Localization of putative GABAergic neurons in the larval tiger salamander retina by immunocytochemical and autoradiographic methods

Putative GABAergic neurons in the larval tiger salamander retina were localized by a comparative analysis of glutamate decarboxylase immunoreactivity (GAD-IR), GABA-like immunoreactivity (GABA-IR), and high-affinity 3H-GABA uptake at the light microscopical level. Preliminary data showed that all GAD-IR neurons were double labeled for GABA-IR. However, because the weak somatic labeling with GAD-IR, we could not determine if the converse were true. Neurons commonly labeled with GABA-IR and 3H-GABA uptake include horizontal cells, type I (outer) and type II (inner) bipolar cells, type I (inner) and type II (outer) amacrine cells, and cell bodies in the ganglion cell layer (GCL). In addition, interplexiform cells were identified with GABA-IR. The presence of GABA-IR ganglion cells was indicated by GABA-IR fibers in the optic fiber layer and optic nerve as well as by a GABA-IR cell in the GCL that included a labeled axon. The percentage of labeled somas in the inner nuclear layer (INL) compared to all cells in each layer was similar for the two methods: 30% in INL 1 (outer layer of somas), 15% in INL 2 (middle layer), 43-52% in INL 3 (inner layer), and about 21-26% in the GCL. Labeled processes were found in three bands in the inner plexiform layer, with the densest band located in the most proximal part. Postembedding labeling of 1-micron Durcupan resin sections for GABA-IR showed the same general pattern as obtained with 10-microns cryostat sections, with additional staining, however, of type II (inner) bipolar cell Landolt's clubs. Extensive colocalization of labeling was indicated, and we conclude that GABA-IR can serve as a valid and reliable marker for GABA-containing neurons in this retina and suggest that GABA serves as a transmitter for horizontal cells, several types of amacrine cell, a type of interplexiform cell, and perhaps a small percentage of type I and type II bipolar cells and ganglion cells.     

Localization of GABA, glycine, glutamate and tyrosine hydroxylase in the human retina.

A light microscope study using postembedding immunocytochemistry techniques to demonstrate the common neurotransmitter candidates gamma-aminobutyric acid (GABA), glycine, glutamate, and tyrosine hydroxylase for dopamine has been done on human retina. By using an antiserum to GABA, we found GABA-immunoreactivity (GABA-IR) to be primarily in amacrine cells lying in the inner nuclear layer (INL) or displaced to the ganglion cell layer (GCL). A few stained cells in the INL, which are probably interplexiform cells, were observed to project thin processes towards the outer plexiform layer (OPL). There were heavily stained bands of immunoreactivity in strata 1, 3 and 5 of the inner plexiform layer (IPL). An occasional ganglion cell was also GABA-IR. By using an antiserum to glycine, stained cells were observed at all levels of the INL. Most of these were amacrines, but a few bipolar cells were also glycine-IR. Displaced amacrine cells and large-bodied cells, which are probably ganglion cells, stained in the GCL. The bipolar cells that stained appeared to include both diffuse and midget varieties. The AII amacrine cell of the rod pathway was clearly stained in our material but at a lower intensity than two other amacrine cell types tentatively identified as A8 and A3 or A4. Again, there was stratified staining in the IPL, with strata 2 and 4 being most immunoreactive. An antiserum to glutamate revealed that most of the neurons of the vertical pathways in the human retina were glutamate-IR. Rod and cone photoreceptor synaptic endings labeled as did the majority of bipolar and ganglion cells. The rod photoreceptor stained more heavily than the cone photoreceptor in our material. While both midget and diffuse cone bipolar cell types were clearly glutamate-IR, rod bipolars were not noticeably stained. The most strongly staining glutamate-IR processes of the IPL lay in the outer half, in sublamina a. The antiserum to tyrosine hydroxylase (TOH) revealed two different amacrine cell types. Strongly immunoreactive cells (TOH1) had their cell bodies in the INL and their dendrites ramified in a dense plexus in stratum 1 of the IPL. Fine processes arising from their cell bodies or from the stratum 1 plexus passed through the INL to reach the OPL but did not produce long-ranging ramifications therein. The less immunoreactive amacrines (TOH2) lay in the INL, the center of the IPL or the GCL and emitted thick dendrites that were monostratified in stratum 3 of the IPL.     

