Neural Circuits Underlying Multifeature Extraction in the Retina.

Classic ON-OFF direction-selective ganglion cells (DSGCs) that encode the four cardinal directions were recently shown to also be orientation-selective. To clarify the mechanisms underlying orientation selectivity, we employed a variety of electrophysiological, optogenetic, and gene knock-out strategies to test the relative contributions of glutamate, GABA, and acetylcholine (ACh) input that are known to drive DSGCs, in male and female mouse retinas. Extracellular spike recordings revealed that DSGCs respond preferentially to either vertical or horizontal bars, those that are perpendicular to their preferred-null motion axes. By contrast, the glutamate input to all four DSGC types measured using whole-cell patch-clamp techniques was found to be tuned along the vertical axis. Tuned glutamatergic excitation was heavily reliant on type 5A bipolar cells, which appear to be electrically coupled via connexin 36 containing gap junctions to the vertically oriented processes of wide-field amacrine cells. Vertically tuned inputs are transformed by the GABAergic/cholinergic "starburst" amacrine cells (SACs), which are critical components of the direction-selective circuit, into distinct patterns of inhibition and excitation. Feed-forward SAC inhibition appears to "veto" preferred orientation glutamate excitation in dorsal/ventral (but not nasal/temporal) coding DSGCs "flipping" their orientation tuning by 90° and accounts for the apparent mismatch between glutamate input tuning and the DSGC's spiking response. Together, these results reveal how two distinct synaptic motifs interact to generate complex feature selectivity, shedding light on the intricate circuitry that underlies visual processing in the retina.

Cholinergic excitation complements glutamate in coding visual information in retinal ganglion cells.

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.

Dynamic tuning of electrical and chemical synaptic transmission in a network of motion coding retinal neurons.

Recently, we demonstrated that gap junction coupling in the population of superior coding ON-OFF directionally selective ganglion cells (DSGCs) genetically labeled in the Hb9::eGFP mouse retina allows the passage of lateral anticipatory signals that help track moving stimuli. Here, we examine the properties of gap junctions in the DSGC network, and address how interactions between electrical and chemical synapses and intrinsic membrane properties contribute to the dynamic tuning of lateral anticipatory signals. When DSGC subtypes coding all four cardinal directions were individually loaded with the gap junction-permeable tracer Neurobiotin, only superior coding DSGCs exhibited homologous coupling. Consistent with these anatomical findings, gap junction-dependent feedback spikelets were only observed in Hb9(+) DSGCs. Recordings from pairs of neighboring Hb9(+) DSGCs revealed that coupling was reciprocal, non-inactivating, and relatively weak, and provided a substrate for an extensive subthreshold excitatory receptive field around each cell. This subthreshold activity appeared to boost coincident light-driven chemical synaptic responses. However, during responses to moving stimuli, gap junction-mediated boosting appeared to be dynamically modulated such that upstream DSGCs primed downstream cells, but not vice versa, giving rise to highly skewed responses in individual cells. We show that the asymmetry in priming arises from a combination of spatially offset GABAergic inhibition and activity-dependent changes in intrinsic membrane properties of DSGCs. Thus, dynamic interactions between electrical and chemical synapses and intrinsic membrane properties allow the network of DSGCs to propagate anticipatory responses most effectively along their preferred direction without leading to runaway excitation.

Hierarchical retinal computations rely on hybrid chemical-electrical signaling.

Bipolar cells (BCs) are integral to the retinal circuits that extract diverse features from the visual environment. They bridge photoreceptors to ganglion cells, the source of retinal output. Understanding how such circuits encode visual features requires an accounting of the mechanisms that control glutamate release from bipolar cell axons. Here, we demonstrate orientation selectivity in a specific genetically identifiable type of mouse bipolar cell-type 5A (BC5A). Their synaptic terminals respond best when stimulated with vertical bars that are far larger than their dendritic fields. We provide evidence that this selectivity involves enhanced excitation for vertical stimuli that requires gap junctional coupling through connexin36. We also show that this orientation selectivity is detectable postsynaptically in direction-selective ganglion cells, which were not previously thought to be selective for orientation. Together, these results demonstrate how multiple features are extracted by a single hierarchical network, engaging distinct electrical and chemical synaptic pathways.

