Uwe D. Behrens

The circadian component of spinule dynamics in teleost retinal horizontal cells is dependent on the dopaminergic system

During the light phase of a light/dark cycle, dendrites of teleost cone horizontal cells display numerous finger-like projections, called spinules, which are formed at dawn and degraded at dusk, and are thought to be involved in chromatic feedback processes. We have studied the oscillations of these spinules during a normal light/dark cycle and during 48 h of constant darkness in two groups of strongly rhythmic, diurnal fish, Aequidens pulcher. In one group the retinal dopaminergic system had been destroyed by the application of 6-OHDA, while in the other (control) group, the dopaminergic system was intact. In control fish, oscillations of spinule numbers were observed under both normal and constant dark conditions, indicating the presence of a robust circadian rhythm. However, spinule dynamics were severely affected by the absence of retinal dopamine. During the normal light phase, the number of spinules in 6-OHDA injected retinae was strongly reduced, and throughout continual darkness, spinule formation was almost completely suppressed. These results indicate that dopamine is essential for both light-evoked and circadian spinule formation; furthermore, we conclude that there is no circadian oscillator within horizontal cells controlling the formation of spinules.

Microanatomy of the dopaminergic system in the rainbow trout retina

We have investigated the morphology of dopaminergic interplexiform cells as well as the distribution of two classes of dopamine receptors in the retina of the rainbow trout. Interplexiform cells were visualized using an antiserum against tyrosine hydroxylase and PAP immunocytochemistry. In whole amounts, these cells have a density of between 91 and 182 cells per mm2 with highest values in the lower temporal quadrant. Their cell bodies lie at the inner margin of the inner nuclear layer with only 12-17 cells per retina displaced to the ganglion cell layer. There are three levels of stratification in the inner plexiform layer, one at the distal and proximal borders respectively, and one in the middle. They arise mostly from a radially oriented, stout primary dendrite. Tangential processes are about 1 micron in diameter and show a number of varicosities. The density of processes is greatest in sublayer 5, but no major difference in the general organization is apparent between the three sublayers. In the outer retina, there are two levels of dense ramification confined to the layer of horizontal cells. Light and electron microscopic analysis shows synaptic input to horizontal cells, but not to photoreceptors. The distribution of D1 receptors was assessed by studying the binding pattern of a specific, fluorescent-labelled antagonist, SCH 23390, in unfixed frozen sections. We found displaceable binding in the inner and outer plexiform layers and in the region of horizontal cell perikarya. We used an anti-peptide antibody directed to an extracellular domain of the rat D2 receptor and a fluorescent secondary antiserum to study the localization of D2 receptors. In addition to marked label in both plexiform layers, the outer, and especially the inner segments of rods and cones show specific immunoreactivity. In addition, there is distinct label at the level of the horizontal cell bodies; in the inner retina, specific fluorescence is found in somata of some amacrine cells. The significance of the connectivity pattern and the distribution of the two receptor types is discussed with respect to the role of dopamine in controlling adaptational processes in the outer retina, such as retinomotor movements and changes in horizontal cell morphology and physiology.

ADAPTATION-DEPENDENT PLASTICITY OF ROD BIPOLAR CELL AXON TERMINAL MORPHOLOGY IN THE RAT RETINA

Abstract We chose synaptic terminals of rat rod bipolar cells as a model system to study activity-related changes in the overall morphology and the fine structure of synaptic sites. Using confocal laser scanning microscopy in conjunction with three-dimensional reconstruction and electron microscopy, we examined the effect of light and dark adaptation on axon terminals identified by protein kinase C (PKC) immunoreactivity. Rod bipolar cell axon terminals consisted of 2–3 polymorphic boutons situated close to the ganglion cell layer and a single ovoid swelling located more distally. Both components of the terminal complex showed adaptation-dependent differences in the distribution of PKC immunoreactivity and in their morphology. In light-adapted rod bipolar cell axon terminals, PKC immunoreactivity was homogeneously distributed throughout the cytoplasm, whereas terminals from dark-adapted animals showed PKC immunoreactivity preferentially localised in the submembrane compartment and a reduced staining of the more central cytoplasm. In three-dimensional reconstructions of optical sections and at the ultrastructural level, the shape of light-adapted axon terminals was round and smooth and exhibited more convexly curved synaptic membranes. In contrast, dark-adapted terminals had irregular contours, numerous dimples and a concave synaptic curvature. No spinules of bipolar cell terminals were observed in dark-adapted material. These observations are discussed in the context of activity-related morphological plasticity of central nervous system synapses and of the functions of PKC in the cycle of vesicle fusion and retrieval at the tonically active ribbon synapses of the rod bipolar axon terminal.

