Ординатура / Офтальмология / Английские материалы / The Retina and its Disorders_Besharse, Bok_2011

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levels, using radioimmunoassays or by high-pressure liquid chromatography.

Low concentrations of exogenously applied NPY stimulate the release of glycine, dopamine, acetylcholine, and 5-hydroxytryptamine from the vertebrate (frog and rabbit) retina. SRIF stimulates release of dopamine from the rat retina, and pharmacological studies indicate this action is mediated by sst receptors. SP also evoked the release of dopamine from rat retina, and the modulation of Ca2þ currents in fish bipolar cells by SP suggests that it may affect transmitter release.

A variant of this experimental approach involves the use of flashing light to stimulate transmitter release to determine if peptides influence light-evoked transmitter release. For example, the m-opioid receptor agonist, (D-Ala2, MePhe4, Gly-ol5)-enkephalin (DAMGO), increases light-evoked release of acetylcholine from rabbit retina. These latter findings are consistent with-opioid- binding sites distributed homogenously over the IPL and GCL of the rat and monkey retina, and the localization of m-opioid receptor immunoreactivity by bistratified ganglion cells of the rat retina. Furthermore in rabbit retina, nociceptin, the endogenous ligand for opioid-receptor- like 1, inhibits acetylcholine release evoked by flickering light. In contrast, SP and SRIF do not change the level of light-evoked release of acetylcholine from rabbit retina.

Peptide Function

Experimental findings are consistent with peptides acting as slow transmitters or modulators in the retina. For instance, NPY, PACAP, SP, SRIF, and VIP, acting through GPCRs, influence adenylate cyclase or phospholipase C activity in retinal homogenates and whole retina. Peptides also have potent modulatory effects on both Ca2þ and Kþ currents in retinal neurons. Furthermore, SRIF acting through sst2A receptors inhibits the release of the excitatory transmitter glutamate from rod bipolar cells. In addition, the action of GABA at GABAA receptors is modulated by SRIF and VIP through phosphorylation of GABAA receptors by protein kinase A in rat rod bipolar, amacrine, and ganglion cells. These examples illustrate the functional role of peptides in regulating both transmitter release and the excitability of retinal neurons.

The concept that peptides act as modulators of retinal circuitry or networks is based on both anatomical and functional findings. Peptide-containing cells in most cases are wide-field amacrine cells that are characterized by a very-low–to-medium cell density. These cells ramify widely and have overlapping processes that cover the entire retinal surface. Therefore, they would have a broad influence on a large number of cells and are unlikely to mediate discrete point-to-point information

processing. Connectivity studies show that most synaptic input onto peptide-containing amacrine cell processes are from amacrine cells, and these cells in turn terminate mainly on amacrine and ganglion cells. Peptides released from these cells could therefore influence multiple cells in a local fashion. In addition, a consistent finding for several peptides is a distinct distribution of peptide-con- taining and peptide-receptor-bearing processes and cells. These findings suggest that peptides are likely to diffuse from their release sites and act at a distance in a paracrine fashion. Together, these observations support the concept that peptides have a widespread effect on multiple types of retinal neurons.

A feature of many wide-field amacrine cells is the coexpression of GABA and a peptide. For example, in rat, cat, and monkey retina, GABA is co-localized with NPY, SP, and VIP in different amacrine cell types. In addition, glutamate, the predominant ganglion cell transmitter, is co-expressed with PACAP in ganglion cells that innervate the suprachiasmatic nucleus of the hypothalamus. These observations are consistent with findings elsewhere in the nervous system reporting the co-expression and co-release of classical transmitters and peptides from the same cell. Interestingly, a differential release of classic transmitters and peptides depending on the frequency and pattern of cell firing has been shown for several neuronal systems. GABA or glutamate and peptides can act together at the same site, or the peptides can diffuse through the tissue and act at more distant cellular sites. There is evidence for both modes of action in the retina. GABA and peptides released from wide-field amacrine cells may act locally at GABAA receptors; for instance, both VIP and GABA act on GABAA receptors expressed by bipolar cell axons and ganglion cells. Peptides can also diffuse from their release site and act in a paracrine manner, as suggested for example, by the different distributions of SRIF immunoreactive processes and sst receptors, which are expressed by multiple retinal cell types.

