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

pathway that reconfigures retinal circuits according to prevailing illumination conditions and signals from the retinal circadian clock. Through dopaminergic signaling, they restructure retinal function by modulation of chemical and electrical synapses, as well as by modification of the functional properties of retinal neurons. Both through direct synaptic contacts from interplexiform processes and through volume transmission, DA neurons influence all levels of retinal circuitry and all major classes of retinal neurons. In particular, dopamine regulates multiple neural circuits in the retina by modulating electrical synaptic transmission through gap junctions. This type of circuit modulation was first described in relation to dopaminergic uncoupling of gap junctions mediating electrical synaptic transmission between horizontal cells in teleost fish retinas, an action thought to reduce the size of retinal receptive fields. Subsequently, the regulatory action of dopamine on retinal gap junctions has been found to be a more general phenomenon, with coupling between rods and cones, AII amacrine cells and ganglion cells also being modulated by dopamine. Dopaminergic regulation of both rod–cone coupling and AII amacrine cell coupling serves to restrict the flow of visual signals from rods to retinal ganglion cells during light-adapted conditions in the day, while allowing flow of rod signals in the dark and at night.

In addition to regulating electrical synaptic transmission, retinal dopamine influences retinal circuit function by modifying chemical synaptic transmission, via modulation of glutamate and gamma aminobutyric acid (GABA) receptors. It also alters intrinsic functional properties of retinal neurons via modulation of membrane ion channels, such as voltage-gated sodium currents in bipolar and ganglion cells. Many of the synaptic and cellular changes induced by dopamine are consistent with a role for this transmitter in mediating light-adaptive changes in retinal function. For example, uncoupling of horizontal cells is consistent with a reduction in receptive field size and increased spatial resolution in light-adapted conditions. Restriction of rod signals from the cone pathways in the light and during the day is consistent with this notion as well. Modulation of sodium currents in bipolar cells and ganglion cells is consistent with accelerating light responses in cone pathways and shifting ganglion cell function from photon detection to contrast signaling, respectively. Finally, although IPCs do not directly synapse on photoreceptors, the outermost cells of the neural retina, they significantly influence their function, presumably through volume transmission of dopamine. In addition to regulating circadian rhythms in rod–cone coupling, dopamine influences regulation of the intracellular second messenger cyclic adenosine monophosphate and the timing of circadian secretion of melatonin by photoreceptors.

Light Drives Interplexiform Neurons via Both Conventional and Novel Pathways

One of the persistent puzzles regarding IPCs has been to understand the precise mechanisms by which they are affected by light and thus provide feedback signals within retinal circuits for background illumination. Studies of retinal dopamine release revealed a surprising heterogeneity in the lighting conditions that evoked dopamine release, including flickering light, steady light, and even prolonged darkness. Although studied intensively for more than three decades, the neural mechanisms by which light influences the activity of dopaminergic interplexiform neurons have remained incompletely understood, in part, because the sparse nature of retinal dopaminergic neurons had prevented analysis within intact retinal circuits with electrophysiological approaches that directly measure neuronal activity. Previous studies with anatomical approaches had suggested that retinal DA neurons are a homogeneous population of cells, that their primary input was from inhibitory GABA/glycine amacrine cells in the OFF sublamina of the IPL, and thus that their light responses could be due to disinhibition of OFF responses. Recently, the limitation on in situ electrophysiological recording of DA neurons was overcome by creating a transgenic mouse in which DA neurons were genetically marked with a fluorescent protein. Electrophysiological studies of dopaminergic interplexiform neuron light responses yielded the surprising finding that despite being considered a morphologically homogenous class, these neurons are functionally heterogeneous, with distinct neuronal subpopulations exhibiting transient and sustained light responses (Figure 3). This cellular heterogeneity is likely the substrate for the observed functional heterogeneity in dopamine regulation by light.

