Nerve Terminals Research Articles

Immunohistochemical analysis of glutamatergic and serotonergic signaling pathways in chemosensory cell clusters in the pharynx and larynx of rats

The present study examined cellular components and the localization of vesicular glutamate transporter (VGLUT) 1 and 2 and serotonin (5-HT) in chemosensory cell clusters in the rat pharynx and larynx. Triple immunolabeling for guanine nucleotide-binding protein G (t), subunit ⍺3 (GNAT3) and nucleoside triphosphate diphosphohydrolase 2 (NTPDase2) with synaptotagmin-1 (Syt1) revealed NTPDase2-immunoreactive type I-like cells in addition to GNAT3-immunoreactive type II-like and Syt1-immunoreactive type III-like cells in pharyngolaryngeal chemosensory cell clusters. Therefore, these clusters appear to comprise similar cell types to those in the lingual taste buds with slight morphological modifications. An immunofluorescence analysis of VGLUT1 or VGLUT2 and GNAT3 with P2X3 purinoceptors revealed that VGLUTs co-localized to P2X3‐immunoreactive spherical nerve terminals closely associated with GNAT3-immunoreactive type II-like cells. Moreover, triple immunolabeling for Syt1/synaptosomal-associated protein, 25 kDa (SNAP25) and P2X3 with VGLUT1 or VGLUT2 revealed punctate immunoreactive products for VGLUT1 and VGLUT2 within P2X3‐immunoreactive flat axon terminals wrapped around Syt1/SNAP25-immunoreactive type III-like cells. The afferent nerve fibers innervating cell clusters may contain glutamate and release it by exocytosis. On the other hand, immunoreactive products for 5-HT and dopa decarboxylase were detected in Syt1-immunoreactive cells, indicating the release of 5-HT by these cells. The present results suggest that chemosensory cell clusters in the pharynx and larynx may be modulated by intrinsic glutamate and 5-HT.

Phospholipid scramblase Xkr8 is required for developmental axon pruning via phosphatidylserine exposure

The mature mammalian brain connectome emerges during development via the extension and pruning of neuronal connections. Glial cells have been identified as key players in the phagocytic elimination of neuronal synapses and projections. Recently, phosphatidylserine has been identified as neuronal “eat‐me” signal that guides elimination of unnecessary input sources, but the associated transduction systems involved in such pruning are yet to be described. Here, we identified Xk‐related protein 8 (Xkr8), a phospholipid scramblase, as a key factor for the pruning of axons in the developing mammalian brain. We found that mouse Xkr8 is highly expressed immediately after birth and required for phosphatidylserine exposure in the hippocampus. Mice lacking Xkr8 showed excess excitatory nerve terminals, increased density of cortico‐cortical and cortico‐spinal projections, aberrant electrophysiological profiles of hippocampal neurons, and global brain hyperconnectivity. These data identify phospholipid scrambling by Xkr8 as a central process in the labeling and discrimination of developing neuronal projections for pruning in the mammalian brain.

Role of magnetic resonance neurography in assessment of lumbosacral radiculo-plexopathy: correlation with electrophysiological studies