Functional studies also support the concept that peptides act as modulators of retinal circuitry. In general, the cellular effects of peptides are slow, and they occur at multiple locations in the retina. Both SRIF and VIP produce excitatory changes in the range of seconds to minutes in the spontaneous activity and neuronal discharge patterns of ganglion cells. Moreover, SRIF affects the center-surround balance of all types of ganglion cells. This action of SRIF suggests a role in light/dark adaptation. The role of other peptides in visual function is less well understood. However, as their wide-ranging actions are also likely to be too slow to mediate fast signaling, these peptides will probably also act in modulatory processes that occur on longer timescales and participate in adaptive mechanisms that globally affect the state of retinal circuits and networks.

Conclusion

The vertebrate retina is richly endowed with multiple peptides and peptide receptors; peptides are often localized to a single or at the most a few amacrine cell types, and their receptors are typically expressed by multiple cell types. Peptides act through GPCRs to modulate voltageand ligand-gated ion channels, and influence neuronal excitability and transmitter release. The pattern of peptide and peptide receptor expression, and the cellular action of peptides being slow in onset, long lasting and potent at low concentrations is congruent with a modulatory role of peptides that would influence multiple cells and cellular networks. This broad modulatory role is consistent with peptides participating in slow signaling events in the retina and influencing adaptive mechanisms.

Acknowledgment

Support for this work was provided by NEI EY 04067 and a Veterans Administration Senior Career Scientist Award.

See also: GABA Receptors in the Retina; Neuropeptides: Localization; Neurotransmitters and Receptors: Dopamine Receptors; Neurotransmitters and Receptors: Melatonin Receptors.

Further Reading

Akopian, A., Johnson, J., Gabriel, R., Brecha, N., and Witkovsky, P. (2000). Somatostatin modulates voltage-gated K(þ) and Ca(2þ) currents in rod and cone photoreceptors of the salamander retina.

Journal of Neuroscience 20: 929–936.

Brecha, N. C. (1983). Retinal neurotransmitters: Histochemical and biochemical studies. In: Emson, P. C. (ed.) Chemical Neuroanatomy, pp. 85–129. New York: Raven.

Brecha, N. C. (2003). Peptide and peptide receptor expression and function in the vertebrate retina. In: Chalupa, L. and Werner, J. (eds.) Visual System, pp. 334–354. Boston, MA: MIT Press.

Casini, G., Catalani, E., Monte, M. D., and Bagnoli, P. (2005). Functional aspects of the somatostatinergic system in the retina and the potential therapeutic role of somatostatin in retinal disease.

Histology and Histopathology 20: 615–632.

Cervia, D., Casini, G., and Bagnoli, P. (2008). Physiology and pathology of somatostatin in the mammalian retina: A current view. Molecular and Cellular Endocrinology 286: 112–122.

D’Angelo, I. and Brecha, N. C. (2004). Y2 receptor expression and inhibition of voltage-dependent Ca2þ influx into rod bipolar cell terminals. Neuroscience 125: 1039–1049.

Feigenspan, A. and Bormann, J. (1994). Facilitation of GABAergic signaling in the retina by receptors stimulating adenylate cyclase.

Proceedings of the National Academy of Sciences of the United States of America 91: 10893–10897.

Ho¨kfelt, T., Broberger, C., Xu, Z. Q., et al. (2000). Neuropeptides – an overview. Neuropharmacology 39: 1337–1356.

Jensen, R. J. (1993). Effects of vasoactive intestinal peptide on ganglion cells in the rabbit retina. Visual Neuroscience 10: 181–189.

Johnson, J., Caravelli, M. L., and Brecha, N. C. (2001). Somatostatin inhibits calcium influx into rat rod bipolar cell axonal terminals. Visual Neuroscience 18: 101–108.

Thermos, K. (2003). Functional mapping of somatostatin receptors in the retina. A review.Vision Research 43: 1805–1815.

Veruki, M. L. and Yeh, H. H. (1992). Vasoactive intestinal polypeptide modulates GABAA receptor function in bipolar cells

and ganglion cells of the rat retina. Journal of Neurophysiology 67: 791–797.

Zalutsky, R. A. and Miller, R. F. (1990). The physiology of somatostatin in the rabbit retina. Journal of Neuroscience 10: 383–393.

Zalutsky, R. A. and Miller, R. F. (1990). The physiology of substance P in the rabbit retina. Journal of Neuroscience 10: 394–402.