An intrinsic neural oscillator in the degenerating mouse retina.

The loss of photoreceptors during retinal degeneration (RD) is known to lead to an increase in basal activity in remnant neural networks. To identify the source of activity, we combined two-photon imaging with patch-clamp techniques to examine the physiological properties of morphologically identified retinal neurons in a mouse model of RD (rd1). Analysis of activity in rd1 ganglion cells revealed sustained oscillatory (∼10 Hz) synaptic activity in ∼30% of all classes of cells. Oscillatory activity persisted after putative inputs from residual photoreceptor, rod bipolar cell, and inhibitory amacrine cell synapses were pharmacologically blocked, suggesting that presynaptic cone bipolar cells were intrinsically active. Examination of presynaptic rd1 ON and OFF bipolar cells indicated that they rested at relatively negative potentials (less than -50 mV). However, in approximately half the cone bipolar cells, low-amplitude membrane oscillation (∼5 mV, ∼10 Hz) were apparent. Such oscillations were also observed in AII amacrine cells. Oscillations in ON cone bipolar and AII amacrine cells exhibited a weak apparent voltage dependence and were resistant to blockade of synaptic receptors, suggesting that, as in wild-type retina, they form an electrically coupled network. In addition, oscillations were insensitive to blockers of voltage-gated Ca(2+) channels (0.5 mm Cd(2+) and 0.5 mm Ni(2+)), ruling out known mechanisms that underlie oscillatory behavior in bipolar cells. Together, these results indicate that an electrically coupled network of ON cone bipolar/AII amacrine cells constitutes an intrinsic oscillator in the rd1 retina that is likely to drive synaptic activity in downstream circuits.

Vsx1 regulates terminal differentiation of type 7 ON bipolar cells.

Although retinal bipolar cells represent a morphologically well defined population of retinal interneurons, very little is known about the developmental mechanisms that regulate their processing. Furthermore, the identity of specific bipolar cell types that function in distinct visual circuits remains poorly understood. Here, we show that the homeobox gene Vsx1 is expressed in Type 7 ON bipolar cells. In the absence of Vsx1, Type 7 bipolar cells exhibit proper morphological specification but show defects in terminal gene expression. Vsx1 is required for the repression of bipolar cell-specific markers, including Calcium-binding protein 5 and Chx10. This contrasts its genetic requirement as an activator of gene expression in OFF bipolar cells. To assess possible ON signaling defects in Vsx1-null mice, we recorded specifically from ON-OFF directionally selective ganglion cells (DSGCs), which cofasciculate with Type 7 bipolar cell terminals. Vsx1-null ON-OFF DSGCs received more sustained excitatory synaptic input, possibly due to Type 7 bipolar cell defects. Interestingly, in Vsx1-null mice, the directionally selective circuit is functional but compromised. Together, these findings indicate that Vsx1 regulates terminal gene expression in Type 7 bipolar cells and is necessary for proper ON visual signaling within a directionally selective circuit.

Intrinsic oscillatory activity arising within the electrically coupled AII amacrine-ON cone bipolar cell network is driven by voltage-gated Na+ channels.

