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Several subtypes of modulatory neurons co‐express vesicular glutamate transporters (VGLUTs) in addition to their cognate vesicular transporters. These neurons are believed to establish new forms of neuronal communication. The atypical VGLUT3 is of particular interest since in the striatum this subtype is found in tonically active cholinergic interneurons (TANs) and in a subset of 5‐HT fibers. The striatum plays a major role in psychomotor effects induced by amphetamine. Whether and how VGLUT3‐operated glutamate/ACh or glutamate/5HT co‐transmissions modulates psychostimulants‐induced maladaptive behaviors is still unknown. Here, we investigate the involvement of VGLUT3 and glutamate co‐transmission in amphetamine‐induced psychomotor effects and stereotypies. Taking advantage of constitutive and cell‐type specific VGLUT3‐deficient mouse lines, we tackled the hypothesis that VGLUT3 could gate psychomotor effects (locomotor activity and stereotypies) induced by acute or chronic administration of amphetamine. Interestingly, VGLUT3‐null mice demonstrated blunted amphetamine‐induced stereotypies as well as reduced striatal ∆FosB expression. VGLUT3‐positive varicosities within the striatum arise in part from 5HT neurons. We tested the involvement of VGLUT3 deletion in serotoninergic neurons in amphetamine‐induced stereotypies. Mice lacking VGLUT3 specifically in 5HT fibers showed no alteration to amphetamine sensitivity. In contrast, specific deletion of VGLUT3 in cholinergic neurons partially phenocopied the effects observed in the constitutive knock‐out mice. Our results show that constitutive deletion of VGLUT3 modulates acute and chronic locomotor effects induced by amphetamine. They point to the fact that the expression of VGLUT3 in multiple brain areas is pivotal in gating amphetamine‐induced psychomotor adaptations. Open Science Badges: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. VGLUT3 is detected in neurons that use other neurotransmitters than glutamate, a neurochemical feature known as vesicular synergy. Here, we investigate the involvement of VGLUT3 in amphetamine‐induced psychomotor effects. We found that deletion of VGLUT3 promoted hyperlocomotor responses whereas blunted stereotypies were observed following amphetamine administration. A reduced expression of ∆FosB in the striatum mirrored such phenotype. In the striatum, VGLUT3 is expressed in cholinergic neurons and 5HT fibers. Cell‐type specific deletion of VGLUT3 in ACh and 5HT neurons revealed a complex dynamic regulation of amphetamine‐related effects. Our study points VGLUT3 as a critical modulator of psychostimulants‐induced adaptations. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/ [ABSTRACT FROM AUTHOR]

Striatal cholinergic interneurons ( CIN) are pivotal for the regulation of the striatal network. Acetylcholine ( ACh) released by CIN is centrally involved in reward behavior as well as locomotor or cognitive functions. Recently, BAC transgenic mice expressing channelrhodopsin-2 (ChR2) protein under the control of the choline acetyltransferase (Ch AT) promoter (Ch AT-ChR2) and displaying almost 50 extra copies of the VAChT gene were used to dissect cholinergic circuit connectivity and function using optogenetic approaches. These mice display over-expression of the vesicular acetylcholine transporter ( VAChT) and increased cholinergic tone. Consequently, Ch AT-ChR2 mice are a valuable model to investigate hypercholinergic phenotypes. Previous experiments established that Ch AT-ChR2 mice display an increased sensitivity to amphetamine induced-locomotor activity and stereotypes. In the present report, we analyzed the impact of VAChT over-expression in the striatum of Ch AT-ChR2 mice. Ch AT-ChR2 mice displayed increased locomotor sensitization in response to low dose of cocaine. In addition, we observed a dramatic remodeling of the morphology of CIN in Ch AT-ChR2 transgenic mice. VAChT immunolabeling was markedly enhanced in the soma and terminal of CIN from Ch AT-ChR2 mice as previously shown (Crittenden et al. 2014). Interestingly, the number of cholinergic varicosities was markedly reduced (−87%) whereas their size was significantly increased (+177%). Moreover, VAChT over-expression dramatically modified its trafficking along the somatodendritic and axonal arbor. These findings demonstrate that Ch AT-ChR2 mice present major alterations of CIN neuronal morphology and increased behavioral sensitization to cocaine, supporting the notion that the increased levels of VAChT observed in these mice make them fundamentally different from wild-type mice. [ABSTRACT FROM AUTHOR]

