Dendro-dendritic cholinergic excitation controls dendritic spike initiation in retinal ganglion cells

Abstract

The retina processes visual images to compute features such as the direction of image motion. Starburst amacrine cells (SACs), axonless feed-forward interneurons, are essential components of the retinal direction-selective circuitry. Recent work has highlighted that SAC-mediated dendro-dendritic inhibition controls the action potential output of direction-selective ganglion cells (DSGCs) by vetoing dendritic spike initiation. However, SACs co-release GABA and the excitatory neurotransmitter acetylcholine at dendritic sites. Here we use direct dendritic recordings to show that preferred direction light stimuli evoke SAC-mediated acetylcholine release, which powerfully controls the stimulus sensitivity, receptive field size and action potential output of ON-DSGCs by acting as an excitatory drive for the initiation of dendritic spikes. Consistent with this, paired recordings reveal that the activation of single ON-SACs drove dendritic spike generation, because of predominate cholinergic excitation received on the preferred side of ON-DSGCs. Thus, dendro-dendritic release of neurotransmitters from SACs bi-directionally gate dendritic spike initiation to control the directionally selective action potential output of retinal ganglion cells.

Figure 1. Starburst amacrine cell-mediated cholinergic excitation and GABAergic inhibition.

(a) Reconstruction of a connected pair of ON-SAC (green) and ON-DSGCs (black). (b) Paired recording revealed the obligatory co-release of ACh and GABA from ON-SACs. Overlain voltage traces show the transformation of SAC-driven synaptic excitation of an ON-DSGC to synaptic inhibition when nAChRs were blocked with Mecamylamine (MMA, 10 μM, blue traces; action potentials (APs) have been truncated for clarity). The lower traces show the somatic current-evoked excitation of the ON-SAC. (c) Cumulative probability distributions of the amplitude of each SAC-evoked PSP recorded under the indicated conditions, from the indicated number of paired recordings. (d) In a different pair of ON-SAC and ON-DSGCs net inhibition was generated under control conditions, which was transformed to excitation by the blockade of GABA_A receptors (SR-95531 (GABAzine), 10 μM, red; morphologies shown in a). (e) Cumulative probability distributions of PSP amplitude under the indicated conditions. (f) Onset latency of pharmacologically isolated excitatory and inhibitory PSPs, recorded in GABAzine (n=7) and MMA (n=7), respectively. Onset latency was measured from the onset of the presynaptic driving current to 5% of the peak of the postsynaptic response. (g) Averaged pharmacologically isolated excitatory and inhibitory PSPs, the vertical dashed line indicates the onset time of the presynaptic driving current. (h) Upper overlain traces show the biphasic waveform (sum, black trace) produced by the arithmetic summation of isolated excitatory and inhibitory PSPs. The lower trace shows an averaged PSP recorded under control conditions, note the biphasic waveform.

Figure 2. The impact of ON-SACs aligns with the directional tuning of postsynaptic ON-DSGCs.

(a) Reconstruction of a connected pair of ON-SAC (green) and ON-DSGCs (black), showing the sites (red symbols), and vectorial angle (red arrow, relative to SAC somata) of close dendro-dendritic appositions. Morphologies have been aligned to the preferred direction vector of ON-DSGC light responses (b inset). In all panels the preferred side of the ON-DSGC, the dendritic field first activated by light stimuli moving in the preferred direction, lies above the horizontal dashed line. Note that the SAC somata and all dendro-dendritic close appositions are positioned on the preferred side of the postsynaptic ON-DSGC. (b) Paired recordings from the illustrated cells (a) revealed that activation of the SAC-evoked excitatory PSPs in the ON-DSGC (grey traces, red trace is a digital average). The somatic current-evoked presynaptic SAC voltage responses are shown below (green trace). (c) Reconstruction of an ON-SAC located in the null side of an ON-DSGC. The morphologies have been aligned to the ON-DSGC light response vector (d inset). (d) Paired recordings from the illustrated cells (c) revealed SAC-mediated inhibitory PSPs (grey traces, blue trace is a digital average). (e) Summary of the impact of SACs located on the preferred and null sides of postsynaptic ON-DSGCs. The morphologies of postsynaptic ON-DSGCs are shown as overlain and spatially filtered (50 μm, black) reconstructions registered to the preferred direction of light responses, shown schematically by the yellow light bars. Nested within this field are perimeter representations of SACs (green). Arrows indicate the vector angle of SAC-DSGC close dendro-dendritic appositions coloured according to their postsynaptic impact (red excitatory, blue inhibitory PSPs). Vector length represents 1 contact per μm. All light stimuli, the direction of which are schematically illustrated, were applied under photopic conditions. Stimulus intensity was twice that of background illumination.