In the rd1 mouse model for retinal degeneration, the loss of photoreceptors results in oscillatory activity (∼10­20 Hz) within the remnant electrically coupled network of retinal ON cone bipolar and AII amacrine cells. We tested the role of hyperpolarization-activated currents (I(h)), voltage-gated Na(+) channels and gap junctions in mediating such oscillatory activity. Blocking I(h) (1 mm Cs(+)) hyperpolarized the network and augmented activity, while antagonizing voltage-dependent Na(+) channels (1 µm TTX) abolished oscillatory activity in the AII amacrine-ON cone bipolar cell network. Voltage-gated Na(+) channels were only observed in AII amacrine cells, implicating these cells as major drivers of activity. Pharmacologically uncoupling the network (200 µm meclofenamic acid (MFA)) blocked oscillations in all cells indicating that Na(+) channels exert their influence over multiple cell types within the network. In wt retina, occluding photoreceptor inputs to bipolar cells (10 µm NBQX and 50 µm l-AP4) resulted in a mild (∼10 mV) hyperpolarization and the induction of oscillatory activity within the AII amacrine-ON cone bipolar cell network. These oscillations had similar properties to those observed in rd1 retina, suggesting that no major degeneration-induced network rewiring is required to trigger spontaneous oscillations. Finally, we constructed a simplified computational model that exhibited Na(+) channel-dependent network oscillations. In this model, mild heterogeneities in channel densities between individual neurons reproduced our experimental findings. These results indicate that TTX-sensitive Na(+) channels in AII amacrine cells trigger degeneration-induced network oscillations, which provide a persistent synaptic drive to downstream remnant neurons, thus appearing to replace photoreceptors as the principal drivers of retinal activity.

Genetically timed, activity-sensor and rainbow transsynaptic viral tools.

We developed retrograde, transsynaptic pseudorabies viruses (PRVs) with genetically encoded activity sensors that optically report the activity of connected neurons among spatially intermingled neurons in the brain. Next we engineered PRVs to express two differentially colored fluorescent proteins in a time-shifted manner to define a time period early after infection to investigate neural activity. Finally we used multiple-colored PRVs to differentiate and dissect the complex architecture of brain regions.

Light-activated channels targeted to ON bipolar cells restore visual function in retinal degeneration.

Genetically encoded optical neuromodulators create an opportunity for circuit-specific intervention in neurological diseases. One of the diseases most amenable to this approach is retinal degeneration, where the loss of photoreceptors leads to complete blindness. To restore photosensitivity, we genetically targeted a light-activated cation channel, channelrhodopsin-2, to second-order neurons, ON bipolar cells, of degenerated retinas in vivo in the Pde6b(rd1) (also known as rd1) mouse model. In the absence of 'classical' photoreceptors, we found that ON bipolar cells that were engineered to be photosensitive induced light-evoked spiking activity in ganglion cells. The rescue of light sensitivity was selective to the ON circuits that would naturally respond to increases in brightness. Despite degeneration of the outer retina, our intervention restored transient responses and center-surround organization of ganglion cells. The resulting signals were relayed to the visual cortex and were sufficient for the animals to successfully perform optomotor behavioral tasks.

Parallel processing in active dendrites during periods of intense spiking activity.

A neuron's ability to perform parallel computations throughout its dendritic arbor substantially improves its computational capacity. However, during natural patterns of activity, the degree to which computations remain compartmentalized, especially in neurons with active dendritic trees, is not clear. Here, we examine how the direction of moving objects is computed across the bistratified dendritic arbors of ON-OFF direction-selective ganglion cells (DSGCs) in the mouse retina. We find that although local synaptic signals propagate efficiently throughout their dendritic trees, direction-selective computations in one part of the dendritic arbor have little effect on those being made elsewhere. Independent dendritic processing allows DSGCs to compute the direction of moving objects multiple times as they traverse their receptive fields, enabling them to rapidly detect changes in motion direction on a sub-receptive-field basis. These results demonstrate that the parallel processing capacity of neurons can be maintained even during periods of intense synaptic activity.

Gain control by sparse, ultra-slow glycinergic synapses.

In the retina, ON starburst amacrine cells (SACs) play a crucial role in the direction-selective circuit, but the sources of inhibition that shape their response properties remain unclear. Previous studies demonstrate that ∼95% of their inhibitory synapses are GABAergic, yet we find that the light-evoked inhibitory currents measured in SACs are predominantly glycinergic. Glycinergic inhibition is extremely slow, relying on non-canonical glycine receptors containing α4 subunits, and is driven by both the ON and OFF retinal pathways. These attributes enable glycine inputs to summate and effectively control the output gain of SACs, expanding the range over which they compute direction. Serial electron microscopic reconstructions reveal three specific types of ON and OFF narrow-field amacrine cells as the presumptive sources of glycinergic inhibition. Together, these results establish an unexpected role for specific glycinergic amacrine cells in the retinal computation of stimulus direction by SACs.

