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Retinal sensitivity to a variety of artificial sweeteners was tested by monitoring changes in internal free calcium in isolated retinal neurons using Fluo3. Several ligands, including aspartame and saccharin elevated internal free calcium. The effects of these ligands were mediated by both ligand-gated membrane channels and G-protein coupled receptors. We explored the receptors responsible for this phenomenon. Surprisingly, mRNA for subunits of the sweet taste receptor dimer (T1R2 and T1R3) were found in retina. Interestingly, knockdown of T1R2 reduced the response to saccharin but not aspartame. But TRPV1 channel antagonists suppressed the responses to aspartame. The results indicate that artificial sweeteners can increase internal free calcium in the retinal neurons through multiple pathways. Furthermore, aspartame reduced the b-wave, but not the a-wave, of the electroretinogram, indicating disruption of communication between photoreceptors and second order neurons., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Yang, Myers and Slaughter.)

The skate retina provides a native all-rod retina suited for investigating a single type of photoreceptor regarding its properties and signaling to second order cells. Using the aspartate-induced isolated A-wave of the skate eyecup electroretinogram (ERG), it has been shown that adaptation in rods remains Weber-Fechner-like over a 6-log unit increase in background light intensity. Zinc, which can block calcium channels, has been found in the rod synaptic terminal and the synaptic cleft. Histidine is a zinc chelator. Voltage signals from neurons post-synaptic to rods indicate that histidine increases the dark release of glutamate and increases the horizontal cell light response. In histidine, the A-wave response to various light intensities in the dark-adapted retina increased more than fifty percent, corresponding to the effect on horizontal cells. In the presence of background light, although histidine-treated rod light responses remained Weber-Fechner-like, their increment threshold was raised significantly. This indicates that endogenous zinc feedback serves to increase rod sensitivity in a light-adapted retina, despite a corresponding reduction of threshold sensitivity in the dark. We propose that the increase in A-wave amplitude is a result of the increased conductance at the synaptic terminal and that the A-wave can be used to monitor changes in rod transmitter release. Furthermore, endogenous zinc may also provide the benefit of reducing metabolic stress and the risk of glutamate toxicity in the dark., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Key Points: Starburst amacrine cells release GABA and ACh. This study explores the coordinated function of starburst-mediated cholinergic excitation and GABAergic inhibition to bistratified retinal ganglion cells, predominantly direction-selective ganglion cells (DSGCs). In rat retina, under our recording conditions, starbursts were found to provide the major excitatory drive to a sub-population of ganglion cells whose dendrites co-stratify with starburst dendrites (putative DSGCs). In mouse retina, recordings from genetically identified DSGCs at physiological temperatures reveal that ACh inputs dominate the response to small spot-high contrast light stimuli, with preferential addition of bipolar cell input shifting the balance towards glutamate for larger spot stimuli In addition, starbursts also appear to gate glutamatergic excitation to DSGCs by postsynaptic and possibly presynaptic inhibitory processes ABSTRACT: Starburst amacrine cells release both GABA and ACh, allowing them to simultaneously mediate inhibition and excitation. However, the precise pre- and postsynaptic targets for ACh and GABA remain under intense investigation. Most previous studies have focused on starburst-mediated postsynaptic GABAergic inhibition and its role in the formation of directional selectivity in ganglion cells. However, the significance of postsynaptic cholinergic excitation is only beginning to be appreciated. Here, we found that light-evoked responses measured in bi-stratified rat ganglion cells with dendrites that co-fasciculate with ON and OFF starburst dendrites (putative direction-selective ganglion cells, DSGCs) were abolished by the application of nicotinic receptor antagonists, suggesting ACh could act as the primary source of excitation. Recording from genetically labelled DSGCs in mouse retina at physiological temperatures revealed that cholinergic synaptic inputs dominated the excitation for high contrast stimuli only when the size of the stimulus was small. Canonical glutamatergic inputs mediated by bipolar cells were prominent when GABA/glycine receptors were blocked or when larger spot stimuli were utilized. In mouse DSGCs, bipolar cell excitation could also be unmasked through the activation of mGluR2,3 receptors, which we show suppresses starburst output, suggesting that GABA from starbursts serves to inhibit bipolar cell signals in DSGCs. Taken together, these results suggest that starbursts amplify excitatory signals traversing the retina, endowing DSGCs with the ability to encode fine spatial information without compromising their ability to encode direction., (© 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.)

Ionotropic receptors are tightly regulated by second messenger systems and are often present along with their metabotropic counterparts on a neuron's plasma membrane. This leads to the hypothesis that the two receptor subtypes can interact, and indeed this has been observed in excitatory glutamate and inhibitory GABA receptors. In both systems the metabotropic pathway augments the ionotropic receptor response. However, we have found that the metabotropic GABA B receptor can suppress the ionotropic GABA A receptor current, in both the in vitro mouse retina and in human amygdala membrane fractions. Expression of amygdala membrane microdomains in Xenopus oocytes by microtransplantation produced functional ionotropic and metabotropic GABA receptors. Most GABA A receptors had properties of α -subunit containing receptors, with ~5% having ρ -subunit properties. Only GABA A receptors with α -subunit-like properties were regulated by GABA B receptors. In mouse retinal ganglion cells, where only α -subunit-containing GABA A receptors are expressed, GABA B receptors suppressed GABA A receptor currents. This suppression was blocked by GABA B receptor antagonists, G-protein inhibitors, and GABA B receptor antibodies. Based on the kinetic differences between metabotropic and ionotropic receptors, their interaction would suppress repeated, rapid GABAergic inhibition., (© 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Key Points: GABA is an inhibitory transmitter but can sometimes produce paradoxical excitatory effects through synaptic networks. We found a novel GABA-mediated excitation within a single retinal cell. It involves a chain of events from receptor stimulation to the sequential modulation of two associated channels, resulting in enhanced neuroexcitability. GABAB receptor activation selectively suppresses N-type calcium channels. The BK-type potassium channels are exclusively linked to the N-type calcium channel. Thus, stimulation of GABAB receptors suppresses an outward current, increasing the excitatory range of single neurons., Abstract: GABAB receptors (GABAB Rs) suppress voltage-gated calcium channels and activate G-protein coupled potassium channels (GIRK and TREK channels), both mechanisms serving to inhibit neurons. In isolated rat retinal spiking neurons, GABAB Rs produce both actions but the net effect is to enhance excitatory signals. This is because GABAB Rs selectively suppress N-type calcium channels, which in turn are specifically linked to BK channels. Consequently, when GABAB Rs are stimulated there is a reduction in outward current, allowing neurons to extend their level of depolarization. Whereas many retinal neurons use L-type channels to stimulate vesicle fusion, the suppression of N-type channels augments dynamic range without affecting transmitter release., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)

