The Bovine Ex Vivo Retina: A Versatile Model for Retinal Neuroscience.

Purpose: The isolated ex vivo retina is the standard model in retinal physiology and neuroscience. During isolation, the retina is peeled from the retinal pigment epithelium (RPE), which plays a key role in the visual cycle. Here we introduce the choroid-attached bovine retina as an in vivo-like model for retinal physiology. We find that-in the bovine eye-the choroid and retina can be peeled from the sclera as a single thin sheet. Importantly, the retina remains tightly associated with the RPE, which is sandwiched between the retina and the choroid. Furthermore, bovine tissue is readily available and cheap, and there are no ethical concerns related to the use of animals solely for research purposes. Methods: We combine multi-electrode array and single-cell patch-clamp recordings to characterize light responses in the choroid-attached bovine ex vivo retina. Results: We demonstrate robust and consistent light responses in choroid-attached preparations. Importantly, light responses adapt to different levels of background illumination and rapidly recover from photobleaching. The choroid-attached retina is also thin enough to permit targeted electrophysiological recording from individual retinal neurons using standard differential interference contrast microscopy. We also characterize light responses and membrane properties of bovine retinal ganglion cells and compare data obtained from bovine and murine retinas. Conclusions: The choroid-attached retinal model retains the advantages of the isolated retina but with an intact visual cycle and represents a useful tool to elucidate retinal physiology.

A visual opsin from jellyfish enables precise temporal control of G protein signalling.

Phototransduction is mediated by distinct types of G protein cascades in different animal taxa: bilateral invertebrates typically utilise the Gαq pathway whereas vertebrates typically utilise the Gαt(i/o) pathway. By contrast, photoreceptors in jellyfish (Cnidaria) utilise the Gαs intracellular pathway, similar to olfactory transduction in mammals1. How this habitually slow pathway has adapted to support dynamic vision in jellyfish remains unknown. Here we study a light-sensing protein (rhodopsin) from the box jellyfish Carybdea rastonii and uncover a mechanism that dramatically speeds up phototransduction: an uninterrupted G protein-coupled receptor - G protein complex. Unlike known G protein-coupled receptors (GPCRs), this rhodopsin constitutively binds a single downstream Gαs partner to enable G-protein activation and inactivation within tens of milliseconds. We use this GPCR in a viral gene therapy to restore light responses in blind mice.

Bipolar cell targeted optogenetic gene therapy restores parallel retinal signaling and high-level vision in the degenerated retina.

Optogenetic gene therapies to restore vision are in clinical trials. Whilst current clinical approaches target the ganglion cells, the output neurons of the retina, new molecular tools enable efficient targeting of the first order retinal interneurons, the bipolar cells, with the potential to restore a higher quality of vision. Here we investigate retinal signaling and behavioral vision in blind mice treated with bipolar cell targeted optogenetic gene therapies. All tested tools, including medium-wave opsin, Opto-mGluR6, and two new melanopsin based chimeras restored visual acuity and contrast sensitivity. The best performing opsin was a melanopsin-mGluR6 chimera, which in some cases restored visual acuities and contrast sensitivities that match wild-type animals. Light responses from the ganglion cells were robust with diverse receptive-field types, inferring elaborate inner retinal signaling. Our results highlight the potential of bipolar cell targeted optogenetics to recover high-level vision in human patients with end-stage retinal degenerations.

Present Molecular Limitations of ON-Bipolar Cell Targeted Gene Therapy.

Recent studies have demonstrated the safety and efficacy of ocular gene therapy based on adeno-associated viral vectors (AAVs). Accordingly, a surge in promising new gene therapies is entering clinical trials, including the first optogenetic therapy for vision restoration. To date, optogenetic therapies for vision restoration target either the retinal ganglion cells (GCs) or presynaptic ON-bipolar cells (OBCs). Initiating light responses at the level of the OBCs has significant advantages over optogenetic activation of GCs. For example, important neural circuitries in the inner retina, which shape the receptive fields of GCs, remain intact when activating the OBCs. Current drawbacks of AAV-mediated gene therapies targeting OBCs include (1) a low transduction efficiency, (2) off-target expression in unwanted cell populations, and (3) a poor performance in human tissue compared to the murine retina. Here, we examined side-by-side the performance of three state-of-the art AAV capsid variants, AAV7m8, AAVBP2, and AAV7m8(Y444F) in combination with the 4xGRM6-SV40 promoter construct in the healthy and degenerated mouse retina and in human post-mortem retinal explants. We find that (1) the 4xGRM6-SV40 promoter is not OBC specific, (2) that all AAV variants possess broad cellular transduction patterns, with differences between the transduction patterns of capsid variants AAVBP2 and AAV7m8 and, most importantly, (3) that all vectors target OBCs in healthy tissue but not in the degenerated rd1 mouse model, potentially limiting the possibilities for an OBC-targeted optogenetic therapy for vision restoration in the blind.

