Drugs that prevent mouse sleep also block light-induced locomotor suppression, circadian rhythm phase shifts and the drop in core temperature.

Exposure of mice to a brief light stimulus during their nocturnal active phase induces several simultaneous behavioral or physiological responses, including circadian rhythm phase shifts, a drop in core body temperature (Tc), suppression of locomotor activity and sleep. Each response is triggered by light, endures for a relatively fixed interval and does not require additional light for expression. The present studies address the ability of the psychostimulant drugs, methamphetamine (MA), modafinil (MOD) or caffeine (CAF), to modify the light-induced responses. Drug or vehicle (VEH) was injected at CT11 into constant dark-housed mice then exposed to 5-min 100µW/cm(2) light or no light at CT13. Controls (VEH/Light) showed approximately 60-min phase delays. In contrast, response was substantially attenuated by each drug (only 12-15min delays). Under a 12-h light:12-h dark (LD12:12) photoperiod, VEH/light-treated mice experienced a Tc drop of about 1.3°C coincident with locomotor suppression and both effects were abolished by drug pre-treatment. Each drug elevated activity during the post-injection interval, but there was also evidence for CAF-induced hypoactivity in the dark prior to the photic test stimulus. CAF acutely elevated Tc; MA acutely lowered it, but both drugs reduced Tc during the early dark (ZT12.5-ZT13). The ability of the psychostimulant drugs to block the several effects of light exposure is not the result of drug-induced hyperactivity. The results raise questions concerning the manner in which drugs, activity, sleep and Tc influence behavioral and physiological responses to light.

Separation of function for classical and ganglion cell photoreceptors with respect to circadian rhythm entrainment and induction of photosomnolence.

Four studies were performed to further clarify the contribution of rod/cone and intrinsically photoreceptive retinal ganglion cells to measures of entrainment, dark preference, light-induced locomotor suppression and photosomnolence. Wild type (WT), retinally degenerate (rd/rd), and melanopsin-less (OPN4⁻/⁻) mouse strains were compared. In Experiment 1, mice were exposed to a graded photoperiod in which approximately 0.26 µW/cm² irradiance diminished to dark over a 6-h interval. This method enabled "phase angle titration," with individual animals assuming activity onsets according to their sensitivity to light. WT and OPN4⁻/⁻ animals entrained with identical phase angles (effective irradiance=0.078 µW/cm²), but rd/rd mice required a more intense irradiance (0.161 µW/cm²) and entrainment occurred about 2.5 h earlier. In Experiment 2, all three strains preferred the dark side of a divided light-dark chamber until the irradiance dropped to 0.5 µW/cm² at which point, rd/rd mice no longer showed a preference. Experiments 3 and 4 determined that WT and rd/rd mice showed equivalent light-induced locomotor suppression, but the response was greatly impaired in OPN4⁻/⁻ mice. Closer examination of open field locomotion using infrared video-based methods and Any-maze(tm) software revealed two opposing effects of light. Locomotor suppression was equivalent in WT and rd/rd mice. Responses by OPN4⁻/⁻ mice varied from being absent (n=17) to normal (similar to WT and rd/rd mice; n=8). Light onset was associated with a significant, but brief, locomotion increase in WT and OPN4⁻/⁻ mice, but not in rd/rd mice. Any-maze(tm) analysis supports the view that light-induced locomotor quiescence is followed by behavioral sleep (photosomnolence), a fact that was visually validated from the raw video files. The data show that (a) classical photoreceptors, most likely rods, allow mice to prefer and entrain to very dim light such as found in natural twilight; (b) the presence of melanopsin photopigment enables light-induced locomotor suppression and photosomnolence; (c) light-induced locomotor suppression/photosomnolence is rod/cone mediated in 36% of mice lacking melanopsin, but not in 64% of the same OPN4⁻/⁻ strain; and (d) light-induced locomotor suppression encompasses an interval of behavioral sleep.

Millisecond light pulses make mice stop running, then display prolonged sleep-like behavior in the absence of light.