One general categorization of retinal ganglion cells is to segregate them into tonically or phasically responding neurons, each conveying discrete aspects of the visual scene. Although best identified in the output signals of the retina, this distinction is initiated at the first synapse: between photoreceptors and the dendrites of bipolar cells. In this study we found that the output synapses of bipolar cells also contribute to separate these pathways. Both transient and sustained ganglion cells can produce maintained spike activity, but bipolar cell glutamate release exhibits a divergence that corresponds to the response characteristics of the ganglion cells. Comparing light intensity coding in the sustained and transient ON pathways revealed that they shared the intensity spectrum. The transient pathway had greater sensitivity but smaller dynamic range, and switched from intensity coding to event detection at light levels where sustained pathway sensitivity began to rise. The distinctive properties of the sustained pathway depended upon inhibition and shifted toward those of the transient pathway in the absence of inhibition. The transient system was comparatively unaffected by the loss of inhibition and this was due to the concomitant activation of perisynaptic NMDA receptors. Overall, the properties of bipolar cell dendritic and axon terminals both contribute to the formation of key aspects of the sustained/transient dichotomy normally associated with ganglion cells.

Glutamate release at bipolar to ganglion cell synapses activates NMDA and AMPA/kainic acid (KA) ionotropic glutamate receptors. Their relative strength determines the output signals of the retina. We found that this balance is tightly regulated by presynaptic inhibition that preferentially suppresses NMDA receptor (NMDAR) activation. In transient ON-OFF neurons, block of GABA and glycine feedback enhanced total NMDAR charge by 35-fold in the ON response and 9-fold in the OFF compared with a 1.7-fold enhancement of AMPA/KA receptors. Blocking only glycine receptors enhanced the NMDAR excitatory postsynaptic current 10-fold in the ON and 2-fold in the OFF pathway. Blocking GABA(A) or GABA(C) receptors (GABA(C)Rs or GABA(A)Rs) produced small changes in total NMDAR charge. When both GABA(A)Rs and GABA(C)Rs were blocked, the total NMDAR charge increased ninefold in the ON and fivefold in the OFF pathway. This exposed a strong GABA(C)R feedback to bipolar cells that was suppressed by serial amacrine cell synapses mediated by GABA(A)Rs. The results indicate that NMDAR currents are large but latent, held in check by dual GABA and glycine presynaptic inhibition. One example of this controlled NMDAR activation is the cross talk between ON and OFF pathways. Blocking the ON pathway increased NMDAR relative strength in the OFF pathway. Stimulus prolongation similarly increased the NMDAR relative strength in the OFF response. This NMDAR enhancement was produced by a diminution in GABA and glycine feedback. Thus the retinal network recruits NMDAR pathways through presynaptic disinhibition., (Copyright © 2014 the American Physiological Society.)

Picrotoxin is a pore blocker that can differentiate ligand-gated inhibitory chloride channels. Even within one receptor type, such as the glycine receptor, picrotoxin block differs between subunits. The effect of subunit gating properties on block of the inhibitory glycine receptor (GlyR) was explored using heteromeric α subunit expression in voltage-clamped HEK293 cells. The α2 GlyR is more sensitive to picrotin block than the α1 GlyR, and this difference was used to explore whether mutations that interfered with gating of the α2 subunit would also interfere with picrotin block. Two mutations were used: one that decreased the glycine sensitivity of α2 by almost two log units and the other that was unresponsive to glycine. In both cases, the sensitivity to picrotin was essentially unaltered. The results indicated that α2 subunits can determine the picrotin sensitivity of α1α2-heteromeric receptors and that direct gating of the α2 subunit is not required for this picrotin inhibition.

GABAergic amacrine cell feedback to bipolar cells in retina has been described, activating both GABA(A) and GABA(C) receptors. We explored whether metabotropic GABA(B) receptors also participate in this feedback pathway. CGP55845, a potent GABA(B) receptor antagonist, was employed to determine the endogenous role of these receptors. Ganglion cell EPSCs and IPSCs were monitored to measure the output of bipolar and amacrine cells. Using the tiger salamander slice preparation, we found that GABA(B) receptor pathways regulate bipolar cell release directly and indirectly. In the direct pathway, the GABA(B) receptor antagonist reduces EPSC amplitude, indicating that GABA(B) receptors cause enhanced glutamate release from bipolar cells to one set of ganglion cells. In the indirect pathway, the GABA(B) receptor antagonist reduces EPSC amplitude in another set of ganglion cells. The indirect pathway is only evident when GABA(A) receptors are inhibited, and is blocked by a glycine receptor antagonist. Thus, this second feedback pathway involves direct glycine feedback to the bipolar cell and this glycinergic amacrine cell is suppressed by GABAergic amacrine cells, through both GABA(A) and GABA(B) but not GABA(C) receptors. Overall, GABA(B) receptors do contribute to feedback regulation of bipolar cell transmitter release. However, unlike the ionotropic GABA receptor pathways, the metabotropic GABA receptor pathways act to enhance bipolar cell transmitter release. Furthermore, there are three discrete subsets of bipolar cell output regulated by GABA(B) receptor feedback (direct, indirect and null), implying three distinct, non-overlapping bipolar cell to ganglion cell circuits.

We found that caffeine is a structural analogue of strychnine and a competitive antagonist at ionotropic glycine receptors (GlyRs). Docking simulations indicate that caffeine and strychnine may bind to similar sites at the GlyR. The R131A GlyR mutation, which reduces strychnine antagonism without suppressing activation by glycine, also reduces caffeine antagonism. GlyR subtypes have differing caffeine sensitivity. Tested against the EC(50) of each GlyR subtype, the order of caffeine potency (IC(50)) is: alpha2beta (248 +/- 32 microm) alpha3beta (255 +/- 16 microm) > alpha4beta (517 +/- 50 microm) > alpha1beta(837 +/- 132 microm). However, because the alpha3beta GlyR is more than 3-fold less sensitive to glycine than any of the other GlyR subtypes, this receptor is most effectively blocked by caffeine. The glycine dose-response curves and the effects of caffeine indicate that amphibian retinal ganglion cells do not express a plethora of GlyR subtypes and are dominated by the alpha1beta GlyR. Comparing the effects of caffeine on glycinergic spontaneous and evoked IPSCs indicates that evoked release elevates the glycine concentration at some synapses whereas summation elicits evoked IPSCs at other synapses. Caffeine serves to identify the pharmacophore of strychnine and produces near-complete inhibition of glycine receptors at concentrations commonly employed to stimulate ryanodine receptors.

