RESUMEN
We previously reported the presence of P2X3 purinoceptors (P2X3)-expressing subserosal afferent nerve endings consisting of net- and basket-like nerve endings in the rat gastric antrum. These nerve endings may morphologically be vagal mechanoreceptors activated by antral peristalsis. The present study investigated immunoreactivities for vesicular glutamate transporter (VGLUT) 1 and VGLUT2 as well as exocytosis-related proteins, i.e., core components of the SNARE complex (SNAP25, Stx1, and VAMP2) and synaptotagmin-1 (Syt1), in whole-mount preparations of the rat gastric antrum using double immunofluorescence. VGLUT1 immunoreactivity was not detected, whereas VGLUT2 immunoreactivity was observed in P2X3-immunoreactive subserosal nerve endings composed of both net- and basket-like endings. In net-like nerve endings, intense VGLUT2 immunoreactivity was localized in polygonal bulges of reticular nerve fibers and peripheral axon terminals. Furthermore, intense immunoreactivities for SNAP25, Stx1, and VAMP2 were localized in net-like nerve endings. Intense immunoreactivities for VAMP2 and Syt1 were observed in VGLUT2-immunoreactive net-like nerve endings. In basket-like nerve endings, VGLUT2 immunoreactivity was localized in pleomorphic terminal structures and small bulges surrounding the subserosal ganglion, whereas immunoreactivities for SNAP25, Stx1, and VAMP2 were weak in these nerve endings. VGLUT2-immunoreactive basket-like nerve endings were weakly immunoreactive for VAMP2 and Syt1. These results suggest that subserosal afferent nerve endings release glutamate by exocytosis mainly from net-like nerve endings to modulate their mechanoreceptor function.
Asunto(s)
Exocitosis , Ácido Glutámico , Terminaciones Nerviosas , Antro Pilórico , Receptores Purinérgicos P2X3 , Proteína 2 de Transporte Vesicular de Glutamato , Animales , Masculino , Ratas , Ácido Glutámico/metabolismo , Inmunohistoquímica , Terminaciones Nerviosas/metabolismo , Antro Pilórico/inervación , Antro Pilórico/metabolismo , Ratas Wistar , Receptores Purinérgicos P2X3/metabolismo , Proteína 25 Asociada a Sinaptosomas/metabolismo , Sinaptotagmina I/metabolismo , Sintaxina 1/metabolismo , Proteína 2 de Membrana Asociada a Vesículas/metabolismo , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Proteína 2 de Transporte Vesicular de Glutamato/metabolismoRESUMEN
A metabotropic glutamate receptor coupled to phospholipase D (PLD-mGluR) was discovered in the hippocampus over three decades ago. Its pharmacology and direct linkage to PLD activation are well established and indicate it is a highly atypical glutamate receptor. A receptor with the same pharmacology is present in spindle primary sensory terminals where its blockade can totally abolish, and its activation can double, the normal stretch-evoked firing. We report here the first identification of this PLD-mGluR protein, by capitalizing on its expression in primary mechanosensory terminals, developing an enriched source, pharmacological profiling to identify an optimal ligand, and then functionalizing it as a molecular tool. Evidence from immunofluorescence, western and far-western blotting indicates PLD-mGluR is homomeric GluK2, since GluK2 is the only glutamate receptor protein/receptor subunit present in spindle mechanosensory terminals. Its expression was also found in the lanceolate palisade ending of hair follicle, also known to contain the PLD-mGluR. Finally, in a mouse model with ionotropic function ablated in the GluK2 subunit, spindle glutamatergic responses were still present, confirming it acts purely metabotropically. We conclude the PLD-mGluR is a homomeric GluK2 kainate receptor signalling purely metabotropically and it is common to other, perhaps all, primary mechanosensory endings.
Asunto(s)
Fosfolipasa D , Receptores de Glutamato Metabotrópico , Animales , Ratones , Hipocampo/metabolismo , Terminaciones Nerviosas/metabolismo , Fosfolipasa D/metabolismo , Receptores de Glutamato/metabolismo , Receptores de Glutamato Metabotrópico/metabolismoRESUMEN
The present study aimed to investigate the immunolocalization of vesicular glutamate transporter (VGLUT) 1 and 2, and proteins associated with exocytosis, i.e., core components of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex (synaptosomal-associated protein of 25 kDa, syntaxin 1, and vesicle-associated membrane protein 2) and synaptotagmin-1 (Syt1), in incisive papillary taste buds of rats using double-indirect immunofluorescence. No VGLUT1 immunoreactivity was observed, whereas VGLUT2-immunoreactive punctate products were closely associated with guanine nucleotide-binding protein G(t) subunit α3-immmunoreactive cells in taste buds. VGLUT2 was immunolocalized in P2X3 purinoceptor-expressing afferent nerve endings. Synaptosomal-associated protein of 25 kDa, syntaxin 1, and vesicle-associated membrane protein 2 were immunolocalized in nerve endings containing VGLUT2-immunoreactive products as well as a few cells in taste buds. VGLUT2 was co-immunolocalized in some intragemmal nerve endings immunoreactive for Syt1, a calcium sensor implicated in vesicle membrane fusion. The present results suggest that afferent nerve endings innervating incisive taste buds release glutamate by exocytosis to modulate taste cell function.
