Retinal remodeling in human retinitis pigmentosa.

Retinitis Pigmentosa (RP) in the human is a progressive, currently irreversible neural degenerative disease usually caused by gene defects that disrupt the function or architecture of the photoreceptors. While RP can initially be a disease of photoreceptors, there is increasing evidence that the inner retina becomes progressively disorganized as the outer retina degenerates. These alterations have been extensively described in animal models, but remodeling in humans has not been as well characterized. This study, using computational molecular phenotyping (CMP) seeks to advance our understanding of the retinal remodeling process in humans. We describe cone mediated preservation of overall topology, retinal reprogramming in the earliest stages of the disease in retinal bipolar cells, and alterations in both small molecule and protein signatures of neurons and glia. Furthermore, while Müller glia appear to be some of the last cells left in the degenerate retina, they are also one of the first cell classes in the neural retina to respond to stress which may reveal mechanisms related to remodeling and cell death in other retinal cell classes. Also fundamentally important is the finding that retinal network topologies are altered. Our results suggest interventions that presume substantial preservation of the neural retina will likely fail in late stages of the disease. Even early intervention offers no guarantee that the interventions will be immune to progressive remodeling. Fundamental work in the biology and mechanisms of disease progression are needed to support vision rescue strategies.

Extreme retinal remodeling triggered by light damage: implications for age related macular degeneration.

PURPOSE: Our objective was to comprehensively assess the nature and chronology of neural remodeling in retinal degenerations triggered by light-induced retinal damage (LIRD) in adult albino rodents. Our primary hypothesis is that all complete photoreceptor degenerations devolve to extensive remodeling. An hypothesis emergent from data analysis is that the LIRD model closely mimics late-stage atrophic age relared macular degeneration (AMD). METHODS: Sprague-Dawley (SD) rats received intense light exposures of varied durations and survival times ranging from 0 to 240 days. Remodeling was visualized by computational molecular phenotyping (CMP) of a small molecule library: 4-aminobutyrate (gamma), arginine (R), aspartate (D), glutamate (E), glutamine (Q), glutathione (J), glycine (G), and taurine (tau). This library was augmented by probes for key proteins such as rod opsin, cone opsin and cellular retinal binding protein (CRALBP). Quantitative CMP was used to profile 160 eyes from 86 animals in over 6,000 sections. RESULTS: The onset of remodeling in LIRD retinas is rapid, with immediate signs of metabolic stress in photoreceptors, the retinal pigmented epithelium (RPE), the choriocapillaris, and Müller cells. In particular, anomalous elevated aspartate levels appear to be an early stress marker in photoreceptors. After the stress phase, LIRD progresses to focal photoreceptor degeneration within 14 days and extensive remodeling by 60 days. RPE and choriocapillaris losses parallel Müller cell distal seal formation, with progressive neuronal migration, microneuroma evolution, fluid channel formation, and slow neuronal death. The remaining retina in advanced light damage can be classified as survivor, light damage (LD), or decimated zones where massive Müller cell and neuronal emigration into the choroid leaves a retina depleted of neurons and Müller cells. These zones and their transitions closely resemble human geographic atrophy. Across these zones, Müller cells manifest extreme changes in the definitive Müller cell tauQE signature, as well as CRALBP and arginine signals. CONCLUSIONS: LIRD retinas manifest remodeling patterns of genetic retinal degeneration models, but involve no developmental complexities, and are ultimately more aggressive, devastating the remaining neural retina. The decimation of the neural retina via cell emigration through the perforated retina-choroid interface is a serious denouement. If focal remodeling in LIRD accurately profiles late stage atrophic age-related macular degenerations, it augurs poorly for simple molecular interventions. Indeed, the LIRD profile in the SD rat manifests more similarities to advanced human atrophic AMD than most genetically or immunologically induced murine models of AMD.

Synaptic organization of enkephalinlike-immunoreactive amacrine cells in the goldfish retina.