Accumulating evidence suggests that the side effects of celecoxib, widely used to treat muscle and joint pain, may be mediated in part through cyclooxygenase-2 (COX-2) independent mechanisms, such as inhibition of ion channels. In this study we report effects of celecoxib on ionic currents and neuronal activity in isolated rat retinal neurons. We found that celecoxib suppressed voltage-gated potassium currents in retinal bipolar cells with an effective concentration to inhibit 50% of function (EC(50)) of 5.5 microM. In retinal amacrine and ganglion cells, celecoxib inhibited voltage-dependent sodium channels with an EC(50) of 5.2 microM, and voltage-dependent transient and sustained potassium currents with EC(50)s of 16.3 and 9.1 microM, respectively. Notably, the rate of spontaneous spike activity was dramatically suppressed in ganglion and amacrine cells with an EC(50) of 0.76 microM. All actions of celecoxib on ionic currents and action potentials occurred from the extracellular side and were completely reversible. These findings indicate that inhibition of ion channels by celecoxib in the CNS may affect neuronal function at clinically relevant concentrations.

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 mum, 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.

Retinal rod photoreceptors are depolarized in darkness to approximately -40 mV, a state in which they maintain sustained glutamate release despite low levels of calcium channel activation. Blocking voltage-gated calcium channels or ryanodine receptors (RyRs) at the rod presynaptic terminal suppressed synaptic communication to bipolar cells. Spontaneous synaptic events were also inhibited when either of these pathways was blocked. This indicates that both calcium influx and calcium release from internal stores are required for the normal release of transmitter of the rod. RyR-independent release can be evoked by depolarization of a rod to a supraphysiological potential (-20 mV) that activates a large fraction of voltage-gated channels. However, this calcium channel-mediated release depletes rapidly if RyRs are blocked, indicating that RyRs support prolonged glutamate release. Thus, the rod synapse couples a small transmembrane calcium influx with a RyR-dependent amplification mechanism to support continuous vesicle release.

In the dark-adapted salamander retina, spikes could be elicited from rods under normal physiological conditions. Spike activity was observed in rods during the recovery phase of the response to saturating light. These action potentials were calcium spikes, blocked by cadmium and L-type calcium channel blockers. In response to light stimuli that saturate the rod peak response, calcium action potentials occurred with a delay that depended on light intensity, with stronger light increasing spike latency. Therefore, these spikes encode rod visual information at light intensities beyond rod saturation. Postsynaptic currents of similar time course were observed in second and third order neurones. Since rods exposed to brighter light stimuli produced more delayed spike activity, these signals might contribute to negative afterimages.

Large-conductance calcium-activated potassium (BK) channels are colocalized with calcium channels at sites of exocytosis at the presynaptic terminals throughout the nervous system. It is expected that their activation would provide negative feedback to transmitter release, but the opposite is sometimes observed. Attempts to resolve this apparent paradox based on alterations in action potential waveform have been ambiguous. In an alternative approach, we investigated the influence of this channel on neurotransmitter release in a nonspiking neuron, the salamander rod photoreceptors. Surprisingly, the BK channel facilitates calcium-mediated transmitter release from rods. The two presynaptic channels form a positive coupled loop. Calcium influx activates the BK channel current, leading to potassium efflux that increases the calcium current. The normal physiological voltage range of the rod is well matched to the dynamics of this positive loop. When the rod is further depolarized, then the hyperpolarizing BK channel current exceeds its facilitatory effect, causing truncation of transmitter release. Thus, the calcium channel-BK channel linkage performs two functions at the synapse: nonlinear potentiator and safety brake.

Glycinergic and GABAergic inhibition are juxtaposed at one retinal synaptic layer yet likely perform different functions. These functions have usually been evaluated using receptor antagonists. In examining retinal glycine receptors, we were surprised to find that commonly used concentrations of GABA antagonists blocked significant fractions of the glycine current. In retinal amacrine and ganglion cells, the competitive GABAA receptor antagonists (bicuculline and SR95531) were also competitive GlyR antagonists. Picrotoxinin produced a noncompetitive inhibition of retinal GlyRs. [1,2,5,6-tetrahydropyridine-4-yl] methylphosphinic acid, the GABACR antagonist, did not inhibit glycine receptors. All three GABAA receptor antagonists were competitive inhibitors of homomeric alpha1 or alpha2 GlyRs expressed in human embryonic kidney cells (HEK293) cells. Interestingly, bicuculline was much more effective at alpha2 GlyRs and might be used to separate glycine receptor subtypes. Thus commonly used concentrations of GABA antagonists do not unambiguously differentiate GABA and glycine pathways. Picrotoxinin inhibition of GABAC receptors requires two amino acids in the second transmembrane region (TM2): 2' serine and 6' threonine. Although TM2 regions in GABA and glycine receptors are highly homologous, neither 2' serine nor 6' threonine is essential for picrotoxinin sensitivity in glycine receptors.

Retinal ganglion cells exhibit fast and slow inhibitory synaptic glycine currents that can be selectively inhibited by strychnine and 5,7-dichlorokynurenic acid (DCKA), respectively. In this study we examined whether strychnine and DCKA selectivity correlated with the subunit composition of the glycine receptor. Homomeric alpha1, alpha2 or alpha2* glycine subunits were in vitro expressed in human embryonic kidney cells (HEK 293). In cells expressing the alpha1 subunit, responses to 200 microm glycine were blocked by 1 microm strychnine but not by 500 microm DCKA. In cells expressing the alpha2 subunit, both 1 microm strychnine and 500 microm DCKA were effective antagonists of 200 microm glycine. In cells expressing alpha2* subunits, which are much less glycine-sensitive, 10 mm glycine was inhibited by 500 microm DCKA but not by 1 microm strychnine. A single amino acid mutation in the alpha1 subunit (R196G), converted this subunit from DCKA-insensitive to DCKA-sensitive. In conclusion, the comparative effectiveness of strychnine and DCKA can be used to distinguish between the alpha1, alpha2 and alpha2* receptor responses. Furthermore, a single amino acid near the glycine receptor's putative agonist binding site may account for differences in DCKA sensitivity amongst the alpha subunits.

