Intestinal infection triggers Parkinson's disease-like symptoms in Pink1-/- mice.

Parkinson's disease is a neurodegenerative disorder with motor symptoms linked to the loss of dopaminergic neurons in the substantia nigra compacta. Although the mechanisms that trigger the loss of dopaminergic neurons are unclear, mitochondrial dysfunction and inflammation are thought to have key roles1,2. An early-onset form of Parkinson's disease is associated with mutations in the PINK1 kinase and PRKN ubiquitin ligase genes3. PINK1 and Parkin (encoded by PRKN) are involved in the clearance of damaged mitochondria in cultured cells4, but recent evidence obtained using knockout and knockin mouse models have led to contradictory results regarding the contributions of PINK1 and Parkin to mitophagy in vivo5-8. It has previously been shown that PINK1 and Parkin have a key role in adaptive immunity by repressing presentation of mitochondrial antigens9, which suggests that autoimmune mechanisms participate in the aetiology of Parkinson's disease. Here we show that intestinal infection with Gram-negative bacteria in Pink1-/- mice engages mitochondrial antigen presentation and autoimmune mechanisms that elicit the establishment of cytotoxic mitochondria-specific CD8+ T cells in the periphery and in the brain. Notably, these mice show a sharp decrease in the density of dopaminergic axonal varicosities in the striatum and are affected by motor impairment that is reversed after treatment with L-DOPA. These data support the idea that PINK1 is a repressor of the immune system, and provide a pathophysiological model in which intestinal infection acts as a triggering event in Parkinson's disease, which highlights the relevance of the gut-brain axis in the disease10.

Dopaminergic neurons establish a distinctive axonal arbor with a majority of non-synaptic terminals.

Chemical neurotransmission typically occurs through synapses. Previous ultrastructural examinations of monoamine neuron axon terminals often failed to identify a pre- and postsynaptic coupling, leading to the concept of "volume" transmission. Whether this results from intrinsic properties of these neurons remains undefined. We find that dopaminergic neurons in vitro establish a distinctive axonal arbor compared to glutamatergic or GABAergic neurons in both size and propensity of terminals to avoid direct contact with target neurons. While most dopaminergic varicosities are active and contain exocytosis proteins like synaptotagmin 1, only ~20% of these are synaptic. The active zone protein bassoon was found to be enriched in dopaminergic terminals that are in proximity to a target cell. Finally, we found that the proteins neurexin-1αSS4- and neuroligin-1A+B play a critical role in the formation of synapses by dopamine (DA) neurons. Our findings suggest that DA neurons are endowed with a distinctive developmental connectivity program.

VGluT2 Expression in Dopamine Neurons Contributes to Postlesional Striatal Reinnervation.

A subset of adult ventral tegmental area dopamine (DA) neurons expresses vesicular glutamate transporter 2 (VGluT2) and releases glutamate as a second neurotransmitter in the striatum, while only few adult substantia nigra DA neurons have this capacity. Recent work showed that cellular stress created by neurotoxins such as MPTP and 6-hydroxydopamine can upregulate VGluT2 in surviving DA neurons, suggesting the possibility of a role in cell survival, although a high level of overexpression could be toxic to DA neurons. Here we examined the level of VGluT2 upregulation in response to neurotoxins and its impact on postlesional plasticity. We first took advantage of an in vitro neurotoxin model of Parkinson's disease and found that this caused an average 2.5-fold enhancement of Vglut2 mRNA in DA neurons. This could represent a reactivation of a developmental phenotype because using an intersectional genetic lineage-mapping approach, we find that >98% of DA neurons have a VGluT2+ lineage. Expression of VGluT2 was detectable in most DA neurons at embryonic day 11.5 and was localized in developing axons. Finally, compatible with the possibility that enhanced VGluT2 expression in DA neurons promotes axonal outgrowth and reinnervation in the postlesional brain, we observed that DA neurons in female and male mice in which VGluT2 was conditionally removed established fewer striatal connections 7 weeks after a neurotoxin lesion. Thus, we propose here that the developmental expression of VGluT2 in DA neurons can be reactivated at postnatal stages, contributing to postlesional plasticity of dopaminergic axons.SIGNIFICANCE STATEMENT A small subset of dopamine neurons in the adult, healthy brain expresses vesicular glutamate transporter 2 (VGluT2) and thus releases glutamate as a second neurotransmitter in the striatum. This neurochemical phenotype appears to be plastic as exposure to neurotoxins, such as 6-OHDA or MPTP, that model certain aspects of Parkinson's disease pathophysiology, boosts VGluT2 expression in surviving dopamine neurons. Here we show that this enhanced VGluT2 expression in dopamine neurons drives axonal outgrowth and contributes to dopamine neuron axonal plasticity in the postlesional brain. A better understanding of the neurochemical changes that occur during the progression of Parkinson's disease pathology will aid the development of novel therapeutic strategies for this disease.

