The development of NADPH-diaphorase and nitric oxide synthase in the visual system of the cichlid fish, Tilapia mariae

The pattern of NADPH-diaphorase expression was studied in the retina and optic tectum of the cichlid fish Tilapia mariae during the first developmental stages. NADPH-diaphorase activity was seen early, at hatching. In the retina a few cell bodies of the retinal inner nuclear layer showed a faint labeling. Scattered labeled cells were found in the stratum periventriculare of the optic tectum, while the optic nerve was unlabeled. Two days after hatching, the number of labeled neurons increased in the inner nuclear layer and a few stained cell bodies were also scattered in the ganglion cell layer. Both the inner and outer plexiform layers showed a diffuse staining and the optic nerve was devoid of labeling. In the optic tectum several positive cells in the periventricular layer, with their dendritic trees extending in the superficial fibrous layer, were found. In 1-month-old Tilapia, NADPH-diaphorase staining and nitric oxide synthase immunoreactivity were found to overlap in both the retina and optic tectum. The density of NADPH-diaphorase labeled neurons in the inner nuclear layer of the retina and in the stratum periventriculare of the optic tectum was largely reduced in comparison with 2 days posthatching embryos. These findings indicated an early and transient production of nitric oxide in the retina and optic tectum of Tilapia, suggesting a functional role for nitric oxide in the development of visual structures in aquatic vertebrates.     

Localization and Synaptic Release of N-acetylaspartylglutamate in the Chick Retina and Optic Tectum

The neuropeptide, N-acetylaspartylglutamate (NAAG), was identified in the chick retina (1.4 nmol/retina) by HPLC, radioimmunoassay and immunohistochemistry. This acidic dipeptide was found within retinal ganglion cell bodies and their neurites in the optic fibre layer of the retina. Substantial, but less intense, immunoreactivity was detected in many amacrine-like cells in the inner nuclear layer and in multiple bands within the inner plexiform layer. In addition, NAAG immunoreactivity was observed in the optic fibre layer and in the neuropil of the superficial layers of the optic tectum, as well as in many cell bodies in the tectum. Using a newly developed, specific and highly sensitive (3 fmol/50 microl) radioimmunoassay for NAAG, peptide release was detected in isolated retinas upon depolarization with 55 mM extracellular potassium. This assay also permitted detection of peptide release from the optic tectum following stimulation of action potentials in retinal ganglion cell axons of the optic tract. Both of these release processes required the presence of extracellular calcium. Electrically stimulated release from the tectum was reversibly blocked by extracellular cadmium. These findings suggest that NAAG serves an extracellular function following depolarization-induced release from retinal amacrine neurons and from ganglion cell axon endings in the chick optic tectum. These data support the hypothesis that NAAG functions in synaptic communication between neurons in the visual system.     

Neurotrophin-3 and TrkC in the frog visual system: changes after axotomy

Neurotrophins are potent regulators of the survival of different neuronal populations in the CNS. Little is known of the immunodistribution of neurotrophin-3 (NT-3) and tyrosine kinase C (TrkC) receptor in the frog visual system, which can successfully regenerate and recover vision after injury. In this study we show that both NT-3 and TrkC are present in the frog retina and tectum, and that their distribution changes after optic nerve transection. Both NT-3 and TrkC are present in the ganglion cell layer, inner nuclear layer, nerve fiber layer and outer plexiform layer, and in Müller cells of control retinas. Quantification of identified RGCs shows that there are only small changes in the proportion, or intensity, of NT-3 immunostained cells surviving after axotomy and regeneration. Müller cell staining, however, is increased. TrkC staining in the retina does not change after axotomy. In the tectum, NT-3 immunoreactivity is present in the retinorecipient layer 9, and in radial processes of neurons and ependymoglia. TrkC is present in ependymoglia and in tectal neurons. After axotomy or colchicine treatment fewer NT-3-immunoreactive processes are present in layer 9 and there is decreased staining of tectal neurons. These data are consistent with the hypothesis that NT-3 is synthesized in the retina and anterogradely transported to the tectum. TrkC immunostaining, on the other hand, increases in tectal cells after optic nerve transection, suggesting that it may be regulated by the supply of NT-3 from the retina.     