Light-responsive DA neurons fall into two distinct subpopulations (ON-transient and ON-sustained), which are driven by separate synaptic circuits. ON-transient cells are driven by rod or cone photoreceptors through ON-bipolar cells, in a conventional retinal ON-pathway circuit. ON-sustained DA neurons, however, are driven by melanopsin-expressing ipRGCs in a novel intraretinal retrograde light pathway in which ganglion cell photic signals are used for network adaptation within the retina. This novel intraretinal feedback pathway completely reverses the canonical direction of visual signaling, with photoreception being initiated in ganglion cells and dopaminergic signaling potentially modulating rod and cone photoreceptors (Figure 3). Thus, the dopaminergic interplexiform system is composed of two functional subpopulations of neurons tuned to distinct aspects of environmental light signals: transient DA neurons (t-DA, Figure 3), driven by rod and cone input through ON-bipolar cells that are tuned to rapidly changing background signals and which may mediate the observed dopamine release to flickering light, and sustained DA neurons (s-DA, Figure 3), driven

by ipRGCs that are tuned to maintained background illumination and which may mediate the observed dopamine release to steady light.

While light stimulates dopamine secretion by dopaminergic retinal neurons, there is also basal release of dopamine in the dark. Studies by Raviola and colleagues showed that isolated DA neurons are spontaneously active, with sodium and calcium currents that support spontaneous spiking and dopamine secretion. Recordings from DA neurons in intact retinas have shown that DA neurons exhibit spontaneous bursts of spikes that are enhanced when input from inhibitory amacrine cells is blocked pharmacologically. In intact retinal circuits, the OFF-channel-driven GABA and glycine input from inhibitory amacrine cells partially suppresses the intrinsic bursting activity of DA neurons. As burst spiking is associated with increased dopamine secretion from midbrain dopaminergic neurons, the input from inhibitory amacrine neurons likely regulates basal secretion of dopamine by retinal DA neurons in the dark.

IPCs Signal Time of Day from the Retinal Clock

The retinal circadian clock shapes retinal function into ‘day’ and ‘night’ states timed to the local day–night cycle. This requires intraretinal signaling mechanisms for both the output of clock signals to the downstream retinal processes and the input of the local light cycle to set the phase of the retinal clock. Retinal dopamine, which is under both circadian and light-evoked control, appears to play a critical role in both of these processes. Among its manifold demonstrated effects on retinal neurons and circuits, dopamine regulates circadian rhythms in rod– cone coupling, and the resulting rhythms in spectral sensitivity and rod–cone balance of retinal responses. Several recent results also suggest that dopamine plays a key role in the signaling of light stimuli to the retinal circadian clock. Recent findings that retinal dopamine is a key component of light resetting of the retinal clock and that retinal dopaminergic neurons receive light input from ipRGCs suggest that retinal circadian photoreception is likely mediated by a novel intraretinal retrograde light-transmission circuit in which light signals originate in the ipRGCs and reset the circadian clock through their influence on sustained DA cells.

DA Neurons and Retinal Degenerative

Disease

Parkinson’s Disease

Parkinson’s disease is a neurodegenerative disease caused by degeneration of doapminergic neurons in striatum and substantia nigra. Visual functions such as contrast

sensitivity detected by pattern electroretinograms (PERGs) are also impaired in Parkinson’s disease since there is concurrent loss of DA neurons in the retina. One possible cause is that dopamine deficiency increases receptive field size of horizontal cells in the outer retina, and of AII amacrine cells in the inner retina, through loss of dopaminergic control of gap junctions, which results in altering receptive field properties of ganglion cells (the primary origin of the PERG response).

Diabetic Retinopathy

Diabetic retinopathy, which is classically defined as a microvasculopathy, is now being viewed as a neurodegenerative disease of the retina. Functional visual deficits detected by electroretinogram (ERG) often occur before the appearance of overt retinal lesions in diabetic retinopathy. The most prominent change of the ERG in early diabetes is a distortion of oscillatory potentials that are thought to derive from the inner retinal neurons including DA cells. Indeed, DA cells initiate programmed cell death in early diabetes, and are lost during the later phases of the disease. Dysfunction of DA cells in diabetic retinas has also been suggested by studies examining retinal dopamine content and tyrosine hydroxylase activity. Together, these studies suggest that DA cells undergo degeneration in diabetes, but the cause remains unclear.