Immunoelectron microscopy was used to examine the synaptic organization of enkephalinlike-immunoreactive amacrine cells in the goldfish retina. Enkephalin-immunostained processes sometimes contained dense-cored vesicles (115-145 nm) in addition to a generally homogeneous population of small, round, clear synaptic vesicles. A total of 194 synaptic relationships were observed that involved the immunostained processes of enkephalin-amacrine cells. The large majority of these were observed in sublayer 5 of the inner plexiform layer. In greater than 95% of the synaptic relationships, the enkephalin-immunostained profile served as the presynaptic element. In 58.8% of these relationships, enkephalin processes synapsed onto amacrine cell processes, while 30.4% of their synapses were onto processes that lacked synaptic vesicles. They also occasionally formed synaptic contacts (6.7%) onto the somas of cells located either in the inner nuclear or in the ganglion cell layers. Enkephalin profiles received synaptic input only from amacrine cells (4.1%), while no direct synaptic interaction was observed between enkephalin processes and bipolar cells. However, in sublayer 1, enkephalin profiles were found to synapse onto amacrine cell processes that were presynaptic to bipolar cell terminals. In the proximal inner plexiform layer, enkephalin processes were presynaptic to amacrine cell processes that as a group surrounded and sometimes provided synaptic input to extremely large and round bipolar cell endings.

The cerebellopontine system in the rat. I. Autoradiographic studies.

This study utilized light microscopic autoradiographic procedures to describe the projections from the three major subdivisions of the deep cerebellar nuclei (DCN) to the basilar pontine nuclei (BPN). Although the vast majority of cerebellopontine axons reached the BPN via the descending limb of the brachium conjunctivum (BC) after crossing the midline within the midbrain, a relatively small number of ipsilaterally projecting fibers was also observed. Fascicles of cerebellopontine axons left the main bundle of descending limb fibers throughout much of the rostrocaudal length of the BPN and passed around and through the medial lemniscus and cerebral peduncle to enter the pontine gray. The lateral cerebellar nucleus gave rise to the largest number of cerebellopontine fibers, whose terminal fields exhibited both diffuse and patchlike labeling patterns within each of the major subdivisions of the BPN including medial, ventral, lateral, and dorsal areas. Projections from the interpositus complex exclusive of its posterior division were fewer and less widely distributed than those from the lateral nucleus. Interpositopontine fibers terminated primarily in the caudal one-half of the BPN in medial, ventral, and lateral regions and overlapped somewhat with projections from the lateral cerebellar nucleus. Pontine projections emanating from the medial cerebellar nucleus were the fewest and most restricted in distribution relative to the other two cerebellar efferent systems. Such fibers formed a patchlike network of terminal fields which extended throughout much of the rostrocaudal length of the BPN in medial and dorsomedial regions. A relatively small but considerable number of ipsilateral cerebellopontine fibers terminated in pontine regions, which often mirrored the typical contralateral projection fields. Although it proved difficult to determine the precise origin of the ipsilateral fiber systems, it appeared that each of the three major DCN subdivisions made some contribution. Also it was apparent that considerable overlap existed between cerebellopontine projection zones and those of other pontine afferents including sensorimotor, visual, and auditory cortices, the superior colliculus, and the mammillary nuclei of the hypothalamus. Moreover, cerebellopontine terminal fields were congruent in some instances with discrete clusters of BPN neurons which serve as the source of pontocerebellar fiber systems, reaching portions of the lateral cerebellar hemispheres, posterior vermis, and the paraflocculus.

Interaction between enkephalin and gamma-aminobutyric acid in the chicken retina: a double-label immunoelectron microscopic analysis.

In the present study, double-label immunoelectron microscopy was used to examine the synaptic relationships between amacrine cell populations in the chicken retina that contain either enkephalin or gamma-aminobutyric acid (GABA) or both enkephalin and GABA. The objectives of the present study were twofold. First, the ultrastructural features and synaptic organization of enkephalin and enkephalin/GABA amacrine cells were compared. Second, the synaptic interactions between these populations and the population of GABA amacrine cells were examined. A total of 475 synaptic arrangements were observed to involved enkephalin or enkephalin/GABA amacrine cell processes. The synaptic relationships of enkephalin and enkephalin/GABA amacrine cells were quite similar. Each population was pre- and postsynaptic to amacrine cells, postsynaptic to bipolar cells, and presynaptic to processes possibly originating from ganglion cells. A substantial percentage of each population's pre- and postsynaptic relationships were with the processes of GABAergic amacrine cells. Moreover, when enkephalin and enkephalin/GABA amacrine cell processes were postsynaptic to bipolar cells, their dyadic partner was observed frequently to be a GABA amacrine cell process. The present study suggests a diversity in the population of chicken enkephalin amacrine cells with respect to their expression of the classical inhibitory transmitter GABA. Moreover, a functional relationship between enkephalinergic and GABAergic pathways is indicated by studies showing that both enkephalin and enkephalin/GABA amacrine cells exhibit substantial synaptic interaction with GABA amacrine cells.