Glossary

Autoradiography – A histochemical technique used to localize the binding site of a ligand in tissue using radiolabeled ligands (usually 131I, 3H, or 35S) and photographic techniques to detect the isotope. BAC – A bacterial artificial-chromosome-generated transgenic animals, which are a relatively potent way to generate knock-in transgenic mice that is thought to better recapitulate normal gene expression due to use of a large, chromosomal amount of regulatory genomic DNA surrounding the protein-coding region of the gene.

In situ hybridization histochemistry –

A histochemical technique used to localize the cellular localization of messenger RNA.

Isoforms – The different forms of a protein, derived from a single gene that result from alternative splicing or from a family of related genes.

Paracrine – Acting by volume transmission of diffuse messengers rather than at chemical synapses in a point-to-point fashion.

Introduction

The vertebrate retina contains numerous transmitters and related signaling molecules, including peptides and growth factors (Table 1). Peptides and growth factors have multiple roles in the retina, including cellular signaling, growth, and maintenance. This article focuses on the localization of peptides that are primarily involved in cellular signaling, including neurotransmission, and participate in retinal circuitry functions mediating visual information processing. Peptides are characterized by their small size ranging from 5 to 35 amino acids and slow actions that are mediated through multiple guanine-nucleotide- binding protein (G-protein)-coupled receptors (GPCRs), which influence intracellular signaling pathways.

The presence of peptides has been documented in both nonmammalian and mammalian retinas. The most studied peptides in the mammalian retina are neuropeptide Y (NPY), somatostatin (somatotropin-release-inhibiting factor, SRIF), the tachykinins (substance P (SP), neurokinin A (NKA) and neurokinin B (NKB)), and vasoactive intestinal polypeptide (VIP). This article primarily uses

these peptides as exemplars of the pattern of peptide expression in the vertebrate retina.

Evidence for abundant peptide expression in the vertebrate retina began with multiple descriptions in the late 1970s of peptide activity in retinal extracts and peptide immunostaining of amacrine cells. These studies established that peptides are usually localized to low-density populations of wide-field amacrine cells; some ganglion cells also express peptides. Consistent with the presence of peptides in the retina is the expression of their receptors, which have a wide distribution, and have been reported in bipolar, amacrine, and ganglion cell populations. Interestingly, there is often a mismatch between the cellular distribution of peptide-containing amacrine cells and their processes compared to the cellular distribution of their receptors. A striking example is the distribution of SRIF and its receptor, SRIF subtype 2A (sst2A, as discussed below), which suggests that peptides mainly act in a paracrine manner, and therefore influence multiple retinal circuits in both the outer and inner retina.

Peptides influence the cellular activity of retinal neurons and circuits by modulating multiple intracellular signaling pathways, which affect transmitter release and intrinsic neuronal properties. Peptide actions are characterized as being slow in onset, long-lasting, and potent at low concentrations, suggestive of a role in adaptive mechanisms. Together, these findings provide strong support for a functional role of peptides in the retina.

Peptide Expression

Bioassays and Radioimmunoassays

In the 1950s, Euler and his colleagues reported the presence of SP bioactivity in dog and bovine retinal extracts. However, it was not until the late 1970s with the development of additional bioassay systems and radioimmunoassays (RIAs) that a rich variety of peptides, including cholecystokinin, the enkephalin peptides, glucagon, SRIF, SP, thyrotropin-releasing hormone, and VIP, was described in vertebrate retinal extracts. At the present time, over 20 peptides have been reported in the vertebrate retina (Table 2) with a greater number of peptides and peptide families in nonmammalian compared to mammalian retinas.

In general, bioassay and RIA studies report low-to- moderate levels of peptides in retinal extracts compared to other tissues and brain regions. In most cases, findings

Table 1 Peptides and peptide receptors in the retina

Table 2 Peptides reported in the vertebrate retina

Angiotensin II

Cholecystokinin Corticotropin-releasing hormone Enkephalin

. Leu5-enkephalin

. Met5-enkephalin ß-Endorphin FMRFamide Glucagon

Luteinizing–hormone-releasing hormone Natriuretic peptides

. Atrial natriuretic peptide (a-ANP and g-ANP)

. Brain natriuretic peptide

. C-type natriuretic peptide Neurotensin

Neuropeptide Y Nociceptin

Pituitary adenylate-cyclase-activating polypeptide Peptide histidine isoleucine

Somatostatin Tachykinin peptides

. Substance P

. Neurokinin A

. Neurokinin B Thyrotropin-releasing hormone Vasoactive intestinal polypeptide

from these investigations are consistent with immunohistochemical studies showing that these peptides are expressed in amacrine and ganglion cells. For instance, SRIF bioactivity and immunoreactivity are in retinal extracts from numerous species, and this peptide is localized to sparsely occurring amacrine and displaced amacrine cells in all species studied to date. Similarly, SP bioactivity and immunoreactivity are reported in the retina of numerous species, and SP immunoreactivity is localized to both amacrine and ganglion cells.