Asunto(s)
Papilas Gustativas , Ratas , Animales , Papilas Gustativas/metabolismo , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo , Proteína 2 de Membrana Asociada a Vesículas/metabolismo , Sintaxina 1/metabolismo , Terminaciones Nerviosas/metabolismo , Exocitosis/fisiologíaRESUMEN
Excessive glutamate release is known to be involved in the pathogenesis of neurological diseases, and suppression of glutamate release from nerve terminals is considered to be a treatment strategy. In this study, we investigated whether isosaponarin, a flavone glycoside isolated from wasabi leaves, could affect glutamate release in rat cerebral cortex nerve terminals (synaptosomes). The release of glutamate was evoked by the K+ channel blocker 4-aminopyridine (4-AP) and measured by an online enzyme-coupled fluorimetric assay. Isosaponarin produced a concentration-dependent inhibition of 4-AP-evoked glutamate release with a half-maximum inhibition of release value of 22 µM. The inhibition caused by isosaponarin was prevented by eliminating extracellular Ca2+ or by using bafilomycin A1, an inhibitor of synaptic vesicle exocytosis. Isosaponarin decreased intrasynaptosomal rises in Ca2+ levels that were induced by 4-AP, without affecting the synaptosomal membrane potential. The isosaponarin-induced inhibition of glutamate release was significantly prevented in synaptosomes that were pretreated with a combination of the calcium channel blockers ω-conotoxin GVIA (N-type) and ω-agatoxin IVA (P/Q-types). The protein kinase C (PKC) pan-inhibitor GF109203X and the Ca2+-dependent PKC inhibitor Go6976 abolished the inhibition of glutamate release by isosaponarin, while the Ca2+-independent PKC inhibitor rottlerin did not show any effect. The results from immunoblotting assays also showed that isosaponarin lowered PKC, PKCα, synaptosomal-associated protein of 25 kDa (SNAP-25), and myristoylated alanine-rich C-kinase substrate (MARCKS) phosphorylation induced by 4-AP. In addition, FM1-43-labeled synaptic vesicles in synaptosomes showed that treatment with isosaponarin resulted in an attenuation of the 4-AP-induced decrease in fluorescence intensity that is consistent with glutamate release. Transmission electron microscopy of synaptosomes also provided evidence that isosaponarin altered the number of synaptic vesicles. These results indicate that isosaponarin suppresses the Ca2+-dependent PKC/SNAP-25 and MARCKS pathways in synaptosomes, causing a decrease in the number of available synaptic vesicles, which inhibits vesicular glutamate release from synaptosomes.
Asunto(s)
Ácido Glutámico , Sinaptosomas , Animales , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Corteza Cerebral/metabolismo , Ácido Glutámico/metabolismo , Potenciales de la Membrana , Terminaciones Nerviosas/metabolismo , Ratas , Ratas Sprague-Dawley , Sinaptosomas/metabolismoRESUMEN
The fungiform papilla (FP) is a gustatory and somatosensory structure incorporating chorda tympani (CT) nerve fibers that innervate taste buds (TB) and also contain somatosensory endings for touch and temperature. Hedgehog (HH) pathway inhibition eliminates TB, but CT innervation remains in the FP. Importantly, after HH inhibition, CT neurophysiological responses to taste stimuli are eliminated, but tactile responses remain. To examine CT fibers that respond to tactile stimuli in the absence of TB, we used Phox2b-Cre; Rosa26LSL-TdTomato reporter mice to selectively label CT fibers with TdTomato. Normally CT fibers project in a compact bundle directly into TB, but after HH pathway inhibition, CT fibers reorganize and expand just under the FP epithelium where TB were. This widened expanse of CT fibers coexpresses Synapsin-1, ß-tubulin, S100, and neurofilaments. Further, GAP43 expression in these fibers suggests they are actively remodeling. Interestingly, CT fibers have complex terminals within the apical FP epithelium and in perigemmal locations in the FP apex. These extragemmal fibers remain after HH pathway inhibition. To identify tactile end organs in FP, we used a K20 antibody to label Merkel cells. In control mice, K20 was expressed in TB cells and at the base of epithelial ridges outside of FP. After HH pathway inhibition, K20 + cells remained in epithelial ridges but were eliminated in the apical FP without TB. These data suggest that the complex, extragemmal nerve endings within and disbursed under the apical FP are the mechanosensitive nerve endings of the CT that remain after HH pathway inhibition.