Distribution of substance P-like immunoreactive retinal ganglion cells and their pattern of termination in the optic tectum of chick (Gallus gallus)

Substance P-like immunoreactive (SP-LI) neurons were identified within the inner nuclear layer and ganglion cell layer of the chick retina. The SP-LI cells in the inner nuclear layer consisted of several subtypes of neurons, differing in soma size and dendritic arborization. In the ganglion cell layer a population of moderately labelled SP-LI neurons was also present. About 6-9 microns in diameter and spaced 50-80 microns apart, they formed a regular array across the entire retina, with a density of about 400 cells/mm2 in the superior temporal retina, declining to less than 100 cells/mm2 in the peripheral retina. The total number of SP-LI cells in the ganglion cell layer was approximately 75,000. Individual axons could be followed toward the optic nerve head. Lesions near the optic nerve head resulted in axotomy of ganglion cells within a limited portion of the retina. Two days of postaxotomy there were numerous SP-LI swellings in the proximal segments of axotomized axons. SP-LI neurons in the axotomized zone were larger, more numerous, and showed increased staining of their processes. Fourteen days following a retinal lesion, there was depletion of all SP-LI cells in the ganglion cell layer within the axotomized zone, but the SP-LI neurons in the inner nuclear layer were not noticeably affected. Following a localized injection of rhodamine-coupled latex beads into the optic tectum, a population of retinal ganglion cells (RGCs) in the contralateral retina was retrogradely labelled. Many of these cells also exhibited SP-like immunoreactivity. Examination of the optic tectum indicated the presence of SP-LI fibres in laminae 2-13 (nomenclature of Cajal: Histologie du Systeme Nerveux. Vol. 2. Paris: Maloine, '11), with immunoreactive terminal regions present mainly in laminae 2-4, 7, and 9-13. SP-LI cell bodies were found predominantly in laminae 10-12 and 13. Fourteen days following a retinal lesion, SP-LI processes and terminals were depleted from laminae 2 and 3. Immunoreactive cells and processes in the remaining laminae of the optic tectum were not noticeably altered. The present report confirms the existence of SP-LI retinal ganglion cells in the chick retina and demonstrates their contribution to lamina specific SP-LI arborization in the optic tectum.     

Thy-1 expression in the retinotectal system of the chick

Previous investigations into the occurrence of Thy-1 in the chick retina have not clearly defined when the antigen first appears and have not adequately described its expression during the relatively early phases of retinal ontogeny. We have investigated these issues, using improved immunohistochemical procedures and show that Thy-1 is associated with the retinal ganglion cells from the time they begin to differentiate by extending their axonal projections. In addition, we have found that its expression reflects the growth of the optic fibre layer and the elaboration of the ganglion cell dendritic processes into the inner plexiform layer. For the first time we describe the appearance and the developmental expression of Thy-1 in the chick tectum. We have found that Thy-1 is associated with retinal axons from the time of their arrival at the tectum and that its expression reflects the elaboration of the stratum opticum. Within the tectum proper Thy-1 appears first in 3 distinct layers all of which are plexiform in nature. By the time that tectal histogenesis is essentially complete the antigen is expressed by all the layers of the tectum. The implications of these findings are discussed in terms of the development of the individual tissues and with respect to the elaboration of the retinotectal pathway.     

Expression of presynaptic proteins is closely correlated with the chronotopic pattern of axons in the retinotectal system of the chick

Newly synthesized presynaptic integral membrane proteins in neurons are transported in precursor vesicles from the site of protein biosynthesis in the cell body by fast axonal flow to the presynaptic terminal. We followed the path that presynaptic proteins travel on the way to their central targets of the highly ordered primary visual pathway of the chick and analyzed the developmental changes in the expression of synaptic vesicle protein 2 (SV2), synaptotagmin, and syntaxin. Immunofluorescences revealed that: (1) the onset of protein expression in the retinal ganglion cells occurs in a central to peripheral developmental pattern from embryonic day 4 (E4) onward; (2) the proteins were found first in the inner and later in the outer plexiform layer of the retina; and (3) they were redistributed from the photoreceptor inner segments and cell bodies to the terminals in the outer plexiform layer. From E4 onward, immunopositive axons for SV2, synaptotagmin, and syntaxin were found in the optic nerve, disappearing after E9 for SV2 and synaptotagmin. The optic tract was stained for SV2 and synaptotagmin between E7 and E12, for syntaxin until the posthatching period. Finally, immunoreactivities for the investigated proteins were present at the surface of the tectum from E8 onward, when first retinal axons arrived there. The present study revealed that SV2 and synaptotagmin, but not syntaxin, are, expressed in a transient wave that follows the advancement of optic axons and the proteins towards the optic tectum.     