Peptides detected in the retina correspond to those detected in other tissues as to their molecular structure based on gel electrophoresis or high-pressure liquid chromatography and, in more limited cases, on peptide sequencing. These studies revealed that both forms of SRIF, SRIF-14 and SRIF-28, are differentially expressed in the mammalian retina; SRIF-14 is the predominant

form in the rat and human retina and SRIF-28, in addition to SRIF-14, is in guinea pig and rabbit retina. Functionally, this finding is likely to be of importance, since SRIF14 and SRIF-28 preferentially bind to different SRIF receptor subtypes.

Peptide Localization

Peptide Messenger RNA

There have been few investigations documenting peptide messenger RNAs (mRNAs) in the retina. Most studies evaluated mouse, rat, and human retinal extracts using Northern blots and reverse transcription polymerase chain reaction (RT-PCR), and rat retinal sections using in situ hybridization histochemistry. A combination of these approaches have been used to show that preprotachykinin (PPT) I mRNA, which generates SP and NKA, and PPT II mRNA, which generates NKB, are expressed in rat retinal extracts. In situ hybridization histochemical studies have extended these findings to establish a differential distribution of the tachykinin mRNAs with PPT I mRNA in cells distributed to the inner nuclear layer (INL), inner plexiform layer (IPL), and ganglion cell layer (GCL), and PPT II mRNA in cells distributed to the GCL. In situ hybridization histochemical studies have also established the presence of sparsely distributed SRIF mRNA-containing cells in the INL and GCL, while another study showed the co-expression of SRIF mRNA and immunoreactivity in amacrine and displaced amacrine cells. Finally, VIP mRNA and immunoreactivity are in sparsely distributed cell bodies in the inner retina. These findings extend earlier biochemical studies showing that the mammalian retina synthesizes multiple peptides, and it is reasonable to assume that other peptides located in the retina by RIA or immunohistochemistry are also synthesized by retinal neurons.

An alternative approach to evaluating the cellular localization of a peptide is the employment of a transgenic mouse line with the peptide promoter driving the expression of a reporter gene. Although this type of genetic approach has been commonly used in other regions of the nervous system, there have been limited findings reported to date in the retina. The best example is the detection of NPY in amacrine cells in the INL and GCL of a mouse line with ß-galactosidase (ß-gal) expression driven by the NPY promoter. ß-Gal-containing amacrine and displaced amacrine cells are distributed to all retinal regions and they have widely ramifying processes. The pattern of ß-gal expression matched the pattern of NPY immunostaining established using a highly characterized antibody to NPY; there is about 85% co-localization between b-gal expression and NPY immunoreactivity. Thus, independent experimental approaches confirm the pattern of NPY expression in the retina.

Peptide Immunostaining

Beginning in the late 1970s and early 1980s, numerous peptide immunolabelings were described in both nonmammalian and mammalian retinas. Peptide immunostaining was usually localized to distinct populations of amacrine cells, and in some cases, to ganglion cells and their central-nervous-system projections.

Peptide immunoreactivity is commonly localized to wide-field amacrine cell populations distributed to both the proximal INL and the GCL. These cells are characterized by processes that ramify in one or more laminae of the IPL. In addition, these processes arborize widely and overlap to form a network across the retinal surface. Some wide-field amacrine cell types that contain peptide immunoreactivity also have axon-like processes. These amacrine cell types are likely to be polyaxonal amacrine cells characterized by a more restricted dendritic field and multiple axons that extend beyond their field of dendrites. Finally, there are several examples of sparse peptidecontaining processes crossing the INL and ramifying in the outer plexiform layer (OPL). These processes are likely to be derived from interplexiform cells, which are characterized by processes that ramify in both the IPL and OPL, and they are often categorized as an amacrine cell variant. These peptide-containing amacrine cell populations are characterized by a very-low-to-moderate cell density. In most cases, their cell bodies are distributed to all retinal regions. In all cases, peptide-containing processes cover the entire retinal surface.

Three examples that illustrate the general features described above are the NPY-, SRIFand VIP-containing amacrine cells in the mammalian retina.

