Giovanni Casini

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.

Mechanisms underlying somatostatin receptor 2 down-regulation of vascular endothelial growth factor expression in response to hypoxia in mouse retinal explants†

Hypoxia is a trigger of VEGF expression, the primary cause of retinal pathologies characterized by neovascularization. During hypoxia, transcription factors such as STAT3 and HIF‐1 promote the increase in VEGF expression. Octreotide, a somatostatin receptor 2 (sst2)‐preferring agonist, reduces retinal VEGF expression and neovascularization. To investigate the intracellular pathways linking sst2 activation to the inhibition of hypoxia‐induced VEGF up‐regulation, we used pharmacological approaches and siRNA in mouse retinal explants cultured in normoxia or hypoxia. In hypoxic explants in which STAT3 or HIF‐1 was inhibited, we observed the existence of reciprocal interactions between STAT3 and HIF‐1, which synergistically induced VEGF expression. Octreotide prevented hypoxia‐induced activation of STAT3 and HIF‐1, and the downstream increase in VEGF expression, as evaluated in hypoxic explants treated with pharmacological inhibitors of STAT3 or HIF‐1 and in normoxic explants in which pharmacological activators of STAT3 or HIF‐1 were used to mimic a hypoxia‐like response. The effect of octreotide on STAT3 activation is in part indirect, through the blockade of VEGFR‐2 phosphorylation. The effect of octreotide on STAT3, HIF‐1, VEGFR‐2, and VEGF required Src homology region 2 domain‐containing phosphatase 1 (SHP‐1). In hypoxic extracts, octreotide induced SHP‐1 phosphorylation and activation, and inhibiting SHP‐1 abolished the octreotide effect on STAT3, HIF‐1, VEGFR‐2, and VEGF. The central role of SHP‐1 in the modulation of STAT3 and HIF‐1 was confirmed in normoxic explants in which pharmacologically activated SHP‐1 prevented the effect of STAT3 or HIF‐1 activation. Immunohistochemical studies showed that under hypoxia sst2 and VEGF are expressed by retinal vessels, thus indicating a possible direct effect of octreotide on VEGF‐containing endothelial cells. These data clarify the mechanism by which octreotide prevents hypoxia‐induced VEGF up‐regulation and support the effectiveness of octreotide in treatment of oxygen‐induced retinopathies. These results may have implications in designing therapies targeting STAT3 and/or HIF‐1 aimed at preventing retinal neovascularization. Copyright

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.

Developmental expression of protein kinase C immunoreactivity in rod bipolar cells of the rabbit retina

Rod bipolar cells constitute the second-order neuron in the rod pathway. Previous investigations of the rabbit retina have evaluated the development of other components of the rod pathway, namely the dopaminergic and AII amacrine cell populations. To gain further insights into the maturation of this retinal circuitry, we studied the development of rod bipolar cells, identified with antibodies directed to the alpha isoform of protein kinase C (PKC), in the rabbit retina. Lightly immunostained PKC-immunoreactive (IR) somata are first observed at postnatal day (PND) 6 in the distal inner nuclear layer (INI.). Immunostaining is also observed in the outer plexiform layer (OPL), indicating the presence of PKC-IR dendrites. PKC-IR axons are present in the INL oriented toward the inner plexiform layer (IPL). Several of them terminate with enlarged structures resembling growth cones. At PND 8, some immunostained terminal bulbs, characteristic of rod bipolar cells, are detected in the proximal IPL. PKC-IR cells at PND 11 (cye opening) display stronger immunostaining and more mature characteristics than at earlier ages. The dendritic arborizations of these cells in the OPL and their axon terminals in the IPL attain mature morphology at later ages (PND 30 or older). The density of PKC-IR cells shows a peak at PND 11 followed by a drastic decrease up to adulthood. The total number of PKC-IR cells increases from PND 6 to PND 11 and then it remains almost unchanged until adulthood. The mosaic of PKC-IR cells is nonrandom in some retinal locations at PND 6, but the overall regularity index at PND 6 is lower than at older ages. The present data provide a comprehensive evaluation of the development of rod bipolar cells in the postnatal rabbit retina and are consistent with those previously reported for dopaminergic and AII amacrine cell populations, indicating that different components of the rod pathway follow a similar pattern of maturation, presumably allowing the rod pathway to be functional at eye opening.