Asunto(s)
Proteínas Hedgehog , Papilas Gustativas , Animales , Nervio de la Cuerda del Tímpano/metabolismo , Proteínas Hedgehog/metabolismo , Ratones , Terminaciones Nerviosas/metabolismo , Gusto/fisiología , Papilas Gustativas/metabolismo , LenguaRESUMEN
To elucidate the efferent functions of sensory nerve endings, the distribution of calretinin and vesicular glutamate transporter 1 (VGLUT1) in laryngeal laminar nerve endings and the immunohistochemical distribution of proteins associated with synaptic vesicle release, i.e., t-SNARE (SNAP25 and syntaxin 1), v-SNARE (VAMP1 and VAMP2), synaptotagmin 1 (Syt1), bassoon, and piccolo, were examined. Subepithelial laminar nerve endings immunoreactive for Na+-K+-ATPase α3-subunit (NKAα3) were largely distributed in the whole-mount preparation of the epiglottic mucosa, and several endings were also immunoreactive for calretinin. VGLUT1 immunoreactivity was observed within terminal part near the outline of the small processes of NKAα3-immunoreactive nerve ending. SNAP25, syntaxin 1, and VAMP1 immunoreactivities were detected in terminal parts of calretinin-immunoreactive endings, whereas VAMP2 immunoreactivity was only observed in a few terminals. Terminal parts immunoreactive for calretinin and/or VGLUT1 also exhibited immunoreactivities for Syt1, Ca2+ sensor for membrane trafficking, and for bassoon and piccolo, presynaptic scaffold proteins. The presence of vesicular release-related proteins, including SNARE proteins, in the terminals of laryngeal laminar endings indicate that intrinsic glutamate modulates their afferent activity in an autocrine-like manner.
Asunto(s)
Epiglotis , Ácido Glutámico , Animales , Epiglotis/metabolismo , Ácido Glutámico/metabolismo , Terminaciones Nerviosas/metabolismo , Ratas , Células Receptoras Sensoriales/metabolismo , Proteína 1 de Transporte Vesicular de Glutamato/metabolismoRESUMEN
Somatosensory low threshold mechanoreceptors (LTMRs) sense innocuous mechanical forces, largely through specialized axon termini termed sensory nerve endings, where the mechanotransduction process initiates upon activation of mechanotransducers. In humans, a subset of sensory nerve endings is enlarged, forming bulb-like expansions, termed bulbous nerve endings. There is no in vitro human model to study these neuronal endings. Piezo2 is the main mechanotransducer found in LTMRs. Recent evidence shows that Piezo1, the other mechanotransducer considered absent in dorsal root ganglia (DRG), is expressed at low level in somatosensory neurons. We established a differentiation protocol to generate, from iPSC-derived neuronal precursor cells, human LTMR recapitulating bulbous sensory nerve endings and heterogeneous expression of Piezo1 and Piezo2. The derived neurons express LTMR-specific genes, convert mechanical stimuli into electrical signals and have specialized axon termini that morphologically resemble bulbous nerve endings. Piezo2 is concentrated within these enlarged axon termini. Some derived neurons express low level Piezo1, and a subset co-express both channels. Thus, we generated a unique, iPSCs-derived human model that can be used to investigate the physiology of bulbous sensory nerve endings, and the role of Piezo1 and 2 during mechanosensation.
Asunto(s)
Células Madre Pluripotentes Inducidas , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Canales Iónicos/genética , Canales Iónicos/metabolismo , Mecanorreceptores/metabolismo , Mecanotransducción Celular , Terminaciones Nerviosas/metabolismo , Células Receptoras Sensoriales/metabolismoRESUMEN
BACKGROUND: In the brain, polyamines are mainly synthesized in neurons, but preferentially accumulated in astrocytes, and are proposed to be involved in neurodegenerative/neuroinflammatory disorders and neuron injury. A transgenic mouse overexpressing spermine oxidase (SMOX, which specifically oxidizes spermine) in the neocortex neurons (Dach-SMOX mouse) was proved to be a model of increased susceptibility to excitotoxic injury. METHODS: To investigate possible alterations in synapse functioning in Dach-SMOX mouse, both cerebrocortical nerve terminals (synaptosomes) and astrocytic processes (gliosomes) were analysed by assessing polyamine levels, ezrin and vimentin content, glutamate AMPA receptor activation, calcium influx, and catalase activity. RESULTS: The main findings are as follows: (i) the presence of functional calcium-permeable AMPA receptors in synaptosomes from both control and Dach-SMOX mice, and in gliosomes from Dach-SMOX mice only; (ii) reduced content of spermine in gliosomes from Dach-SMOX mice; and (iii) down-regulation and up-regulation of catalase activity in synaptosomes and gliosomes, respectively, from Dach-SMOX mice. CONCLUSIONS: Chronic activation of SMOX in neurons leads to major changes in the astrocyte processes including reduced spermine levels, increased calcium influx through calcium-permeable AMPA receptors, and stimulation of catalase activity. Astrocytosis and the astrocyte process alterations, depending on chronic activation of polyamine catabolism, result in synapse dysregulation and neuronal suffering.
