulus, but had much less effect on the response to the second stimulus (Fig. 8C). Thus, glycine increased the P 2 /P 1 ratio. This suggests that glycine, acting at the bipolar cell synaptic terminal, reduces vesicle depletion during the first stimulus and allows for more release during the second stimulus. Forskolin suppresses the effect of glycine on metabotropic receptors and would be expected to also block glycine s effect on the paired-pulse ratio. Forskolin (100 μm) did block the enhancement of P 2 /P 1 by 10 μm glycine strychnine (Fig. 8C and E), while on its own forskolin did not alter the ratio (Fig. 8D). Taken together, these experiments indicate that glycine-sensitive metabotropic receptors suppress bipolar cell glutamate release. Discussion These experiments demonstrate that glycine activates metabotropic as well as ionotropic receptors. The receptor action of glycine on calcium channel current. E, glycine plus strychnine (both 10 μm) suppressed the calcium current. 8-Bromo-cAMP (100 μm) alone (grey trace) produced a small enhancement of the control calcium channel current (black trace). Concurrent application of 8-bromo-cAMP with 10 μm glycine strychnine prevented most of glycine s effect on the calcium current. F, glycine and strychnine (both 10 μm) suppressed the control calcium current. After washout and recovery, application of 50 μm camp-rp, a PKA antagonist, reduced the calcium current (black trace) and occluded the action of glycine strychnine (grey trace superimposed with the response to camp-rp).

10 2922 M. Hou and others J Physiol Figure 7. Metabotropic glycine receptors suppressed bipolar cell synaptic signalling A, light-evoked EPSCs originating from bipolar cells were recorded in a ganglion cell clamped at 80 mv. Glycine plus strychnine (both 10 μm) suppressed the light-evoked EPSCs in response to green light and red light illumination. Glycine strychnine (10 μm each) did not modify a ganglion cell s response to application of 100 μm glutamate (inset, black trace is the response to glutamate under control conditions, the grey trace is glutamate applied in the presence of 10 μm glycine strychnine). The bar above the trace represents the timing of glutamate application. B, glycine plus strychnine (both 10 μm) did not suppress the light-evoked current in an ON-bipolar cell (black trace is control light response, grey trace is the light response in the presence of 10 μm glycine strychnine). C, control inward calcium current was elicited by voltage ramp from 100 to +50 mv. Bath solution contained 40 mm TEA, 1 μm TTX and 10 mm barium. In the presence of a potent antagonist of glycine binding at the NMDA receptor, DCKA (1 μm), 10 μm glycine strychinine still suppressed the calcium current. D, DCKA (1 μm) did not block the inhibitory action of 10 μm glycine strychnine on light-evoked EPSCs in a ganglion cell. E, glycine uptake inhibitors were used to examine the time course of depletion of presynaptic glycine vesicular pools. A ganglion cell was clamped at 10 mv in the presence of 50 μm picrotoxin. Under these conditions light stimulation elicited a glycinergic IPSC (black trace). After application of 100 μm sarcosine and 100 μm amoxapine for 30 s, the IPSC was enhanced (blue trace). After application of sarcosine and amoxapine for 2 min the IPSC was reduced (red trace). The grey trace indicates the partial recovery from treatment with sarcosine and amoxapine. F, to gauge the effect of endogenous metabotropic GlyRs on bipolar cell output, a ganglion cell s EPSCs were monitored during treatment with sarcosine and amoxapine. The ganglion cell was clamped at 80 mv in the presence of 50 μm picrotoxin and 10 μm strychnine. After application of 100 μm sarcosine and 100 μm amoxapine for 2 min, the inward light-evoked current was enhanced.