Neurokinin 1 receptor expression and substance p physiological actions are developmentally regulated in the rabbit retina

We investigated the expression of the substance P (SP) receptor (the neurokinin 1 receptor, NK1 receptor) and SP functional effects in developing rabbit retinas. NK1 receptors in adult retinas were in a population of cone bipolar cells and in dopaminergic amacrine cells, as previously described. In contrast, at birth and at postnatal day (PND) 6, NK1 receptors were exclusively expressed by cholinergic amacrine and displaced amacrine cells. NK1 receptor expression in cholinergic cells was still observed at PND10 (eye opening), while at PND21 it was confined to cholinergic cells of the inner nuclear layer. Starting at PND10, NK1 receptors were also in bipolar cells and in dopaminergic amacrine cells. A fully mature NK1 receptor expression pattern was observed at PND35. Dopamine release was assessed in isolated retinas in the presence of SP, the NK1 receptor agonist GR73632 or the NK1 receptor antagonist GR82334. At PND35, extracellular dopamine was significantly increased by 10 microM SP or 0.01-100 microM GR73632, and it was decreased by 0.01-10 microM GR82334. No effects were detected in developing retinas up to PND21. Ca2+ imaging experiments were performed in single cholinergic cells identified by their starburst morphology in perinatal retinas. Intracellular Ca2+ levels were significantly increased by 1 microM SP or GR73632. This effect was reversibly inhibited by 1 microM GR82334. These data demonstrate that both NK1 receptor expression and SP physiological actions are developmentally regulated in the retina. SP neurotransmission in the immature retina may subserve developmental events, and SP is likely to represent an important developmental factor for the maturation of retinal neurons and circuitries.

Expression of substance P, neurokinin 1 receptors (NK1) and neurokinin 3 receptors in the developing mouse retina and in the retina of NK1 knockout mice.

To complete a series of studies on the expression of substance P and neurokinin receptors in mammalian retinas, we investigated the occurrence of these molecules in developing mouse retinas and in retinas of mice with genetic deletion of the neurokinin 1 receptor, the preferred substance P receptor. Using semi-quantitative reverse transcription-polymerase chain reaction, we measured detectable levels of the gamma isoform of preprotachykinin A (a substance P precursor) mRNA at postnatal day 4. Neurokinin 1 receptor and neurokinin 3 receptor mRNAs were also detected at postnatal day 4. While gamma preprotachykinin A and neurokinin 1 receptor mRNA levels significantly increased up to eye opening (postnatal day 11), neurokinin 3 receptor mRNA levels remained constant throughout development. Substance P, neurokinin 1 receptor and neurokinin 3 receptor immunoreactivities were present at postnatal day 5. Substance P was in amacrine cells, neurokinin 1 receptor in developing amacrine and bipolar cells and neurokinin 3 receptor in OFF-type cone bipolar cells. Interestingly, a transient increase in the density of neurokinin 1 receptor immunoreactive processes was observed at eye opening in lamina 3 of the inner plexiform layer, suggesting a role of substance P and neurokinin 1 receptor in this developmental phase. However, in neurokinin 1 receptor knockout retinas, besides a significant increase of the gamma preprotachykinin A mRNA levels, no major changes were detected: neurokinin 3 receptor mRNA levels as well as substance P and neurokinin 3 receptor immunostainings were similar to wild types. Together with previous studies, these observations indicate that there are major differences in neurokinin 1 receptor expression patterns among developing mammalian retinas. The observations in neurokinin 1 receptor knockout mice may not be applicable to rats or rabbits, and substance P and neurokinin 1 receptor may play different developmental roles in different species.

Morphologic maturation of tachykinin peptide-expressing cells in the postnatal rabbit retina.