Asunto(s)
Gliosis/metabolismo , Gliosis/patología , Poliaminas/metabolismo , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Astrocitos/patología , Calcio/metabolismo , Catalasa/metabolismo , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Proteínas del Citoesqueleto/metabolismo , Modelos Animales de Enfermedad , Ratones , Ratones Transgénicos , Terminaciones Nerviosas/efectos de los fármacos , Terminaciones Nerviosas/metabolismo , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Receptores AMPA/metabolismo , Espermina/análogos & derivados , Espermina/metabolismo , Espermina/farmacología , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismo , Vimentina/metabolismo , Poliamino OxidasaRESUMEN
Mechanical and metabolic signals associated with skeletal muscle contraction stimulate the sensory endings of thin fiber muscle afferents and produce reflex increases in sympathetic nerve activity and blood pressure during exercise (i.e., the exercise pressor reflex; EPR). The EPR is exaggerated in patients and animals with heart failure with reduced ejection fraction (HF-rEF) and its activation contributes to reduced exercise capacity within this patient population. Accumulating evidence suggests that the exaggerated EPR in HF-rEF is partially attributable to a sensitization of mechanically activated channels produced by thromboxane A2 receptors (TxA2 -Rs) on those sensory endings; however, this has not been investigated. Accordingly, the purpose of this investigation was to determine the role played by TxA2 -Rs on the sensory endings of thin fiber muscle afferents in the exaggerated EPR in rats with HF-rEF induced by coronary artery ligation. In decerebrate, unanesthetized rats, we found that injection of the TxA2 -R antagonist daltroban (80 µg) into the arterial supply of the hindlimb reduced the pressor response to 30 s of electrically induced 1 Hz dynamic hindlimb muscle contraction in HF-rEF (n = 8, peak ∆MAP pre: 22 ± 3; post: 14 ± 2 mmHg; p = 0.01) but not sham (n = 10, peak ∆MAP pre: 13 ± 3; post: 11 ± 2 mmHg; p = 0.68) rats. In a separate group of HF-rEF rats (n = 4), we found that the systemic (intravenous) injection of daltroban had no effect on the EPR (peak ΔMAP pre: 26 ± 7; post: 25 ± 7 mmHg; p = 0.50). Our data suggest that TxA2 -Rs on thin fiber muscle afferents contribute to the exaggerated EPR evoked in response to dynamic muscle contraction in HF-rEF.
Asunto(s)
Presión Sanguínea , Insuficiencia Cardíaca/metabolismo , Actividad Motora , Receptores de Tromboxano A2 y Prostaglandina H2/metabolismo , Reflejo , Animales , Insuficiencia Cardíaca/fisiopatología , Masculino , Contracción Muscular , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiología , Terminaciones Nerviosas/metabolismo , Terminaciones Nerviosas/fisiología , Ratas , Ratas Sprague-Dawley , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/fisiologíaRESUMEN
Decreasing synaptic release of glutamate may counteract glutamate excitotoxicity in many neurological diseases. In this study, we investigated the effect of albanin A, a constituent in the root bark of Morus alba L., on the release of glutamate in rat cerebral cortex nerve endings (synaptosomes). We found that albanin A at 5-30µM suppressed 4-aminopyridine (4-AP)-induced release of glutamate. This phenomenon was abolished by extracellular calcium removal or by vesicular transporter inhibition, and was associated with a decrease in intrasynaptosomal Ca2+ levels. However, albanin A had no effect on the synaptosomal membrane potential. The inhibition of N- and P/Q-type Ca2+ channels, calmodulin, adenylate cyclase (AC), and protein kinase A, abolished the effect of albanin A on the glutamate release evoked by 4-AP. Moreover, the albanin A-mediated inhibition of glutamate release was prevented by the Ca2+/calmodulin-stimulated AC1 inhibitor. Western blot showed that AC1, but not AC8, was presented in the synaptosomes, and albanin A reduced 4-AP-induced increases in synaptosomal cyclic adenosine monophosphate content. In addition, albanin A pretreatment substantially attenuated neuronal damage in a rat model of kainic acid-induced glutamate excitotoxicity. Our data reveal that albanin A suppresses glutamate release by decreasing Ca2+/calmodulin/AC1 activation in synaptosomes and exerts neuroprotective effect in vivo.