Histochemical localization of cytochrome oxidase in the retina and optic tectum of normal goldfish: a combined cytochrome oxidase-horseradish peroxidase study

Cytochrome oxidase (C.O.) was histochemically localized in the normal retina and optic tectum of goldfish in order to examine the laminar and cellular oxidative metabolic organization of these structures. In the optic tectum, C.O. exhibited a distinct laminar, regional, and cellular distribution. The laminae with highest C.O. levels were those that receive optic input, suggesting a dominant role for visual activity in tectal function. This was demonstrated by colocalizing C.O. and HRP-filled optic fibers in the same section. However, the distribution of C.O. within the optic laminae was not uniform. Within the main optic layers, the SFGS, four metabolically distinct sublaminae were distinguished and designated from superficial to deep as sublaminae a, b, c, and d. The most intense reactivity was localized within SFGSa and SFGSd, followed by SFGSb, then SFGSc. In SFGSd, intense reactivity was found to occur specifically within a class of large diameter axons and terminals that were apparently optic since these were also labeled with HRP and cobaltous lysine applied to the optic nerve. Regional C.O. differences across the tectum were also noted. Low levels were found in neurons and optic terminals along the growing immature medial, lateral, and posterior edges of tectum, but were higher at the more mature anterior pole and central regions of tectum. This suggests that the oxidative metabolic activity is initially low in newly formed tectal neurons and optic axons, but gradually increases with neuronal growth and functional axon terminal maturation. Most C.O. staining was localized within neuropil, whereas the perikarya of most tectal neurons were only lightly reactive. Only a few neuron classes, mostly the relatively larger projection neurons, had darkly reactive perikarya. In the retina, intense C.O. reactivity was localized within the inner segments of photoreceptors, the inner and outer plexiform layers, and within certain classes of bipolar and ganglion cells. The large ganglion cells in particular were intensely reactive. Like the large diameter optic terminals in SFGSd, the large ganglion cells were preferentially filled with HRP, suggesting that they may project to tectum and are the source of the darkly reactive large diameter axons and terminals in sublamina SFGSd. We propose a new scheme to describe tectal lamination that integrates laminar differences in C.O. reactivity with classical histological work.(ABSTRACT TRUNCATED AT 400 WORDS)     

Further study of the outward displacement of retinal ganglion cells during optic nerve regeneration, with a note on the normal cells of Dogiel in the adult frog

In a previous study we observed massive retinal ganglion cell death in adult Rana pipiens after periods of optic nerve regeneration, and reported that large numbers of the surviving cells had become displaced bodily into the inner plexiform layer of the affected eye (Scalia et al.: Brain Research 344:267-280, 1985). The outwardly displaced cells could be identified as retinal ganglion cells because they could be back-filled with horseradish peroxidase (HRP) injected into the regenerated optic nerve. Quantitative observations on the abnormal outward displacement of ganglion cells are reported here. Parallel observations on normally displaced ganglion cells (cells of Dogiel) are also reported to clarify the distinctions between these two classes of cells. For the present work, injections of HRP of varying size were placed in the optic tectum bilaterally in 3 normal frogs and 9 frogs sustaining unilateral optic nerve regeneration. Most injections were centered at loci mapping the middle region of the nasal retina. The retinas were examined as flat-mounts and in-section. In 8 other frogs sustaining optic nerve regeneration, the HRP was administered bilaterally directly to the optic nerves in the orbit. Ganglion cells were labeled by retrograde transport of the HRP in the retinal ganglion cell layer in both the normal and affected eyes in areas topographically isomorphic with the tectal areas subtended by the injections. In the normal eyes, the orthotopic ganglion cells formed a strict monolayer, and virtually no cells existed in the inner plexiform layer. In the retinas sustaining optic nerve regeneration, the retinal ganglion cells abnormally displaced into the inner plexiform layer were also labeled topographically in correspondence with the injection sites. The abnormally displaced cells comprised 5.5% of the total population of surviving neurons in the retinal ganglion cell and inner plexiform layers. The mean outward dislocation of the displaced cells, as measured in one frog surviving optic nerve crush for 8 weeks, was 69.9 +/- 2.4% of the distance across the inner plexiform layer, which itself was uniformly 14.3 +/- 0.39 microns thick. Cells of Dogiel, which were embedded within the inner nuclear layer, were also labeled when the injections of HRP spread to include the area of representation of the optic disc. The labeled cells were restricted to a dorsal, peripapillary locus capping the optic disc. Therefore, some cells of Dogiel project to the tectum normally, but only from the central retina.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distribution of CB2 cannabinoid receptor in adult rat retina