Neonatal 6-OHDA lesion of the SNc induces striatal compensatory sprouting from surviving SNc dopaminergic neurons without VTA contribution.

Dopamine (DA) neurons of the substantia nigra pars compacta (SNc) are uniquely vulnerable to neurodegeneration in Parkinson's disease (PD). We hypothesize that their large axonal arbor is a key factor underlying their vulnerability, due to increased bioenergetic, proteostatic and oxidative stress. In keeping with this model, other DAergic populations with smaller axonal arbors are mostly spared during the course of PD and are more resistant to experimental lesions in animal models. Aiming to improve mouse PD models, we examined if neonatal partial SNc lesions could lead to adult mice with fewer SNc DA neurons that are endowed with larger axonal arbors because of compensatory mechanisms. We injected 6-hydroxydopamine (6-OHDA) unilaterally in the SNc at an early postnatal stage at a dose selected to induce loss of approximately 50% of SNc DA neurons. We find that at 10 and 90 days after the lesion, the axons of SNc DA neurons show massive compensatory sprouting, as revealed by the proportionally smaller decrease in tyrosine hydroxylase (TH) in the striatum compared with the loss of SNc DA neuron cell bodies. The extent and origin of this axonal sprouting was further investigated by AAV-mediated expression of eYFP in SNc or ventral tegmental area (VTA) DA neurons of adult mice. Our results reveal that SNc DA neurons have the capacity to substantially increase their axonal arbor size and suggest that mice designed to have reduced numbers of SNc DA neurons could potentially be used to develop better mouse models of PD, with elevated neuronal vulnerability.

Segregation of dopamine and glutamate release sites in dopamine neuron axons: regulation by striatal target cells.

Dopamine (DA) is a key regulator of circuits controlling movement and motivation. A subset of midbrain DA neurons has been shown to express the vesicular glutamate transporter (VGLUT)2, underlying their capacity for glutamate release. Glutamate release is found mainly by DA neurons of the ventral tegmental area (VTA) and can be detected at terminals contacting ventral, but not dorsal, striatal neurons, suggesting the possibility that target-derived signals regulate the neurotransmitter phenotype of DA neurons. Whether glutamate can be released from the same terminals that release DA or from a special subset of axon terminals is unclear. Here, we provide in vitro and in vivo data supporting the hypothesis that DA and glutamate-releasing terminals in mice are mostly segregated and that striatal neurons regulate the cophenotype of midbrain DA neurons and the segregation of release sites. Our work unveils a fundamental feature of dual neurotransmission and plasticity of the DA system.-Fortin, G. M., Ducrot, C., Giguère, N., Kouwenhoven, W. M., Bourque, M.-J., Pacelli, C., Varaschin, R. K., Brill, M., Singh, S., Wiseman, P. W., Trudeau, L.-E. Segregation of dopamine and glutamate release sites in dopamine neuron axons: regulation by striatal target cells.

Comparative analysis of Parkinson's disease-associated genes in mice reveals altered survival and bioenergetics of Parkin-deficient dopamine neurons.

