Corelease of dopamine and GABA by a retinal dopaminergic neuron.

Numerous neurons release two transmitters of low molecular mass, but it is controversial whether they are localized within the same synaptic vesicle, with the single exception of GABA and glycine because they are ferried into the vesicle by the same transporter. Retinal dopaminergic (DAergic) amacrine cells synthesize both dopamine (DA) and GABA. Both transmitters are released over the entire cell surface and act on neighboring and distant neurons by volume transmission, but, in addition, DAergic cells establish GABAergic synapses onto AII amacrine cells, the neurons that transfer rod signals to cone bipolars. By combining recordings of DA and GABA release from isolated, genetically identified perikarya of DAergic cells from the mouse retina, we observed that a proportion of the events of DA and GABA exocytosis were simultaneous, suggesting corelease. Furthermore, a proportion of the secretory organelles in the perikaryon and synaptic endings of DAergic cells contained both vesicular transporters for DA [vesicular monoamine transporter 2 (VMAT2)] and GABA [vesicular GABA transporter (VGAT)]. Because the majority of the DA release events concerned a single transmitter and organelles were present that contained a single transporter, either VMAT2 or VGAT, we conclude that the secretory organelles of DAergic cells contain variable concentrations of the two transmitters, which are in turn determined by a variable mixture of the two transporter molecules in their limiting membrane. This variability can be explained if the relative numbers of transporter molecules is determined stochastically during the budding of the somatic organelles from the trans-Golgi network or the retrieval of the vesicular membrane from the plasmalemma after exocytosis.

Unexpected expression of alpha- and beta-globin in mesencephalic dopaminergic neurons and glial cells.

The mesencephalic dopaminergic (mDA) cell system is composed of two major groups of projecting cells in the substantia nigra (SN) (A9 neurons) and the ventral tegmental area (VTA) (A10 cells). A9 neurons form the nigrostriatal pathway and are involved in regulating voluntary movements and postural reflexes. Their selective degeneration leads to Parkinson's disease. Here, we report that gene expression analysis of A9 dopaminergic neurons (DA) identifies transcripts for alpha- and beta-chains of hemoglobin (Hb). Globin immunoreactivity decorates the majority of A9 DA, a subpopulation of cortical and hippocampal astrocytes and mature oligodendrocytes. This pattern of expression was confirmed in different mouse strains and in rat and human. We show that Hb is expressed in the SN of human postmortem brain. By microarray analysis of dopaminergic cell lines overexpressing alpha- and beta-globin chains, changes in genes involved in O(2) homeostasis and oxidative phopshorylation were observed, linking Hb expression to mitochondrial function. Our data suggest that the most famed oxygen-carrying globin is not exclusively restricted to the blood, but it may play a role in the normal physiology of the brain and neurodegenerative diseases.

In vivo physiological transdifferentiation of adult adipose cells.

Grafts of adipose tissue from adult Rosa26 mice from different sites of the body, irrespective of the sex of the donor, share with the mammary fat the property of giving rise to milk-secreting epithelial cells when exposed to the microenvironment of the mammary gland in pregnant and lactating females. To rule out the possibility that the labeled mammary glandular tissue was derived from stem cells associated with the stroma vascular part of the grafts, we injected into the mammary gland a pure suspension of adipocytes obtained by treating a fragment of adipose tissue with collagenase. X-gal-positive cells were inserted into the alveoli of the native gland, and electron microscopy showed that the labeled cells had transformed into milk-secreting glandular cells. At the site of the adipocyte injection, the labeled alveoli contained a mixture of X-gal-positive and X-gal-negative cells, and a single epithelial cell was occasionally stained in an otherwise unlabeled alveolus. This suggests that growing ducts individually recruit adjacent adipocytes that transdifferentiate into secretory epithelial cells as they became part of the glandular alveoli. After dissociation, the isolated adipocytes retained the morphology and protein markers typical of differentiated fat cells but expressed high levels of stem cell genes and the reprogramming transcription factor Klf4. Thus, the well-documented osteogenic, chondrogenic, myogenic, and angiogenic transformation of preadipocytes associated with the stroma vascular component of the adipose tissue may reflect an intrinsic capability of adipocytes to reprogram their gene expression and transform into different cytotypes.

Light-dependent photoreceptor orientation in mouse retina.

