Maureen A. McCall

The nob2 mouse, a null mutation in Cacna1f : Anatomical and functional abnormalities in the outer retina and their consequences on ganglion cell visual responses

Glutamate release from photoreceptor terminals is controlled by voltage-dependent calcium channels (VDCCs). In humans, mutations in the Cacna1f gene, encoding the alpha1F subunit of VDCCs, underlie the incomplete form of X-linked congenital stationary night blindness (CSNB2). These mutations impair synaptic transmission from rod and cone photoreceptors to bipolar cells. Here, we report anatomical and functional characterizations of the retina in the nob2 (no b-wave 2) mouse, a naturally occurring mutant caused by a null mutation in Cacna1f. Not surprisingly, the b-waves of both the light- and dark-adapted electroretinogram are abnormal in nob2 mice. The outer plexiform layer (OPL) is disorganized, with extension of ectopic neurites through the outer nuclear layer that originate from rod bipolar and horizontal cells, but not from hyperpolarizing bipolar cells. These ectopic neurites continue to express mGluR6, which is frequently associated with profiles that label with the presynaptic marker Ribeye, indicating potential points of ectopic synapse formation. However, the morphology of the presynaptic Ribeye-positive profiles is abnormal. While cone pedicles are present their morphology also appears compromised. Characterizations of visual responses in retinal ganglion cells in vivo, under photopic conditions, demonstrate that ON-center cells have a reduced dynamic range, although their basic center-surround organization is retained; no alteration in the responses of OFF-center cells was evident. These results indicate that nob2 mice are a valuable model in which to explore the pathophysiological mechanisms associated with Cacna1f mutations causing CSNB2, and the subsequent effects on visual information processing. Further, the nob2 mouse represents a model system in which to define the signals that guide synapse formation and/or maintenance in the OPL.

Mutations in TRPM1 Are a Common Cause of Complete Congenital Stationary Night Blindness

Congenital stationary night blindness (CSNB) is a clinically and genetically heterogeneous group of retinal disorders characterized by nonprogressive impaired night vision and variable decreased visual acuity. We report here that six out of eight female probands with autosomal-recessive complete CSNB (cCSNB) had mutations in TRPM1, a retinal transient receptor potential (TRP) cation channel gene. These data suggest that TRMP1 mutations are a major cause of autosomal-recessive CSNB in individuals of European ancestry. We localized TRPM1 in human retina to the ON bipolar cell dendrites in the outer plexifom layer. Our results suggest that in humans, TRPM1 is the channel gated by the mGluR6 (GRM6) signaling cascade, which results in the light-evoked response of ON bipolar cells. Finally, we showed that detailed electroretinography is an effective way to discriminate among patients with mutations in either TRPM1 or GRM6, another autosomal-recessive cCSNB disease gene. These results add to the growing importance of the diverse group of TRP channels in human disease and also provide new insights into retinal circuitry.

Failure to Maintain Eye-Specific Segregation in nob, a Mutant with Abnormally Patterned Retinal Activity

Axon terminals from the two eyes initially overlap in the dorsal-lateral geniculate nucleus (dLGN) but subsequently refine to occupy nonoverlapping territories. Retinal activity is required to establish and maintain this segregation. We show that despite the presence of retinal activity, segregated projections desegregate when the structure of activity is altered. Early in development, spontaneous retinal activity in the no b-wave (nob) mouse is indistinguishable from that of wild-type mice, and eye-specific segregation proceeds normally. But, around eye-opening, spontaneous and visually evoked activity in nob retinas become abnormal, coincident with a failure to preserve precise eye-specific territories. Dark-rearing studies suggest that altered visual experience is not responsible. Transgenic rescue of the mutated protein (nyctalopin) within nob retinal interneurons, without rescuing expression in either retinal projection neurons or their postsynaptic targets in the dLGN, restores spontaneous retinal activity patterns and prevents desegregation. Thus, normally structured spontaneous retinal activity stabilizes newly refined retinogeniculate circuitry.

Presynaptic inhibition modulates spillover, creating distinct dynamic response ranges of sensory output.