Gonadotropin-releasing hormone, a neuropeptide of efferent projections to the teleost retina induces light-adaptive spinule formation on horizontal cell dendrites in dark-adapted preparations kept in vitro

The teleost retina receives efferent projections from neurons of the nucleus olfactoretinalis at the base of the olfactory bulbs. These fibres contain gonadotropin-releasing hormone (GnRH) immunoreactive material and are presynaptic to retinal dopaminergic interplexiform cells. We have incubated isolated dark-adapted retinae and eyecup preparations of roach with salmon-GnRH and found an increase in horizontal cell spinule numbers to 70% light-adaptive levels. This effect was blocked by addition of haloperidol to the incubation medium suggesting that GnRH acts via stimulation of the dopaminergic interplexiform cells. We conclude that GnRH containing efferent fibres are capable of inducing light-adaptive changes in the retina and discuss their implication in the control of endogenous rhythms.

Effect of melatonin agonists and antagonists on horizontal cell spinule formation and dopamine release in a fish retina

Abstract. The crucian carp retina was used to study the effects of the melatonin antagonist DH97 (N-pentanoyl 2-benzyltryptamine) and the melatonin agonists [+]- and [–]-AMMTC (N-acetyl-4-aminomethyl-6-methoxy-9-methyl-1,2,3,4-tetrahydrocarbazole) on horizontal cell spinule formation, an indicator of the state of retinal adaptation. DH97 was capable of both counteracting dark-adaptive spinule degradation and inducing light-adaptive spinule formation at the beginning of the dark phase. Addition of dopamine receptor blockers opposed the action of DH97 on spinules, with SCH 23930, a D1 dopamine receptor antagonist, being more effective than the D2 receptor antagonist sulpiride. DH97 induced a twofold increase in dopamine release. We conclude that melatonin acts as a dark signal within the teleost retina by inhibiting the dopaminergic system. In accordance with this, both enantiomers of AMMTC prevented light-induced spinule formation, and reduced dopamine release to below dark-adaptive baseline levels. We suggest that the suppression of spinule formation by AMMTC may be due to either a direct inhibitory interaction between the melatonin agonist and horizontal cell dopamine D1 receptors, or an inhibitory effect on the activity of the dopamine-releasing interplexiform cells.

Adaptation-dependent changes of bipolar cell terminals in fish retina: Effects on overall morphology and spinule formation in Ma and Mb cells

We have investigated the effects of light and dark adaptation on the overall morphology of bipolar cell (BC) terminals in sublaminae a and b of the inner plexiform layer after labelling with Lucifer Yellow (LY) and PKC immunostaining using confocal laser scanning microscopy and serially sectioned material for electron microscopy. Three-dimensional reconstructed terminals showed marked adaptation-dependent changes of their morphology. Terminals of mixed rod-cone BCs in sublamina a (Ma BC) were irregular and scalloped in light adapted, but smooth and regular in dark-adapted specimens. Terminals from mixed rod-cone BCs in sublamina b (Mb BCs) exhibited an opposite behaviour. At the ultrastructural level, bipolar terminals in both sublaminae showed fingerlike extensions (spinules) invaginating presynaptic amacrine cell (AC) processes. Sixty-two percent of the dark-adapted Mb terminals in sublamina b showed spinules, whereas 21% of the light-adapted terminals had spinules. By contrast, 50.6% of the light-adapted Ma terminals in sublamina a formed spinules, compared to 17.8% of the dark-adapted Ma terminals in this sublamina. These observations reflect the functional subdivision of the inner plexiform layer in an inner ON-and an outer OFF-centre lamina. Our findings suggest that the synaptic plasticity of BC axon terminals may be due to differences of BC membrane potential, or the activity of AC input onto bipolar terminals. They may contribute to processes of fine tuning regulating the efficiency of AC-BC interaction under varying adaptation conditions.

cAMP-mediated second messenger mechanisms are involved in spinule formation in teleost cone horizontal cells

A number of light adaptive changes of teleost horizontal cells are mediated by dopamine D1 receptors coupled positively with the cAMP second messenger system. Spinules, finger-like extensions from horizontal cell dendrites directed towards the cone pedicle cytoplasm, are formed in response to a stimulation of D1 receptors. We studied the second messenger mechanism associated with this process using isolated dark-adapted cyprinid retinae. Increasing intracellular cAMP concentrations by adding a membrane permeable analogue, or by stimulating the adenylate cyclase and simultaneously blocking the degradation of cAMP, resulted in a significant increase of spinule numbers in spite of the absence of light. In contradistinction to using isolated retinae for pharmacological experiments, injection of drugs into the vitreous had inconsistent or negative results.

