Ground Squirrel Retina Research Articles

Characterization of Retinal Development in 13-Lined Ground Squirrels.

The cone-dominant, 13-lined ground squirrel (13-LGS) retina mimics the human central retina, but a thorough examination of retinal development in this species has not been reported. Here, the embryonic and postnatal development of the 13-LGS retina was studied to further characterize 13-LGS as a practical alternative animal model for investigating cone-based vision in health and disease. The spatiotemporal expression of key progenitor and cell type markers was examined in retinas from defined embryonic and postnatal stages using immunohistochemistry. Postnatal gene expression changes were validated by quantitative PCR. The 13-LGS neuroblastic layer expressed key progenitor markers (Sox2, Vsx2, Pax6, and Lhx2) at E18. Sequential cell fate determination evidenced by the first appearance of cell-type-specific marker labeling was at embryonic stage 18 (E18) with ganglion cells (Brn-3A, HuC/D) and microglia (Iba1); at E22.5 with photoreceptor progenitors (Otx2, recoverin) followed shortly by horizontal and amacrine cells (Lhx1, Oc1) at E24 to E25.5; and at postnatal stage 15 (P15) with bipolar cells (Vsx1, CaBP5) and Müller glia cells (GS, Rlbp1). Photoreceptor maturation indicated by opsin-positive outer segments and peanut agglutinin (PNA) labeling of cone sheaths was completed at the time of eye opening (P21-P24). The timeline and order of retinal cell development in the 13-LGS generally matches that recorded from other mammalian models but with a stark variation in the proportion of various cell types due to cone-dense photoreceptors. This thorough examination of an emerging translationally relevant cone-dominant specie provides a baseline for future disease modeling and stem cell approach studies of human vision.

Establishing the ground squirrel as a superb model for retinal ganglion cell disorders and optic neuropathies

Retinal ganglion cell (RGC) death occurs after optic nerve injury due to acute trauma or chronic degenerative conditions such as optic neuropathies (e.g., glaucoma). Currently, there are no effective therapies to prevent permanent vision loss resulting from RGC death, underlining the need for research on the pathogenesis of RGC disorders. Modeling human RGC/optic nerve diseases in non-human primates is ideal because of their similarity to humans, but has practical limitations including high cost and ethical considerations. In addition, many retinal degenerative disorders are age-related making the study in primate models prohibitively slow. For these reasons, mice and rats are commonly used to model RGC injuries. However, as nocturnal mammals, these rodents have retinal structures that differ from primates - possessing less than one-tenth of the RGCs found in the primate retina. Here we report the diurnal thirteen-lined ground squirrel (TLGS) as an alternative model. Compared to other rodent models, the number and distribution of RGCs in the TLGS retina are closer to primates. The TLGS retina possesses ~600,000 RGCs with the highest density along the equatorial retina matching the location of the highest cone density (visual streak). TLGS and primate retinas also share a similar interlocking pattern between RGC axons and astrocyte processes in the retina nerve fiber layer (RNFL). In addition, using TLGS we establish a new partial optic nerve injury model that precisely controls the extent of injury while sparing a portion of the retina as an ideal internal control for investigating the pathophysiology of axon degeneration and RGC death. Moreover, in vivo optical coherence tomography (OCT) imaging and ex vivo microscopic examinations of the retina in optic nerve injured TLGS confirm RGC loss precedes proximal axon degeneration, recapitulating human pathology. Thus, the TLGS retina is an excellent model, for translational research in neurodegeneration and therapeutic neuroprotection.This study characterizes the composition of the retinal ganglion cell (RGC) layer and the distribution of RGCs in the cone-dominant ground squirrel retina. The RGC density and the anatomical features of axon-astrocyte interaction in the ground squirrel resemble primates, rendering it an excellent alternative model for RGC neurodegeneration and neuroprotection.