Human Health Implications of the Retinal Clock

Retinal circadian clock signaling modulates human vision, is associated with retinal degenerative diseases, and modifies photoreceptor survival in animal models of human ocular disease. Humans have daily psychophysical rhythms in absolute visual sensitivity, as well as the temporal resolution of vision, with peak scotopic sensitivity occurring at night and peak temporal resolution occurring during the day. These visual rhythms are mediated by corresponding rhythmic changes in retinal function with scotopic ERG b-wave sensitivity, and implicit time decreased during the day. Interestingly, these ERG rhythms are altered in patients with retinitis pigmentosa, indicating a link between circadian regulation of retinal function and this disease, which is a major cause of adult blindness. In addition, macular edema in diabetic retinopathy, intraocular pressure associated with glaucoma, and refractive errors associated with myopia in primates also exhibit circadian regulation. Finally, photoreceptors exhibit circadian-dependent vulnerability to light damage, being more susceptible in the night phase, and that dopamine and melatonin, two neurochemical messengers of the circadian clock, modulate photoreceptor survival in retinal degeneration animal models.

Summary

Retinal interplexiform neurons are a specialized subclass of amacrine cells that mediate intraretinal feedback of photic signals from photoreceptors, ganglion cells, and from brain nuclei to reconfigure retinal circuits according to prevailing illumination and circadian factors. They act primarily through the secretion of the neurotransmitter dopamine, both synaptically and via volume transmission. DA neurons and interplexiform neurons play critical roles in overall retinal function and in visual health by modulating retinal circuits, synchronizing the retinal clock and regulating eye and photoreceptor tropism.

See also: The Circadian Clock in the Retina Regulates Rod and Cone Pathways; Circadian Photoreception; Neurotransmitters and Receptors: Dopamine Receptors.

Further Reading

Besharse, J. C. and Luvone, P. M. (1992). Is dopamine a light-adaptive or a dark-adaptive modulator in retina? Neurochemistry International 20: 193–199.

Boelen, M. K., Boelen, M. G., and Marshak, D. W. (1998). Lightstimulated release of dopamine from the primate retina is blocked by 1-2-amino-4-phosphonobutyric acid (APB). Visual Neuroscience 15: 97–103.

Dacey, D. M. (1990). The dopaminergic amacrine cell. The Journal of Comparative Neurology 301: 461–489.

Djamgoz, M. B. A., Hankins, M. W., Hirano, J., and Archer, S. N. (1997). Neurobiology of retinal dopamine in relation to degenerative states of the tissue. Vision Research 37: 3509–3529.

Dowling, J. E. and Ehinger, B. (1975). Synaptic organization of the amine-containing interplexiform cells of the goldfish and Cebus monkey retinas. Science 188: 270–273.

Gustincich, S., Feigenspan, A., Wu, D. K., Koopman, L. J., and Raviola, E. (1997). Control of dopamine release in the retina: A transgenic approach to neural networks. Neuron 18: 723–736.

Hokoc, J. N. and Mariani, A. P. (1987). Tyrosine hydroxylase immunoreactivity in the rhesus monkey retina reveals synapses from bipolar cells to dopaminergic amacrine cells. Journal of Neuroscience 7: 2785–2793.

McMahon, D. G., Knapp, A. G., and Dowling, J. E. (1989). Horizontal cell gap junctions: Single-channel conductance and modulation by dopamine. Proceedings of the National Academy of Sciences USA

86: 7639–7643.

Mills, S. L. and Massey, S. C. (1995). Differential properties of two gap junctional pathways made by AII amacrine cells. Nature 377: 734–737.

Ribelayga, C., Cao, Y., and Mangel, S. C. (2008). The circadian clock in the retina controls rod–cone coupling. Neuron 59: 790–801.