Masking, measured as a decrease in nocturnal rodent wheel running, is a visual system response to rod/cone and retinal ganglion cell photoreception. Here, the authors show that a few milliseconds of light are sufficient to initiate masking, which continues for many minutes without additional photic stimulation. C57J/B6 mice were tested using flash stimuli previously shown to elicit large circadian rhythm phase shifts. Ten flashes, 2 msec each and equally distributed over 5 min, activate locomotor suppression that endures for an additional 25 to 35 min in the dark and does not differ in magnitude or duration from that elicited by 5-min saturating light pulse. Locomotor activity by mice without access to running wheels is also suppressed by light flashes. The effects of various light flash patterns on mouse locomotor suppression are similar to those previously described for hamster phase shifts. Video analysis of active mice indicates that light flashes initiated at ZT13 rapidly induce an interval of behavioral quiescence that lasts about 10 min at which time the animals assume a typical sleep posture that is maintained for an additional 25 min. Thus, the period coincident with light-induced wheel running suppression appears to consist of two distinct behavioral states, one interval during which locomotor quiescence is initiated and maintained, followed by a second interval characterized by behavioral sleep. Given this sequence effected by light stimulation, we suggest that it be referred to as "photosomnolence," the term reflecting upon both the nature of the stimulus and the associated behavioral change.

Localization of the AMPA subunit GluR2 in the outer plexiform layer of goldfish retina.

L-glutamate, the photoreceptor neurotransmitter, depolarizes horizontal cells and OFF bipolar cells by ionotropic AMPA-glutamate receptors. The AMPA-receptor subunit (GluR4) is localized to dendrites of OFF bipolar cells in goldfish retina. Here, we used immunohistochemical techniques to identify AMPA-receptor subunits on horizontal cell dendrites. A monoclonal antibody against rat GluR2, with high sequence homology to the recently cloned goldfish GluR2a receptor, was used for light- and electron-microscopical immunocytochemistry. Light- and dark-adapted retinas were analyzed, with no major difference in results. GluR2-immunoreactivity (IR) was restricted to a narrow band in the outer plexiform layer, in which it appeared as bright dome-shaped structures amidst numerous puncta. At the ultrastructural level, GluR2-IR was found in horizontal cell dendrites that invaginated cones and rods. Dendrites of OFF bipolar cells were not labeled. GluR2-IR was present mostly in horizontal cell dendrites that were the lateral elements of the triad, rather than in dendrites that were the central elements. In light-adapted retinas, GluR2-IR was found in many horizontal cell spinules. GluR2-IR was observed, on occasion, in a mixed rod/cone (Mb) ON bipolar cell process that innervated rod spherules. Verification of the Mb ON bipolar cell was made by protein kinase C and metabotropic mGluR1alpha immunolabeling. The presence of GluR2-IR in lateral elements suggests that lateral horizontal cell dendrites are postsynaptic to cones rather than only sites of feedback inhibition. All horizontal cell types express the GluR2 subunit, uniquely differentiating themselves from OFF bipolar cells that express the GluR4 subunit. This differentiation most likely has a major influence on the glutamate pharmacology and response kinetics of these cell types to glutamate.

Neurochemical anatomy of the zebrafish retina as determined by immunocytochemistry.

The zebrafish retina is rapidly becoming a major preparation for the study of molecular genetic mechanisms underlying neural development and visual behavior. Studies utilizing retinal mutants would benefit by the availability of a data base on the distribution of neurotransmitter systems in the wild-type fish. To this end, the neurochemical anatomy of the zebrafish retina was surveyed by light microscopic immunocytochemistry. An extensive series of 60 separate antibodies were used to describe the distribution of major transmitter systems and a variety of neuron-associated membrane channels and proteins. These include markers (i.e., antibodies against enzymes, receptors, transporters) for transmitters: GABA, glycine, glutamate, biogenic amines, acetylcholine, cannabinoids and neuropeptides; as well as a sample of voltage-gated channels and synapse associated membrane proteins. Discussion of the comparative localization of these antibodies is restricted to other teleost fishes, particularly goldfish. Overall, there was great similarity in the distribution of the various markers, as might be expected. However, there were some notable differences, including several antibodies that did not label zebrafish at all, even though goldfish retinas that were processed in parallel, labeled beautifully. This survey is extensive, but not exhaustive, and hopefully will serve as a valuable resource for future studies of the zebrafish retina.