Colocalization of GAD-like immunoreactivity and 3H-GABA uptake in amacrine cells of rabbit retina

Rabbit retinas were double labeled to determine the degree of colocalization of glutamic-acid-decarboxylase-like immunoreactivity (GAD-like IR) and 3H-GABA uptake using light (LM) and electron microscopic (EM) autoradiography. Both GAD-like IR and 3H-GABA uptake were found in amacrine cell bodies in the inner nuclear layer (INL) as well as in cell bodies in the ganglion cell layer (GCL), and throughout the inner plexiform layer. GAD-like IR was found in 32% of the amacrine cells in the INL, 86% of which also showed 3H-GABA uptake; 3H-GABA uptake was observed in 38% of the amacrine cells. However, only 72% of these cells showed GAD-like IR. Labeled cells in the GCL were only 10-15% as common as similarly labeled cells in the INL. As in the INL, all GAD-positive cells in the GCL were double labeled, but only 53% of the cells taking up 3H-GABA were double labeled. We suggest that labeled cells in the GCL were ganglion cells rather than displaced amacrine cells. Cells, in both the INL and GCL, that showed 3H-GABA uptake but no GAD-like IR had a higher average grain density than double-labeled cells, indicating that uptake by these cells was specific. The relevance to GABAergic function of 3H-GABA uptake without an indication of GAD-like IR is yet to be determined. Statistical analysis at the EM level showed that one-third of the GAD-positive synaptic terminals of amacrine cells were double labeled after a 4-month exposure. Longer exposures at the EM level should reveal a higher percentage of GAD-positive terminals because at the LM level, one-half of the double-labeled cell bodies were "lightly" labeled with grains. The high degree of colocalization of GAD-like IR and 3H-GABA uptake suggests that both markers may be useful for labeling GABAergic neurons in the rabbit retina.     

Multiple cell types form the VIP amacrine cell population

Amacrine cells are a heterogeneous group of interneurons that form microcircuits with bipolar, amacrine and ganglion cells to process visual information in the inner retina. This study has characterized the morphology, neurochemistry and major cell types of a VIP-ires-Cre amacrine cell population. VIP-tdTomato and -Confetti (Brainbow2.1) mouse lines were generated by crossing a VIP-ires-Cre line with either a Cre-dependent tdTomato or Brainbow2.1 reporter line. Retinal sections and whole-mounts were evaluated by quantitative, immunohistochemical, and intracellular labeling approaches. The majority of tdTomato and Confetti fluorescent cell bodies were in the inner nuclear layer (INL) and a few cell bodies were in the ganglion cell layer (GCL). Fluorescent processes ramified in strata 1, 3, 4, and 5 of the inner plexiform layer (IPL). All tdTomato fluorescent cells expressed syntaxin 1A and GABA-immunoreactivity indicating they were amacrine cells. The average VIP-tdTomato fluorescent cell density in the INL and GCL was 535 and 24 cells/mm², respectively. TdTomato fluorescent cells in the INL and GCL contained VIP-immunoreactivity. The VIP-ires-Cre amacrine cell types were identified in VIP-Brainbow2.1 retinas or by intracellular labeling in VIP-tdTomato retinas. VIP-1 amacrine cells are bistratified, wide-field cells that ramify in strata 1, 4, and 5, VIP-2A and 2B amacrine cells are medium-field cells that mainly ramify in strata 3 and 4, and VIP-3 displaced amacrine cells are medium-field cells that ramify in strata 4 and 5 of the IPL. VIP-ires-Cre amacrine cells form a neuropeptide-expressing cell population with multiple cell types, which are likely to have distinct roles in visual processing.     

Glutamate, GABA, and glycine in the human retina: an immunocytochemical investigation.