Many mutations in genes encoding proteins such as Parkin, PTEN-induced putative kinase 1 (PINK1), protein deglycase DJ-1 (DJ-1 or PARK7), leucine-rich repeat kinase 2 (LRRK2), and α-synuclein have been linked to familial forms of Parkinson's disease (PD). The consequences of these mutations, such as altered mitochondrial function and pathological protein aggregation, are starting to be better understood. However, little is known about the mechanisms explaining why alterations in such diverse cellular processes lead to the selective loss of dopamine (DA) neurons in the substantia nigra (SNc) in the brain of individuals with PD. Recent work has shown that one of the reasons for the high vulnerability of SNc DA neurons is their high basal rate of mitochondrial oxidative phosphorylation (OXPHOS), resulting from their highly complex axonal arborization. Here, we examined whether axonal growth and basal mitochondrial function are altered in SNc DA neurons from Parkin-, Pink1-, or DJ-1-KO mice. We provide evidence for increased basal OXPHOS in Parkin-KO DA neurons and for reduced survival of DA neurons that have a complex axonal arbor. The surviving smaller neurons exhibited reduced vulnerability to the DA neurotoxin and mitochondrial complex I inhibitor MPP+, and this reduction was associated with reduced expression of the DA transporter. Finally, we found that glial cells play a role in the reduced resilience of DA neurons in these mice and that WT Parkin overexpression rescues this phenotype. Our results provide critical insights into the complex relationship between mitochondrial function, axonal growth, and genetic risk factors for PD.

Characterization of a Human Point Mutation of VGLUT3 (p.A211V) in the Rodent Brain Suggests a Nonuniform Distribution of the Transporter in Synaptic Vesicles.

The atypical vesicular glutamate transporter type 3 (VGLUT3) is expressed by subpopulations of neurons using acetylcholine, GABA, or serotonin as neurotransmitters. In addition, VGLUT3 is expressed in the inner hair cells of the auditory system. A mutation (p.A211V) in the gene that encodes VGLUT3 is responsible for progressive deafness in two unrelated families. In this study, we investigated the consequences of the p.A211V mutation in cell cultures and in the CNS of a mutant mouse. The mutation substantially decreased VGLUT3 expression (-70%). We measured VGLUT3-p.A211V activity by vesicular uptake in BON cells, electrophysiological recording of isolated neurons, and its ability to stimulate serotonergic accumulation in cortical synaptic vesicles. Despite a marked loss of expression, the activity of the mutated isoform was only minimally altered. Furthermore, mutant mice displayed none of the behavioral alterations that have previously been reported in VGLUT3 knock-out mice. Finally, we used stimulated emission depletion microscopy to analyze how the mutation altered VGLUT3 distribution within the terminals of mice expressing the mutated isoform. The mutation appeared to reduce the expression of the VGLUT3 transporter by simultaneously decreasing the number of VGLUT3-positive synaptic vesicles and the amount of VGLUT3 per synapses. These observations suggested that VGLUT3 global activity is not linearly correlated with VGLUT3 expression. Furthermore, our data unraveled a nonuniform distribution of VGLUT3 in synaptic vesicles. Identifying the mechanisms responsible for this complex vesicular sorting will be critical to understand VGLUT's involvement in normal and pathological conditions.SIGNIFICANCE STATEMENT VGLUT3 is an atypical member of the vesicular glutamate transporter family. A point mutation of VGLUT3 (VGLUT3-p.A211V) responsible for a progressive loss of hearing has been identified in humans. We observed that this mutation dramatically reduces VGLUT3 expression in terminals (∼70%) without altering its function. Furthermore, using stimulated emission depletion microscopy, we found that reducing the expression levels of VGLUT3 diminished the number of VGLUT3-positive vesicles at synapses. These unexpected findings challenge the vision of a uniform distribution of synaptic vesicles at synapses. Therefore, the overall activity of VGLUT3 is not proportional to the level of VGLUT3 expression. These data will be key in interpreting the role of VGLUTs in human pathologies.

Dynamic SERS nanosensor for neurotransmitter sensing near neurons.