Ruan, G. X., Allen, G. C., Yamazaki, S., and McMahon, D. G. (2008). An autonomous circadian clock in the inner mouse retina regulated by dopamine and GABA. PLoS Biology 6: e249.

Witkovsky, P. and Schu¨tte, M. (1991). The organization of dopaminergic neurons in vertebrate retinas. Visual Neuroscience 7: 113–124.

Zhang, D. Q., Stone, J. F., Zhou, T., Ohta, H., and McMahon, D. G. (2004). Characterization of genetically labeled catecholamine neurons in the mouse retina. Neuroreport 15: 1761–1765.

Zhang, D. Q., Zhou, T. R., and McMahon, D. G. (2007). Functional heterogeneity of retinal dopaminergic neurons underlying their multiple roles in vision. Journal of Neuroscience 27: 692–699.

Zhang, D. Q., Wong, K. Y., Sollars, P. J., Berson, D. M., Pickard, G. E., and McMahon, D. G. (2008). Intraretinal signaling by ganglion cell photoreceptors to dopaminergic amacrine neurons. Proceedings of the National Academy of Sciences USA 105: 14181–14186.

Glossary

Calcium imaging physiology – An experimental technique used for detecting and measuring calcium (Ca2þ) levels in cells or tissues. Calcium imaging techniques take advantage of calcium indicator dyes, which are molecules that respond to the binding of Ca2þ ions by changing their spectral properties.

G-protein-coupled receptor – A large protein family of seven-pass transmembrane receptors that bind molecules and activate intracellular signal transduction pathways to generate cellular responses by coupling to heterotrimeric guanine-triphosphate-binding proteins.

Ion channels – The transmembrane protein complexes that form a water-filled channel across the plasma membrane through which specific inorganic ions can diffuse across their electrochemical gradients.

Paracrine – Acting by volume transmission of transmitters or modulators rather than at chemical synapses in a point-to-point fashion.

Patch-clamp electrophysiology – The study of the electrical properties of biological cells and tissues using electrodes with a high-impedance (gigaohm) seal that permits measurements of small (pA) current and voltage changes.

Introduction

The vertebrate retina contains numerous transmitters and related signaling molecules, including peptides and growth factors, which have multiple roles in the retina, including cellular signaling, growth, and maintenance. The presence, expression, and distribution of peptides, and their receptors in the retina are discussed elsewhere in the encyclopedia. This article mainly focuses on the cellular actions of peptides in the retina. Peptides are characterized by their small size, ranging from 5 to 35 amino acids, and their actions are mediated through guanine-nucleotide-binding protein (G-protein)-coupled receptors (GPCRs) to influence multiple intracellular effectors, including cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), Ca2þ, protein kinases, and phosphatases. These intracellular

effectors modulate ion channels, ligand-gated channels, gap junctions, transporters, and receptors, and affect nuclear transcription factors.

The presence of peptides in the retina was firmly established in the 1970s on the basis of peptide bioactivity and immunoreactivity in retinal extracts, and peptide immunostaining, mainly of amacrine cells. Consistent with the action of peptides in the retina, peptide receptor binding sites have been reported in retinal extracts, and later the expression of their receptors by different retinal cell populations was demonstrated. There is a wide distribution of receptors, with bipolar, amacrine, and ganglion cell populations, expressing different complements of peptide receptors. Interestingly, there is often a difference between the cellular distribution of peptide-containing amacrine cells and their processes compared to the cellular distribution of their receptors, which suggests that peptides can act in a paracrine manner to influence multiple retinal circuits in both the outer and inner retina.