BackgroundLumbosacral plexus is a collection of nerves that converge and diverge and finally merge into terminal nerves that innervate the pelvis and lower limbs. Lumbosacral plexopathy is a clinical disease characterized by motor and sensory abnormalities that can result from damage to the lumbosacral plexus by different types of insults. Electrophysiological studies are used to demonstrate the presence of lumbosacral radiculo-plexopathy, but it is restricted owing to deep position of nerves and regional differences in muscle nerve supply. High-resolution MR neurography can simply show the precise site, size, etiology, and type of the lumbosacral radiculo-plexopathy. The aim of this study was to assess the various diseases affecting the lumbosacral plexus with their 1.5-T MR neurography imaging findings and to correlate these findings with electrophysiological studies.ResultsFifty adult patients with clinical presentation of lumbosacral radiculo-plexopathy were included in this cross-sectional study. Based on clinical diagnosis, the sensitivity of electrophysiological studies in diagnosing lumbosacral radiculo-plexopathy was 80% and the sensitivity of MR neurography in diagnosing lumbosacral radiculo-plexopathy was 90%. While based on electrophysiological studies, the sensitivity of MR neurography in diagnosing lumbosacral radiculo-plexopathy was 97.5% and the specificity was 75.86%. There was a high statistically significant relation between the side affected in electrophysiological studies and the side affected in MR neurography and between the stage of the lesion in the electrophysiological studies and in MR neurography among patients (p ≤ 0.001, p ≤ 0.001) , respectively.ConclusionsMR neurography of the lumbosacral plexus is a helpful non-invasive approach for the assessment of patients with inconclusive lumbar spine MR imaging because of its ability for diagnosis of neuromuscular lesions and determining their causes. Electrophysiological studies assess the nerve function and MR neurography assesses the nerve anatomy, and therefore, the correlation between electrophysiological studies and MR neurography are complementary for detection of lumbosacral radiculo-plexopathy.

Cynarin, a caffeoylquinic acid derivative in artichoke, inhibits exocytotic glutamate release from rat cortical nerve terminals (synaptosomes)

The purpose of this study was to evaluate the effect of cynarin, a caffeoylquinic acid derivative in artichoke, on glutamate release elicited by 4-aminopyridine (4-AP) in rat cortical nerve terminals (synaptosomes). We observed that cynarin decreased 4-aminopyridine-elicited glutamate release, which was prevented by the removal of external free Ca2+ with ethylene glycol bis (β-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA) or the blockade of P/Q-type calcium channels with ω-agatoxin IVA. Molecular docking also revealed that cynarin formed a hydrogen bond with the P/Q-type Ca2+ channel, indicating a mechanism of action involving Ca2+ influx inhibition. Additionally, the inhibitory effect of cynarin on glutamate release is associated with a change in the available synaptic vesicles, as cynarin decreased 4-AP-elicited FM1-43 release or hypertonic sucrose-evoked glutamate release from synaptosomes. Furthermore, the suppression of protein kinase A (PKA) prevented the effect of cynarin on 4-AP-elicited glutamate release. 4-AP-elicited PKA and synapsin I or synaptosomal-associated protein of 25 kDa (SNAP-25) phosphorylation at PKA-specific residues were also attenuated by cynarin. Our data indicate that cynarin, through the suppression of P/Q-type Ca2+ channels, inhibits PKA activation and attenuates synapsin I and SNAP-25 phosphorylation at PKA-specific residues, thus decreasing synaptic vesicle availability and contributing to glutamate release inhibition in cerebral cortex terminals.

Anatomical analysis of the intramuscular distribution patterns of the levator scapulae and the clinical implications for pain management.

The present study aimed to demonstrate the intramuscular nerve distribution of the levator scapulae muscle that is responsible for pain and to use this anatomic data to propose possible injection sites. Twenty levator scapulae muscles were dissected from 16 Korean embalmed cadavers. The intramuscular nerve distribution of the levator scapulae muscle was identified by whole-mount nerve staining to preserve and stain the nerve fibers without damage. The posterior ramus of spinal nerves C3, C4, and C5 innervated the levator scapulae muscles. When the origin and insertion of the muscle were set to 0% and 100%, respectively, most of the intramuscular nerve terminals were located between 30 and 70%. This area may correspond to the cricoid cartilage of the sixth cervical vertebra. Most intramuscular nerve terminals can be found in the middle and distal portions of the levator scapulae muscle. Our findings improve our understanding of the intramuscular nerve distribution of the levator scapulae muscle and will aid in pain management in clinical practice.