Cannabinoid effects are mediated through two receptors, CB1 and CB2. In the retina CB1 has been reported in bipolar cells, gabaergic amacrine cells, horizontal cells, and inner plexiform layer. CB2 receptor mRNA localization was shown in photoreceptors, inner nuclear layer, and ganglion cell layer by using RT‐PCR. The aim of this work was to localize CB2 receptor in the rat retina by using immunocytochemistry. Our results showed that CB2 receptor was localized in retinal pigmentary epithelium, inner photoreceptor segments, horizontal and amacrine cells, cells localized in ganglion cell layer, and in fibers of inner plexiform layer. These results are in agreement with studies using RT‐PCR and provide some additional information about the distribution of CB2 receptor. Further studies are needed to clarify the role of this cannabinoid receptor in the retina. Synapse, 2010. © 2010 Wiley‐Liss, Inc.     

Glutamate-immunoreactivity in the retina and optic tectum of goldfish

Glutamate was immunohistochemically localized in the goldfish retina and tectum at the light and electron microscopic (E.M.) levels using double affinity purified antisera against glutaraldehyde conjugated L-glutamate. In retina, glutamate-immunoreactivity (Glu+) was observed in cone inner segments, cone pedicles, bipolar cells, a small number of amacrine cells and the majority of cells in the ganglion cell layer. The latter were shown to be ganglion cells by simultaneous retrograde labeling. Centrally, Glu+ was observed in axons in the optic nerve and tract, and in stratum opticum and stratum fibrosum et griseum superficialis (SFGS) of the tectum. The Glu+ in the optic pathway disappeared four days after optic denervation and was restored by regeneration without affecting the Glu+ of intrinsic tectal neurons. In tectum, Glu+ was also observed in torus longitudinalis granule cells, toral terminals in stratum marginale, some pyramidal neurons in the SFGS, multipolar and fusiform neurons in stratum griseum centrale, large multipolar and pyriform projection neurons in stratum album centrale, and many periventricular neurons. Glu+ was also localized within unidentified puncta throughout the tectum and within radially oriented dendrites of periventricular neurons. At the E.M. level, a variety of Glu+ terminals were observed. Glu+ toral terminals formed axospinous synapses with dendritic spines of pyramidal neurons. Ultrastructurally identifiable Glu+ putative optic terminals formed synapses with either Glu+ or Glu- dendritic profiles, and with Glu- vesicle-containing profiles, presumed to be GABAergic. These findings are consistent with the hypothesis that a number of intrinsic and projection neurons in the goldfish retinotectal system, including most ganglion cells, may use glutamate as a neurotransmitter.     

Changes in brain-derived neurotrophic factor and trkB receptor in the adult Rana pipiens retina and optic tectum after optic nerve injury

In this study we used immunocytochemistry to investigate the distribution of brain-derived neurotrophic factor (BDNF) and its receptor tyrosine kinase (trkB) in retina and optic tectum of the frog Rana pipiens during regeneration after axotomy. We also measured changes in BDNF mRNA in retina and tectum. Retrograde labeling was used to identify retinal ganglion cells (RGCs) prior to quantification of the BDNF immunoreactivity. In control animals, BDNF was found in the majority of RGCs and displaced amacrine cells and in some cells in the inner nuclear layer (INL). After axotomy, BDNF immunoreactivity was reduced in RGCs but increased in the INL. BDNF mRNA levels in the retina remained high before and after axotomy. Three months after axotomy, after reconnection to the target, the staining intensity of many of the surviving RGCs had partially recovered. In the control tectum, BDNF staining was present in ependymoglial cells and in neurons throughout layers 4, 6, 8, and 9. After axotomy, BDNF staining in tectal neurons became more intense, even though mRNA synthesis was transiently down-regulated. In control retinas, trkB receptor immunostaining was present in most RGCs; no significant changes were observed after axotomy. In control tectum, trkB was detected only in ependymoglial cells. After axotomy, many neuronal cell bodies were transiently labeled. Our data are consistent with the hypothesis that a considerable fraction of the BDNF normally present in RGCs is acquired from their targets in the tectum. However, there are also intraretinal sources of BDNF that could contribute to the survival of RGCs.     