For decades, neuroscientists have used enriched preparations of synaptic particles called synaptosomes to study synapse function. However, the interpretation of corresponding data is problematic as synaptosome preparations contain multiple types of synapses and non-synaptic neuronal and glial contaminants. We established a novel Fluorescence Activated Synaptosome Sorting ( FASS) method that substantially improves conventional synaptosome enrichment protocols and enables high-resolution biochemical analyses of specific synapse subpopulations. Employing knock-in mice with fluorescent glutamatergic synapses, we show that FASS isolates intact ultrapure synaptosomes composed of a resealed presynaptic terminal and a postsynaptic density as assessed by light and electron microscopy. FASS synaptosomes contain bona fide glutamatergic synapse proteins but are almost devoid of other synapse types and extrasynaptic or glial contaminants. We identified 163 enriched proteins in FASS samples, of which FXYD6 and Tpd52 were validated as new synaptic proteins. FASS purification thus enables high-resolution biochemical analyses of specific synapse subpopulations in health and disease. [ABSTRACT FROM AUTHOR]

In many brain areas, few cholinergic synapses are identified. Acetylcholine is released into the extracellular space and acts through diffuse transmission. Motoneurons, however, are contacted by numerous cholinergic terminals, indicating synaptic cholinergic transmission on them. The muscarinic m2 receptor is the major acetylcholine receptor subtype of motoneurons; therefore, we analyzed the localization of the m2 receptor in correlation with synapses by electron microscopic immunohistochemistry in the mouse trigeminal, facial, and hypoglossal motor nuclei. In all nuclei, m2 receptors were localized at the membrane of motoneuronal perikarya and dendrites. The m2 receptors were concentrated at cholinergic synapses located on the perikarya and most proximal dendrites. However, m2 receptors at cholinergic synapses represented only a minority (<10%) of surface m2 receptors. The m2 receptors were also enriched at glutamatergic synapses in both motoneuronal perikarya and dendrites. A relatively large proportion (20-30%) of plasma membrane-associated m2 receptors were located at glutamatergic synapses. In conclusion, the effect of acetylcholine on motoneuron populations might be mediated through a synaptic as well as diffuse type of transmission. J. Comp. Neurol. 521:2008-2024, 2013. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Understanding the trafficking of G-protein-coupled receptors (GPCRs) is of particular importance, especially when modifications of the neurochemic environment occur as in pathological or therapeutic circumstances. In the central nervous system, although some GPCRs were reported to internalize in vivo, little is known about their trafficking downstream of the endocytic event. To address this issue, distribution and expression pattern of the major somatostatin receptor subtype, the somatostatin type 2 (sst2), was monitored in the hippocampus using immunofluorescence, autoradiographic and immunogold experiments from 10 minutes to 7 days after in vivo injection of the receptor agonist octreotide. We then analyzed whether postendocytic trafficking of the receptor was dependent upon integrity of the microtubule network using colchicine-injected animals. Together, our results suggest that upon agonist stimulation, dendritic receptors are retrogradely transported through a microtubule-dependent mechanism to a trans Golgi domain enriched in the t-SNARE syntaxin 6 and trans Golgi network 38 proteins, before recycling. Because we show that the exit rate from the trans Golgi apparatus back to the plasma membrane (hours) is slower than the entry rate (minutes), the neuronal postendocytic trafficking of sst2 receptor is likely to have functional consequences in several neurological diseases in which an increase in somatostatin release occurs. [ABSTRACT FROM AUTHOR]

Increasing evidence suggests that somatostatin may control neuronal excitability during epileptogenesis. In the hippocampus, sst2A receptors are likely to mediate somatostatin inhibitory actions but little is known about their status in kindled tissues. In the present study, sst2A receptor and somatostatin immunoreactivity were examined by confocal microscopy in the hippocampus during and after kindling acquisition. In control rats, somatostatin-positive axon terminals were mainly found in the stratum lacunosum moleculare of CA1 area and in the outer molecular layer of the dentate gyrus. sst2A receptor immunoreactivity was diffusely distributed in the strata radiatum and oriens of CA1 and in the stratum moleculare of the dentate gyrus. Immunogold electron microscopy revealed that sst2A receptors were predominantly localized postsynaptically, at the plasma membrane of dendritic shafts and spines of principal neurons. During kindling epileptogenesis, qualitative and semiquantitative analysis revealed a progressive decrease of sst2A immunoreactivity in the outer molecular layer, which was spatially associated with an increase in somatostatin immunoreactivity. No obvious changes in sst2A receptor immunoreactivity were observed in other hippocampal subfields. These results suggest that the decrease of sst2A receptor immunoreactivity in the outer molecular layer reflects receptor down-regulation in distal dendrites of granule cells in response to chronic somatostatin release. Because the sst2A receptor appears to mediate anticonvulsant and antiepileptogenic effects of somatostatin, this may represent a pivotal mechanism contributing to epileptogenesis. [ABSTRACT FROM AUTHOR]

