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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]

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]

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]

Abstract: By using immunohistochemical approaches at the light and electron microscopic levels, we have shown that aging modifies the subcellular distribution of the m2 muscarinic autoreceptor (m2R) differentially at somato-dendritic postsynaptic sites and at axonal presynaptic sites in cholinergic basalocortical neurons, in vivo. In cholinergic perikarya and dendrites of the nucleus basalis magnocellularis (NBM), aging is associated with a decrease of the density of m2R at the plasma membrane and in the cytoplasm, suggesting a decrease of the total number of m2R in the somato-dendritic field. In contrast, the number of substance P receptors per somato-dendritic surface was not affected. In the frontal cortex (FC), we have shown a decrease of cytoplasmic m2R density also leading to a decrease of the number of m2R per surface of varicosities but with no change of the density of m2R at the membrane. Our results suggest that the decrease of m2R in the somato-dendritic field of the NBM, but not a modification of the number of presynaptic m2 autoreceptors at the plasma membrane in the FC, could contribute to the decrease of the efficacy of cholinergic transmission observed with aging in the rat. [Copyright &y& Elsevier]

Exposure to stressors has profound effects on sleep that have been linked to serotonin (5-HT) neurons of the dorsal raphe nucleus (DR). However, the DR also comprises glutamatergic neurons expressing vesicular glutamate transporter type 3 (DR VGLUT3 ), leading us to examine their role. Cell-type-specific tracing revealed that DR VGLUT3 neurons project to brain areas regulating arousal and stress. We found that chemogenetic activation of DR VGLUT3 neurons mimics stress-induced sleep perturbations. Furthermore, deleting VGLUT3 in the DR attenuated stress-induced sleep perturbations, especially after social defeat stress. In the DR, VGLUT3 is found in subsets of 5-HT and non-5-HT neurons. We observed that both populations are activated by acute stress, including those projecting to the ventral tegmental area. However, deleting VGLUT3 in 5-HT neurons minimally affected sleep regulation. These findings suggest that VGLUT3 expression in the DR drives stress-induced sleep perturbations, possibly involving non-5-HT DR VGLUT3 neurons., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cholinergic striatal interneurons (ChIs) express the vesicular glutamate transporter 3 (VGLUT3) which allows them to regulate the striatal network with glutamate and acetylcholine (ACh). In addition, VGLUT3-dependent glutamate increases ACh vesicular stores through vesicular synergy. A missense polymorphism, VGLUT3-p.T8I, was identified in patients with substance use disorders (SUDs) and eating disorders (EDs). A mouse line was generated to understand the neurochemical and behavioral impact of the p.T8I variant. In VGLUT3 T8I/T8I male mice, glutamate signaling was unchanged but vesicular synergy and ACh release were blunted. Mutant male mice exhibited a reduced DA release in the dorsomedial striatum but not in the dorsolateral striatum, facilitating habit formation and exacerbating maladaptive use of drug or food. Increasing ACh tone with donepezil reversed the self-starvation phenotype observed in VGLUT3 T8I/T8I male mice. Our study suggests that unbalanced dopaminergic transmission in the dorsal striatum could be a common mechanism between SUDs and EDs., (© 2024. The Author(s).)

Fear is an emotional mechanism that helps to cope with potential hazards. However, when fear is generalized, it becomes maladaptive and represents a core symptom of posttraumatic stress disorder (PTSD). Converging lines of research show that dysfunction of glutamatergic neurotransmission is a cardinal feature of trauma and stress related disorders such as PTSD. However, the involvement of glutamatergic co-transmission in fear is less well understood. Glutamate is accumulated into synaptic vesicles by vesicular glutamate transporters (VGLUTs). The atypical subtype, VGLUT3, is responsible for the co-transmission of glutamate with acetylcholine, serotonin, or GABA. To understand the involvement of VGLUT3-dependent co-transmission in aversive memories, we used a Pavlovian fear conditioning paradigm in VGLUT3 -/- mice. Our results revealed a higher contextual fear memory in these mice, despite a facilitation of extinction. In addition, the absence of VGLUT3 leads to fear generalization, probably because of a pattern separation deficit. Our study suggests that the VGLUT3 network plays a crucial role in regulating emotional memories. Hence, VGLUT3 is a key player in the processing of aversive memories and therefore a potential therapeutic target in stress-related disorders., Competing Interests: The authors declare no competing financial interests., (Copyright © 2023 de Almeida et al.)