Peptides modulate the cellular activity of retinal neurons and circuits by influencing intracellular signaling pathways. For instance, several different peptides reported in the retina have an effect on cAMP and Ca2þ levels, and somatostatin (somatotropin-release-inhibiting factor, SRIF) modulates Kþ and Ca2þ currents. SRIF and vasoactive intestinal polypeptide (VIP) also modulate gamma aminobutyric acid-A (GABAA) receptor currents. Through these actions, peptides can change the efficacy of synaptic transmission in the retina by regulating cellular excitability as well as by modulating the release of the fast-acting transmitters, GABA and glutamate, from presynaptic axonal terminals. The overall actions of peptides on retinal neurons are generally characterized as being slow in onset, long lasting and potent at low concentrations, suggestive of a role in adaptive mechanisms. Together, these findings provide support for multiple physiological roles for peptides in the retina.

Peptide Receptor Expression

In general, peptide actions are mediated by GPCRs that influence intracellular signaling pathways, which in turn modulate neuronal excitability and transmitter secretion. Furthermore, there are multiple peptide receptor subtypes for most peptides that are differentially coupled to these intracellular effectors, which markedly increase the diversity of peptide action. For instance, numerous

receptor subtypes mediate the action of neuropeptide Y (NPY), somatostatin (SRIF), and VIP in the nervous system. In retina, there is a similar situation, with peptides expressed by a limited number of rarely occurring amacrine cell populations and multiple peptide receptor subtypes expressed by multiple and distinct retinal cell populations.

Numerous studies report the presence of numerous peptides in the vertebrate retina (Table 1). There are also multiple peptide receptors, including those for atrial natriuretic peptide, angiotensin II of the renin-angiotensin system, corticotropin-releasing hormone, NPY, opioid (enkephalin) and opioid-related peptides, SRIF, the takykinin (TK) peptides (substance P (SP), neurokinin A (NKA), neurokinin B (NKB)), and VIP in some vertebrate retinas. These observations are based on biochemical, molecular biological, and immunohistochemical findings. The best-documented peptides and peptide receptors in the mammalian retina are currently, NPY, SRIF, the TK peptides, and VIP.

Peptide-Binding Sites and Localization

In the late 1980s and early 1990s, the presence of highaffinity peptide-binding sites was reported for several peptides in the vertebrate retina. Biochemical studies showed peptide-binding sites in retinal homogenates, and autoradiographic techniques reported their localization to the plexiform layers. These findings were suggestive of peptide-mediated actions through specific receptors in the retina, although at that time very few peptide receptors had been identified and cloned.

Peptide Receptor messenger RNAs

The identification and cloning of the key peptide receptor genes in the early to mid-1990s provided the tools for determining the expression of specific peptide receptors in the retina. NPY, SRIF, and the TK peptide receptors

Table 1 Peptides and peptide receptors in the retina

were reported in the mammalian retina. There are typically multiple subtypes of these receptors, based on both molecular and immunohistochemical findings, and these different subtypes vary in their abundance. These findings confirm that the mammalian retina, as indicated by binding and autoradiographic studies, synthesizes multiple peptide receptors.

Peptide Receptor Localization

Peptide receptors, including those that mediate the cellular actions of NPY, SRIF, and the TKs, have been principally studied in the mouse and rat retinas. Several receptor subtypes are expressed in the retina, often by one or more distinct cell types, including photoreceptors, bipolar, amacrine, and ganglion cells. For instance, in rat retina, the neurokinin-1 (NK-1) receptor, which is the preferred receptor for SP, is expressed by GABAergic amacrine cells, while, the NK-3 receptor, which is the preferred receptor for NKB, is expressed by OFF-type bipolar cells.

A frequent observation that has emerged from immunostaining studies is the mismatch between the distribution and number of peptide-containing processes and their receptors. For example, there is a differential distribution of SRIF processes and SRIF subtype (sst) receptors in the inner plexiform layer (IPL). There are examples of several peptides acting at receptors that are located away from the peptide-containing processes, which support the notion of a paracrine mode of action, although this does not rule out direct transmitter actions occurring at or near synaptic specializations where there is a close apposition of peptideand peptide-receptor-expressing processes.

Intracellular Signaling

The activation of signal transduction cascades by peptides and related analogs in retinal extracts also supports the idea that endogenous peptides are present in the retina and their actions are mediated by specific receptors.

