Presynaptic and postsynaptic localization of GABAB receptors in neurons of the rat retina

The recently cloned GABA_B receptors were localized in rat retina using specific antisera. Immunolabelling was detected in the inner and outer plexiform layers (IPL, OPL), and in a number of cells in the inner nuclear layer and the ganglion cell layer. Double-labelling experiments for GABA (γ-aminobutyric acid) and GABA_B receptors, respectively, demonstrated a co-localization in horizontal cells and amacrine cells. Electron microscopy showed that GABA_B receptors of the OPL were localized presynaptically in horizontal cell processes invaginating into photoreceptor terminals. In the IPL, GABA_B receptors were present presynaptically in amacrine cells, as well as postsynaptically in amacrine and ganglion cells. The postnatal development of GABA_B receptors was also studied, and immunoreactivity was observed well before morphological and synaptic differentiation of retinal neurons. The present results suggest a presynaptic (autoreceptor) as well as postsynaptic role for GABA_B receptors. In addition, the extrasynaptic localization of GABA_B receptors could indicate a paracrine function of GABA in the retina.     

Serotonin-like immunoreactivity in the adult and developing retina of the leopard frog Rana pipiens

Recent work in nonmammalian vertebrate retinas has suggested that other cell types besides the generally accepted amacrine cells may contain serotonin. We have used immunocytochemical methods to study serotonin-like immunoreactivity (5-HTLI) in the retina of the developing and mature frog Rana pipiens. In the adult, two types of serotonin immunoreactive (5-HT-ir) cells were found in the inner nuclear layer (INL) of the retina. Additionally, a large population of cells in the retinal ganglion cell layer (RGCL) had 5-HTLI. These cells were grouped into three types based on their soma size and their primary dendritic branching pattern. The optic nerve fiber layer was also intensely stained with serotonin antisera although staining intensity decreased progressively as the fibers approached the optic nerve head. Severing the optic nerve resulted in 5-HT-ir elements that extended up the optic nerve shaft from the lesion site toward the retina. Both regional and temporal changes in the pattern of 5-HTLI were seen. In middle regions of retina, approximately 30% of the cells in the RGCL were 5-HT-ir. Nasal and temporal regions of central retina had significantly fewer 5-HT-ir cells. Early in development only scattered cells in the RGCL were 5-HT-ir. As the animals matured there was an increase in both the proportion and the staining intensity of these cells. Our results suggest that in studying the function and development of the visual system in this animal, the role of serotonin must be examined. © 1993 Wiley-Liss, Inc.     

Ontogeny of somatostatin immunoreactivity in the cat retina.

In the ganglion cell layer of the adult cat retina, subgroups of displaced amacrine cells and alpha ganglion cells are immunoreactive for somatostatin or a somatostatinlike substance. Both types of immunoreactive cells are found preferentially in inferior retina. We studied the development of somatostatin immunoreactivity in the prenatal and postnatal cat retina to determine how such unusual distributions of immunoreactive cells arise. Somatostatin-immunoreactive profiles were first observed at embryonic day (E) 30, within the inner retina in a central region that included the optic disk and the area centralis. By E36, immunoreactivity had virtually disappeared from the central retina but was present throughout the periphery. The immunoreactive profiles could not be classified morphologically because of their immaturity but were most likely retinal ganglion cells, the earliest born cells of the inner retina. Of the two types of immunoreactive cell observed in the adult, the first to be recognized morphologically was the displaced amacrine cell, at E45. These cells were virtually adultlike in morphology and number by E51, two weeks before birth. In contrast, immunoreactive alpha ganglion cells were not apparent until five days after birth and did not achieve their mature numbers and immunoreactive staining characteristics until more than a month later. From the time they could initially be recognized, both immunoreactive displaced amacrine cells and alpha cells were distributed mainly in the inferior retina. A third type of somatostatin-immunoreactive cell was transiently observed in the superior and inferior retina during prenatal and early postnatal development. These cells were characterized by granular staining in irregular shapes and few, if any, faintly stained processes. Injections of retrograde tracers into retinorecipient targets revealed that many of these cells were retinal ganglion cells. They disappeared by postnatal day 38. Our results indicate that somatostatin immunoreactivity initially follows a central-to-peripheral pattern of development, as is typical of other developmental events in the mammalian retina. They also indicate that the two types of somatostatin-immunoreactive neurons present in the adult cat retina (displaced amacrine and alpha ganglion cells) attain their mature immunocytochemical properties with very different timecourses. Finally, the observation that somatostatin immunoreactivity appears transiently in the granular-staining ganglion cells, distributed throughout the superior and inferior retina, suggests that the peptide may play a regulatory role in the development of the retina and/or retinofugal pathways.     