Choline acetyltransferase immunoreactivity suggests that ganglion cells in the goldfish retina are not cholinergic

Published evidence that ganglion cells in the retinae of nonmammalian species are cholinergic is strong but indirect. In this paper we report results of attempts to demonstrate choline acetyltransferase immunoreactivity in ganglion cells of goldfish retina using two different antisera against choline acetyltransferase (ChAT), the acetylcholine-synthesizing enzyme. We obtained ChAT-immunoreactive staining of amacrine and displaced amacrine cells in the retina and type XIV cells in the tectum, but we obtained no direct immunocytochemical evidence that ganglion cells in the goldfish retina are cholinergic. Thus, ganglion cells identified by retrograde transport of propidium iodide were never ChAT-immunoreactive. Intraocular injections of colchicine did not result in the appearance of a population of ChAT-immunoreactive neurons in the ganglion cell layer. ChAT-immunoreactive axons were not observed in intact, ligated, or transected optic nerves. And finally, the ChAT immunoreactivity of cells and fibers in the optic tectum was unaffected by deafferentation. These experiments provide no positive evidence that any ganglion cells in goldfish retina contain the acetylcholine-synthesizing enzyme, ChAT. While it is possible that our method is too insensitive to detect the enzyme in ganglion cell somata or too specific to recognize the form of ChAT present in these cells, the fact that we can stain putatively cholinergic retinal amacrine cells and tectal neurons makes these alternative explanations improbable. We conclude that it is unlikely that any of the ganglion cells in the retina are cholinergic and that alternative explanations should be sought for previously published results that suggest that they are.     

Evidence for shifting connections during development of the chick retinotectal projection

The pattern in which optic axons invade the tectum and begin synaptogenesis was studied in the chick. The anterogradely transported marker, horseradish peroxidase, was injected into one eye of embryos between 5 and 16 days of development (E5 to E16). This labeled the optic axons in the brain. The first retinal axons arrived in the most superficial lamina of the tectum on E6. They entered the tectum at the rostroventral margin. During the next 6 days of development the axons grew over the tectal surface. First they filled the rostral tectum, the oldest portion of the tectum, and then they spread to the caudal pole. Shortly after the first axons entered the tectum on E6, labeled retinal axons were found penetrating from the surface into deeper tectal layers. In any given area of the tectum, optic axons were seen penetrating deeper layers shortly after arriving in that area. Electron microscopic examination showed that at least some of the labeled axons in rostral tectum formed synapses with tectal cells by E7. These results show two things which contrast with results from previous studies. First, there is no delay between the time the retinal axons enter the tectum and the time they penetrate into synaptic layers of the tectum. Second, the first retinotectal connections are formed in rostral tectum and not central tectum. Retrograde tracing showed the first optic axons that arrived in the tectum were from ganglion cells in central retina. Previous studies have shown that the ganglion cells of central retina project to the central tectum in the mature chick. This opens the possibility that the optic axons from central retina, which connect to rostral tectum in the young embryo, shift their connections to central tectum during subsequent development. As they enter the tectum the growth cones of retinal axons appear to be associated with the external limiting membrane. During the time that connections would begin to shift in the tectum a second population of axons appears at the bottom of stratum opticum, some with characteristics of growth cones. This late-appearing population may represent axons shifting their connections. These results have implications for theories on how the retinotopic pattern of retinotectal connections develops.     