Almost a century ago, Stiles and Crawford reported that the human eye is more sensitive to light entering through the pupil center than through its periphery (Stiles-Crawford effect). This psychophysical phenomenon, later found to correlate with photoreceptor orientation toward the pupil, was dynamically phototropic, adjustable within days to an eccentrically displaced pupil. For decades, this phototropism has been speculated to involve coordinated movements of the rectilinear photoreceptor outer and inner segments. We report here that, unexpectedly, the murine photoreceptor outer segment has a seemingly light-independent orientation, but the inner segment's orientation undergoes light-dependent movement, giving rise to nonrectilinear outer and inner segments in adult mice born and reared in darkness. Light during an early critical period (~P0 to P8), however, largely sets the correct photoreceptor orientation permanently afterward. Unexpectedly, abolishing rod and cone phototransductions did not mimic darkness in early life, suggesting photosignaling extrinsic to rods and cones is involved.

Roles of subthreshold calcium current and sodium current in spontaneous firing of mouse midbrain dopamine neurons.

We used a preparation of acutely dissociated neurons to quantify the ionic currents driving the spontaneous firing of substantia nigra pars compacta neurons, isolated from transgenic mice in which the tyrosine hydroxylase promoter drives expression of human placental alkaline phosphatase (PLAP) on the outer surface of the cell membrane. Dissociated neurons identified by fluorescent antibodies to PLAP showed firing properties similar to those of dopaminergic neurons in brain slice, including rhythmic spontaneous firing of broad action potentials and, in some cells, rhythmic oscillatory activity in the presence of tetrodotoxin (TTX). Spontaneous activity in TTX had broader, smaller spikes than normal pacemaking and was stopped by removal of external calcium. Normal pacemaking was also consistently silenced by replacement of external calcium by cobalt and was slowed by more specific calcium channel blockers. Nimodipine produced a slowing of pacemaking frequency. Pacemaking was also slowed by the P/Q-channel blocker omega-Aga-IVA, but the N-type channel blocker omega-conotoxin GVIA had no effect. In voltage-clamp experiments, using records of pacemaking as command voltage, cobalt-sensitive current and TTX-sensitive current were both sizeable at subthreshold voltages between spikes. Cobalt-sensitive current was consistently larger than TTX-sensitive current at interspike voltages from -70 to -50 mV, with TTX-sensitive current larger at voltages positive to -45 mV. These results support previous evidence for a major role of voltage-dependent calcium channels in driving pacemaking of midbrain dopamine neurons and suggest that multiple calcium channel types contribute to this function. The results also show a significant contribution of subthreshold TTX-sensitive sodium current.

Synaptogenesis and outer segment formation are perturbed in the neural retina of Crx mutant mice.

BACKGROUND: In Leber's congenital amaurosis (LCA), affected individuals are blind, or nearly so, from birth. This early onset suggests abnormal development of the neural retina. Mutations in genes that affect the development and/or function of photoreceptor cells have been found to be responsible in some families. These examples include mutations in the photoreceptor transcription factor, Crx. RESULTS: A Crx mutant strain of mice was created to serve as a model for LCA and to provide more insight into Crx's function. In this study, an ultrastructural analysis of the developing retina in Crx mutant mice was performed. Outer segment morphogenesis was found to be blocked at the elongation stage, leading to a failure in production of the phototransduction apparatus. Further, Crx-/- photoreceptors demonstrated severely abnormal synaptic endings in the outer plexiform layer. CONCLUSIONS: This is the first report of a synaptogenesis defect in an animal model for LCA. These data confirm the essential role this gene plays in multiple aspects of photoreceptor development and extend our understanding of the basic pathology of LCA.

Extrasynaptic release of GABA and dopamine by retinal dopaminergic neurons.

In the mouse retina, dopaminergic amacrine (DA) cells synthesize both dopamine and GABA. Both transmitters are released extrasynaptically and act on neighbouring and distant retinal neurons by volume transmission. In simultaneous recordings of dopamine and GABA release from isolated perikarya of DA cells, a proportion of the events of dopamine and GABA exocytosis were simultaneous, suggesting co-release. In addition, DA cells establish GABAergic synapses onto AII amacrine cells, the neurons that transfer rod bipolar signals to cone bipolars. GABAA but not dopamine receptors are clustered in the postsynaptic membrane. Therefore, dopamine, irrespective of its site of release-synaptic or extrasynaptic-exclusively acts by volume transmission. Dopamine is released upon illumination and sets the gain of retinal neurons for vision in bright light. The GABA released at DA cells' synapses probably prevents signals from the saturated rods from entering the cone pathway when the dark-adapted retina is exposed to bright illumination. The GABA released extrasynaptically by DA and other amacrine cells may set a 'GABAergic tone' in the inner plexiform layer and thus counteract the effects of a spillover of glutamate released at the bipolar cell synapses of adjacent OFF and ON strata, thus preserving segregation of signals between ON and OFF pathways.

