Nonlinear interactions between excitatory and inhibitory retinal synapses control visual output

The visual system is highly sensitive to dynamic features in the visual scene. However, it is not known how or where this enhanced sensitivity first occurs. We investigated this phenomenon by studying interactions between excitatory and inhibitory synapses in the second synaptic layer of the mouse retina. We found that these interactions showed activity-dependent changes that enhanced signaling of dynamic stimuli. Excitatory signaling from cone bipolar cells to ganglion cells exhibited strong synaptic depression, attributable to reduced glutamate release from bipolar cells. This depression was relieved by amacrine cell inhibitory feedback that activated presynaptic GABA(C) receptors. We found that the balance between excitation and feedback inhibition depended on stimulus frequency; at short interstimulus intervals excitation was enhanced, attributable to reduced inhibitory feedback. This dynamic interplay may enrich visual processing by enhancing retinal responses to closely spaced temporal events, representing rapid changes in the visual environment

Local and global interneuron function in the retina

Brain regions consist of intricate neuronal circuits with diverse interneuron types. In order to gain mechanistic insights into brain function, it is essential to understand the computational purpose of the different types of interneurons. How does a single interneuron type shape the input-output transformation of a given brain region? Here I investigated how different interneuron types of the retina contribute to retinal computations. I developed approaches to systematically and quantitatively investigate the function of retinal interneurons by combining precise circuit perturbations with a system-wide read-out of activity. I studied the functional roles of a locally acting interneuron type, starburst amacrine cells, and of a globally acting type, horizontal cells. In Chapter 1, I show how a defined genetic perturbation in starburst amacrine cells, the mutation of the FRMD7 gene, leads to specific effects in the direction-selective output channels of the retina. Our findings provide a link between a specific neuronal computation and a human disease, and present an entry point for understanding the molecular pathways responsible for generating neuronal circuit asymmetries. Chapter 2 addresses how mutated FRMD7 in starburst cells and the genetic ablation of starburst cells affect the computation of visual motion in the retina and in primary visual cortex. Chapter 3 addresses how horizontal cells mediate rod depolarization under bright daylight conditions. In Chapter 4, I combined the precise, yet retina-wide, perturbation of horizontal cells with a system-level readout of the retinal output. I uncovered that horizontal cells can differentially shape the response dynamics of individual retinal output channels. Our combined experimental and theoretical work shows how the inhibitory feedback at the first visual synapse shapes functional diversity in the retina

The diverse roles of inhibition in identified neural circuits

Inhibitory interneurons represent a diverse population of cell types in the central nervous system, whose general role is to suppress activity of target neurons. The timing of spikes in principal neurons has millisecond precision, and I asked what are the roles of inhibition in shaping the temporal codes that emerge from different parallel local neural circuits. First I investigated the local circuitry of melanopsin-containing ganglion cells in the mouse retina, which are intrinsically photosensitive and responsible for circadian photoentrainment. Using transsynaptic viral tracing, I identified three types of melanopsin-containing ganglion cell, and found that inhibitory (GABAergic) dopaminergic amacrine cells are presynaptic to one of these types. These results provided a direct circuitry link between the medium time scale process of light-dark adaptation, which involves dopamine, and the longer time scale of the circadian rhythm. Next I characterised a subpopulation of genetically-identified neurons in the mouse retina, in order to compare the precise timing of inhibition in different circuits at a high temporal resolution. I identified eight physiologically and morphologically distinct ganglion cell types and found that each circuit could be described by a 'motif' that represented the inhibitory-excitatory interactions that lead to cell-type-specific firing patterns. The cell would fire only when the change in excitation was faster than the change in inhibition. Therefore the role of inhibition is to detect 'irrelevance' in the visual scene, only allowing the ganglion cell to fire at specific time points relating to functions that are both parallel and complementary to the other cell types. Finally, I looked deeper within the neural circuitry of one of the genetically-identified cell types, to study the mechanism of 'fast inhibition' in detecting approaching objects. Through two-photon targeted paired recordings of postsynaptic ganglion cells and presynaptic amacrine cells, I found evidence that the AII amacrine cell - a well-characterised glycinergic inhibitory interneuron known to be involved in night vision circuits - conveys fast inhibitory information to the ganglion cell via an electrical synapse with an excitatory neuron of day vision circuitry only during non-approach motion. Therefore, it appears that the role of inhibition is to dynamically interact with direct excitatory neural pathways during 'irrelevant' stimulation, suppressing or completely blocking activity, resulting in precisely timed spikes that occur in the brief moments when excitation changes faster than inhibition

Transient receptor potential cation channel, subfamilies V, member 1 (TRPV1) and M, member 1 (TRPM1) contribute to neural signaling in mouse retina.

