Roy A. Jacoby

How do tonic glutamatergic synapses evade receptor desensitization

Photoreceptor output synapses are the best known tonic chemical synapses in the nervous system, in which glutamate is continuously released in darkness, activating AMPA/kainate receptors in postsynaptic neurons. It has been shown that glutamate receptors in certain types of second‐order retinal cells are largely desensitized in darkness, leading to small postsynaptic currents and reduced response dynamic ranges. Here we show that the tonic glutamatergic synapses between photoreceptors and rod‐dominated hyperpolarizing bipolar cells (HBCRs) in the salamander retina evade postsynaptic receptor desensitization by using (1) multiple invaginating ribbon junctions as releasing sites for low‐frequency, synchronized multiquantal release at each site; and (2) the GluR4 AMPA receptors as the postsynaptic receptors. The multiquantal events exhibit faster decay time than the GluR4 receptor desensitization time constant and therefore self‐desensitization is minimized, and the average inter‐event duration in darkness is much longer than the GluR4 desensitization recovery time and thus mutual desensitization is avoided. Consequently, the HBCRs are not desensitized in darkness, allowing light signals to be encoded by the full operating range of the glutamate‐gated postsynaptic currents. Our study illustrates for the first time how a tonic glutamatergic synapse avoids postsynaptic receptor desensitization, a strategy that may be shared by many other synapses in the nervous system that need extended operation capacity.

Light- and dopamine-regulated receptive field plasticity in primate horizontal cells

Center‐surround antagonistic receptive fields (CSARFs) are building blocks for spatial vision and contrast perception. Retinal horizontal cells (HCs) are the first lateral elements along the visual pathway, and are thought to contribute to receptive field surrounds of higher order neurons. Primate HC receptive fields have not been found to change with light, and dopaminergic modulation has not been investigated. Recording intracellularly from HCs in dark‐adapted macaque retina, we found that H1‐HCs had large receptive fields (λ = 1,158 ± 137 μm) that were reduced by background light (−45%), gap junction closure (−53%), and D1 dopamine receptor activation (−48%). Tracer coupling was modulated in a correlative manner, suggesting that coupling resistance plays a dominant role in receptive field formation under low light conditions. The D1 antagonist SCH23390 increased the size of receptive fields (+13%), suggesting tonic dopamine release in the dark. Because light elevates dopamine release in primate retina, our results support a dopaminergic role in post‐receptoral light adaptation by decreasing HC receptive field diameters, which influences the center‐surround receptive field organization of higher‐order neurons and thereby spatial contrast sensitivity. J. Comp. Neurol. 519:2125–2134, 2011.

Morphological and electrophysiological properties of dissociated primate retinal cells.

Although isolated retinal cell preparations have been used widely to study retinal function in lower vertebrates, dissociated cells from primate retina have not been developed for routine physiological experiments. In this study, we demonstrated the feasibility of obtaining viable and identifiable dissociated cells from the primate retina. In addition, we characterized voltage-dependent membrane currents in each type of primate retinal cell with the whole-cell patch-clamp technique. Multiple types of ionic conductance with distinctive current profiles were recorded in various types of primate retinal neurons. Photoreceptors exhibited an inward I(H) activated by membrane hyperpolarization and an outward current activated at depolarized potentials. Two types of potassium currents (transient potassium current, I(K(A)), and delayed rectifier potassium current, I(K(V))) were recorded from bipolar cells. I(K(A)) dominated the current response in putative midget bipolar cells, and I(K(V)) was mainly associated with putative rod bipolar cells. L-type calcium currents (I(Ca)) were observed in primate bipolar cells with axon terminals, but not in axotomized bipolar cells. Large voltage-dependent sodium currents (I(Na)) were only recorded from ganglion cells. Muller cells exhibited I(K(V)) and large potassium inward rectifier current (I(K(IR))), and occasionally a small I(Na). Neurons with electrophysiological signatures of amacrine cells and horizontal cells were also studied even though their morphological features were lost during cell dissociation. By using both morphological and physiological criteria outlined in this report, it is possible to use the dissociated retinal cell preparation as an in vitro system for physiological, biochemical and pharmacological studies of the primate visual system.