Electroretinogram of the Cone-Dominant Thirteen-Lined Ground Squirrel during Euthermia and Hibernation in Comparison with the Rod-Dominant Brown Norway Rat

PurposeThe majority of small animal species used in research are nocturnal, with retinae that are anatomically and functionally dissimilar from humans, complicating their use as disease models. Herein we characterize the retinal structure and electrophysiological function of the diurnal, cone-dominant 13-lined ground squirrel (13-LGS) retina during euthermia and in hibernation.MethodsFull-field electroretinography (ERG) was performed in 13-LGS and Brown Norway (BN) rat models to establish baseline values for retinal function in each species, including following intravitreal injection of pharmacologic agents to selectively block the contributions of ON- and OFF-bipolar cells. The effect of hibernation-associated retinal remodeling on electrophysiological function was assessed in 13-LGS during torpor and emergence, with correlative histology performed using transmission electron microscopy.ResultsUnder light-adapted conditions, the a-, b-, and d-wave amplitude of the 13-LGS was significantly greater than that of the BN rat. Retinal function was absent in the 13-LGS during hibernation and correlated to widespread disruption of photoreceptor and RPE structure. Remarkably, both retinal function and structure recovered rapidly on emergence from hibernation, with ERG responses reaching normal amplitude within 6 hours.ConclusionsERG responses for both BN rats and 13-LGS reflect the relative proportions of cone photoreceptors present within the retinae, indicating that the cone-dominant 13-LGS may be a potentially useful model for studying human central retinal function and disease. That retinal remodeling and restoration of electrophysiological function occurs rapidly on emergence from hibernation implies the 13-LGS may also be a useful tool for studying aspects of retinal physiology and recovery from injury.

Using Transcriptomic Analysis to Assess Double-Strand Break Repair Activity: Towards Precise in vivo Genome Editing.

Mutations in more than 200 retina-specific genes have been associated with inherited retinal diseases. Genome editing represents a promising emerging field in the treatment of monogenic disorders, as it aims to correct disease-causing mutations within the genome. Genome editing relies on highly specific endonucleases and the capacity of the cells to repair double-strand breaks (DSBs). As DSB pathways are cell-cycle dependent, their activity in postmitotic retinal neurons, with a focus on photoreceptors, needs to be assessed in order to develop therapeutic in vivo genome editing. Three DSB-repair pathways are found in mammalian cells: Non-homologous end joining (NHEJ); microhomology-mediated end joining (MMEJ); and homology-directed repair (HDR). While NHEJ can be used to knock out mutant alleles in dominant disorders, HDR and MMEJ are better suited for precise genome editing, or for replacing entire mutation hotspots in genomic regions. Here, we analyzed transcriptomic in vivo and in vitro data and revealed that HDR is indeed downregulated in postmitotic neurons, whereas MMEJ and NHEJ are active. Using single-cell RNA sequencing analysis, we characterized the dynamics of DSB repair pathways in the transition from dividing cells to postmitotic retinal cells. Time-course bulk RNA-seq data confirmed DSB repair gene expression in both in vivo and in vitro samples. Transcriptomic DSB repair pathway profiles are very similar in adult human, macaque, and mouse retinas, but not in ground squirrel retinas. Moreover, human-induced pluripotent stem-cell-derived neurons and retinal organoids can serve as well suited in vitro testbeds for developing genomic engineering approaches in photoreceptors. Our study provides additional support for designing precise in vivo genome-editing approaches via MMEJ, which is active in mature photoreceptors.

Stereotyped Synaptic Connectivity Is Restored during Circuit Repair in the Adult Mammalian Retina

Proper function of the central nervous system (CNS) depends on the specificity of synaptic connections between cells of various types. Cellular and molecular mechanisms responsible for the establishment and refinement of these connections during development are the subject of an active area of research [1-6]. However, it is unknown if the adult mammalian CNS can form new type-selective synapses following neural injury or disease. Here, we assess whether selective synaptic connections can be reestablished after circuit disruption in the adult mammalian retina. The stereotyped circuitry at the first synapse in the retina, as well as the relatively short distances new neurites must travel compared to other areas of the CNS, make the retina well suited to probing for synaptic specificity during circuit reassembly. Selective connections between short-wavelength sensitive cone photoreceptors (S-cones) and S-cone bipolar cells provides the foundation of the primordial blue-yellowvision, common to all mammals [7-18]. Wetake advantage of the ground squirrel retina, which has a one-to-one S-cone-to-S-cone-bipolar-cellconnection, to test if this connectivity can bereestablished following local photoreceptor loss [8, 19]. We find that after invivo selective photoreceptor ablation, deafferented S-cone bipolar cells expand their dendritic trees. The new dendrites randomly explore the proper synaptic layer, bypass medium-wavelength sensitive cone photoreceptors (M-cones), and selectively synapse with S-cones. However, non-connected dendrites are not pruned back to resemble unperturbed S-cone bipolar cells. We show, for the first time, that circuit repair in the adult mammalian retina can recreate stereotypic selective wiring.