The retina processes light information through parallel pathways in order to extract and encode the visual scene. Light information is transmitted to the brain through approximately 30 ganglion cells (GCs), the retinal output neurons. Trp channels modulate the responses of retinal neurons within specific pathways. The study of the expression and function of the majority of Trp channels in the retina is largely in its infancy. My dissertation first investigated the expression and function of the transient receptor potential vanilloid-1 (TRPV1) receptor/channel in the retina. TRPV1, the first cloned and most highly studied Trp channel in the peripheral nervous system, is a non-selective cation channel with an affinity for Ca2+. The channel can be activated by capsaicin, acid, endovanilloids, noxious heat or pressure (Moreira et al., 2012). Located on the peripheral and central terminals of nociceptive fibers in the PNS and in limited areas of the CNS (Cavanaugh et al, 2011b). TRPV1 plays a role in inflammation, chronic pain, nociceptor sensitization and desensitization, long-term depression and potentiation, and apoptosis. The role of TRPV1 in the retina is not known. Using the electroretinogram (ERG), a mass potential that assesses the function of photoreceptors and bipolar cells, the TRPV1 knockout mouse appears normal. However, TRPV1 is thought to play a role in calcium regulation and glaucoma (Sappington et al., 2009 & Leonelli et al., 2010) so we investigated its role in normal visual transduction in the inner retina. To investigate TRPV1 modulation, I recorded GC spiking responses to light stimuli from mice which either express or lack TRPV1 protein. I found that TRPV1 is critical for: 1. GC responses to dim light. 2. Sustained responses to light 3. Surround suppression of GCs to large spots. Further, I investigate the specific retinal cells that express TRPV1. I used TRPV1cre mice with genetic or viral methods to fluorescently label neurons that express TRPV1. I determined TRPV1 is expressed in four classes of amacrine and three classes of ganglion cells in the inner retina. My results indicate TRPV1 activity in the amacrine cells enhances the sustained spiking responses in GCs. In this way, TRPV1 likely enhances the perception of subtle details in the visual world. TRPV1 also is expressed in subsets of intrinsically photosensitive GCs, which are known to play a role in circadian photoentrainment. TRPV1 therefore has the potential to modulate circadian photoentrainment or other non-image forming visual functions as well. The role of TRPM1 in the retina is well known. It is required for signaling through the ON pathway, which detects light increments. Responses through the vii ON pathway are initiated by synapses between rod and cone photoreceptors with ON bipolar cells (BCs). The human disease, complete congenital stationary night blindness (cCSNB) results from a disruption in signaling within the ON BC mGluR6 G-protein coupled cascade, which culminates in the opening of the TRPM1 channel and signaling through ON BCs. I helped expand our understanding of the role of TRPM1 in the retina by investigating the expression and function of leucine rich repeat immunoglobulin like transmembrane protein 3 (LRIT3), a novel protein component in the mGluR6-TRPM1 signalplex that was found mutated within cCSNB patients and a knockout mouse (Zeitz et al., 2013; Neuillé et al., 2014). The function of LRIT3 within the cascade remains unknown. To better understand the role of LRIT3, we examined retinal structure and function. We compared the structure of the pre and postsynaptic elements in the OPL of WT and Lrit3-/- mice using a variety of antibodies and with confocal microscopy. We assessed overall retinal function with ERG and GC spontaneous and visually evoked activity with single cell and multielectrode array recordings. The overall laminar structure of the Lrit3-/- retina is similar to WT. Consistent with published results and other cCSNB mouse models, Lrit3-/- mouse dark- and lightadapted ERGs have a normal a-wave, but lack a b-wave. The dendritic terminals of Lrit3-/- ON BCs lack expression of nyctalopin and TRPM1. Lrit3-/- mice significantly differ from other cCSNB mutants. Cone ON BCs lack expression of mGluR6, GPR179 and RGS11, whereas rod BCs maintain expression of these proteins. LRIT3 is necessary for expression and localization of nyctalopin and TRPM1 to the ON BC dendrites. As expected there are no ON responses, but surprisingly very few (~22%) Lrit3-/- GCs have even OFF responses. Lrit3-/- OFF BCs express functional kainate glutamate receptors. However, Lrit3-/- OFF BC and OFF GCs have significantly smaller response to light decrements than WT. Like all other mouse models of cCSNB, LRIT3 is critical to signaling in ON BCs, however, unlike all other cCSNB models, LRIT3 also has a trans-synaptic role in enhancing glutamate transmission from cones to BCs