Current electrophysiology and electrochemistry techniques have provided unprecedented understanding of neuronal activity. However, these techniques are suited to a small, albeit important, panel of neurotransmitters such as glutamate, GABA and dopamine, and these constitute only a subset of the broader range of neurotransmitters involved in brain chemistry. Surface-enhanced Raman scattering (SERS) provides a unique opportunity to detect a broader range of neurotransmitters in close proximity to neurons. Dynamic SERS (D-SERS) nanosensors based on patch-clamp-like nanopipettes decorated with gold nanoraspberries can be located accurately under a microscope using techniques analogous to those used in current electrophysiology or electrochemistry experiments. In this manuscript, we demonstrate that D-SERS can measure in a single experiment ATP, glutamate (glu), acetylcholine (ACh), GABA and dopamine (DA), among other neurotransmitters, with the potential for detecting a greater number of neurotransmitters. The SERS spectra of these neurotransmitters were identified with a barcoding data processing method and time series of the neurotransmitter levels were constructed. The D-SERS nanosensor was then located near cultured mouse dopaminergic neurons. The detection of neurotransmitters was performed in response to a series of K+ depolarisations, and allowed the detection of elevated levels of both ATP and dopamine. Control experiments were also performed near glial cells, showing only very low basal detection neurotransmitter events. This paper demonstrates the potential of D-SERS to detect neurotransmitter secretion events near living neurons, but also constitutes a strong proof-of-concept for the broad application of SERS to the detection of secretion events by neurons or other cell types in order to study normal or pathological cell functions.

Glutamate corelease promotes growth and survival of midbrain dopamine neurons.

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.

Synaptotagmin-1-dependent phasic axonal dopamine release is dispensable for basic motor behaviors in mice.

In Parkinson's disease (PD), motor dysfunctions only become apparent after extensive loss of DA innervation. This resilience has been hypothesized to be due to the ability of many motor behaviors to be sustained through a diffuse basal tone of DA; but experimental evidence for this is limited. Here we show that conditional deletion of the calcium sensor synaptotagmin-1 (Syt1) in DA neurons (Syt1 cKODA mice) abrogates most activity-dependent axonal DA release in the striatum and mesencephalon, leaving somatodendritic (STD) DA release intact. Strikingly, Syt1 cKODA mice showed intact performance in multiple unconditioned DA-dependent motor tasks and even in a task evaluating conditioned motivation for food. Considering that basal extracellular DA levels in the striatum were unchanged, our findings suggest that activity-dependent DA release is dispensable for such tasks and that they can be sustained by a basal tone of extracellular DA. Taken together, our findings reveal the striking resilience of DA-dependent motor functions in the context of a near-abolition of phasic DA release, shedding new light on why extensive loss of DA innervation is required to reveal motor dysfunctions in PD.

Conditional deletion of neurexins dysregulates neurotransmission from dopamine neurons.

Midbrain dopamine (DA) neurons are key regulators of basal ganglia functions. The axonal domain of these neurons is highly complex, with a large subset of non-synaptic release sites and a smaller subset of synaptic terminals from which in addition to DA, glutamate or GABA are also released. The molecular mechanisms regulating the connectivity of DA neurons and their neurochemical identity are unknown. An emerging literature suggests that neuroligins, trans-synaptic cell adhesion molecules, regulate both DA neuron connectivity and neurotransmission. However, the contribution of their major interaction partners, neurexins (Nrxns), is unexplored. Here, we tested the hypothesis that Nrxns regulate DA neuron neurotransmission. Mice with conditional deletion of all Nrxns in DA neurons (DAT::NrxnsKO) exhibited normal basic motor functions. However, they showed an impaired locomotor response to the psychostimulant amphetamine. In line with an alteration in DA neurotransmission, decreased levels of the membrane DA transporter (DAT) and increased levels of the vesicular monoamine transporter (VMAT2) were detected in the striatum of DAT::NrxnsKO mice, along with reduced activity-dependent DA release. Strikingly, electrophysiological recordings revealed an increase of GABA co-release from DA neuron axons in the striatum of these mice. Together, these findings suggest that Nrxns act as regulators of the functional connectivity of DA neurons.