Spatiotemporal properties of glutamate input support direction selectivity in the dendrites of retinal starburst amacrine cells.

The asymmetric summation of kinetically distinct glutamate inputs across the dendrites of retinal 'starburst' amacrine cells is one of the several mechanisms that have been proposed to underlie their direction-selective properties, but experimentally verifying input kinetics has been a challenge. Here, we used two-photon glutamate sensor (iGluSnFR) imaging to directly measure the input kinetics across individual starburst dendrites. We found that signals measured from proximal dendrites were relatively sustained compared to those measured from distal dendrites. These differences were observed across a range of stimulus sizes and appeared to be shaped mainly by excitatory rather than inhibitory network interactions. Temporal deconvolution analysis suggests that the steady-state vesicle release rate was ~3 times larger at proximal sites compared to distal sites. Using a connectomics-inspired computational model, we demonstrate that input kinetics play an important role in shaping direction selectivity at low stimulus velocities. Taken together, these results provide direct support for the 'space-time wiring' model for direction selectivity.

Rapid multi-directed cholinergic transmission in the central nervous system.

In many parts of the central nervous system, including the retina, it is unclear whether cholinergic transmission is mediated by rapid, point-to-point synaptic mechanisms, or slower, broad-scale 'non-synaptic' mechanisms. Here, we characterized the ultrastructural features of cholinergic connections between direction-selective starburst amacrine cells and downstream ganglion cells in an existing serial electron microscopy data set, as well as their functional properties using electrophysiology and two-photon acetylcholine (ACh) imaging. Correlative results demonstrate that a 'tripartite' structure facilitates a 'multi-directed' form of transmission, in which ACh released from a single vesicle rapidly (~1 ms) co-activates receptors expressed in multiple neurons located within ~1 µm of the release site. Cholinergic signals are direction-selective at a local, but not global scale, and facilitate the transfer of information from starburst to ganglion cell dendrites. These results suggest a distinct operational framework for cholinergic signaling that bears the hallmarks of synaptic and non-synaptic forms of transmission.

The functional organization of excitation and inhibition in the dendrites of mouse direction-selective ganglion cells.

Recent studies indicate that the precise timing and location of excitation and inhibition (E/I) within active dendritic trees can significantly impact neuronal function. How synaptic inputs are functionally organized at the subcellular level in intact circuits remains unclear. To address this issue, we took advantage of the retinal direction-selective ganglion cell circuit, where directionally tuned inhibition is known to shape non-directional excitatory signals. We combined two-photon calcium imaging with genetic, pharmacological, and single-cell ablation methods to examine the extent to which inhibition 'vetoes' excitation at the level of individual dendrites of direction-selective ganglion cells. We demonstrate that inhibition shapes direction selectivity independently within small dendritic segments (<10µm) with remarkable accuracy. The data suggest that the parallel processing schemes proposed for direction encoding could be more fine-grained than previously envisioned.

Neogenin neutralization prevents photoreceptor loss in inherited retinal degeneration.

Inherited retinal degenerations (IRDs) are characterized by the progressive loss of photoreceptors and represent one of the most prevalent causes of blindness among working-age populations. Cyclic nucleotide dysregulation is a common pathological feature linked to numerous forms of IRD, yet the precise mechanisms through which this contributes to photoreceptor death remain elusive. Here we demonstrate that cAMP induced upregulation of the dependence receptor neogenin in the retina. Neogenin levels were also elevated in both human and murine degenerating photoreceptors. We found that overexpressing neogenin in mouse photoreceptors was sufficient to induce cell death, whereas silencing neogenin in degenerating murine photoreceptors promoted survival, thus identifying a pro-death signal in IRDs. A possible treatment strategy is modeled whereby peptide neutralization of neogenin in Rd1, Rd10, and Rho P23H-knockin mice promotes rod and cone survival and rescues visual function as measured by light-evoked retinal ganglion cell recordings, scotopic/photopic electroretinogram recordings, and visual acuity tests. These results expose neogenin as a critical link between cAMP and photoreceptor death, and identify a druggable target for the treatment of retinal degeneration.

