Absence of VGLUT3 Expression Leads to Impaired Fear Memory in Mice.

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.

Nanoscopic distribution of VAChT and VGLUT3 in striatal cholinergic varicosities suggests colocalization and segregation of the two transporters in synaptic vesicles.

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.

Nicotinic Acetylcholine Receptors Expressed by Striatal Interneurons Inhibit Striatal Activity and Control Striatal-Dependent Behaviors.

Acetylcholine is an important modulator of striatal activity, and it is vital to controlling striatal-dependent behaviors, including motor and cognitive functions. Despite this significance, the mechanisms determining how acetylcholine impacts striatal signaling are still not fully understood. In particular, little is known about the role of nAChRs expressed by striatal interneurons. In the present study, we used FISH to determine which neuronal types express the most prevalent beta2 nicotinic subunit in the mouse striatum. Our data support a common view that nAChR expression is mostly restricted to striatal interneurons. Surprisingly though, cholinergic interneurons were identified as a population with the highest expression of beta2 nicotinic subunit. To investigate the functional significance of beta2-containing nAChRs in striatal interneurons, we deleted them by injecting the AAV-Cre vector into the striatum of beta2-flox/flox male mice. The deletion led to alterations in several behavioral domains, namely, to an increased anxiety-like behavior, decrease in sociability ratio, deficit in discrimination learning, and increased amphetamine-induced hyperlocomotion and c-Fos expression in mice with beta2 deletion. Further colocalization analysis showed that the increased c-Fos expression was present in both medium spiny neurons and presumed striatal interneurons. The present study concludes that, despite being relatively rare, beta2-containing nAChRs are primarily expressed in striatal neurons by cholinergic interneurons and play a significant role in behavior.SIGNIFICANCE STATEMENT A large variety of nAChRs are expressed in the striatum, a brain region that is crucial in the control of behavior. The complexity of receptors with different functions is hindering our understanding of mechanisms through which striatal acetylcholine modulates behavior. We focused on the role of a small population of beta2-containing nAChRs. We identified neuronal types expressing these receptors and determined their impact in the control of explorative behavior, anxiety-like behavior, learning, and sensitivity to stimulants. Additional experiments showed that these alterations were associated with an overall increased activity of striatal neurons. Thus, the small population of nicotinic receptors represents an interesting target for a modulation of response to stimulant drugs and other striatal-based behavior.

A proline-rich motif on VGLUT1 reduces synaptic vesicle super-pool and spontaneous release frequency.

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.

VGLUT3 gates psychomotor effects induced by amphetamine.

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/.

Endocytosis of Activated Muscarinic m2 Receptor (m2R) in Live Mouse Hippocampal Neurons Occurs via a Clathrin-Dependent Pathway.

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.

Structural and Functional Characterization of the Interaction of Snapin with the Dopamine Transporter: Differential Modulation of Psychostimulant Actions.

The importance of dopamine (DA) neurotransmission is emphasized by its direct implication in several neurological and psychiatric disorders. The DA transporter (DAT), target of psychostimulant drugs, is the key protein that regulates spatial and temporal activity of DA in the synaptic cleft via the rapid reuptake of DA into the presynaptic terminal. There is strong evidence suggesting that DAT-interacting proteins may have a role in its function and regulation. Performing a two-hybrid screening, we identified snapin, a SNARE-associated protein implicated in synaptic transmission, as a new binding partner of the carboxyl terminal of DAT. Our data show that snapin is a direct partner and regulator of DAT. First, we determined the domains required for this interaction in both proteins and characterized the DAT-snapin interface by generating a 3D model. Using different approaches, we demonstrated that (i) snapin is expressed in vivo in dopaminergic neurons along with DAT; (ii) both proteins colocalize in cultured cells and brain and, (iii) DAT and snapin are present in the same protein complex. Moreover, by functional studies we showed that snapin produces a significant decrease in DAT uptake activity. Finally, snapin downregulation in mice produces an increase in DAT levels and transport activity, hence increasing DA concentration and locomotor response to amphetamine. In conclusion, snapin/DAT interaction represents a direct link between exocytotic and reuptake mechanisms and is a potential target for DA transmission modulation.

KCC3 loss-of-function contributes to Andermann syndrome by inducing activity-dependent neuromuscular junction defects.

Loss-of-function mutations in the potassium-chloride cotransporter KCC3 lead to Andermann syndrome, a severe sensorimotor neuropathy characterized by areflexia, amyotrophy and locomotor abnormalities. The molecular events responsible for axonal loss remain poorly understood. Here, we establish that global or neuron-specific KCC3 loss-of-function in mice leads to early neuromuscular junction (NMJ) abnormalities and muscular atrophy that are consistent with the pre-synaptic neurotransmission defects observed in patients. KCC3 depletion does not modify chloride handling, but promotes an abnormal electrical activity among primary motoneurons and mislocalization of Na+/K+-ATPase α1 in spinal cord motoneurons. Moreover, the activity-targeting drug carbamazepine restores Na+/K+-ATPase α1 localization and reduces NMJ denervation in Slc12a6-/- mice. We here propose that abnormal motoneuron electrical activity contributes to the peripheral neuropathy observed in Andermann syndrome.

