Elio Raviola, M.D.
Bullard Professor of Neurobiology, Emeritus
Theories of brain function as well as interpretations of electrophysiological experiments are suffering from incomplete knowledge of the microcircuitry of the nervous system. In this respect, the retina is a privileged organ, because its physical location, the distinctive morphology of its neurons, the regularity of its architecture, and the properties of its inputs and outputs favor a unique variety of experimental approaches that are not possible elsewhere in the brain. In addition, the retina is one of the few sites in which the shape of neurons is the visible signature of its connections, because each neuronal type has a unique morphology, dictated by the rigorous stratification of their synaptic connections and the need for ordered sampling of the visual world. During the past few years, as a result of the efforts of a number of laboratories including ours, a complete inventory of the anatomical types of horizontal, bipolar, amacrine and ganglion cells has become available, at least for the retina of a few species of mammals. The problem now is to learn the contribution of each type of neuron to the internal economy of the retina or its coding of the visual world. In tackling such a task, first and foremost, one has to identify in the living state the cell type that is the object of physiological or molecular studies. To solve the problem of cell type identification, we pioneered the use of genetic methods to express a visible marker in specific neuronal populations of the mouse retina. As an object of our experiments, we chose retinal dopaminergic neurons (DA cells), which fulfill a fundamental role in vision because they release dopamine, a catecholamine modulator responsible for many of the events that lead to neural adaptation to light. We targeted PLAP to this cell type by linking its cDNA to a regulatory sequence of the gene that codes for tyrosine hydroxylase, the rate-limiting enzyme for dopamine biosynthesis.
In these transgenic animals, we studied the synaptic connections of DA cells and investigated their ligand- and voltage-gated channels with the patch clamp technique. We observed that DA cells possess a pacemaker activity and thus fire in vitro action potentials in a slow, rhythmic pattern, a property also found in other catecholaminergic neurons of the CNS. We studied the constellation of voltage-gated channels responsible for this behavior, as well as the effects of GABA, glycine and glutamate/kainate on the firing pattern. To analyze the composition of the GABAA receptors of DA cells, we developed an RT-PCR assay to identify the transcripts of the various GABAA receptor subunits at a single cell level. We showed that the cells express multiple subunits of the GABAA receptor, assembled into multiple receptors, and we analyzed by patch clamp their pharmacological properties. Next, we studied dopamine release in solitary DA cells by amperometry with carbon fiber electrodes and showed that, as a result of the spontaneous generation of action potentials, quanta of dopamine are released by exocytosis over the surface of the cell body. Since the perikaryon of DA cells does not contain presynaptic active zones, this release is by necessity extrasynaptic and represents one of the sources of the dopamine that acts by volume transmission on distant targets in the retina. In addition to paracrine secretion of dopamine, we have recently shown that DA cells also release GABA extrasynaptically. It is known that DA cells establish synapses onto AII amacrine cells, a neuron that transfers rod signals to cone bipolar endings. By using triple-label immunocytochemistry and confocal microscopy, we showed that these contacts are probably GABAergic, because GABAA receptors are clustered at the postsynaptic active zone. Another accomplishment of our laboratory was the development of a technique to study global gene expression in single DA cells. Solitary DA cells were patch clamped and individually harvested. Their mRNA was amplified by a novel method, SMARTT7, based on the combination of the PCR-based SMART technique with the T7 RNA polymerase. The single cell probes were then used to screen the original RIKEN cDNA microarrays. We found that DA cells secrete insulin, the neuropeptide CART, the cytokine interferon a and the chemokine monocyte chemoattractant protein-1. They contain the COP9/Signalosome, a 500 kDa nuclear protein complex that acts as a transcriptional repressor in the cascade of events regulated by light during seedling development: it was the first time that the presence of this macromolecular complex was described in the nervous system and its functional significance remains to be elucidated. Finally, DA cells contain the most common circadian clock-related proteins, supporting the idea that DA cells have a role in the retinal internal clock.
Thus, DA cells appear to carry out four main functions in the retina, each characterized by a different time course: 1) through their fast GABAergic synapses on AII amacrine cells, DA cells control the transfer of rod signals to ganglion cells on a time scale of the order of the millisecond. 2) They release dopamine over their entire surface. This modulator acts at a distance by volume transmission on a large number of retinal neurons, presiding over the process of transition from the dark-adapted to the light-adapted state over a time scale of seconds to minutes. 3) They contain the most common circadian clock-related proteins suggesting a role in the circadian regulation of retinal function over a time scale of hours.
Corelease of dopamine and GABA by a retinal dopaminergic neuron. September 19, 2012. The Journal of neuroscience : the official journal of the Society for Neuroscience.
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Synaptic input of ON-bipolar cells onto the dopaminergic neurons of the mouse retina. June 1, 2010. The Journal of comparative neurology.
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Unexpected expression of alpha- and beta-globin in mesencephalic dopaminergic neurons and glial cells. August 26, 2009. Proceedings of the National Academy of Sciences of the United States of America.
Extrasynaptic release of GABA by retinal dopaminergic neurons. April 29, 2009. Journal of neurophysiology.
Intrinsic circadian clock of the mammalian retina: importance for retinal processing of visual information. August 24, 2007. Cell.
Expression of circadian clock genes in retinal dopaminergic cells. August 17, 2007. Visual neuroscience.
Roles of subthreshold calcium current and sodium current in spontaneous firing of mouse midbrain dopamine neurons. January 17, 2007. The Journal of neuroscience : the official journal of the Society for Neuroscience.
Physiological importance of a circadian clock outside the suprachiasmatic nucleus. January 1, 2007. Cold Spring Harbor symposia on quantitative biology.