Expression and function of the neuronal gap junction protein connexin 36 in developing mammalian retina

With the advent of transgenic mice, much has been learned about the expression and function of gap junctions. Previously, we reported that retinal ganglion cells in mice lacking the neuronal gap junction protein connexin 36 (Cx36) have nearly normal firing patterns at postnatal day 4 (P4) but many more asynchronous action potentials than wild-type mice at P10 (Torborg et al. [2005] Nat. Neurosci. 8:72-78). With the goal of understanding the origin of this increased activity in Cx36-/- mice, we used a transgenic mouse (Deans et al. [2001] Neuron 31:477-485) to characterize the developmental expression of a Cx36 reporter in the retina. We found that Cx36 was first detected weakly at P2 and gradually increased in expression until it reached an adult pattern at P14. Although the onset of expression varied by cell type, we identified Cx36 in the glycinergic AII amacrine cell, glutamatergic cone bipolar cell, and retinal ganglion cells (RGCs). In addition, we used calcium imaging and multielectrode array recording to characterize further the firing patterns in Cx36-/- mice. Both correlated and asynchronous action potentials in P10 Cx36-/- RGCs were significantly inhibited by bath application of an ionotropic glutamate receptor antagonist, indicating that the increase in activity was synaptically mediated. Hence, both the expression patterns and the physiology suggest an increasing role for Cx36-containing gap junctions in suppressing RGC firing between waves during postnatal retinal development.     

Cellular localization of cyclooxygenase-1 and cyclooxygenase-2 in the normal mouse,rat, and human retina

Prostaglandins, synthesized by cyclooxygenase (COX), regulate diverse neurophysiological actions such as regulation of autonomic responses, transmission of pain, generation of fever, control of sleep-wake cycle, synaptic signaling, and cross-talk between neurons and glia in the central nervous system. Although prostaglandins have been widely studied in the anterior segment tissues of the eye, relatively little is known about prostaglandins in the neural retina. By using immunohistochemistry, we have compared the cellular expression and localization of COX-1 and COX-2 in the normal mouse, rat, and human retina. In the normal mouse retina, COX-1 immunoreactivity is present in the outer segments of photoreceptor cells, horizontal cells, microglia, retinal ganglion cells, and displaced amacrine cells. In the normal rat retina, COX-1 immunoreactivity is present in microglia, retinal ganglion cells, and displaced amacrine cells. In the normal human retina, COX-1 immunoreactivity is present in microglia, astrocytes, retinal ganglion cells, and displaced amacrine cells. In the normal mouse and rat retina, COX-2 immunoreactivity is present in processes of the outer plexiform layer and in certain amacrine cells and retinal ganglion cells. In the normal human retina, COX-2 immunoreactivity is only present in processes of the outer plexiform layer. These results suggest that prostaglandins, synthesized by COX-1 or COX-2, may contribute to normal physiological and homeostatic functions in the retina.     