The cerebellopontine system in the rat. II. Electron microscopic studies.

Cerebellopontine axonal boutons in the neuropil of the basilar pontine nuclei (BPN) were marked for ultrastructural identification by producing unilateral electrolytic lesions of the superior cerebellar peduncle (SCP) as it exited from the cerebellum and before its decussation in the caudal midbrain. Three varieties of degenerating boutons were distinguished on the basis of size, type of degeneration, and postsynaptic locus. A relatively large variety of bouton (2.5-6.0 microns) that exhibited filamentous degeneration throughout the range of survival times employed (1-14 days) was the most frequently observed type of degenerating cerebellopontine bouton. Such boutons formed synaptic contacts with several small, dendritic, spinelike profiles as well as the shafts of intermediate or proximal dendrites. A second, far less numerous and somewhat smaller type of bouton (1.5-4.5 microns) was distinguished by the fact that it exhibited advanced dark degenerative changes after a 2-day survival period, formed multiple spine contacts (but not shafts), and was no longer apparent in the neuropil after a postlesion survival time of 6 days. The third variety of degenerating bouton was small (0.8-2.0 microns), exhibited dark degeneration with a 2-6 day survival period, contacted primarily shafts of small-diameter dendrites, and was observed more frequently than the larger dark boutons but less often than the large filamentous boutons. All three types of degenerating boutons contained round, clear, synaptic vesicles and formed only asymmetric synaptic active sites. It is suggested that the three types of degenerating axon terminals arise from at least three varieties of neurons in the deep cerebellar nuclei. Further it is suggested that such boutons originate from cerebellar efferent axons which distribute in collateral fashion to the thalamus, red nucleus, and/or the inferior olive.

Localization of serotoninlike-immunoreactive amacrine cells in the larval tiger salamander retina.

Light microscopic immunocytochemistry was used to study the populations of serotoninlike-immunoreactive cells in the larval tiger salamander retina. Of 1,135 serotonin-immunostained cells observed in transverse cryosections, 87% were identified as amacrine cells, whereas 13% were tentatively designated as displaced amacrine cells. The somas of the vast majority of serotonin-amacrine cells were situated in the innermost cell row of the inner nuclear layer. Only a few serotonin-immunostained amacrine cell somas were observed in the second row of cells from the inner nuclear layer. Serotonin-immunoreactive processes generally appeared as a diffuse plexus distributed evenly throughout all levels of the inner plexiform layer. As determined in whole-mount preparations, serotonin-amacrine cells were divisible into two populations on the basis of the diameters of their somas. Large cells (45%) ranged from 16 to 19 microns in diameter with the vast majority measuring 17-18 microns. Smaller and sometimes less intensely stained cells ranged from 14 to 16 microns in diameter with the large majority measuring 15 microns. The diameters of serotonin-displaced amacrine cells ranged from 19 to 22 microns with the large majority measuring 20 microns in diameter. An examination of whole-mount retinas revealed that serotonin-immunoreactive amacrine and displaced amacrine cells were distributed throughout the center and the periphery of the retina. The density of serotonin-amacrine cells (large and small combined) was calculated to be 173 +/- 4.5 (mean +/- standard error) cells per mm2.

Projections to the basilar pontine nuclei from face sensory and motor regions of the cerebral cortex in the rat.