The distribution of the neuroactive amino acids glutamate, GABA, and glycine in the human retina was examined in consecutive semithin sections treated with antisera specific for fixed glutamate, GABA, and glycine, respectively. Glutamate immunoreactivity was conspicuous in all photoreceptor cells (rods more strongly labelled than cones), and in a majority (85-89%) of the cells in the inner nuclear layer (INL). Rod spherules and cone pedicles showed a greater enrichment of glutamate immunoreactivity than the parent cell bodies and inner segments. Also, structures of the inner plexiform layer (IPL) were labelled. A large majority (83-91%) of cells in the ganglion cell layer (GCL) was strongly stained, as were most axons in the nerve fibre layer. Müller cell processes appeared unstained. GABA immunoreactivity was present in presumed amacrine but not in bipolar-like cells. The stained cells were restricted to the inner 1/3 of the INL and were more frequent in central than in peripheral retina (40% and 26% of all cells in the inner 1/2 of INL, respectively). GABA positive cell processes, probably originating from interplexiform cells, appeared to traverse the INL and end in the outer plexiform layer. Dense immunolabeling was found in the IPL. GABA immunoreactive cells (some also stained for glutamate) comprised 23% of all GCL cells in the peripheral retina, but only 5% in the central retina. Most of them were localized adjacent to the IPL. A few GABA positive (possibly ganglion) cells extended a single fibre toward the nerve fibre layer. Solitary GABA positive fibres were seen in this layer and in the optic nerve. Glycine immunoreactivity was observed in cells with the location typical of amacrine and bipolar (peripheral retina) cells, as well as in punctate structures of the IPL. In contrast to the GABA positive cells, the glycine positive cells were more frequent in the peripheral than in the central retina (42% and 23% of all cells in inner 1/3 of INL, respectively). A few cells in the GCL (0.5-1.5%) were glycine positive. Glutamate colocalized with GABA or glycine in a majority of the cells stained for either of these inhibitory transmitters (90-95% of the GABA positive cells, and 80-86% of the glycine positive cells, in the INL). Some bipolar cells were stained for both glutamate and glycine. Colocalization of GABA and glycine occurred in a subpopulation (3-4%) of presumed amacrine cells, about half of which was also glutamate positive.     

Distribution and synaptic connectivity of neuropeptide Y-immunoreactive amacrine cells in the rat retina

Neuropeptide Y (NPY) is a potent bioactive peptide that is widely expressed in the nervous system, including the retina. Here we show that specific NPY immunoreactivity was localized to amacrine and displaced amacrine cells in the rat retina. Immunoreactive cells had a regular distribution across the retina and an overall cell density of 280 cells/mm(2) in the inner nuclear layer (INL) and 90 cells/mm(2) in the ganglion cell layer (GCL). In the INL, most immunoreactive cells were characterized by small cell bodies and fine processes that appeared to ramify primarily in stratum 1 of the inner plexiform layer (IPL). A few cells in the INL also ramified in stratum 3 of the IPL. In the GCL, small to medium immunoreactive cells appeared to ramify primarily in stratum 5 of the IPL. A few immunoreactive processes, originating from somata in the INL and processes in the IPL, ramified in the OPL. NPY-immunoreactive cells contained GABA immunoreactivity, and some amacrine cells also contained tyrosine hydroxylase immunoreactivity. NPY-immunostained processes were most frequently presynaptic to nonimmunostained amacrine and ganglion cell processes and postsynaptic to nonimmunostained amacrine cell processes and cone bipolar cell axonal terminals. These findings indicate that NPY immunoreactivity is present in two populations of amacrine cells, one located in the INL and the other in the GCL, and that these cells mainly form synaptic contacts with other amacrine cells. These observations suggest that NPY-immunoreactive cells participate in multiple circuits mediating visual information processing in the inner retina.     

Is taurine a neurotransmitter in rabbit retina?

Rabbit retina was used as a model to study the possible role of taurine in the retina. The taurine-synthesizing enzyme, cysteine sulfinic acid decarboxylase (CSAD), is localized immunohistochemically using specific antibodies against CSAD. The CSAD-immunoreactivity appears to be most prominent in the inner nuclear layer (INL) and ganglion cell layer (GCL). The inner plexiform layer (IPL), the outer nuclear layer and outer plexiform layer are sporadically stained. The CSAD-positive neurons include some amacrine cells and probably the bipolar cells in the INL and some large and small ganglion cells in the GCL. Autoradiographic studies reveal that the uptake of [3H]taurine is most prominent in the INL. The IPL and GCL, as well as the Müller cells, also show a moderate degree of [3H]taurine accumulation. In conclusion, we have demonstrated the presence of the taurine-synthesizing enzyme and uptake systems in rabbit retina. Based on the above evidence, we propose that taurine may be used by some neurons, presumably amacrine cells, as a transmitter in the rabbit retina.     