The human brain contains billions of nerve cells, known as neurons, which receive input from the outside world and process this information in the brain. Neurons communicate with each other by releasing chemical messengers from specialized structures, called axon terminals, some of which form junctions known as synapses. These messengers then generate signals in the target neurons. Based on the type of chemical they release, neurons can be classified into different types. For example, neurons releasing dopamine are considered to act as key regulators of learning, movements and motivation. Such neurons establish very large numbers of axon terminals, but very few of them form synapses. Specific sets of proteins, including neurexins and neuroligins, are thought to help regulate the activity of the connexions between these neurons. Previous research has shown that when neuroligins were removed from the neurons of worms or mice, it affected the ability of the animals to move. So far, the role of neurexins in managing the connectivity of regulatory neurons, such as those releasing dopamine, has received much less attention. To bridge this knowledge gap, Ducrot et al. explored how removing neurexins from dopamine neurons in mice affected their behaviour. The experiments revealed that eliminating neurexins did not affect their motor skills on a rotating rod, but it did reduce their movements in response to the psychostimulant amphetamine, a molecule known to enhance dopamine-associated behaviours. The cellular structure of dopamine neurons lacking neurexins was the same as in neurons containing this protein. But dopamine neurons without neurexins were slower to recycle dopamine, and they released a higher amount of the inhibitory messenger GABA. This suggests that neurexin acts as an important suppressor of GABA secretion to help regulate the signals released by dopamine neurons. These findings set the stage for further research into the role of neurexins in regulating dopamine and other populations of neurons in conditions such as Parkinson's disease, where movement and coordination are affected.

Somatodendritic dopamine release requires synaptotagmin 4 and 7 and the participation of voltage-gated calcium channels.

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.

Developmental and target-dependent regulation of vesicular glutamate transporter expression by dopamine neurons.

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.

Block Copolymer Brush Layer-Templated Gold Nanoparticles on Nanofibers for Surface-Enhanced Raman Scattering Optophysiology.

A nanothin block copolymer (BCP) brush-layer film adsorbed on glass nanofibers is shown to address the long-standing challenge of forming a template for the deposition of dense and well-dispersed nanoparticles on highly curved surfaces, allowing the development of an improved nanosensor for neurotransmitters. We employed a polystyrene- block-poly(4-vinylpyridine) BCP and plasmonic gold nanoparticles (AuNPs) of 52 nm in diameter for the fabrication of the nanosensor on pulled fibers with diameters down to 200 nm. The method is simple, using only solution processes and a plasma cleaning step. The templating of the AuNPs on the nanofiber surprisingly gave rise to more than 1 order of magnitude improvement in the surface-enhanced Raman scattering (SERS) performance for 4-mercaptobenzoic acid compared to the same AuNPs aggregated on identical fibers without the use of a template. We hypothesize that a wavelength-scale lens formed by the nanofiber contributes to enhancing the SERS performance to the extent that it can melt the glass nanofiber under moderate laser power. We then show the capability of this nanosensor to detect the corelease of the neurotransmitters dopamine and glutamate from living mouse brain dopaminergic neurons with a sensitivity 1 order of magnitude greater than with aggregated AuNPs. The simplicity of fabrication and the far superior performance of the BCP-templated nanofiber demonstrates the potential of this method to efficiently pattern nanoparticles on highly curved surfaces and its application as molecular nanosensors for cell physiology.

Histamine H3 Receptors Decrease Dopamine Release in the Ventral Striatum by Reducing the Activity of Striatal Cholinergic Interneurons.

Histamine H3 receptors are widely distributed Gi-coupled receptors whose activation reduces neuronal activity and inhibits release of numerous neurotransmitters. Although these receptors are abundantly expressed in the striatum, their modulatory role on activity-dependent dopamine release is not well understood. Here, we observed that histamine H3 receptor activation indirectly diminishes dopamine overflow in the ventral striatum by reducing cholinergic interneuron activity. Acute brain slices from C57BL/6 or channelrhodopsin-2-transfected DAT-cre mice were obtained, and dopamine transients evoked either electrically or optogenetically were measured by fast-scan cyclic voltammetry. The H3 agonist α-methylhistamine significantly reduced electrically- evoked dopamine overflow, an effect blocked by the nicotinic acetylcholine receptor antagonist dihydro-ß-erythroidine, suggesting involvement of cholinergic interneurons. None of the drug treatments targeting H3 receptors affected optogenetically evoked dopamine overflow, indicating that direct H3-modulation of dopaminergic axons is unlikely. Next, we used qPCR and confirmed the expression of histamine H3 receptor mRNA in cholinergic interneurons, both in ventral and dorsal striatum. Activation of H3 receptors by α-methylhistamine reduced spontaneous firing of cholinergic interneurons in the ventral, but not in the dorsal striatum. Resting membrane potential and number of spontaneous action potentials in ventral-striatal cholinergic interneurons were significantly reduced by α-methylhistamine. Acetylcholine release from isolated striatal synaptosomes, however, was not altered by α-methylhistamine. Together, these results indicate that histamine H3 receptors are important modulators of dopamine release, specifically in the ventral striatum, and that they do so by decreasing the firing rate of cholinergic neurons and, consequently, reducing cholinergic tone on dopaminergic axons.