Orthograde axonal transport tracing methods were used to describe the projections to the basilar pontine nuclei (BPN) which arise within the face representation of motor or somatosensory cerebral cortex. Injections centered in motor face (MF) cortex resulted in the labeling of several corticopontine terminal fields which exhibit a rostrocaudal columnar arrangement within the ipsilateral BPN. The location of such terminal zones is consistent with the somatotopic pattern of termination previously described for limb sensorimotor cortices. In contrast, the projections from somatosensory face (SF) cortical regions largely terminate in BPN areas separate from those receiving either limb sensorimotor or MF inputs. Both MF and SF cortices also give rise to projections to the contralateral BPN; those from SF cortex are less extensive than those of MF origin. In addition to their relationship with limb sensorimotor corticopontine terminations, the MF projections to the BPN also seem to partially overlap the projection zones of the cerebellopontine system, particularly the regions projected upon by the lateral cerebellar nucleus. The SF projections, on the other hand, appear to terminate in BPN areas that also receive input from either the dorsal column nuclei or the spinal trigeminal complex. There is only minimal potential overlap between MF and SF projections in the BPN. With regard to the pontocerebellar system, the projections from MF cortex terminate among BPN neurons which project to the cerebellar hemispheres, particularly lobus simplex, crus I and crus II. The SF projections also overlap BPN neurons which project to the lateral hemispheres in addition to the paraflocculus and vermal lobules VII and IXa,b. Taken together these observations suggest that subsets of BPN neurons might exist such that some receive convergent inputs from systems whose function can generally be regarded as motor (sensorimotor cortex, cerebellopontine) while another population of BPN neurons might integrate signals from systems which transmit somatosensory information (dorsal column nuclei, spinal trigeminal).

Localization of tyrosine-hydroxylase-like-immunoreactive amacrine cells in the larval tiger salamander retina.

Immunocytochemistry was used to localize the populations of tyrosine-hydroxylase-like (TH)-immunoreactive cells in the tiger salamander retina. Ninety percent of these cells possessed somas that were situated in the innermost cell row of the inner nuclear layer and were classified as amacrine cells. Ten percent of TH-immunoreactive somas were located in the ganglion cell layer and were tentatively designated as those of displaced amacrine cells. The processes of TH-immunoreactive cells ramified most heavily in sublayer 1 of the inner plexiform layer, while a relatively small number of TH-labelled processes distributed in sublayers 3 and 5. Less than 1% of TH-immunoreactive cells in the amacrine cell layer exhibited a short process of somal origin that extended distally toward the outer plexiform layer. However, these processes did not cross the whole of the inner nuclear layer, and no immunolabelling was observed in the outer plexiform layer. An examination of retinal whole-mounts revealed that TH-immunoreactive amacrine and displaced amacrine cells were distributed throughout the center and periphery of the retina. The density of TH-immunolabelled amacrine cells was calculated to be 49 +/- 13 (mean +/- standard error) cells per mm2. The vast majority of TH-immunoreactive amacrine and displaced amacrine cells exhibited a stellate appearance and gave rise to three or more primary dendrites. A few TH-amacrine and displaced amacrine cells possessed two primary dendrites that emerged from opposite sides of their somas. The processes of TH-immunoreactive cells were generally poorly branched and varicose with terminal branches sometimes appearing thin and beaded. Because some TH-immunolabelled processes were very long, there was considerable overlap between the dendritic fields of neighboring TH-cells. Lastly, individual TH-immunoreactive amacrine and displaced amacrine cells were often observed in whole-mounts to provide processes that ramified at more than one level of the inner plexiform layer.

Localization of enkephalin-like immunoreactive amacrine cells in the larval tiger salamander retina: a light and electron microscopic study.

Immunohistochemistry was utilized to examine the light and electron microscopic localization of enkephalin-like (enk) immunoreactive amacrine cells in the larval tiger salamander retina. The vast majority of enk-immunoreactive cells were typical amacrine cells whose round or oval cell bodies (14-16 microns) were situated in the innermost cell row of the inner nuclear layer. A relatively small number of enk-stained oval cell bodies (14-22 microns) were located in the ganglion cell layer and were designated as those of displaced amacrine cells. Enkephalin immunostaining was observed in the inner plexiform layer as a fine plexus in sublamina 1 and as a dense network of fibers in sublamina 5. In both the center and periphery of the retina the density of typical enk-amacrine cells was determined to be 250 +/- 16.36 cells per mm2 surface area of the retina. At the ultrastructural level typical enk-stained amacrine cells possessed a round, indented nuclear membrane. Enk-immunoreactive processes sometimes contained dense-core vesicles (60-115 nm) in addition to a rather homogeneous population of small, round, agranular synaptic vesicles (25-35 nm). In sublamina 1 the processes of enk-amacrine cells were presynaptic to amacrine and bipolar cells. They also contacted processes devoid of synaptic vesicles which possibly arise from ganglion cells. As the postsynaptic element in sublamina 1, they received synaptic input from amacrine cells. In sublamina 5 the processes of enk-amacrine cells were presynaptic to amacrine cells, bipolar cells, and the somas of cells situated in the ganglion cell layer.(ABSTRACT TRUNCATED AT 250 WORDS)

