Andrew T. Ishida

VOLTAGE-GATED NA+ CURRENT AVAILABILITY AFTER STEP- AND SPIKE-SHAPED CONDITIONING DEPOLARIZATIONS OF RETINAL GANGLION CELLS

Abstract We used two conditioning voltage protocols to assess inactivation of voltage-gated Na+ current in retinal ganglion cells. The first protocol tested the possibility, raised by published activation and steady-state inactivation curves, that Na+ ions carry a ”window” current in these cells. The second protocol was used, because these cells spike repetitively in situ, to measure the Na+ current available for activation following spikes. Na+ current activated at test potentials more positive than –65 mV. At test potentials more positive than –55 mV, Na+ current peaked and then declined along a time course that could be fit by the sum of a large, rapidly decaying component, a small, slowly decaying component and a non-decaying component. Both step- and spike-shaped conditioning depolarizations reduced the amount of current available for subsequent activation, sparing the non-decaying ”persistent” component. Most of the Na+ current recovered from this inactivation along a rapid exponential time course (τ=3 ms). The remaining recovery was complete within at least 4 s (at –70 mV). Our use of step depolarizations has identified a current component not anticipated from previous measurements of steady-state inactivation in retinal ganglion cells. Our use of spike-shaped depolarizations shows that Na+ current density at 1 ms after a single spike is roughly 25% of that activated by the conditioning spike, and that recovery from inactivation is 50–90% complete within 10 ms thereafter. Na+ current amplitude declines during spikes repeated at relatively low frequencies, consistent with a slow component of full recovery from inactivation.

Colocalization of hyperpolarization-activated, cyclic nucleotide-gated channel subunits in rat retinal ganglion cells

The current‐passing pore of mammalian hyperpolarization‐activated, cyclic nucleotide‐gated (HCN) channels is formed by subunit isoforms denoted HCN1–4. In various brain areas, antibodies directed against multiple isoforms bind to single neurons, and the current (Ih) passed during hyperpolarizations differs from that of heterologously expressed homomeric channels. By contrast, retinal rod, cone, and bipolar cells appear to use homomeric HCN channels. Here, we assess the generality of this pattern by examining HCN1 and HCN4 immunoreactivity in rat retinal ganglion cells, measuring Ih in dissociated cells, and testing whether HCN1 and HCN4 proteins coimmunoprecipitate. Nearly half of the ganglion cells in whole‐mounted retinae bound antibodies against both isoforms. Consistent with colocalization and physical association, 8‐bromo‐cAMP shifted the voltage sensitivity of Ih less than that of HCN4 channels and more than that of HCN1 channels, and HCN1 coimmunoprecipitated with HCN4 from membrane fraction proteins. Finally, the immunopositive somata ranged in diameter from the smallest to the largest in rat retina, the dendrites of immunopositive cells arborized at various levels of the inner plexiform layer and over fields of different diameters, and Ih activated with similar kinetics and proportions of fast and slow components in small, medium, and large somata. These results show that different HCN subunits colocalize in single retinal ganglion cells, identify a subunit that can reconcile native Ih properties with the previously reported presence of HCN4 in these cells, and indicate that Ih is biophysically similar in morphologically diverse retinal ganglion cells and differs from Ih in rods, cones, and bipolar cells. J. Comp. Neurol. 519:2546–2573, 2011.

Inhibition of Adult Rat Retinal Ganglion Cells by D1-Type Dopamine Receptor Activation

The spike output of neural pathways can be regulated by modulating output neuron excitability and/or their synaptic inputs. Dopaminergic interneurons synapse onto cells that route signals to mammalian retinal ganglion cells, but it is unknown whether dopamine can activate receptors in these ganglion cells and, if it does, how this affects their excitability. Here, we show D1a receptor-like immunoreactivity in ganglion cells identified in adult rats by retrogradely transported dextran, and that dopamine, D1-type receptor agonists, and cAMP analogs inhibit spiking in ganglion cells dissociated from adult rats. These ligands curtailed repetitive spiking during constant current injections and reduced the number and rate of rise of spikes elicited by fluctuating current injections without significantly altering the timing of the remaining spikes. Consistent with mediation by D1-type receptors, SCH-23390 [R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine] reversed the effects of dopamine on spikes. Contrary to a recent report, spike inhibition by dopamine was not precluded by blocking I h. Consistent with the reduced rate of spike rise, dopamine reduced voltage-gated Na+ current (I Na) amplitude, and tetrodotoxin, at doses that reduced I Na as moderately as dopamine, also inhibited spiking. These results provide the first direct evidence that D1-type dopamine receptor activation can alter mammalian retinal ganglion cell excitability and demonstrate that dopamine can modulate spikes in these cells by a mechanism different from the presynaptic and postsynaptic means proposed by previous studies. To our knowledge, our results also provide the first evidence that dopamine receptor activation can reduce excitability without altering the temporal precision of spike firing.