11 J Physiol Metabotropic glycine signal 2923 Figure 8. Forskolin blocks the effects of glycine on bipolar cell excitation of ganglion cells A, light-evoked EPSCs were elicited by red and green lights in a ganglion cell clamped at 80 mv (black trace). Forskolin alone did not change the light-evoked EPSCs (grey trace superimposed on control EPSCs). In the presence of 100 μm forskolin, 10 μm glycine strychnine did not suppress light responses in the ganglion cell (second grey trace, also superimposed on control trace). B, using the same experimental procedures as in A, 10 μm glycine strychnine suppressed the light-evoked EPSCs (control light response in black, effect of 10 μm glycine strychnine in grey). Forskolin (100 μm) then reversed the action of glycine strychnine (grey trace superimposed on black control trace). C, glycine changed the response to paired-pulse light stimulation. Light-evoked EPSCs were elicited at light onset in ganglion cells clamped at 80 mv by a pair of 500 ms light stimuli separated by a 3 s interval (black, control response). The second EPSC (P 2 ) was smaller than the first one (P 1 ). Glycine plus strychnine (both 10 μm) suppressed the amplitude of the first EPSC more than the second (grey traces) and increased the P 2 /P 1 ratio. Forskolin (100 μm) reversed the action of glycine strychnine (grey trace superimposed on black control trace). D, application of 100 μm forskolin alone (grey trace) did not change the amplitude of the first and second EPSCs. E, a summary of the effects of 10 μm glycine strychnine on the paired-pulse ratio (P 2 /P 1 ). Glycine (n = 12) enhanced the paired-pulse ratio. Forskolin (n = 7), when co-applied with 10 μm glycine strychnine, recovered the paired-pulse ratio to the control level (Wilcoxon paired rank test: P < 0.01).

12 2924 M. Hou and others J Physiol has a tenfold lower EC 50 than the retinal ionotropic glycine receptor, is G-protein coupled, is mimicked by inhibitors of PKA and overcome by agents that enhance intracellular camp. This suggests a model (Fig. 9) in which stimulation of glycine-sensitive metabotropic receptors activates G i which suppresses adenylyl cyclase, leading to a reduction of PKA-induced enhancement of the calcium channel. This is consistent with numerous studies showing that L-type calcium channel currents are enhanced by protein kinase A (Hille, 1994; Catterall, 2000; Keef et al. 2001). It is not clear, without direct isolation of the gene and the protein, if the metabotropic response is due to a specific glycine receptor. However, several of the results in this study suggest a unique receptor. One is the high sensitivity to glycine, similar to that of other metabotropic receptors in retina. In addition, the most likely alternative is that glycine activates a metabotropic GABA receptor; but the GABA B R mediates an increase in L-type calcium channel current (Shen & Slaughter, 1999). The ineffectiveness of metabotropic GABA antagonists and the opposing effects on calcium current do not support this alternative explanation. Bipolar cells possess T-type and L-type calcium channels (Kaneko et al. 1989; Maguire et al. 1989; Tachibana et al. 1993; Pan, 2000). The calcium channel current regulated by glycine is high-voltage activated, therefore the L-type. Ganglion cells also possess an L-type calcium channel current, but they possess other high-voltage-activated calcium channels (Zhang et al. 1997a; Kamphuis & Hendriksen, 1998; Bieda & Copenhagen, 2004). We have not determined which calcium channel types are suppressed by glycine s metabotropic action in ganglion cells, but it is likely to include the L-type. Bipolar cells receive both GABAergic and glycinergic feedback, acting at ionotropic and metabotropic receptors. The present experiments indicate that metabotropic glycine receptors suppress L-type calcium channels and glutamate release in bipolar cells. We have found that GABA B Rs increase bipolar cell transmitter release (Shen & Slaughter, 1999). Thus, these two metabotropic receptor systems have opposing actions on transmitter release at the bipolar cell axon terminal. Ionotropic GABA and glycine receptors also mediate opposing actions at the bipolar cell terminal because their activations are out of phase. In ON bipolar cells, ionotropic GABA receptors reduce the light-driven output while glycine receptor disinhibition serves to augment the output (Molnar & Werblin, 2007). Ganglion cells also receive both GABAergic and glycinergic amacrine cell innervation from amacrine cells. GABA B Rs mediate an enhancement of L-type calcium channels in ganglion cells (Shen & Slaughter, 2001), while mglyrs may suppress these channels. The balance between these two inhibitory pathways determines the net current through the L-type calcium channel. In third order retinal neurons the L-type calcium current is associated with the BK channel (Mitra & Slaughter, 2002). Thus, glycine suppression of L-type channels in ganglion cells can reduce the inhibitory effect of the BK channels. Activation of glycine-sensitive metabotropic receptors reduces calcium currents in about two-thirds of ganglion cells. It also reduces glutamate release into about two-thirds of the ganglion cells tested. We have not tested whether there is a correlation between the metabotropic glycine-sensitive inputs and the glycine-sensitive ganglion cells, but the similar ratio raises the possibility that there are selective circuits controlled by these receptors. Figure 9. A model of glycine s transduction pathway Glycine activates metabotropic receptors to suppress calcium current through a G-protein-coupled pathway, in which the activation of G i α inhibits adenylyl cyclase. The intracellular camp level and PKA activity decrease. The PKA-induced phosphorylation of the calcium channels is suppressed, thus reducing calcium influx.

