Malcolm M. Slaughter

Excitatory amino acid receptors of the retina: diversity of subtypes and conductance mechanisms

Abstract Excitatory amino acid (EAA) receptors underlie major pathways of synaptic communication in the vertebrate retina, including neurotransmission from photoreceptors in the outer plexiform layer (OPL), and from bipolar cells and perhaps some amacrines in the inner plexiform layer (IPL) 1 . Pharmacological studies, combined with intracellular electrophysiological recordings, have provided new insights into EAA receptor subtypes associated with identified neurons. The results obtained have established the retina as a model system for studying both the diverse nature of EAA receptors on different identified neurons and the unique conductance mechanisms that underlie the separation of ON and OFF channels within the OPL. There is now strong evidence for an EAA receptor which, when activated, hyperpolarizes the cell by closing ionic channels 2,3 , and for a new receptor subtype in the carp retina 4,5 , which hyperpolarizes by opening channels to either K + or Cl − ions. This brief review describes the retinal pathways that utilize EAA and the evidence for diversity in pharmacology and ionic mechanisms.

Serial inhibitory synapses in retina

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.

Intensity-Dependent, Rapid Activation of Presynaptic Metabotropic Glutamate Receptors at a Central Synapse

Synaptic signals from retinal bipolar cells were monitored by measuring EPSCs in ganglion cells voltage-clamped at −70 mV. Spontaneous EPSCs were strongly suppressed byl-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 μA). 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)-α-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.

Synaptic Transmission Mediated by Internal Calcium Stores in Rod Photoreceptors

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.

Large-conductance calcium-activated potassium channels facilitate transmitter release in salamander rod synapse.

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.

Metabotropic and ionotropic glutamate receptors regulate calcium channel currents in salamander retinal ganglion cells

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 ω‐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.

Metabotropic GABA receptors facilitate L-type and inhibit N-type calcium channels in single salamander 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.

Amacrine and ganglion cell contributions to the electroretinogram in amphibian retina.

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.

Multireceptor GABAergic regulation of synaptic communication in amphibian retina

1 The synaptic output of retinal bipolar cells was monitored by recording light‐evoked EPSCs in ganglion cells. 2 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. 3 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. 4 GABA receptor antagonists blocked the effect of ATPA. This indicates that ATPA stimulated an endogenous inhibitory feedback pathway that suppressed bipolar cell output. 5 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. 6 (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. 7 SR95531 produced a very small suppression of the ATPA effect. Thus, GABAA receptors provide a negligible component to this feedback pathway. 8 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. 9 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.

GABAb receptors in the vertebrate retina

Abstract The experiments summarized here indicate that baclofen affects almost all classes of retinal neuron. Photoreceptors, bipolar cells and ganglion cells all possess voltage-dependent calcium channels which are down-regulated when GABAb receptors are activated. This has ramifications for transmitter release, as well as for the movement of other ions through calcium-dependent channels. In both photoreceptor cones and bipolar cells in the amphibian retina, only about 20% of the cells possess putative GABAb receptors. Thus, these receptors modulate very specific visual pathways. In amacrine and ganglion cells, there also appear to be potassium channels activated by GABAb receptors. These receptors are more ubiquitous, almost all third-order cells are affected by GABAb agonists. Under standard experimental conditions, only a small portion of the available GABAb receptor population is synaptically regulated. Whether this represents a potential that can be stimulated under unique circumstances, or whether this reserve simply reflects non-synaptic receptors, is uncertain. GABAb receptors clearly modulate the balance between sustained and transient signals in the retina. This has been observed in the calcium channel kinetics of goldfish ganglion cells, in the light responses of amacrine and ganglion cells and in the feedback pathway to bipolar cells. The functional significance of this transition is uncertain, although it has been incorporated into models of directional selectivity and selective attention. The models are intriguing but as yet unsubstantiated. The GABAb receptor is finding a place within an integrative view of GABA function. There are several GABA receptor classes in the retina. Each cell type discussed in this review has at least two of these receptors. They may work in parallel, for example GABAb receptors may suppress presynaptic transmitter release while GABAa receptors shunt the postsynaptic neurons ability to respond. Or they may work serially, for example low extracellular GABA concentrations may only activate GABAb receptors, thereby reducing sustained, but not transient, light responses. Higher GABA concentrations would activate the GABAa receptor, providing a powerful shunt which would suppress all light responses. The actual significance of GABA receptor diversity will no doubt be much richer than this simple introduction, but this retinal receptor is clearly a work in progress.