GABA and GAD-like immunoreactivity in the primate retina

Summary GABA immunoreactivity was studied and compared with GAD immunoreactivity in the retinae of baboon, cynomolgus monkey and man. The central and peripheral parts of the retinae were investigated separately in cynomolgus monkey and in man. The same kinds of structures were stained with both antisera. Cells with a position corresponding to amacrine cells were stained, as well as processes in the inner plexiform layer and some cells in the ganglion cell layer. The outer plexiform layer and some cells with the position and configuration of horizontal cells also appeared immunoreactive. Staining was also observed in bipolar-like cells, in man most clearly when using the GABA antiserum in sections from the central parts of the retina. It is possible that horizontal cells, as well as bipolar-like cells, may play a previously unsuspected role in GABAergic transmission in the primate retina.     

Localization of GAD-like immunoreactivity in the pancreas and stomach of the rat and mouse

Summary The aim of this study was to localize cells immunoreactive for glutamate decarboxylase (GAD), the enzyme of GABA synthesis, in pyloric and oxyntic regions of the rat stomach as well as in the rat and mouse pancreas. GAD immunocytochemistry was carried out on polyethylene glycol or cryostat sections of alkaline paraformaldehyde fixed tissue, with simultaneous immunolabelling of various gastro-pancreatic hormones for topographical comparison. In the rat stomach, nerve fibers displaying intense GAD-like immunoreactivity were seen in the myenteric plexus, the circular muscular layer, the submucosa and the lamina propria of the mucosa. But, they were absent from the submucous plexus. Colchicine treatment of the rats allowed to detect some labelled perikarya in the myenteric plexus suggesting that the GABAergic innervation is at least partly intrinsic to the stomach. In the oxyntic and pyloric mucosa, endocrine cells appeared immunostained for GAD. However, the nature of their hormones remained unknown since double immunodetections revealed that they were immunoreactive neither for gastrin nor for somatostatin. In the rat and mouse pancreas, GAD-like immunoreactivity was found in islet cells which corresponded only to insulin-secreting cells. Somatostatin-, glucagon- and pancreatic polypeptide-immunopositive cells were devoid of GAD immunolabelling. No GAD-like immunoreactivity was detected in the exocrine tissue and innervation. These results strenghten the hypothesis that GABA is not only a neurotransmitter in the stomach but that it could also be an endocrine or paracrine factor in the stomach and pancreas.     

Existence of immunoreactivity to GABA in dopaminergic amacrine cells of the rat retina

Two different immunohistochemical double labelling techniques have allowed the demonstration of a GABA-immunoreactivity in the dopamine amacrine cells of the rat retina. The functional implications of such a colocalization are of importance because antagonist effects of these two neurotransmitters on retinal horizontal cells have been demonstrated.     

Localization of GAD-like immunoreactivity in the pancreas and stomach of the rat and mouse.

The aim of this study was to localize cells immunoreactive for glutamate decarboxylase (GAD), the enzyme of GABA synthesis, in pyloric and oxyntic regions of the rat stomach as well as in the rat and mouse pancreas. GAD immunocytochemistry was carried out on polyethylene glycol or cryostat sections of alkaline paraformaldehyde fixed tissue, with simultaneous immunolabelling of various gastro-pancreatic hormones for topographical comparison. In the rat stomach, nerve fibers displaying intense GAD-like immunoreactivity were seen in the myenteric plexus, the circular muscular layer, the submucosa and the lamina propria of the mucosa. But, they were absent from the submucous plexus. Colchicine treatment of the rats allowed to detect some labelled perikarya in the myenteric plexus suggesting that the GABAergic innervation is at least partly intrinsic to the stomach. In the oxyntic and pyloric mucosa, endocrine cells appeared immunostained for GAD. However, the nature of their hormones remained unknown since double immunodetections revealed that they were immunoreactive neither for gastrin nor for somatostatin. In the rat and mouse pancreas, GAD-like immunoreactivity was found in islet cells which corresponded only to insulin-secreting cells. Somatostatin-, glucagon- and pancreatic polypeptide-immunopositive cells were devoid of GAD immunolabelling. No GAD-like immunoreactivity was detected in the exocrine tissue and innervation. These results strenghten the hypothesis that GABA is not only a neurotransmitter in the stomach but that it could also be an endocrine or paracrine factor in the stomach and pancreas.     