Glutamate receptor currents were examined in horizontal cells from cultured human retina using whole-cell recording procedures. Horizontal cells possess both AMPA and kainate receptors and both produce significant sustained currents. The kainate-induced current did not show significant desensitization and was not enhanced by concanavalin A. The sustained AMPA current was smaller than the kainate current, but the difference was almost entirely due to pronounced desensitization. The horizontal cell AMPA current was enhanced by cyclothiazide but not by PEPA, indicating the presence of the flip receptor variant. GYKI-52466 blocked the AMPA response (IC50 = 5 microM against 100 microM AMPA) but also blocked the kainate response (IC50 = 45 microM against 100 microM kainate). The diversity of glutamate receptors in human horizontal cells suggests that synaptic input to these neurons may be multiplexed through both kainate and AMPA channels.

Although there are many glutamate receptors in the retina, 2-amino-4-phosphonobutyrate (L-AP4) is an agonist that acts selectively at metabotropic glutamate receptors (mGluR6) of ON bipolar cells. We explored the properties of agonists that activate this receptor. The effects of various glutamate analogs on the b-wave of the electroretinogram (ERG) were used as a measure of their activity. Conformational comparisons among agonists suggest that ligands in an extended conformation preferentially bind to the ON bipolar synaptic receptor. But this property is insufficient to explain the selectivity of mGluR6 because some inactive glutamate analogs could also match this extended conformation. Comparative molecular field analysis (CoMFA) was used to compare the electrostatic and steric potentials of agonists with their action at the ON bipolar synapse. Steric potentials beneath a plane defined by the three putative binding sites plays a key role in determining agonist activity. The CoMFA model was used to predict the activity of glutamate analogs and correlations between predicted and measured activity support the model.

A subtype of retinal amacrine cells displayed a distinctive array of K(+) currents. Spontaneous miniature outward currents (SMOCs) were observed in the narrow voltage range of -60 to -40 mV. Depolarizations above approximately -40 mV were associated with the disappearance of SMOCs and the appearance of transient (I(to)) and sustained (I(so)) outward K(+) currents. I(to) appeared at about -40 mV and its apparent magnitude was biphasic with voltage, whereas I(so) appeared near -30 mV and increased linearly. SMOCs, I(to), and a component of I(so) were Ca(2+) dependent. SMOCs were spike shaped, occurred randomly, and had decay times appreciably longer than the time to peak. In the presence of cadmium or cobalt, SMOCs with pharmacologic properties identical to those seen in normal Ringer's could be generated at voltages of -20 mV and above. Their mean amplitude was Nernstian with respect to [K(+)](ext) and they were blocked by tetraethylammonium. SMOCs were inhibited by iberiotoxin, were insensitive to apamin, and eliminated by nominally Ca(2+)-free solutions, indicative of BK-type Ca(2+)-activated K(+) currents. Dihydropyridine Ca(2+) channel antagonists and agonists decreased and increased SMOC frequencies, respectively. Ca(2+) permeation through the kainic acid receptor had no effect. Blockade of organelle Ca(2+) channels by ryanodine, or intracellular Ca(2+) store depletion with caffeine, eradicated SMOCs. Internal Ca(2+) chelation with 10 mM BAPTA eliminated SMOCs, whereas 10 mM EGTA had no effect. These results suggest a mechanism whereby Ca(2+) influx through L-type Ca(2+) channels and its subsequent amplification by Ca(2+)-induced Ca(2+) release via the ryanodine receptor leads to a localized elevation of internal Ca(2+). This amplified Ca(2+) signal in turn activates BK channels in a discontinuous fashion, resulting in randomly occurring SMOCs.

Spontaneous miniature outward currents (SMOCs) occur in a subset of retinal amacrine cells at membrane potentials between -60 and -40 mV. At more depolarized potentials, a transient outward current (I(to)) appears and SMOCs disappear. Both SMOCs and the I(to) are K(+) currents carried by BK channels. They both arise from Ca(2+) influx through high voltage-activated (HVA) Ca(2+) channels, which stimulates release of internal Ca(2+) from caffeine- and ryanodine-sensitive stores. An increase in Ca(2+) influx resulted in an increase in SMOC frequency, but also led to a decline in SMOC mean amplitude. This reduction showed a temporal dependence: the effect being greater in the latter part of a voltage step. Thus, Ca(2+) influx, although required to generate SMOCs, also produced a negative modulation of their amplitudes. Increasing Ca(2+) influx also led to a decline in the first latency to SMOC occurrence. A combination of these effects resulted in the disappearance of SMOCs, along with the concomitant appearance of the I(to) at high levels of Ca(2+) influx. Therefore, low levels of Ca(2+) influx, arising from low levels of activation of the HVA Ca(2+) channels, produce randomly occurring SMOCs within the range of -60 to -40 mV. Further depolarization leads to greater activation of the HVA Ca(2+) channels, larger Ca(2+) influx, and the disappearance of discontinuous SMOCs, along with the appearance of the I(to). Based on their characteristics, SMOCs in retinal neurons may function as synaptic noise suppressors at quiescent glutamatergic synapses.

Retinal ganglion cells are driven by glutamatergic synapses, but they are also very susceptible to glutamate toxicity. Whereas the conventional excitotoxicity model of glutamate-induced cell death requires membrane depolarization, we have found that glutamate toxicity need not be linked with excitation. A large subset of ganglion cells possesses high-affinity kainate receptors that are calcium permeable. At 1-5 microM, kainate produced elevation of internal calcium but did not significantly depolarize ganglion cells. This low concentration of kainate caused ganglion cell death, which could be inhibited by specific kainate receptor antagonists. The toxic effect of kainate may be associated with calcium influx, because toxicity was reduced by polyamines that suppress calcium influx and by an inhibitor of calcium phosphatase. Thus activation of ionotropic glutamate receptors can produce neurotoxicity uncoupled from neuroexcitation.

The electrical signals resulting from phototransduction are decomposed by bipolar cells and then encoded into spike trains by ganglion cells. The signal decomposition by bipolar cells includes formation of ON and OFF pathways and separation of tonic and phasic signals. The decomposition is accomplished by post-synaptic receptors in the ON and OFF bipolar cells. This chapter focuses on these phenomena in ON bipolar cells and the role of metabotropic glutamate receptors in these processes.