Homeostatic regulation of excitatory synapses on striatal medium spiny neurons expressing the D2 dopamine receptor.

Striatal medium spiny neurons (MSNs) are contacted by glutamatergic axon terminals originating from cortex, thalamus and other regions. The striatum is also innervated by dopaminergic (DAergic) terminals, some of which release glutamate as a co-transmitter. Despite evidence for functional DA release at birth in the striatum, the role of DA in the establishment of striatal circuitry is unclear. In light of recent work suggesting activity-dependent homeostatic regulation of glutamatergic terminals on MSNs expressing the D2 DA receptor (D2-MSNs), we used primary co-cultures to test the hypothesis that stimulation of DA and glutamate receptors regulates the homeostasis of glutamatergic synapses on MSNs. Co-culture of D2-MSNs with mesencephalic DA neurons or with cortical neurons produced an increase in spines and functional glutamate synapses expressing VGLUT2 or VGLUT1, respectively. The density of VGLUT2-positive terminals was reduced by the conditional knockout of this gene from DA neurons. In the presence of both mesencephalic and cortical neurons, the density of synapses reached the same total, compatible with the possibility of a homeostatic mechanism capping excitatory synaptic density. Blockade of D2 receptors increased the density of cortical and mesencephalic glutamatergic terminals, without changing MSN spine density or mEPSC frequency. Combined blockade of AMPA and NMDA glutamate receptors increased the density of cortical terminals and decreased that of mesencephalic VGLUT2-positive terminals, with no net change in total excitatory terminal density or in mEPSC frequency. These results suggest that DA and glutamate signaling regulate excitatory inputs to striatal D2-MSNs at both the pre- and postsynaptic level, under the influence of a homeostatic mechanism controlling functional output of the circuit.

Role of calcium in neurotensin-evoked enhancement in firing in mesencephalic dopamine neurons.

Neurotensin (NT) increases neurotransmission within the mesolimbic dopamine system by enhancing the firing rate of dopaminergic (DAergic) neurons and by acting at the nerve terminal level. The signal transduction pathways involved in these effects have not been characterized, but NT receptors are coupled to the phospholipase C pathway and Ca(2+) mobilization. However, an enhancement of intracellular Ca(2+) concentration ([Ca(2+)](i)) evoked by NT in DAergic neurons has yet to be demonstrated. Furthermore, the hypothesis that the excitatory effects of NT in DAergic neurons are Ca(2+) dependent is currently untested. In whole-cell recording experiments, DAergic neurons in culture were identified by their selective ability to express a cell-specific green fluorescent protein reporter construct. These experiments confirmed that NT increases firing rate in cultured DAergic neurons. This effect was Ca(2+) dependent because it was blocked by intracellular dialysis with BAPTA. Using Ca(2+) imaging, we showed that NT caused a rapid increase in [Ca(2+)](i) in DAergic neurons. Most of the Ca(2+) originated from the extracellular medium. NT-induced excitation and Ca(2+) influx were blocked by SR48692, an antagonist of the type 1 NT receptor. Blocking IP(3) receptors using heparin prevented the excitatory effect of NT. Moreover, Zn(2+) and SKF96365 both blocked the excitatory effect of NT, suggesting that nonselective cationic conductances are involved. Finally, although NT can also induce a rise in [Ca(2+)](i) in astrocytes, we find that NT-evoked excitation of DAergic neurons can occur independently of astrocyte activation.

Elevated Mitochondrial Bioenergetics and Axonal Arborization Size Are Key Contributors to the Vulnerability of Dopamine Neurons.