Cannabinoid receptors on goldfish retinal bipolar cells: electron-microscope immunocytochemistry and whole-cell recordings.

Cannabinoid CB1 receptors are distributed throughout the CNS and interact with GABA, glutamate, and dopamine systems. Cannabinoids have effects on the visual system, some of which may have a retinal component, particularly the enhancement of photosensitivity. We used immunocytochemistry and whole-cell recording to study cannabinoids in the goldfish retina. Immunoblots of an antiserum against amino acids (1-14) of the rat CB1 receptor produced a single band in goldfish retina at about 70 kDa. Light microscope immunocytochemistry of CB1 receptor immunoreactivity (CB1R-IR) revealed intense staining of Müller cells and weaker staining of ON bipolar cells (verified with double labeling with PKC-IR) and the outer and inner plexiform layers. Ultrastructural analysis revealed that CB1R-IR was localized intracellularly as well as on the plasma membrane of photoreceptor terminals, bipolar cell terminals and, rarely, amacrine cell boutons. Membrane-associated CB1R-IR was restricted to cone pedicles at sites removed from the synaptic ribbon. Regarding bipolar cells, membrane-associated CB1R-IR was found at 93% of the synaptic terminals in sublamina b (ON-type) and only at 33% of the synaptic terminals in sublamina a (OFF-type). Whole-cell recordings from large ON-type Mb bipolar cells showed that the delayed rectifier (I(K(V))) was rapidly and reversibly inhibited by 1 microM of the cannabinoid agonists CP 54490 and (+)-WIN 55212-2, effects blocked completely by the antagonist SR 141716A (1 microM). Inhibition of I(K(V)) in the Mb bipolar cells by cannabinoids should result in a more tonic ON response to increments of light. As such, cannabinoids may play a role in modulating the temporal aspects of signaling in the retina.

Immunocytochemical localization of the glutamate transporter GLT-1 in goldfish (Carassius auratus) retina.

Glutamate is the major excitatory neurotransmitter in the retina of vertebrates. Electrophysiological experiments in goldfish and salamander have shown that neuronal glutamate transporters play an important role in the clearance of glutamate from cone synaptic clefts. In this study, the localization of the glutamate transporter GLT-1 has been investigated immunocytochemically at the light and electron microscopical levels in the goldfish retina using a GLT-1-specific antibody. GLT immunoreactivity (IR) was observed at the light microscopical level in Müller cells, bipolar cells, the outer plexiform layer (OPL), and the inner plexiform layer (IPL). At the electron microscopical level, membrane-bound and cytoplasmic GLT-IR in the OPL was located in finger-like protrusions of the cone terminal located near the invaginating postsynaptic processes of bipolar and horizontal cells. GLT-IR was not observed in the vicinity of synaptic ribbons. This location of GLT-1 allows modulation of the glutamate concentration in the synaptic cleft, thereby shaping the dynamics of synaptic transmission between cones and second-order neurons. In the inner IPL, GLT-IR was observed in the cytoplasm and was membrane bound in mixed rod/cone bipolar cell terminals and cone bipolar cell terminals. The membrane-bound GLT-1 was generally observed at some distance from the synaptic ribbon. The morphology of the bipolar cell terminal together with the localization of GLT-1 suggests that at least these glutamate transporters are not primarily involved in rapid uptake of glutamate release by the bipolar cells. The GLT-IR in the cytoplasm of Müller cells was located throughout the entire goldfish retina from the outer limiting membrane to the inner limiting membrane. The location of GLT-1 in Müller cells is consistent with the role of Müller cells in converting glutamate to glutamine.

Immunocytochemical localization of cannabinoid CB1 receptor and fatty acid amide hydrolase in rat retina.

