RESUMEN
Pudendal motoneurons are located in the ventral horn of the caudal lumbar spinal cord and innervate striated pelvic muscles implicated in sexual and eliminative functions. In rats they are distributed in the dorsomedial (DM) and dorsolateral (DL) nucleus. In male rats, dorsomedial motoneurons innervate the bulbocavernosus, the levator ani and the external anal sphincter, whereas dorsolateral motoneurons control the ischiocavernosus and external urethral sphincter. Using spinal cord slices of young male rats and whole-cell patch-clamp recordings, we investigated the sensitivity of pudendal motoneurons to nicotinic cholinergic agonists. Motoneurons were identified following 1,1'-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine, 4-chlorobenzenesulphonate retrograde labelling. In the presence of atropine, both dorsomedial and dorsolateral motoneurons responded to acetylcholine (ACh) by generating a rapidly activating inward current. By using selective nicotinic antagonists and a nicotinic positive allosteric modulator, we found that nicotinic ACh receptors present in dorsomedial and dorsolateral motoneurons display distinct pharmacological profiles. Whereas the former are of the heteromeric type, the latter are predominantly of the alpha7-containing type. These data were confirmed by light microscopic autoradiography. In young rats, a ligand for heteromeric nicotinic receptors labelled all laminae of the central grey matter, whereas in the ventral part of the central grey, a ligand selective for alpha7-containing nicotinic receptors labelled the DL but not the DM. Dorsolateral and dorsomedial motoneurons innervate two distinct groups of pelvic muscles. A difference in their nicotinic pharmacology may be clinically relevant, as it might allow a selective pharmacological intervention in view of influencing the activity of one or the other set of muscles.
Asunto(s)
Acetilcolina/metabolismo , Neuronas Motoras/metabolismo , Receptores Nicotínicos/metabolismo , Médula Espinal/metabolismo , Transmisión Sináptica/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Genitales Masculinos/inervación , Masculino , Neuronas Motoras/citología , Neuronas Motoras/efectos de los fármacos , Músculo Esquelético/inervación , Agonistas Nicotínicos/farmacología , Antagonistas Nicotínicos/farmacología , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Diafragma Pélvico/inervación , Ratas , Ratas Sprague-Dawley , Receptores Nicotínicos/efectos de los fármacos , Médula Espinal/citología , Médula Espinal/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
The lateral septal area is rich in vasopressin V(1A) receptors and is densely innervated by vasopressinergic axons, originating mainly from the bed nucleus of the stria terminalis and the amygdala. Genetic and behavioral studies provide evidence that activation of vasopressin receptors in this area plays a determinant role in promoting social recognition. What could be the neuronal mechanism underlying this effect? Using rat brain slices and whole-cell recordings, we found that lateral septal neurons are under the influence of a basal GABAergic inhibitory input. Vasopressin, acting via V(1A) but not V(1B) receptors, greatly enhanced this input in nearly all neurons. The peptide had no effect on miniature inhibitory postsynaptic currents, indicating that it acted on receptors located in the somatodendritic membrane, rather than on axon terminals, of GABAergic interneurons. Cell-attached recordings showed that vasopressin can cause a direct excitation of a subpopulation of lateral septal neurons by acting via V(1A) but not V(1B) receptors. The presence in the lateral septum of V(1A) but not of V(1B) receptors was confirmed by competition binding studies using light microscopic autoradiography. In conclusion, vasopressin appears to act in the lateral septum in a dual mode: (i) by causing a direct excitation of a subpopulation of neurons, and (ii) by causing an indirect inhibition of virtually all lateral septal neurons. This modulation by vasopressin of the lateral septal circuitry may be part of the neuronal mechanism by which the peptide, acting via V(1A) receptors, promotes social recognition.