Synaptic organization of serotonin-like immunoreactive amacrine cells in the larval tiger salamander retina.

Immunoelectron microscopy was used to investigate the ultrastructural features and synaptic relationships of serotonin-like immunoreactive amacrine cells in the larval tiger salamander retina. Serotonin-positive somas exhibited an evenly distributed peroxidase reaction product throughout their cytoplasm. Their nuclei were unstained and possessed indented nuclear membranes. Serotonin-immunoreactive processes were generally stained throughout with the exception of their mitochondria, whose morphology was often disrupted by the staining reaction. They were further characterized by an occasional dense-cored vesicle/s in addition to a generally homogeneous population of small, round, clear synaptic vesicles. Serotonin-immunoreactive amacrine cell processes formed conventional synapses that were characterized by symmetrical synaptic membrane densities. A total of 222 synaptic arrangements were observed that involved the immunostained processes of serotonin-amacrine cells. As presynaptic elements, they primarily contacted amacrine cells processes (37.8%). They also provided substantial synaptic input to processes that lacked synaptic vesicles (16.2%) and whose origin was unidentified. Serotonin-processes provided a far fewer number of synaptic contacts onto the processes of bipolar cells (1.4%) and the somas of cells in the amacrine cell layer (0.5%). As postsynaptic elements, they received synaptic inputs from amacrine cells (27.9%) and bipolar cells (16.2%). With the exception of their synapses onto bipolar cells and the somas of cells in the amacrine cell layer, each of the synaptic relationships of serotonin-amacrine cells was observed in each of sublayers 1-5 of the inner plexiform layer.

Synaptic organization of dopaminergic amacrine cells in the larval tiger salamander retina.

The ultrastructural features and synaptic interactions of tyrosine hydroxylase-like-immuno-reactive amacrine cells in the larval tiger salamander retina were examined using routine immunoelectron microscopy. The somas of tyrosine hydroxylase-like-immunoreactive amacrine cells were immunostained evenly throughout their cytoplasm. Their nuclei were generally unstained and possessed indented nuclear membranes. The processes of tyrosine hydroxylase-like-immunoreactive amacrine cells were homogeneously stained with the exception of their mitochondria, whose morphology was often disrupted by the staining procedure. Tyrosine hydroxylase-like-immunoreactive amacrine cell processes were characterized by an occasional dense-cored vesicle(s), in addition to a generally homogeneous population of small, round, agranular synaptic vesicles. They formed conventional synaptic junctions that were characterized by symmetrical synaptic membrane densities. A total of 168 synapses were observed that involved tyrosine hydroxylase-like-immunoreactive amacrine cell processes. A large percentage (79.8%) of these synaptic arrangements were found in sublayer 1 of the inner plexiform layer, while substantially lower percentages were observed in sublayers 3 (9.5%) and 5 (10.7%). They served as pre- and postsynaptic elements 63.1 and 36.9% of the time, respectively. Tyrosine hydroxylase-like-immunoreactive amacrine cell processes were presynaptic to amacrine cell processes (36.9% of total synaptic involvement) and processes that lack synaptic vesicles and whose origin remains uncertain (26.2%). They received synaptic input primarily from amacrine cell processes (31.0%). Tyrosine hydroxylase-like-immunoreactive amacrine cell processes also received a few ribbon synapses from bipolar cells (5.9%). Each of these synaptic relationships were observed in each of sublayers 1, 3 and 5 of the inner plexiform layer, with the majority of each arrangement being found in sublayer 1.

Localization of classical neurotransmitters in interneurons of the larval tiger salamander retina.

