Organotypic Culture of Physiologically Functional Adult Mammalian Retinas

BACKGROUND: The adult mammalian retina is an important model in research on the central nervous system. Many experiments require the combined use of genetic manipulation, imaging, and electrophysiological recording, which make it desirable to use an in vitro preparation. Unfortunately, the tissue culture of the adult mammalian retina is difficult, mainly because of the high energy consumption of photoreceptors. METHODS AND FINDINGS: We describe an interphase culture system for adult mammalian retina that allows for the expression of genes delivered to retinal neurons by particle-mediated transfer. The retinas retain their morphology and function for up to six days— long enough for the expression of many genes of interest—so that effects upon responses to light and receptive fields could be measured by patch recording or multielectrode array recording. We show that a variety of genes encoding pre- and post-synaptic marker proteins are localized correctly in ganglion and amacrine cells. CONCLUSIONS: In this system the effects on neuronal function of one or several introduced exogenous genes can be studied within intact neural circuitry of adult mammalian retina. This system is flexible enough to be compatible with genetic manipulation, imaging, cell transfection, pharmacological assay, and electrophysiological recordings

A genetically encoded reporter of synaptic activity in vivo

To image synaptic activity within neural circuits, we tethered the genetically encoded calcium indicator (GECI) GCaMP2 to synaptic vesicles by fusion to synaptophysin. The resulting reporter, SyGCaMP2, detected the electrical activity of neurons with two advantages over existing cytoplasmic GECIs: it identified the locations of synapses and had a linear response over a wider range of spike frequencies. Simulations and experimental measurements indicated that linearity arises because SyGCaMP2 samples the brief calcium transient passing through the presynaptic compartment close to voltage-sensitive calcium channels rather than changes in bulk calcium concentration. In vivo imaging in zebrafish demonstrated that SyGCaMP2 can assess electrical activity in conventional synapses of spiking neurons in the optic tectum and graded voltage signals transmitted by ribbon synapses of retinal bipolar cells. Localizing a GECI to synaptic terminals provides a strategy for monitoring activity across large groups of neurons at the level of individual synapses

Inhibition decorrelates visual feature representations in the inner retina

The retina extracts visual features for transmission to the brain. Different types of bipolar cell split the photoreceptor input into parallel channels and provide the excitatory drive for downstream visual circuits. Mouse bipolar cell types have been described at great anatomical and genetic detail, but a similarly deep understanding of their functional diversity is lacking. Here, by imaging light-driven glutamate release from more than 13,000 bipolar cell axon terminals in the intact retina, we show that bipolar cell functional diversity is generated by the interplay of dendritic excitatory inputs and axonal inhibitory inputs. The resulting centre and surround components of bipolar cell receptive fields interact to decorrelate bipolar cell output in the spatial and temporal domains. Our findings highlight the importance of inhibitory circuits in generating functionally diverse excitatory pathways and suggest that decorrelation of parallel visual pathways begins as early as the second synapse of the mouse visual system

Expression of SPIG1 Reveals Development of a Retinal Ganglion Cell Subtype Projecting to the Medial Terminal Nucleus in the Mouse