Tachykinin (TK) peptides, which include substance P, neurokinin A, two neurokinin A-related peptides and neurokinin B, are widely present in the nervous system, including the retina, where they act as neurotransmitters/modulators as well as growth factors. In the present study, we investigated the maturation of TK-immunoreactive (IR) cells in the rabbit retina with the aim of further contributing to the knowledge of the development of transmitter-identified retinal cell populations. In the adult retina, the pattern of TK immunostaining is consistent with the presence of TK peptides in amacrine, displaced amacrine, interplexiform and ganglion cells. In the newborn retina, intensely immunostained TK-IR somata are located in the ganglion cell layer (GCL) and in the inner nuclear layer (INL) adjacent to the inner plexiform layer (IPL). They are characterized by an oval-shaped cell body originating a single process without ramifications. TK-IR processes are occasionally observed in the IPL and in the outer plexiform layer (OPL). Long TK-IR fiber bundles are observed in the ganglion cell axon layer. TK-IR profiles resembling small somata are rarely observed in the INL adjacent to the OPL. At postnatal day (PND) 2, some TK-IR cells display more complex morphologic features, including processes with secondary ramifications. Long TK-IR processes in the IPL are often seen to terminate with growth cones. Between PND 6 and PND 11 (eye opening), there is a dramatic increase in the number of immunolabeled processes with growth cones both in the IPL and in the OPL and the mature lamination of TK-IR fibers in laminae 1, 3 and 5 of the IPL is established. TK-IR cells attain mature morphological characteristics and the rare, putative TK-IR somata in the distal INL are no longer observed. After eye opening, growth cones are not present and the pattern typical of the adult is reached. These observations indicate that the development of TK-IR cells can be divided into an early phase (from birth to PND 6) in which these cells establish their morphological characteristics, and a later phase (from PND 6 to eye opening) in which they are involved in active growth of their processes and likely in synapse formation. Since TK peptides are thought to play neurotrophic actions in the developing nervous system and they are consistently present in the retina throughout postnatal development, they may also act as growth factors during retinal maturation.

Transient somatostatin-immunoreactive ganglion cells in the developing rat retina

The peptide somatostatin (SRIF) is likely to play important roles in neuronal differentiation and maturation. In the mammalian retina, it is reported to be expressed by populations of amacrine and/or displaced amacrine cells and, in some species, by some ganglion cells. Previous studies have shown that in the rat retina the maturation of somatostatinergic systems encompasses late prenatal and early postnatal periods, suggesting a role of SRIF in maturative events of the retina. SRIF-expressing ganglion cells have not been reported in the rat retina at any developmental age. In the present study, we re-evaluated the postnatal development of SRIF-containing neurons of the rat retina focusing on the analysis of SRIF-containing cells in the ganglion cell layer (GCL), to test the possibility that SRIF is expressed by some ganglion cells during development. To this aim we combined immunocytochemical staining of SRIF-positive neurons with retrograde tracing of ganglion cell bodies through Fluoro-Gold injections into the superior colliculus. Double-labelling experiments revealed the presence of SRIF-containing ganglion cells at postnatal day (PND) 10. They accounted for 14% of the total SRIF-containing cells in the GCL. Such double-labelled cells were not observed either before (PND 7) or after (PND 15 and PND 45) this period. This transient expression of SRIF in retinal ganglion cells suggests that SRIF may be a factor regulating the maturation of retinocollicular projections in a restricted period of postnatal development.

Preproenkephalin messenger RNA-containing amacrine cells in the chicken retina identified with in situ hybridization histochemistry

Enkephalin peptides are present in the retina of several vertebrate species. In the avian retina, enkephalin immunoreactivity is primarily localized to a population of amacrine cells. In the present study, we determined the localization of cells expressing preproenkephalin (PPE) mRNA, which encodes the precursor of enkephalin peptides, in adult as well as in embryonic chicken retinas. The localization of PPE mRNA-expressing cells to the proximal inner nuclear layer (INL) in the adult chicken retina is similar to that of enkephalin-immunoreactive cells observed in previous studies, indicating that amacrine cells expressing PPE mRNA synthesize Met5- and Leu5-enkephalin peptides and related extended forms. Specific hybridization signal is absent in retinas at embryonic day (E) 11, but it is detected in retinas at E 15 and at hatching. PPE mRNA-expressing cells at these ages are located in the proximal INL, and they can be classified as amacrine cells on the basis of their soma size and laminar position. These findings confirm and extend previous observations on the presence of opioid peptides in amacrine cells of the chicken retina. The presence of PPE mRNA at embryonic ages, together with the evidence that enkephalins influence developmental processes, suggests that these peptides modulate retinal maturation in birds.