Cannabinoids have major effects on central nervous system function. Recent studies indicate that cannabinoid effects on the visual system have a retinal component. Immunocytochemical methods were used to localize cannabinoid CB1 receptor immunoreactivity (CB1R-IR) and an endocannabinoid (anandamide and 2-arachidonylglycerol) degradative enzyme, fatty acid amide hydrolase (FAAH)-IR, in the rat retina. Double labeling with neuron-specific markers permitted identification of cells that were labeled with CB1R-IR and FAAH-IR. CB1R-IR was observed in all cells that were protein kinase C-immunoreactive (rod bipolar cells and a subtype of GABA-amacrine cell) as well as horizontal cells (identified by calbindin-IR). There was also punctate CB1R-IR in the distal one-third of the inner plexiform layer (IPL) that could not be assigned to a cell type. FAAH-IR was most prominent in large ganglion cells, whose dendrites projected to a narrow band in the proximal IPL. Weaker FAAH-IR was observed in the soma of horizontal cells (identified by calbindin-IR); the soma of large, but not small, dopamine amacrine cells (identified by tyrosine hydroxylase-IR); and dendrites of orthotopic- and displaced-starburst amacrine cells (identified by choline acetyltransferase-IR) but in less than 50% of the starburst amacrine cell somata. The extensive distribution of CB1R-IR on horizontal cells and rod bipolar cells indicates a role of endocannabinoids in scotopic vision, whereas the more widespread distribution of FAAH-IR indicates a complex control of endocannabinoid release and degradation in the retina.

Co-localization of Shaker A-type K+ channel (Kv1.4) and AMPA-glutamate receptor (GluR4) immunoreactivities to dendrites of OFF-bipolar cells of goldfish retina.

Immunocytochemical methods were used to determine the comparative distribution of Shaker Kv1.4 and Shal Kv4.2 A-type voltage-gated K(+) channels and AMPA-type GluR4 glutamate receptors in the goldfish retina. Kv1.4-immunoreactivity (IR) was restricted to a very narrow band of bright puncta and filamentous processes in the outer plexiform layer (OPL), whereas GluR4-IR was found in radial processes of Müller cells in addition to a narrow band in the OPL. Kv4.2-IR was most prominent over cell bodies of horizontal cell, amacrine cells and ganglion cells, with very weak labeling over the synaptic terminal of cone photoreceptors. Double label experiments revealed complete co-localization of Kv1.4-IR and GluR4-IR in the OPL and showed that the Kv1.4 puncta in the OPL appeared enclosed by the Kv4.2-IR cone terminals. Electron microscopical immunocytochemistry showed that Kv1.4-IR and GluR4-IR were restricted to the dendrites of OFF-bipolar cells that innervated cone photoreceptor terminals and thin processes that coursed between the rod and cone terminals in the OPL. These data are consistent with other studies demonstrating the selective clustering of A-type voltage-gated K(+) channels and ionotropic glutamate receptors. However, they differ from mammalian preparations in which Shal-like Kv4.2 rather than Shaker-like Kv1.4 co-localize postsynaptically with glutamate receptors.

Dopamine D1-receptor immunolocalization in goldfish retina.

Dopamine, a neuromodulator in the vertebrate retina, is involved in numerous functions related to light adaptation. However, unlike in mammals, localization of retinal D1-dopamine receptors in nonmammalian vertebrates has been hampered due to a lack of antisera. To address this problem, an antiserum against the 18 C-terminal amino acids of the goldfish D1 receptor (gfD1r) was generated in chicken eggs and tested in retinae of goldfish and rat, and rat caudate putamen, by using immunoblots and light microscopic immunocytochemistry. No labeling was observed in any tissue or immunoblots with preabsorbed gfD1r antiserum. Immunoblot analysis of goldfish retina revealed a single band at about 101 kDa. The patterns of gfD1r immunoreactivity (gfD1r-IR), found in rat caudate putamen and rat retina were virtually identical to that previously reported with other D1-receptor ligands and antisera. In goldfish retina, gfD1r-IR was most intense over cell bodies in the ganglion cell layer, amacrine cells in the proximal inner nuclear layer (INL), and bipolar cells in the distal INL. Weaker gfD1r-IR was observed over horizontal cell bodies and both plexiform layers. Müller cells and axons of cone photoreceptors were labeled as well. Double labeling showed that all protein kinase C-immunoreactive bipolar cells (ON type) were gfD1r-IR on the soma, axon terminal, and dendrites. All glutamate decarboxylase-immunoreactive (i.e., gamma-aminobutyric acid utilizing) amacrine cells and horizontal cells were gfD1r-IR. Retinal D1r distribution is more extensive than dopamine neuron innervation, but is consistent with physiologic estimates of dopamine function, suggestive of both wiring and volume transmission of dopamine in the retina. The gfD1r antiserum displays cross-reactivity to dopamine receptors in a mammal and a nonmammal and should prove useful in future studies of dopaminergic systems.