Asunto(s)
Ácido Glutámico , Morus , Adenilil Ciclasas , Animales , Calcio/metabolismo , Calmodulina , Corteza Cerebral/metabolismo , Terminaciones Nerviosas/metabolismo , Corteza de la Planta , Ratas , Ratas Sprague-Dawley , Sinaptosomas/metabolismoRESUMEN
We previously reported the immunoreactivity for the vesicular glutamate transporter 2 (VGLUT2) in afferent nerve terminals attached to chemoreceptor type I cells of the carotid body (CB), suggesting that glutamate is released from afferent terminals to stimulate these cells. In the present study, we examined the immunoreactivity for the glutamate-binding subunits of N-methyl-D-aspartate (NMDA) receptors, GluN2A and GluN2B in the rat CB, and the immunohistochemical relationships between these subunits and VGLUT2. Immunoreactivities for GluN2A and GluN2B were predominant in a subpopulation of tyrosine hydroxylase-immunoreactive type I cells rather than those of dopamine beta-hydroxylase-immunoreactive cells. Punctate VGLUT2-immunoreactive products were attached to GluN2A- and GluN2B-immunoreactive type I cells. Bassoon-immunoreactive products were localized between VGLUT2-immunoreactive puncta and type I cells immunoreactive for GluN2A and GluN2B. These results suggest that afferent nerve terminals release glutamate by exocytosis to modulate chemosensory activity of a subpopulation of type I cells via GluN2A- and GluN2B subunits-containing NMDA receptors.
Asunto(s)
Cuerpo Carotídeo/metabolismo , Terminaciones Nerviosas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo , Animales , Cuerpo Carotídeo/química , Ácido Glutámico/metabolismo , Masculino , Terminaciones Nerviosas/química , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/análisisRESUMEN
Fingolimod, a sphingosine-1-phosphate (S1P) receptor modulator approved for treating multiple sclerosis, is reported to prevent excitotoxic insult. Because excessive glutamate release is a major cause of neuronal damage in various neurological disorders, the effect of fingolimod on glutamate release in rat cerebrocortical nerve terminals (synaptosomes) was investigated in the current study. Fingolimod decreased 4-aminopyridine (4-AP)-stimulated glutamate release and calcium concentration elevation. Fingolimod-mediated inhibition of 4-AP-induced glutamate release was dependent on extracellular calcium, persisted in the presence of the glutamate transporter inhibitor DL-TBOA or intracellular Ca2+-releasing inhibitors dantrolene and CGP37157, and was prevented by blocking vesicular transporters or N- and P/Q-type channels. Western blot and immunocytochemical analysis revealed the presence of S1P1 receptor proteins in presynaptic terminals. Fingolimod-mediated inhibition of 4-AP-induced glutamate release was also abolished by the sphingosine kinase inhibitor DMS, selective S1P1 receptor antagonist W146, Gi/o protein inhibitor pertussis toxin, and G protein ßγ subunit inhibitor gallein; however, it was unaffected by the adenylyl cyclase inhibitor SQ22536, protein kinase A inhibitor H89, and phospholipase C inhibitor U73122. These data indicate that fingolimod decreases glutamate release from rat cerebrocortical synaptosomes by suppressing N- and P/Q-type Ca2+ channel activity; additionally, the activation of presynaptic S1P1 receptors and the G protein ßγ subunit participates in achieving the effect.
Asunto(s)
Corteza Cerebral/metabolismo , Clorhidrato de Fingolimod/farmacología , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Ácido Glutámico/metabolismo , Moduladores de los Receptores de fosfatos y esfingosina 1/farmacología , Receptores de Esfingosina-1-Fosfato/metabolismo , Animales , Corteza Cerebral/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Antagonistas de Aminoácidos Excitadores/farmacología , Subunidades beta de la Proteína de Unión al GTP/agonistas , Subunidades gamma de la Proteína de Unión al GTP/agonistas , Masculino , Terminaciones Nerviosas/efectos de los fármacos , Terminaciones Nerviosas/metabolismo , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Esfingosina-1-Fosfato/agonistasRESUMEN
The long history of studies on the effect of catecholamines on synaptic transmission does not answer the main question about the mechanism of their action on quantal release in the neuromuscular junction. Currently, interest in catecholamines has increased not only because of their widespread use in the clinic for the treatment of cardiovascular and pulmonary diseases but also because of recent data on their possible use for the treatment of certain neurodegenerative diseases, muscle weakness and amyotrophic sclerosis. Nevertheless, the effects and mechanisms of catecholamines on acetylcholine release remain unclear. We investigated the action of noradrenaline and adrenaline on the spontaneous and evoked quantal secretion of acetylcholine in the neuromuscular junction of the rat soleus muscle. Noradrenaline (10 µM) did not change the spontaneous acetylcholine quantal release, the number of released quanta after nerve stimulation, or the timing of the quantal secretion. However, adrenaline at the same concentration increased spontaneous secretion by 40%, increased evoked acetylcholine quantal release by 62%, and synchronized secretion. These effects differ from those previously described by us in the synapses of the frog cutaneous pectoris muscle and mouse diaphragm. This indicates specificity in catecholamine action that depends on the functional type of muscle and the need to take the targeted type of muscle into account in clinical practice.