NPY immunoreactivity is localized to wide-field amacrine cells that are located in both the proximal INL and the GCL. NPY cells have a similar appearance in different mammalian retinas. In the INL, most immunoreactive

cells were characterized by small cell bodies and fine processes that ramify primarily in lamina 1 of the IPL. A few cells also ramified in lamina 3 of the IPL. In the GCL, small-to-medium immunoreactive cells ramify primarily in lamina 5 of the IPL. A few immunoreactive processes, originating from somata in the INL and processes in the IPL, ramified in the OPL (Figure 1). In rat retina, immunoreactive cells had a regular distribution

across the retina and an overall cell density of 280 cells mm–2 in INL and 90 cells mm–2 in GCL.

SRIF immunoreactivity is localized to sparsely distributed, wide-field amacrine and displaced amacrine cells (Figure 2). Immunoreactive somata give rise to thin varicose fibers that form a narrow and continuous plexus in lamina 1 of the IPL. In many species, there is also a narrow plexus of varicose fibers in laminae 3 and 5 of the IPL. Frequently, a smooth, thin-caliber, axon-like process is observed to arise from a cell body or a primary process. A few immunoreactive fibers also cross the INL to ramify in the OPL in both ventral and dorsal retina. These fibers can be traced to the plexus in lamina 1 of the IPL. Finally, in rabbit and cat retinas, there is a dense accumulation of SRIF-immunoreactive fibers along the retinal margin, which form a circumferential band in all retinal regions.

A major feature of the SRIF expression in the mouse, rabbit, cat, and human retina is the predominant distribution of SRIF-containing cell bodies to the ventral retina. These cells form a very low-density cell population. For example, in rabbit retina, cell density ranges from 6 cells mm–2 in ventral retina to 11 cells mm–2 at the retinal margins. In addition, the total number of SRIF-immunoreactive amacrine cells in these retinas is correspondingly very low.

VIP immunoreactivity is localized to sparsely distributed wide-field amacrine cells, mainly located in the proximal INL. VIP cells have a similar appearance and

distribution in different mammalian retinas. VIP immunoreactivity is distributed to multiple varicose processes and collaterals that ramify in laminae 1, 3, and 5 of the IPL in all retinal regions. VIP mRNA-containing and immunoreactive cells also form low-density cell populations in both the INL and GCL. For example, VIPimmunoreactive cells have an overall cell density of 25 cells mm–2 in rabbit retina, and there is an overall cell density of 50 cells mm–2 in the INL and 12 cells mm–2 in the GCL in the monkey retina.

Tachykinin and pituitary adenylate-cyclase-activating polypeptide (PACAP), a peptide in the VIP family, are reported in ganglion cells. These peptide-containing ganglion cells innervate multiple retinorecipient targets.

SP-immunoreactive ganglion cells are present in the frog, rat, hamster, rabbit, monkey, and human retina. These cells have been identified in co-staining experiments following either retrograde labeling of ganglion cells after fluorescent tracers are injected into retinorecipient nuclei or by the loss of ganglion cell immunostaining, following optic nerve section. In hamster, a small number of ganglion cells contain SP immunoreactivity; SP immunoreactivity in the lateral geniculate nucleus (LGN) is eliminated following optic nerve section. In rabbit, about 30% of the ganglion cells contain SP immunoreactivity. These cells have medium-to-large somata and dendrites that ramify extensively in the IPL. Their axons terminate in several retinorecipient nuclei, including the LGN, superior colliculus, and the accessory optic nuclei. In human retina, weakly staining SP-containing ganglion cells have also been identified on the basis of their morphology. Finally, in the Macaca monkey retina, the presence of SP-containing ganglion cells is suggested by the partial loss of SP immunostaining in the pregeniculate nucleus and the olivary pretectal nucleus, following bilateral eye enucleation.

There have been a modest number of ultrastructural investigations concerning the connectivity of peptide immunoreactive cells in rat, guinea pig, and primate retina. In general, the main input to peptide immunoreactive

processes is from amacrine cells, whereas the major output formed by conventional synapses is onto amacrine and ganglion cells. There is a smaller percentage of input and output connections with bipolar cell axonal terminals. The large number of synaptic connections with amacrine cells indicates that peptide-containing cells are influenced principally by other amacrine cells and to a lesser degree by bipolar cells. There is more limited information about the connectivity of peptide-containing cells in other species, although overall the pattern of connectivity of these cells appears to be quite similar to that observed in monkey retina.

