Dennis W. Rickman

Expression of the proto-oncogene, trk, receptors in the developing rat retina.

The neurotrophins, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and NT-4/5 are important in a variety of developmental processes in the peripheral and central nervous systems. These molecules bind to a low-affinity receptor and to distinct high-affinity receptors. The high-affinity receptor for NGF is the proto-oncogene product, p140trkA(trkA). Isoforms of p140trkA, p145trkB(trkB), and p140trkC(trkC), are the primary high-affinity receptors for BDNF and NT-3, respectively. We evaluated the developmental regulation of the high-affinity neurotrophin receptors in the rat retina using polyclonal antibodies directed to a highly conserved region of the C-terminus of the p140trkA isoforms (pantrk) and antibodies directed to unique amino-acid sequences of p140trkA, p145trkB, and p140trkC. Immunoreactivities for trkA and trkB, as well as pantrk, were detected in the developing retina and showed similar distributions. At similar antibody concentrations, trkC immunoreactivity was not detected. In the embryo, immunoreactivties were present in cells located throughout the neuroblastic retina, especially in the inner retinal layers, and in fibers in the nerve fiber layer and optic nerve. In the newborn retina, immunoreactivities for these two receptor isoforms were localized to numerous somata in the inner nuclear layer (INL), as well as to cells in the ganglion cell layer (GCL) and axons in the nerve fiber layer and optic nerve. A similar pattern of immunostaining persisted throughout the first postnatal week. By postnatal day-10, immunostaining was confined to large-diameter cells in the GCL, both heavily stained and lightly stained cells in the INL and a plexus of processes in the inner plexiform layer (IPL).(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple γ‐aminobutyric acid plasma membrane transporters GAT‐1, GAT‐2, GAT‐3 in the rat retina

γ‐Aminobutyric acid (GABA) plasma membrane transporters (GATs) influence synaptic neurotransmission by high‐affinity uptake and release of GABA. The distribution and cellular localization of GAT‐1, GAT‐2, and GAT‐3 in the rat retina have been evaluated by using affinity‐purified polyclonal antibodies directed to the C terminus of each of these GAT subtypes. Small GAT‐1‐immunoreactive cell bodies were located in the proximal inner nuclear layer (INL) and ganglion cell layer (GCL), and processes were distributed to all laminae of the interplexiform layer (IPL). Varicose processes were in the optic fiber layer (OFL) and the outer plexiform layer (OPL). Weak GAT‐1 immunostaining surrounded cells in the INL and GCL, and it was found in the OFL and OPL and in numerous processes in the outer nuclear layer (ONL) that ended at the outer limiting membrane. GAT‐1 is therefore strongly expressed by amacrine, displaced amacrine, and interplexiform cells and weakly expressed by Müller cells. GAT‐2 immunostaining was observed in the retina pigment epithelium and the nonpigmented ciliary epithelium. GAT‐3 immunoreactivity was distributed to the OFL, to all laminae of the IPL, GCL and INL, and to processes in the ONL that ended at the outer limiting membrane. Small GAT‐3‐immunoreactive cell bodies were in the proximal INL and GCL. GAT‐3 is therefore strongly expressed by Müller cells, and by some amacrine and displaced amacrine cells. Together, these observations demonstrate a heterologous distribution of GATs in the retina. These transporters are likely to take up GABA from, and perhaps release GABA into, the synaptic cleft and extracellular space. This suggests that GATs regulate GABA levels in these areas and thus influence synaptic neurotransmission.

Developmental expression of neurokinin‐1 and neurokinin‐3 receptors in the rat retina

Tachykinin (TK) peptides act on retinal neurons through neurokinin (NK) receptors. We examined the expression of neurokinin‐1 (NK1; the substance P receptor), NK3 [the neurokinin B (NKB) receptor], and TK peptides in developing rat retinas. NK1 immunolabeling was found in newborn retinas in rare amacrine cells and in putative ganglion cells. At postnatal day 2 (PND 2), NK1 immunostaining was reduced greatly among ganglion cells, and it appeared in many amacrine cells and in fibers in the inner plexiform layer (IPL), with the highest density in laminae 1, 3, and 5. A similar pattern was found at PND 7. At PND 12, interplexiform NK1‐immunoreactive (‐IR) cells were detected, and NK1‐IR fibers in the IPL were concentrated in lamina 2, similar to what was seen in adults. NK3 was expressed mainly by OFF‐cone bipolar cells, and the developmental pattern of NK3 was compared with that of cone bipolar cells that were labeled with antibodies to recoverin. Immature recoverin‐IR cone bipolar cells were seen at PND 2. NK3 immunolabeling was detected first in the outer plexiform layer and in sparse bipolar cell somata at PND 10, when recoverin‐IR cone bipolar cells are nearly mature. By PND 15, both the NK3 immunostaining pattern and the recoverin immunostaining pattern were similar to the patterns seen in adults. TK immunoreactivity was present at PND 0 in amacrine cells and displaced amacrine cells. By PND 10, the morphologic maturation of TK‐IR cells was complete. These findings indicate that, in early postnatal retinas, substance P may act on NK1 receptors, whereas NKB/NK3 interactions are unlikely, suggesting that there are different levels of importance for different TK peptides in the developing retina. J. Comp. Neurol. 421:275–287, 2000.