Morphology and distribution of neurons in the retina of the American garter snake Thamnophis sirtalis

It is confirmed that cone photoreceptors observed in flatmounts of the American garter snake Thamnophis sirtalis, retina correspond to the retinal mosaic viewed in the living eye (Land and Snyder, Vision Res. 11:105-114, '85). The garter snake has three major morphological types of cones; large single cones, small single cones, and double cones. The brightly reflecting components seen in the living eye are large single cones and principle cones of double cones, whereas irregularly spaced dark regions within this mosaiac mark the positions of small single cones. The "sparkle" of the retinal mosaic originates from the ellipsoid region of the cones where microdroplets of high refractive index are densely packed. Unlike conventional oil droplets, these microdroplets reside adjacent to mitochondrial cristae within the ellipsoid. However, the microdroplets may function collectively as a single large oil droplet to increase the angular sensitivity of the inner segments, thus reducing a potentially large Stiles-Crawford effect predicted for this geometrically small eye. The ganglion cell layer of the garter snake comprises two morphologically distinct populations of presumed neurons; classical neurons and microneurons. Density distribution maps for neurons in the ganglion cell layer and the photoreceptor layer reveal the presence of a putative area centralis and a horizontal visual streak. The topography of large cones parallels that of classical neurons. Small single cones have a more circular distribution, but also peak in density at the area centralis. The convergence of cones to classical neurons is lowest at the area centralis, 2.5:1, and highest, 4:1, at the retinal edge. With its interesting structural features, the garter snake retina provides helpful insight into different strategies in eye design.     

Comparative expression profiles of Trk receptors and Shc-related phosphotyrosine adapters during retinal development: potential roles of N-Shc/ShcC in brain-derived neurotrophic factor signal transduction and modulation

Neurotrophins (NTs) have multiple roles in retinal development and survival, which are mediated through their specific receptors and signaling molecules. An emerging family of adapter protein, Shc (Src homology and collagen)-related molecules, i.e., Shc/ShcA, Sck/ShcB, and N-Shc/ShcC, has been implicated in various phosphotyrosine signal transduction mechanisms, including that for NTs. To explore the potential role(s) of Shc-related adapters in NT signaling in the retina, we compared the developmental changes of the mRNA expression of TrkA -B, and -C in the rat retina, on one hand and, on the other hand, studied which members of the Shc family were activated after brain-derived neurotrophic factor (BDNF) application in axotomized rat retinas. Early in development, both TrkA and ShcA were highly expressed, whereas, in late development to adulthood, TrkB/C and ShcB/C were highly expressed. In the mature retinal ganglion cell layer, the expression of ShcB/C and TrkB/C was evident. Immunoreactivity of ShcC was located in the retinal ganglion cells, amacrine cells, and inner plexiform layer. The response of ShcC following retinal axotomy was most profound with the administration of BDNF, and there was some response with neurotrophin-3. These results indicate that ShcC could be a potential phosphotyrosine adapter among the Shc family members for BDNF signaling and function during retinal development and regeneration in vivo.     

Dendritic plasticity in the early postnatal feline retina: quantitative characteristics and sensitive period

Retinal lesions were made in kittens between 3 and 60 days postnatal age and in adult cats. After postlesion survival times ranging from 4 to 11 months the dendritic morphology of retinal ganglion cells was revealed by retrograde labeling with horseradish peroxidase or with neurofibrillar staining techniques. After retinal lesions on the third postnatal day changes of dendritic morphology were observed in retinal ganglion cells adjacent to regions of retrograde degeneration. Originating from eccentrically positioned somata the dendritic fields extended into the regions that were free of neighboring cells. The dendrites oriented toward the ganglion-cell-free region were elongated and thicker than normal. The density of dendrites per unit area was increased in this part of the dendritic trees. Lesions on the 20th, 38th, and 56th postnatal days elicited increasingly weaker changes of dendritic morphology. The sensitive period for the type of dendritic plasticity described ends between 40 and 60 days postnatally.     

Influence of the visual cortex on responses of retinal ganglion cells in the rat

The objective of the present investigation was to answer the following question: Does the visual cortex affect the neuronal firing of retinal ganglion cells in the rat? To test this hypothesis, the visual cortex was inactivated by a reversible cryoblockade. Action potentials of a ganglion cell were recorded from its axon at the optic tract level prior to, during, and following cortical blockade. The results indicated that indeed the visual cortex influenced the retinal output since its inactivation led to a modification of the firing pattern evoked in response to a flash of light. In most cases the modification was an increase of the bursting pattern of the evoked discharges. By contrast cooling nonvisual areas failed to modify ganglion cells' discharge. A comparison between cortico-geniculate and cortico-retinal feedback loops seems to suggest that the first path is involved mostly with the spatial organization of center-surround receptive fields, whereas the second path is associated with temporal aspects of the retinal responses in the rat.     