Network Deficiency Exacerbates Impairment in a Mouse Model of Retinal Degeneration

Neural oscillations play an important role in normal brain activity, but also manifest during Parkinson’s disease, epilepsy, and other pathological conditions. The contribution of these aberrant oscillations to the function of the surviving brain remains unclear. In recording from retina in a mouse model of retinal degeneration (RD), we found that the incidence of oscillatory activity varied across different cell classes, evidence that some retinal networks are more affected by functional changes than others. This aberrant activity was driven by an independent inhibitory amacrine cell oscillator. By stimulating the surviving circuitry at different stages of the neurodegenerative process, we found that this dystrophic oscillator further compromises the function of the retina. These data reveal that retinal remodeling can exacerbate the visual deficit, and that aberrant synaptic activity could be targeted for RD treatment

Glycine receptor expression across identified retinal ganglion cell types.

Retinal ganglion cells (RGCs) represent the culmination of all retinal signaling and their output forms the substrate for vision throughout the rest of the brain. About 40 different RGC types have been defined by differences in their visually evoked responses, morphology, and genetic makeup. These responses arise from interactions between inhibition and excitation throughout the retinal circuit (Franke et al., 2017; Masland, 2012; Sanes & Masland, 2015; Werblin, 2011). Unlike most other areas of the central nervous system (CNS), the retina utilizes both GABA and glycine inhibitory neurotransmitters to refine glutamatergic excitatory signals (Franke & Baden, 2017; Werblin, 2011; C. Zhang, Nobles, & McCall, 2015). Glycine receptors (GlyRs) are heteromers composed of a single β subunit and one of four α subunits, with a stoichiometry of 3β:2α (Grudzinska et al., 2005; Heinze, Harvey, Haverkamp, & Wassle, 2007; Lynch, 2004). All four GlyRα subunits (α1, α2, α3, or α4) are differentially expressed in the retina and subunit specific expression has been defined for bipolar, some amacrine cells and vi RGCs (Haverkamp, Muller, Zeilhofer, Harvey, & Wassle, 2004; Heinze et al., 2007). The roles for GlyRα subunit specific inhibition are unknown, although glycinergic input is generally linked to temporal response tuning (Murphy & Rieke, 2006; Nobles, Zhang, Muller, Betz, & McCall, 2012; van Wyk, Wassle, & Taylor, 2009; Wassle et al., 2009; Werblin, 2010). We have surveyed GlyRα subunit expression in a variety of identified RGC types, using GlyRα knockout mice and an rAAV-mediated RNAi to knockdown GlyRα subunit specific expression. We find that the four α RGCs only express GlyRα1. All of the other RGCs we studied express at least two GlyRα subunits. In some RGCs, the GlyR kinetics are similar, whereas in others the kinetics differs. We propose that this diversity will contribute to the richness of retinal inhibitory processing

Characterization Of High-Voltage-Activated Calcium Channels In Retinal Bipolar Cells