Morphology of neuromuscular junction in patients with peripheral artery disease

Peripheral artery disease (PAD) is primarily caused by occlusion of arteries that supply blood to the lower limbs and presents significantly pathological changes of calf muscles, include degenerating and regenerating fibers, reduction of the skeletal muscle area, connective tissue proliferation and fibrosis, fat accumulation, and abnormal mitochondria, all of which are predictors of muscle functional decline. Additionally, skeletal muscles also require the motor nerve innervation for the signal transmission through the neuromuscular junction (NMJ) to induce skeletal muscle contraction. To observe whether the NMJ is changed in the PAD patients, gastrocnemius muscles were collected from 2 health subjects and 6 PAD stage III/IV patients. The motor nerve terminals were labeled with anti-neurofilament 200 and anti-synaptophysin antibodies, the nicotinic acetylcholine receptors (nAChRs) were labeled with α-Bungarotoxin. Labeled NMJs were imaged using a laser scanning confocal microscope with Z-stack. The image was captured in 65 NMJs from health subjects and 140 NMJs from PAD patients, respectively. The total nAChR area and the number of discrete fragments of each nAChR cluster, the area per fragment, the percentage of fragmented nAChR clusters, the nerve innervation and the nerve terminal occupancy were calculated. The results were represented with mean ± standard error (SE). The nAChR area was 354.9 ± 12.7 and 209.4 ± 8.2, the average number of fragment/ nAChR cluster was 2.6 ± 0.2 and 3.1 ± 0.2, 6.2% and 19.3% of the nAChR clusters were fragmented, and the nerve terminal occupancy was 61.5 ± 1.4 % and 52.0 61.5 ± 1.8 in the health subjects and PAD patients, respectively. All nAChR clusters were innervated by the motor nerve terminal in the health subjects, whereas 8.6% of nAChR clusters were denervated in PAD Patients. These data indicate that the total nAChR area per nAChR cluster and the area of each discrete fragment are reduced and fragmented nAChR clusters are increased in PAD patients. Additionally, the denervation of nAChR clusters is increased in PAD patients. These results suggest that the neuromuscular transmission is compromised in PAD patients, which limits patients’ physical activities. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

BAR Domain Proteins As Putative Regulators of the Protein Liquid Phase in Nerve Terminals in the Central Nervous System

BAR Domain Proteins As Putative Regulators of the Protein Liquid Phase in Nerve Terminals in the Central Nervous System

Regional differences of extrinsic and intrinsic cholinergic innervation and alterations by chronic opioid-induced constipation in the porcine colonic enteric nerve system

The colon is richly innervated by extrinsic (ext) and intrinsic (int) cholinergic nerves from central and enteric nervous system (CNS and ENS) modulating colonic motility, secretion and inflammation. However, a challenge lies in our inability to anatomically distinguish such innervation in the colon. The porcine colon is regarded as a useful translational research model due to many similarities with human. The present study developed an approach to differentiate ext- and int-cholinergic innervation of the porcine colon using double immunolabeling with a novel mouse antibody against human peripheral choline acetyltransferase (hpChAT) combined with a rabbit antibody anti-common ChAT (cChAT) respectively. The spatial configuration of ext- and int- cholinergic innervation in the ENS was assessed on 3D images generated from CLARITY-cleared porcine colonic samples. Their densities were computationally quantitated using Imaris 9 within the inner and outer submucosal and myenteric plexuses (ISP, OSP, MP) along the proximal, transverse and distal colonic regions (pC, tC, dC) collected from 18 adult Yucatan minipigs including 6 naïve (3 of each sex), 4 vehicle controls (2 of each sex), 8 treated with loperamide, a μ opioid receptor agonist (LOP, 0.4 or 3 mg/kg/day, po, 4 weeks, 4/group, 2 of each sex). The specificities of cChAT and hpChAT antibodies were confirmed with porcine cervical vagal trunk (CVT), sacral spinal cord (SSC, S2-S4) and colonic ENS. The double labeling showed the strong cChAT immunoreactive (+) fibers in the CVT and neuronal somata and fibers in the SSC, but no hpChAT+ labeling was visualized in these structures. In the colonic MP, the dense hpChAT+ neurons and fibers and varicose cChAT+ fibers were simultaneously visualized. 3D images demonstrated that int- cholinergic neurons (hpChAT+) were closely surrounded by ext- cholinergic varicose fibers and dot like structures (cChAT+), presumably nerve terminals. The density of cChAT+ but not hpChAT+ fibers+somata showed significant differences among three segments (dC>tC>pC) within three enteric plexuses (MP>OSP>ISP) in naïve pigs. Compared to control groups, the high dose LOP induced a 1.3-fold decrease of cChAT+ fiber density in males (p<0.05) and 1.5-fold in females (p<0.05), and 2.2-fold decrease of hpChAT+ fibers+somata density in males (p<0.05) and 3.6-fold in female (p<0.01) with a 1.5-fold greater decrease in females than males (p<0.05) in the MP of the dC. This study demonstrates for the first time the ext- and int- cholinergic innervation, the spatial configuration in 3D structure, regional variations in the porcine colonic ENS and the reduction by chronic opiate use with greater in females than males. These findings provide neuroanatomical evidence of CNS-gut cross-talk via cholinergic innervation of the colon and point to the neuromodulation of this pathway to alleviate functional colonic disorders like chronic opioid-induced constipation. Supported by NIH/SPARC OT2OD024899. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Chronic Pelvic Pain Develops in Sensory Neuron Conditional Asic3 Null Mice Subjected to Chemical Injury