Visual information is transmitted to the brain by roughly a dozen distinct types of retinal ganglion cells (RGCs) defined by a characteristic morphology, physiology, and central projections. However, our understanding about how these parallel pathways develop is still in its infancy, because few molecular markers corresponding to individual RGC types are available. Previously, we reported a secretory protein, SPIG1 (clone name; D/Bsp120I #1), preferentially expressed in the dorsal region in the developing chick retina. Here, we generated knock-in mice to visualize SPIG1-expressing cells with green fluorescent protein. We found that the mouse retina is subdivided into two distinct domains for SPIG1 expression and SPIG1 effectively marks a unique subtype of the retinal ganglion cells during the neonatal period. SPIG1-positive RGCs in the dorsotemporal domain project to the dorsal lateral geniculate nucleus (dLGN), superior colliculus, and accessory optic system (AOS). In contrast, in the remaining region, here named the pan-ventronasal domain, SPIG1-positive cells form a regular mosaic and project exclusively to the medial terminal nucleus (MTN) of the AOS that mediates the optokinetic nystagmus as early as P1. Their dendrites costratify with ON cholinergic amacrine strata in the inner plexiform layer as early as P3. These findings suggest that these SPIG1-positive cells are the ON direction selective ganglion cells (DSGCs). Moreover, the MTN-projecting cells in the pan-ventronasal domain are apparently composed of two distinct but interdependent regular mosaics depending on the presence or absence of SPIG1, indicating that they comprise two functionally distinct subtypes of the ON DSGCs. The formation of the regular mosaic appears to be commenced at the end of the prenatal stage and completed through the peak period of the cell death at P6. SPIG1 will thus serve as a useful molecular marker for future studies on the development and function of ON DSGCs

Expression Pattern of Kv11 (Ether à-go-go-Related Gene; erg) K+ Channels in the Mouse Retina

In response to light, most retinal neurons exhibit gradual changes in membrane potential. Therefore K+ channels that mediate threshold currents are well-suited for the fine-tuning of signal transduction. In the present study we demonstrate the expression of the different Kv11 (ether-à-go-go related gene; erg) channel subunits in the human and mouse retina by RT PCR and quantitative PCR, respectively. Immunofluorescence analysis with cryosections of mouse retinae revealed the following local distribution of the three Kv11 subunits: Kv11.1 (m-erg1) displayed the most abundant expression with the strongest immunoreactivity in rod bipolar cells. In addition, immunoreactivity was found in the inner part of the outer plexiform layer (OPL), in the inner plexiform layer (IPL) and in the inner segments of photoreceptors. Immunoreactivity for Kv11.2 (m-erg2) was observed in the outer part of the OPL and throughout the IPL. Double-labeling for vGluT1 or synaptophysin indicated a mainly presynaptic localization of Kv11.2. While no significant staining for Kv11.3 (m-erg3) was detected in the neuronal retina, strong Kv11.3 immunoreactivity was present in the apical membrane of the retinal pigment epithelium. The different expression levels were confirmed by real-time PCR showing almost equal levels of Kv11.1 and Kv11.2, while Kv11.3 mRNA expression was significantly lower. The two main splice variants of Kv11.1, isoforms a and b were detected in comparable levels suggesting a possible formation of cGMP/cGK-sensitive Kv11.1 channels in photoreceptors and rod bipolar cells. Taken together, the immunohistological results revealed different expression patterns of the three Kv11 channels in the mouse retina supposing distinct physiological roles

Visual Properties of Transgenic Rats Harboring the Channelrhodopsin-2 Gene Regulated by the Thy-1.2 Promoter

Channelrhodopsin-2 (ChR2), one of the archea-type rhodopsins from green algae, is a potentially useful optogenetic tool for restoring vision in patients with photoreceptor degeneration, such as retinitis pigmentosa. If the ChR2 gene is transferred to retinal ganglion cells (RGCs), which send visual information to the brain, the RGCs may be repurposed to act as photoreceptors. In this study, by using a transgenic rat expressing ChR2 specifically in the RGCs under the regulation of a Thy-1.2 promoter, we tested the possibility that direct photoactivation of RGCs could restore effective vision. Although the contrast sensitivities of the optomotor responses of transgenic rats were similar to those observed in the wild-type rats, they were enhanced for visual stimuli of low-spatial frequency after the degeneration of native photoreceptors. This result suggests that the visual signals derived from the ChR2-expressing RGCs were reinterpreted by the brain to form behavior-related vision

