RESUMEN
Botulinum neurotoxin (BNT) injection into the cricopharyngeus muscle (CPM) under ultrasound (US) guidance is a minimally invasive technique performed to relieve cricopharyngeal dysphagia by reducing CPM spasticity. This technique is basically accessible only to both lateral sides of the CPM. This cadaveric study aimed to evaluate whether US-guided injection could effectively deliver BNT to abundant areas of gross nerve endings within the CPM. We utilized a newly modified Sihler's staining method to identify regions with abundant neural endings within the CPM while preserving the three-dimensional morphology of the muscle in 10 sides of 5 fresh cadavers. A mixture of 0.2 mL dye was injected into the 16 sides of CPM under US guidance in 8 cadavers. Nerve endings were abundant in posterolateral areas of the CPM; the injected dye was identified at the posterolateral area on 12 sides (12/16 side, 75%) without diffusion into the posterior cricoarytenoid muscle. The injection failed on four sides (two sides of the prevertebral fascia and two sides of the esophagus below the CPM). These results suggest that US-guided injection could be a feasible technique as it can deliver BNT to the most abundant nerve distribution areas within the CPM in most cases.
Asunto(s)
Cadáver , Estudios de Factibilidad , Espasticidad Muscular , Ultrasonografía Intervencional , Humanos , Espasticidad Muscular/tratamiento farmacológico , Masculino , Femenino , Toxinas Botulínicas Tipo A/administración & dosificación , Terminaciones Nerviosas/efectos de los fármacos , Músculos Faríngeos/efectos de los fármacos , Músculos Faríngeos/diagnóstico por imagen , Anciano , Anciano de 80 o más Años , Inyecciones Intramusculares , Toxinas Botulínicas/administración & dosificaciónRESUMEN
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
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
Lateral ligament tears, also known as high-grade ankle sprains, are common, debilitating, and usually heal slowly. Ten to thirty percent of patients continue to suffer from chronic pain and ankle instability even after 3 to 9 months. Previously, we showed that the recombinant human amelogenin (rHAM+ ) induced regeneration of fully transected rat medial collateral ligament, a common proof-of-concept model. Our aim was to evaluate whether rHAM+ can regenerate torn ankle calcaneofibular ligament (CFL), an important component of the lateral ankle stabilizers. Right CFLs of Sabra rats were transected and treated with 0, 0.5, or 1 µg/µL rHAM+ dissolved in propylene glycol alginate (PGA). Results were compared with the normal group, without surgery. Healing was evaluated 12 weeks after treatment by mechanical testing (ratio between the right and left, untransected ligaments of the same rat), and histology including immunohistochemical staining of collagen I and S100. The mechanical properties, structure, and composition of transected ligaments treated with 0.5 µg/µL rHAM+ (experimental) were similar to untransected ligaments. PGA (control) treated ligaments were much weaker, lax, and unorganized compared with untransected ligaments. Treatment with 1 µg/µL rHAM+ was not as efficient as 0.5 µg/µL rHAM+ . Normal arrangement of collagen I fibers and of proprioceptive nerve endings, parallel to the direction of the force, was detected in ligaments treated with 0.5 µg/µL rHAM+ , and scattered arrangement, resembling scar tissue, in control ligaments. In conclusion, we showed that rHAM+ induced significant mechanical and structural regeneration of torn rat CFLs, which might be translated into treatment for grades 2 and 3 ankle sprain injuries.
Asunto(s)
Amelogenina/uso terapéutico , Traumatismos del Tobillo/tratamiento farmacológico , Ligamentos Laterales del Tobillo/efectos de los fármacos , Regeneración/efectos de los fármacos , Amelogenina/farmacología , Animales , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Femenino , Terminaciones Nerviosas/efectos de los fármacos , Ratas , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéuticoRESUMEN
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.