DARPP-32-like immunoreactivity in AII amacrine cells of rat retina

Previous studies demonstrated that the dopamine‐ and adenosine 3′,5′‐monophosphate‐regulated phosphatase inhibitor known as “DARPP‐32” is present in rat, cat, monkey, and human retinas. We have followed up these studies by asking what specific cell subtypes contain DARPP‐32. Using a polyclonal antibody directed against a peptide sequence of human DARPP‐32, we immunostained adult rat retinas that were either transretinally sectioned or flat mounted and found DARPP‐32‐like immunoreactivity in some cells of the amacrine cell layer across the entire retinal surface. We report here, based on the shape and spatial distribution of these cells, their staining by an anti‐parvalbumin antibody, and their juxtaposition with processes containing tyrosine hydroxylase, that DARPP‐32‐like immunoreactivity is present in AII amacrine cells of rat retina. These results suggest that the response of AII amacrine cells to dopamine is not mediated as simply as previously supposed. J. Comp. Neurol. 480:251–263, 2004.

Dopamine and full-field illumination activate D1 and D2-D5-type receptors in adult rat retinal ganglion cells.

Dopamine can regulate signal generation and transmission by activating multiple receptors and signaling cascades, especially in striatum, hippocampus, and cerebral cortex. Dopamine modulates an even larger variety of cellular properties in retina, yet has been reported to do so by only D1 receptor‐driven cyclic adenosine monophosphate (cAMP) increases or D2 receptor‐driven cAMP decreases. Here, we test the possibility that dopamine operates differently on retinal ganglion cells, because the ganglion cell layer binds D1 and D2 receptor ligands, and displays changes in signaling components other than cAMP under illumination that should release dopamine. In adult rat retinal ganglion cells, based on patch‐clamp recordings, Ca2+ imaging, and immunohistochemistry, we find that 1) spike firing is inhibited by dopamine and SKF 83959 (an agonist that does not activate homomeric D1 receptors or alter cAMP levels in other systems); 2) D1 and D2 receptor antagonists (SCH 23390, eticlopride, raclopride) counteract these effects; 3) these antagonists also block light‐induced rises in cAMP, light‐induced activation of Ca2+/calmodulin‐dependent protein kinase II, and dopamine‐induced Ca2+ influx; and 4) the Ca2+ rise is markedly reduced by removing extracellular Ca2+ and by an IP3 receptor antagonist (2‐APB). These results provide the first evidence that dopamine activates a receptor in adult mammalian retinal neurons that is distinct from classical D1 and D2 receptors, and that dopamine can activate mechanisms in addition to cAMP and cAMP‐dependent protein kinase to modulate retinal ganglion cell excitability. J. Comp. Neurol. 520:4032–4049, 2012.

Dissociation of Retinal Ganglion Cells Without Enzymes

We describe here methods for dissociating retinal ganglion cells from adult goldfish and rat without proteolytic enzymes, and show responses of ganglion cells isolated this way to step-wise voltage changes and fluctuating current injections. Taking advantage of the laminar organization of vertebrate retinas, photoreceptors and other cells were lifted away from the distal side of freshly isolated goldfish retinas, after contact with pieces of membrane filter. Likewise, cells were sliced away from the distal side of freshly isolated rat retinas, after these adhered to a membrane filter. The remaining portions of retina were incubated in an enzyme-free, low Ca2+ solution, and triturated. After aliquots of the resulting cell suspension were plated, ganglion cells could be identified by dye retrogradely transported via the optic nerve. These cells showed no obvious morphological degeneration for several days of culture. Perforated-patch whole-cell recordings showed that the goldfish ganglion cells spike tonically in response to depolarizing constant current injections, that these spikes are temporally precise in response to fluctuating current injections, and that the largest voltage-gated Na+ currents of these cells were larger than those of ganglion cells isolated with a neutral protease.