Differential distribution of Shaker-like and Shab-like K+-channel subunits in goldfish retina and retinal bipolar cells.

The distributions of Shaker subfamily Kv1.1 and Kv1.2 and Shab subfamily Kv2.1 subunits of voltage-gated K+ channels were determined in the retina and ON bipolar cells of goldfish by using double-label light and electron microscopic immunocytochemistry. All labeling to be described was blocked by preabsorption of the primary antibodies with antigen. The retina was labeled throughout with all three antibodies. However, labeling was densest in the inner plexiform layer for Kv1.1, more concentrated in the outer nuclear layer for Kv2.1, and uniform throughout for Kv1.2. All ON mixed rod/cone (mb) and cone (cb) bipolar somata and the proximal portions of their axons and dendrites were labeled for anti-Kv1.1, Kv1.2, and Kv2.1. Labeling of axons rarely extended over the mb axon terminal. Only Kv1.2 antibodies labeled mb bipolar cell dendrites in the outer plexiform layer. No evidence for Kv1.1, 1.2, or 2.1 antibody labeling of OFF bipolar cells was found. Ultrastructurally, Kv1.2-immunoreactivity was associated with the plasma membrane of bipolar cell bodies and with dendrites that make narrow-cleft junctions with cone terminals (ON-type). Kv immunoreactivity was not found associated with presynaptic membranes in the inner plexiform layer and was found only rarely with membranes, postsynaptic to an amacrine cell process. Although both Shaker and Shab subfamilies include delayed rectifiers, their activation properties differ, suggesting differential modulation of K+ conductances in bipolar cells based not only on the presence or absence of rod photoreceptor input but also whether the bipolar cells are of the ON or OFF type.

Differential reinnervation of retinal bipolar cell dendrites and axon terminals by dopamine interplexiform cells following dopamine depletion with 6-OHDA.

Depletion of retinal dopamine in goldfish increases light sensitivity at photopic backgrounds. As horizontal cells appear not to be involved with this effect (Yazulla and Studholme [1995] Vis. Neurosci. 12:827-837), we investigated the innervation patterns of the ON rod/cone bipolar cells (ON-BC) by dopaminergic interplexiform cells (DA-IPCs) normally and during the period of neogeneration of new DA-IPCs at the marginal zone following DA-IPC destruction. DA-IPCs were destroyed via intraocular injection of 6-hydroxydopamine over 2 successive days. Controls and 1 year post-injection retinas were double labeled for protein kinase C and tyrosine hydroxylase (TH) immunocytochemistry to identify the ON-BCs and the DA-IPCs, respectively. Double-labeled 25 microns tissue sections were examined on a confocal laser scanning microscope by using dual channel immunofluorescence acquisition. Image stacks were analyzed for DA-IPC/ON-BC contacts in the distal inner nuclear layer (INL) and inner plexiform layer (IPL). Image stacks were rotated 180 degrees with respect to each other and reanalyzed to determine potential randomness of the contacts. For control retinas there were 1.8 contacts/axon terminal in the IPL (n = 165) and 9.4 contacts/ON-BC in the distal INL (n = 28). At 1 year after injection, reinnervation of TH-immunoreactive boutons in the retina recovered to 16% of control in the IPL but only 10% in the distal INL. Establishment of DA-IPC/ON-BC contacts recovered to 36% of control for ON-BC axon terminals (n = 103), whereas there was no recovery of contacts in the distal INL (n = 30). Reinnervation of ON-BC by DA-IPCs preferentially targets the axon terminals. The absence of reinnervation of bipolar cell dendrites by DA-IPCs may account for the persistence of the increased light sensitivity following retinal dopamine depletion. Thus, dopamine input to ON-BCs in the outer retina maybe involved in setting background sensitivity under photopic conditions.