Striatal cholinergic interneurons (CINs) use acetylcholine (ACh) and glutamate (Glut) to regulate the striatal network since they express vesicular transporters for ACh (VAChT) and Glut (VGLUT3). However, whether ACh and Glut are released simultaneously and/or independently from cholinergic varicosities is an open question. The answer to that question requires the multichannel detection of vesicular transporters at the level of single synaptic vesicle (SV). Here, we used super-resolution STimulated Emission Depletion microscopy (STED) to characterize and quantify the distribution of VAChT and VGLUT3 in CINs SVs. Nearest-neighbor distances analysis between VAChT and VGLUT3-immunofluorescent spots revealed that 34% of CINs SVs contain both VAChT and VGLUT3. In addition, 40% of SVs expressed only VAChT while 26% of SVs contain only VGLUT3. These results suggest that SVs from CINs have the potential to store simultaneously or independently ACh and/or Glut. Overall, these morphological findings support the notion that CINs varicosities can signal with either ACh or Glut or both with an unexpected level of complexity., Competing Interests: SDu was employed by Oramacell. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Cristofari, Desplanque, Poirel, Hébert, Dumas, Herzog, Danglot, Geny, Gilles, Geeverding, Bolte, Canette, Trichet, Fabre, Daumas, Pietrancosta, El Mestikawy and Bernard.)

Glutamate secretion at excitatory synapses is tightly regulated to allow for the precise tuning of synaptic strength. Vesicular Glutamate Transporters (VGLUT) accumulate glutamate into synaptic vesicles (SV) and thereby regulate quantal size. Further, the number of release sites and the release probability of SVs maybe regulated by the organization of active-zone proteins and SV clusters. In the present work, we uncover a mechanism mediating an increased SV clustering through the interaction of VGLUT1 second proline-rich domain, endophilinA1 and intersectin1. This strengthening of SV clusters results in a combined reduction of axonal SV super-pool size and miniature excitatory events frequency. Our findings support a model in which clustered vesicles are held together through multiple weak interactions between Src homology three and proline-rich domains of synaptic proteins. In mammals, VGLUT1 gained a proline-rich sequence that recruits endophilinA1 and turns the transporter into a regulator of SV organization and spontaneous release., Competing Interests: XZ, UF, KS, MA, MF, MM, CM, VB, FC, MD, SP, UM, AB, SW, SE, YH, EH No competing interests declared, (© 2019, Zhang et al.)

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/., (© 2018 International Society for Neurochemistry.)

Our aim was to examine the dynamics of the muscarinic m2 receptor (m2R), a G-protein coupled receptor (GPCR), after agonist activation in living hippocampal neurons, and especially clathrin dependency endocytosis. We have previously shown that the m2R undergoes agonist-induced internalization in vivo . However, the nature of the endocytotic pathway used by m2R after activation is still unknown in living neurons. Using live cell imaging and quantitative analyses, we have monitored the effect of stimulation on the fate of the membrane-bound m2R and on its redistribution in intraneuronal compartments. Shortly (6 min) after activation, m2R is internalized into clathrin immunopositive structures. Furthermore, after clathrin-dependent endocytosis, m2R associates with early and late endosomes and with subcellular organelles involved in degradation. Together, these results provide, for the first time, a description of m2R trafficking in living neurons and prove that m2R undergoes clathrin-dependent endocytosis before being degraded.

Dymeclin is a Golgi-associated protein whose deficiency causes Dyggve-Melchior-Clausen syndrome (DMC, MIM #223800), a rare recessively inherited spondyloepimetaphyseal dysplasia consistently associated with postnatal microcephaly and intellectual disability. While the skeletal phenotype of DMC patients has been extensively described, very little is known about their cerebral anomalies, which result in brain growth defects and cognitive dysfunction. We used Dymeclin-deficient mice to determine the cause of microcephaly and to identify defective mechanisms at the cellular level. Brain weight and volume were reduced in all mutant mice from postnatal day 5 onward. Mutant mice displayed a narrowing of the frontal cortex, although cortical layers were normally organized. Interestingly, the corpus callosum was markedly thinner, a characteristic we also identified in DMC patients. Consistent with this, the myelin sheath was thinner, less compact and not properly rolled, while the number of mature oligodendrocytes and their ability to produce myelin basic protein were significantly decreased. Finally, cortical neurons from mutant mice and primary fibroblasts from DMC patients displayed substantially delayed endoplasmic reticulum to Golgi trafficking, which could be fully rescued upon Dymeclin re-expression. These findings indicate that Dymeclin is crucial for proper myelination and anterograde neuronal trafficking, two processes that are highly active during postnatal brain maturation., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Cannabis is the most consumed illicit substance in France, and its use can lead to dependency. Lille university hospital, le Pari association, offers patients wanting to stop using cannabis a support therapy based on positive feedback led by nurses, as well as symptomatic treatment of anxiety and sleep disorders.