Laminar asymmetry in the distribution of choline acetyltransferase-immunoreactive neurons in the retina of the tree shrew (Tupaia belangeri)

Cholinergic neurons in the retina of the tree shrew were identified immunocytochemically using a monoclonal antibody directed against choline acetyltransferase (ChAT). The chief result is that roughly 4 times as many ChAT-immunoreactive neurons are found in the inner nuclear layer (INL) as in the ganglion cell layer (GCL). In the INL, two classes of cholinergic neuron can be distinguished on the basis of soma size, one large and one small. The large neurons correspond closely in size and number to the displaced cholinergic neurons in the GCL, suggesting that these are the matching populations of cholinergic amacrine cells reported in other species. The small ChAT-immunoreactive neurons, on the other hand, which make up 60% of the total number of ChAT-positive neurons in the retina, appear to have no counterpart in the GCL. Whether these small neurons are a separate class of amacrine cell or some other cell type (e.g. bipolar, interplexiform, etc.) remains to be determined.     

Glycine high-affinity uptake labels a subpopulation of somatostatin-like immunoreactive cells in theRana pipiens retina

Somatostatin-like immunoreactivity (Som-LI) and glycine high-affinity uptake have been characterized in the Rana pipiens retina. These labels are found in both the outer and inner plexiform layers (OPL and IPL), suggesting that interplexiform cells (IPCs) contain both Som and glycine in this retina. In double-label experiments these labels colocalize to an abundant population of cells in the mid-inner nuclear layer (INL), in the second or third cell layer distal from the IPL. These cells have medium sized spherical or oval somas, each with a single thin descending dendrite which ramifies in the distal IPL. Processes ascending from cells at this location were not visualized by immunocytochemistry, but could be seen by autoradiography of tissue processed for glycine high-affinity uptake. In autoradiographs apparent IPCs were the most intensely labeled cell type in this retina. Som-LI is also found in two types of probable amacrine cells in the proximal INL adjacent to the IPL, neither of which is labeled by glycine high-affinity uptake. One of these is rare (about 10 cells/mm ²), and has a large pyriform soma with a thick dendrite that branches in the proximal IPL. The other type is more common (324 ± 20cells/mm²), has medium-sized spherical or horizontally elongated elliptical somas, and has multiple thin dendrites projecting into the distal IPL. In addition to the above cell types, faint Som-LI was seen in cells of the ganglion cell layer, possibly indicating the presence of somatostaninergic ganglion cells or displaced amacrine cells.     

Glycine high-affinity uptake labels a subpopulation of somatostatin-like immunoreactive cells in the Rana pipiens retina

Somatostatin-like immunoreactivity (Som-LI) and glycine high-affinity uptake have been characterized in the Rana pipiens retina. These labels are found in both the outer and inner plexiform layers (OPL and IPL), suggesting that interplexiform cells (IPCs) contain both Som and glycine in this retina. In double-label experiments these labels colocalize to an abundant population of cells in the mid-inner nuclear layer (INL), in the second or third cell layer distal from the IPL. These cells have medium sized spherical or oval somas, each with a single thin descending dendrite which ramifies in the distal IPL. Processes ascending from cells at this location were not visualized by immunocytochemistry, but could be seen by autoradiography of tissue processed for glycine high-affinity uptake. In autoradiographs apparent IPCs were the most intensely labeled cell type in this retina. Som-LI is also found in two types of probable amacrine cells in the proximal INL adjacent to the IPL, neither of which is labeled by glycine high-affinity uptake. One of these is rare (about 10 cells/mm2), and has a large pyriform soma with a thick dendrite that branches in the proximal IPL. The other type is more common (324 +/- 20 cells/mm2), has medium-sized spherical or horizontally elongated elliptical somas, and has multiple thin dendrites projecting into the distal IPL. In addition to the above cell types, faint Som-LI was seen in cells of the ganglion cell layer, possibly indicating the presence of somatostatinergic ganglion cells or displaced amacrine cells.     