Asunto(s)
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
Acute cutaneous exposure to allergen often leads to itch, but seldom pain. The effect of mast cell activation on cutaneous C-fibers was studied using innervated isolated mouse skin preparation that allows for intra-arterial delivery of chemicals to the nerve terminals in the skin. Allergen (ovalbumin) injection into the isolated skin of actively sensitized mice strongly stimulated chloroquine (CQ)-sensitive C-fibers (also referred to as "itch" nerves); on the other hand, CQ-insensitive C-fibers were activated only modestly, if at all. The histamine H1 receptor antagonist pyrilamine abolished itch C-fibers response to histamine, but failed to significantly reduce the response to ovalbumin. Ovalbumin also strongly activated itch C-fibers in skin isolated from Mrgpr-cluster Δ-/- mice. When pyrilamine was studied in the Mrgpr-cluster Δ-/- mice thereby eliminating the influence of both histamine H1 and Mrgpr receptors (MrgprA3 and C11 are selectively expressed by itch nerves), the ovalbumin response was very nearly eliminated. The data indicate that the acute activation of itch C-fibers in mouse skin is largely secondary to the combined effect of activation of histamine H1 and Mrpgr receptors.
Asunto(s)
Alérgenos/toxicidad , Histamina/metabolismo , Terminaciones Nerviosas/metabolismo , Fibras Nerviosas Amielínicas/metabolismo , Prurito/metabolismo , Piel/metabolismo , Animales , Antagonistas de los Receptores Histamínicos H1/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Terminaciones Nerviosas/efectos de los fármacos , Fibras Nerviosas Amielínicas/efectos de los fármacos , Técnicas de Cultivo de Órganos , Prurito/inducido químicamente , Piel/efectos de los fármacos , Piel/inervaciónRESUMEN
Perinatal hypoxia can lead to multiple chronic neurological deficits, e.g., mental retardation, behavioral abnormalities, and epilepsy. Levetiracetam (LEV), 2S-(2-oxo-1-pyrrolidiny1) butanamide, is an anticonvulsant drug with proven efficiency in treating patients with focal and generalized seizures. Rats were underwent hypoxia and seizures at the age of 10-12 postnatal days (pd). The ambient level and depolarization-induced exocytotic release of [3H]GABA (γ-aminobutyric acid) were analyzed in nerve terminals in the hippocampus and cortex during development at the age of pd 17-19 and pd 24-26 (infantile stage), pd 38-40 (puberty) and pd 66-73 (young adults) in norm and after perinatal hypoxia. LEV had no effects on the ambient [3H]GABA level. The latter increased during development and was further elevated after perinatal hypoxia in nerve terminals in the hippocampus during the whole period and in the cortex in young adults. Exocytotic [3H]GABA release from nerve terminals increased after perinatal hypoxia during development in the hippocampus and cortex, however this effect was preserved at all ages during blockage of GABA transporters by NO-711 in the hippocampus only. LEV realized its anticonvulsant effects at the presynaptic site through an increase in exocytotic release of GABA. LEV exerted more significant effect after perinatal hypoxia than in norm. Action of LEV was strongly age-dependent and can be registered in puberty and young adults, but the drug was inert at the infantile stage.
Asunto(s)
Envejecimiento/patología , Exocitosis/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/patología , Hipoxia/metabolismo , Levetiracetam/farmacología , Terminaciones Nerviosas/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Animales Recién Nacidos , Masculino , Terminaciones Nerviosas/efectos de los fármacos , Terminaciones Nerviosas/patología , Ratas WistarRESUMEN
Evidence indicates that indirect inhibitory regulation of glutamatergic transmission, via reducing glutamate release, may induce neuroprotection. The present work was designed to examine whether allicin, a major component of garlic with neuroprotective effects, affected the release of glutamate evoked by 4-aminopyridine in rat cerebrocortical nerve terminals (synaptosomes). Allicin caused a potent inhibition on the release of glutamate evoked by 4-aminopyridine, and this inhibitory effect was abolished in the presence of Ca2+-free medium and vesicular transporter inhibitor. Allicin decreased the 4-aminopyridine-evoked elevation of intrasynaptosomal Ca2+ levels, but had no effect on the synaptosomal plasma membrane potential. The allicin-mediated inhibition of glutamate release was prevented by the N- and P/Q-type channel blocker and the protein kinase C (PKC) inhibitor, but was not affected by the intracellular Ca2+-release inhibitors, mitogen-activated protein kinase inhibitor, and protein kinase A inhibitor. Western blotting data also showed that allicin significantly reduced the phosphorylation of PKC. Together, these data indicate that in rat cerebrocortical nerve terminals, allicin depresses glutamate release and appears to decrease N- and P/Q-type Ca2+ channel and PKC activity.