Motoneurons in the spinal nucleus of the bulbocavernosus (SNB) and their target bulbocavernosus (BC) and levator ani (LA) muscles play a role in male copulation and fertility. Testosterone (T) induces sexual differentiation of this SNB neuromuscular system during development and maintains its activation in adulthood. In the rat, T-induced effects mostly involve the androgen receptor (AR). However, the role of central AR in T-induced effects remains to be studied with pertinent genetic models. We addressed this question by using specific motoneuron immunolabeling and retrograde tracing in mice selectively disrupted for AR in the nervous system. This work reveals that nervous system AR is not required either for T-induced development of BC-LA muscles and perinatal sparing of SNB motoneurons from atrophy or for adult sensitivity of BC-LA muscles to T. By contrast, loss of AR expression in the nervous system resulted in SNB motoneurons having smaller somata and shorter dendrites than controls. We studied the effects of adult castration and T supplementation on SNB cell morphology in control and mutant males; these experiments showed that central AR is involved in the developmental regulation of soma size and dendritic length and in the adult maintenance of soma size of SNB motoneurons. T seemed to act indirectly through BC-LA muscles to maintain dendritic length in adulthood. Our results also suggest that central AR functions may contribute to normal activity of SNB motoneurons and perineal muscles because mutant mice displayed diminished copulatory behavior and fertility.

Acetylcholinesterase (AChE) terminates the action of acetylcholine at cholinergic synapses thereby preventing rebinding of acetylcholine to nicotinic postsynaptic receptors at the neuromuscular junction. Here we show that AChE is not localized close to these receptors on the postsynaptic surface, but is instead clustered along the presynaptic membrane and deep in the postsynaptic folds. Because AChE is anchored by ColQ in the basal lamina and is linked to the plasma membrane by a transmembrane subunit (PRiMA), we used a genetic approach to evaluate the respective contribution of each anchoring oligomer. By visualization and quantification of AChE in mouse strains devoid of ColQ, PRiMA or AChE, specifically in the muscle, we found that along the nerve terminus the vast majority of AChE is anchored by ColQ that is only produced by the muscle, whereas very minor amounts of AChE are anchored by PRiMA that is produced by motoneurons. In its synaptic location, AChE is therefore positioned to scavenge ACh that effluxes from the nerve by non-quantal release. AChE-PRiMA, produced by the muscle, is diffusely distributed along the muscle in extrajunctional regions., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Neuropeptides play a major role in the modulation of information processing in neural networks. Somatostatin, one of the most concentrated neuropeptides in the brain, is found in many sensory systems including the olfactory pathway. However, its cellular distribution in the mouse main olfactory bulb (MOB) is yet to be characterized. Here we show that approximately 95% of mouse bulbar somatostatin-immunoreactive (SRIF-ir) cells describe a homogeneous population of interneurons. These are restricted to the inner lamina of the external plexiform layer (iEPL) with dendritic field strictly confined to the region. iEPL SRIF-ir neurons share some morphological features of Van Gehuchten short-axon cells, and always express glutamic acid decarboxylase, calretinin, and vasoactive intestinal peptide. One-half of SRIF-ir neurons are parvalbumin-ir, revealing an atypical neurochemical profile when compared to SRIF-ir interneurons of other forebrain regions such as cortex or hippocampus. Somatostatin is also present in fibers and in a few sparse presumptive deep short-axon cells in the granule cell layer (GCL), which were previously reported in other mammalian species. The spatial distribution of somatostatin interneurons in the MOB iEPL clearly outlines the region where lateral dendrites of mitral cells interact with GCL inhibitory interneurons through dendrodendritic reciprocal synapses. Symmetrical and asymmetrical synaptic contacts occur between SRIF-ir dendrites and mitral cell dendrites. Such restricted localization of somatostatin interneurons and connectivity in the bulbar synaptic network strongly suggest that the peptide plays a functional role in the modulation of olfactory processing., ((c) 2010 Wiley-Liss, Inc.)