A feature of many wide-field amacrine cells is the coexpression of gamma aminobutyric acid (GABA) and a peptide. For example, in rat, cat, and monkey retina, GABA is co-localized with NPY, SRIF, and VIP. In addition, glutamate, the predominant ganglion cell transmitter, is reported to be co-expressed with PACAP in ganglion cells that innervate the suprachiasmatic nucleus of the hypothalamus. The co-expression of glutamate and SP in ganglion cells is also likely, although not formally demonstrated to date. These observations are consistent with findings elsewhere in the nervous system, reporting the co-expression and co-release of classical transmitters and peptides from the same cell. Interestingly, a differential release of classic transmitters and peptides, depending on the frequency and pattern of cell firing, has been shown in several systems. GABA or glutamate and peptides can act together at the same site, or the peptides can diffuse through the tissue and act at more distant cellular sites. There is evidence for both modes of action in the retina. GABA and peptides released from wide-field amacrine cells may act locally at GABAA receptors; for instance, both VIP and GABA act on GABAA receptors expressed by bipolar cell axons and ganglion cells. Peptides can also diffuse from their release site and act in a paracrine manner, as suggested, for example, by the differential distribution of SRIF-containing amacrine cell processes and SRIF receptors, which are expressed by multiple retinal cell types located away from the SRIF-containing processes.

Peptide Receptor Expression

As mentioned above, peptide actions are mediated by multiple GPCRs that influence intracellular signaling pathways, which regulate transmitter secretion and neuronal excitability. For instance, SRIF’s cellular actions are mediated by five distinct GPCRs, sst1–sst5. There are also two sst2 isoforms, sst2A and sst2B, from alternative splicing. The cellular actions of the tachykinin peptides are mediated by three receptors, known as NK-1, NK-2, and NK-3, whose preferred ligands are SP, NKA, and NKB, respectively.

Peptide-Binding Sites and Localization

The distribution of peptide receptors was initially evaluated using binding sites with autoradiographic approaches. In the late 1980s and early 1990s, the presence of high-affinity peptide-binding sites for several peptides, including SRIF, tachykinins, and VIP, was reported using radiolabeled peptides. Autoradiographic techniques were also used to define the regional distribution of peptidebinding sites. For example, SRIF-binding sites, identified with radiolabeled SRIF or a radiolabeled SRIF analog, were over the photoreceptor and both plexiform layers of the mouse and rat retina. High-affinity SPand VIPbinding sites were homogeneously distributed over the IPL and GCL. In addition, NKAand NKB-binding sites were evenly distributed over the IPL of the guinea pig retina. However, there are major difficulties in defining the cell types associated with these binding sites because of the low resolution of the autoradiographic technique. Furthermore, there are very few selective peptide agonists or antagonists available to distinguish among the different receptor isoforms. For example, octreotide binds to several SRIF receptor subtypes, and there is a high likelihood that multiple receptor subtypes were detected in these autoradiographic studies.

Peptide receptor mRNAs

The identification and cloning of peptide receptor genes in the 1990s have provided the tools for determining the expression of specific peptide receptors in the retina. NPY, SRIF, TK, and VIP receptors have been described most often in the mouse and rat retina. Multiple subtypes of these receptor mRNAs are found in the retina and they vary in their abundance; for example, in rat retina, the sst2 and sst4 mRNAs are the most abundant compared to the other sst mRNAs. Similarly, mRNAs of NPY and NK isoforms are present in retinal extracts with different levels of expression. Finally, mRNAs of the selective PACAP receptor, PAC1, and the VIP/PACAP-preferring receptors, VPAC1 and VPAC2, have been reported in retinal extracts. These findings illustrate that multiple

peptide receptors are synthesized by the mammalian retina, in agreement with binding, autoradiographic, and immunohistochemical studies.

Pharmacological Studies

The activation of intracellular signaling pathways by peptides and related analogs in retinal extracts also supports the notion that endogenous peptides are present in the retina and they have a functional role that is mediated by specific receptors. For instance, both NPY and SRIF potently inhibit forskolin-induced cyclic adenosine monophosphate (AMP) accumulation, and VIP potently stimulates adenylate cyclase activity in retinal extracts. In addition, PACAP-27 and PACAP-38 are positively coupled to adenylate cyclase activity, and their actions are more potent than VIP, indicating the presence of PAC1. Furthermore, in rat retinal homogenates, PACAP-27 and PACAP-38, but not VIP, show a dose-dependent stimulation of inositol phosphate levels, suggesting multiple signal transduction pathways for PACAP peptides. Finally, the presence of functional NK receptors has been shown by the dose-dependent stimulation of inositol phosphate accumulation and [Ca2þ]i mobilization by the tachykinin peptides, SP, NKA, and NKB.