Autoradiography was used to visualize the neurons in the tiger salamander retina that exhibit high-affinity uptake of 3H-dopamine, [3H]-serotonin, [3H]-glycine, and [3H]-GABA. Both [3H]-dopamine and [3H]-serotonin were accumulated by amacrine cells and by displaced amacrine cells. [3H]-glycine was taken up by amacrine cells, displaced amacrine cells, bipolar cells, and displaced bipolar cells. [3H]-GABA was accumulated by amacrine cells and by cells in the ganglion cell layer that may be displaced amacrine or ganglion cells. [3H]-GABA was also taken up by horizontal cells, bipolar cells, and displaced bipolar cells.

The production and characterization of monoclonal antibodies against enkephalins.

The hybridoma technology of Kohler and Milstein (1975) was utilized to produce monoclonal antibodies against the enkephalins. Two hybridomas, AD4 and DB4, produced monoclonal antibodies of the IgG type 1 class against Leu(5)-enkephalin that were highly specific for Leu(5)- and Met(5)-enkephalin. AD4 exhibited almost equal reactivity with either Leu(5)- or Met(5)-enkephalin, whereas DB4 exhibited only a 20% cross-reactivity with Met(5)-enkephalin. The IC(50) of these monoclonal antibodies were approximately two orders of magnitude greater than the IC(50) a polyclonal antiserum against enkephalins (A206; Miller et al 1978) used routinely in many immunochemical and immunocytochemical studies. The monoclonal antibodies, AD4 and DB4, exhibited specific sequence and size requirements for binding enkephalin-related peptides. The amino acid sequence Gly-Gly-Phe-Leu or Gly-Gly-Phe-Met was essential for recognition by AD4 and DB4. However, Tyr-Gly-Gly-Phe which lacks Leu or Met in the fifth position did not react with our monoclonal antibodies. Moreover, enkephalin-related peptides in which the enkephalin sequence was situated at the amino terminus and which contained six or more amino acids did not react significantly with AD4 or DB4. In particular, unlike the polyclonal antiserum A206, our monoclonal antibodies do not react with dynorphins 1-6 or 1-13. However, when the monoclonal antibody (AD4) was used to localize immunohistochemically the population of enkephalinergic amacrine cells in the chicken retina, it provided a staining pattern quite comparable to that observed in previous studies (Watt et al., 1983) using the polyclonal enkephalin antiserum A206. This finding therefore demonstrates that the immunoreactive products visualized in the enkephalin-immunoreactive amacrine cells of the chicken retina with the polyclonal antiserum correspond to authentic enkephalin or peptides very closely related to the enkephalins.

A double-label study demonstrating that all serotonin-like immunoreactive amacrine cells in the larval tiger salamander retina express GABA-like immunoreactivity.

A previous study localized serotonin-like immunoreactivity to amacrine cell populations in the larval tiger salamander retina. The present double-label immunocytochemical analysis of the tiger salamander retina was performed to determine if gamma-aminobutyric acid (GABA)-like immunoreactivity is expressed by serotonin-immunoreactive amacrine cells. More than 3,000 serotonin-amacrine cells were observed in double-label preparations, and all were found to express GABA-like immunoreactivity. This finding extends previous studies of serotonin-GABA coexistence in the retina by providing the first report of the co-localization of endogenous serotonin and GABA-like compounds in a retinal neuron.

A re-examination of enkephalin's coexistence with gamma-aminobutyric acid in amacrine cells of the larval tiger salamander retina.

Double-label immunocytochemistry was utilized to re-examine the colocalization of enkephalin and gamma-aminobutyric acid (GABA) in amacrine cells of the larval tiger salamander retina. A total of 465 enkephalin-immunoreactive amacrine cells were identified and in all cases these cells were GABA-immunoreactive. This finding corroborates a previous study that showed greater than 96% of enkephalin-amacrine cells in the tiger salamander retina to specifically accumulate [3H]GABA and provides additional evidence for the GABAergic nature of these enkephalin-amacrine cells.

Interaction between enkephalin and GABA in the chicken retina: further analyses of coexisting relationships.