Assessment of Outer Retinal Remodeling in the Hibernating 13-Lined Ground Squirrel.

PurposeWe examined outer retinal remodeling of the euthermic and torpid cone-dominant 13-lined ground squirrel (13-LGS) retina using optical coherence tomography (OCT) imaging and histology.MethodsRetinas and corneas of living 13-LGSs were imaged during euthermic and torpid physiological states using OCT. Retinal layer thickness was measured at the visual streak from registered and averaged vertical B-scans. Following OCT, some retinas were collected immediately for postmortem histologic comparison using light microscopy, immunofluorescence, or transmission electron microscopy.ResultsCompared to OCT images from euthermic retinae, OCT images of torpid retinae revealed significantly thicker inner and outer nuclear layers, as well as increases in the distances between outer retinal reflectivity bands 1 and 2, and bands 3 and 4. A significant decrease in the distance between bands 2 and 3 also was seen, alongside significant thinning of the choriocapillaris and choroid. OCT image quality was reduced in torpid eyes, partly due to significant thickening of the corneal stroma during this state.ConclusionsThe torpid retina of the hibernating 13-LGS undergoes structural changes that can be detected by OCT imaging. Comparisons between in vivo OCT and ex vivo histomorphometry may offer insight to the origin of hyperreflective OCT bands within the outer retina of the cone-dominant 13-LGS.

Integrated transcriptomic and metabolomic analysis reveals adaptive changes of hibernating retinas.

Hibernation is a seasonally adaptive strategy that allows hibernators to live through extremely cold conditions. Despite the profound reduction of blood flow to the retinas, hibernation causes no lasting retinal injury. Instead, hibernators show an increased tolerance to ischemic insults during the hibernation period. To understand the molecular changes of the retinas in response to hibernation, we applied an integrative transcriptome and metabolome analysis to explore changes in gene expression and metabolites of 13-lined ground squirrel retinas during hibernation. Metabolomic analysis showed a global decrease of ATP synthesis in hibernating retinas. Decreased glucose and galactose, increased beta-oxidation of carnitine and decreased storage of some amino acids in hibernating retinas indicated a shift of fuel use from carbohydrates to lipids and alternative usage of amino acids. Transcriptomic analysis revealed that the down-regulated genes were enriched in DNA-templated transcription and immune-related functions, while the up-regulated genes were enriched in mitochondrial inner membrane and DNA packaging-related functions. We further showed that a subset of genes underwent active alternative splicing events in response to hibernation. Finally, integrative analysis of the transcriptome and metabolome confirmed the shift of fuel use in the hibernating retina by the regulation of catabolism of amino acids and lipids. Through transcriptomic and metabolomic data, our analysis revealed the altered state of mitochondrial oxidative phosphorylation and the shift of energy source in the hibernating retina, advancing our understanding of the molecular mechanisms employed by hibernators. The data will also serve as a useful resource for the ocular and hibernation research communities.

DSCAM localization and function at the mouse cone synapse

The Down syndrome cell adhesion molecule (DSCAM) is required for regulation of cell number, soma spacing, and cell type-specific dendrite avoidance in many types of retinal ganglion and amacrine cells. In this study we assay the organization of cells making up the outer plexiform layer of the retina in the absence of Dscam. Some types of OFF bipolar cells, type 3b and type 4 bipolar cells, had defects in dendrite arborization in the Dscam mutant retina, whereas other cell types appeared similar to wild type. The cone synapses that these cells project their dendrites to were intact, as visualized by electron microscopy, and had a distribution and density that was not significantly different from that of wild type. The spacing of type 3b bipolar cell dendrites was further analyzed by Voronoi domain analysis, density recovery profiling (DRP) analysis, and nearest neighbor analysis. Spacing was found to be significantly different when wild-type and mutant type 3b bipolar cell dendrites were compared. Defects in arborization of these bipolar cells could not be attributed to the disorganization of inner plexiform layer cells that occurs in the Dscam mutant retina or an increase in cell number, as they arborized when Dscam was targeted in retinal ganglion cells only or in the bax null retina. Localization of DSCAM was assayed and the protein was localized near to cone synapses in mouse, macaque, and ground squirrel retinas. DSCAM protein was detected in several types of bipolar cells, including type 3b and type 4 bipolar cells.