Form deprivation modulates retinal neurogenesis in primate experimental myopia. March 13, 2006. Proceedings of the National Academy of Sciences of the United States of America.
Spontaneous activity of isolated dopaminergic periglomerular cells of the main olfactory bulb. July 20, 2005. Journal of neurophysiology.
Synaptogenesis and outer segment formation are perturbed in the neural retina of Crx mutant mice. January 27, 2005. BMC neuroscience.
The population of bipolar cells in the rabbit retina. April 19, 2004. The Journal of comparative neurology.
Gene discovery in genetically labeled single dopaminergic neurons of the retina. March 26, 2004. Proceedings of the National Academy of Sciences of the United States of America.
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GABAergic synapses made by a retinal dopaminergic neuron. January 23, 2003. Proceedings of the National Academy of Sciences of the United States of America.
A molecular approach to retinal neural networks. July 1, 2002. Functional neurology.
Extrasynaptic release of dopamine in a retinal neuron: activity dependence and transmitter modulation. April 1, 2001. Neuron.
Pharmacology of GABA(A) receptors of retinal dopaminergic neurons. October 1, 2000. Journal of neurophysiology.
Real-time amperometric measurements of zeptomole quantities of dopamine released from neurons. February 1, 2000. Analytical chemistry.
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The shapes and numbers of amacrine cells: matching of photofilled with Golgi-stained cells in the rabbit retina and comparison with other mammalian species. October 18, 1999. The Journal of comparative neurology.
Composition of the GABA(A) receptors of retinal dopaminergic neurons. September 15, 1999. The Journal of neuroscience : the official journal of the Society for Neuroscience.
Spontaneous activity of solitary dopaminergic cells of the retina. September 1, 1998. The Journal of neuroscience : the official journal of the Society for Neuroscience.
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Light responses from one type of ON-OFF amacrine cells in the rabbit retina. December 1, 1995. Journal of neurophysiology.
Cone bipolar cells as interneurons in the rod pathway of the rabbit retina. September 1, 1994. The Journal of comparative neurology.
Synaptic connections of the narrow-field, bistratified rod amacrine cell (AII) in the rabbit retina. November 8, 1992. The Journal of comparative neurology.
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Axonless horizontal cells of the rabbit retina: synaptic connections and origin of the rod aftereffect. October 1, 1990. Journal of neurocytology.
Synaptic connections of rod bipolar cells in the inner plexiform layer of the rabbit retina. May 15, 1990. The Journal of comparative neurology.
Physiology of HI horizontal cells in the primate retina. March 22, 1990. Proceedings of the Royal Society of London. Series B, Containing papers of a Biological character. Royal Society (Great Britain).
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Excitatory dyad synapse in rabbit retina. October 1, 1987. Proceedings of the National Academy of Sciences of the United States of America.
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The rod pathway in the rabbit retina: a depolarizing bipolar and amacrine cell. February 1, 1986. The Journal of neuroscience : the official journal of the Society for Neuroscience.
Structure of the sinus-lining cells in the popliteal lymph node of the rabbit. August 1, 1985. The Anatomical record.
An animal model of myopia. June 20, 1985. The New England journal of medicine.
Rod cells dissociated from mature salamander retina: ultrastructure and uptake of horseradish peroxidase. January 1, 1985. The Journal of cell biology.
Variations in structure and response properties of horizontal cells in the retina of the rabbit. January 1, 1983. Vision research.
Horizontal cells in the retina of the rabbit. October 1, 1982. The Journal of neuroscience : the official journal of the Society for Neuroscience.
Structure of the synaptic membranes in the inner plexiform layer of the retina: a freeze-fracture study in monkeys and rabbits. August 10, 1982. The Journal of comparative neurology.
Membrane specializations in the outer plexiform layer of the turtle retina. January 20, 1982. The Journal of comparative neurology.
Paracellular route of aqueous outflow in the trabecular meshwork and canal of Schlemm. A freeze-fracture study of the endothelial junctions in the sclerocorneal angel of the macaque monkey eye. July 1, 1981. Investigative ophthalmology & visual science.
Structure of rapidly frozen gap junctions. October 1, 1980. The Journal of cell biology.
Increase in axial length of the macaque monkey eye after corneal opacification. December 1, 1979. Investigative ophthalmology & visual science.
Membrane recycling in the cone cell endings of the turtle retina. December 1, 1978. The Journal of cell biology.
Intercellular junctions in the ciliary epithelium. October 1, 1978. Investigative ophthalmology & visual science.
Effect of dark-rearing on experimental myopia in monkeys. June 1, 1978. Investigative ophthalmology & visual science.
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Ultrastructure of perfusion-fixed fetal capillaries in the human placenta. September 14, 1976. Cell and tissue research.
Ultrastructural analysis of functional changes in the synaptic endings of turtle cone cells. January 1, 1976. Cold Spring Harbor symposia on quantitative biology.
Intramembrane organization of specialized contacts in the outer plexiform layer of the retina. A freeze-fracture study in monkeys and rabbits. April 1, 1975. The Journal of cell biology.
Differences in membrane structure between excitatory and inhibitory components of the reciprocal synapse in the olfactory bulb. May 1, 1974. The Journal of comparative neurology.
Gap junctions between photoreceptor cells in the vertebrate retina. June 1, 1973. Proceedings of the National Academy of Sciences of the United States of America.
Evidence for a blood-thymus barrier using electron-opaque tracers. September 1, 1972. The Journal of experimental medicine.
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Harvard Medical School
Dept of Neurobiology, B2-242
220 Longwood Ave
Boston MA 02115