Asunto(s)
Red Nerviosa/metabolismo , Vías Nerviosas/metabolismo , Receptores de Vasopresinas/metabolismo , Núcleos Septales/metabolismo , Vasopresinas/fisiología , Animales , Antagonistas de los Receptores de Hormonas Antidiuréticas , Unión Competitiva/efectos de los fármacos , Unión Competitiva/fisiología , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Potenciales Postsinápticos Inhibidores/fisiología , Red Nerviosa/efectos de los fármacos , Inhibición Neural/efectos de los fármacos , Inhibición Neural/fisiología , Vías Nerviosas/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Receptores de Vasopresinas/agonistas , Receptores de Vasopresinas/efectos de los fármacos , Núcleos Septales/efectos de los fármacos , Conducta Social , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Vasopresinas/farmacología , Ácido gamma-Aminobutírico/metabolismoRESUMEN
As a hormone, vasopressin binds to three distinct receptors: V1a and V1b receptors, which induce phospholipase-Cbeta (PLCbeta) activation and Ca2+ mobilization; and V2 receptors, which are coupled to adenylyl cyclase. V1a and V1b receptors are also present in neurons. In particular, hypoglossal (XII) and facial (VII) motoneurons are excited following vasopressin-V1a receptor binding. The aim of the present study was double: (i) to determine whether V1b receptors contribute to the excitatory effect of vasopressin in XII and VII motoneurons; and (ii) to establish whether the action of vasopressin on motoneurons is mediated by Ca2+ signalling. Patch-clamp recordings were performed in brainstem slices of young rats. Vasopressin depolarized the membrane or generated an inward current. By contrast, [1-deamino-4-cyclohexylalanine] arginine vasopressin (d[Cha4]AVP), a V1b agonist, had no effect. The action of vasopressin was suppressed by Phaa-D-Tyr(Et)-Phe-Gln-Asn-Lys-Pro-Arg-NH2, a V1a antagonist, but not by SSR149415, a V1b antagonist. Thus, the vasopressin-induced excitation of brainstem motoneurons was exclusively mediated by V1a receptors. Light microscopic autoradiography failed to detect V1b binding sites in the facial nucleus. In motoneurons loaded with GTP-gamma-S, a non-hydrolysable analogue of GTP, the effect of vasopressin was suppressed, indicating that neuronal V1a receptors are G-protein-coupled. Intracellular Ca2+ chelation suppressed a Ca2+-activated potassium current, but did not affect the vasopressin-evoked current. H7 and GF109203, inhibitors of protein kinase C, were without effect on the vasopressin-induced excitation. U73122 and D609, PLCbeta inhibitors, were also without effect. Thus, excitation of brainstem motoneurons by V1a receptor activation is probably mediated by a second messenger distinct from that associated with peripheral V1a receptors.
Asunto(s)
Nervio Facial/fisiología , Nervio Hipogloso/fisiología , Neuronas Motoras/efectos de los fármacos , Receptores de Vasopresinas/fisiología , Vasopresinas/farmacología , Animales , Animales Recién Nacidos , Antagonistas de los Receptores de Hormonas Antidiuréticas , Arginina Vasopresina/análogos & derivados , Arginina Vasopresina/farmacología , Autorradiografía/métodos , Tronco Encefálico/citología , Señalización del Calcio/fisiología , Agonistas de Aminoácidos Excitadores/farmacología , Técnicas In Vitro , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Neuronas Motoras/fisiología , Oligopéptidos/farmacología , Oxitocina/análogos & derivados , Oxitocina/farmacología , Técnicas de Placa-Clamp/métodos , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Ratas , Ratas Sprague-Dawley , Receptores de Vasopresinas/agonistas , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacologíaRESUMEN
The paraventricular nucleus of the hypothalamus contains three classes of neurones: (i) magnocellular and (ii) parvocellular neurosecretory neurones and (iii) nonendocrine projection neurones. The present study aimed to determine whether functional GABA(B) receptors are present on axon terminals that synapse with parvocellular neurosecretory and nonendocrine paraventricular neurones and to determine how activation of GABA(B) receptors control GABAergic input to these neurones. Whole-cell recordings were performed in coronal hypothalamic slices of the rat containing the paraventricular nucleus. GABA(A) receptor-mediated inhibitory postsynaptic currents (i.p.s.c.) were isolated pharmacologically in the presence of antagonists of glutamatergic ionotropic receptors. We found that baclofen, an agonist of GABA(B) receptors, decreased the frequency of spontaneous and miniature i.p.s.c. It also decreased the amplitude of evoked i.p.s.c. These effects were suppressed by CGP55845A, a competitive antagonist of GABA(B) receptors. CGP55845A also increased the frequency of miniature i.p.s.c. and the amplitude of evoked i.p.s.c., suggesting that, in physiological conditions, presynaptic GABA(B) receptors exert a tonic inhibition on GABA release. Baclofen had no effect on GABA-evoked postsynaptic currents, suggesting that the baclofen-dependent suppression of GABAergic i.p.s.c. was exclusively due to a presynaptic action of the agonist. Our data indicate that GABA(B) receptors are present on axon terminals of GABAergic presynaptic neurones contacting parvocellular neurosecretory and nonendocrine paraventricular neurones, and suggest that GABA(B) receptors exert a tonic inhibition of GABA release from GABAergic terminals. Activation of these receptors causes disinhibition of parvocellular neurosecretory and nonendocrine paraventricular neurones.
Asunto(s)
Agonistas de Receptores GABA-B , Hipotálamo/efectos de los fármacos , Neuronas/efectos de los fármacos , Núcleo Hipotalámico Paraventricular/efectos de los fármacos , Transmisión Sináptica , Animales , Baclofeno/farmacología , Agonistas del GABA/farmacología , Antagonistas del GABA/farmacología , Antagonistas de Receptores de GABA-B , Hipotálamo/citología , Hipotálamo/metabolismo , Inmunohistoquímica , Técnicas In Vitro , Masculino , Neuronas/metabolismo , Núcleo Hipotalámico Paraventricular/citología , Núcleo Hipotalámico Paraventricular/metabolismo , Ácidos Fosfínicos/farmacología , Propanolaminas/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de GABA-B/metabolismoRESUMEN
The aim of the present study was to determine whether, in young rats, spinal motoneurons possess functional nicotinic acetylcholine receptors. Motoneurons were identified either by retrograde labelling or by choline acetyltransferase immunohistochemistry. Whole-cell recordings were performed in spinal cord slices cut at the lumbar level. In voltage clamp, acetylcholine evoked a rapidly activating inward current. In current clamp, it depolarized the motoneuron membrane and induced action potential firing. The acetylcholine-evoked current was strongly reduced by d-tubocurarine or dihydro-beta-erythroidine, broad spectrum nicotinic antagonists, but was almost insensitive to methyllycaconitine, a nicotinic antagonist selective for receptors containing the alpha7 subunit. Moreover, exo-2-(2-pyridyl)-7-azabicyclo[2.2.1]heptane, an alpha7-specific agonist, was without effect. In young animals, light-microscopic autoradiography showed that in the central grey matter all laminae were intensely and equally labelled by [3H]epibatidine. A dense [125I]-alpha-bungarotoxin binding was also found in all laminae, with slightly lower levels in the superficial layers of the dorsal horns and in the ventral part of the grey matter. In adults, the density of [3H]epibatidine binding sites was much lower in the entire grey matter, except in layer 2 of the dorsal horn, and [125I]-alpha-bungarotoxin binding sites were present only in some selected areas. Our data indicate that spinal motoneurons possess functional nicotinic receptors of the heteromeric type and suggest that nicotinic cholinergic transmission may play a significant role in the developing spinal cord.