Synaptic signals from retinal bipolar cells were monitored by measuring EPSCs in ganglion cells voltage-clamped at -70 mV. Spontaneous EPSCs were strongly suppressed by l-2-amino-4-phosphonobutyrate (AP-4), an agonist at group III metabotropic glutamate receptors (mGluRs). Agonists of group I or II mGluRs were ineffective. AP-4 also suppressed ganglion cell EPSCs evoked by bipolar cell stimulation using potassium puffs, sucrose puffs, or zaps of current (0.5-1 microA). In addition, AP-4 suppressed Off EPSCs evoked by dim-light stimuli. This indicates that group III mGluRs mediate a direct suppression of bipolar cell transmitter release. An mGluR antagonist, (RS)-alpha-cyclopropyl-4-phosphonophenylyglycine (CPPG), blocked the action of AP-4. When bipolar cells were weakly stimulated, AP-4 produced a large suppression of the EPSC, but CPPG alone had little effect. Conversely, when bipolar cells were strongly stimulated, CPPG produced an enhancement of the EPSC, but AP-4 alone had little effect. This indicates that endogenous feedback regulates bipolar cell transmitter release and that the dynamic range of the presynaptic metabotropic autoreceptor is similar to that of the postsynaptic ionotropic receptor. Furthermore, the feedback is rapid and intensity-dependent. Hence, concomitant activation of presynaptic and postsynaptic glutamate receptors shapes the responses of ganglion cells.

The neuronal generators of the b- and d-waves of the electroretinogram (ERG) were investigated in the tiger salamander retina to determine if amacrine and ganglion cells contribute to this field potential. Several agents were used that affect third-order neurons, such as tetrodotoxin, baclofen, and NMDA agonists and antagonists. Baclofen, an agent that enhances light responses in third-order neurons, increased the d-wave and reduced the b-wave. In contrast, agents that decrease light responses in third-order neurons had the opposite effect of enhancing the b-wave and depressing the d-wave. The effect on the d-wave was particularly pronounced. The results indicate that third-order neuronal activity influences b- and d-waves of the ERG. The opposing actions suggest that the b-wave to d-wave ratio might serve as an measure of ganglion cell function.

The synaptic output of retinal bipolar cells was monitored by recording light-evoked EPSCs in ganglion cells. Application of (RS)-2-amino-3-(3-hydroxy-5-tert-butyl-4-isoxazolyl (ATPA), a selective agonist at kainate receptors, depolarized amacrine cells and reduced the light-evoked excitatory current (L-EPSC) in ganglion cells. ATPA had only a slight effect on the light responses of bipolar cells. Therefore, ATPA suppresses bipolar cell synaptic output to ganglion cells. ATPA reduced the transient L-EPSC, but had comparatively little effect on sustained L-EPSC, of ganglion cells. The transient ON L-EPSC was more suppressed than the transient OFF L-EPSC. Thus, ATPA preferentially suppressed transient output from bipolar cells.GABA receptor antagonists blocked the effect of ATPA. This indicates that ATPA stimulated an endogenous inhibitory feedback pathway that suppressed bipolar cell output.CGP55845 and CGP35348 reduced the ATPA-induced suppression of L-EPSCs in ganglion cells, signifying that part of the feedback pathway is mediated by metabotropic GABA receptors.(1,2,5,6-Tetrahydropyridine-4-yl)-methylphosphinic acid (TPMPA) and picrotoxin, GABAC receptor antagonists, reduced the ATPA effect. Picrotoxin was more effective than ATPA. However, picrotoxin blocked only a part of this GABAC effect, while imidazole-4-acetic acid (I4AA) blocked another segment of the effect. This indicates that two pharmacologically distinct GABAC receptors mediate feedback to bipolar cells. SR95531 produced a very small suppression of the ATPA effect. Thus, GABAA receptors provide a negligible component to this feedback pathway. The experiments indicate that endogenous GABAergic feedback to bipolar cells suppresses their output, and that this feedback is mediated by at least three types of GABA receptor, both metabotropic and ionotropic.In conjunction with previous studies, the results indicate that feedback inhibition is the predominant factor regulating transient signalling in ganglion cells, while feedforward inhibition is the primary regulator of tonic ganglion cell signals.

Phasic and tonic light responses provide a fundamental division of visual information that is thought to originate in the inner retina. However, evidence presented here indicates that this duality originates in the outer retina. In response to a steady light stimulus, the temporal responses of On-bipolar cells fell into two groups. In one group, the light response peaked and then rapidly declined (tau approximately 400 msec) close to the resting membrane potential. At light offset, these cells exhibited a transient afterhyperpolarization. In the second group of On-bipolar cells, the light response declined 10-fold more slowly and reached a steady depolarization that was approximately 40% of the peak response. These neurons had a slowly decaying afterhyperpolarization at light offset. A metabotropic glutamate antagonist, (RS)-alpha-cyclopropyl-4-phosphonophenylyglycine (CPPG), blocked light responses in both types of On-bipolar cell. CPPG only slightly depolarized transient On-bipolar cells, whereas sustained On-bipolar cells were significantly depolarized. Inorganic calcium channel blockers disclosed that these distinct On-bipolar responses were inherent to the bipolar cell and not attributable to synaptic feedback. CPPG had distinct effects on sustained and transient ganglion cells, similar to its action on bipolar cells. The antagonist depolarized and blocked the light responses of sustained ganglion cells. In transient ganglion cells, CPPG suppressed the On light response but did not depolarize the cell or block the Off light response. These results suggest that transient and sustained light responses in ganglion cells result from selective bipolar cell input and that these two fundamental visual channels originate at the dendritic terminals of bipolar cells.

The metabotropic GABA receptor (GABA(B)R) regulates calcium influx in neurons. Whole cell voltage-clamp techniques were employed to determine the effects of internal calcium on the activity of GABA(B)Rs. GABA(B)R receptor apparent affinity was maximal when bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) maintained internal calcium below 70 nM. Apparent affinity was reduced as internal calcium increased. EGTA did not produce similar effects, suggesting that localized increases in calcium influenced GABA(B)R apparent affinity. Confocal imaging disclosed relatively high internal calcium just below the plasma membrane of isolated neurons. BAPTA, but not EGTA, reduced this ring of high calcium. Heparin, dantrolene, and ryanodine increased GABA(B)R apparent affinity, effects similar to that of BAPTA. Calmodulin inhibitors also increased receptor apparent affinity. These results suggest that internally released calcium activates calmodulin, which reduces GABA(B)R apparent affinity. This identifies a reciprocal system in which the metabotropic GABA receptor can reduce calcium influx, but internal calcium can suppress this receptor pathway. Metabotropic glutamate receptors linked to inositol 1,4,5 trisphosphate (InsP(3)) raised internal calcium and suppressed the action of GABA(B)Rs. Thus negative feedback systems control the balance between excitatory and inhibitory metabotropic receptor pathways in retinal neurons.