AbstractGlutamatergic neurotransmission in the neostriatum and the globus pallidus is mediated through NMDA-type as well as other glutamate receptors and is critical in the expression of basal ganglia function. In order to characterize the cellular, subcellular and subsynaptic localization of NMDA receptors in the neostriatum and globus pallidus, multiple immunocytochemical techniques were applied using antibodies that recognize the NR1 subunit of the NMDA receptor. In order to determine the spatial relationship between NMDA receptors and AMPA receptors, double labelling was performed with the NR1 antibodies and an antibody that recognizes the GluR2 and 3 subunits of the AMPA receptor. In the neostriatum all neurons with characteristics of spiny projection neurons, some interneurons and many dendrites and spines were immunoreactive for NR1. In the globus pallidus most perikarya and many dendritic processes were immunopositive. Immunogold methods revealed that most NR1 labelling is associated with asymmetrical synapses and, like the labelling for GluR2/3, is evenly spread across the synapse. Double immunolabelling revealed that in neostriatum, over 80% of NR1-positive axospinous synapses are also positive for GluR2/3. In the globus pallidus most NR1-positive synapses are positive for GluR2/3. In both regions many synapses labelled only for GluR2/3 were also detected. These results, together with previous data, suggest that NMDA and AMPA receptor subunits are expressed by the same neurons in the neostriatum and globus pallidus and that NMDA and AMPA receptors are, at least in part, colocalized at individual asymmetrical synapses. The synaptic responses to glutamate in these regions are thus likely be mediated by both AMPA and NMDA receptors at the level of individual synapses. [ABSTRACT FROM AUTHOR]

Maitotoxin, a potent marine toxin extracted from peredinians, was found to mimic fertilization in Xenopus oocytes and to trigger the breakdown of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2, the precursor of inositol 1,4,5-trisphosphate], an increase of intracellular pCa and the cortical reaction, including the exocytosis of cortical granules and a wave-like propagation of contraction in the animal hemisphere. All these effects of maitotoxin required the presence of external calcium. Moreover, the toxin considerably increased 24 influx in amphibian oocytes arrested at first meiotic prophase, due to the permanent activation of voltage-dependent Ca2+ channels. Nevertheless it is doubtful that maitotoxin acts primarily as a Ca2+ ionophore or at the level of Ca2+ channels. Indeed no stimulation of Ca2+ uptake was observed in metaphase-II-arrested oocytes, although maitotoxin readily triggered the breakdown of PtdIns(4,5)P2 as well as the cortical reaction in such cells. On the other hand, PtdIns(4,5)P2 breakdown was not reduced in oocytes rnicroinjected with EGTA, although the calcium chelator prevented the oocytes from undergoing the cortical reaction. Taken together, these findings support the view that the toxin might act primarily by increasing PtdIns(4,5)P2 phosphodiesterase activity. [ABSTRACT FROM AUTHOR]

We have studied "in vivo" neurochemically identified striatal neurons to analyze the localisation and the trafficking of dopamine and acetylcholine G protein coupled receptors (GPCR) (D1R, D2R, m2R and m4R) under the influence of neurotransmitter environment. We have identified receptors in tissue sections through immunohistochemical detection at the light and electron microscopic level. We have identified receptors in normal animals and after acute and chronic stimulations. We have quantified receptors through image analysis at the electron microscopic level in relation to various subcellular compartments. Our results demonstrate that, in normal conditions, GPCRs are mostly associated with plasma membrane of the striatal neurons, mostly at extra-synaptic sites. In certain instances (m4R; D2R), receptors have prominent localisation inside the rough endoplasmic reticulum. Our results also show that two distinct receptors for a same neurotransmitter may have distinct subcellular localisation in a same neuronal population (m2R versus m4R) and that the same neurotransmitter receptor (m4R) can have distinct localisation in distinct neuronal populations (cytoplasm versus cell surface). After acute stimulation, cell surface receptors undergo dramatic subcellular changes that involve plasma membrane depletion, internalisation in endosomes and in multivesicular bodies. Such changes are reversible after the end of the stimulation and are blocked by antagonist action. Chronic stimulation also provokes changes in subcellular localisation with specific pattern: plasma membrane depletion, and exaggerated storage of receptors in rough endoplasmic reticulum and eventually Golgi complex (D1R; m2R and m4R). Decreasing chronic receptor stimulation reverses such changes. These results demonstrate that, "in vivo", in the striatum, GPCRs undergo complex intraneuronal trafficking under the influence of neurochemical environment in conditions that dramatically modulate the number of cell surface receptors available for interaction with neurotransmitters or drugs. This confirms that "in vivo", the trafficking and the subcellular compartmentalization of GPCRs may contribute to regulate neuronal sensitivity and neuronal interactions in physiological, experimental and pathological conditions, including in therapeutic conditions.