A Synaptic Mechanism for Temporal Filtering of Visual Signals

The visual system transmits information about fast and slow changes in light intensity through separate neural pathways. We used in vivo imaging to investigate how bipolar cells transmit these signals to the inner retina. We found that the volume of the synaptic terminal is an intrinsic property that contributes to different temporal filters. Individual cells transmit through multiple terminals varying in size, but smaller terminals generate faster and larger calcium transients to trigger vesicle release with higher initial gain, followed by more profound adaptation. Smaller terminals transmitted higher stimulus frequencies more effectively. Modeling global calcium dynamics triggering vesicle release indicated that variations in the volume of presynaptic compartments contribute directly to all these differences in response dynamics. These results indicate how one neuron can transmit different temporal components in the visual signal through synaptic terminals of varying geometries with different adaptational properties

Ambient light modulation of exogenous attention to threat

Planet Earth’s motion yields a 50 % day–50 % night yearly balance in every latitude or longitude, so survival must be guaranteed in very different light conditions in many species, including human. Cone- and rod-dominant vision, respectively specialized in light and darkness, present several processing differences, which are—at least partially—reflected in event-related potentials (ERPs). The present experiment aimed at characterizing exogenous attention to threatening (spiders) and neutral (wheels) distractors in two environmental light conditions, low mesopic (L, 0.03 lx) and high mesopic (H, 6.5 lx), yielding a differential photoreceptor activity balance: rod > cone and rod < cone, respectively. These distractors were presented in the lower visual hemifield while the 40 participants were involved in a digit categorization task. Stimuli, both targets (digits) and distractors, were exactly the same in L and H. Both ERPs and behavioral performance in the task were recorded. Enhanced attentional capture by salient distractors was observed regardless of ambient light level. However, ERPs showed a differential pattern as a function of ambient light. Thus, significantly enhanced amplitude to salient distractors was observed in posterior P1 and early anterior P2 (P2a) only during the H context, in late P2a during the L context, and in occipital P3 during both H and L contexts. In other words, while exogenous attention to threat was equally efficient in light and darkness, cone-dominant exogenous attention was faster than rod-dominant, in line with previous data indicating slower processing times for rod- than for cone-dominant visionThis research was supported by the Grants PSI2014-54853-P and PSI2012-37090 from the Ministerio de Economía y Competitividad of Spain (MINECO

A First- and Second-Order Motion Energy Analysis of Peripheral Motion Illusions Leads to Further Evidence of “Feature Blur” in Peripheral Vision

Anatomical and physiological differences between the central and peripheral visual systems are well documented. Recent findings have suggested that vision in the periphery is not just a scaled version of foveal vision, but rather is relatively poor at representing spatial and temporal phase and other visual features. Shapiro, Lu, Huang, Knight, and Ennis (2010) have recently examined a motion stimulus (the “curveball illusion”) in which the shift from foveal to peripheral viewing results in a dramatic spatial/temporal discontinuity. Here, we apply a similar analysis to a range of other spatial/temporal configurations that create perceptual conflict between foveal and peripheral vision.To elucidate how the differences between foveal and peripheral vision affect super-threshold vision, we created a series of complex visual displays that contain opposing sources of motion information. The displays (referred to as the peripheral escalator illusion, peripheral acceleration and deceleration illusions, rotating reversals illusion, and disappearing squares illusion) create dramatically different perceptions when viewed foveally versus peripherally. We compute the first-order and second-order directional motion energy available in the displays using a three-dimensional Fourier analysis in the (x, y, t) space. The peripheral escalator, acceleration and deceleration illusions and rotating reversals illusion all show a similar trend: in the fovea, the first-order motion energy and second-order motion energy can be perceptually separated from each other; in the periphery, the perception seems to correspond to a combination of the multiple sources of motion information. The disappearing squares illusion shows that the ability to assemble the features of Kanisza squares becomes slower in the periphery.The results lead us to hypothesize “feature blur” in the periphery (i.e., the peripheral visual system combines features that the foveal visual system can separate). Feature blur is of general importance because humans are frequently bringing the information in the periphery to the fovea and vice versa