Retinal bipolar cells, conveying visual information from photoreceptors to ganglion cells, segregate visual information into multiple parallel pathways through their diversified cell types and physiological properties. Voltage-gated Ca2+ channels could be particularly important underlying the diversified physiological properties of different BCs. In this dissertation, I investigated the high-voltage-activated (HVA) calcium current in retinal bipolar cells in mice. In the first part of my dissertation, I characterized multiple bipolar cell-expressing GFP and/or Cre transgenic mouse lines. In the second part of my dissertation, by performing whole-cell patch-clamp recordings, I examined the electrophysiological properties of HVA calcium currents among CBCs and between CBCs and CBCs. In particular, the second part of my study focused on the investigation of electrophysiological, pharmacological, and molecular properties of HVA calcium currents in RBCs. The results of my studies showed that the HVA Ca2+ currents with different electrophysiological properties were observed among CBCs, and between CBCs and RBCs. First, large HVA Ca2+ currents were observed in OFF CBCs but not in ON-CBCs. Second, HVA Ca2+ currents among different bipolar cells were found to show different activation potentials. Furthermore, the HVA Ca2+ currents in RBCs exhibited two components, a sustained and a transient component with the latter activated at more negative potentials. My pharmacological results indicated the sustained and transient HVA Ca2+ currents are originated from L- and P/Q type Ca2+ channels, respectively. Using L-type Ca2+ channel knockout or deficient mouse lines, my results showed that the L-type Ca2+ currents in RBCs are mediated mainly by alpha1C Ca2+ channels with a minor component from alpha1F Ca2+ channels. The studies will advance our understanding of the role of voltage-gated Ca2+ channels in basic visual information processing in the retina as well as Ca2+ signaling and Ca2+ channel deficit-related diseases in the visual system

Optimal electrical activation of retinal ganglion cells

Retinal prostheses are emerging as a viable therapy option for those blinded by degenerative eye conditions that destroy the photoreceptors of the retina but spare the retinal ganglion cells (RGCs). My research sought to address the issue of how a retinal prosthesis might best activate these cells by way of electrical stimulation. Whole-cell patch clamp recordings were made in explanted retinal wholemount preparations from normally-sighted rats. Stimulating electrodes were fabricated from nitrogen-doped ultra-nanocrystalline diamond (N-UNCD) and placed on the epiretinal surface, adjacent to the cell soma. Electrical stimuli were delivered against a distant monopolar return electrode. Using rectangular, biphasic constant current waveforms as employed by modern retinal prostheses, I examined which waveform parameters had the greatest effect on RGC activation thresholds. In a second set of experiments intracellular current injection was employed to assess the effectiveness of sinusoidal current waveforms in selectively activating different RGC subsets. These recordings were also used to validate a biophysical model of RGC activation. Where possible, recorded cells were identified and classified based on 3D confocal reconstruction of their morphology. Electrodes fabricated from N-UNCD were able to electrically activate RGCs while remaining well within the electrochemical limits of the material. They were found to exhibit high electrochemical stability and were resistant to morphological and electrochemical changes over one week of continuous pulsing at charge injection limits. Retinal ganglion cells invariably favoured cathodic-first biphasic current pulses of short first-phase duration, with a small interphase interval. The majority of cells (63\%) were most sensitive to a highly asymmetric waveform: a short-cathodic phase followed by a longer duration, lower amplitude anodic phase. Using the optimal interphase interval led to median charge savings of 14\% compared to the charge required in the absence of any inter-phase interval. Optimising phase duration resulted in median charge savings of 22\%. All RGCs became desensitised to repetitive electrical stimulation. The efficacy of a given stimulus delivered repeatedly decreased after the first stimulus, stabilising at a lower efficacy by the thirtieth pulse. This asymptotic efficacy decreased with increasing stimulus frequency. Cells with smaller somas and dendritic fields were better able to sustain repetitive activation at high frequency. Intracellular sinusoidal stimulation was used to demonstrate that certain RGC subsets, defined on the basis of morphological type, stratification, and size, were more responsive to high frequency stimulation. Simulated RGC responses were validated by experimental data, which confirmed that ON cell responses were heavily suppressed by stimulus frequencies of 20 Hz and higher. OFF cells, on the other hand, were able to sustain repetitive activation over all tested frequencies. Additional simulations suggest this difference may be plausibly attributed to the presence of low-voltage-activated calcium channels in OFF but not ON RGCs. The results of my work demonstrate that (a) N-UNCD is a suitable material for retinal prosthesis applications; (b) a careful choice of electrical waveform parameters can significantly improve prosthesis efficacy; and (c) it may be possible to bias neural activation for certain RGC populations by varying the frequency of stimulation