Asunto(s)
Acetilcolina/metabolismo , Epinefrina/farmacología , Neuronas Motoras/metabolismo , Terminaciones Nerviosas/metabolismo , Transmisión Sináptica/fisiología , Animales , Potenciales Evocados/efectos de los fármacos , Placa Motora/efectos de los fármacos , Placa Motora/metabolismo , Neuronas Motoras/efectos de los fármacos , Terminaciones Nerviosas/efectos de los fármacos , Norepinefrina/farmacología , Ratas Wistar , Transmisión Sináptica/efectos de los fármacosRESUMEN
The present study investigated the morphological characteristics of subserosal afferent nerve endings with immunoreactivity for the P2X3 purinoceptor (P2X3) in the rat stomach by immunohistochemistry of whole-mount preparations using confocal scanning laser microscopy. P2X3 immunoreactivity was observed in subserosal nerve endings proximal and lateral to the gastric sling muscles in the distal antrum of the lesser curvature. Parent axons ramified into several lamellar processes to form net-like complex structures that extended two-dimensionally in every direction on the surface of the longitudinal smooth muscle layer. The axon terminals in the periphery of P2X3-immunoreactive net-like structures were flat and looped or leaf-like in shape. Some net-like lamellar structures and their axon terminals with P2X3 immunoreactivity were also immunoreactive for P2X2. P2X3-immunoreactive nerve fibers forming net-like terminal structures were closely surrounded by S100B-immunoreactive terminal Schwann cells, whereas axon terminals twined around these cells and extended club-, knob-, or thread-like protrusions in the surrounding tissues. Furthermore, a retrograde tracing method using fast blue dye indicated that most of these nerve endings originated from the nodose ganglia of the vagus nerve. These results suggest that P2X3-immunoreactive subserosal nerve endings have morphological characteristics of mechanoreceptors and contribute to sensation of a mechanical deformation of the distal antral wall associated with antral peristalsis.
Asunto(s)
Terminaciones Nerviosas/metabolismo , Terminaciones Nerviosas/ultraestructura , Neuronas Aferentes/citología , Antro Pilórico/inervación , Receptores Purinérgicos P2X3/metabolismo , Animales , Masculino , Microscopía Confocal , Neuronas Aferentes/metabolismo , Ratas , Ratas WistarRESUMEN
Purpose: The purpose of this work was to test whether palisade endings express structural and molecular features of exocytotic machinery, and are associated with acetylcholine receptors, and enzymes for neurotransmitter breakdown. Methods: Extraocular rectus muscles from six cats were studied. Whole-mount preparations of extraocular muscles (EOMs) were immunolabeled with markers for exocytotic proteins, including synaptosomal-associated protein of 25 kDa (SNAP25), syntaxin, synaptobrevin, synaptotagmin, and complexin. Acetylcholine receptors (AChRs) were visualized with α-bungarotoxin and with an antibody against AChRs, and acetylcholinesterase (AChE) was tagged with anti-AChE. Molecular features of multicolor labeled palisade endings were analyzed in the confocal scanning microscope, and their ultrastructural features were revealed in the transmission electron microscope. Results: All palisade endings expressed the exocytotic proteins SNAP25, syntaxin, synaptobrevin, synaptotagmin, and complexin. At the ultrastructural level, vesicles docked at the plasma membrane of terminal varicosities of palisade endings. No AChRs were associated with palisade endings as demonstrated by the absence of α-bungarotoxin and anti-AChR binding. AChE, the degradative enzyme for acetylcholine exhibited low, if any, activity in palisade endings. Axonal tracking showed that axons with multiple en grappe motor terminals were in continuity with palisade endings. Conclusions: This study demonstrates that palisade endings exhibit structural and molecular characteristics of exocytotic machinery, suggesting neurotransmitter release. However, AChRs were not associated with palisade endings, so there is no binding site for acetylcholine, and, due to low/absent AChE activity, insufficient neurotransmitter removal. Thus, the present findings indicate that palisade endings belong to an effector system that is very different from that found in other skeletal muscles.
