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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.)

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.

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.

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 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.

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.

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.