Cloning and expression of a second photoreceptor-specific membrane retina guanylyl cyclase (RetGC), RetGC-2.

One of the membrane guanylyl cyclases (GCs), RetGC, is expressed predominantly in photoreceptors. No extracellular ligand has been described for RetGC, but it is sensitive to activation by a soluble 24-kDa protein (p24) and is inhibited by Ca2+. This enzyme is, therefore, thought to play a role in resynthesizing cGMP for photoreceptor recovery or adaptation. By screening a human retinal cDNA library at low stringency with the cytoplasmic domains from four cyclases, we cloned cDNAs encoding a membrane CG that is most closely related to RetGC. We have named this GC RetGC-2, and now term the initially described RetGC RetGC-1. By in situ hybridization, mRNA encoding RetGC-2 is found only in the outer nuclear layer and inner segments of photoreceptor cells. By using synthetic peptide antiserum specific for each RetGC subtype, RetGC-2 can be distinguished from RetGC-1 as a slightly smaller protein in immunoblots of bovine rod outer segments. Membrane GC activity of recombinant RetGC-2 expressed in human embryonic kidney 293 cells is stimulated by the activator p24 and is inhibited by Ca2+ with an EC50 value of 50-100 nM. Our data reveal a previously unappreciated diversity of photoreceptor GCs.

Characterization of subtype-specific antibodies to the human D5 dopamine receptor: studies in primate brain and transfected mammalian cells.

To achieve a better understanding of how D5 dopamine receptors mediate the actions of dopamine in brain, we have developed antibodies specific for the D5 receptor. D5 antibodies reacted with recombinant baculovirus-infected Sf9 cells expressing the D5 receptor but not with the D1 receptor or a variety of other catecholaminergic and muscarinic receptors. Epitope-tagged D5 receptors expressed in mammalian cells were reactive with both D5 antibodies and an epitope-specific probe. A mixture of N-linked glycosylated polypeptides and higher molecular-mass species was detected on immunoblots of membrane fractions of D5-transfected cells and also of primate brain. D5 receptor antibodies intensely labeled pyramidal neurons in the prefrontal cortex, whereas spiny medium-sized neurons and aspiny large interneurons of the caudate nucleus were relatively lightly labeled. Antibodies to the D5 dopamine receptor should prove important in experimentally determining specific roles for the D5 and D1 receptors in cortical processes and diseases.

Feedback from luminosity horizontal cells mediates depolarizing responses of chromaticity horizontal cells in the Xenopus retina.

It has been proposed that the depolarizing responses of chromaticity horizontal cells (C-HCs) to red light depend on a feedback signal from luminosity horizontal cells (L-HCs) to short-wavelength-sensitive cones in the retinas of lower vertebrates. In this regard we studied the C-HCs of the Xenopus retina. C-HCs and L-HCs were identified by physiological criteria and then injected with neurobiotin. The retina then was incubated with peanut agglutinin, which stains red-but not blue-sensitive cones. Electron microscopic examination revealed that L-HCs contact all cone classes, whereas C-HCs contact only blue-sensitive cones. Simultaneous recordings from C-HC/L-HC pairs established that when the L-HC was saturated by a steady bright red light, C-HCs alone responded to a superimposed blue stimulus. In response to red test flashes, the C-HC response was delayed by approximately 30 msec with respect to the L-HC response. Isolated HCs of both subtypes were examined by whole-cell patch clamp. Both responded to kainate with sustained inward currents and to quisqualate or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) with desensitizing currents from a negative holding potential; i.e., both have AMPA-type glutamate receptors. gamma-Aminobutyric acid or glycine opened a chloride channel in the L-HC, whereas the C-HC was unresponsive to either inhibitory amino acid. Since glycine has been shown to abolish selectively the depolarizing response of the C-HC, this finding and other pharmacological data strongly implicate the L-HC in the underlying circuit. Moreover, because the C-HC does not respond to gamma-aminobutyric acid, the neurotransmitter of the L-HC, by elimination, a feedback synapse from L-HC to blue cone is the most plausible mechanism for the creation of depolarizing responses in C-HCs.

Radial and tangential dispersion patterns in the mouse retina are cell-class specific.

The retina is derived from a pseudostratified germinal zone in which the relative position of a progenitor cell is believed to determine the position of the progeny aligned in the radial axis. Such a developmental mechanism would ensure that radial arrays of cells which comprise functional units in the mature central nervous system are also clonally related. The present study has tested this hypothesis by using X chromosome-inactivation transgenic mosaic mice. We report that the retina shows a conspicuous distinction for clonally related neuroblasts of different laminar and functional fates: the rod photoreceptor, Müller, and bipolar cells are aligned in the radial axis, whereas the cone photoreceptor, horizontal, amacrine, and ganglion cells are tangentially displaced with respect to them. These results indicate that the dispersion of cell classes across the retinal surface is differentially constrained. Some classes of retinal neuroblast exhibit a significant tangential, as well as radial, component in their dispersion from the germinal zone, whereas others disperse only in the radial dimension. Consequently, the majority of radial columns within the mature retina must be derived from multiple progenitors. Because the cone photoreceptor, horizontal, amacrine, and ganglion cells establish nonrandom matrices in their cellular distributions within the respective retinal layers, tangential dispersion may be the means by which these matrices are constructed.