However, there are differences in observations concerning peptide actions on intracellular signaling pathways that may be due to the experimental approaches and sensitivity of the assays, as well as species differences. For instance, both NPY and SRIF inhibit forskolin-induced cAMP accumulation in some, but not all, vertebrate retinas. In contrast, VIP and VIP-related peptides potently stimulate adenylate cyclase activity in many vertebrate retinas. For example, SRIF is reported to stimulate cAMP accumulation in chick and ovine retinas, but SRIF does not affect cAMP accumulation in fish, pigeon, mouse, and rabbit retinas. Furthermore, SRIF inhibits VIP-stimulated adenylate cyclase activity in sheep retina. There is further complexity in evaluating SRIF’s action in

retinal homogenates, based on a recent report using both wild-type and sst receptor knock-out mouse lines. This study reported that interactions of sst receptor subtypes as well as levels of sst receptors influenced SRIF potency on adenylate cyclase activity. Finally, the sst2 receptor is reported to mediate SRIF inhibition of adenylate cyclase activity through a G protein of the Goa type in both mouse and rabbit retina.

In contrast, VIP’s potent stimulation of adenylate cyclase activity is well established in many vertebrate retinas. Peptide histidine isoleucine, which is coexpressed with VIP, also stimulates adenylate cyclase activity. In addition, the VIP-related peptides, pituitary adenylate-cyclase-activating polypeptide-27 (PACAP-27) and PACAP-38, are positively coupled to adenylate cyclase activity, and their actions are more potent than VIP, indicating the presence of the PAC1 receptor in retinal homogenates. 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.

SRIF acting through the sst2 receptor is also reported to induce nitric oxide production in the retina. Nitric oxide, in turn, would activate soluble guanylate cyclase and increase levels of cGMP. The presence of functional NK receptors has been shown by the dose-dependent stimulation of inositol phosphate accumulation and intracellular Ca2þ ([Ca2þ]i) mobilization by the TK peptides, SP, NKA, and NKB.

Cellular Signaling

Ca2þ Imaging and Ion Channel Physiology

Several peptides, including met5-enkephalin, NPY, SP, and SRIF, have an action at the cellular level in both nonmammalian and mammalian retinas. For instance, in goldfish retina, met5-enkephalin, SP, and SRIF partially inhibit voltage-dependent Ca2þ currents in isolated mixed bipolar cell axon terminals. In the mammalian retina, low concentrations of NPY, SRIF, or VIP modulate both voltageand ligand-gated channels in multiple cell types, as discussed below.

Pharmacological studies used isolated rat rod bipolar cells in acute preparations, coupled with Ca2þ imaging techniques to characterize the cellular actions of NPY and SRIF. Low concentrations of NPY did not result in detectable changes in [Ca2þ]i levels in rod bipolar cell axon terminals, suggesting that NPY alone does not influence [Ca2þ]i levels. In contrast, there is a dose-dependent inhibition of Kþ-evoked increases of [Ca2þ]i with NPY; inhibition is maximal with 1 mM NPYand is also observed with 0.1 nM NPY. Maximal inhibition is also seen with 1 mM C2-NPY and NPY(13–36), selective Y2 receptor agonists.

In contrast, no inhibition is observed with Y1, Y4, and Y5 agonists. These findings indicate that NPY acts presynaptically through Y2 receptors to regulate glutamate release from rod bipolar cell axon terminals (Figure 1).

There have been several studies of SRIF action at the cellular level. In both isolated rat and rabbit rod bipolar cells, there is no detectable change in [Ca2þ]i levels following direct application of SRIF. However, SRIF strongly inhibits a Kþ-stimulated increase of [Ca2þ]i through L-type Ca2þ channels in a dose-dependent manner in rat and rabbit rod bipolar cell axon terminals (Figure 2). SRIF also enhances GABA-evoked whole-cell currents in amacrine cells likely due to GABAA receptor phosphorylation, following activation of adenylate cyclase. In rabbit retina, SRIF and octreotide, a SRIF agonist, reduce a Kþ- stimulated increase in [Ca2þ]i in rod bipolar cell axon terminals. In addition, SRIF inhibits Caþ-activated Kþ currents (IBK) in these cells. The octreotide effect is prevented by L-Tyr8Cyanamid 154806, an sst2 receptor antagonist, indicating that these SRIF effects are likely to be mediated by sst2 receptor activation.