Structures with GABA-like and GAD-like immunoreactivity in the cervical sympathetic ganglion complex of adult rats

Summary The distribution of gamma-aminobutyric acid (GABA)-like and glutamate decarboxylase (GAD)-like immunoreactivity was studied in the cervical sympathetic ganglion complex of rats, including the intermediate and inferior cervical ganglia and the uppermost thoracic ganglion. GABA-positive axons may enter the ganglion complex via its caudal end. Others apparently arise from small GABA-positive cell bodies which are scattered among principal neurons, within clusters of SIF cells and in bundles of GABA-negative axons. The majority of these cells is located in the lower half of the ganglion complex. Principal neurons did not react with antibodies against GABA or GAD. An unevenly distributed meshwork of GABA-immunoreactive axons was seen in each of the ganglia. Immunoreactive axons formed numerous varicosities. Some of them were aggregated in a basket-like form around a subpopulation of GABA-negative principal ganglion cell bodies. Electron-microscopic immunocytochemistry revealed that GABA-positive nerve fibers establish asymmetric synaptic junctions with dendritic and somatic spines of principal neurons, whereas postsynaptic densities are inconspicous or absent on dendritic shafts and somata. The results suggest that in the cervical sympathetic ganglion complex principal neurons are not GABAergic, but are innervated by axons which react with both antibodies against GAD and/ or GABA antibodies and originate from a subpopulation of small neurons.     

Phase-contrast microscopy of the primate retina

The study of hematoxylin and eosin stained thick sections (15 microns) of the primate retina with the phase-contrast microscope provided a means for the selective demonstration of many cellular structures that could not be resolved with the same degree of detail which was possible when bright-field microscopy was used, or when phase-contrast microscopy was employed to examine unstained material. The H & E-stain greatly enhanced the phase-contrast image, so that cytoplasmic structure, fiber trajectories, and gross synaptic detail of the retina could be demonstrated to better advantage.     

Thyrotropin-like immunoreactivity in the developing chicken retina.

In this paper we analysed the presence and localisation of thyrotropin during retinal development in Gallus domesticus. Specific thyrotropin-like immunohistochemical staining was observed from the beginning of the second incubation week to one day post-hatching in chicken retina. Thyrotropin is a 28.3 KDa glycoprotein, synthesised by the anterior pituitary gland, and it is implicated in the stimulation of the synthesis and release of thyroid hormones. Until now, the action of thyrotropin has been established exclusively in hormonal terms. Recently, this glycoprotein has been localised in synaptic processes in the human retina by using a specific antiserum (Fdez-Trujillo et al., 1995). To the best of our knowledge this report is the first time that thyrotropin has been immunocytochemically demonstrated in the chicken retina. The pattern of thyrotropin-like immunoreactivity suggests that this glycoprotein could act as modulator of synaptic transmission, but it may also play a much broader role in regulating trophic functions.     

Cells presenting GABA immunoreactivity in the hypothalamus of the cat

The distribution of GABA-immunoreactive (IR) cells was studied by immunohistochemistry in conjunction with highly specific antiserum GABA in the cat hypothalamus. Colchicine pretreatment made it possible to visualize a large number of labeled cells in the medial preoptic and dorsal hypothalamic areas. In contrast, the ventromedial and anterior hypothalamic nuclei contained only a few labeled cells, and the paraventricular and supraoptic nuclei were devoid of them. A very dense network of GABA-IR presumptive terminals was seen in the ventrolateral posterior hypothalamus where labeled cells could be recognized. The possibility of an involvement of the GABAergic neuronal system in the regulation of sleep-waking cycle is discussed.     

Localization of CD15 immunoreactivity in the rat retina

Using immunocytochemistry, we have investigated the localization of CD15 in the rat retina. In the present study, two types of amacrine cell in the inner nuclear layer (INL) and some cells in the ganglion cell layer were labeled with anti-CD15 antisera. Type 1 amacrine cells have large somata located in the INL, with long and branched processes ramifying mainly in stratum 3 of the inner plexiform layer (IPL). Type 2 cells have a smaller soma and processes branching in stratum 1 of the IPL. A third population showing CD15 immunoreactivity was a class of displaced amacrine cells in the ganglion cell layer. The densities of type 1 and type 2 amacrine cells were 166/mm(2) and 190/mm(2) in the central retina, respectively. The density of displaced amacrine cells was 195/mm(2). Colocalization experiments demonstrated that these CD15-immunoreactive cells exhibit gamma-aminobutyric acid and neuronal nitric oxide synthase (nNOS) immunoreactivities. Thus, the same cells of the rat retina are labeled by anti-CD15 and anti-nNOS antisera and these cells constitute a subpopulation of GABAergic amacrine cells.     

