Physiological studies of neurons of the inner retina, e.g., of amacrine cells, are now possible in a mammalian retinal slice preparation. The present anatomical study characterizes glycinergic amacrine cells of the rat retina and thus lays the ground for such future physiological and pharmacological experiments. Rat retinae were immunolabeled with antibodies against glycine and the glycine transporter-1 (GLYT-1), respectively. Glycine immunoreactivity was found in approximately 50% of the amacrine and 25% of the bipolar cells. GLYT-1 immunoreactivity was restricted to glycinergic amacrine cells. They were morphologically characterized by the intracellular injection of Lucifer Yellow followed by GLYT-1 immunolabeling. Eight different types of glycinergic amacrine cells could be distinguished. They were all small-field amacrine cells with bushy dendritic trees terminating at different levels within the inner plexiform layer. The well-known AII amacrine cell was encountered most frequently. From our measurements of the dendritic field sizes and the density of glycinergic cells, we estimate that there are enough glycinergic amacrine cells available to make sure that all eight types and possibly more tile the retina regularly with their dendritic fields.

In addition to the well-studied AII amacrine cell, there is another amacrine cell type participating in the rod pathway of the mammalian retina. In cat, this cell is called the A17 amacrine cell, and in rabbits, it is called the indoleamine-accumulating amacrine cell (S1 and S2); however, the presence of the corresponding cell type has not yet been described in detail for the rat retina. To this end, we injected amacrine cells with Neurobiotin in vertical retinal slices. After histological processing, we were able to reconstruct the morphology of a wide-field amacrine cell which showed characteristics of A17 and S1/S2 amacrine cells. The rat wide-field amacrine cells exhibited the same stratification pattern, their dendrites bore varicosities and ramified in sublamina 5 of the inner plexiform layer (IPL), and they were dye-coupled to other amacrine cells. To determine whether those amacrine cells shared electrophysiological characteristics as well, we performed whole-cell patch-clamp recordings and examined their voltage-activated currents and neurotransmitter-induced currents. We never observed voltage-gated Na+ currents and spike-like potentials upon depolarization by current injection in these cells. We identified GABA- and glycine-sensitive Cl- currents that could be blocked by bicuculline and strychnine, respectively. We also observed kainate- and AMPA-activated currents, which could be inhibited by the application of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Finally, a 400-ms full-field light stimulus was used to characterize the light responses of A17 amacrine cells. The light ON-induced inward current could be suppressed by the application of 2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulphonamide (NBQX), while the majority of the light OFF-induced current was inhibited by bicuculline and reduced to a smaller extent by NBQX. CPP, an NMDA blocker, had no effect on the light response of rat A17 amacrine cells.

These findings suggest autoimmune retinopathy, mediated by anti-GAD65 autoantibodies as the underlying cause of visual loss.

Caldendrin is a novel calcium-binding protein confined to the somatodendritic compartment of neurons. Here we have studied the expression pattern of caldendrin in the rat retina. First we assessed the distribution of caldendrin transcripts in the adult and developing retina by in situ hybridization. In the adult retina, transcripts are expressed mainly in the inner half of the inner nuclear layer (INL) and to a lesser extent in the ganglion cell layer (GCL). During development labeling of the inner part of the cytoblast layer, where amacrine cells reside, is already present at postnatal day 1 (P1). The intensity of hybridization signal in this sublamina of the developing INL increases up to P8, whereas significant labeling in the GCL was first found at P14, coinciding with eye opening. Immunodetection with a polyclonal antibody revealed intensive staining of cells in the inner retina, which are presumably mainly amacrine and significantly fewer bipolar and ganglion cells. All parvalbumin-containing All amacrines were immunopositive for caldendrin. Colocalization with calbindin was found in cone bipolar cells, the majority of AII amacrines, and calbindin-positive cells in the GCL. In the GCL, caldendrin was also colocalized with calretinin-immunopositive cells. Most caldendrin-positive amacrine cells in the adult rat retina were glycinergic and only a few were GABAergic. In retinal flat mounts, it was confirmed that less than 10% of retrogradely labeled retinal ganglion cells (RGC) are caldendrin-positive. Caldendrin immunoreactivity does not colocalize with tyrosine hydroxylase, VIP, substance P and somatostatin immunoreactivity. In summary, caldendrin expression is regulated differentially in retinal cell types during development and is restricted to a subpopulation of amacrine, bipolar, and ganglion cells, suggesting specific functions in the developing and mature retina.