Figure 3. Cholinergic signalling powerfully controls the light-evoked action potential output of ON-DSGCs.

(a) Physiological activation of the retinal microcircuit (summarized in the inset schematic) by light bars moved across the receptive field of an ON-DSGCs (inset morphological reconstruction) leads to the generation of powerful action potential (AP) firing when moved in a preferred direction, but sparse AP output when moved in a null direction (control, black traces, APs have been truncated for clarity). The antagonism of nAChRs leads to a reduction of preferred direction light-evoked AP firing and the generation of pure inhibitory responses when light stimuli are moved in the null direction (hexamethonium (Hex); red traces). (b) Peri-stimulus histogram of preferred and null direction light-evoked AP firing under the indicated conditions (bin size 20 μm). (c) Quantification of the reduction of preferred direction light-evoked AP firing by nAChR antagonists (mecamylamine (MMA); blue symbols). (d) Quantification of the voltage integral of median filtered (10 ms) preferred and null direction light responses under the indicated conditions (control versus Hex: preferred: P<0.0001, T=10.19; null: P<0.0001, T=11.47; control versus MMA: preferred: P<0.0001, T=15.86; null: P=0.0038, T=6.02). All light stimuli were applied under photopic conditions. Stimulus intensity was twice that of background illumination.

Figure 4. Selective pre or postsynaptic manipulation of cholinergic signalling controls light responses.

(a) Blockade of acetylcholinesterase activity powerfully augments action potential (AP) firing evoked by preferred and null direction light bars (APs have been truncated for clarity). (b) Peri-stimulus histograms of light-evoked AP firing under the indicated conditions (ambenonium (ABN); bin size 20 μm). (c) Ambenonium augments pharmacologically isolated SAC-mediated excitatory PSPs (recorded in tetrodotoxin (TTX); 1 μM and GABAzine (10 μM)), but not inhibitory PSPs (recorded in TTX and mecamylamine (MMA); 10 μM). (d) Pooled data showing the selective augmentation of the area of excitatory PSPs by the blockade of acetylcholinesterase activity (area measured over a 100 ms time-window). (e) Blockade of the vesicular ACh transporter attenuates preferred direction light-evoked AP output, and unmasks null direction membrane hyperpolarization. (f) Peri-stimulus histograms of light-evoked AP firing under the indicated conditions (vesamicol (VES); bin size 20 μm). (g) Vesamicol attenuates pharmacologically isolated SAC-mediated excitatory PSPs, but not inhibitory PSPs. (h) Pooled data showing the selective reduction of excitatory PSPs by the pharmacological blockade of the vesicular ACh transporter (PSP amplitude has been normalized). All light stimuli were applied under photopic conditions. Stimulus intensity was twice that of background illumination. Data in d,h represent mean±s.e.m.

Figure 5. Cholinergic signalling controls the stimulus sensitivity and dynamic range of ON-DSGCs.

(a,b) Antagonism of nAChRs (hexamethonium (Hex); red symbols and traces) attenuates action potential (AP) firing evoked by preferred direction light bars moved across the receptive field over a wide-range of speeds (a), and at the optimal speed across a range of light intensities (b; speed=0.24 mm/s). (c) Impact of cholinergic signalling on the computation of direction selectivity, and preferred direction light-evoked AP firing over the stimulus range shown in a,b. Note the decreased stimulus sensitivity, attenuation of AP firing rate, but preservation of directional tuning in hexamethonium. All light stimuli were applied under photopic conditions. Stimulus intensity is relative to background illumination.

Figure 6. Cholinergic signalling controls receptive field structure.

(a) The tight spatial relationship between the onset of action potential (AP) firing and the peripheral edge of the dendritic arbour of ON-DSGCs is disrupted by nAChR antagonism (control, black traces; hexamethonium (Hex); 100 μM, red traces, * indicates first AP). The dashed reference line shows the edge of the partially reconstructed dendritic tree. Light stimuli were applied under photopic conditions (stimulus intensity 50% greater than background illumination). (b,c) Dendritic edge aligned peri-stimulus histogram of preferred direction light-evoked AP firing under control (black) and in the presence of nAChR antagonists (red; Hex; 100 μM, bin size 20 μm, photopic 50% stimuli). (d) Cholinergic excitation controls receptive field size across a wide range of light intensities. Graphs summarize the relationship between the mean (±s.e.m.) spatial position of the first AP evoked by preferred direction light stimuli relative to the edge of the preferred dendritic subtree under control (black) and in nAChR antagonists (red). The percentage stimulus intensity, relative to background, is shown above each graph. Each row represents the results from a single cell (>=5 trials under each condition), the grey arrows indicate ON-DSGCs that did not generate AP output in the presence of the nAChR antagonist. (e) Cumulative probability plots of the spatial position of the first AP evoked by preferred direction light stimuli relative to the edge of the preferred dendritic subtree in single trials, data are illustrated over the stimulus intensity range indicated in d.