Kainate receptor subunit diversity underlying response diversity in retinal Off bipolar cells

Postsynaptic kainate receptors mediate excitatory synaptic transmission over a broad range of temporal frequencies. In heterologous systems, the temporal responses of kainate receptors vary when different channel-forming and auxiliary subunits are co-expressed but how this variability relates to the temporal differences at central synapses is incompletely understood. The mammalian cone photoreceptor synapse provides advantages for comparing the different temporal signalling roles of kainate receptors, as cones release glutamate over a range of temporal frequencies, and three functionally distinct Off bipolar cell types receive cone signals at synapses that contain either AMPA or kainate receptors, all with different temporal properties. A disadvantage is that the different receptor subunits are not identified. We used in situ hybridization, immunocytochemistry, and pharmacology to identify the kainate receptor and auxiliary subunits in ground squirrel (Ictidomys tridecimlineatus) cb1a/b, cb2, and cb3a/b Off bipolar cell types. As expected, the types showed distinct subunit expression patterns. Kainate receptors mediated ∼80% of the synaptic response in cb3a/b cells and were heteromers of GluK1 and GluK5. Cb3a/b cells contained message for GluK1 and GluK5, and also GluK3 and the auxiliary subunit Neto1. The synaptic responses in cb1a/b cells were mediated by GluK1-containing kainate receptors that behaved differently from the receptors expressed by cb3a/b cells. AMPA receptors mediated the entire synaptic response in cb2 cells and the remaining synaptic response in cb3a/b cells. We conclude that GluK1 is the predominant kainate receptor subunit in cb1 and cb3 Off bipolar cells. Different temporal response properties may result from selective association with GluK3, GluK5, or Neto1.

Glutamate Spillover between Mammalian Cone Photoreceptors

Cone photoreceptors transmit signals at high temporal frequencies and mediate fine spatial vision. High-frequency transmission requires a high rate of glutamate release, which could promote spillover to neighboring cells, whereas spatial vision requires that cones within a tightly packed array signal light to postsynaptic bipolar cells with minimal crosstalk. Glutamate spread from the cone terminal is thought to be limited by presynaptic transporters and nearby glial processes. In addition, there is no ultrastructural evidence for chemical synapses between mammalian cones, although such synapses have been described in lower vertebrate retinas. We tested for cone-cone glutamate diffusion by recording from adjacent cone pairs in the ground squirrel retina, and instead found that the glutamate released by one cone during electrical stimulation activates glutamate transporter Cl(-) conductances on neighboring cones. Unlike in other systems, where crosstalk is diminished by increasing the temperature and by moving to a more intact preparation, glutamate spread persisted at physiological temperatures (37°C) and in retinal flat mounts. The glutamate-gated anion conductance in cones has a reversal potential of ∼-30 mV compared with a cone resting potential of ∼-50 mV; thus, crosstalk should have a depolarizing effect on the cone network. Cone-cone glutamate spread is regulated by the physiological stimulus, light, and under physiological conditions can produce a response of ∼2 mV, equivalent to 13-20% of a cone's light response. We conclude that in the absence of discrete chemical synapses, glutamate flows between cones during a light response and may mediate a spatially distributed positive feedback.

Bipolar cells of the ground squirrel retina

Parallel processing of an image projected onto the retina starts at the first synapse, the cone pedicle, and each cone feeds its light signal into a minimum of eight different bipolar cell types. Hence, the morphological classification of bipolar cells is a prerequisite for analyzing retinal circuitry. Here we applied common bipolar cell markers to the cone-dominated ground squirrel retina, studied the labeling by confocal microscopy and electron microscopy, and compared the resulting bipolar cell types with those of the mouse (rod dominated) and primate retina. Eight different cone bipolar cell types (three OFF and five ON) and one rod bipolar cell were distinguished. The major criteria for classifying the cells were their immunocytochemical identity, their dendritic branching pattern, and the shape and stratification level of their axons in the inner plexiform layer (IPL). Immunostaining with antibodies against Gγ13, a marker for ON bipolar cells, made it possible to separate OFF and ON bipolars. Recoverin-positive OFF bipolar cells partly overlapped with ON bipolar axon terminals at the ON/OFF border of the IPL. Antibodies against HCN4 labeled the S-cone selective (bb) bipolar cell. The calcium-binding protein CaB5 was expressed in two OFF and two ON cone bipolar cell types, and CD15 labeled a widefield ON cone bipolar cell comparable to the DB6 in primate.