Sensitization of primary afferents is an important underlying mechanism for visceral hypersensitivity and pain. We investigated the contribution of acid-sensing ion channels (ASICs) in sensory neurons to the development of pain in a model of chemical-induced cystitis. ASICs are assembled as heterotrimers in bladder sensory neurons; however, the deletion of the ASIC3 subunit results in a loss-of-function phenotype with sensory neurons unable to discharge action potentials in response to acidification. In this study, conditional sensory neuron Asic3 knockout (KO) mice ( Asic3 fl/fl ;Avil-Cre +/- ) and control littermates ( Asic3 fl/fl ;Avil-Cre -/- ) received cyclophosphamide (CYP) (IP 80 mg/kg), or saline, every other day for five days. Experimental observations were made after one day (acute) or 14 days (chronic) after the last dose of CYP. In the acute setting, both control and conditional Asic3 KO mice treated with CYP exhibited an irritated bladder phenotype with a high number of voiding events of small volume. However, voiding activity normalized in both groups within two weeks of receiving CYP. In contrast, conditional Asic3 KO mice treated with CYP developed pelvic allodynia that persisted for at least two weeks, whereas control mice had no pain phenotype. In the chronic setting, no apparent edema or inflammatory cells were observed in bladders of control or conditional Asic3 KO mice, indicating that the pelvic allodynia seen in conditional Asic3 KO mice treated with CYP is likely driven by abnormal functioning of the nervous system and not by inflammation. To assess whether, in the chronic setting, the referred pelvic allodynia seen in conditional Asic3 KO mice treated with CYP is caused by sensitized primary afferents, we examined the firing evoked by sustained suprathreshold electrical stimulation of acutely isolated bladder sensory neurons. Sensory neurons were classified based of the sensitivity of the action potential to tetrodotoxin (TTX), as TTX-resistant (TTX-R) or TTX-sensitive (TTX-S). Strikingly, ~ 40% (11/17) of the bladder sensory neurons with TTX-R action potentials from conditional Asic3 KO mice treated with CYP exhibited aberrant firing (i.e., sensitization) in response to suprathreshold stimulation, compared to 3% (1/33) in control mice. These findings indicate that the pelvic allodynia seen in conditional Asic3 KO mice is driven in part by sensitized bladder afferents. Taken together, our studies support the notion that ASICs operate at the nerve terminals to modulate nociceptor excitability and sensitization. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The substantia nigra modulates proximal colon tone and motility in a vagally-dependent manner in the rat