1. Whole-cell voltage clamp experiments were performed on isolated spiking retinal neurons from the salamander retina. Calcium channel currents were studied using barium as the charge carrier while potassium and sodium currents were suppressed with TEA and TTX, respectively. 2. Baclofen, a metabotropic GABA receptor agonist, both enhanced and suppressed high-voltage-activated calcium channel current. Baclofen facilitated an L-type channel current, and this effect was not voltage dependent. As reported previously, baclofen inhibited an N-type channel current and this action was voltage dependent. 3. While the suppressive effect was mediated by a fast-acting, direct G-protein action, the facilitatory effect was slower and was blocked by inhibitors of protein kinase C (PKC), either GF-109203x or the PKC (19-36) sequence fragment. 4. The pharmacology of the inhibitory and facilitatory responses differed. Commonly used antagonists of metabotropic GABA receptors, CGP35348 and CGP55845, were more potent antagonists of the inhibitory response. Similarly, a selective agonist at the metabotropic GABA receptor, APMPA, was also more effective in eliciting the inhibitory response. 5. These observations indicate that there may be two baclofen-sensitive metabotropic GABA receptors with opposing effects on calcium channel current. This is the first description of a facilitatory action of GABAB receptors and indicates that GABA may not function exclusively as an inhibitory transmitter.

Neuronal discriminator formed by metabotropic gamma-aminobutyric acid receptors. J. Neurophysiol. 80: 3365-3368, 1998. Neurotransmitters function in one of two modes, promoting either inhibition or excitation. However, the metabotropic gamma-aminobutyric acid receptor (GABABR) system can switch between these modes. In the presence of a small excitatory stimulus, the GABABR mediates a shunting inhibition that suppresses excitation. However, in the presence of a strong excitatory stimulus, the GABABR potentiates the response. This bipartite action is accomplished by linking the GABABR to two electrogenic mechanisms; one activates an outward current and another reduces an outward current. As a consequence, the GABABR serves as a discriminator that reduces the influence of weak signals while augmenting responses to strong signals. In retinal ganglion cells, this mechanism acts to promote the communication of phasic information.

1. Glutamate suppressed high-voltage-activated barium currents (IBa, HVA) in tiger salamander retinal ganglion cells. Both ionotropic (iGluR) and metabotropic (mGluR) receptors contributed to this calcium channel inhibition. 2. Trans-ACPD (1-aminocyclopentane-trans-1S,3R-dicarboxylic acid), a broad-spectrum metabotropic glutamate receptor agonist, suppressed a dihydropyridine-sensitive barium current. Kainate, an ionotropic glutamate receptor agonist, reduced an omega-conotoxin GVIA-sensitive current. 3. The relative effectiveness of selective agonists indicated that the predominant metabotropic receptor was the L-2-amino-4-phosphonobutyrate (L-AP4)-sensitive, group III receptor. This receptor reversed the action of forskolin, but this was not responsible for calcium channel suppression. l-AP4 raised internal calcium concentration. Antagonists of phospholipase C, inositol trisphosphate (IP3) receptors and ryanodine receptors inhibited the action of metabotropic agonists, indicating that group III receptor transduction was linked to this pathway. 4. The action of kainate was partially suppressed by BAPTA, by calmodulin antagonists and by blockers of calmodulin-dependent phosphatase. Suppression by kainate of the calcium channel current was more rapid when calcium was the charge carrier, instead of barium. The results indicate that calcium influx through kainate-sensitive glutamate receptors can activate calmodulin, which stimulates phosphatases that may directly suppress voltage-sensitive calcium channels. 5. Thus, ionotropic and metabotropic glutamate receptors inhibit distinct calcium channels. They could act synergistically, since both increase internal calcium. These pathways provide negative feedback that can reduce calcium influx when ganglion cells are depolarized.

1. Protein kinase modulation of glycine-activated currents was examined in acutely dissociated ganglion cells from tiger salamander retina using whole-cell voltage-clamp techniques. 2. Glycine (100 microM) induced an outward chloride current in cells clamped at 0 mV. Co-application of 50 microM forskolin made the glycine-induced current more transient. The combination of forskolin and glycine reduced the later portion of current response without changing the initial peak amplitude. 3. 3-Isobutyl-1-methylxanthine (IBMX) or 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) produced effects similar to those of forskolin. H-89, a protein kinase A (PKA) inhibitor, blocked the effect of forskolin. 4. A protein kinase C (PKC) activator, OAG (1-oleoyl-2-acetyl-sn-glycerol), also made the glycine response more transient. Unlike PKA analogues, OAG enhanced the glycine peak response without changing the glycine late response. OAG effects were blocked by 1 microM GF-109203X, a PKC inhibitor. 5. Nanomolar concentrations of strychnine selectively blocked the fast phase of the glycine current and reversed the effect of OAG, but not that of forskolin. Conversely, forskolin occluded the effect of 5,7-dichlorokynurenic acid, which selectively suppresses the late phase of the glycine current. The action of OAG was not blocked by 5,7-dichlorokynurenic acid. 6. Thus, through a differential modulation, both protein kinase A and C shorten the decay time of the glycine current. PKA suppresses the slow component, while PKC potentiates the fast component.

Metabotropic gamma-aminobutyric acid (GABA) receptors were studied in amphibian retinal ganglion cells using whole cell current and voltage clamp techniques. The aim was to identify the types of receptor present and their mechanisms of action and modulation. Previous results indicated that ganglion cells possess two ionotropic GABA receptors: GABAAR and GABACR. This study demonstrates that they also possess two types of metabotropic GABAB receptor: one sensitive to baclofen and another to cis-aminocrotonic acid (CACA). The effects of these selective agonists were blocked by GDP-beta-S. Baclofen suppressed an omega-conotoxin-GVIA-sensitive barium current, and this action was reversed by prepulse facilitation, indicative of a direct G-protein pathway. The effect of baclofen was also partially occluded by agents that influence the protein kinase A (PKA) pathway. But the effect of PKA activation was unaffected by prepulse facilitation, indicating PKA acted through a parallel pathway. Calmodulin antagonists reduced the action of baclofen, whereas inhibitors of calmodulin phosphatase enhanced it. Antagonists of internal calcium release, such as heparin and ruthenium red; did not affect the baclofen response. Thus, the baclofen-sensitive receptor may respond to influx of calcium. The CACA-sensitive GABA receptor reduced current through dihydropyridine-sensitive channels. Sodium nitroprusside and 8-bromo-cGMP enhanced the action of CACA, indicating that a nitric oxide system can up-regulate this receptor pathway. CACA-sensitive and baclofen-sensitive GABAB receptors reduced spike activity in ganglion cells. Overall, retinal ganglion cells possess four types of GABA receptor, two ionotropic and two metabotropic. Each has a unique electrogenic profile, providing a wide range of neural integration at the final stage of retinal information processing.