Pharmacological studies with isolated retinal cells and peptide receptor agonists and, in more limited cases, antagonists have also indicated the presence of pharmacological subtypes of peptide receptors. For instance, Y2, an NPY receptor, and sst2A modulate L-type calcium channels expressed by rod bipolar cells. These investigations along with peptide binding and autoradiographic studies have been valuable in establishing the presence of functional peptide receptors in the retina.

Peptide Receptor Localization

Peptide Receptor mRNAs

In situ hybridization histochemical experiments have documented the cellular expression of NK-1 and NK-3 mRNAs in the rat retina. NK-1 mRNA is located in the INL and GCL, and NK-3 mRNA is mainly distributed to the middle and outer regions of the INL, corresponding to the location of bipolar cell bodies. This pattern of expression matches the NK-1- and NK-3-immunostain- ing patterns in the mouse and rat retina.

Peptide Receptor Localization

Peptide receptors, including those that mediate the actions of NPY, SRIF, and the TKs, have been mainly studied in the mouse and rat retinas. Several receptor subtypes are expressed in the retina, often by one or

more distinct cell types, including bipolar, amacrine, and ganglion cells.

Sst and NK receptor expressions illustrate the general pattern of cellular localization of peptide receptors, although many details of individual receptor expression remain to be determined in future studies. In some cases, there are marked differences in the pattern of expression of these receptors, when studied using different, wellcharacterized antibodies. These disparate observations suggest that antibody specificity is a major factor influencing the understanding of peptide receptor expression in the retina. To date, animal models using genetic reporters driven by peptide receptor promoters to localize the cellular expression of peptide receptors have not been reported in the retina. The use of molecular approaches and knock-out lines will be needed to fully characterize the antibodies used for the immunostaining studies, and bacterial artificial chromosome (BAC) transgenics and knock-in mouse lines will be important for establishing the cellular localization of these receptors, independent of immunostaining approaches.

There are major differences in the reported pattern of SRIF receptor expression. For instance, some groups report that numerous amacrine cells in the rat and rabbit retina express sst1 immunoreactivity. In contrast, other groups using a different antibody report sst1 immunoreactivity in a distinct population of ganglion cells based on their size, and distribution of dendrites to lamina 3 of the IPL. Several independent studies, using different antibodies, agree that sst2A immunoreactivity is the most abundant SRIF receptor subtype, and it is mainly localized to bipolar cells in mouse, rat, and rabbit retinas (Figure 3). Sst2A immunoreactivity is also reported in some widefield amacrine cells, photoreceptor terminals, and horizontal cells in some species. All investigators agree that ganglion cells express sst4 immunoreactivity. Sst4 immunoreactivity is in multiple ganglion cells and numerous multistratified processes in the IPL. Consistent with the immunostaining studies are in situ hybridization histochemical experiments describing sst4 mRNA expression in cells distributed to the INL and GCL. Together, these findings provide strong evidence for the expression of SRIF receptors by ganglion cells. There are no reports of the cellular localization of sst3 to retinal cells and there is a single report of sst5 expression in nearly all amacrine cells.

NK-1 immunoreactivity is in numerous amacrine cell bodies in the INL and in some small and large cell bodies in the GCL in the rat retina (Figure 4). Immunoreactivity is prominent in all IPL laminae, and in addition there are fine caliber and varicose processes in the OPL, and a few processes in the ganglion cell axon layer. NK-1 immunoreactivity is observed in tyrosine-hydroxylase-containing amacrine cell bodies and their processes. Finally, most NK-1immunoreactive amacrinecellscontainGABA immunoreactivity. The large number of NK-1 immunoreactive

OPL

INL

IPL

Figure 3 – Sst2A immunoreactive rod bipolar cells in the mouse retina. Sst2A immunostaining of rod bipolar cells, as well as some amacrine cells. Note very high level of expression (bright green) in rod bipolar cell terminals in laminae 5 of the IPL. Vertical section: scale ¼15 mm.