Previous studies have shown that a monosynaptic pathway connects the substantia nigra pars compacta (SNpc) to neurons of the dorsal motor nucleus of the vagus (DMV). This monosynaptic pathway modulates the vagal control of gastric motility. The present study was designed to test the hypothesis that this nigro-vagal pathway also modulates the tone and motility of the proximal colon.In rats, concurrent microinjection of a retrograde tracer in the proximal colon and an anterograde tracer in the SNpc (n=4) showed that bilaterally labelled colonic-projecting preganglionic vagal motoneurons in the DMV received inputs from SNpc neurons. Activation of SNpc neurons via microinjection of the ionotropic glutamate selective agonist, NMDA, increased proximal colonic motility (187±52.6% of baseline, n=13) and tone (323±97.2mg, n=14) as measured via a strain gauge aligned with the colonic circular smooth muscle (paired t test, p<0.0001). Following transfection of SNpc neurons with pAAV-hSyn-hM3D(Gq)-mCherry, chemogenetic activation of nigro-vagal nerve terminals by brainstem application of CNO increased proximal colon motility (157±24.5% of baseline; n=3; unpaired t test; p=0.0399). The increase in colon motility following SNpc neuronal activation was abolished by brainstem pretreatment with the dopaminergic DA1-like antagonist SCH23390 (n=4; one-way ANOVA, p=0.9762 vs baseline; p=0.0159 vs NMDA alone) as well as by acute subdiaphragmatic vagotomy (94±47.9% of baseline; n=3; One-way ANOVA, p=0.9816 vs baseline and p=0.0105 vs NMDA alone). In contrast to the excitatory effects observed upon activation of brainstem D1R following SNpc stimulation, DMV microinjection of DA decreased both the tone (-81±27.3mg, n=7) and motility (49±21.6% of baseline; n=7; paired t test: p=0.0004) of the proximal colon. These inhibitory DA effects were attenuated by application of the DA2-like antagonist L741646 (tone -100±58.9mg vs 99±100.1mg; n=4; paired t test: t3=2.567, p=0.0414 and motility 63±15% vs 144±38% of baseline; n=5; one-way ANOVA, p=0462 in the absence and presence of L741646).These data suggest that a monosynaptic nigro-vagal pathway activates brainstem D1 receptors to increase proximal colon tone and motility in a vagally-dependent manner. This study provides the anatomical and mechanistic basis to explain how loss of SNpc neurons may lead to gastrointestinal dysfunction, including constipation, observed in patients with Parkinson’s Disease. no disclose This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Lack of Sod1 induces disruption of innervation accompanied by Schwann cell proliferation in skeletal muscle