Physiological and pharmacological properties of possible subtypes of the native glycine receptor were investigated in retinal neurons using whole-cell voltage-clamp techniques. Two discrete inhibitory glycine responses were identified in ganglion cells. The responses could be distinguished pharmacologically: one was sensitive to strychnine and the other to 5,7-dichlorokynurenic acid. The two responses had different kinetics: the former had a fast onset and fast desensitization, whereas the latter had a slower onset and was much more sustained. The physiological and pharmacological distinctions suggest that the responses are mediated by different receptors. These receptors transduce glycinergic synaptic signals to ganglion cells, where they serve as low- and high-pass filters, respectively, of EPSPs.

Whole-cell voltage clamp in the retinal slice and intracellular current clamp in the intact retina were used to study inhibitory interactions in the inner plexiform layer. Picrotoxin or strychnine reduced inhibitory, light-evoked currents in a majority of ganglion cells. However, in nearly a third of the ganglion cells, each of these antagonists enhanced the inhibitory synaptic current. All inhibitory current was blocked by the addition of the other antagonist. This indicates a cross-inhibition between GABAergic and glycinergic feedforward pathways. Blocking of GABAARs with SR95531 shortened the time course of both excitatory and inhibitory synaptic currents in ganglion cells. Application of picrotoxin, which blocked both GABAARs and GABACRs, produced the opposite effect. Recordings in the intact retina indicated that the light responses of ON bipolar cells, sustained ON, and transient ON-OFF third-order neurons were all made more transient by SR95531 and made more sustained by picrotoxin. The data suggest that a GABAC feedback pathway to bipolar cells makes light responses more phasic and that this feedback is inhibited through a GABAAR pathway. Consequently, the balance between GABAAR and GABACR inhibition regulates the time course of inputs to ganglion cells.

1. Electrophysiological recordings were obtained from neurons in the amphibian intact retina and retinal slice preparations. The effects of gamma-aminobutyric acid (GABA) were evaluated in the presence of bicuculline or SR95531, which block the GABAA receptor, and baclofen, which saturates the GABAB receptor. 2. Under these conditions, GABA preferentially reduced ON light responses in amacrine and ganglion cells, apparently through a presynaptic mechanism that reduced bipolar cell input. GABA also produced a small hyperpolarization in the resting membrane potential of ganglion cells. 3. Picrotoxin blocked these effects of GABA. The action of GABA was duplicated by muscimol and by trans-aminocrotonic acid. Cis-aminocrotonic acid was neither a potent nor selective agonist. This pharmacology is indicative of the GABAC receptor. 4. In voltage-clamp recordings of ganglion cells in the slice preparation, GABA produced a large chloride conductance that was blocked by bicuculline or SR95531, and a smaller chloride conductance that was not blocked by these GABAA receptor antagonists, but was blocked by picrotoxin. This indicates that ganglion cells possess both GABAA and GABAC receptors. 5. The GABAC receptor current was relatively nondesensitized. Consequently, whereas the peak GABAA receptor current was more than fivefold larger than the GABAC receptor current, after desensitization the latter current was larger. Both currents reversed near the chloride equilibrium potential.

Perforated patch-voltage and current-clamp recordings were obtained from ON bipolar cells in the amphibian retinal slice preparation. The currents produced by the photoreceptor transmitter were compared to the currents produced by selective metabotropic glutamate agonists: L-2-amino-4-phosphonobutyrate (L-AP4, APB) and IS,3R 1-amino-1,3 cyclopentanedicarboxylic acid (1S, 3R ACPD). Both agonists produced currents that were very similar to that produced by the photoreceptor transmitter in terms of conductance and reversal potential. The similarities suggest that the metabotropic glutamate receptors are functionally localized to the synaptic region of ON bipolar dendrites. The synaptic conductance rarely exceeded the non-synaptic conductance. The mean input resistance of ON bipolar neurons was 770 M omega in the light and 1.2 G omega in the dark. The average light-regulated synaptic conductance was 57% of the non-synaptic conductance. The L-AP4 regulated conductance averaged 77% of the non-synaptic conductance, while the 1S, 3R ACPD regulated conductance averaged 95% of the non-synaptic conductance. This balance between synaptic and non-synaptic conductance indicates that the synapse will not shunt the cell and the conductance ratio serves to maximize incremental gain at the photoreceptor to ON bipolar synapse. This conductance mechanism makes the ON bipolar cell well equipped to relay rod signals.

2-Amino-4-phosphonobutyrate (APB) is known to selectively suppress the light response of ON bipolar cells in the vertebrate retina, and reduce the b-wave of the electroretinogram (ERG) as a consequence. Using slow drug application, the progressive effect of APB was used to compare the relative response amplitudes of the b-wave and the ON bipolar cell. Simultaneous ERG recordings and ON bipolar intracellular recordings were performed in the amphibian retina. The results indicate that there is a strong positive correlation between these two waveforms. This supports the possibility that the b-wave of the ERG is the direct result of ON bipolar cell activity.