Sensory information is thought to be modulated by presynaptic inhibition. Although this form of inhibition is a well-studied phenomenon, it is still unclear what role it plays in shaping sensory signals in intact circuits. By visually stimulating the retinas of transgenic mice lacking GABAc receptor-mediated presynaptic inhibition, we found that this inhibition regulated the dynamic range of ganglion cell (GC) output to the brain. Presynaptic inhibition acted differentially upon two major retinal pathways; its elimination affected GC responses to increments, but not decrements, in light intensity across the visual scene. The GC dynamic response ranges were different because presynaptic inhibition limited glutamate release from ON, but not OFF, bipolar cells, which modulate the extent of glutamate spillover and activation of perisynaptic NMDA receptors at ON GCs. Our results establish a role for presynaptic inhibitory control of spillover in determining sensory output in the CNS.

GABAC receptor-mediated inhibition in the retina

Inhibition at bipolar cell axon terminals regulates excitatory signaling to ganglion cells and is mediated, in part, by GABAC receptors. We investigated GABAC receptor-mediated inhibition using pharmacological approaches and genetically altered mice that lack GABAC receptors. Responses to applied GABA showed distinct time courses in various bipolar cell classes, attributable to different proportions of GABAA and GABAC receptors. The elimination of GABAC receptors in GABAC null mice reduced and shortened GABA-activated currents and light-evoked inhibitory synaptic currents (L-IPSCs) in rod bipolar cells. ERG measurements and recordings from the optic nerve showed that inner retinal function was altered in GABAC null mice. These data suggest that GABAC receptors determine the time course and extent of inhibition at bipolar cell terminals that, in turn, modulates the magnitude of excitatory transmission from bipolar cells to ganglion cells.

Generation of an Inbred Miniature Pig Model of Retinitis Pigmentosa

PURPOSE The Pro23His (P23H) rhodopsin (RHO) mutation underlies the most common form of human autosomal dominant retinitis pigmentosa (adRP). The objective of this investigation was to establish a transgenic miniature swine model of RP using the human P23H RHO gene. METHODS Somatic cell nuclear transfer (SCNT) was used to create transgenic miniature pigs that expressed the human P23H RHO mutation. From these experiments, six transgenic founders were identified whose retinal function was studied with full-field electroretinography (ffERG) from 3 months through 2 years. Progeny from one founder were generated and genotyped to determine transgene inheritance pattern. Retinal mRNA was isolated, and the ratio of P23H to wild-type pig RHO was measured. RESULTS A single transgene integration site was observed for five of the six founders. All founders had abnormal scotopic and photopic ffERGs after 3 months. The severity of the ffERG phenotype was grouped into moderately and severely affected groups. Offspring of one founder inherited the transgene as an autosomal dominant mutation. mRNA analyses demonstrated that approximately 80% of total RHO was mutant P23H. CONCLUSIONS Expression of the human RHO P23H transgene in the retina creates a miniature swine model with an inheritance pattern and retinal function that mimics adRP. This large-animal model can serve as a novel tool for the study of the pathogenesis and therapeutic intervention in the most common form of adRP.

Presynaptic inhibition differentially shapes transmission in distinct circuits in the mouse retina

Diverse retinal outputs are mediated by ganglion cells that receive excitatory input from distinct classes of bipolar cells (BCs). These classes of BCs separate visual signals into rod, ON and OFF cone pathways. Although BC signalling is a major determinant of the ganglion cell‐mediated retinal output, it is not fully understood how light‐evoked, presynaptic inhibition from amacrine cell inputs shapes BC outputs. To determine whether differences in presynaptic inhibition uniquely modulate BC synaptic output to specific ganglion cells, we assessed the inhibitory contributions of GABAA, GABAC and glycine receptors across the BC pathways. Here we show that different proportions of GABAA and GABAC receptor‐mediated inhibition determined the kinetics of GABAergic presynaptic inhibition across different BC classes. Large, slow GABAC and small, fast GABAA receptor‐mediated inputs to rod BCs prolonged light‐evoked inhibitory postsynaptic currents (L‐IPSCs), while smaller GABAC and larger GABAA receptor‐mediated contributions produced briefer L‐IPSCs in ON and OFF cone BCs. Glycinergic inhibition also varied across BC class. In the rod‐dominant conditions studied here, slow glycinergic inputs dominated L‐IPSCs in OFF cone BCs, attributable to inputs from the rod pathway via AII amacrine cells, while rod and ON cone BCs received little and no glycinergic input, respectively. As these large glycinergic inputs come from rod signalling pathways, in cone‐dominant conditions L‐IPSCs in OFF cone bipolar cells will probably be dominated by GABAA receptor‐mediated input. Thus, unique presynaptic receptor combinations mediate distinct forms of inhibition to selectively modulate BC outputs, enhancing the distinctions among parallel retinal signals.