Previous studies have indicated an interactive relationship between enkephalin and gamma-aminobutyric acid (GABA) in the vertebrate retina. Among these studies are those that have demonstrated the colocalization of enkephalin and GABA in retinal amacrine cells. In the present study, enkephalin immunocytochemistry was combined with either autoradiography of tritiated GABA high-affinity uptake or GABA immunocytochemistry to further investigate the coexistence of GABA in enkephalin-amacrine cells of the chicken retina. A regional analysis revealed that the percentage colocalization of GABA high-affinity uptake in enkephalin-amacrine cells did not vary appreciably throughout the retina. Overall, 15.2% of enkephalin-amacrine cells exhibited high-affinity GABA uptake. Double-label immunofluorescence histochemistry revealed that 15.1% of enkephalin-amacrine cells express endogenous GABA-like immunoreactivity. These double-labelled cells were observed throughout central and peripheral regions of the retina. In each of the double-label analyses, only less intensely labelled enkephalin-amacrine cells expressed markers of GABA activity. The two double-label analyses reveal almost identical percentages of coexistence of GABA markers in chicken enkephalin-amacrine cells and therefore, provide supportive evidence for the GABAergic nature of these cells. These results suggest a functional diversity in the population of chicken enkephalin-amacrine cells and imply the possibility of multiple signalling through amacrine cells which contain enkephalin and GABA.

Double-label analyses demonstrating the non-coexistence of enkephalin and glycine in amacrine cells of the larval tiger salamander retina.

Enkephalin immunocytochemistry was combined with either glycine immunocytochemistry or autoradiography of high-affinity glycine uptake to examine for colocalization of enkephalin and glycine in amacrine cells of the larval tiger salamander retina. A total of 995 enkephalin-immunoreactive amacrine cells were visualized in double-label preparations. None of the enkephalin-labelled cells was observed to co-label for markers of glycinergic activity.

Interaction between enkephalin and dopamine in the avian retina.

Biochemical and pharmacological techniques were utilized to investigate the interaction between the enkephalinergic and dopaminergic systems in the chicken retina. Exogenously applied enkephalin and its analogues were observed to inhibit the release of preloaded dopamine from the retina. This inhibition was concentration-dependent and was suppressed by the opiate antagonist, naloxone. The relationship between enkephalinergic and dopaminergic amacrine cells was studied in retinas which were subjected to 6-hydroxydopamine (6-OHDA) treatments. 6-OHDA degenerated approximately 80-90% of those cells which exhibit high affinity uptake of [3H]dopamine. In 6-OHDA-treated retinas, the capacity of 3H-labelled [D-Ala2]methionine enkephalinamide to bind specifically to opiate receptors was substantially reduced (only 70-75% of the control). Scatchard analyses and ligand displacement studies indicated that this decrease in binding was due to a reduction in the number of opiate receptors. Taken together, these observations strongly indicate that a fraction of the opiate receptors in the chicken retina (25-30%) are closely associated with the population of dopaminergic amacrine cells.

Double-label analyses of the coexistence of somatostatin with GABA and glycine in amacrine cells of the larval tiger salamander retina.

To investigate the possible GABAergic nature of somatostatin-immunoreactive neurons of the larval tiger salamander retina, somatostatin immunocytochemistry was combined with either gamma-aminobutyric acid (GABA) immunocytochemistry or autoradiography of GABA high-affinity uptake. A total of 1,062 somatostatin cells were visualized in these studies. Double-label immunocytochemistry revealed that 96.3% of somatostatin-immunoreactive cells expressed GABA immunoreactivity. Double-label studies combining somatostatin immunocytochemistry with autoradiography of GABA high-affinity uptake revealed a slightly lower percentage (93%) of colocalization. Double-labelled cells were identified as Type 1, Type 2 and displaced amacrine cells. The small percentage of somatostatin-immunoreactive cells that did not co-label for GABA were identified as Type 1 amacrine cells. An analysis of retinal sections processed for double-label immunocytochemistry revealed that approximately 5% of GABA-immunoreactive cells in the amacrine and ganglion cell layers co-label for somatostatin. Somatostatin immunocytochemistry was combined with autoradiography of glycine high-affinity uptake to examine whether tiger salamander somatostatin-amacrine cells express this glycine marker. A total of 100 somatostatin-immunoreactive amacrine cells were visualized in double-label preparations. None of these cells were observed to exhibit glycine high-affinity uptake.