In salamander photoreceptors, low concentrations of SRIF modulate both voltage-activated Kþ and L-type Ca2þ currents. SRIF enhances a delayed outwardly rectifying Kþ current in both rod and cone photoreceptors (Figure 3). It differentially modulates L-type Ca2þ channels currents: SRIF reduces the Ca2þ current in rods and increases the Ca2þ current in cones (Figure 4). Ca2þ imaging experiments of isolated rod and cone photoreceptors produce findings consistent with the electrophysiological findings. Together, these observations suggest that SRIF has a role in the regulation of glutamate release from photoreceptors based on its modulation of both voltage-gated Kþ and Ca2þ currents.

Membrane currents are not altered following direct application of VIP to isolated rat rod bipolar and ganglion cells. However, VIP does influence GABAs action at GABAA receptors. It potentiates GABA-evoked wholecell currents in bipolar cells by 65% and ganglion cells by 54% (Figure 5). The onset of VIP action is slow with a long recovery period. VIP-induced potentiation of wholecell GABA currents is mediated through phosphorylation of GABAA receptors, following adenylate cyclase activation. The activation of a cAMP-dependent pathway is consistent with biochemical findings that VIP increases cAMP levels in the mammalian retina. In contrast to these observations in rat, VIP reduces GABA-evoked wholecell currents of rabbit bipolar cells by about 40%. Finally, in rat amacrine cells, VIP, SRIF, and enkephalin also enhance GABA-evoked whole-cell currents through GABAA receptor phosphorylation by protein kinase A to increase affinity of GABAA receptor for GABA. Thus, the peptides appear to fine-tune the inhibition by GABA.

These findings of NPY, SRIF, and VIP action at the cellular level are consistent with biochemical studies

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reporting the presence of peptide receptors and the activation of intracellular effectors in retinal homogenates. They are also consistent with the localization of peptide receptors to different retinal cell types. Together, they

show that peptides have multiple cellular actions including modulation of voltageand ligand-gated ion channels, which would influence intrinsic cellular properties and transmitter release from retinal cells.

Figure 4 Excitatory effects of somatostatin (SRIF) on the Ca2+ current in cones. (a) Cones were held at 70 mV, and depolarizing pulses were applied from 40 to +40 mV in 10 mV steps. (b) Current–voltage plot of the data in (a) is shown.

(c) A summary of the changes induced in the peak Ca current of rods and cones by SRIF is shown. From Figure 6 in Akopian, A., Johnson, J., Gabriel, R., Brecha, N., and Witkovsky, P. (2000)

Somatostatin modulates voltage-gated K(+) and Ca(2+) currents in rod and cone photoreceptors of the salamander retina. Journal of Neuroscience 20: 929–936.

Functional Studies

Electroretinogram Recording

A limited action of peptides in the retina has been shown using electroretinogram (ERG), which reflects the contribution of electrical activity from the different retinal cell types. The initial a-wave appears to arise from photoreceptors; the b-wave, the activity of primarily second-order bipolar cells and Mu¨ller glial cells; the slower c-wave, the activity of the retinal pigment epithelium and Mu¨ller cells; and the d-waves, the activity of the OFF pathway. In rabbit retina eyecups, low concentrations of SRIF decreased the amplitude of the b-wave in rabbit eyecup preparations, but several other peptides, including cholecystokinin (CCK) and SP do not appear to affect the ERG.