13 J Physiol Metabotropic glycine signal 2925 References Akopian A & Witkovsky P (1996). Activation of metabotropic glutamate receptors decreases a high-threshold calcium current in spiking neurons of the Xenopus retina. Vis Neurosci 13, Awatramani GB & Slaughter MM (2001). Intensity-dependent, rapid activation of presynaptic metabotropic glutamate receptors at a central synapse. JNeurosci21, Beindl W, Mitterauer T, Hohenegger M, Ijzerman AP, Nanoff C & Freissmuth M (1996). Inhibition of receptor/g protein coupling by suramin analogues. Mol Pharmacol 50, Bieda MC & Copenhagen DR (2004). N-type and L-type calcium channels mediate glycinergic synaptic inputs to retinal ganglion cells of tiger salamanders. Vis Neurosci 21, Brandstatter JH, Koulen P, Kuhn R, van der Putten H & Wassle H (1996). Compartmental localization of a metabotropic glutamate receptor (mglur7): two different active sites at a retinal synapse. JNeurosci16, Catterall WA (2000). Structure and regulation of voltage-gated Ca 2+ channels. Annu Rev Cell Dev Biol 16, Davies CH, Pozza MF & Collingridge GL (1993). CGP 55845A: a potent antagonist of GABA B receptors in the CA1 region of rat hippocampus. Neuropharmacology 32, Gao F & Wu SM (1998). Characterization of spontaneous inhibitory synaptic currents in salamander retinal ganglion cells. J Neurophysiol 80, Grenningloh G (1987). The strychnine-binding subunit of the glycine receptor shows homology with nicotinic acetylcholine receptors. Nature 328, Hartveit E, Brandstatter JH, Enz R & Wassle H (1995). Expression of the mrna of seven metabotropic glutamate receptors (mglur1 7) in the rat retina. An in situ hybridization study on tissue sections and isolated cells. Eur J Neurosci 7, Hille B (1994). Modulation of ion-channel function by G-protein-coupled receptors. Trends Neurosci 17, Jacoby RA & Wu SM (2001). AMPA-preferring receptors mediate excitatory non-nmda responses of primate retinal ganglion cells. Vis Neurosci 18, Kamphuis W & Hendriksen H (1998). Expression patterns of voltage-dependent calcium channel α 1 subunits (α 1A α 1E ) mrna in rat retina. Brain Res Mol Brain Res 55, Kaneko A, Pinto LH & Tachibana M (1989). Transient calcium current of retinal bipolar cells of the mouse. J Physiol 410, Keef KD, Hume JR & Zhong J (2001). Regulation of cardiac and smooth muscle Ca 2+ channels (CaV1.2a,b) by protein kinases. Am J Physiol Cell Physiol 281, C1743 C1756. Koulen P, Malitschek B, Kuhn R, Bettler B, Wassle H & Brandstatter JH (1998). Presynaptic and postsynaptic localization of GABA B receptors in neurons of the rat retina. Eur J Neurosci 10, Lukasiewicz PD, Maple BR & Werblin FS (1994). A novel GABA receptor on bipolar cell terminals in the tiger salamander retina. JNeurosci14, Lukasiewicz PD, Wilson JA & Lawrence JE (1997). AMPA-preferring receptors mediate excitatory synaptic inputs to retinal ganglion cells. J Neurophysiol 77, Maguire G, Maple B, LukasiewiczP&WerblinF(1989). γ -Aminobutyrate type B receptor modulation of L-type calcium channel current at bipolar cell terminals in the retina of the tiger salamander. ProcNatlAcadSciUSA86, Marc RE (1989). The role of glycine in the mammalian retina. In Progress in Retinal Research, vol. 8, ed. Osborne N, Chader G, pp Pergamon Press, London. Marc RE & Liu WL (1985). (3H) glycine-accumulating neurons of the human retina. J Comp Neurol 232, MitraP&Slaughter MM (2002). Mechanism of generation of spontaneous miniature outward currents (SMOCs) in retinal amacrine cells. J Gen Physiol 