The neuromuscular junction (NMJ) is an excitatory synapse that constitutes the primary site of communication between the nervous system and skeletal muscle and therefore any degenerative changes at the NMJ have the potential to impair muscle function. The primary cellular components of the NMJ are the motor neuron, the muscle fiber and terminal Schwann cells (tSC) that surround the NMJ. The importance of redox homeostasis for the maintenance of NMJ integrity is supported by work from our group showing degenerative changes at NMJs in mice lacking copper zinc superoxide dismutase (CuZnSOD; Sod1 -/- ) as early as 4 months of age. Prior work from others suggests that tSCs contribute trophic support for the NMJ and facilitate motor unit remodeling, but changes in tSC structure and function under conditions of elevated oxidative stress have not been thoroughly explored. Using in vitro assays, we previously showed that primary Schwann cells treated with a sublethal dose of the reactive oxygen species inducing agent Paraquat (PQ) resulted in increased Schwann cell number, ki67+ staining, and gene expression of trophic factors and the critical NMJ organizing protein Agrin. Furthermore, differentiated C2C12 myotubes treated with conditioned media from PQ treated Schwann cell cultures showed increased number of acetylcholine receptor (AChR) plaques. The goal of the current study was to characterize NMJs in young Sod1 -/- mice (2-months) to determine the early effects of elevated oxidative stress on NMJ structure in vivo with a specific focus on tSCs. We hypothesized that loss of Sod1 would lead to aberrant tSC and NMJ structure associated with decreased muscle function. We assessed maximum isometric force, NMJ morphology, and number of Schwann cells in gastrocnemius muscles of WT (n = 3) and Sod1 -/- (n = 3) mice. In Sod1 -/- mice, muscle force was reduced by 26% ( p = 2e-4) with nerve stimulation compared to direct muscle stimulation, and overlap between motor nerve terminals and AChRs was reduced by nearly 60% compared to WT controls. Post-synaptic AChR area was increased by 37% in Sod1 -/- mice, and tSC area and numbers per NMJ were increased by 60% and 155%, respectively in Sod1 -/- mice. Finally, Schwann cells proximal to NMJs showed increased Ki67+ labeling in Sod1 -/- mice compared to WT mice. This analysis demonstrates that early changes in both pre- and post-synaptic NMJ components and reduced neurotransmission due to loss of Sod1 are associated with marked increases in tSC proliferation. Our data implicate a protective role of muscle-resident Schwann cells in preserving NMJ integrity in response to alterations in redox homeostasis that may be mediated in part through activation and proliferation of these cells. These findings indicate that further investigation of the dynamics between Schwann cells and NMJ function is important to increase understanding of neuromuscular degenerativeconditions that involve altered redox homeostasis such as sarcopenia. Supported by the University of Michigan Medical School Office of Research, the American Physiological Society, and NIH AR069620. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A novel RRGW derived peptide is a promising inhibitor of BoNT/A

Clostridium botulinum neurotoxin type A (BoNT/A) is one of the most potent biotoxins ever known. Its entry into neurons could block vesicle exocytosis to abolish the release of neurotransmitters from nerve terminals, thus leading to muscle paralysis. Although there are so many peptides, antibodies and chemical compounds claimed to have anti-toxin activity, no drug is available in the clinical application except equine antitoxin serum. In the present work, a short peptide inhibitor RRGW of BoNT/A was firstly identified by computer-aided ligand-receptor binding simulation, then an RRGW derived peptide was rational designed based on the fragment of SNAP-25 (141–206 aa). Proteolytic assay showed that the anti-toxin activity of the RRGW derived peptide was much higher than that of RRGW. Digit abduction score assay demonstrated that the derived peptide delayed BoNT/A-induced muscle paralysis at a lower concentration by 20-fold than RRGW. The results supported that RRGW derived peptide can be a potential BoNT/A inhibitor candidate for further treating botulism.

Neuronal Cannabinoid CB1 Receptors Suppress the Growth of Melanoma Brain Metastases by Inhibiting Glutamatergic Signalling.

Melanoma is one of the deadliest forms of cancer. Most melanoma deaths are caused by distant metastases in several organs, especially the brain, the so-called melanoma brain metastases (MBMs). However, the precise mechanisms that sustain the growth of MBMs remain elusive. Recently, the excitatory neurotransmitter glutamate has been proposed as a brain-specific, pro-tumorigenic signal for various types of cancers, but how neuronal glutamate shuttling onto metastases is regulated remains unknown. Here, we show that the cannabinoid CB1 receptor (CB1R), a master regulator of glutamate output from nerve terminals, controls MBM proliferation. First, in silico transcriptomic analysis of cancer-genome atlases indicated an aberrant expression of glutamate receptors in human metastatic melanoma samples. Second, in vitro experiments conducted on three different melanoma cell lines showed that the selective blockade of glutamatergic NMDA receptors, but not AMPA or metabotropic receptors, reduces cell proliferation. Third, in vivo grafting of melanoma cells in the brain of mice selectively devoid of CB1Rs in glutamatergic neurons increased tumour cell proliferation in concert with NMDA receptor activation, whereas melanoma cell growth in other tissue locations was not affected. Taken together, our findings demonstrate an unprecedented regulatory role of neuronal CB1Rs in the MBM tumour microenvironment.