Quantitative morphology of the central fovea in the primate retina

Electron micrograph composites of tangenital sections of the fovea centralis of three cynomolgus monkeys (Macaca irus) and one baboon (Papio anubis) were used to determine the spatial density of the principal retinal cells. In the center of the foveola, the density of cones ranged from 113,000 to 230,000/mm2, and pigment epithelial cells from 4,900 to 7,000/mm2. At a distance of 500 microns from the foveolar center the density of the cone cell pedicles ranged from 29,000 to 36,300/mm2, and the density of horizontal cells ranged from 19,000 to 25,100/mm2. Densities of bipolar, Müller, and amacrine cells were determined in only two monkeys and in the baboon. The fact that the cone cell pedicles have a larger diameter than the foveolar cones explains the geometry of the fovea. The morphology of the junction between foveolar cone outer segments and the pigment epithelium reflects the complex metabolism of this functional unit. The comparison with the peripheral primate retina suggests that the densities of horizontal and bipolar cells, but not of amacrine and Müller cells, are correlated with the density of cone cell pedicles.     

Nitric oxide synthase immunoreactivity in the developing and adult human retina

Nitric oxide synthase (NOS) catalyzes the formation of nitric oxide (NO) from L-arginine. In this study, the cellular localization of neuronal NOS (nNOS) activity in the human retina since fetal development was examined by immunohistochemistry. No detectable staining in the fetal retina was present at 14 weeks of gestation (wg), the earliest age group examined. A centro-peripheral gradient of development of nNOS immunoreactivity was evident at 16–17 wg, with the midperipheral retina showing nNOS immunoreactivity in most of the cell types and the inner plexiform layer while the peripheral part demonstrated moderate immunoreactivity only in the ganglion cell layer and photoreceptor precursors. A transient increase in nNOS immunoreactivity in the ganglion cells and Müller cell endfeet between 18–19 and 24–25 wg was observed at the time when programmed cell death in the ganglion cell layer, loss of optic nerve fibres as well as increase in glutamate immunoreactivity and parvalbumin (a calcium binding protein) immunoreactivity in the ganglion cells was reported. These observations indicate that programmed cell death of ganglion cells in the retina may be linked to glutamate toxicity and NO activity, as also suggested by others in the retina and cerebral cortex. The presence of nNOS immunoreactivity in the photoreceptors from 16–17 weeks of fetal life to adulthood indicates other functions, besides their involvement in photoreceptor function of transduction and information processing.     

Immunocytochemical and autoradiographic localization of GABA system in the vertebrate retina

Summary The localization of -aminobutyric acid (GABA) neurons in the goldfish and the rabbit retina has been studied by immunocytochemical localization of the GABA-synthesizing enzyme L-glutamate decarboxylase (GAD, L-glutamate 1-carboxy-lase, EC 4.1.1.15) and by [³H] GABA uptake autoradiography. In the goldfish retina, GAD is localized in some horizontal cells (H1 type), a few amacrine cells and sublamina b of the inner plexiform layer. Results from immunocytochemical studies of GAD-containing neurons and autoradiographic studies of GABA uptake reveals a marked similarity in the labeling pattern suggesting that in goldfish retina, the neurons which possess a high-affinity system for GABA uptake also contain significant levels of GAD. In the rabbit retina, when Triton X-100 was included in immunocytochemical incubations with a modified protein A-peroxidase-antiperoxidase method, reaction product was found in four broad, evenly spaced laminae within the inner plexiform layer. In the absence of the detergent, these laminae were seen to be composed of small, punctate deposits. When colchicine was injected intravitreally before glutamate decarboxylase staining, cell bodies with the characteristic shape and location of amacrine cells were found to be immunochemically labeled. Electron microscopic examination showed that these processes were presynaptic to ganglion cell dendrites (infrequently), amacrine cell telodendrons, and bipolar cell terminals. Often, bipolar cell terminals were found which were densely innervated by several GAD-positive processes. No definite synapses were observed in which a GAD-positive process represented the postsynaptic element. In autoradiographic studies by intravitreal injection of [³H] GABA a diffuse labeling of the inner plexiform layer and a dense labeling of certain amacrine cell bodies in the inner nuclear layer was observed. Both immunocytochemical and autoradiographic results support the notion that certain, if not all, amacrine cells use GABA as their neurotransmitter.