Variable phenotypic expressivity in inbred retinal degeneration mouse lines: A comparative study of C3H/HeOu and FVB/N rd1 mice.

PURPOSE: Recent advances in optogenetics and gene therapy have led to promising new treatment strategies for blindness caused by retinal photoreceptor loss. Preclinical studies often rely on the retinal degeneration 1 (rd1 or Pde6b(rd1)) retinitis pigmentosa (RP) mouse model. The rd1 founder mutation is present in more than 100 actively used mouse lines. Since secondary genetic traits are well-known to modify the phenotypic progression of photoreceptor degeneration in animal models and human patients with RP, negligence of the genetic background in the rd1 mouse model is unwarranted. Moreover, the success of various potential therapies, including optogenetic gene therapy and prosthetic implants, depends on the progress of retinal degeneration, which might differ between rd1 mice. To examine the prospect of phenotypic expressivity in the rd1 mouse model, we compared the progress of retinal degeneration in two common rd1 lines, C3H/HeOu and FVB/N. METHODS: We followed retinal degeneration over 24 weeks in FVB/N, C3H/HeOu, and congenic Pde6b(+) seeing mouse lines, using a range of experimental techniques including extracellular recordings from retinal ganglion cells, PCR quantification of cone opsin and Pde6b transcripts, in vivo flash electroretinogram (ERG), and behavioral optokinetic reflex (OKR) recordings. RESULTS: We demonstrated a substantial difference in the speed of retinal degeneration and accompanying loss of visual function between the two rd1 lines. Photoreceptor degeneration and loss of vision were faster with an earlier onset in the FVB/N mice compared to C3H/HeOu mice, whereas the performance of the Pde6b(+) mice did not differ significantly in any of the tests. By postnatal week 4, the FVB/N mice expressed significantly less cone opsin and Pde6b mRNA and had neither ERG nor OKR responses. At 12 weeks of age, the retinal ganglion cells of the FVB/N mice had lost all light responses. In contrast, 4-week-old C3H/HeOu mice still had ERG and OKR responses, and we still recorded light responses from C3H/HeOu retinal ganglion cells until the age of 24 weeks. These results show that genetic background plays an important role in the rd1 mouse pathology. CONCLUSIONS: Analogous to human RP, the mouse genetic background strongly influences the rd1 phenotype. Thus, different rd1 mouse lines may follow different timelines of retinal degeneration, making exact knowledge of genetic background imperative in all studies that use rd1 models.

Restoring the ON Switch in Blind Retinas: Opto-mGluR6, a Next-Generation, Cell-Tailored Optogenetic Tool.

Photoreceptor degeneration is one of the most prevalent causes of blindness. Despite photoreceptor loss, the inner retina and central visual pathways remain intact over an extended time period, which has led to creative optogenetic approaches to restore light sensitivity in the surviving inner retina. The major drawbacks of all optogenetic tools recently developed and tested in mouse models are their low light sensitivity and lack of physiological compatibility. Here we introduce a next-generation optogenetic tool, Opto-mGluR6, designed for retinal ON-bipolar cells, which overcomes these limitations. We show that Opto-mGluR6, a chimeric protein consisting of the intracellular domains of the ON-bipolar cell-specific metabotropic glutamate receptor mGluR6 and the light-sensing domains of melanopsin, reliably recovers vision at the retinal, cortical, and behavioral levels under moderate daylight illumination.

Distinct roles for inhibition in spatial and temporal tuning of local edge detectors in the rabbit retina.