AMPA-preferring receptors mediate excitatory non-NMDA responses of primate retinal ganglion cells.

Glutamate and kainate-induced currents of primate ganglion cells were studied using the whole-cell patch-clamp technique in a retinal slice preparation. Antagonists and allosteric modulators of desensitization selective for either alpha-amino-3-hydroxy-5-methyl-4-isoazoleprionic acid (AMPA)- or kainate-preferring receptors were used to determine the contributions of each type of receptor to excitatory responses. With synaptic transmission and NMDA receptors blocked, the AMPA-preferring receptor antagonist GYKI 52466 (30 microM-100 microM) reversibly blocked most of the glutamate-induced current in ganglion cells. GYKI 52466 also blocked the response in ganglion cells to focally applied kainate, suggesting that the current response to kainate arises from activation of AMPA-preferring receptors, and not kainate-preferring receptors. Both cyclothiazide (10 microM-100 microM) and the novel drug 4-[2-(phenylsulfonylamino)ethylthio]-2,6-difluoro-phenoxyacetamide (PEPA, 20 microM-100 microM), which selectively enhance responses mediated by AMPA-preferring receptors, enhanced glutamate-induced responses of ganglion cells. Since these drugs preferentially inhibit desensitization of the flip and flop splice variants, respectively, of AMPA-preferring receptors, it is likely that both splice variants are present on these ganglion cells. Concanavalin A, which selectively suppresses the desensitization of kainate-preferring receptors, had no effect on the glutamate-induced responses of ganglion cells. We conclude that the non-NMDA component of the excitatory, glutamatergic input to primate ganglion cells is mediated largely by AMPA-preferring receptors, with little, if any, kainate-preferring receptor-mediated response.

Synaptic connections of amacrine cells containing vesicular glutamate transporter 3 in baboon retinas

The goals of these experiments were to describe the morphology and synaptic connections of amacrine cells in the baboon retina that contain immunoreactive vesicular glutamate transporter 3 (vGluT3). These amacrine cells had the morphology characteristic of knotty bistratified type 1 cells, and their dendrites formed two plexuses on either side of the center of the inner plexiform layer. The primary dendrites received large synapses from amacrine cells, and the higher-order dendrites were both pre- and postsynaptic to other amacrine cells. Based on light microscopic immunolabeling results, these include AII cells and starburst cells, but not the polyaxonal amacrine cells tracer-coupled to ON parasol ganglion cells. The vGluT3 cells received input from ON bipolar cells at ribbon synapses and made synapses onto OFF bipolar cells, including the diffuse DB3a type. Many synapses from vGluT3 cells onto retinal ganglion cells were observed in both plexuses. At synapses where vGluT3 cells were presynaptic, two types of postsynaptic densities were observed; there were relatively thin ones characteristic of inhibitory synapses and relatively thick ones characteristic of excitatory synapses. In the light microscopic experiments with Neurobiotin-injected ganglion cells, vGluT3 cells made contacts with midget and parasol ganglion cells, including both ON and OFF types. Puncta containing immunoreactive gephyrin, an inhibitory synapse marker, were found at appositions between vGluT3 cells and each of the four types of labeled ganglion cells. The vGluT3 cells did not have detectable levels of immunoreactive γ-aminobutyric acid (GABA) or immunoreactive glycine transporter 1. Thus, the vGluT3 cells would be expected to have ON responses to light and make synapses onto neurons in both the ON and the OFF pathways. Taken with previous results, these findings suggest that vGluT3 cells release glycine at some of their output synapses and glutamate at others.

Expression pattern of connexins in the corneal and limbal epithelium of a primate.