3H-adenosine uptake selectively labels rod horizontal cells in goldfish retina.

There are four types of horizontal cell in the goldfish retina, three cone- and one rod-type. The neurotransmitter of only one type, the H1 (cone) horizontal cell, has been identified as GABA. 3H-adenosine uptake was examined as a possible marker for the other classes of horizontal cell. Isolated goldfish retinae were incubated in 3H-adenosine (10-40 microCi) in HEPES-buffered saline for 30 min, then fixed, embedded in plastic, and processed for light-microscopic autoradiography (ARG). For double-label immuno/ARG studies, 1-micron-thick sections were processed for GABA postembed immunocytochemistry, then for ARG. 3H-adenosine uptake was localized to cone photoreceptors, presumed precursor cells in the proximal outer nuclear layer, and to a single, continuous row of horizontal cell bodies in the inner nuclear layer. No uptake was localized to the region of horizontal cell axon terminals. 3H-adenosine uptake did not colocalize with GABA-IR in H1 horizontal cells, but it did colocalize with adenosine deaminase immunoreactivity. It is concluded that 3H-adenosine uptake selectively labels rod horizontal cells in the goldfish retina based on position and staining pattern, which are similar to rod horizontal cells stained by Golgi or HRP injection methods. The use of 3H-adenosine uptake may provide a useful tool to study other properties of rod horizontal cells (i.e. development) as well as provide clues as to the transmitter used by these interneurons.

Light adaptation affects synaptic vesicle density but not the distribution of GABAA receptors in goldfish photoreceptor terminals.

GABA is a likely feedback transmitter from H1 horizontal cells to cone photoreceptors in fish retinas. Spinules arise from H1 cell dendrites in light-adapted retinas, are correlated with responses attributed to feedback, and have been proposed to be the GABA release sites. We used mAb 62-3G1, an antibody against the beta 2/beta 3 subunits of the GABAA receptor complex, to visualize GABAA receptor immunoreactivity (GABAr-IR) in photoreceptors as a function of light and dark adaptation at the electron microscopical level. Regardless of adaptation, GABAr-IR was restricted to the synaptic terminals of all cones and most rods; synaptic vesicular membrane and plasma membrane, exhibited GABAr-IR. Contrary to expectations, the density of GABAr-IR was least on the plasma membrane within the invagination, regardless of the presence or absence of spinules. Dense GABAr-IR was observed on the lateral surface of cone pedicles, on cone processes proximal to the invagination, and on presumed telodendria from nearby cones. There was no difference in GABAr-IR of rod plasma membranes within or outside of the invagination or with adaptation. The only novel effect of adaptation was in regards to the density of synaptic vesicles. Cones showed a 29% increase in vesicle density with dark adaptation, whereas rods showed a 17% decrease. We conclude that all goldfish photoreceptors will be GABA-sensitive and that the sensitivity is distributed over the surface of the synaptic terminal rather than localized to within the invagination. The role of spinules in GABA release remains to be determined, but we conclude that spinules are not related to the GABA sensitivity of goldfish photoreceptors.

Differences in the retinal GABA system among control, spastic mutant and retinal degeneration mutant mice.