Development of the retinotectal system in normal quail embryos: cytoarchitectonic development and optic fiber innervation

The development of the optic tectum and the establishment of retinotectal projections were investigated in the quail embryo from day E2 to hatching day (E16) with Cresyl violet-thionine, silver staining and anterograde axonal tracing methods. Both tectal cytodifferentiation and retinotectal innervation occur according to a rostroventral-caudodorsal gradient. Radial migration of postmitotic neurons starts on day E4. At E14, the tectum is fully laminated. Optic fibers reach the tectum on day E5 and cover its surface on day E10. 'Golgi-like' staining of optic fibers with HRP injected in vitro on the surface of the tectum reveals that: growing fronts are formed exclusively by axons extending over the tectal surface; fibers penetrating the outer tectal layers are always observed behind the growing fronts; the penetrating fibers are either the tip of the optic axons or collateral branches; as they penetrate the tectum, optic fibers give off branches which may extend for long distances within their terminal domains; the optic fiber terminal arbors acquire their mature morphology by day E14. The temporal sequence of retinotectal development in the quail was compared to that already established for the chick, thus providing a basis for further investigation of the development of the retinotectal system in chimeric avian embryos obtained after xenoplastic transplantation of quail tectal primordia into the chick neural tube.     

Immunocytochemical localization of L-glutamate decarboxylase, gamma-aminobutyric acid transaminase, cysteine sulfinic acid decarboxylase, aspartate aminotransferase and somatostatin in rat retina

The regional distribution and cellular location of GABA-synthesizing enzyme, L-glutamate decarboxylase (GAD), GABA degrading enzyme, GABA-transaminase (GABA-T), taurine synthesizing enzyme, cysteine sulfinic acid decarboxylase (CSAD), aspartate and glutamate converting enzyme, aspartate aminotransferase (AAT), and somatostatin have been visualized in the rat retina by immunocytochemical methods. GAD immunoreactivity was found to be concentrated in the inner plexiform layer. A moderate to weak staining of GAD was found in the inner nuclear layer. The distribution of GABA-T immunoreactivity was similar to that of GAD with the exception that a weak to moderate staining of GABA-T was also observed in the outer plexiform layer. CSAD immunoreactivity was seen in every layer with the heaviest staining in the inner plexiform layer, and moderate staining in the inner and outer nuclear layers and ganglion cell layer. AAT immunoreactivity was mostly concentrated in the outer nuclear layer; there was weak staining in the inner nuclear layer and inner and outer plexiform layer. Dense somatostatin staining was seen in the inner plexiform layer and moderate staining was present in the inner nuclear layer, outer plexiform layer and ganglion cell layer. These findings suggest that in rat retina, GABA-containing cells occur in some types of amacrine cells only, while taurine and somatostatin appear in both amacrine and horizontal cells. AAT immunoreactivity was primarily associated with the photoreceptor cells suggesting that AAT may be used as a marker for aspartergic/glutamergic cells and their endings in the central nervous system.     

Immunocytochemical localization of l-glutamate decar☐ylase, gamma-aminobutyric acid transaminase, cysteine sulfinic acid decar☐ylase, aspartate aminotransferase and somatostatin in rat retina

The regional distribution and cellular location of GABA-synthesizing enzyme, l-glutamate decar☐ylase (GAD), GABA degradating enzyme, GABA-transaminase (GABA-T), taurine synthesizing enzyme, cysteinesulfinic acid decar☐ylase (CSAD), aspartate and glutamate converting enzyme, aspartate aminotrasferase (AAT), and somatostatin have been visualized in the rat retina by immunocytochemical methods. GAD immunoreactivity was found to be concentrated in the inner plexiform layer. A moderate to weak staining of GAD was found in the inner nuclear layer. The distribution of GABA-T immunoreactivity was similar to that of GAD with the exception that a weak to moderate staining of GABA-T was also observed in the outer plexiform layer. CSAD immunoreactivity was seen in every layer with the heaviest staining in the inner plexiform layer, and moderate staining in the inner and outer nuclear layers and ganglion cell layer. AAT immunoreactivity was mostly concentrated in the outer nuclear layer; there was weak staining in the inner nuclear layer and inner and outer plexiform layer. Dense somatostatin staining was seen in the inner plexiform layer and moderate staining was present in the inner nuclear layer, outer plexiform layer and ganglion cell layer. These findings suggest that in rat retina, GABA-containing cells occur in some types of amacrine cells only, while taurine and somatostatin appear in both amacrine and horizontal cells. AAT immunoreactivity was primarily associated with the photoreceptor cells suggesting that AAT may be used as a marker for aspartegic/glutamergic cells and their endings in the central nervous system.     

Localization of aspartate-like immunoreactivity in the retina of the turtle (Pseudemys scripta).