Somatostatin-like immunoreactivity and glycine high-affinity uptake colocalize to an interplexiform cell of the Xenopus laevis retina

Antibodies directed against somatostatin have been used to label a population of interplexiform cells (IPCs) in the Xenopus laevis retina. These cells have spherical soma which lie in the inner nuclear layer (INL), adjacent to or one cell distal to the inner plexiform layer (IPL). Processes from these cells project throughout the IPL, with a fairly dense accumulation of labeled dendrites in the upper two-fifths of the IPL and a dense, narrow band of labeled dendrites adjacent to the ganglion cell layer. These cells also have finer processes, originating at the cell body, that traverse the INL and ramify in the outer plexiform layer (OPL). Double label experiments show that all of the cells that contain somatostatin-like immunoreactivity (SOM-LI) in the INL are also labeled by high-affinity uptake with 3H-glycine. Immunocytochemistry of retinal whole mounts shows that these cells are evenly distributed across the retina at a density of 542 +/- 65 cells/mm2. On the basis of the colocalization experiments and the morphological homogeneity of these cells, we suggest that they represent a single cell type. Interplexiform cell processes were further characterized by electron microscopy after immunocytochemistry or 3H-glycine autoradiography. In the IPL, IPC processes are seen to be postsynaptic at both ribbon and conventional synapses. This input is found almost entirely in the distal two-fifths of the IPL. Interplexiform cell processes are presynaptic to unlabeled processes in both the distal and proximal IPL. In the OPL, labeled processes are found near or contiguous with photoreceptor bases, and are often presynaptic to small-diameter processes. The postsynaptic processes have been identified as bipolar cell dendrites in six cases. Interplexiform cell processes may also contact horizontal cell processes in the OPL.     

Immunocytochemical staining of cholinergic amacrine cells in rabbit retina

Cholinergic neurons of rabbit retina were labelled with an antibody against choline acetyltransferase, the synthesizing enzyme for acetylcholine. Two populations of cells are immunoreactive. Type a cell bodies lie in the inner nuclear layer (INL), their dendrites branching narrowly in sublamina a of the inner plexiform layer (IPL), while type b cell bodies lie in the ganglion cell layer (GCL) with dendrites branching in sublamina b of the IPL. The irregular networks of clustered immunoreactive dendrites are similar, but not identical, in the two sublaminae. Type b cells are more numerous than type a cells in central retina. No axons were stained. It appears that the immunoreactive neurons are normally placed and displaced starburst/cholinergic amacrine cells.     

Tracer coupling of neurons in the rat retina inner nuclear layer labeled by Fluorogold

A subpopulation of neurons in the inner nuclear layer (INL) of the rat retina were labeled 9-13 weeks after application of Fluorogold (FG) to the superior colliculus. Neurobiotin injection of FG-labeled cells in the INL of flatmounted living retina revealed that these cells consisted of both displaced ganglion cells and a subset of amacrine cells. Fluorogold-labeled amacrine cells in the INL showed tracer coupling to other presumptive amacrine cells in the INL, but there was no evidence of coupling to neurons in the ganglion cell layer (GCL). As the labeling of amacrine cells by FG may be due to gap junction coupling between ganglion and amacrine cells, these data add to the evidence that tracer coupling between these cells can be unidirectional. Some of the FG-labeled displaced ganglion cells in the INL injected with Neurobiotin also showed tracer coupling to neurons in the INL or GCL.     

Localization of GAD- and GABA-like immunoreactivity in ground squirrel retina: retrograde labeling demonstrates GAD-positive ganglion cells.

Glutamic acid decarboxylase (GAD)- and gamma-aminobutyric acid (GABA)-like immunoreactivity was examined in the retina of the 13-lined ground squirrel (Spermophilus tridecemlineatus). Labeling was observed in the inner nuclear layer (INL), inner plexiform layer (IPL) and ganglion cell layer (GCL). The immunoreactive cell bodies in the inner third of the INL were 6-13 microns in diameter and, because of their size and location it was considered that these were amacrine cells. Labeling in the IPL was concentrated in 5 bands corresponding to laminae 1a, 1c, 2, 4 and 5. In the GCL a heterogeneous population of neurons exhibited GAD- and GABA-like immunoreactivity. The soma diameters of the GCL cells ranged from 5 to 17 microns. These may represent displaced amacrines and/or ganglion cells. To determine if any of the immunoreactive cells in the GCL were ganglion cells, double labeling experiments were performed using rhodamine latex microspheres ('beads') as retrograde neuronal tracers. Rhodamine beads were injected into the superior colliculus, and retinas with retrogradely labeled ganglion cells were subsequently incubated with the anti-GAD antiserum. These experiments revealed a small population of GAD-positive ganglion cells, setting a lower limit for the total number of GABAergic ganglion cells.     