Asunto(s)
Calcio/metabolismo , Corteza Cerebral/metabolismo , Ácido Glutámico/metabolismo , Terminaciones Nerviosas/metabolismo , Proteína Quinasa C/metabolismo , Ácidos Sulfínicos/farmacología , Animales , Corteza Cerebral/efectos de los fármacos , Disulfuros , Masculino , Terminaciones Nerviosas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Proteína Quinasa C/genética , Ratas , Ratas Sprague-Dawley , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismoRESUMEN
Electroencephalogram monitoring during propofol (PRO) anesthesia typically features low-frequency oscillations, which may be involved with thalamic reticular nucleus (TRN) modulation. TRN receives noradrenergic inputs from the locus coeruleus (LC). We hypothesized that specific noradrenergic connections in the TRN may contribute to the emergence from PRO anesthesia. Intranuclei norepinephrine (NE) injections (n = 10) and designer receptors exclusively activated by designer drugs (DREADDs) (n = 10) were used to investigate the role of noradrenergic inputs from the LC to the TRN during PRO anesthesia. Whole-cell recording in acute brain slice preparations was used to identify the type of adrenoceptor that regulates noradrenergic innervation in the TRN. An intracerebral injection of NE into the TRN delays arousal in mice recovering from PRO anesthesia (means ± sd; 486.6 ± 57.32 s for the NE injection group vs. 422.4 ± 48.19 s for the control group; P = 0.0143) and increases the cortical-δ (0.1-4 Hz, 25.4 ± 2.9 for the NE injection group vs. 21.0 ± 1.7 for the control group; P = 0.0094) oscillation. An intra-TRN injection of NE also decreased the EC50 of PRO-induced unconsciousness (57.05 ± 1.78 mg/kg for the NE injection group vs. 72.44 ± 3.23 mg/kg for the control group; P = 0.0096). Moreover, the activation of LC-noradrenergic nerve terminals in the TRN using DREADDs increased the recovery time [466.1 ± 44.57 s for the clozapine N-oxide (CNO) injection group vs. 426.1 ± 38.75 s for the control group; P = 0.0033], decreased the EC50 of PRO-induced unconsciousness (64.77 ± 3.40 mg/kg for the CNO injection group vs. 74.00 ± 2.08 mg/kg for the control group; P = 0.0081), and increased the cortical-δ oscillation during PRO anesthesia (23.29 ± 2.58 for the CNO injection group vs. 19.56 ± 1.9 for the control group; P = 0.0213). In addition, whole-cell recording revealed that NE augmented the inhibitory postsynaptic currents in the TRN neurons via the α1-adrenoceptor. Our data indicated that enhanced NE signaling at the noradrenergic terminals of the LC-TRN projection delays arousal from general anesthesia, which is likely mediated by the α1-adrenoceptor activation. Our findings open a door for further understanding of the functions of various LC targets in both anesthesia and arousal.-Zhang, Y., Fu, B., Liu, C., Yu, S., Luo, T., Zhang, L., Zhou, W., Yu, T. Activation of noradrenergic terminals in the reticular thalamus delays arousal from propofol anesthesia in mice.