Asunto(s)
Acetilcolinesterasa/metabolismo , Exocitosis , Terminaciones Nerviosas/metabolismo , Músculos Oculomotores/inervación , Receptores Colinérgicos/metabolismo , Animales , Gatos , Técnica del Anticuerpo Fluorescente , Microscopía Electrónica de Transmisión , Terminaciones Nerviosas/fisiología , Terminaciones Nerviosas/ultraestructura , Músculos Oculomotores/enzimología , Músculos Oculomotores/metabolismo , Músculos Oculomotores/ultraestructura , PropiocepciónRESUMEN
Neferine, a bisbenzylisoquinoline alkaloid present in Nelumbo nucifera, has been reported to exhibit neuroprotective effects. Because reduced glutamatergic transmission through inhibition of glutamate release has been proposed as a mechanism of neuroprotection, we investigated whether and how neferine inhibits glutamate release in the nerve terminals of the cerebral cortex of rats. The results demonstrated that neferine inhibits the glutamate release that is evoked by the potassium channel blocker 4-aminopyridine, doing so in a dose-dependent manner. This effect was prevented by removing extracellular calcium and blocking vesicular transporters or N- and P/Q-type calcium channels but not by blocking glutamate transporters. Neferine decreased the 4-aminopyridine-stimulated elevation in intrasynaptosomal calcium concentration; however, it had no effect on the synaptosomal membrane potential. The inhibition of glutamate release by neferine was also eliminated by the selective 5-hydroxytryptamine 1A (5HT1A) receptor antagonist WAY100635, Gi/o protein inhibitor pertussis toxin, adenylyl cyclase inhibitor MDL12330A, and protein kinase A inhibitor H89. Moreover, immunocytochemical analysis revealed the presence of 5-HT1A receptor proteins in the vesicular transporter of glutamate type 1 positive synaptosomes. The molecular docking study also demonstrated that neferine exhibited the highest binding affinity with 5-HT1A receptors (Autodock scores for 5-HA1A = -11.4 kcal/mol). Collectively, these results suggested that neferine activates 5-HT1A receptors in cortical synaptosomes, which decreases calcium influx and glutamate release through the activation of Gi/o protein and the inhibition of adenylyl cyclase/cAMP/protein kinase A cascade.
Asunto(s)
Bencilisoquinolinas/farmacología , Corteza Cerebral/metabolismo , Ácido Glutámico/metabolismo , Nelumbo , Terminaciones Nerviosas/metabolismo , Receptor de Serotonina 5-HT1A/metabolismo , Alcaloides/aislamiento & purificación , Alcaloides/farmacología , Animales , Bencilisoquinolinas/química , Bencilisoquinolinas/aislamiento & purificación , Corteza Cerebral/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Medicamentos Herbarios Chinos/aislamiento & purificación , Medicamentos Herbarios Chinos/farmacología , Antagonistas de Aminoácidos Excitadores/química , Antagonistas de Aminoácidos Excitadores/aislamiento & purificación , Antagonistas de Aminoácidos Excitadores/farmacología , Masculino , Simulación del Acoplamiento Molecular , Terminaciones Nerviosas/efectos de los fármacos , Estructura Secundaria de Proteína , Ratas , Ratas Sprague-Dawley , Receptor de Serotonina 5-HT1A/químicaRESUMEN
BACKGROUND: Purine receptors play roles in peripheral and central sensitization and are associated with migraine headache. We investigated the possibility that ATP plays a permissive role in the activation of AMPA receptors thus inducing Glu release from nerve terminals isolated from the rat trigeminal caudal nucleus (TCN). METHODS: Nerve endings isolated from the rat TCN were loaded with [3H]D-aspartic acid ([3H]D-ASP), layered into thermostated superfusion chambers, and perfused continuously with physiological medium, alone or with various test drugs. Radioactivity was measured to assess [3H]D-ASP release under different experimental conditions. RESULTS: Synaptosomal [3H]D-ASP spontaneous release was stimulated by ATP and to an even greater extent by the ATP analogue benzoylbenzoylATP (BzATP). The stimulation of [3H]D-ASP basal release by the purinergic agonists was prevented by the selective P2X7 receptor antagonist A438079. AMPA had no effect on basal [3H]D-ASP release, but the release observed when synaptosomes were exposed to AMPA plus a purinoceptor agonist exceeded that observed with ATP or BzATP alone. The selective AMPA receptor antagonist NBQX blocked this "excess" release. Co-exposure to AMPA and BzATP, each at a concentration with no release-stimulating effects, evoked a significant increase in [3H]D-ASP basal release, which was prevented by exposure to a selective AMPA antagonist. CONCLUSIONS: P2X7 receptors expressed on glutamatergic nerve terminals in the rat TCN can mediate Glu release directly and indirectly by facilitating the activation of presynaptic AMPA receptors. The high level of glial ATP that occurs during chronic pain states can promote widespread release of Glu as well as can increase the function of AMPA receptors. In this manner, ATP contributes to the AMPA receptor activation involved in the onset and maintenance of the central sensitization associated with chronic pain.