Nuclear Magnetic Resonance and Biochemical Measurements of Glucose Utilization in the Cone-Dominant Ground Squirrel Retina

To provide quantitative information on glucose utilization in cone-dominant ground squirrel retinas. Ground squirrel eyecups were incubated in medium containing (14)C-glucose, and the production of (14)CO(2) was measured. Measurements were also made of lactic acid production (glycolysis). Nuclear magnetic resonance (NMR) was used to track metabolites generated from (13)C-1 glucose. Ground squirrel eyecups produced lactate at a high rate and exhibited normal histology. Light-adaptation reduced glycolysis by 20%. Ouabain decreased glycolysis by 25% and decreased (14)CO(2) production by 60%. Blockade of glutamate receptors had little effect on the glycolysis and (14)CO(2) produced. When metabolic responses were restricted to photoreceptors, light caused a 33% decrease in (14)CO(2) production. The rate of (14)CO(2) production was less than 10% of lactate production. Lactate was the major product formed from (13)C-glucose. Other (13)C-labeled compounds included glutamate, aspartate, glutamine, alanine, taurine, and GABA. Lactate was the only product detected in the medium bathing the ground squirrel retinas. The rod-dominant rat retina exhibited a similar pattern of metabolites formed from glucose. Lactate, not CO(2), is the major product of glucose metabolism in both ground squirrel and rat retinas. Active Na(+) transport, however, depends more on ATP produced by mitochondria than by glycolysis. A relatively high fraction of ATP production from glycolysis and glucose oxidation continues in the absence of active Na(+) pumping and glutamatergic transmission. Major neurotransmitters are synthesized from the aerobic metabolism of glucose; anoxia-induced impairment in retinal synaptic transmission may be due to depletion of neurotransmitters.

Biocytin wide‐field bipolar cells in rabbit retina selectively contact blue cones

The biocytin wide-field bipolar cell in rabbit retina has a broad axonal arbor in layer 5 of the inner plexiform layer and a wide dendritic arbor that does not contact all cones in its dendritic field. The purpose of our study was to identify the types of cones that this cell contacts. We identified the bipolar cells by selective uptake of biocytin, labeled the cones with peanut agglutinin, and then used antibodies against blue cone opsin and red-green cone opsin to identify the individual cone types. The biocytin-labeled cells selectively contacted cones whose outer segments stained for blue cone opsin and avoided cones that did not. We conclude that the biocytin wide-field bipolar cell is an ON blue cone bipolar cell in the rabbit retina and is homologous to the blue cone bipolar cells that have been previously described in primate, mouse, and ground squirrel retinas.

Serca isoform expression in the mammalian retina

The sarcoplasmic–endoplasmic reticulum calcium ATPase (SERCA) is a key intracellular calcium transporter, which regulates cellular calcium concentration [Ca 2+] by transporting Ca 2+ ions from the cytosol into the endoplasmic reticulum. SERCA-mediated Ca 2+ sequestration controls proper folding of newly synthesized proteins within the ER as well as the timing and spatial patterning of depolarization-evoked Ca 2+ responses in the cytoplasm. To understand the spatial and temporal properties of Ca 2+ homeostasis in retinal neurons better, I studied expression and distribution of all three SERCA isoforms in the mouse retina using isoform-specific antibodies. No immunostaining was observed with the SERCA1 antibody. SERCA2 was expressed in photoreceptor inner segments, amacrine and ganglion cells of the mouse retina. Similar SERCA2 localization was observed in adult rat, macaque and ground squirrel retinas. Analysis of distribution of SERCA2 immunofluorescence in the developing mouse retina revealed prominent SERCA2 signals throughout postnatal development. The N89 antibodys used to identify the SERCA3 isoforms labelled cone outer segments, inner segments of photoreceptors and cell processes in the inner nuclear layer of the mouse retina. These results imply that the SERCA2 isoform controls Ca 2+ sequestration into the endoplasmic reticulum in most classes of retinal neuron. A potential role for SERCA3 in cone function is suggested.