ONL

OPL

INL

IPL

GCL

Figure 4 – NK-1 immunoreactivity in amacrine cells of the rat retina. NK-1 immunostaining of multiple amacrine cell

bodies in the INL and heavy staining of processes in all laminae of the IPL. The pattern of immunostaining suggests that multiple amacrine cell types express this receptor. Vertical section: scale bar ¼50 mm. Adapted from figure 1 in Casini, G., Rickman, D. W., Sternini, C., and Brecha, N. C. (1997) Neurokinin 1 receptor expression in the rat retina. Journal of Comparative Neurology

389(3): 496–507. Copyright 1997, John Wiley & Sons, Inc. Reprinted with permission of John Wiley & Sons, Inc.

cell bodies and the distribution of processes to the IPL suggest that this receptor is expressed by multiple amacrine cell populations. Some ganglion cells, based on cell body size, appearance, and position, are also likely to express NK-1 immunoreactivity.

In contrast, NK-3 immunoreactivity is in bipolar cell bodies distributed to the middle of the INL, and in their dendritic and axonal processes in the OPL and IPL, respectively (Figure 5). In the IPL, they ramify in

OPL

INL

IPL

Figure 5 – NK-3 immunoreactivity in bipolar cells of the mouse retina. (a) NK-3 immunoreactive bipolar cells (red) are localized to the INL and labeled processes are in the OPL and laminae

1 and 2 of the IPL. PKC immunoreactivity in rod bipolar cells (green) that ramify in lamina 5 of the IPL. (b) The cell bodies of the NK-3 immunoreactive bipolar cells are clearly distinct from those of the rod bipolar cells. The pattern of NK-3 expression indicates OFF-type cone bipolar cells express this receptor. Vertical section: scale ¼10 mm.

laminae 1 and 2, distal to where the type 3 cone bipolar cells stratify, suggesting NK-3 expression in OFF-type bipolar cells. A subset of the NK-3 immunoreactive bipolar cells expresses synaptotagmin 2. This same subgroup of NK-3 immunoreactive bipolar cells contains recoverin, a marker for type 2 bipolar cells. Together, these findings show that NK-3 immunoreactivity is localized to at least two types of OFF-type bipolar cells. In addition, NK-3 immunoreactivity is in tyrosine-hydroxylase-containing amacrine cells. There are no reports of specific NK-2 immunostaining in the retina, although NK-2 mRNA is detected in retinal extracts.

A frequent observation from these studies is the mismatch between the distribution of peptide-containing processes and their receptors. This mismatch supports the notion of a paracrine mode of action for peptides acting at receptors that are located away from the

peptide-containing processes, although this does not rule out direct transmitter actions that could occur at synaptic specializations in areas of the IPL where there is apposition of peptide and peptide receptor processes.

Acknowledgment

Support for this work was provided by NEI EY 04067 and a Veterans Administration Senior Career Scientist Award.

See also: Information Processing: Amacrine Cells; Morphology of Interneurons: Amacrine Cells; Neuropeptides: Function.

Further Reading

Brecha, N. C. (1983). Retinal neurotransmitters: Histochemical and biochemical studies. In: Emson, P. C. (ed.) Chemical Neuroanatomy, pp. 85–129. New York: Raven.

Brecha, N. C. (2003). Peptide and peptide receptor expression and function in the vertebrate retina. In: Chalupa, L. and Werner, J. (eds.) Visual System, pp. 334–354. Boston, MA: MIT Press.

Brecha, N., Johnson, D., Bolz, J., et al. (1987). Substance P-immunoreactive retinal ganglion cells and their central axon terminals in the rabbit. Nature 32: 155–158.

Casini, G., Catalani, E., Dal Monte, M., and Bagnoli, P. (2005). Functional aspects of the somatostatinergic system in the retina and the potential therapeutic role of somatostatin in retinal disease.

Histology and Histopathology 20: 615–632.

Casini, G., Rickman, D. W., Sternini, C., and Brecha, N. C. (1997). Neurokinin 1 receptor expression in the rat retina. Journal of Comparative Neurology 389: 496–507.

Cervia, D., Casini, G., and Bagnoli, P. (2008). Physiology and pathology of somatostatin in the mammalian retina: A current view. Molecular and Cellular Endocrinology 286: 112–122.

Marshak, D. W. (1989). Peptidergic neurons of the macaque monkey retina. Neuroscience Research.Supplement 10: S117–S130.

Thermos, K. (2003). Functional mapping of somatostatin receptors in the retina: A review. Vision Research 43: 1805–1815.

Zalutsky, R. A. and Miller, R. F. (1990). The physiology of somatostatin in the rabbit retina. Journal of Neuroscience 10: 383–393.