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Terminaciones Nerviosas/efectos de los fármacos , Terminaciones Nerviosas/metabolismo , Receptores AMPA/metabolismo , Receptores Presinapticos/metabolismo , Receptores Purinérgicos P2X7/fisiología , Núcleo Caudal del Trigémino/metabolismo , Animales , Antagonistas de Aminoácidos Excitadores/farmacología , Masculino , Agonistas del Receptor Purinérgico P2X , Antagonistas del Receptor Purinérgico P2X/farmacología , Ratas , Transmisión Sináptica , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismoRESUMEN
We studied telocytes/CD34+ stromal cells in the normal and pathological peripheral nervous system (PNS), for which we reviewed the literature and contributed our observations under light and electron microscopy in this field. We consider the following aspects: (A) general characteristics of telocytes and the terminology used for these cells (e.g., endoneurial stromal cells) in PNS; (B) the presence, characteristics and arrangement of telocytes in the normal PNS, including (i) nerve epi-perineurium and endoneurium (e.g., telopodes extending into the endoneurial space); (ii) sensory nerve endings (e.g., Meissner and Pacinian corpuscles, and neuromuscular spindles); (iii) ganglia; and (iv) the intestinal autonomic nervous system; (C) the telocytes in the pathologic PNS, encompassing (i) hyperplastic neurogenic processes (neurogenic hyperplasia of the appendix and gallbladder), highly demonstrative of telocyte characteristics and relations, (ii) PNS tumours, such as neurofibroma, schwannoma, granular cell tumour and nerve sheath myxoma, and interstitial cell of Cajal-related gastrointestinal stromal tumour (GIST), (iii) tumour-invaded nerves and (iv) traumatic, metabolic, degenerative or genetic neuropathies, in which there are fewer studies on telocytes, e.g., neuroinflammation and nerves in undescended testicles (cryptorchidism), Klinefelter syndrome, crush injury, mucopolysaccharidosis II (Hunter's syndrome) and Charcot-Marie-Tooth disease.
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Susceptibilidad a Enfermedades , Enfermedades del Sistema Nervioso Periférico/etiología , Enfermedades del Sistema Nervioso Periférico/metabolismo , Sistema Nervioso Periférico/metabolismo , Telocitos/metabolismo , Animales , Biomarcadores , Humanos , Inmunohistoquímica , Terminaciones Nerviosas/metabolismo , Sistema Nervioso Periférico/patología , Enfermedades del Sistema Nervioso Periférico/diagnóstico , Telocitos/ultraestructuraRESUMEN
Numerous genes whose mutations cause, or increase the risk of, Parkinson's disease (PD) have been identified. An inactivating mutation (R258Q) in the Sac inositol phosphatase domain of synaptojanin 1 (SJ1/PARK20), a phosphoinositide phosphatase implicated in synaptic vesicle recycling, results in PD. The gene encoding Sac2/INPP5F, another Sac domain-containing protein, is located within a PD risk locus identified by genome-wide association studies. Knock-In mice carrying the SJ1 patient mutation (SJ1RQKI) exhibit PD features, while Sac2 knockout mice (Sac2KO) do not have obvious neurologic defects. We report a "synthetic" effect of the SJ1 mutation and the KO of Sac2 in mice. Most mice with both mutations died perinatally. The occasional survivors had stunted growth, died within 3 wk, and showed abnormalities of striatal dopaminergic nerve terminals at an earlier stage than SJ1RQKI mice. The abnormal accumulation of endocytic factors observed at synapses of cultured SJ1RQKI neurons was more severe in double-mutant neurons. Our results suggest that SJ1 and Sac2 have partially overlapping functions and are consistent with a potential role of Sac2 as a PD risk gene.
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Inositol Polifosfato 5-Fosfatasas/genética , Enfermedad de Parkinson/enzimología , Animales , Dopamina/metabolismo , Estudio de Asociación del Genoma Completo , Humanos , Inositol Polifosfato 5-Fosfatasas/deficiencia , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Terminaciones Nerviosas/metabolismo , Enfermedad de Parkinson/genética , Fenotipo , Sinapsis/metabolismoRESUMEN
Sensory nerve endings respond to various stimuli and subsequently transmit afferent informations to central nervous system, but their responsibility has been suggested to be modulated by glutamate. In the present study, we examined the immunohistochemical localization of vesicular glutamate transporter 1 (vGLUT1) and vGLUT2 in baroreceptor nerve endings immunoreactive for P2X2 and P2X3 purinoceptors in the rat carotid sinus by immunohistochemistry of whole-mount preparations with confocal scanning laser microscopy. P2X3-immunoreactive flat leaf-like axon terminals were immunoreactive to vGLUT2, but not to vGLUT1. Among members of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex, immunoreactivities for synaptosomal-associated protein, 25â¯kDa, Syntaxin1, and vesicle-associated membrane protein 2 (VAMP2) were localized in P2X2- and P2X3-immunoreactive axon terminals. Punctate immunoreactive products for VAMP2 and vGLUT2 were co-localized in axon terminals. These results suggest that vGLUT2 is localized in P2X3-immunoreactive baroreceptor terminals in the carotid sinus, and these terminals may release glutamate by exocytosis in order to modulate baroreceptor function in the carotid sinus.