Presence of thyrotropin-releasing-hormone-immunoreactive (TRHir) amacrine cells in the retina of anuran and urodele amphibians

The presence of thyrotropin-releasing-hormone-immunoreactive (TRH-ir) amacrine cells in the retina of amphibians is reported for the first time. The anuran and urodele retinas studied exhibit major differences in the distribution of TRH-ir cells. In the two urodele species investigated, most TRH-ir amacrine cells were located in the ganglion cell layer (GCL). These pear-shaped cells originate a dense TRH-ir dendritic plexus in strata 4-5 of the inner plexiform layer (IPL). A small number of TRH-ir amacrine cells were observed in the inner nuclear layer (INL). Most of these INL TRH-ir cells were multipolar neurons with radiating dendrites that originate a loose plexus in the IPL stratum 1. In the three anuran species investigated, most TRH-ir amacrine cells were located in the INL. Distribution of TRH-ir processes in the IPL of anurans was not so clearly layered as in urodeles, dendrites being observed throughout strata 1-5. In the toad retina THR-ir material was also observed in the outer plexiform layer, which suggests that toads may have some TRH-ir interplexiform neurons. In the frog and toad, TRH-ir fibers were also observed in the optic nerve, although their origin could not be ascertained. The number of TRH-ir amacrine cells per whole retina was higher in anurans than in urodeles, though urodeles have higher cell densities. The marked differences in distribution of TRH-ir amacrine cells observed between anurans and urodeles, and among the three anuran species, suggest different functions of TRH in retinal processing, perhaps related to the different specializations of the visual systems of these species.     

Immunocytochemical analysis of cholinergic amacrine cells in the tiger salamander retina

Cholinergic amacrine cells in the tiger salamander retina were observed for the first time by using antibodies against choline acetyltransferase (ChAT). ChAT-immunoreactive cells were present in the inner nuclear layer (INL) and in the ganglion cell layer (GCL), and the somas of the former population (average diameter = 15.13 microm) were slightly smaller than those of the latter population (average diameter = 16.42 microm). The processes of these cells form two distinct narrow bands in the inner plexiform layer (IPL), one located near 0.2 inner plexiform units (IU) and the other near 0.65-0.7 IU. Soma size, cell density and spatial distribution of ChAT-positive cells were quantitatively analyzed. Our results suggest that cholinergic amacrine cells in the salamander retina are very similar to their counter parts in other species, and they can be used as a model system for studying cholinergic functions in the visual system.     

Neurokinin 1 receptor expression in the rat retina

Tachykinin (TK) peptides influence neuronal activity in the inner retina of mammals. The aim of this investigation was to determine the cellular localization of the neurokinin 1 receptor (NK1), whose preferred ligand is the TK peptide substance P (SP), in the rat retina. These studies used a polyclonal antiserum directed to the C-terminus of rat NK1. The majority of NK1-immunoreactive (IR) cells were located in the proximal inner nuclear layer (INL), and very rarely they were found in the distal INL. Some small and large NK1-IR somata were present in the ganglion cell layer. NK1-IR processes were densely distributed across the inner plexiform layer (IPL) with a maximum density over lamina 2 of the IPL. Immunoreactive processes also crossed the INL and ramified in the outer plexiform layer where they formed a sparse meshwork. NK1-IR processes were rarely observed in the optic nerve fiber layer. Double-label immunofluorescence studies with different histochemical markers for bipolar cells indicated that NK1 immunoreactivity was not present in bipolar cells. Together, these observations indicate that NK1 immunoreactivity is predominantly expressed by amacrine, displaced amacrine, interplexiform, and some ganglion cells. Double-label immunofluorescence experiments were also performed to characterize NK1-containing amacrine cells. Sixty-one percent of the γ-aminobutyric acid (GABA)-IR cells, 71% of the large tyrosine hydroxylase (TH)-IR cells, and 100% of the small TH-IR cells contained NK1 immunoreactivity. In addition, most (91%) of the NK1-IR cells had GABA immunoreactivity. In contrast, vasoactive intestinal polypeptide-, TK-, choline acetyltransferase-, and parvalbumin-IR amacrine cells did not express NK1 immunoreactivity. Overall, the present findings suggest that SP acts directly upon several cell populations, including GABA-containing amacrine cells and ganglion cells, to influence visual information processing in the inner retina. J. Comp. Neurol. 389:496–507, 1997. © 1997 Wiley-Liss, Inc.     