Purpose: To detect the expression pattern of connexins in epithelial cells of the central cornea and limbus of the macaque. Methods: Total RNA was extracted from the central corneal and limbal epithelia of Macaca fascicularis and processed by reverse transcriptase-polymerase chain reaction with isoform primers to detect the expression of 16 connexin (Cx). Immunofluorescent staining of frozen sections of corneal tissue confirmed and localized connexin proteins expression. Results: Transcripts encoding 10 Cx isoforms (Cx26, Cx30, Cx30.3, Cx31, Cx31.1, Cx32, Cx43, Cx45, Cx50, and Cx58) were detected by reverse transcriptase-polymerase chain reaction in both central and peripheral corneal epithelium. Six (Cx26, Cx31, Cx32, Cx43, Cx45, and Cx58) were confirmed by laser scanning confocal microscopy. Cx26 was detected throughout the central corneal epithelium and in the mid and superficial layers of the limbal epithelium. Cx43 and Cx45 were localized to the basal and suprabasal epithelial cells. Cx58 was expressed in the superficial epithelium throughout the cornea, whereas Cx31 and Cx32 were mainly expressed in the central corneal epithelium and weakly in the limbal area. Conclusions: The complex distribution pattern of the connexins suggests that selected isoforms play important roles in maintaining corneal homeostasis.

Blue cone bipolar cells of the macaque retina

Antisera which recognize glycine-extended precursors of the peptide chole-cystokin in labeled neurons in the macaque retina with dendrites that ran obliquely and had diffuse dendritic terminals as is the case for blue cone bipolar cells described previously. They clearly formed a single population since the density and sizes of their perikarya varied monotonically with eccentricity, while the levels of their perikarya and axon terminals were constant. Short-wavelength cones were also labeled in some preparations, either with Procion Black or anti-blue opsin, and the labeled bipolar cells received input exclusively from these cones. The dendrites formed the central elements at ribbon synapses in the short-wavelength cones, and the axons of the labeled bipolar cells branched in the fifth stratum of the inner plexiform layer. Taken together, these findings indicate that the labeled bipolar cells were different from the invaginating midget bipolar cells that contact middle- and long-wavelength cones and suggest that the labeled bipolar cells depolarize in response to increments in short-wavelength stimuli. There were numerous, unlabeled bipolar cell dendrites receiving inputs from short-wavelength cones at other types of contacts, however, and these presumably belonged to bipolar cells with hyperpolarizing light responses.

Physiological and morphological characterization of ganglion cells in the salamander retina

Retinal ganglion cells (RGCs) integrate visual information from the retina and transmit collective signals to the brain. A systematic investigation of functional and morphological characteristics of various types of RGCs is important to comprehensively understand how the visual system encodes and transmits information via various RGC pathways. This study evaluated both physiological and morphological properties of 67 RGCs in dark-adapted flat-mounted salamander retina by examining light-evoked cation and chloride current responses via voltage-clamp recordings and visualizing morphology by Lucifer yellow fluorescence with a confocal microscope. Six groups of RGCs were described: asymmetrical ON-OFF RGCs, symmetrical ON RGCs, OFF RGCs, and narrow-, medium- and wide-field ON-OFF RGCs. Dendritic field diameters of RGCs ranged 102-490 μm: narrow field (<200 μm, 31% of RGCs), medium field (200-300 μm, 45%) and wide field (>300 μm, 24%). Dendritic ramification patterns of RGCs agree with the sublamina A/B rule. 34% of RGCs were monostratified, 24% bistratified and 42% diffusely stratified. 70% of ON RGCs and OFF RGCs were monostratified. Wide-field RGCs were diffusely stratified. 82% of RGCs generated light-evoked ON-OFF responses, while 11% generated ON responses and 7% OFF responses. Response sensitivity analysis suggested that some RGCs obtained separated rod/cone bipolar cell inputs whereas others obtained mixed bipolar cell inputs. 25% of neurons in the RGC layer were displaced amacrine cells. Although more types may be defined by more refined classification criteria, this report is to incorporate more physiological properties into RGC classification.