Modulation of transmitter release by presynaptic resting potential and background calcium levels.

Activation of presynaptic ion channels alters the membrane potential of nerve terminals, leading to changes in transmitter release. To study the relationship between resting potential and exocytosis, we combined pre- and postsynaptic electrophysiological recordings with presynaptic Ca(2+) measurements at the calyx of Held. Depolarization of the membrane potential to between -60 mV and -65 mV elicited P/Q-type Ca(2+) currents of < 1 pA and increased intraterminal Ca(2+) by < 100 nM. These small Ca(2+) elevations were sufficient to enhance the probability of transmitter release up to 2-fold, with no effect on the readily releasable pool of vesicles. Moreover, the effects of mild depolarization on release had slow kinetics and were abolished by 1 mM intraterminal EGTA, suggesting that Ca(2+) acted through a high-affinity binding site. Together, these studies suggest that control of resting potential is a powerful means for regulating synaptic function at mammalian synapses.

Retinal direction selectivity in the absence of asymmetric starburst amacrine cell responses.

In the mammalian retina, direction-selectivity is thought to originate in the dendrites of GABAergic/cholinergic starburst amacrine cells, where it is first observed. However, here we demonstrate that direction selectivity in downstream ganglion cells remains remarkably unaffected when starburst dendrites are rendered non-directional, using a novel strategy combining a conditional GABAA α2 receptor knockout mouse with optogenetics. We show that temporal asymmetries between excitation/inhibition, arising from the differential connectivity patterns of starburst cholinergic and GABAergic synapses to ganglion cells, form the basis for a parallel mechanism generating direction selectivity. We further demonstrate that these distinct mechanisms work in a coordinated way to refine direction selectivity as the stimulus crosses the ganglion cell's receptive field. Thus, precise spatiotemporal patterns of inhibition and excitation that determine directional responses in ganglion cells are shaped by two 'core' mechanisms, both arising from distinct specializations of the starburst network.

Selective reduction of weak synaptic activity awakens dormant synapses.

Slutsky et al. (this issue of Neuron) report that by selectively filtering out low-level uncorrelated synaptic activity at NMDA receptors in hippocampal cultures they can unlock a large reserve of quiescent synapses and make them available for potentiation with theta burst stimulation. These findings differ from previously reported activity-dependent mechanisms in that inactivity does not necessarily increase synaptic activity globally.

An Old Neuron Learns New Tricks: Redefining Motion Processing in the Primate Retina.

Motion sensitivity requires the comparison of neural responses activated by nearby points in visual space. In this issue of Neuron, Manookin et al. (2018) find that in the primate retina, such comparisons are already manifest in second-order retinal bipolar cells, relying on lateral excitation mediated by gap junctions.

Gap Junctions Contribute to Differential Light Adaptation across Direction-Selective Retinal Ganglion Cells.

Direction-selective ganglion cells (DSGCs) deliver signals from the retina to multiple brain areas to indicate the presence and direction of motion. Delivering reliable signals in response to motion is critical across light levels. Here we determine how populations of DSGCs adapt to changes in light level, from moonlight to daylight. Using large-scale measurements of neural activity, we demonstrate that the population of DSGCs switches encoding strategies across light levels. Specifically, the direction tuning of superior (upward)-preferring ON-OFF DSGCs becomes broader at low light levels, whereas other DSGCs exhibit stable tuning. Using a conditional knockout of gap junctions, we show that this differential adaptation among superior-preferring ON-OFF DSGCs is caused by connexin36-mediated electrical coupling and differences in effective GABAergic inhibition. Furthermore, this adaptation strategy is beneficial for balancing motion detection and direction estimation at the lower signal-to-noise ratio encountered at night. These results provide insights into how light adaptation impacts motion encoding in the retina.