This paper examines the role of inhibition in generating the receptive-field properties of local edge detector (LED) ganglion cells in the rabbit retina. We confirm that the feed-forward inhibition is largely glycinergic but, contrary to a recent report, our data demonstrate that the glycinergic inhibition contributes to temporal tuning for the OFF and ON inputs to the LEDs by delaying the onset of spiking; this delay was more pronounced for the ON inputs (∼ 340 ms) than the OFF inputs (∼ 12 ms). Blocking glycinergic transmission reduced the delay to spike onset and increased the responses to flickering stimuli at high frequencies. Analysis of the synaptic conductances indicates that glycinergic amacrine cells affect temporal tuning through both postsynaptic inhibition of the LEDs and presynaptic modulation of the bipolar cells that drive the LEDs. The results also confirm that presynaptic GABAergic transmission contributes significantly to the concentric surround antagonism in LEDs; however, unlike presumed LEDs in the mouse retina, the surround is only partly generated by spiking amacrine cells.

A comparative evaluation of NB30, NB54 and PTC124 in translational read-through efficacy for treatment of an USH1C nonsense mutation.

Translational read-through-inducing drugs (TRIDs) promote read-through of nonsense mutations, placing them in the spotlight of current gene-based therapeutic research. Here, we compare for the first time the relative efficacies of new-generation aminoglycosides NB30, NB54 and the chemical compound PTC124 on retinal toxicity and read-through efficacy of a nonsense mutation in the USH1C gene, which encodes the scaffold protein harmonin. This mutation causes the human Usher syndrome, the most common form of inherited deaf-blindness. We quantify read-through efficacy of the TRIDs in cell culture and show the restoration of harmonin function. We do not observe significant differences in the read-through efficacy of the TRIDs in retinal cultures; however, we show an excellent biocompatibility in retinal cultures with read-through versus toxicity evidently superior for NB54 and PTC124. In addition, in vivo administration of NB54 and PTC124 induced recovery of the full-length harmonin a1 with the same efficacy. The high biocompatibilities combined with the sustained read-through efficacies of these drugs emphasize the potential of NB54 and PTC124 in treating nonsense mutation-based retinal disorders.

Synaptic inputs and timing underlying the velocity tuning of direction-selective ganglion cells in rabbit retina.

There are two types of direction-selective ganglion cells (DSGCs) identified in the rabbit retina, which can be readily distinguished both morphologically and physiologically. The well characterized ON-OFF DSGCs respond to a broad range of image velocities whereas the less common ON DSGCs are tuned to slower image velocities. This study examined how the synaptic inputs shape the velocity tuning of DSGCs in an isolated preparation of the rabbit retina. The receptive-field properties were mapped by extracellular spike recordings and compared with the light-evoked excitatory and inhibitory synaptic conductances that were measured under voltage-clamp. The synaptic mechanisms underlying the generation of direction selectivity appear to be similar in both cell types in that preferred-direction image motion elicits a greater excitatory input and null-direction image motion elicits a greater inhibitory input. To examine the temporal tuning of the DSGCs, the cells were stimulated with either a grating drifted over the receptive-field centre at a range of velocities or with a light spot flickered at different temporal frequencies. Whereas the excitatory and inhibitory inputs to the ON-OFF DSGCs are relatively constant over a wide range of temporal frequencies, the ON DSGCs receive less excitation and more inhibition at higher temporal frequencies. Moreover, transient inhibition precedes sustained excitation in the ON DSGCs, leading to slowly activating, sustained spike responses. Consequently, at higher temporal frequencies, weaker excitation combines with fast-rising inhibition resulting in lower spike output.

Visual function in mice with photoreceptor degeneration and transgenic expression of channelrhodopsin 2 in ganglion cells.