Asunto(s)
Neuronas Adrenérgicas/fisiología , Anestesia General , Nivel de Alerta/fisiología , Retraso en el Despertar Posanestésico/fisiopatología , Núcleos Talámicos Intralaminares/fisiopatología , Terminaciones Nerviosas/fisiología , Receptores Adrenérgicos alfa 1/fisiología , Neuronas Adrenérgicas/efectos de los fármacos , Anestésicos Intravenosos , Animales , Clozapina/análogos & derivados , Clozapina/farmacología , Drogas de Diseño/farmacología , Electroencefalografía , Vectores Genéticos/administración & dosificación , Núcleos Talámicos Intralaminares/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Terminaciones Nerviosas/efectos de los fármacos , Norepinefrina/farmacología , Técnicas de Placa-Clamp , Propofol , Distribución Aleatoria , Receptor Muscarínico M3/efectos de los fármacos , Receptor Muscarínico M3/fisiología , Receptores Adrenérgicos alfa 1/efectos de los fármacos , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Reflejo de Enderezamiento/efectos de los fármacos , Método Simple Ciego , Organismos Libres de Patógenos Específicos , Potenciales Sinápticos/efectos de los fármacos , Potenciales Sinápticos/fisiologíaRESUMEN
RATIONALE: Mephedrone is a commonly abused constituent of "bath salts" and has many pharmacological effects in common with methamphetamine. Despite their structural similarity, mephedrone differs significantly from methamphetamine in its effects on core body temperature and dopamine nerve endings. The reasons for these differences remain unclear. OBJECTIVES: Mephedrone elicits a transient hypothermia which may provide intrinsic neuroprotection against methamphetamine-like toxicity to dopamine nerve endings. Furthermore, evidence in the literature suggests that this hypothermia is mediated by serotonin. By utilizing transgenic mice devoid of brain serotonin, we determined the contribution of this neurotransmitter to changes in core body temperature as well as its possible role in protecting against neurotoxicity. The effects of methcathinone and 4-methyl-methamphetamine, two structural analogs of mephedrone and methamphetamine, were also evaluated in these mice. RESULTS: The hypothermia induced by mephedrone and methcathinone in wild-type mice was not observed in mice lacking brain serotonin. Despite preventing drug-induced hypothermia, the lack of serotonin did not alter the neurotoxic profiles of the test drugs. CONCLUSIONS: Serotonin is a key mediator of pharmacological hypothermia induced by mephedrone and methcathinone, but these body temperature effects do not contribute to dopamine nerve ending damage observed in mice following treatment with mephedrone, methcathinone or 4-methyl-methamphetamine. Thus, the key component of methamphetamine neurotoxicity lacking in mephedrone remains to be elucidated.
Asunto(s)
Hipotermia/metabolismo , Drogas Ilícitas/toxicidad , Metanfetamina/análogos & derivados , Síndromes de Neurotoxicidad/metabolismo , Propiofenonas/toxicidad , Triptófano Hidroxilasa/deficiencia , Animales , Femenino , Hipotermia/inducido químicamente , Hipotermia/fisiopatología , Metanfetamina/toxicidad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Terminaciones Nerviosas/efectos de los fármacos , Terminaciones Nerviosas/metabolismo , Síndromes de Neurotoxicidad/etiología , Síndromes de Neurotoxicidad/fisiopatología , Triptófano Hidroxilasa/genéticaRESUMEN
Presynaptic mGlu2/3 autoreceptors exist in rat spinal cord nerve terminals as suggested by the finding that LY379268 inhibited the 15â¯mM KCl-evoked release of [3H]D-aspartate ([3H]D-Asp) in a LY341495-sensitive manner. Spinal cord glutamatergic nerve terminals also possess presynaptic release-regulating 5-HT2A heteroreceptors. Actually, the 15â¯mM KCl-evoked [3H]D-Asp exocytosis from spinal cord synaptosomes was reduced by the 5-HT2A agonist (±)DOI, an effect reversed by the 5-HT2A antagonists MDL11,939, MDL100907, ketanserin and trazodone (TZD). We investigated whether mGlu2/3 and 5-HT2A receptors colocalize and cross-talk in these terminals and if 5-HT2A ligands modulate the mGlu2/3-mediated control of glutamate exocytosis. Western blot analysis and confocal microscopy highlighted the presence of mGlu2/3 and 5-HT2A receptor proteins in spinal cord VGLUT1 positive synaptosomes, where mGlu2/3 and 5-HT2A receptor immunoreactivities largely colocalize. Furthermore, mGlu2/3 immunoprecipitates from spinal cord synaptosomes were also 5-HT2A immunopositive. Interestingly, the 100 pM LY379268-induced reduction of the 15â¯mM KCl-evoked [3H]D-Asp overflow as well as its inhibition by 100â¯nM (±)DOI became undetectable when the two agonists were concomitantly added. Conversely, 5-HT2A antagonists (MDL11,939, MDL100907, ketanserin and TZD) reinforced the release-regulating activity of mGlu2/3 autoreceptors. Increased expression of mGlu2/3 receptor proteins in synaptosomal plasmamembranes paralleled the gain of function of the mGlu2/3 autoreceptors elicited by 5-HT2A antagonists. Based on these results, we propose that in spinal cord glutamatergic terminals i) mGlu2/3 and 5-HT2A receptors colocalize and interact one each other in an antagonist-like manner, ii) 5-HT2A antagonists are indirect positive allosteric modulator of mGlu2/3 autoreceptors controlling glutamate exocytosis.