HCN4-like immunoreactivity in rat retinal ganglion cells

Antisera directed against hyperpolarization-activated, cyclic nucleotide-sensitive (HCN) channels bind to somata in the ganglion cell layer of rat and rabbit retinas, and mRNA for different HCN channel isoforms has been detected in the ganglion cell layer of mouse retina. However, previous studies neither provided evidence that any of the somata are ganglion cells (as opposed to displaced amacrine cells) nor quantified these cells. We therefore tested whether isoform-specific anti-HCN channel antisera bind to ganglion cells labeled by retrograde transport of fluorophore-coupled dextran. In flat-mounted adult rat retinas, the number of dextran-backfilled ganglion cells agreed with cell densities reported in previous studies, and anti-HCN4 antisera bound to the somata of approximately 40% of these cells. The diameter of these somata ranged from 7 to 30 microm. Consistent with localization to cell membranes, the immunoreactivity formed a thin line that circumscribed individual somata. Optic fiber layer axon fascicles, and the proximal dendrites of some ganglion cells, also displayed binding of anti-HCN4 antisera. These results suggest that the response of some mammalian retinal ganglion cells to hyperpolarization may be modulated by changes in intracellular cAMP levels, and could thus be more complex than expected from previous voltage and current recordings.

Thy1 Associates with the Cation Channel Subunit HCN4 in Adult Rat Retina

PURPOSE The membrane expression and gene promoter of the glycosylphosphatidylinositol (GPI)-anchored protein Thy1 have been widely used to examine the morphology and distribution of retinal ganglion cells in normal eyes and disease models. However, it is not known how adult mammalian retinal neurons use Thy1. Because Thy1 is not a membrane-spanning protein and, instead, complexes with structural and signaling proteins in other tissues, the aim of this study was to find protein partners of retinal Thy1. METHODS Coimmunoprecipitation, immunohistochemistry, confocal imaging, and patch-clamp recording were used to test for association of Thy1 and HCN4, a cation channel subunit, in adult rat retina. RESULTS Hyperpolarization of cells immunopanned by an anti-Thy1 antibody activated HCN channels. Confocal imaging showed that individual somata in the ganglion cell layer bound antibodies against Thy1 and HCN4, that the majority of these bindings colocalized, and that some of the immunopositive cells also bound antibody against a ganglion cell marker (Brn3a). Consistent with these results, Thy1 and HCN4 were coimmunoprecipitated by magnetic beads coated with either anti-Thy1 antibody or anti-HCN4 antibody. In control experiments, beads coated with these antibodies did not immunoprecipitate a photoreceptor rim protein (ABCR) and uncoated beads did not immunoprecipitate either Thy1 or HCN4. CONCLUSIONS This is the first report that Thy1 colocalizes and coimmunoprecipitates with a membrane-spanning protein in retina, that Thy1 complexes with an ion channel protein in any tissue, and that a GPI-anchored protein associates with an HCN channel subunit protein.

ω-Conotoxin-MVIID Blocks an ω-Conotoxin-GVIA-sensitive, High-threshold Ca2+ Current in Fish Retinal Ganglion Cells

Abstract Reduction of Ca 2+ current amplitude by the Conus peptide ω-conotoxin-MVIID (ω-CTx-MVIID) was measured in voltage-clamped, goldfish retinal ganglion cells. Effects of depolarizing shifts in holding potential, and sequential applications of ω-CTx-MVIID, ω-CTx-GVIA, and BAY-K-8644, together with effects of Ni 2+ and ω-Aga-IIA, indicate that ω-CTx-MVIID may target Ca 2+ channels differing from those termed T, L, N, P, Q and R. Copyright

Fixation strategies for retinal immunohistochemistry

Immunohistochemical and ex vivo anatomical studies have provided many glimpses of the variety, distribution, and signaling components of vertebrate retinal neurons. The beauty of numerous images published to date, and the qualitative and quantitative information they provide, indicate that these approaches are fundamentally useful. However, obtaining these images entailed tissue handling and exposure to chemical solutions that differ from normal extracellular fluid in composition, temperature, and osmolarity. Because the differences are large enough to alter intercellular and intracellular signaling in neurons, and because retinae are susceptible to crush, shear, and fray, it is natural to wonder if immunohistochemical and anatomical methods disturb or damage the cells they are designed to examine. Tissue fixation is typically incorporated to guard against this damage and is therefore critically important to the quality and significance of the harvested data. Here, we describe mechanisms of fixation; advantages and disadvantages of using formaldehyde and glutaraldehyde as fixatives during immunohistochemistry; and modifications of widely used protocols that have recently been found to improve cell shape preservation and immunostaining patterns, especially in proximal retinal neurons.