The progression of rod and cone degeneration in retinally degenerate (rd) mice ultimately results in a complete loss of photoreceptors and blindness. The inner retinal neurons survive and several recent studies using genetically targeted, light activated channels have made these neurons intrinsically light sensitive. We crossbred a transgenic mouse line expressing channelrhodopsin2 (ChR2) under the control of the Thy1 promoter with the Pde6b(rd1) mouse, a model for retinal degeneration (rd1/rd1). Approximately 30-40% of the ganglion cells of the offspring expressed ChR2. Extracellular recordings from ChR2-expressing ganglion cells in degenerated retinas revealed their intrinsic light sensitivity which was approximately 7 log U less sensitive than the scotopic threshold and approximately 2 log U less sensitive than photopic responses of normal mice. All ChR2-expressing ganglion cells were excited at light ON. The visual performance of rd1/rd1 mice and ChR2 rd1/rd1 mice was compared. Behavioral tests showed that both mouse strains had a pupil light reflex and they were able to discriminate light fields from dark fields in the visual water task. Cortical activity maps were recorded with optical imaging. The ChR2rd1/rd1 mice did not show a better visual performance than rd1/rd1 mice. In both strains the residual vision was correlated with the density of cones surviving in the peripheral retina. The expression of ChR2 under the control of the Thy1 promoter in retinal ganglion cells does not rescue vision.

Receptive field properties of ON- and OFF-ganglion cells in the mouse retina.

There are two subclasses of alpha cell in the mammalian retina, which are morphologically identical in plain view but have opposite responses to a luminance change: one is ON center and the other is OFF center. Recent studies have shown that the neural circuitries, which underlie light responses in such ON- and OFF-ganglion cell pairs, are not mirror symmetric with respect to the ON and OFF pathways (Pang et al., 2003; Zaghloul et al., 2003; Murphy & Rieke, 2006). This study examines alpha-cell homologues in the mouse retina and elucidates the synaptic mechanisms that generate their light responses. Morphological analysis of recorded cells revealed three subclasses that were essentially identical in plan view but had distinct vertical stratification levels. We refer to these cells as the sustained ON (ON-S), sustained OFF (OFF-S), and transient OFF (OFF-T) cells (Murphy & Rieke, 2006; Margolis & Detwiler, 2007). Both ON-S and OFF-S cells were largely driven through the ON pathway via changes in excitatory and inhibitory inputs, respectively. Light responses of OFF-T cells were driven by transient changes in excitatory and inhibitory inputs. Light responses of OFF-S cells were also measured in connexin 36 knockout mice in order to dissect glycinergic input arising from AII amacrine cells. At photopic/mesopic intensities, peak glycinergic input to OFF-S cells in the connexin 36 knockout mouse was reduced by ~85% compared to OFF-S cells in the wild-type retina. This is consistent with the idea that AII cells receive their input from ON-cone bipolar cells through gap junctions and in turn provide glycinergic inhibition to OFF-S cells.

Local edge detectors: a substrate for fine spatial vision at low temporal frequencies in rabbit retina.

Visual acuity is limited by the size and density of the smallest retinal ganglion cells, which correspond to the midget ganglion cells in primate retina and the beta-ganglion cells in cat retina, both of which have concentric receptive fields that respond at either light-On or light-Off. In contrast, the smallest ganglion cells in the rabbit retina are the local edge detectors (LEDs), which respond to spot illumination at both light-On and light-Off. However, the LEDs do not predominate in the rabbit retina and the question arises, what role do they play in fine spatial vision? We studied the morphology and physiology of LEDs in the isolated rabbit retina and examined how their response properties are shaped by the excitatory and inhibitory inputs. Although the LEDs comprise only approximately 15% of the ganglion cells, neighboring LEDs are separated by 30-40 microm on the visual streak, which is sufficient to account for the grating acuity of the rabbit. The spatial and temporal receptive-field properties of LEDs are generated by distinct inhibitory mechanisms. The strong inhibitory surround acts presynaptically to suppress both the excitation and the inhibition elicited by center stimulation. The temporal properties, characterized by sluggish onset, sustained firing, and low bandwidth, are mediated by the temporal properties of the bipolar cells and by postsynaptic interactions between the excitatory and inhibitory inputs. We propose that the LEDs signal fine spatial detail during visual fixation, when high temporal frequencies are minimal.

Differential effects of nicotine on the activity of substantia nigra and ventral tegmental area dopaminergic neurons in vitro.