A mouse strain with a deleted acetylcholinesterase (AChE) gene (AChE knockout) shows a decreased inspiration time and increased tidal volume and ventilation .To investigate the respective roles of AChE in brain and muscle, we recorded respiration by means of whole-body plethysmography in knockout mice with tissue selective deletions in AChE expression. A mouse strain with the anchoring domains of AChE deleted (del E5+6 knockout mice) has very low activity in the brain and neuromuscular junction, but increased monomeric AChE in serum. A mouse strain with deletion of the muscle specific region of AChE (del i1RR knockout mice) exhibits no expression in muscle, but unaltered expression in the central nervous system. Neither strain exhibits the pronounced phenotypic traits observed in the complete AChE knockout strain. A third strain lacking the anchor molecule PRiMA, has no functional AChE and butyrylcholinesterase (BChE) in brain and an unaltered respiratory function. BChE inhibition by bambuterol decreases tidal volume and body temperature in del E5+6 and i1RR knockout strains, but not in PRiMA deletion or wild-type controls. We find that: (1) deletion of the full AChE gene is required for a pronounced alteration in respiratory phenotype, (2) BChE is involved in respiratory muscles contraction and temperature control in del E5+6 and i1RR knockout mice, and (3) AChE expression requiring a gene product splice to either exons 5 and 6 or regulated by intron1 influences temperature control.

Schwartz-Jampel syndrome (SJS) is a recessive neuromyotonia with chondrodysplasia. It results from hypomorphic mutations of the gene encoding perlecan, leading to a decrease in the levels of this heparan sulphate proteoglycan in basement membranes (BMs). It has been suggested that SJS neuromyotonia may result from endplate acetylcholinesterase (AChE) deficiency, but this hypothesis has never been investigated in vivo due to the lack of an animal model for neuromyotonia. We used homologous recombination to generate a knock-in mouse strain with one missense substitution, corresponding to a human familial SJS mutation (p.C1532Y), in the perlecan gene. We derived two lines, one with the p.C1532Y substitution alone and one with p.C1532Y and the selectable marker Neo, to down-regulate perlecan gene activity and to test for a dosage effect of perlecan in mammals. These two lines mimicked SJS neuromyotonia with spontaneous activity on electromyogramm (EMG). An inverse correlation between disease severity and perlecan secretion in the BMs was observed at the macroscopic and microscopic levels, consistent with a dosage effect. Endplate AChE levels were low in both lines, due to synaptic perlecan deficiency rather than major myofibre or neuromuscular junction disorganization. Studies of muscle contractile properties showed muscle fatigability at low frequencies of nerve stimulation and suggested that partial endplate AChE deficiency might contribute to SJS muscle stiffness by potentiating muscle force. However, physiological endplate AChE deficiency was not associated with spontaneous activity at rest on EMG in the diaphragm, suggesting that additional changes are required to generate such activity characteristic of SJS.

At the neuromuscular junction (NMJ) acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) can hydrolyze acetylcholine (ACh). Released ACh quanta are known to diffuse rapidly across the narrow synaptic cleft and pairs of ACh molecules cooperate to open endplate channels. During their diffusion through the cleft, or after being released from muscle nicotinic ACh receptors (nAChRs), most ACh molecules are hydrolyzed by AChE highly concentrated at the NMJ. Advances in mouse genomics offered new approaches to assess the role of specific cholinesterases involved in synaptic transmission. AChE knockout mice (AChE-KO) provide a valuable tool for examining the complete abolition of AChE activity and the role of BChE. AChE-KO mice live to adulthood, and exhibit an increased sensitivity to BChE inhibitors, suggesting that BChE activity facilitated their survival and compensated for AChE function. Our results show that BChE is present at the endplate region of wild-type and AChE-KO mature muscles. The decay time constant of focally recorded miniature endplate currents was 1.04 +/- 0.06 ms in wild-type junctions and 5.4 ms +/- 0.3 ms in AChE-KO junctions, and remained unaffected by BChE-specific inhibitors, indicating that BChE is not limiting ACh duration on endplate nAChRs. Inhibition of BChE decreased evoked quantal ACh release in AChE-KO NMJs. This reduction in ACh release can explain the greatest sensitivity of AChE-KO mice to BChE inhibitors. BChE is known to be localized in perisynaptic Schwann cells, and our results strongly suggest that BChE's role at the NMJ is to protect nerve terminals from an excess of ACh.