119, Molnar A & Werblin F (2007). Inhibitory feedback shapes bipolar cell responses in the rabbit retina. J Neurophysiol 98, Moss SJ & Smart TG (2001). Constructing inhibitory synapses. Nat Rev Neurosci 2, Pan ZH (2000). Differential expression of high- and two types of low-voltage-activated calcium currents in rod and cone bipolar cells of the rat retina. J Neurophysiol 83, Pan ZH & Lipton SA (1995). Multiple GABA receptor subtypes mediate inhibition of calcium influx at rat retinal bipolar cell terminals. JNeurosci15, Pow DV (1998). Transport is the primary determinant of glycine content in retinal neurons. JNeurochem70, Rothe T, BiglV&GrantynR(1994). Potentiating and depressant effects of metabotropic glutamate receptor agonists on high-voltage-activated calcium currents in cultured retinal ganglion neurons from postnatal mice. Pflugers Arch 426, Schofield PR, Darlison MG, Fujita N, Burt DR, Stephenson FA, Rodriguez H et al. (1987). Sequence and functional expression of the GABA A receptor shows a ligand-gated receptor super-family. Nature 328, ShenW&Jiang Z (2007). Characterization of glycinergic synapses in vertebrate retinas. J Biomed Sci 14, ShenW&Slaughter MM (1999). Metabotropic GABA receptors facilitate L-type and inhibit N-type calcium channels in single salamander retinal neurons. J Physiol 516, ShenW&Slaughter MM (2001). Multireceptor GABAergic regulation of synaptic communication in amphibian retina. J Physiol 530, Stevens ER, Esguerra M, Kim PM, Newman EA, Snyder SH, Zahs KR & Miller RF (2003). d-serine and serine racemase are present in the vertebrate retina and contribute to the physiological activation of NMDA receptors. Proc Natl Acad SciUSA100, Tachibana M (1999). Regulation of transmitter release from retinal bipolar cells. Prog Biophys Mol Biol 72, Tachibana M, Okada T, Arimura T, Kobayashi K & Piccolino M (1993). Dihydropyridine-sensitive calcium current mediates neurotransmitter release from bipolar cells of the goldfish retina. JNeurosci13, TaylorWR,ChenE&Copenhagen DR (1995). Characterization of spontaneous excitatory synaptic currents in salamander retinal ganglion cells. J Physiol 486,

14 2926 M. Hou and others J Physiol Tian N, Hwang TN & Copenhagen DR (1998). Analysis of excitatory and inhibitory spontaneous synaptic activity in mouse retinal ganglion cells. J Neurophysiol 80, WangP&Slaughter MM (2005). Effects of GABA receptor antagonists on retinal glycine receptors and on homomeric glycine receptor alpha subunits. J Neurophysiol 93, Wu SM (1987). Synaptic connections between neurons in living slices of the larval tiger salamander retina. J Neurosci Methods 20, Wu SM (1992). Feedback connections and operation of the outer plexiform layer of the retina. Curr Opin Neurobiol 2, Zhang J, Jung CS & Slaughter MM (1997a). Serial inhibitory synapses in retina. Vis Neurosci 14, Zhang J, Li W, Trexler EB & Massey SC (2002). Confocal analysis of reciprocal feedback at rod bipolar terminals in the rabbit retina. JNeurosci22, Zhang J, ShenW&Slaughter MM (1997b). Two metabotropic γ -aminobutyric acid receptors differentially modulate calcium currents in retinal ganglion cells. J Gen Physiol 110, Zhang J, Tian N & Slaughter MM (1998). Neuronal discriminator formed by metabotropic γ -aminobutyric acid receptors. J Neurophysiol 80, Acknowledgements This work was supported by National Eye Institute Grant EY Author s present address M. Hou: Harvard Medical School, Department of Neurobiology, 220 Longwood Ave, Boston, MA 02115, USA.