Asunto(s)
Exocitosis/fisiología , Ácido Glutámico/metabolismo , Terminaciones Nerviosas/metabolismo , Receptor de Serotonina 5-HT2A/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Transducción de Señal/fisiología , Médula Espinal/ultraestructura , Animales , Biotinilación , Corteza Cerebral/metabolismo , Relación Dosis-Respuesta a Droga , Fármacos actuantes sobre Aminoácidos Excitadores/farmacología , Exocitosis/efectos de los fármacos , Femenino , Ácido Glutámico/farmacología , Inmunoprecipitación , Masculino , Microscopía Confocal , Terminaciones Nerviosas/efectos de los fármacos , Ratas , Serotoninérgicos/farmacología , Transducción de Señal/efectos de los fármacos , Estadísticas no Paramétricas , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismo , Proteína 1 de Transporte Vesicular de Glutamato/metabolismoRESUMEN
AMN082 is a selective metabotropic glutamate mGlu7 receptor agonist reported to exhibit antidepressant activity. Considering that excessive glutamate release is involved in the pathogenesis of depression, the effect of N,N'-dibenzyhydryl-ethane-1,2-diamine dihydrochloride (AMN082) on glutamate release in rat cerebrocortical nerve terminals and the possible underlying mechanism were investigated. In this study, we observed here that AMN082 inhibited 4-aminopyridine-evoked glutamate release and this phenomenon was blocked by the metabotropic glutamate mGlu7 receptor antagonist MMPIP. Moreover, western blot analysis and immunocytochemistry confirmed the presence of presynaptic metabotropic glutamate mGlu7 receptor proteins. The effect of AMN082 on the 4-aminopyridine-evoked release of glutamate was prevented by chelating the extracellular Ca2+ ions and the vesicular transporter inhibitor; however, the effect of AMN082 was unaffected by the glutamate transporter inhibitor. AMN082 reduced the elevation of 4-aminopyridine-evoked intrasynaptosomal Ca2+ concentration, but did not alter the synaptosomal membrane potential. In the presence of the Cav2.2 (N-type) and Cav2.1 (P/Q-type) channel blocker, the adenylate cyclase inhibitor, and the protein kinase A inhibitor, the action of AMN082 on the 4-aminopyridine-evoked glutamate release was markedly reduced. These results suggest that the activation of the metabotropic glutamate mGlu7 receptors by AMN082 reduces adenylate cyclase/protein kinase A activation, which subsequently reduces the entry of Ca2+ through voltage-dependent Ca2+ channels and decreases evoked glutamate release. Additionally, fluoxetine, a clinically effective antidepressant, completely occluded the inhibitory effect of AMN082 on glutamate release, thus indicating the existence of a common intracellular mechanism for these two compounds to inhibit glutamate release from the cerebrocortical nerve terminals.
Asunto(s)
Compuestos de Bencidrilo/farmacología , Corteza Cerebral/efectos de los fármacos , Ácido Glutámico/metabolismo , Terminaciones Nerviosas/efectos de los fármacos , Terminaciones Nerviosas/metabolismo , Receptores de Glutamato Metabotrópico/agonistas , Animales , Transporte Biológico/efectos de los fármacos , Calcio/metabolismo , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
γ-Aminobutyric acid (GABA) and glutamate (Glu) are the main inhibitory and excitatory neurotransmitters in the central nervous system (CNS), respectively. Fine tuning regulation of extracellular levels of these amino acids is essential for normal brain activity. Recently, we showed that neocortical nerve terminals from patients with epilepsy express higher amounts of the non-desensitizing ionotropic P2X7 receptor. Once activated by ATP released from neuronal cells, the P2X7 receptor unbalances GABAergic vs. glutamatergic neurotransmission by differentially interfering with GABA and Glu uptake. Here, we investigated if activation of the P2X7 receptor also affects [3H]GABA and [14C]Glu release measured synchronously from isolated nerve terminals (synaptosomes) of the rat cerebral cortex. Data show that activation of the P2X7 receptor consistently increases [14C]Glu over [3H]GABA release from cortical nerve terminals, but the GABA/Glu ratio depends on extracellular Ca2+ concentrations. While the P2X7-induced [3H]GABA release is operated by a Ca2+-dependent pathway when external Ca2+ is available, this mechanism shifts towards the reversal of the GAT1 transporter in low Ca2+ conditions. A different scenario is verified regarding [14C]Glu outflow triggered by the P2X7 receptor, since the amino acid seems to be consistently released through the recruitment of connexin-containing hemichannels upon P2X7 activation, both in the absence and in the presence of external Ca2+. Data from this study add valuable information suggesting that ATP, via P2X7 activation, not only interferes with the high-affinity uptake of GABA and Glu but actually favors the release of these amino acids through distinct molecular mechanisms amenable to differential therapeutic control.