MEF2D drives photoreceptor development through a genome-wide competition for tissue-specific enhancers.

Organismal development requires the precise coordination of genetic programs to regulate cell fate and function. MEF2 transcription factors (TFs) play essential roles in this process but how these broadly expressed factors contribute to the generation of specific cell types during development is poorly understood. Here we show that despite being expressed in virtually all mammalian tissues, in the retina MEF2D binds to retina-specific enhancers and controls photoreceptor cell development. MEF2D achieves specificity by cooperating with a retina-specific factor CRX, which recruits MEF2D away from canonical MEF2 binding sites and redirects it to retina-specific enhancers that lack the consensus MEF2-binding sequence. Once bound to retina-specific enhancers, MEF2D and CRX co-activate the expression of photoreceptor-specific genes that are critical for retinal function. These findings demonstrate that broadly expressed TFs acquire specific functions through competitive recruitment to enhancers by tissue-specific TFs and through selective activation of these enhancers to regulate tissue-specific genes.

The population of bipolar cells in the rabbit retina.

The population of bipolar cells in the rabbit retina was studied using Golgi impregnation and photocatalyzed filling of single cells with dihydrorhodamine, a quantitative sampling technique. The Golgi method revealed the morphology and stratification of cells in detail. The photofilling method allowed us to estimate the frequency of the cell types. From a sample of 243 Golgi-impregnated bipolar cells and 107 photofilled cells, we identified 1 type of rod bipolar cell and 12 types of cone bipolar cells. An analysis based on retinal coverage indicates that this number of types could be contained within the number of bipolar cells known to exist. The dendrites of most cone bipolars contacted all the cones within the individual cone bipolar cell's dendritic field. Types of bipolar cell were encountered at roughly similar frequency, without any one type predominating. The rabbit retina thus contains about a dozen parallel and roughly equipotent through-pathways.

A molecular approach to retinal neural networks.

Our understanding of the computations carried out by neural networks in the central nervous system is limited by our incomplete knowledge of the diversity of cell types and the multiplicity of their functions. In the retina, over fifty cell types encode the spatial, temporal, and chromatic parameters of the incoming light stimuli to generate the messages of action potentials that travel to the brain along the fibers of the optic nerve. We have combined molecular techniques with microscopy and electrophysiology to study a rare cell type in the retina, the dopaminergic amacrine (interplexiform cells). These neurons were labeled in transgenic mice with human placental alkaline phosphatase so that they could be identified in vitro for patch-clamping and analysis of gene expression by single-cell RT-PCR and cDNA array profiling. We have thus demonstrated that the retinal dopaminergic neurons spontaneously generate action potentials in a rhythmic fashion, release dopamine over their entire surface, establish GABAergic synapses and contain proteins whose existence went undetected with more conventional techniques.

The diffuse nervous network of Camillo Golgi: facts and fiction.

The name of Camillo Golgi is inextricably associated, in the mind of most neuroscientists, with the theory that nerve cells communicate with one another by means of an intricate network of anastomosing axonal branches contained in the neuropil intervening between cell bodies in the gray matter of the brain and spinal cord. Examination, however, of Golgi's drawings in the papers published in the decade intervening between publication of his method (1873) and the beginning of his studies on malaria (1885) shows that axonal arborization in the cerebellar cortex and olfactory bulb are depicted as independent of one other. This is in striking contrast with the drawings included by Golgi in his 1906 Nobel lecture where the entire granular layer of the cerebellar cortex is occupied by a network of branching and anastomosing nerve processes. Thus, Golgi in his original papers on the cerebellum represents nerve cells as discrete units and only later in life merges axonal arborizations in the context of a lecture in defense of the reticular theory.

Synaptic input of ON-bipolar cells onto the dopaminergic neurons of the mouse retina.

In the retina, dopamine fulfills a crucial role in neural adaptation to photopic illumination, but the pathway that carries cone signals to the dopaminergic amacrine (DA) cells was controversial. We identified the site of ON-cone bipolar input onto DA cells in transgenic mice in which both types of catecholaminergic amacrine (CA) cells were labeled with green fluorescent protein or human placental alkaline phosphatase (PLAP). In confocal Z series of retinal whole mounts stained with antibodies to tyrosine hydroxylase (TH), DA cells gave rise to varicose processes that descended obliquely through the scleral half of the inner plexiform layer (IPL) and formed a loose, tangential plexus in the middle of this layer. Comparison with the distribution of the dendrites of type 2 CA cells and examination of neurobiotin-injected DA cells proved that their vitreal processes were situated in stratum S3 of the IPL. Electron microscope demonstration of PLAP activity showed that bipolar cell endings in S3 established ribbon synapses onto a postsynaptic dyad in which one or both processes were labeled by a precipitate of lead phosphate and therefore belonged to DA cells. In places, the postsynaptic DA cell processes returned a reciprocal synapse onto the bipolar endings. Confocal images of sections stained with antibodies to TH, kinesin Kif3a, which labels synaptic ribbons, and glutamate or GABA(A) receptors, confirmed that ribbon-containing endings made glutamatergic synapses onto DA cells processes in S3 and received from them GABAergic synapses. The presynaptic ON-bipolar cells most likely belonged to the CB3 (type 5) variety.