Regulation of the Hippocampal Network by VGLUT3-Positive CCK- GABAergic Basket Cells.

Hippocampal interneurons release the inhibitory transmitter GABA to regulate excitation, rhythm generation and synaptic plasticity. A subpopulation of GABAergic basket cells co-expresses the GABA/glycine vesicular transporters (VIAAT) and the atypical type III vesicular glutamate transporter (VGLUT3); therefore, these cells have the ability to signal with both GABA and glutamate. GABAergic transmission by basket cells has been extensively characterized but nothing is known about the functional implications of VGLUT3-dependent glutamate released by these cells. Here, using VGLUT3-null mice we observed that the loss of VGLUT3 results in a metaplastic shift in synaptic plasticity at Shaeffer's collaterals - CA1 synapses and an altered theta oscillation. These changes were paralleled by the loss of a VGLUT3-dependent inhibition of GABAergic current in CA1 pyramidal layer. Therefore presynaptic type III metabotropic could be activated by glutamate released from VGLUT3-positive interneurons. This putative presynaptic heterologous feedback mechanism inhibits local GABAergic tone and regulates the hippocampal neuronal network.

Vesicular acetylcholine transporter (VAChT) over-expression induces major modifications of striatal cholinergic interneuron morphology and function.

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 (ChAT) promoter (ChAT-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, ChAT-ChR2 mice are a valuable model to investigate hypercholinergic phenotypes. Previous experiments established that ChAT-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 ChAT-ChR2 mice. ChAT-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 ChAT-ChR2 transgenic mice. VAChT immunolabeling was markedly enhanced in the soma and terminal of CIN from ChAT-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 ChAT-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.

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.

A selective and sensitive near-infrared fluorescent probe for acetylcholinesterase imaging.

Two near infra-red (NIR) fluorescent probes HupNIR1 and HupNIR2 based on the huprine scaffold and cyanine 5.0 dye have been synthesised and evaluated in situ for the detection of acetylcholinesterases in different tissues. As anticipated by the initial properties of huprine, both probes displayed a high affinity and selectivity for AChE toward BChE, with IC50 values in the nanomolar range and without any non-specific binding in the tissues. HupNIR2 appears the best probe for AChE with a great selectivity and sensitivity for AChE even in the brain region displaying a low AChE concentration as striatum. Moreover, the binding of HupNIR2 is affected when AChE is inhibited with toxic molecules such as organophosphates. This work provides a new tool to visualize active AChE in biological applications.

Trans-Modulation of the Somatostatin Type 2A Receptor Trafficking by Insulin-Regulated Aminopeptidase Decreases Limbic Seizures.

Within the hippocampus, the major somatostatin (SRIF) receptor subtype, the sst2A receptor, is localized at postsynaptic sites of the principal neurons where it modulates neuronal activity. Following agonist exposure, this receptor rapidly internalizes and recycles slowly through the trans-Golgi network. In epilepsy, a high and chronic release of somatostatin occurs, which provokes, in both rat and human tissue, a decrease in the density of this inhibitory receptor at the cell surface. The insulin-regulated aminopeptidase (IRAP) is involved in vesicular trafficking and shares common regional distribution with the sst2A receptor. In addition, IRAP ligands display anticonvulsive properties. We therefore sought to assess by in vitro and in vivo experiments in hippocampal rat tissue whether IRAP ligands could regulate the trafficking of the sst2A receptor and, consequently, modulate limbic seizures. Using pharmacological and cell biological approaches, we demonstrate that IRAP ligands accelerate the recycling of the sst2A receptor that has internalized in neurons in vitro or in vivo. Most importantly, because IRAP ligands increase the density of this inhibitory receptor at the plasma membrane, they also potentiate the neuropeptide SRIF inhibitory effects on seizure activity. Our results further demonstrate that IRAP is a therapeutic target for the treatment of limbic seizures and possibly for other neurological conditions in which downregulation of G-protein-coupled receptors occurs. SIGNIFICANCE STATEMENT: The somatostatin type 2A receptor (sst2A) is localized on principal hippocampal neurons and displays anticonvulsant properties. Following agonist exposure, however, this receptor rapidly internalizes and recycles slowly. The insulin-regulated aminopeptidase (IRAP) is involved in vesicular trafficking and shares common regional distribution with the sst2A receptor. We therefore assessed by in vitro and in vivo experiments whether IRAP could regulate the trafficking of this receptor. We demonstrate that IRAP ligands accelerate sst2A recycling in hippocampal neurons. Because IRAP ligands increase the density of sst2A receptors at the plasma membrane, they also potentiate the effects of this inhibitory receptor on seizure activity. Our results further demonstrate that IRAP is a therapeutic target for the treatment of limbic seizures.