Asunto(s)
Corteza Cerebral/metabolismo , Ácido Glutámico/metabolismo , Terminales Presinápticos/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Estrés Psicológico/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Animales , Corteza Cerebral/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Terminaciones Nerviosas/efectos de los fármacos , Terminaciones Nerviosas/metabolismo , Terminales Presinápticos/efectos de los fármacos , Ratas , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismoRESUMEN
The effects of nanomolar cocaine concentrations, possibly not blocking the dopamine transporter activity, on striatal D2-σ1 heteroreceptor complexes and their inhibitory signaling over Gi/o, have been tested in rat striatal synaptosomes and HEK293T cells. Furthermore, the possible role of σ1 receptors (σ1Rs) in the cocaine-provoked amplification of D2 receptor (D2R)-induced reduction of K+-evoked [3H]-DA and glutamate release from rat striatal synaptosomes, has also been investigated. The dopamine D2-likeR agonist quinpirole (10nM-1µM), concentration-dependently reduced K+-evoked [3H]-DA and glutamate release from rat striatal synaptosomes. The σ1R antagonist BD1063 (100nM), amplified the effects of quinpirole (10 and 100nM) on K+-evoked [3H]-DA, but not glutamate, release. Nanomolar cocaine concentrations significantly enhanced the quinpirole (100nM)-induced decrease of K+-evoked [3H]-DA and glutamate release from rat striatal synaptosomes. In the presence of BD1063 (10nM), cocaine failed to amplify the quinpirole (100nM)-induced effects. In cotransfected σ1R and D2LR HEK293T cells, quinpirole had a reduced potency to inhibit the CREB signal versus D2LR singly transfected cells. In the presence of cocaine (100nM), the potency of quinpirole to inhibit the CREB signal was restored. In D2L singly transfected cells cocaine (100nM and 10µM) exerted no modulatory effects on the inhibitory potency of quinpirole to bring down the CREB signal. These results led us to hypothesize the existence of functional D2-σ1R complexes on the rat striatal DA and glutamate nerve terminals and functional D2-σ1R-DA transporter complexes on the striatal DA terminals. Nanomolar cocaine concentrations appear to alter the allosteric receptor-receptor interactions in such complexes leading to enhancement of Gi/o mediated D2R signaling.
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Cocaína/administración & dosificación , Cuerpo Estriado/metabolismo , Receptores de Dopamina D2/metabolismo , Receptores sigma/metabolismo , Animales , Cuerpo Estriado/efectos de los fármacos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Dopamina/metabolismo , Agonistas de Dopamina/administración & dosificación , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/genética , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Ácido Glutámico/metabolismo , Células HEK293 , Humanos , Complejos Multiproteicos/efectos de los fármacos , Complejos Multiproteicos/genética , Terminaciones Nerviosas/efectos de los fármacos , Terminaciones Nerviosas/metabolismo , Quinpirol/administración & dosificación , Ratas , Receptores de Dopamina D2/química , Receptores de Dopamina D2/genética , Receptores sigma/química , Receptores sigma/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismo , Receptor Sigma-1RESUMEN
Knowledge of the molecular mechanisms underlying signaling of mechanical stimuli by muscle spindles remains incomplete. In particular, the ionic conductances that sustain tonic firing during static muscle stretch are unknown. We hypothesized that tonic firing by spindle afferents depends on sodium persistent inward current (INaP) and tested for the necessary presence of the appropriate voltage-gated sodium (NaV) channels in primary sensory endings. The NaV1.6 isoform was selected for both its capacity to produce INaP and for its presence in other mechanosensors that fire tonically. The present study shows that NaV1.6 