Extrasynaptic release of GABA by retinal dopaminergic neurons.

GABA release by dopaminergic amacrine (DA) cells of the mouse retina was detected by measuring Cl- currents generated by isolated perikarya in response to their own neurotransmitter. The possibility that the Cl- currents were caused by GABA release from synaptic endings that had survived the dissociation of the retina was ruled out by examining confocal Z series of the surface of dissociated tyrosine hydroxylase-positive perikarya after staining with antibodies to pre and postsynaptic markers. GABA release was caused by exocytosis because 1) the current events were transient on the millisecond time scale and thus resembled miniature synaptic currents; 2) they were abolished by treatment with a blocker of the vesicular proton pump, bafilomycin A1; and 3) their frequency was controlled by the intracellular Ca2+ concentration. Because DA cell perikarya do not contain presynaptic active zones, release was by necessity extrasynaptic. A range of depolarizing stimuli caused GABA exocytosis, showing that extrasynaptic release of GABA is controlled by DA cell electrical activity. With all modalities of stimulation, including long-lasting square pulses, segments of pacemaker activity delivered by the action-potential-clamp method and high-frequency trains of ramps, discharge of GABAergic currents exhibited considerable variability in latency and duration, suggesting that coupling between Ca2+ influx and transmitter exocytosis is extremely loose in comparison with the synapse. Paracrine signaling based on extrasynaptic release of GABA by DA cells and other GABAergic amacrines may participate in controlling the excitability of the neuronal processes that interact synaptically in the inner plexiform layer.

Expression of circadian clock genes in retinal dopaminergic cells.

The mammalian neural retina contains single or multiple intrinsic circadian oscillators that can be directly entrained by light cycles. Dopaminergic amacrine (DA) cells represent an especially interesting candidate as a site of the retinal oscillator because of the crucial role of dopamine in light adaptation, and the widespread distribution of dopamine receptors in the retina. We hereby show by single-cell, end-point RT-PCR that retinal DA cells contain the transcripts for six core components of the circadian clock: Bmal1, Clock, Cry1, Cry2, Per1, and Per2. Rod photoreceptors represented a negative control, because they did not appear to contain clock transcripts. We finally confirmed that DA cells contain the protein encoded by the Bmal1 gene by comparing immunostaining of the nuclei of DA cells in the retinas of wildtype and Bmal1-/- mice. It is therefore likely that DA cells contain a circadian clock that anticipates predictable variations in retinal illumination.

Intrinsic circadian clock of the mammalian retina: importance for retinal processing of visual information.

Circadian clocks are widely distributed in mammalian tissues, but little is known about the physiological functions of clocks outside the suprachiasmatic nucleus of the brain. The retina has an intrinsic circadian clock, but its importance for vision is unknown. Here we show that mice lacking Bmal1, a gene required for clock function, had abnormal retinal transcriptional responses to light and defective inner retinal electrical responses to light, but normal photoreceptor responses to light and retinas that appeared structurally normal by light and electron microscopy. We generated mice with a retina-specific genetic deletion of Bmal1, and they had defects of retinal visual physiology essentially identical to those of mice lacking Bmal1 in all tissues and lacked a circadian rhythm of inner retinal electrical responses to light. Our findings indicate that the intrinsic circadian clock of the retina regulates retinal visual processing in vivo.

Form deprivation modulates retinal neurogenesis in primate experimental myopia.