Nonsynaptic localization of the excitatory amino acid transporter 4 in photoreceptors

Excitatory amino acid transporters (EAATs) are involved in regulating extracellular glutamate levels at synaptic regions in the CNS. EAAT1, 2, 3, and 5 have been found in the mammalian retina, but the presence of EAAT4 has remained controversial. Recently, we found a high level of EAAT4 mRNA in the human retina, and this observation lead us to examine whether EAAT4 was expressed in the mammalian retina. Immunoblotting studies showed the presence of EAAT4-immunoreactive proteins in human and mouse retinas, corresponding to EAAT4 monomers and dimers. Immunohistochemistry revealed that EAAT4 was localized in rod and cone photoreceptor outer segments in the human retina, and in the outer and inner segments of mouse and ground squirrel retinas. In no case was EAAT4 found in the outer plexiform layer or in any other layer in the retina. EAAT4 expression by photoreceptors was confirmed by immunoblotting a purified rod outer segment preparation, which showed the presence of a 50-kDa EAAT4-immunoreactive protein. In addition, the EAAT4-associated protein, GTRAP41, was found in the human, mouse, and squirrel retinas as well as in the rod outer segment preparation. Further immunocytochemical and co-immunoprecipitation experiments demonstrated that GTRAP41 was colocalized and interacted in vivo with EAAT4. Importantly, glutamate uptake and drug inhibition experiments showed that an EAAT4-like glutamate uptake system is present in the rod outer segments. Finally, we examined whether glutamate signaling mediated by EAAT4 can modulate rod outer segment phagocytosis by the retinal pigment epithelium. Results of the present study show that EAAT4 is present in the outer segments, a nonsynaptic region of photoreceptors, where it might provide a feedback mechanism for sensing extracellular glutamate or serve as an outer barrier to prevent glutamate from escaping from the retina.

In intact mammalian photoreceptors, Ca2+-dependent modulation of cGMP-gated ion channels is detectable in cones but not in rods.

In the mammalian retina, cone photoreceptors efficiently adapt to changing background light intensity and, therefore, are able to signal small differences in luminance between objects and backgrounds, even when the absolute intensity of the background changes over five to six orders of magnitude. Mammalian rod photoreceptors, in contrast, adapt very little and only at intensities that nearly saturate the amplitude of their photoresponse. In search of a molecular explanation for this observation we assessed Ca2+-dependent modulation of ligand sensitivity in cyclic GMP–gated (CNG) ion channels of intact mammalian rods and cones. Solitary photoreceptors were isolated by gentle proteolysis of ground squirrel retina. Rods and cones were distinguished by whether or not their outer segments bind PNA lectin. We measured membrane currents under voltage-clamp in photoreceptors loaded with Diazo-2, a caged Ca2+ chelator, and fixed concentrations of 8Br-cGMP. At 600 nM free cytoplasmic Ca2+ the midpoint of the cone CNG channels sensitivity to 8BrcGMP, 8BrcGMPK1/2, is ∼2.3 μM. The ligand sensitivity is less in rod than in cone channels. Instantly decreasing cytoplasmic Ca2+ to <30 nM activates a large inward membrane current in cones, but not in rods. Current activation arises from a Ca2+ -dependent modulation of cone CNG channels, presumably because of an increase in their affinity to the cyclic nucleotide. The time course of current activation is temperature dependent; it is well described by a single exponential process of ∼480 ms time constant at 20–21°C and 138 ms at 32°C. The absence of detectable Ca2+-dependent CNG current modulation in intact rods, in view of the known channel modulation by calmodulin in-vitro, affirms the modulation in intact rods may only occur at low Ca2+ concentrations, those expected at intensities that nearly saturate the rod photoresponse. The correspondence between Ca2+ dependence of CNG modulation and the ability to light adapt suggest these events are correlated in photoreceptors.

Choline acetyltransferase is expressed by non-starburst amacrine cells in the ground squirrel retina

We have used immunostaining techniques to reveal a new type of amacrine cell that is immunoreactive for choline acetyltransferase (ChAT), the acetylcholine synthesizing enzyme, in the Ground Squirrel ( Spermophilus beecheyi) retina. Cryostat sections and double immunostained wholemount preparations were examined by confocal microscopy. This new ChAT type III cell is distinct in morphology and neurotransmitter content from the well know ‘starburst’ amacrine cells (types I and II) that are so well represented in the ground squirrel retina [J. Comp. Neurol. 365 (1996) 173–216]. The type III cell colocalizes glycine with the acetylcholine and does not appear to be GABAergic or exhibit calcium-binding proteins like the well-known starburst type. As well, type III cells do not occur as a mirror-symmetric pair with normally placed and displaced varieties. The type III cell is probably a small field amacrine type branching broadly in upper sublamina b of the inner plexiform layer, and is most likely A6 of the Ground Squirrel retina [J. Comp. Neurol. 365 (1996) 173–216]. Type III cells are ideally placed in the architecture of the Ground Squirrel retina to influence ON directionally selective ganglion cell types.