Somatostatin and somatostatin subtype 2A expression in the mammalian retina.

This review discusses the expression and cellular localization of the neuropeptide somatostatin (SRIF) and one of the SRIF subtype (sst) receptors, sst2A in the mammalian retina. SRIF immunoreactivity is predominantly localized to a sparse population of amacrine and displaced amacrine cells in the ganglion cell layer in several mammalian retinas including the rat, rabbit, cat, and primate. These cells, characterized by multiple processes, form a sparse network in the inner plexiform layer (IPL) in all retinal regions. Very few processes are also in the outer plexiform layer. In contrast to the predominant distribution of SRIF processes to the IPL, there is a widespread distribution of sst2A immunoreactivity to both the inner and outer retina in all mammalian retinas studied to date. In rabbit retina, sst2A immunoreactivity is predominant in rod bipolar cells and in sparse wide‐field amacrine cells. In the rat retina, sst2A immunoreactivity is localized to several neuronal cell types—cone photoreceptors, horizontal cells, rod and cone bipolar cells, and amacrine cells. Reverse‐transcriptase–polymerase chain reaction analysis found that sst2A mRNA is expressed in the rat retina, while sst2B mRNA is not detected. Finally, in the primate retina sst2 immunoreactivity is predominant in cone photoreceptors, with additional immunostained cell bodies and processes in the inner retina. These findings indicate that SRIF may modulate several neuronal cell types in the retina, and that it has a broad influence on both scotopic and photopic visual pathways. Microsc. Res. Tech. 50:103–111, 2000.

Neurokinin 1 receptor expression in the rat retina.

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

POSTNATAL DEVELOPMENT OF PARVALBUMIN IMMUNOREACTIVE AMACRINE CELLS IN THE RABBIT RETINA

In the adult rabbit, rat and cat retina, parvalbumin (PV) immunoreactivity is primarily localized to a population of narrow-field, bistratified amacrine cells, the AII amacrine cells-major interneurons of the rod pathway. This investigation examines the postnatal development of PV immunoreactivity in order to better understand the ontogeny of the AII amacrine cell population and the formation of the rod pathway. Rabbit retinas at various postnatal ages were processed for immunohistochemistry using a monoclonal antibody directed to PV and analyzed morphometrically. On the day of birth, PV immunoreactive cell bodies are numerous in the proximal inner nuclear layer (INL) in all retinal regions. These cells have a primary process directed towards the inner plexiform layer (IPL). At postnatal day (PND) 2, a few faint immunoreactive processes are observed in the IPL. At PND 4, well-stained processes are observed to ramify mainly in the proximal IPL. At PND 6, strongly immunoreactive processes are present in both the distal and proximal IPL, and at PND 10 they form a continuous, dense plexus in both levels of the IPL. By PND 10, the morphology of PV immunoreactive cells is similar to PV immunoreactive cells in adult retinas. The density of PV immunoreactive cells in the proximal INL increases from PND 2 to PND 5, then it gradually decreases to adult values, while the total number of PV immunoreactive cell bodies increases until PND 10. PV immunoreactive amacrine cells at PND 2, as in the adult, are nonrandomly distributed across the retinal surface. These studies show that PV immunoreactive amacrine cells have a developmental profile that is similar to several other amacrine cell types. This includes the elaboration of processes in the IPL during the first postnatal week and a mature appearance towards the end of the second week of life, about the time of eye opening. These observations indicate that the AII amacrine cell may participate in the processing of visual information at eye opening.

AII amacrine cells in the mammalian retina show disabled-1 immunoreactivity.