Aspartate has been reported to be a putative excitatory neurotransmitter in the retina, but little detailed information is available concerning its anatomical distribution. We used an antiserum directed against an aspartate-albumin conjugate to analyze the anatomy, dendritic stratification, and regional distribution of cell types with aspartate-like immunoreactivity in the turtle retina. The results showed dramatic differences in immunoreactivity in the peripheral versus the central retina. Strong aspartate-like immunoreactivity was shown in the peripheral retina, with many well-labeled processes in the inner plexiform layer. Many bipolar, horizontal, amacrine, and ganglion cells, some photoreceptors, and some unidentified cells were strongly immunoreactive in the peripheral retina. In contrast, although the central retina showed well-labeled horizontal cells, there was only light labeling in the inner plexiform layer with weakly immunoreactive amacrine and ganglion cells and no labeled bipolar cells. There were several strongly immunoreactive efferent nerve fibers which left the optic nerve head and arborized extensively in the retina. At the electron microscopic level, electron-dense reaction product was associated with synaptic vesicles at bipolar and amacrine cell synapses in the inner plexiform layer. These results suggest that aspartate may be involved in many diverse synaptic interactions in both the outer plexiform layer and the inner plexiform layer of the turtle retina.     

Observations on the afferent and efferent connections of the avian isthmo-optic nucleus

The efferent and afferent connections of the avian isthmo-optic nucleus (ION) were studied using light microscopic techniques. Injections of [³H]proline into the nucleus resulted in labeling of centrifugal endings in the retina at the junction of the inner plexiform layer and inner nuclear layer, but produced no other transported label to any thalamic or mesencephalic nucleus. The origin of the tectal afferents to the ION was demonstrated by means of injections of [³H]proline into the most superficial layers of the optic tectum and by stereotaxic injections of horseradish peroxidase into the ION. The tectal efferent cell bodies were located in lamina h of the optic tectum and at the junction of laminae h and i.     

GABA-like immunoreactive cells containing nicotinic acetylcholine receptors in the chick retina.

The possibility that GABA-like immunoreactive cells of the chick retina also contain neuronal nicotinic acetylcholine receptors was investigated by means of immunohistochemical techniques. Double-labeled cell bodies containing GABA-like immunoreactivity and nicotinic receptor-like immunoreactivity were seen in the inner third of the inner nuclear layer and were presumably amacrine cells. Approximately 29-36% of the GABA-positive cells in the inner nuclear layer contained nicotinic receptor immunoreactivity. Their soma sizes ranged from 5-12 microns. Some double-labeled cells ranging from 7-21 microns were observed in the ganglion cell layer as well. Between 9-37% of the GABA-positive cells in this layer contained nicotinic receptor-like immunoreactivity. Following injection of a retrograde tracer into the optic tectum, some of the retrogradely labeled cells were also double labeled with antibodies against GABA and nicotinic receptors. This indicates that at least some of the GABA-positive cells containing nicotinic acetylcholine receptors in the ganglion cell layer are indeed ganglion cells. The present data appear to represent the first demonstration of the presence of acetylcholine receptors in GABA-containing cells in the retina, thus providing a basis for a possible influence of acetylcholine upon those presumptive GABAergic cells.     

Postnatal development of dopamine D1 receptor immunoreactivity in the rat retina.

Dopamine, an important neuromodulator in the retina, controls the balance of rod cone photoreceptor activity and influences the activity of several interneurons. The postnatal development of dopaminergic neurons, visualized immunocytochemically, was compared to the development of dopamine D1 receptor immunoreactivity. Expression of D1 receptors was monitored throughout the postnatal development of the rat retina using a subtype-specific monoclonal antibody. D1 receptors are expressed in the inner plexiform layer beginning at birth. Labeling of the inner plexiform layer changed from a diffuse pattern, staining the entire layer, to the typical adult punctate staining, that was organized in layered bands and occurred in the second postnatal week. The staining did not co-localize with dopaminergic cells; instead, it colocalized with cells in the inner nuclear layer or the ganglion cell layer. Within these cells, D1 receptors were most heavily expressed in processes stratifying in the inner plexiform layer. Staining in the outer plexiform layer and in horizontal cells was found beginning in the second postnatal week. Clustering of the D1 receptor within plexiform layers, a process typical for the well-described function of dopamine modulation in the adult, occurred late in postnatal development. A possible function of D1 receptors in neuronal development is discussed.