Figure 7. Cholinergic signalling controls light-evoked dendritic spike initiation.

(a) Reconstruction of an ON-DSGC showing the placement of recording electrodes and the preferred direction movement of a light bar; the blue coloured section of the dendritic tree feeds to the dendritic recording site. (b) The generation of dendritic spikes (dendritic recording (blue traces), positions 1 and 2 in a), and back-propagating action potentials (positions 3 and 4 in a) were attenuated by antagonism of nAChRs (hexamethonium (Hex); 100 μM) when a preferred direction light bar was swept across the receptive field. Traces are aligned to the peak of somatically recorded action potentials (APs). (c) Summary of the reduction of AP output and dendritic spike generation by hexamethonium. (d) Spatial pattern of somato-dendritic spike delay under control (black symbols) and following the blockade of nAChRs (Hex; red symbols). (e) Reconstruction of simultaneously recorded ON-SAC and ON-DSGCs, showing the placement of recording electrodes. (f) ON-DSGC somatic (black traces) and dendritic (blue traces) recordings show that the presynaptic SAC drives dendritic spike generation (reconstruction in a; recordings were made in GABAzine (10 μM); five consecutive SAC-evoked supra-threshold excitatory responses are shown). The lower overlain traces show the dendro-somatic attenuation of a sub-threshold voltage response evoked by SAC activation (green trace). (g) Quantification of the sites of SAC-DSGC close apposition in recorded DSGC dendritic subtrees (left graph, vertical line represents average dendritic recording site; 145±5 μm from the soma; n=5). Note that all detected appositions were distal to dendritic recording sites. The right graph shows that dendritic spikes preceded action potentials in all supra-threshold trials.

Figure 8. Cholinergic signalling drives a cascade of dendritic spike generators.

(a) Dramatic attenuation of light spot-evoked terminal dendritic (marked by *) and large-amplitude dendritic spike generation by the blockade of nAChRs (Hex; 100 μM). The inset shows ON-DSGC morphology, and the placement of recording electrodes and light spot stimuli. (b) Peri-stimulus time histogram of AP firing evoked by dendritic light spots (ON time=0–0.5 s) under the indicated conditions. (c) Amplitude distributions of light spot-evoked dendritic regenerative activity under control (black) and in hexamethonium show the nAChR-mediated control of terminal and large-amplitude dendritic spike generation.

Figure 9. Cholinergic signalling positively gates terminal dendritic spike generation.

(a) Light spot-evoked terminal dendritic (marked by *) and large-amplitude dendritic spike generation were powerfully attenuated by nAChR antagonism (upper traces, hexamethonium; Hex). This reduction could be rescued by pairing light stimuli with sub-threshold dendritic depolarization (lower left traces). The insets show light spot-evoked dendritic spikes (blue traces) and somatically recorded activity (black traces). A section of the ON-DSGC morphology, the placement of electrodes, and the position of the light spot stimuli are also shown. (b) Peri-stimulus time histogram of light spot-evoked (ON time=0–0.5 s) action potential (AP) output under the indicated conditions. (c) Amplitude distribution of dendritically recorded spikes under the indicated conditions. Note that pairing of light spot stimuli with dendritic depolarization increased the generation of both terminal and large-amplitude dendritic spikes. (d) Number of terminal and large-amplitude dendritic spikes evoked by light spot stimuli under the indicated conditions (data represent mean±s.e.m). (e) When presynaptic ACh release was depleted (vesamicol), the pairing of light spot stimuli with the local dendritic iontophoretic delivery of ACh augmented dendritic spike generation. The insets show overlain spikes, a section of the ON-DSGC morphology, the placement of recording and iontophoresis electrodes, and the position of light spot stimuli. (f) Number of terminal and large-amplitude dendritic spikes evoked by light spot stimuli per trial under the indicated conditions (data represent mean±s.e.m).

Figure 10. Schematic summary of the role of SAC-mediated control of active dendritic integration compartments of ON-DSGCs.

(a) SAC-mediated cholinergic excitation and GABAergic inhibition gate terminal dendritic integration to control action potential output under control conditions. (b) In the absence of cholinergic excitation terminal dendritic spike generation is weakly driven by bipolar cell-mediated excitation.