Juvenile primates develop myopia when their visual experience is degraded by lid fusion. In response to this abnormal visual input, retinal neural networks cause an excessive growth of the postequatorial segment of the eye, but the mechanism underlying this axial elongation is unknown. After fusion of the lids in one eye of juvenile rhesus macaques and green monkeys, we combined cDNA subtractions, microarray profiling, and real-time PCR to compare gene expression in the retinas of the closed and open eyes. This molecular analysis showed up-regulation of a number of genes associated with cell division in the retina of the closed eye and differential expression of six genes localized to chromosomal loci linked to forms of human hereditary myopia. In addition, it substantiated a previous observation, based on immunocytochemistry, that synthesis of vasoactive intestinal polypeptide was increased upon lid fusion. Injection of 5-bromo-2'-deoxyuridine and immunocytochemistry showed that the primate retinal periphery harbors mitotically active neuroprogenitor cells that increase in number when the visual experience is altered by lid fusion. Furthermore, the number of dividing cells is highly correlated with axial elongation of the eye and the resulting myopic refractive error. Thus, the retina undergoes active growth during the postnatal development of the primate eye. This growth is modulated by the visual input and accelerates considerably when the eye develops axial myopia. Vasoactive intestinal polypeptide may be the molecule that stimulates retinal growth.

Spontaneous activity of isolated dopaminergic periglomerular cells of the main olfactory bulb.

We examined the electrophysiological properties of a population of identified dopaminergic periglomerular cells of the main olfactory bulb using transgenic mice in which catecholaminergic neurons expressed human placental alkaline phosphatase (PLAP) on the outer surface of the plasma membrane. After acute dissociation, living dopaminergic periglomerular cells were identified by a fluorescently labeled monoclonal antibody to PLAP. In current-clamp mode, dopaminergic periglomerular cells spontaneously generated action potentials in a rhythmic fashion with an average frequency of 8 Hz. The hyperpolarization-activated cation current (Ih) did not seem important for pacemaking because blocking the current with ZD 7288 or Cs+ had little effect on spontaneous firing. To investigate what ionic currents do drive pacemaking, we performed action-potential-clamp experiments using records of pacemaking as voltage command in voltage-clamp experiments. We found that substantial TTX-sensitive Na+ current flows during the interspike depolarization. In addition, substantial Ca2+ current flowed during the interspike interval, and blocking Ca2+ current hyperpolarized the neurons and stopped spontaneous firing. These results show that dopaminergic periglomerular cells have intrinsic pacemaking activity, supporting the possibility that they can maintain a tonic release of dopamine to modulate the sensitivity of the olfactory system during odor detection. Calcium entry into these neurons provides electrical drive for pacemaking as well as triggering transmitter release.

GABAergic synapses made by a retinal dopaminergic neuron.

In the retina, dopaminergic amacrine (interplexiform) cells establish multiple synapses on the perikarya of AII amacrines, the neurons that distribute rod signals to on- and off-cone bipolars. We used triple-label immunocytochemistry and confocal microscopy to identify the receptors contained within the postsynaptic active zone of these synapses in both mouse and rat retinas. We found that at the interface between the dendrites of the dopaminergic neurons and the AII amacrine cell perikarya clusters of postsynaptic gamma-aminobutyric acid type A (GABA(A)) receptors are situated in register with aggregates of presynaptic organelles immunoreactive for GABA, the GABA vesicular transporter, and the vesicular monoamine transporter-2. D1 and D23 dopamine receptors, on the other hand, do not form clusters on the surface of the perikarya of AII amacrine cells. We suggest that the synapses between retinal dopaminergic neurons and AII amacrine cells are GABAergic and that both GABA and dopamine are released by the presynaptic endings. GABA acts on the ionotropic receptors clustered at the postsynaptic active zone, whereas dopamine diffuses to more distant, slower-acting metabotropic receptors.

Gene discovery in genetically labeled single dopaminergic neurons of the retina.

In the retina, dopamine plays a central role in neural adaptation to light. Progress in the study of dopaminergic amacrine (DA) cells has been limited because they are very few (450 in each mouse retina, 0.005% of retinal neurons). Here, we applied transgenic technology, single-cell global mRNA amplification, and cDNA microarray screening to identify transcripts present in DA cells. To profile gene expression in single neurons, we developed a method (SMART7) that combines a PCR-based initial step (switching mechanism at the 5' end of the RNA transcript or SMART) with T7 RNA polymerase amplification. Single-cell targets were synthesized from genetically labeled DA cells to screen the RIKEN 19k mouse cDNA microarrays. Seven hundred ninety-five transcripts were identified in DA cells at a high level of confidence, and expression of the most interesting genes was confirmed by immunocytochemistry. Twenty-one previously undescribed proteins were found in DA cells, including a chloride channel, receptors and other membrane glycoproteins, kinases, transcription factors, and secreted neuroactive molecules. Thirty-eight percent of transcripts were ESTs or coding for hypothetical proteins, suggesting that a large portion of the DA cell proteome is still uncharacterized. Because cryptochrome-1 mRNA was found in DA cells, immunocytochemistry was extended to other components of the circadian clock machinery. This analysis showed that DA cells contain the most common clock-related proteins.