Nifedipine Ameliorates Cellular Differentiation Defects of Smn-Deficient Motor Neurons and Enhances Neuromuscular Transmission in SMA Mice.

In spinal muscular atrophy (SMA), mutations in or loss of the Survival Motor Neuron 1 (SMN1) gene reduce full-length SMN protein levels, which leads to the degeneration of a percentage of motor neurons. In mouse models of SMA, the development and maintenance of spinal motor neurons and the neuromuscular junction (NMJ) function are altered. Since nifedipine is known to be neuroprotective and increases neurotransmission in nerve terminals, we investigated its effects on cultured spinal cord motor neurons and motor nerve terminals of control and SMA mice. We found that application of nifedipine increased the frequency of spontaneous Ca2+ transients, growth cone size, cluster-like formations of Cav2.2 channels, and it normalized axon extension in SMA neurons in culture. At the NMJ, nifedipine significantly increased evoked and spontaneous release at low-frequency stimulation in both genotypes. High-strength stimulation revealed that nifedipine increased the size of the readily releasable pool (RRP) of vesicles in control but not SMA mice. These findings provide experimental evidence about the ability of nifedipine to prevent the appearance of developmental defects in SMA embryonic motor neurons in culture and reveal to which extent nifedipine could still increase neurotransmission at the NMJ in SMA mice under different functional demands.

Control of a hippocampal recurrent excitatory circuit by cannabinoid receptor-interacting protein Gap43

The type-1 cannabinoid receptor (CB1R) is widely expressed in excitatory and inhibitory nerve terminals, and by suppressing neurotransmitter release, its activation modulates neural circuits and brain function. While the interaction of CB1R with various intracellular proteins is thought to alter receptor signaling, the identity and role of these proteins are poorly understood. Using a high-throughput proteomic analysis complemented with an array of in vitro and in vivo approaches in the mouse brain, we report that the C-terminal, intracellular domain of CB1R interacts specifically with growth-associated protein of 43 kDa (GAP43). The CB1R-GAP43 interaction occurs selectively at mossy cell axon boutons, which establish excitatory synapses with dentate granule cells in the hippocampus. This interaction impairs CB1R-mediated suppression of mossy cell to granule cell transmission, thereby inhibiting cannabinoid-mediated anti-convulsant activity in mice. Thus, GAP43 acts as a synapse type-specific regulatory partner of CB1R that hampers CB1R-mediated effects on hippocampal circuit function.

Marco Capogna, a pioneering neuroscientist and true European

Marco Capogna, a pioneering neuroscientist and true European

Secretome mediated interactions between sensory neurons and breast cancer cells.

The role of the nervous system in aiding cancer progression and metastasis is an important aspect of cancer pathogenesis. Interaction between cancer cells and neurons in an in vitro platform is a simple and robust method to further understand this phenomenon. In our study, we aimed to examine in vitro reciprocal effect between breast cancer cells and cancer-sensitized peripheral primary sensory neurons. Secretome obtained from either cultured DRG neurons from tumor-burdened rats, or MRMT1 breast cancer cells were used to study neuronal and cancer cell reciprocity. We utilized neurite analysis, modified cell migration assay and cell signaling pathway inhibitors to determine neurite growth patterns and cell migration in PC12/DRG neurons and MRMT1 cells, respectively. MRMT1 secretome was found to induce significant neurite outgrowth in PC12 and primary sensory neurons. Secretome-induced neurite growth in PC12 cells was partly mediated by PI3K and ERK pathways, but not by adenylyl cyclase. Conversely, secretome from tumor-sensitized sensory neuron cultures induced increased rate of migration in cultured MRMT1 cells. Results from our study provide additional support to the hypothesis that both breast cancer cells and nerve terminals secrete signaling messengers that have a reciprocal effect on each other.