Organotypic Tissue Culture of Adult Rodent Retina Followed by Particle-Mediated Acute Gene Transfer In Vitro

BACKGROUND: Organotypic tissue culture of adult rodent retina with an acute gene transfer that enables the efficient introduction of variable transgenes would greatly facilitate studies into retinas of adult rodents as animal models. However, it has been a difficult challenge to culture adult rodent retina. The purpose of this present study was to develop organotypic tissue culture of adult rodent retina followed by particle-mediated acute gene transfer in vitro. METHODOLOGY/PRINCIPAL FINDINGS: We established an interphase organotypic tissue culture for adult rat retinas (>P35 of age) which was optimized from that used for adult rabbit retinas. We implemented three optimizations: a greater volume of Ames' medium (>26 mL) per retina, a higher speed (constant 55 rpm) of agitation by rotary shaker, and a greater concentration (10%) of horse serum in the medium. We also successfully applied this method to adult mouse retina (>P35 of age). The organotypic tissue culture allowed us to keep adult rodent retina morphologically and structurally intact for at least 4 days. However, mouse retinas showed less viability after 4-day culture. Electrophysiologically, ganglion cells in cultured rat retina were able to generate action potentials, but exhibited less reliable light responses. After transfection of EGFP plasmids by particle-mediated acute gene transfer, we observed EGFP-expressing retinal ganglion cells as early as 1 day of culture. We also introduced polarized-targeting fusion proteins such as PSD95-GFP and melanopsin-EYFP (hOPN4-EYFP) into rat retinal ganglion cells. These fusion proteins were successfully transferred into appropriate locations on individual retinal neurons. CONCLUSIONS/SIGNIFICANCE: This organotypic culture method is largely applicable to rat retinas, but it can be also applied to mouse retinas with a caveat regarding cell viability. This method is quite flexible for use in acute gene transfection in adult rodent retina, replacing molecular biological bioassays that used to be conducted in isolated cultured cells

Glycine receptor alpha subunit (GlyRa) specific inhibition contributes to ganglion cell signaling in mouse retina.

In the retina, numerous types of neurons are wired together in a highly specific albeit complex pattern. This sophisticated retinal network allows extraction and encoding of more than 20 representations of the visual scene in its output neurons, the retinal ganglion cells (RGCs). Within the inner plexiform layer (IPL) of retina, glycine receptors (GlyRs) are expressed on different cell classes and modulate RGC visual activity to light onset (ON RGCs) and to light offset (OFF RGCs), for example, their temporal precision and gain control. There are four GlyR alpha subunits (GlyRα1-4) with differential expression patterns in IPL. Each mediates spontaneous inhibitory postsynaptic currents (sIPSCs) with different decay kinetics. Moreover, GlyR alpha subunit-specific expression was discovered across different RGC types. This evidence suggests subunit-specific roles for glycinergic inhibitory circuits to modulate the RGC visual outputs. However, the details remain largely unknown. To investigate glycinergic subunit-specific modulation, I used GlyRα subunit knockout (KO) mouse lines, which lack GlyRα2 (Glra2-/-), GlyRα3 (Glra3-/-) or both (Glra2/3-/-). I found that GlyRα2 and GlyRα3 enhance ON RGCs visual responses whereas only GlyRα2 enhances OFF RGCs visual responses. Second, I used viral tools to manipulate the expression of the GlyRα1 subunit on RGCs to examine its role in visual processing. Adeno-associated viruses (AAVs) were injected into dorsal lateral geniculate nucleus and transported retrogradely to infect RGCs and generate shRNA to selectively knockdown GlyRα1 expression. In OFFαTransient RGCs, which predominantly express GlyRα1, shRNA almost completely eliminated all glycinergic input and I showed that this input increases signal to noise ratio of OFFαTransient RGC visual responses. I expanded our understanding of subunit-specificity by surveying subunit specific expression and currents across eight identified RGCs in the PVcre mouse. By comparing co-localization of GlyR α subunit puncta on identified RGC dendrites with the decay kinetics of their sIPSCs, I showed that there is subunit-specific expression of GlyRs. My data not only support the hypothesis of subunit-specific glycinergic inhibitory modulation in retinal signaling, but provide new tools to further explore their individual roles in shaping RGC visual function