Electrical Coupling between Mammalian Cones

Background: Cone photoreceptors are noisy because of random fluctuations of photon absorption, signaling molecules, and ion channels. However, each cone's noise is independent of the others, whereas their signals are partially shared. Therefore, electrically coupling the synaptic terminals prior to forward transmission and subsequent nonlinear processing can appreciably reduce noise relative to the signal. This signal-processing strategy has been demonstrated in lower vertebrates with rather coarse vision, but its occurrence in mammals with fine acuity has been doubted (even though gap junctions are present) because coupling would blur the neural image.Results: In ground squirrel retina, whose triangular cone lattice resembles the human fovea, paired electrical recordings from adjacent cones demonstrated electrical coupling with an average conductance of approximately 320 pS. Blur caused by this degree of coupling had a space constant of approximately 0.5 cone diameters. Psychophysical measurements employing laser interferometry to bypass the eye's optics suggest that human foveal cones experience a similar degree of neural blur and that it is invariant with light intensity. This neural blur is narrower than the eye's optical blur, and we calculate that it should improve the signal-to-noise ratio at the cone terminal by about 77%.Conclusions: We conclude that the gap junctions observed between mammalian cones, including those in the human fovea, represent genuine electrical coupling. Because the space constant of the resulting neural blur is less than that of the optical blur, the signal-to-noise ratio can be markedly improved before the nonlinear stages with little compromise to visual acuity.

Experimental retinal detachment in the cone-dominant ground squirrel retina: morphology and basic immunocytochemistry.

The cellular responses of the cone-dominant ground squirrel retina to retinal detachment were examined and compared to those in rod-dominant species. Retinal detachments were made in California ground squirrels. The retinas were prepared for light, electron, and confocal microscopy. Tissue sections were labeled with antibodies to cone opsins, rod opsin, glial fibrillary acidic protein (GFAP), vimentin, synaptophysin, cytochrome oxidase, and calbindin D 28K. Wax sections were probed with the MIB-1 antibody to detect proliferating cells. By 10 h postdetachment many photoreceptor cells in the ground squirrel already show structural signs of apoptosis. At 1 day many photoreceptors have collapsed inner segments (IS), yet others still have short stacks of outer segment discs. At 3 days there is a marked increase in the number of dying photoreceptors. Rod and medium-/long-wavelength opsins are redistributed in the cell membrane to their synaptic terminals. At 7 days photoreceptor cell death has slowed. Some regions of the outer nuclear layer (ONL) have few photoreceptor somata. IS remnants are rare on surviving photoreceptors. At 28 days these trends are even more dramatic. Retinal pigmented epithelium (RPE) cells do not expand into the subretinal space. The outer limiting membrane (OLM) appears flat and uninterrupted. Müller cells remain remarkably unreactive; they show essentially no proliferation, only negligible hypertrophy, and there is no increase in their expression of GFAP or vimentin. Horizontal cells show no dendritic sprouting in response to detachment. The speed and extent of photoreceptor degeneration in response to detachment is greater in ground squirrel than in cat retina-only a small number of rods and cones survive at 28 days of detachment. Moreover, the almost total lack of Müller cell and RPE reactivity in the ground squirrel retina is a significant difference from results in other species.

An animal model for studying cone function in retinal detachment.

In people, retinal detachment often leads to a significant loss in cone-based vision. Most of the animal models commonly used for studying the consequences of retinal detachment have rod-dominated retinas. The purpose of this investigation was to evaluate the possibility that the ground squirrel, a rodent with a heavily cone-dominated retina, might provide a useful model for studying cone function in retinal detachment. Corneal ERGs were recorded from ground squirrels for large-field temporal modulations presented on a computer-controlled color monitor. Modulations were chosen to selectively stimulate either of the two classes of cone found in the ground squirrel retina. Under these test conditions, large and reliable cone ERGs could be readily recorded. In animals in which the retina had been surgically detached, the loss of cone signal was directly related to the number of cones in the detachment zone relative to the total cone population and that relationship did not differ for short-wavelength sensitive (S) and middle-wavelength sensitive (M) cones. Surgical reattachment produced a progressive recovery of cone-based signals. The ground squirrel seems likely to provide a useful animal model for studying the dynamics of cone function in retinal detachment and subsequent events.