immunoreactivity (IR) is concentrated in heminodes, presumably where tonic firing is generated, and we were surprised to find NaV1.6 IR strongly expressed also in the sensory terminals, where mechanotransduction occurs. This spatial pattern of NaV1.6 IR distribution was consistent for three mammalian species (rat, cat, and mouse), as was tonic firing by primary spindle afferents. These findings meet some of the conditions needed to establish participation of INaP in tonic firing by primary sensory endings. The study was extended to two additional NaV isoforms, selected for their sensitivity to TTX, excluding TTX-resistant NaV channels, which alone are insufficient to support firing by primary spindle endings. Positive immunoreactivity was found for NaV1.1, predominantly in sensory terminals together with NaV1.6 and for NaV1.7, mainly in preterminal axons. Differential distribution in primary sensory endings suggests specialized roles for these three NaV isoforms in the process of mechanosensory signaling by muscle spindles.NEW & NOTEWORTHY The molecular mechanisms underlying mechanosensory signaling responsible for proprioceptive functions are not completely elucidated. This study provides the first evidence that voltage-gated sodium channels (NaVs) are expressed in the spindle primary sensory ending, where NaVs are found at every site involved in transduction or encoding of muscle stretch. We propose that NaVs contribute to multiple steps in sensory signaling by muscle spindles as it does in other types of slowly adapting sensory neurons.
Asunto(s)
Potenciales de Acción/efectos de los fármacos , Músculo Esquelético/citología , Terminaciones Nerviosas/fisiología , Células Receptoras Sensoriales/efectos de los fármacos , Bloqueadores de los Canales de Sodio/farmacología , Tetrodotoxina/farmacología , Potenciales de Acción/fisiología , Vías Aferentes/efectos de los fármacos , Vías Aferentes/fisiología , Animales , Anticuerpos/farmacología , Gatos , Ganglios Espinales/citología , Técnicas In Vitro , Ratones , Ratones Noqueados , Proteína Básica de Mielina/metabolismo , Terminaciones Nerviosas/efectos de los fármacos , Proteínas de Neurofilamentos/metabolismo , Ratas , Canales de Sodio Activados por Voltaje/genética , Canales de Sodio Activados por Voltaje/inmunología , Canales de Sodio Activados por Voltaje/metabolismoRESUMEN
Mephedrone (MEPH) is a ß-ketoamphetamine stimulant drug of abuse that is often a constituent of illicit bath salts formulations. Although MEPH bears remarkable similarities to methamphetamine (METH) in terms of chemical structure, as well as its neurochemical and behavioral effects, it has been shown to have a reduced neurotoxic profile compared with METH. The addition of a ß-keto moiety and a 4-methyl ring substituent to METH yields MEPH, and a loss of direct neurotoxic potential. In the present study, two analogs of METH, methcathinone (MeCa) and 4-methylmethamphetamine (4MM), were assessed for their effects on mouse dopamine (DA) nerve endings to determine the relative contribution of each individual moiety to the loss of direct neurotoxicity in MEPH. Both MeCa and 4MM caused significant alterations in core body temperature as well as locomotor activity and stereotypy, but 4MM was found to elicit minimal dopaminergic toxicity only at the highest dose. By contrast, MeCa caused significant reductions in all markers of DA nerve-ending damage over a range of doses. These results lead to the conclusion that ring substitution at the 4-position profoundly reduces the neurotoxicity of METH, whereas the ß-keto group has much less influence on this property. Although the mechanism(s) by which the 4-methyl substituent reduces METH-induced neurotoxicity remains unclear, it is speculated that this effect is mediated by a loss of DA-releasing action in MEPH and 4MM at the synaptic vesicle monoamine transporter, an effect that is thought to be critical for METH-induced neurotoxicity.
