No abstract

Mesencephalic dopamine (DA) neurons have been suggested to use glutamate as a cotransmitter. Here, we suggest a mechanism for this form of cotransmission by showing that a subset of DA neurons both in vitro and in vivo expresses vesicular glutamate transporter 2 (VGluT2). Expression of VGluT2 decreases with age. Moreover, when DA neurons are grown in isolation using a microculture system, there is a marked upregulation of VGluT2 expression. We provide evidence that expression of this transporter is normally repressed through a contact-dependent interaction with GABA and other DA neurons, thus providing a partial explanation for the highly restricted expression of VGluT2 in DA neurons in vivo. Our results demonstrate that the neurotransmitter phenotype of DA neurons is both developmentally and dynamically regulated. These findings may have implications for a better understanding of the fast synaptic action of DA neurons as well as basal ganglia circuitry.

Recent studies have proposed that glutamate corelease by mesostriatal dopamine (DA) neurons regulates behavioral activation by psychostimulants. How and when glutamate release by DA neurons might play this role remains unclear. Considering evidence for early expression of the type 2 vesicular glutamate transporter in mesencephalic DA neurons, we hypothesized that this cophenotype is particularly important during development. Using a conditional gene knock-out approach to selectively disrupt the Vglut2 gene in mouse DA neurons, we obtained in vitro and in vivo evidence for reduced growth and survival of mesencephalic DA neurons, associated with a decrease in the density of DA innervation in the nucleus accumbens, reduced activity-dependent DA release, and impaired motor behavior. These findings provide strong evidence for a functional role of the glutamatergic cophenotype in the development of mesencephalic DA neurons, opening new perspectives into the pathophysiology of neurodegenerative disorders involving the mesostriatal DA system.

Glial cell line-derived neurotrophic factor (GDNF) is known to promote the survival and differentiation of dopaminergic neurons of the midbrain. GDNF also causes an enhancement of dopamine release by a mechanism which is presently unclear. Using isolated dopaminergic neurons of the rat ventral tegmental area in culture, we have tested the hypothesis that GDNF regulates the establishment and functional properties of synaptic terminals. Previous studies have shown that single dopaminergic neurons in culture can co-release glutamate in addition to dopamine, leading to the generation of a fast excitatory autaptic current via glutamate receptors. Using excitatory autaptic currents as an assay for the activity of synapses established by identified dopaminergic neurons, we found that chronically applied GDNF produced a threefold increase in the amplitude of excitatory autaptic currents. This action was specific for dopaminergic neurons because GDNF had no such effect on ventral tegmental area GABAergic neurons. The enhancement of excitatory autaptic current amplitude caused by GDNF was accompanied by an increase in the frequency of spontaneous miniature excitatory autaptic currents. These observations confirmed a presynaptic locus of change. We identified synaptic terminals by using synapsin-1 immunofluorescence. In single tyrosine hydroxylase-positive neurons, the number of synapsin-positive puncta which represent putative synaptic terminals was found to be approximately doubled in GDNF-treated cells at 5, 10 and 15 days in culture. The number of such morphologically identified terminals in isolated GABAergic neurons was unchanged by GDNF. These results suggest that one mechanism through which GDNF may enhance dopamine release is through promoting the establishment of new functional synaptic terminals.

Somatodendritic (STD) dopamine (DA) release is a key mechanism for the autoregulatory control of DA release in the brain. However, its molecular mechanism remains undetermined. We tested the hypothesis that differential expression of synaptotagmin (Syt) isoforms explains some of the differential properties of terminal and STD DA release. Down-regulation of the dendritically expressed Syt4 and Syt7 severely reduced STD DA release, whereas terminal release required Syt1. Moreover, we found that although mobilization of intracellular Ca 2؉ stores is inefficient, Ca 2؉ influx through N-and P/Q-type voltage-gated channels is critical to trigger STD DA release. Our findings provide an explanation for the differential Ca 2؉ requirement of terminal and STD DA release. In addition, we propose that not all sources of intracellular Ca 2؉ are equally efficient to trigger this release mechanism. Our findings have implications for a better understanding of a fundamental cell biological process mediating transcellular signaling in a system critical for diseases such as Parkinson disease. Dopamine (DA),4 like other monoamine neurotransmitters, is released from the cell body and dendrites in addition to axon terminals (1). This process, called somatodendritic (STD) release, is important in the ventral tegmental area (VTA) for induction of behavioral sensitization to amphetamine through activation of local D1 receptors (2, 3) and in the substantia nigra (SN) for control of motor performance (4, 5). In addition, STD DA release modulates DA neuron firing activity through D2 autoreceptor activation (6, 7) and increases firing activity of SN pars reticulata ␥-aminobutyric acid-releasing neurons, a process that might activate feedback signals regulating DA neuron activity (8), thereby influencing axonal DA release.Two mechanisms have been proposed to mediate STD DA release: reversal of the DA transporter (9) and a vesicular exocytotic-like mechanism. In agreement with the second mechanism, STD DA release is activity-dependent (6, 10), sensitive to depletion of vesicular stores with reserpine (6, 11, 12), and Ca 2ϩ -dependent (6, 10, 12, 13). Moreover, disruption of SNARE proteins with botulinum toxins blocks STD DA release (10, 13). Vesicular exocytosis requires the concerted action of SNARE proteins and a synaptotagmin (Syt). During release, SNAREs have a direct role in vesicle-membrane fusion, and Syt acts as a Ca 2ϩ sensor. Of the 15 Syt isoforms identified so far, Syt1, 2, 3, 5, 6, 7, 9, and 10 have been reported to drive Ca 2ϩ -dependent vesicular fusion (14), and only Syt1, 2, and 9 are confirmed as Ca 2ϩ sensors for synaptic neurotransmitter release from axon terminals (15).One of the hallmarks of STD DA release is its relative persistence at reduced levels of extracellular Ca 2ϩ concentrations: although release from axon terminals is drastically reduced at extracellular Ca 2ϩ levels lower than 1 mM, STD DA release persists at Ca 2ϩ levels between 0.5 and 1 mM (Refs. 10, 12, and 13; but see also Ref. 16). This differential Ca 2...