The somatostatin subtype 2A (sst2A) receptor, a member of the G protein-coupled receptor superfamily, mediates many of the neuroendocrine and neuromodulatory actions of somatostatin, and it represents the primary target for somatostatin analogs used in cancer therapy and tumor localization. Agonist stimulation leads to the rapid phosphorylation, endocytosis, and desensitization of the sst2A receptor; however, little is known about the role of phosphorylation in sst2A regulation. sst2A phosphorylation occurs on serine and threonine residues in the third intracellular loop and carboxyl terminus. Therefore, we generated mutant receptors in which serine (SerϪ), threonine (ThrϪ), or both (SerϪ/ThrϪ) residues in these regions were mutated to alanine. In contrast to the wild-type receptor, somatostatin treatment did not stimulate the phosphorylation of the SerϪ/ThrϪ mutant, and it did not produce desensitization. Furthermore, internalization of the SerϪ/ ThrϪ mutant occurred 5 times more slowly than with the wildtype receptor. Mutating only the Ser residues did not inhibit either internalization or desensitization. In contrast, mutating only the Thr residues inhibited receptor endocytosis to the same extent as in the full mutant, but it did not affect receptor desensitization. In both the wild-type and SerϪ receptors, agonist binding produced a stable arrestin-receptor complex that was maintained during receptor trafficking, whereas arrestin was not recruited to either the ThrϪ or the SerϪ/ThrϪ receptors. These results demonstrate that agonist-stimulated receptor phosphorylation is necessary for both desensitization and rapid internalization of the sst2A receptor. However, sst2A receptor internalization and uncoupling can occur independently, involve different receptor phosphorylation sites, and exhibit different requirements for stable arrestin association.

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.

The goals of this study were to localize the neuropeptide Cocaine- and Amphetamine-Regulated Transcript (CART) in primate retinas and to describe the morphology, neurotransmitter content and synaptic connections of the neurons that contain it. Using in situ hybridization, light and electron microscopic immunolabeling, CART was localized to GABAergic amacrine cells in baboon retinas. The CART-positive cells had thin, varicose dendrites that gradually descended through the inner plexiform layer and ramified extensively in the innermost stratum. They resembled two types of wide-field diffuse amacrine cells that had been described previously in macaque retinas using the Golgi method and also A17, serotonin-accumulating and waterfall cells of other mammals. The CART-positive cells received synapses from rod bipolar cell axons and made synapses onto the axons in a reciprocal configuration. The CART-positive cells also received synapses from other amacrine cells. Some of these were located on their primary dendrites, and the presynaptic cells there included dopaminergic amacrine cells. Although some CART-positive somas were localized in the ganglion cell layer, they did not contain the ganglion cell marker RNA binding protein with multiple splicing (RBPMS). Based on these results and electrophysiological studies in other mammals, the CART-positive amacrine cells would be expected to play a major role in the primary rod pathway of primates, providing feedback inhibition to rod bipolar cells.