Asunto(s)
Neuronas Dopaminérgicas/efectos de los fármacos , Metanfetamina/análogos & derivados , Terminaciones Nerviosas/efectos de los fármacos , Síndromes de Neurotoxicidad , Propiofenonas , Animales , Estimulantes del Sistema Nervioso Central/química , Estimulantes del Sistema Nervioso Central/farmacología , Relación Dosis-Respuesta a Droga , Drogas Ilícitas/química , Drogas Ilícitas/farmacología , Metanfetamina/química , Metanfetamina/farmacología , Ratones , Síndromes de Neurotoxicidad/etiología , Síndromes de Neurotoxicidad/metabolismo , Propiofenonas/química , Propiofenonas/farmacologíaRESUMEN
Recently, we have shown that new fluorinated analogues of γ-aminobutyric acid (GABA), bioisosters of pregabalin (ß-i-Bu-GABA), i.e. ß-polyfluoroalkyl-GABAs (FGABAs), with substituents: ß-CF3-ß-OH (1), ß-CF3 (2); ß-CF2CF2H (3), are able to increase the initial rate of [3H]GABA uptake by isolated rat brain nerve terminals (synaptosomes), and this effect is higher than that of pregabalin. So, synthesized FGABAs are structural but not functional analogues of GABA. Herein, we assessed the effects of synthesized FGABAs (100µM) on the ambient level and exocytotic release of [3H]GABA in nerve terminals and compared with those of pregabalin (100µM). It was shown that FGABAs 1-3 did not influence the ambient level of [3H]GABA in the synaptosomal preparations, and this parameter was also not altered by pregabalin. During blockage of GABA transporters GAT1 by specific inhibitor NO-711, FGABAs and pregabalin also did not change ambient [3H]GABA in synaptosomal preparations. Exocytotic release of [3H]GABA from synaptosomes decreased in the presence of FGABAs 1-3 and pregabalin, and the effects of FGABAs 1 &3 were more significant than those of FGABAs 2 and pregabalin. FGABAs 1-3/pregabalin-induced decrease in exocytotic release of [3H]GABA from synaptosomes was not a result of changes in the potential of the plasma membrane. Therefore, new synthesized FGABAs 1 &3 were able to decrease exocytotic release of [3H]GABA from nerve terminals more effectively in comparison to pregabalin. Absence of unspecific side effects of FGABAs 1 &3 on the membrane potential makes these compounds perspective for medical application.
Asunto(s)
Encéfalo/efectos de los fármacos , Terminaciones Nerviosas/efectos de los fármacos , Pregabalina/farmacología , Radiofármacos/farmacología , Tritio/química , Animales , Encéfalo/metabolismo , Relación Dosis-Respuesta a Droga , Exocitosis/efectos de los fármacos , Halogenación , Masculino , Estructura Molecular , Terminaciones Nerviosas/metabolismo , Pregabalina/síntesis química , Pregabalina/química , Radiofármacos/síntesis química , Radiofármacos/química , Ratas , Ratas Wistar , Relación Estructura-ActividadRESUMEN
OBJECTIVE: The aim of this study was to review the preclinical data showing the role of both estrogens and androgens in the physiology of the vagina, and, most likely, in vulvovaginal atrophy of menopause. METHODS: Mass spectrometry-based assays (validated according to the FDA guidelines) for the measurement of sex steroids, their precursors, and metabolites were used. In addition to fixation of the vagina for morphological examination, histomorphometry, immunocytochemistry, immunofluorescence, and quantitative reverse transcription polymerase chain reaction were performed. RESULTS: The vaginal epithelium of the animals receiving dehydroepiandrosterone (DHEA) was made of large multilayered columnar mucous cells showing distended cytoplasmic vacuoles representative of an androgenic effect. DHEA also stimulates collagen fiber compactness of the lamina propria (second layer)-an effect essentially due to an androgenic effect, whereas stimulation by DHEA of the muscularis in the third vaginal layer is approximately 70% due to the androgenic conversion of DHEA. Stimulation of the surface area of the nerve endings, on the contrary, is exclusively androgenic. Vaginal weight stimulation by DHEA is about 50% androgenic and 50% estrogenic. CONCLUSIONS: Practically all studies on the influence of steroid hormones in the vagina have focused on luminal epithelial cells. Since all estrogens and androgens in postmenopausal women are made intracellularly and derive from the conversion of circulating DHEA, it is of interest to observe from these preclinical data that DHEA exerts both estrogenic and androgenic activity in the three layers of the vagina, the stimulatory effect on nerve density being 100% androgenic. Taking vaginal weight as a global parameter, the stimulatory effect of DHEA in the rat vagina is about equally estrogenic and androgenic, thus illustrating the importance of androgens in vaginal morphology and function, and the likely importance of androgens in vulvovaginal atrophy of menopause.