Whole cell voltage and current clamp recordings were obtained from third order neurons isolated from the salamander retina. Using cross desensitization, the structure-function relationship of short chain amino acids on the glycine receptor were examined. L-Serine, L-alanine, beta-alanine and taurine all cross desensitized with glycine, but did not show significant cross desensitization with GABA. This indicates that these amino acids act at the glycine receptor. The order of potency was glycine >> beta-alanine > taurine >> L-alanine > L-serine. TAG, a reputed selective taurine antagonist, was equally effective in blocking taurine and glycine currents. There is no evidence for distinct receptors for taurine. Amino acids with larger moieties at the alpha carbon, such as threonine and valine, produced inactive ligands. Placing a methyl group on the amine of glycine or esterification of the carboxyl group also greatly reduced activity. Based on these modifications of the glycine molecule, it appears that selectivity at the glycine receptor results in part from steric restrictions at all three sites in the glycine chain. The steric interference is most critical at the carboxyl and amino ends, and less limiting at the alpha carbon. Doses of L-serine that had only slight effects in voltage clamp experiments, nevertheless produced large effects in current clamp experiments. This indicates that several endogenous amino acids can have significant effects on membrane voltage, even when their shunting activity may be small. High concentrations of agonists produced desensitization in the voltage clamp records, but there was little evidence of desensitization in the current clamp experiments. These results indicate that several endogenous amino acids can activate the glycine receptor, but there is no evidence for a discrete receptor for taurine, beta-alanine, L-alanine or L-serine. Since all these endogenous amino acids have similar amino and acid terminals, reduction in potency results from steric interference around the alpha carbon. This graded potency may have functional significance in mediating inhibition.

Amacrine and ganglion cells in the amphibian retina contain GABAB, as well as GABAA, receptors. Baclofen, a GABAB agonist, hyperpolarizes the dark membrane potential of these third order neurons and makes their light responses more transient. GABAB receptors in the retina have a similar agonist profile to GABAB receptors described at other sites in the brain. Namely, preferential activation by the R-enantiomer of baclofen, and agonist sensitivity in the order 3-aminopropylphosphinic acid > baclofen >> 3-aminopropylphosphonic acid. The GABAB receptor was not activated by 4-aminobutylphosphonic acid. Several antagonists, such as phaclofen, saclofen, and 2-hydroxysaclofen, were ineffective in the amphibian retina. However, CGP35348 blocked the action of applied baclofen and produced effects on the light response that were opposite to those of baclofen. Applied agonists and antagonists support the hypothesis that GABAB receptors serve to regulate the balance of sustained and transient signals to the inner retina.

1. We performed current-clamp and voltage-clamp experiments in the amphibian retina to examine the effects of 1-amino-1,3-cyclopentanedicarboxylic acid (1S,3R ACPD), which is a selective agonist for the family of metabotropic glutamate receptors. 2. 1S,3R ACPD was found to selectively block the light responses of ON bipolar cells. It did not suppress the responses of horizontal cells of OFF bipolar cells. It blocked ON but not OFF responses of third-order retinal neurons. 3. 1S,3R ACPD mimicked the effect of the photoreceptor transmitter at the ON bipolar synapse. It reduced an inward current by a decrease in conductance. 4. The action of 1S,3R ACPD was very similar to that of 2-amino-4-phosphonobutyrate (APB) both in terms of effects on the ON bipolar cell potential and conductance and in terms of the retinal network. This suggests that the APB receptor is a predominant synaptic metabotropic glutamate receptor in the retina. 5. The rank-order potency at the retinal APB receptor is APB > 1S,3R ACPD > ibotenate. Quisqualate appears to be inactive at this receptor. The pharmacology of the retinal APB receptor matches that of the cloned mGluR4 and mGluR6 metabotropic glutamate receptors. On the basis of the in situ localization of mGluR6 to the inner nuclear layer of the retina, the retinal APB receptor may be this cloned receptor protein. 6. The effects of the three other ACPD stereo isomers were examined. 1S,3S ACPD was a weak agonist at the APB receptor. 1R,3R ACPD was a potent agonist in the inner retina, but inactive in the outer retina. This fits the profile of N-methyl-D-aspartate agonists. 1R,3S ACPD was inactive.

The b-wave of the electroretinogram (ERG) is believed to result largely from the light responses of ON bipolar neurons. One apparent inconsistency is that the b-wave is a phasic signal, more similar to ON responses of transient third-order neurons than to the ON bipolar light responses. However, when selective glutamate agonists or antagonists were used to isolate the b-wave, it was found to be tonic and resemble the light response waveform of the ON bipolar cell.

Writing a chapter on retinal GABAB receptors is premature, as evidenced by the paucity of citations more than two years old. Despite that, this area of retinal pharmacology has made significant strides and, although it is a story without an ending, it has had an exciting beginning. To date, the experiments indicate that horizontal cell feedback to cones is mediated, at least in part, by the GABAB receptor system which probably regulates a potassium conductance. In the inner retina, GABAB receptors are found on bipolar cells, amacrines, and ganglion cells. Here, the actions are a subtle regulation of channel conductance, but the effects are a dramatic reorganization of a fundamental coding property of the retina, namely the distinction between tonic and phasic responses to light. In both the distal and proximal retina, the GABAB receptor does not appear to work alone, but instead works in concert with the GABAA receptor. The full significance of these interactions has yet to be determined. Although the discovery of the GABAB receptor has led to the resolution of several retinal mysteries, the case is far from closed. At this juncture, what can be said is that the GABAB receptor represents a unique and ubiquitous system that reveals the power of regulating calcium and potassium conductances, as opposed to the more familiar properties of the glutamate/acetylcholine regulated cationic conductances or the GABAA/glycine controlled chloride channels.

1. The effects of inhibitory amino acid transmitters on horizontal cells in the superfused amphibian retina were studied by the use of conventional intracellular recording techniques. 2. Gamma-aminobutyric acid (GABA) caused a calcium-independent depolarization of horizontal cells in mud puppy and tiger salamander. This action was mimicked by muscimol but not baclofen (BAC) and blocked by bicuculline and picrotoxin (PTX), matching the GABAa receptor profile. 3. The purported GABA uptake inhibitors nipecotate (NPA) and guvacine (GUV) acted as GABAa agonists, having pharmacological properties very similar to GABA itself. These agents also activated receptors of amacrine and ganglion cells, causing membrane polarizations similar to GABA. Concentrations of these analogues that did not activate the GABAa receptor (submillimolar) did not lower the effective dose of GABA, even after prolonged application. 4. Glycine (GLY) also depolarized horizontal cells, but only in approximately 25% of the horizontal cells was the amplitude of the depolarization as great as GABA. The glycine response was blocked by both strychnine (STR, 10 microM) and PTX (100 microM). In contrast, the action of GABA was unaffected by STR. 5. Ion substitution and channel-blocking agents indicated that the effects of applied GABA and GLY were independent of both external sodium and calcium. 6. The results suggest that GABA receptors on horizontal cells may act 1) as a positive feedback system to modulate the light response and 2) as a mechanism for chemical coupling between horizontal cells.