Disabled 1 (Dab1) is an adapter molecule in a signaling pathway, stimulated by Reelin, which controls cell positioning in the developing brain. It has been localized to AII amacrine cells in the mouse and guinea pig retinas. This study was conducted to identify whether Dab1 is commonly localized to AII amacrine cells in the retinas of other mammals. We investigated Dab1‐labeled cells in human, rat, rabbit, and cat retinas in detail by immunocytochemistry with antisera against Dab1. Dab1 immunoreactivity was found in certain populations of amacrine cells, with lobular appendages in the outer half of the inner plexiform layer (IPL) and a bushy, smooth dendritic tree in the inner half of the IPL. Double‐labeling experiments demonstrated that all Dab1‐immunoreactive amacrine cells were immunoreactive to antisera against calretinin or parvalbumin (i.e., other markers for AII amacrine cells in the mammalian retina) and that they made contacts with the axon terminals of the rod bipolar cells in the IPL close to the ganglion cell layer. Furthermore, all Dab1‐labeled amacrine cells showed glycine transporter‐1 immunoreactivity, indicating that they are glycinergic. The peak density was relatively high in the human and rat retinas, moderate in the cat retina, and low in the rabbit retina. Together, these morphological and histochemical observations clearly indicate that Dab1 is commonly localized to AII amacrine cells and that antiserum against Dab1 is a reliable and specific marker for AII amacrine cells of diverse mammals. J. Comp. Neurol. 470:372–381, 2004.

Parvalbumin immunoreactivity is enhanced by brain-derived neurotrophic factor in organotypic cultures of rat retina.

The rodent retina undergoes considerable postnatal neurogenesis and phenotypic differentiation, and it is likely that diffusible neurotrophic factors contribute to this development and to the subsequent formation of functional retinal circuitry. Accordingly, perturbation of specific neurotrophin ligand-receptor interactions has provided valuable information as to the fundamental processes underlying this development. In the present studies we have built upon our previous observation that suppression of expression of trk(B), the high-affinity receptor for brain-derived neurotrophic factor (BDNF), in the postnatal rat retina results in the alteration of a specific interneuron in the rod pathway-the parvalbumin (PV)-immunoreactive AII amacrine cell. Here, we isolated retinas from newborn rats and maintained them in organotypic culture for up to 14 days (approximating the time of eye opening, in vivo) in the presence of individual neurotrophins [BDNF or nerve growth factor (NGF)]. We then examined histological sections of cultures for PV immunoreactivity. In control cultures, only sparse PV-immunostained cells were observed. In cultures supplemented with NGF, numerous lightly immunostained somata were present in the inner nuclear layer (INL) at the border of the inner plexiform layer (IPL). Many of these cells had rudimentary dendritic arborizations in the IPL. Cultures supplemented with BDNF displayed numerous well-immunostained somata at the INL/IPL border that gave rise to elaborate dendritic arborizations that approximated the morphology of mature AII amacrine cells in vivo. These observations indicate that neurotrophins have specific effects upon the neurochemical and, perhaps, morphological differentiation of an important interneuron in a specific functional retinal circuit.

Somatostatin‐immunoreactive neurons in the adult rabbit retina

Somatostatin (SRIF) is a neuroactive peptide that is distributed throughout the nervous system, including the retina. This peptide has been localized to populations of amacrine cells in a variety of vertebrate species. In the rabbit retina, SRIF immunoreactivity is present in a sparse population of medium to large neurons (13.72 μm in diameter, or 147.84 μ2) in the ganglion cell layer and in a small number of neurons in the inner nuclear layer. These cells display a preferential distribution to the inferior retina, with the highest density near the ventral and ventrolateral retinal margins (11.33 cells/mm2). SRIF‐immunoreactive cells have two to five primary processes that arborize in the proximal inner plexiform layer (IPL). These give rise to a plexus of finer processes in the distal IPL. Occasional immunoreactive processes are also present in the outer plexiform layer. In the IPL, these laminar networks are present in all retinal regions. In addition, SRIF‐immunoreactive cells often have a fine‐caliber axonlike process that eminates from the soma or perisomal region. These processes travel for great distances across the retina in either the nerve fiber layer or in the distal IPL but are never seen to enter the optic nerve head. In addition, the number of SRIF‐immunoreactive somata remains unchanged following transection of the optic nerve. Taken together, these data indicate that SRIF‐immunoreactive neurons of the rabbit retina are displaced amacrine cells. Furthermore, the sparse distribution of SRIF‐immunoreactive somata, the wide‐ranging, asymmetric arborization of their cellular processes, and previous pharmacological studies suggest that these neurons mediate a broad modulatory role in retinal function.

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

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