Although the mechanisms underlying the loss of neurons in Parkinson's disease are not well understood, impaired mitochondrial function and pathological protein aggregation are suspected as playing a major role. Why DA (dopamine) neurons and a select small subset of brain nuclei are particularly vulnerable to such ubiquitous cellular dysfunctions is presently one of the key unanswered questions in Parkinson's disease research. One intriguing hypothesis is that their heightened vulnerability is a consequence of their elevated bioenergetic requirements. Here, we show for the first time that vulnerable nigral DA neurons differ from less vulnerable DA neurons such as those of the VTA (ventral tegmental area) by having a higher basal rate of mitochondrial OXPHOS (oxidative phosphorylation), a smaller reserve capacity, a higher density of axonal mitochondria, an elevated level of basal oxidative stress, and a considerably more complex axonal arborization. Furthermore, we demonstrate that reducing axonal arborization by acting on axon guidance pathways with Semaphorin 7A reduces in parallel the basal rate of mitochondrial OXPHOS and the vulnerability of nigral DA neurons to the neurotoxic agents MPP(+) (1-methyl-4-phenylpyridinium) and rotenone. Blocking L-type calcium channels with isradipine was protective against MPP(+) but not rotenone. Our data provide the most direct demonstration to date in favor of the hypothesis that the heightened vulnerability of nigral DA neurons in Parkinson's disease is directly due to their particular bioenergetic and morphological characteristics.

Presynaptic mu-opioid receptors regulate a late step of the secretory process in rat ventral tegmental area GABAergic neurons.

Gamma-aminobutyric acid (GABA)-containing interneurons of the ventral tegmental area (VTA) regulate the activity of dopaminergic neurons. These GABAergic interneurons are known to be innervated by synaptic terminals containing enkephalin, an endogenous ligand of mu-opioid receptors. Bath application of mu-opioid receptor agonists inhibits the activity of VTA GABAergic neurons but the mechanism whereby mu-opioid receptors regulate synaptic GABA release from these neurons has not been directly identified. Using cultured VTA neurons we have confirmed that mu-opioid receptor agonists inhibit synaptic GABA release. DAMGO, a selective mu-opioid receptor agonist, had four distinct effects on GABAergic IPSCs: (1) it inhibited the frequency and amplitude of spontaneous IPSCs (sIPSCs), (2) it reduced the amplitude of IPSCs evoked by single action potentials, (3) it inhibited the frequency, but not the amplitude of miniature IPSCs (mIPSCs), and (4) DAMGO inhibited mIPSCs evoked by ionomycin, a Ca(2+) ionophore. The inhibition of action potential-evoked IPSCs and of spontaneous and ionomycin-evoked mIPSCs by DAMGO was prevented by the K(+) channel blocker, 4-aminopyridine (4-AP). In conclusion, our work shows that one of the mechanisms through which mu-opioid receptors inhibit GABA release by VTA neurons is through inhibition of the secretory process at the nerve terminal level. In addition, considering that ionomycin stimulates exocytosis through a mechanism that should be insensitive to membrane polarization, our experiments with 4-AP suggest that K(+) channels are implicated in the inhibition of the efficacy of the secretory process by mu-opioid receptors.

Evaluation of D1 and D2 dopamine receptor segregation in the developing striatum using BAC transgenic mice.

The striatum is predominantly composed of medium spiny neurons (MSNs) that send their axons along two parallel pathways known as the direct and indirect pathways. MSNs from the direct pathway express high levels of D1 dopamine receptors, while MSNs from the indirect pathway express high levels of D2 dopamine receptors. There has been much debate over the extent of colocalization of these two major dopamine receptors in MSNs of adult animals. In addition, the ontogeny of the segregation process has never been investigated. In this paper, we crossed bacterial artificial chromosome drd1a-tdTomato and drd2-GFP reporter transgenic mice to characterize these models and estimate D1-D2 co-expression in the developing striatum as well as in striatal primary cultures. We show that segregation is already extensive at E18 and that the degree of co-expression further decreases at P0 and P14. Finally, we also demonstrate that cultured MSNs maintain their very high degree of D1-D2 reporter protein segregation, thus validating them as a relevant in vitro model.