Immunocytochemical methods were used to compare the GABA system in control mice and two mutant strains: spastic which has reduced glycine receptors and retinal degeneration mutant in which the photoreceptors degenerate and reportedly have increased GABA and GAD levels. We found that the spastic mutant retina had reduced GABA-immunoreactivity (IR) in the proximal retina, reduced staining for GAD-1440 in the OPL, and reduced GABAA receptor staining in the OPL, compared to control. The retinal degeneration mutant retinas had enhanced GABA-IR throughout the retina, particularly in Müller cells, bipolar cells and IPL, and enhancement of GABAA receptor staining in the OPL, compared to control. The distributions of GABA-IR, GAD-1440-IR and GABAA receptor-IR in retinas of spastic mutant mice that also expressed the retinal degeneration phenotype resembled those found in retinas of mice that expressed only the retinal degeneration phenotype rather than those that expressed only the spastic mutation. No differences were observed among the conditions for GAD-65, GAD-67 or GABA-T. Our results with the spastic and retinal degeneration mutant mice demonstrate that attenuation in the glycinergic system and photoreceptor degeneration, respectively, is accompanied by alterations in different aspects of the GABA system, giving impetus for caution in the interpretation of experiments involving genetic manipulation of complex phenotypes.

Dopaminergic control of light-adaptive synaptic plasticity and role in goldfish visual behavior.

Dopamine has been implicated in processes of retinal light and dark adaptation. In goldfish retina, horizontal cell dendrites elaborate neurite processes (spinules) into cone terminals, in a light- and dopamine-dependent manner. However, the functions of retinal dopamine and the horizontal cell spinules in visual behavior are unknown. These issues were addressed in behavioral, electroretinographic, and anatomical studies of normal fish and those with unilateral depletion of retinal dopamine induced by intraocular (i.o.) injections with 6-hydroxydopamine (6-OHDA). Dopamine interplexiform cells (DA-IPC) disappear within 2 weeks after 6-OHDA injection; cell bodies appear at the marginal zone within 6 weeks at which time neurites slowly reinnervate the retina with a sparse plexus over the next 12 months. We found that dopamine depletion increased light sensitivity at photopic but not scotopic backgrounds by 2.5 log units, an effect mimicked by i.o. injections of dopamine D1 and D2 antagonists. The ERG b-wave increment thresholds were the same for control and dopamine depleted eyes, indicating a normal transition from rod to cone systems in the ON pathway. Light-dependent spinule formation was reduced by about 60% in dopamine-depleted retinas, but returned to normal by 3 months and 9 months after injection in the entire retina, even areas not directly innervated with DA-IPC processes. Spinule formation in vivo was inhibited 50% with i.o. injection of SCH 23390 in control retinas as well as throughout 3 month 6-OHDA injected retinas, including DA-IPC free areas. This latter result indicates a volume effect of dopamine, diffusing laterally through the retina over several millimeters, in regulating spinules. We conclude that DA-IPCs regulate sensitivity to background at photopic levels not via the ON pathway, but perhaps the OFF pathway. Goldfish display both increased sensitivity to light and a normal Purkinje shift in the ERG b-wave whether or not horizontal cell spinules are present, indicating that dopamine control of photopic vision in fish is not mediated through light-induced spinule formation of horizontal cell dendrites.

Volume transmission of dopamine may modulate light-adaptive plasticity of horizontal cell dendrites in the recovery phase following dopamine depletion in goldfish retina.

We investigated the recovery of light-adaptive spinule formation following dopamine depletion with intraocular injection of 6-hydroxydopamine (6-OHDA) and subsequent neogeneration of dopamine interplexiform cells (DA-IPC) at the marginal zone. DA-IPCs were gone by 2 weeks postinjection and appeared at the marginal zone by 6 weeks postinjection, at which time DA-IPC neurites grew toward the central retina, reaching within 0.5 mm of the central retina by 1 year. Retinas from day time, light-adapted fish at 2 weeks, 4 weeks, 3 months, and 1 year postinjection with 6-OHDA were processed for pre-embedding tyrosine hydroxylase immunoreactivity (TOH-IR) and compared to sham-injected and control retinas at the electron-microscopical (EM) level. Only 6-OHDA fish that tilted markedly toward the injected eye were used for these experiments. The tilt mimics the dorsal light reaction, indicating a 2-2.5 log unit increase in the photopic sensitivity of the 6-OHDA eye. Spinule formation was reduced by about 60% in the 2- and 4-week 6-OHDA retinas, but returned to control levels throughout the entire retina of 3-month and 1 year 6-OHDA retinas even though the central region of these retinas contained no detectable TOH-IR. Intraocular injection with 10 microM SCH 23390 (a D1 antagonist) reduced light-adaptive spinule formation by 50% both in control eyes as well as those eyes that were 3 months post 6-OHDA injected. The full return of spinule formation with only partial reinnervation of the retina with DA-IPC processes and their subsequent inhibition by SCH 23390 indicates that dopamine diffused large distances within the retina to regulate this synaptic plasticity (i.e. displayed volume transmission). Also, since all 6-OHDA injected fish displayed an increased photopic sensitivity in the injected eye when sacrificed, we suggest that horizontal cell spinules are not required for photopic luminosity coding in the outer retina.