Postnatal development of 3H-GABA-accumulating cells in rabbit retina

Light and electron microscopic autoradiography demonstrates that 3H-GABA is accumulated by horizontal cells in neonatal rabbit retina but not in the adult. A specific population of horizontal cells appears to be mature at birth and they avidly accumulate 3H-GABA during a 15-minute incubation period in vitro. Uptake into horizontal cells is not observed after the fifth postnatal day; 3H-GABA-accumulating horizontal cell bodies and their processes are the first identifiable components that clearly mark the future location of the outer plexiform layer at birth and as such, may be considered pioneering elements. Our observations raise the interesting possibility that the pioneering horizontal cell may provide structural and/or chemical factors necessary for the subsequent development of the outer plexiform layer of the retina. Labeling patterns of other retinal cells also show varying degrees of change during development. A population of amacrine cells accumulate 3H-GABA at birth. These cells show little change in their morphological or 3H-GABA uptake properties from birth to adulthood. Müller cells show weak accumulation of 3H-GABA at birth. Subsequent to this time, labeling of Müller cells is significantly more robust, resulting in Müller cell domination of retinal autoradiographic patterns in more mature retinas. Every cell body in the ganglion cell layer accumulates 3H-GABA at birth. The number of labeled cells declines during postnatal development, resulting in a very limited adult population. We conclude that the ability of retinal cells to accumulate 3H-GABA does not remain constant during postnatal development; rather each cell population displays a unique maturation sequence that results in a dramatic developmental shift in the number and types of GABA-accumulating cells present in the retina.     

Plasmalemmal and vesicular gamma-aminobutyric acid transporter expression in the developing mouse retina

Plasmalemmal and vesicular gamma-aminobutyric acid (GABA) transporters influence neurotransmission by regulating high-affinity GABA uptake and GABA release into the synaptic cleft and extracellular space. Postnatal expression of the plasmalemmal GABA transporter-1 (GAT-1), GAT-3, and the vesicular GABA/glycine transporter (VGAT) were evaluated in the developing mouse retina by using immunohistochemistry with affinity-purified antibodies. Weak transporter immunoreactivity was observed in the inner retina at postnatal day 0 (P0). GAT-1 immunostaining at P0 and at older ages was in amacrine and displaced amacrine cells in the inner nuclear layer (INL) and ganglion cell layer (GCL), respectively, and in their processes in the inner plexiform layer (IPL). At P10, weak GAT-1 immunostaining was in Müller cell processes. GAT-3 immunostaining at P0 and older ages was in amacrine cells and their processes, as well as in Müller cells and their processes that extended radially across the retina. At P10, Müller cell somata were observed in the middle of the INL. VGAT immunostaining was present at P0 and older ages in amacrine cells in the INL as well as processes in the IPL. At P5, weak VGAT immunostaining was also observed in horizontal cell somata and processes. By P15, the GAT and VGAT immunostaining patterns appear similar to the adult immunostaining patterns; they reached adult levels by about P20. These findings demonstrate that GABA uptake and release are initially established in the inner retina during the first postnatal week and that these systems subsequently mature in the outer retina during the second postnatal week.     

Immunocytochemical localization of three vesicular glutamate transporters in the cat retina