Critical Role of the Presynaptic Protein CAST in Maintaining the Photoreceptor Ribbon Synapse Triad.

The cytomatrix at the active zone-associated structural protein (CAST) and its homologue, named ELKS, being rich in glutamate (E), leucine (L), lysine (K), and serine (S), belong to a family of proteins that organize presynaptic active zones at nerve terminals. These proteins interact with other active zone proteins, including RIMs, Munc13s, Bassoon, and the β subunit of Ca2+ channels, and have various roles in neurotransmitter release. A previous study showed that depletion of CAST/ELKS in the retina causes morphological changes and functional impairment of this structure. In this study, we investigated the roles of CAST and ELKS in ectopic synapse localization. We found that the involvement of these proteins in ribbon synapse distribution is complex. Unexpectedly, CAST and ELKS, in photoreceptors or in horizontal cells, did not play a major role in ribbon synapse ectopic localization. However, depletion of CAST and ELKS in the mature retina resulted in degeneration of the photoreceptors. These findings suggest that CAST and ELKS play critical roles in maintaining neural signal transduction in the retina, but the regulation of photoreceptor triad synapse distribution is not solely dependent on their actions within photoreceptors and horizontal cells.

The cholinergic anti-inflammatory pathway inhibits inflammation without lymphocyte relay.

The magnitude of innate inflammatory immune responses is dependent on interactions between peripheral neural and immune cells. In particular, a cholinergic anti-inflammatory pathway (CAP) has been identified in the spleen whereby noradrenaline (NA) released by splenic nerves binds to ß2-adrenergic receptors (β2-AR) on CD4+ T cells which, in turn, release acetylcholine (ACh). The binding of ACh to α7 acetylcholine receptors (α7-AChR) expressed by splenic macrophages inhibits the production of inflammatory cytokines, including tumor necrosis factor (TNF). However, the role of ACh-secreting CD4+ T-cells in the CAP is still controversial and largely based on the absence of this anti-inflammatory pathway in mice lacking T-cells (nude, FoxN1-/-). Using four conscious, non-lymphopenic transgenic mouse models, we found that, rather than acting on CD4+ T-cells, NA released by splenic nerve terminals acts directly onto β2-AR on splenic myeloid cells to exert this anti-inflammatory effect. We also show that, while larger doses of LPS are needed to trigger CAP in nude mouse strain compared to other strains, TNF production can be inhibited in these animals lacking CD4+ T-cell by stimulating either the vagus or the splenic nerve. We demonstrate that CD4+ T-cells are dispensable for the CAP after antibody-mediated CD4+ T-cell depletion in wild type mice. Furthermore, we found that NA-mediated inhibition of in vitro LPS-induced TNF secretion by human or porcine splenocytes does not require α7-AChR signaling. Altogether our data demonstrate that activation of the CAP by stimulation of vagus or splenic nerves in mice is mainly mediated by direct binding of NA to β2-AR on splenic macrophages, and suggest that the same mechanism is at play in larger species.

GABABR silencing of nerve terminals.

Control of neurotransmission efficacy is central to theories of how the brain computes and stores information. Presynaptic G-protein coupled receptors (GPCRs) are critical in this problem as they locally influence synaptic strength and can operate on a wide range of time scales. Among the mechanisms by which GPCRs impact neurotransmission is by inhibiting voltage-gated calcium (Ca2+) influx in the active zone. Here, using quantitative analysis of both single bouton Ca2+ influx and exocytosis, we uncovered an unexpected non-linear relationship between the magnitude of action potential driven Ca2+ influx and the concentration of external Ca2+ ([Ca2+]e). We find that this unexpected relationship is leveraged by GPCR signaling when operating at the nominal physiological set point for [Ca2+]e, 1.2 mM, to achieve complete silencing of nerve terminals. These data imply that the information throughput in neural circuits can be readily modulated in an all-or-none fashion at the single synapse level when operating at the physiological set point.