Biocytin: intracellular staining, dye-coupling and immunocytochemistry in carp retina.

CORRELATION of electrophysiological and morphological, including ultrastructural, characteristics of neurones is important for understanding the functional organization of neuronal systems. Further correlation with neuro-transmitter content is essential for determining the ncurochemical(s) used by a given neurone for propagating its signal. The two main neuronal markers presently available (lucifer yellow and horseradish peroxidase) are not satisfactory for correlating all three aspects. We have devised a new simple procedure whereby retinal interneurones can be labelled with biocytin by positive ionophoresis of an unbuffered solution. Biocytin readily crosses gap junctions thus revealing extensive networks of coupled cells. In the case of H1 horizontal cells, which are known to be GABAergic, the neurotransmitter can also be demonstrated by superimposed immunocytochemistry.

Quantitative anatomy, synaptic connectivity and physiology of amacrine cells with glucagon-like immunoreactivity in the turtle retina

SummaryAlthough a wide variety of neuropeptides have been localized in vertebrate retinas, many questions remain about the function of these peptides and the amacrine cells that contain them. This is because many of these peptidergic amacrine cells have been studied using only immunocytochemical techniques. To address this limitation, the present study used a combination of quantitative anatomy, biochemistry and electrophysiology to examine amacrine cells in the turtle retina that contain the neuropeptide glucagon. In the turtle retina, there is a small population of 2500 glucagonergic amacrine cells, which probably represents <1% of the total number of amacrine cells. Circular distribution statistics indicated that many of these tristratified amacrine cells had asymmetric dendritic arborizations that were radially oriented toward the retinal periphery. The cells were found to have similar dendritic coverage factors, to be distributed in a non-random arrangement in all regions of the retina, and to peak in density in the visual streak region. Electron microscopic studies indicated that glucagonergic amacrine cells made synaptic contacts primarily with other amacrine cells, and small numbers of bipolar cells. The synaptic inputs and outputs were balanced in the inner strata of the inner plexiform layer, and were biased toward synaptic outputs in the outer strate of the inner plexiform layer. These contacts involved small unlabelled synaptic vesicles, and not the large labelled dense core vesicles also found in these neurons. The biochemical studies indicated that glucagon could be released from the retina in a calcium dependent manner by high potassium stimulation. The electrophysiology found no color opponency, and the glucagonergic amacrine cells gave sustained hyperpolarizing responses to small stimulation spots and had antagonistic surrounds. The results of these studies suggest that there are significant regional specializations of glucagonergic amacrine cells, and that they may provide OFF-modulation in interactions between the ON- and OFF centre visual pathways in the turtle retina.

Distribution of phosphorylated protein kinase C alpha in goldfish retinal bipolar synaptic terminals: control by state of adaptation and pharmacological treatment

Protein kinase C (PKC) is a signalling enzyme critically involved in many aspects of synaptic plasticity. In cyprinid retinae, the PKC alpha isoform is localized in a subpopulation of depolarizing bipolar cells that show adaptation-related morphological changes of their axon terminals. We have studied the subcellular localization of phosphorylated PKC alpha (pPKC alpha) in retinae under various conditions by immunohistochemistry with a phosphospecific antibody. In dark-adapted retinae, pPKC alpha immunoreactivity is weak in the cytoplasm of synaptic terminals, labelling being predominantly associated with the membrane compartment. In light-adapted cells, immunoreactivity is diffusely distributed throughout the terminal. Western blot analysis has revealed a reduction of pPKC alpha immunoreactivity in cytosolic fractions of homogenized dark-adapted retinae compared with light-adapted retinae. Pharmacological experiments with the isoform-specific PKC blocker Goe6976 have shown that inhibition of the enzyme influences immunolabelling for pPKC alpha, mimicking the effects of light on the subcellular distribution of immunoreactivity. Our findings suggest that the state of adaptation modifies the subcellular localization of a signalling molecule (PKC alpha) at the ribbon-type synaptic complex. We propose that changes in the subcellular distribution of PKC alpha immunoreactivity might be one component regulating the strength of the signal transfer of the bipolar cell terminal.