Vesicular transporters play an essential role in the packaging of glutamate for synaptic release and so are of particular importance in the retina, where glutamate serves as the neurotransmitter for photoreceptors, bipolar cells, and ganglion cells. In the present study, we have examined the distribution of the three known isoforms of vesicular glutamate transporter (VGLUT) in the cat retina. VGLUT1 was localized to all photoreceptor and bipolar cells, whereas VGLUT2 was found in ganglion cells. This basic pattern of complementary distribution for the two transporters among known populations of glutamatergic cells is similar to previous findings in the brain and spinal cord. However, the axon terminals of S-cone photoreceptors were found to express both VGLUT1 and VGLUT2 and some ganglion cells labeled for both VGLUT2 and VGLUT3. Such colocalizations suggest the existence of dual modes of regulation of vesicular glutamate transport in these neurons. Staining for VGLUT2 was also present in a small number of varicose processes, which were seen to ramify throughout the inner plexiform layer. These fibers may represent axon collaterals of ganglion cells. The most prominent site of VGLUT3 immunoreactivity was in a population of amacrine cells; the axon terminals of B-type horizontal cells were also labeled at their contacts with rod spherules. The presence of the VGLUT3 transporter at sites not otherwise implicated in glutamate release may indicate novel modes of glutamate signaling or additional roles for the transporter molecule.     

Subgroup of alpha ganglion cells in the adult cat retina is immunoreactive for somatostatin.

We have previously shown that two types of cells in the ganglion cell layer of the adult cat retina are immunoreactive for somatostatin (White et al., '90). One of the types was identified by morphological criteria as a wide-field amacrine cell. The other cell type had a large, angular soma that resembled the alpha ganglion cell, but evidence was not available to identify it definitively as a ganglion cell. Both cell types were distributed preferentially in the inferior retina. In this report, we demonstrate that the two types of cell are, indeed, displaced amacrine cells and alpha ganglion cells. First, when retrograde tracers were injected into central visual targets, the immunoreactive large cells but not the displaced amacrine cells were found to be labeled. Second, after unilateral section of the optic nerve, the immunoreactive large cells disappeared from the retina on the lesioned side, but the displaced amacrine cells occurred in the same numbers in both retinae. In the periphery, the large cells ranged in diameter from 33 to 47 microns, comparable only to alpha ganglion cells (Boycott and W?ssle, '74). An antiserum to parvalbumin was used to visualize the dendrites (R?hrenbeck and W?ssle, '88) of somatostatin-immunoreactive large cells. Based on dendritic stratification within the inner plexiform layer (Famiglietti and Kolb, '76), the somatostatin-immunoreactive large cells were found to include both on-center cells and off-center cells, but were predominantly of the off-center type. Within a local region, they were found to be arrayed with greater regularity than the overall population of alpha ganglion cells. These results indicate that alpha ganglion cells of the cat retina can be subdivided on the basis of their immunoreactive staining for somatostatin and suggest that the diversity of ganglion cells in the cat retina may be greater than has been recognized on the basis of morphological criteria alone.     

Normal chiasmatic routing of uncrossed projections from the ventrotemporal retina in albino Xenopus frogs

Albino mammals lacking melanin in the embryonic retinal pigment epithelium (RPE) have abnormal retinal decussation patterns at the optic chiasm: their uncrossed projections are smaller and arise from fewer, more peripheral temporal retinal ganglion cells than in con-specific wild-types. To determine whether these abnormalities generalize to nonmammalian mutants, we used anterograde and retrograde labeling methods to compare the distribution of retinal projections to the thalamus in adult normal and albino Xenopus frogs. In both pigmentation phenotypes, crossed retinal terminations covered approximately 80% of the neuropil of Bellonci (nB) and corpus geniculatum thalamicum (cgt) and uncrossed inputs occupied, respectively, approximately 75% and 25% of these two main visual centers. In the wild-type frogs and in the albinos, ganglion cells giving rise to the crossed projections were distributed throughout the retina, whereas ipsilaterally projecting cells were confined to a specific ventrotemporal retinal division. This region comprised approximately 40% of the total retinal area, was bordered by a well-defined line of decussation, and contained an average of approximately 3,000 ipsilaterally projecting ganglion cells of equivalent soma sizes in the two pigmentation phenotypes. In summary, we found no evidence of chiasmatic misrouting in the uncrossed retinothalamic projections of albino Xenopus, even though these pathways are substantial in normal frogs and share features in common with mammalian retinogeniculate projections. Our findings suggest that congenital RPE melanin deficiency results in major defects in the development of the retina and its central projections only in mammals.     