Characterization of the cell death promoter, Bad, in the developing rat retina and forebrain.

Neuronal programmed cell death, or apoptosis, occurs during development, following injury or in certain disease processes, and is regulated by members of the B-cell leukemia-2 (Bcl-2) protein family. These molecules include both positive and negative regulators of cell death and act by selective dimerization that results in permissive or inhibitory effects on a cascade of cellular events, including mitochondrial release of cytochrome c, stimulation of cysteine protease activity and subsequent cellular deterioration. Here, we have characterized the expression of the cell death agonist, Bad, in the postnatal rat retina and forebrain. Isolation, subsequent amplification by RT-PCR and DNA sequence analysis revealed that retinal Bad was identical to Bad expressed in the developing and adult rat brain. Using a polyclonal antibody to Bad, we determined that, in the retina, on the day of birth (postnatal day-0, PND-0) Bad immunoreactivity was expressed primarily by retinal ganglion cells, some cells in the inner neuroblastic layer (NBL) and an indistinct plexus of processes in the inner plexiform layer (IPL). On PND-7, Bad immunoreactivity was observed in most cells in the ganglion cell layer (GCL), numerous cells scattered throughout the inner nuclear layer (INL), a lightly stained IPL and in a distinct band of immunostained fibers in the forming outer plexiform layer (OPL). By PND-15, Bad immunoreactivity was present in cells in the GCL, in some cells in the proximal INL and in horizontal cell processes in the OPL. The IPL was only faintly labeled. In the adult retina, specific Bad immunostaining was confined to large cells in the ganglion cell layer (presumed ganglion cells), occasional lightly stained horizontal cells and their processes in the OPL and to occasional small, lightly stained cells in the proximal INL (presumed amacrine cells) and GCL (presumed displaced amacrine cells). Again, the interposed IPL was faintly labeled. In the brain, Bad immunoreactive cells were scattered throughout the forebrain parenchyma but were particularly concentrated in neurons of the cerebral cortex, hippocampus and amygdala. Bad immunoreactivity was heaviest in these cells at PND-7, distinctly weaker at PND-10 and absent by PND-24. At all time points examined, Bad immunoreactivity was present in epithelial cells of the choroid plexus, as previously reported in the adult rat brain. These data suggest that Bad is transiently expressed by various cell types in the perinatal retina, particularly ganglion cells, and in discrete forebrain regions. In the context of corroborative observations, Bad expression may be regulated in response to acute ischemia and may act as a control point for retinal neuronal apoptosis.     

Localization of choline acetyltransferase in the developing and adult turtle retinas

Acetylcholine has important epigenetic roles in the developing retina. In this study, cells that expressed choline acetyltransferase (ChAT), the enzyme that synthesizes acetylcholine, were investigated in embryonic, postnatal, and adult turtle retinas by using immunofluorescence histochemistry. ChAT was present at stage 15 (S15) in cells near the vitreal surface. With the formation of the inner plexiform layer (IPL) at S18, ChAT-immunoreactive (-IR) cells were located in the inner nuclear layer (INL) and the ganglion cell layer (GCL). In the INL, presumed starburst amacrine cells were homogenous in appearance and formed a single row next to the IPL: This pattern was conserved until adulthood. In the GCL, however, there were multiple rows of ChAT-IR cells early in development, and this high density of labeled cells continued during the embryonic stages, until around birth. The high density of ChAT-IR cells in the GCL was due in part to a population of cells that expressed ChAT transiently. In postnatal stages and adult retinas, the presumed starburst amacrine ChAT-IR cells formed two mirror-like rows of homogenous cells on both borders of the IPL. Two cholinergic dendritic strata that were continuous with these cells were observed as early as S18, and their depths in the IPL were relatively stable throughout development. A third population of ChAT-IR cells was observed toward the middle of the INL around S25 and persisted into adulthood. Finally, cells in the outer nuclear layer (ONL) were ChAT-IR during the embryonic stages, were less immunoreactive during the postnatal stages, and were not immunoreactive in the adult retinas.