Light-dependent plasticity of the synaptic terminals of Mb bipolar cells in goldfish retina.

We recently described spine-like protrusions (spinules) from the synaptic terminals of mixed rod-cone (Mb) bipolar cells of goldfish retina that invaginated about 5% of the presynaptic amacrine cell processes (Yazulla and Studholme, J Comp Neurol 310:11, 1991). In view of reports of a light/dark dependent-plasticity on the formation of dendritic spinules on goldfish horizontal cells (Raynauld et al., Science 204:1436, 1979; Wagner, J Neurocytol 9:573, 1980), we investigated the possibility that Mb terminal spinules also might show light/dark-dependent plasticity. Retinas were obtained at noon time from light-adapted and 3-hour dark-adapted goldfish and processed for electron microscopy using conventional histological procedures and by preembedding immunocytochemistry to detect protein kinase C immunoreactivity. Two effects of light adaptation on Mb terminal morphology were observed. First, the surface of Mb terminals was significantly more irregular after dark adaptation than light adaptation. With dark adaptation, Mb terminals appeared "amoeboid," with numerous cytoplasmic extensions between the presynaptic processes. Second, spinules were sevenfold more frequent after dark adaptation than light adaptation (8% vs. 1.2% of the presynaptic processes were invaginated by spinules). We suggest that the increased frequency of spinules during dark adaptation is related to an enhancement of synaptic transmission from a minor amacrine cell input when the major input from GABAergic amacrine cells is reduced. Also, the irregular surface of dark-adapted Mb terminals may be related to the reduction of synaptic input during dark adaptation.

Glycine-receptor immunoreactivity in retinal bipolar cells is postsynaptic to glycinergic and GABAergic amacrine cell synapses.

Glycinergic innervation of the synaptic terminals of mixed rod-cone bipolar cells in the goldfish retina was investigated by electron microscopical immunocytochemistry with presynaptic and postsynaptic markers for glycinergic neurons: a monoclonal antibody (mAb 7A) against the 93 kDa subunit of the strychnine-sensitive glycine receptor and polyclonal antisera against a glycine/BSA conjugate. Conventional "glycinergic" synaptic contacts, made by amacrine cell processes, accounted for 7-10% of the input to the bipolar cell terminals, whether determined by glycine receptor immunoreactivity (GlyR-IR) or glycine-IR. In addition to the conventional synapses, the large bipolar cell terminals in the proximal inner plexiform layer (type Mb) gave rise to spinules (spine-like protrusions) that invaginated into presynaptic amacrine cell processes. Although 85% of the spinules were GlyR-IR, no spinules were postsynaptic to glycine-IR processes; yet 86% of the spinules were postsynaptic to GAD-IR processes, suggesting that the GlyR-IR spinules were postsynaptic to GABAergic terminals. Furthermore, a single amacrine cell process could make two synapses with an Mb terminal: a GlyR-IR contact onto a spinule and a conventional synapse that was not GlyR-IR. We suggest that glycinergic innervation of bipolar cell terminals involves conventional glycinergic synapses as well as an unconventional situation in which GABA and glycine may interact in as yet undetermined manner, perhaps by potentiation.