The formation of the axonal pattern in the embryonic avian retina

Both the polarity of the axonal growth and the formation of the optic fiber pattern early in retinal morphogenesis were studied in silver stained whole mounts of embryonic chick, quail, and pigeon retinae. The surface area of the retina and of the optic fiber layer increases in size exponentially, the optic fiber layer expanding faster than the retina. The optic fiber layer covers the retinal surface at E5 in quail and at E6 in chick and pigeon. In all species studied, the retinal fiber layer does not expand homogeneously with the optic nerve head as the center. Instead, the retinal fiber layer enlarges with polarities in the dorsal to ventral and nasal to temporal direction. The very first axon bearing ganglion cells appear at stage 16 in the dorsal and central portion of the retina and grow ventrally to merge at the optic disk. From stage 23 on, the optic fiber layer expands faster in the temporal than in the nasal side. Measurements on the initial polarization of young axonal processes show that the axonal growth is directed toward the optic fissure and the optic nerve head. This growth polarization is found at the onset of growth cone formation and in axons far from the nearest ganglion cells or ganglion cell axons. Therefore axon-axon interaction cannot be involved in the initial axon orientation early in retinal morphogenesis.     

Expression of the somatostatin subtype 2A receptor in the rabbit retina

In the retina, somatostatin influences neuronal activity likely by acting at one or more somatostatin subtype (sst) receptors. Somatostatin and somatostatin-binding sites are distributed predominantly to the inner retina. The present study has investigated the cellular expression of one of the sst receptors, the sst_2A receptor isoform, in the rabbit retina. These studies have used a new polyclonal antibody directed to the predicted C-terminus of mouse sst_2A(361–369) receptor. Antibody specificity was tested by preadsorption of the primary antibody with a peptide corresponding to sst_2A(361–369). sst_2A Receptor immunoreactivity was localized mainly to the plasma membrane of rod bipolar cells and to sparsely occurring, wide-field amacrine cells. Immunostaining in rod bipolar cells was strongest in the axon and axon terminals in lamina 5 of the inner plexiform layer (IPL) and was weakest in the cell body and dendrites. Double-labeling experiments using a monoclonal antibody against protein kinase C (PKC; α and β), a rod bipolar cell-selective marker, showed complete colocalization. In horizontal sections of retina, immunostained bipolar cell bodies had a dense distribution, which is in agreement with the reported distribution of rod bipolar cell bodies. Immunoreactive amacrine cell bodies were located at the border of the inner nuclear layer and the IPL, and thin varicose processes ramified mainly in laminae 2 and 4 of the IPL. These observations indicate that somatostatin influences visual information processing in the retina 1) by acting presynaptically on rod bipolar cell axon terminals and b) by influencing the activity of sparsely occurring amacrine cells. J. Comp. Neurol. 393:93–101, 1998. © 1998 Wiley-Liss, Inc.     

Changing course of growing axons in the optic chiasm of the mouse

The distribution of retinofugal fibres has been studied by electron microscopy throughout the extent of the developing mouse optic nerve and chiasm at embryonic day (E) 16, in order to determine the course of fibre growth. Growth cones and mature axons, which are randomly distributed in bundles in the extracranial optic nerve, segregate in the juxtachiasmatic optic nerve. Here, growth cones accumulate in subpial regions amongst the endfeet of radial glia, whereas axons lie in the depths of the nerve. Surprisingly, however, growth cones move away from this region toward the ventricular zone in the lateral and midline parts of the chiasm, only to return to subpial regions once more before entering the optic tract, where fibres are again in an age-related order. Superficially, mature axons mingle with growth cones in the chiasm and near the beginning of the optic tract, suggesting that the age-related order begins to be reestablished before growth cones enter the tract. Deep and superficial regions of the pathway were examined in different planes of section. Specialised membrane relationships between retinofugal fibres and radial glial cells were also studied in deep and superficial regions of the lateral part of the chiasm. In addition, the distribution of retinofugal fibre bundles in the adult mouse was looked at by using light microscopy. The changing fibre positions noted in the embryo are maintained in the adult. J. Comp. Neurol. 379:495–514, 1997. © 1997 Wiley-Liss, Inc.