GPR179 is required for depolarizing bipolar cell function and is mutated in autosomal-recessive complete congenital stationary night blindness

Complete congenital stationary night blindness (cCSNB) is a clinically and genetically heterogeneous group of retinal disorders characterized by nonprogressive impairment of night vision, absence of the electroretinogram (ERG) b-wave, and variable degrees of involvement of other visual functions. We report here that mutations in GPR179, encoding an orphan G protein receptor, underlie a form of autosomal-recessive cCSNB. The Gpr179(nob5/nob5) mouse model was initially discovered by the absence of the ERG b-wave, a component that reflects depolarizing bipolar cell (DBC) function. We performed genetic mapping, followed by next-generation sequencing of the critical region and detected a large transposon-like DNA insertion in Gpr179. The involvement of GPR179 in DBC function was confirmed in zebrafish and humans. Functional knockdown of gpr179 in zebrafish led to a marked reduction in the amplitude of the ERG b-wave. Candidate gene analysis of GPR179 in DNA extracted from patients with cCSNB identified GPR179-inactivating mutations in two patients. We developed an antibody against mouse GPR179, which robustly labeled DBC dendritic terminals in wild-type mice. This labeling colocalized with the expression of GRM6 and was absent in Gpr179(nob5/nob5) mutant mice. Our results demonstrate that GPR179 plays a critical role in DBC signal transduction and expands our understanding of the mechanisms that mediate normal rod vision.

Rod vision is controlled by dopamine-dependent sensitization of rod bipolar cells by GABA

Dark and light adaptation of retinal neurons allow our vision to operate over an enormous light intensity range. Here we report a mechanism that controls the light sensitivity and operational range of rod-driven bipolar cells that mediate dim-light vision. Our data indicate that the light responses of these cells are enhanced by sustained chloride currents via GABA(C) receptor channels. This sensitizing GABAergic input is controlled by dopamine D1 receptors, with horizontal cells serving as a plausible source of GABA release. Our findings expand the role of dopamine in vision from its well-established function of suppressing rod-driven signals in bright light to enhancing the same signals under dim illumination. They further reveal a role for GABA in sensitizing the circuitry for dim-light vision, thereby complementing GABAs traditional role in providing dynamic feedforward and feedback inhibition in the retina.

Stimulus size and intensity alter fundamental receptive-field properties of mouse retinal ganglion cells in vivo

The receptive field (RF) of most retinal ganglion cells (RGCs) is comprised of an excitatory center and an antagonistic surround. Interactions between these RF elements shape most of the visual responses of RGCs. To begin to investigate center-surround interactions of mouse RGCs quantitatively, we characterized their responses in an in vivo preparation to a variety of spot and full-field stimuli. When RGCs were stimulated with a spot that matched the cells RF center diameter (optimal spot), all RGCs could be categorized as either ON- or OFF-center. In all RGCs, full-field stimulation significantly reduced both the peak and the mean firing rates evoked with an optimal spot stimulus. Full-field stimulation revealed differences in other response properties between ON- and OFF-center RGCs. With a full-field stimulus, the duration of the OFF-center RGCs response was reduced making them more transient, while the duration of the ON-center RGCs increased making them more sustained. Of most interest, full-field stimulation altered the RF center response sign in approximately half of the OFF-center RGCs, which became either OFF/ON or ON only. In contrast, all ON-center and the other OFF-center cells conserved their RF response sign in the presence of the full-field stimulus. We propose that sign-altering OFF-center RGCs possess an additional RF surround mechanism that underlies this alteration in their response. Of general interest these results suggest that the sole use of full-field stimulation to categorize visual response properties of RGCs does not adequately reflect their RF organization and, therefore, is not an optimal strategy for their classification.