Extracellular Recordings

In contrast, extracellular recordings have shown that SP, SRIF, and VIP influence multiple retinal cell types in both nonmammalian and mammalian retinal eyecup preparations. Typically, low concentrations of peptides increase the general excitability of cells. In the mudpuppy eyecup preparation, neurotensin and SP increased the firing activity of ganglion cells; however, met5-enkephalin

Figure 5 VIP potentiation of GABA-evoked Cl– current in a bipolar cell (a) and a ganglion cell (b). The whole-cell GABAevoked currents in both the bipolar and ganglion cells were potentiated after a 11-s application of VIP. From Figure 4 in 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.

inhibited them. However, the distribution of m-opioid receptors is unknown in mudpuppy retina and the action of met5-enkephalin could be an indirect effect on amacrine cells. These actions are characterized as slow and long lasting. There is also some specificity in peptide action. For example, using a rabbit eyecup preparation as described below, SRIF acts on all ganglion cell types; in contrast, SP acts on most brisk ganglion cells and VIP acts on ONand OFF-center brisk ganglion cell types, respectively. Overall, peptide actions in this preparation are characterized as being modulatory and too slow to participate in fast, light-evoked responses.

Application of SP at low-to-moderate concentrations excites most brisk ganglion cells, including ONand OFF-center and ON–OFF directionally selective ganglion cells in the rabbit retina (Figure 6). SP exerts these excitatory effects without affecting ganglion cell receptive field properties. The latency of the SP response

was shorter than that of SRIF. Furthermore, intracellular recordings show that SP depolarizes some amacrine cells, including GABA-containing amacrine cells. These experiments are consistent with the expression of the SPspecific NK-1 receptor by GABA-containing amacrine cells. SP did not affect horizontal cell activity, consistent with the lack of TK-binding sites and NK-1- or NK-3- receptor immunostaining of horizontal cells. Investigations also report that SP excites most ganglion cells in mudpuppy and fish retina. Together, these findings indicate that TK peptides act in the inner retina and affect the general excitability of ganglion cells and their level of spontaneous activity, rather than altering the characteristics of ganglion cell receptive field properties, as reported for SRIF and VIP in the rabbit retina.

In rabbit retina, low concentrations of SRIF excite all ganglion cell types, change their signal-to-noise ratio discharge activity and shift their center-surround balance toward a more dominant center (Figure 7). Similar to other peptides, SRIF actions are characterized as being slow in onset and having a long latency. SRIF is also reported to act on multiple cells in the inner and outer retina; it directly affects bipolar, amacrine, and ganglion cells and influences the horizontal cell network. Moreover, SRIF is reported to increase input resistance of amacrine and bipolar cells, suggesting an action on ion channels or gap junctions. This would be consistent with patch-clamp experiments showing that SRIF affects Kþ and Ca2þ currents in retinal neurons. In addition, modulation of gap junctions could be mediated by SRIFinduced dopamine release or nitric oxide production, as suggested by pharmacological studies. Together, these

observations indicate that SRIF acts on multiple retinal circuits to produce long-lasting changes in ganglion cell activity and receptive field organization, consistent with the idea that this peptide acts as a modulator to mediate the effects of light adaptation in the retina.

VIP excites ONand OFF-center brisk ganglion cells in the rabbit retina. Similar to observations using SRIF and SP, VIP is potent at low concentrations and there is a delay in the onset of its action on ganglion cells. The maintained activity of ONand OFF-center ganglion cells is increased by VIP and their excitatory responses to flashes of light are unaffected or slightly reduced in the presence of VIP. In contrast, VIP has little effect on the maintained activity of ON/OFF directionally selective ganglion cells, and the response of these cells to a moving stimulus. Finally, the action of VIP on ganglion cells in rabbit retina is congruent with other reports of the action of VIP on GABA currents on isolated ganglion cells in the rat retina.

Peptide Influence on Transmitter Release

Several investigators have evaluated the influence of peptides on transmitter release from the retina. The general experimental paradigm is to preload the retina with a radiolabeled transmitter, such as GABA, glycine, or dopamine, and, following a washout period, to add the peptide and measure changes of radiolabeled transmitter levels induced by the peptide. An alternative experimental design is to measure endogenously released transmitter