Despite resembling each other in many respects, dopaminergic neurons of the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) exhibit dissimilar responses to nicotine in vivo. To investigate this in an in vitro model, the acute effects of nicotine on the firing of SNc and VTA neurons were compared in transverse juvenile rat midbrain sections (300-350 microm) using extracellular recording. Levels of nicotine comparable with those encountered in smokers (0.2-1.0 microM, 3 min) not only increased firing rate, but also evoked prolonged irregular firing, as indicated by the increase in the coefficient of variation of discharge frequencies. Pre- and postsynaptic nicotinic cholinergic receptors (nAChRs) were involved, as both effects persisted, although at an attenuated level, in low Ca2+ / high Mg2+. Only the nicotine-induced elevation of firing rate was sensitive to the glutamate receptor antagonists APV and CNQX, implying that enhanced glutamate release and glutamate receptor activation are involved in the effects of nicotine on discharge frequency but not pattern. Furthermore, nicotine (1.0 microM) exerted a greater increase in the firing frequency of VTA neurons relative to SNc neurons, suggesting that the differential effects on the two populations previously reported in vivo were due to a difference in the postsynaptic nAChR response and/or local synaptic circuits. Low concentrations of nicotine can thus profoundly modulate the activity of dopaminergic mesencephalic neurons through a local action within the ventral midbrain in vitro, and, similarly to in vivo conditions, evoke stronger effects in the VTA.

Dendritic projections and dye-coupling in dopaminergic neurons of the substantia nigra examined in horizontal brain slices from young rats.

We examined the rostro-caudal dendritic spread of striatally projecting dopaminergic neurons of the Substantia Nigra pars compacta (SNc) and investigated the presence of dye-coupling after labeling these cells with a mixture of lucifer yellow (LY) and neurobiotin (NB) or with LY alone. Whole cell recordings were made from horizontal brain slices (400 microm) obtained from P5-P20 rats. SNc neurons retrogradely labeled with Fluoro-Gold and located in the region containing tyrosine hydroxylase-immunoreactive cells displayed Ih current and other properties characteristic of SNc neurons. To prevent extracellular leakage, dyes were introduced into patch pipettes after the establishment of whole cell configuration, and cells were filled under visual control. In contrast to previous studies conducted in coronal sections that identified dendritic projections of SNc neurons mainly in the medio-lateral and ventral directions, almost all neurons labeled in our study (53/54) additionally displayed a large rostro-caudal dendritic span (649 +/- 219 microm). Dye-coupling between SNc neurons was not observed under basal conditions, in the presence of gap junction "openers" (forskolin, trimethylamine), or after neurons were filled with LY using sharp intracellular microelectrodes. As a "positive control," dye-coupling was demonstrated in four hippocampal dentate gyrus neurons that were filled using the same patch pipette technique. In addition, none of the tested SNc cells (n = 12) showed expression of connexin 36 (the "neuronal" connexin) when tested with single-cell RT-PCR. In conclusion, this study revealed extensive rostro-caudal dendritic projections of SNc neurons. Under our in vitro conditions, no evidence was found for dye-coupling among these neurons.

The network vs. pacemaker theory of the activity of RVL presympathetic neurons--a comparison with another putative pacemaker system.

Intracellular studies previously conducted in our laboratory on adult rats indicate that the activity of spinally projecting RVL neurons (neurons located in the Rostral Ventrolateral Medulla) results from synaptic inputs. The data obtained by others in medullary slices suggest that the firing of these neurons (RVL C1 and/or non-C1 type, depending on experimental conditions) is mainly determined by their 'beating' pacemaker properties. Interestingly, there is an analogy between the contrasting views on the role of the network vs. pacemakers in the generation of sympathetic tone, and a debate regarding the relative role of such mechanisms in other types of 'spontaneously' active neurons, including dopaminergic neurons of the Substantia Nigra/Ventral Tegmental Area (in ventral mesencephalon). This short review discusses our previous in vivo studies and more recent data obtained in vitro after acute cell isolation, showing that under both experimental conditions, the RVL neurons display no clear pacemaker-like properties. Interestingly, pacemaker activity of dopaminergic mesencephalic neurons can be easily demonstrated in brain slices and after acute isolation, but not in vivo. These findings strongly suggest that under normal in vivo conditions, individual neurons belonging to these two neural systems function as elements of networks.