[Outpatient cannabis withdrawal programme]. / Sevrage ambulatoire du cannabis.

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.

Dymeclin deficiency causes postnatal microcephaly, hypomyelination and reticulum-to-Golgi trafficking defects in mice and humans.

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.

Schwann cells sense and control acetylcholine spillover at the neuromuscular junction by α7 nicotinic receptors and butyrylcholinesterase.

Terminal Schwann cells (TSCs) are key components of the mammalian neuromuscular junction (NMJ). How the TSCs sense the synaptic activity in physiological conditions remains unclear. We have taken advantage of the distinct localization of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) at the NMJ to bring out the function of different ACh receptors (AChRs). AChE is clustered by the collagen Q in the synaptic cleft and prevents the repetitive activation of muscle nicotinic AChRs. We found that BChE is anchored at the TSC by a proline-rich membrane anchor, the small transmembrane protein anchor of brain AChE. When BChE was specifically inhibited, ACh release was significant depressed through the activation of α7 nAChRs localized on the TSC and activated by the spillover of ACh. When both AChE and BChE were inhibited, the spillover increased and induced a dramatic reduction of ACh release that compromised the muscle twitch triggered by the nerve stimulation. α7 nAChRs at the TSC may act as a sensor for spillover of ACh adjusted by BChE and may represent an extrasynaptic sensor for homeostasis at the NMJ. In myasthenic rats, selective inhibition of AChE is more effective in rescuing muscle function than the simultaneous inhibition of AChE and BChE because the concomitant inhibition of BChE counteracts the positive action of AChE inhibition. These results show that inhibition of BChE should be avoided during the treatment of myasthenia and the pharmacological reversal of residual curarization after anesthesia.

Proteomic screening of glutamatergic mouse brain synaptosomes isolated by fluorescence activated sorting.

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.

Peripheral nerve hyperexcitability with preterminal nerve and neuromuscular junction remodeling is a hallmark of Schwartz-Jampel syndrome.

Schwartz-Jampel syndrome (SJS) is a recessive disorder with muscle hyperactivity that results from hypomorphic mutations in the perlecan gene, a basement membrane proteoglycan. Analyses done on a mouse model have suggested that SJS is a congenital form of distal peripheral nerve hyperexcitability resulting from synaptic acetylcholinesterase deficiency, nerve terminal instability with preterminal amyelination, and subtle peripheral nerve changes. We investigated one adult patient with SJS to study this statement in humans. Perlecan deficiency due to hypomorphic mutations was observed in the patient biological samples. Electroneuromyography showed normal nerve conduction, neuromuscular transmission, and compound nerve action potentials while multiple measures of peripheral nerve excitability along the nerve trunk did not detect changes. Needle electromyography detected complex repetitive discharges without any evidence for neuromuscular transmission failure. The study of muscle biopsies containing neuromuscular junctions showed well-formed post-synaptic element, synaptic acetylcholinesterase deficiency, denervation of synaptic gutters with reinnervation by terminal sprouting, and long nonmyelinated preterminal nerve segments. These data support the notion of peripheral nerve hyperexcitability in SJS, which would originate distally from synergistic actions of peripheral nerve and neuromuscular junction changes as a result of perlecan deficiency.

CLIPR-59: a protein essential for neuromuscular junction stability during mouse late embryonic development.

CLIPR-59 is a new member of the cytoplasmic linker proteins (CLIP) family mainly localized to the trans-Golgi network. We show here that Clipr-59 expression in mice is restricted to specific pools of neurons, in particular motoneurons (MNs), and progressively increases from embryonic day 12.5 (E12.5) until the first postnatal days. We generated a Clipr-59 knockout mouse model that presents perinatal lethality due to respiratory defects. Physiological experiments revealed that this altered innervation prevents the normal nerve-elicited contraction of the mutant diaphragm that is reduced both in amplitude and fatigue-resistance at E18.5, despite unaffected functional muscular contractility. Innervation of the mutant diaphragm is not altered until E15.5, but is then partially lost in the most distal parts of the muscle. Ultrastructural observations of neuromuscular junctions (NMJs) in the distal region of the diaphragm reveal a normal organization, but a lower density of nerve terminals capped by terminal Schwann cells in E18.5 mutant when compared with control embryos. Similar defects in NMJ stability, with a hierarchy of severity along the caudo-rostral axis, are also observed in other muscles innervated by facial and spinal MNs in Clipr-59 mutant mice. Clipr-59 deficiency therefore affects axon maintenance but not axon guidance toward muscle targets. Thus, CLIPR-59 is involved in the stabilization of specific motor axons at the NMJ during mouse late embryogenesis and its role is crucial for mouse perinatal development.

Postsynaptic muscarinic m2 receptors at cholinergic and glutamatergic synapses of mouse brainstem motoneurons.

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.