Asunto(s)
Aconitina/análogos & derivados , Lisina/análogos & derivados , Neuronas Motoras/metabolismo , Receptores Nicotínicos/metabolismo , Médula Espinal/citología , Acetilcolina/farmacología , Aconitina/farmacología , Factores de Edad , Animales , Animales Recién Nacidos , Autorradiografía/métodos , Sitios de Unión , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacocinética , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Bungarotoxinas/farmacocinética , Colina O-Acetiltransferasa/metabolismo , Dihidro-beta-Eritroidina/farmacología , Inmunohistoquímica/métodos , Técnicas In Vitro , Isótopos de Yodo/farmacocinética , Lisina/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Neuronas Motoras/efectos de los fármacos , Agonistas Nicotínicos/farmacocinética , Antagonistas Nicotínicos/farmacología , Técnicas de Placa-Clamp/métodos , Piridinas/farmacocinética , Piridinas/farmacología , Ratas , Ratas Sprague-Dawley , Receptores Nicotínicos/clasificación , Receptores Nicotínicos/efectos de los fármacos , Tritio/farmacocinética , Tubocurarina/farmacologíaRESUMEN
We have examined the properties of nicotinic acetylcholine receptors in embryonic chick retinal ganglion cells. Ganglion cells, identified according to morphological and physiological criteria, displayed spontaneous or induced action potentials. In 94/99 cells acetylcholine pulses evoked responses. In current clamp mode, acetylcholine provoked membrane depolarization and triggered action potentials. Under voltage clamp conditions, acetylcholine evoked inward currents that were readily blocked by d-tubocurarine. Antagonists specific for homomeric (alpha-bungarotoxin) and heteromeric (dihydro-beta-erythroidine) receptors revealed that ganglion cells express multiple functional receptor subtypes. These findings demonstrate that ACh modulates the electrical activity of these cells and is likely to mediate synaptic transmission. The presence of multiple receptor subtypes may contribute to processing and transmission of information in the retina.
Asunto(s)
Acetilcolina/metabolismo , Receptores Nicotínicos/metabolismo , Retina/metabolismo , Células Ganglionares de la Retina/metabolismo , Transmisión Sináptica/fisiología , Acetilcolina/farmacología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Bungarotoxinas/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Embrión de Pollo , Dihidro-beta-Eritroidina/farmacología , Antagonistas Nicotínicos/farmacología , Técnicas de Placa-Clamp , Receptores Nicotínicos/efectos de los fármacos , Retina/citología , Retina/embriología , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Tubocurarina/farmacologíaRESUMEN
The distribution in the rat brain of high affinity nicotinic heteromeric acetylcholine receptors and of low affinity nicotinic, alpha7-containing, homomeric receptors was studied using in vitro light microscopic autoradiography. As ligands, we used [3H]epibatidine, or [125I]epibatidine, and [125I]alpha-bungarotoxin, respectively. In adult animals, the two types of binding sites were widely distributed in many different brain structures, including the brainstem, cerebellum, mesencephalic structures, limbic system and cortex, but their anatomical distribution differed markedly. Only in rare instances could a co-localization be observed, for example in the superficial layer of the superior colliculus. In developing animals, both types of labeling were strongly expressed during embryonic and postnatal phases. Their distributions were qualitatively similar to those observed in adult animals, with a few noticeable exceptions in the cerebral cortex, hippocampus and brain stem. In aging animals, neither the distribution nor the density of nicotinic binding sites was significantly altered. Our conclusions are the following. (a) There is little overlap in the distribution of heteromeric and alpha7-containing homomeric nicotinic receptors in the rat brain. (b) The abundance of neuronal nicotinic receptors during embryonic and postnatal development suggests that they may play a role in the establishment of neuronal connectivity. (c) The expression of neuronal nicotinic receptors is unaltered in middle aged animals, suggesting that in the rat these receptors do not play any major role in aging process.
Asunto(s)
Envejecimiento/metabolismo , Encéfalo/metabolismo , Receptores Nicotínicos/metabolismo , Factores de Edad , Animales , Animales Recién Nacidos , Autorradiografía , Sitios de Unión , Encéfalo/anatomía & histología , Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Química Encefálica , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacocinética , Bungarotoxinas/farmacocinética , Embrión de Mamíferos , Femenino , Isótopos de Yodo/farmacocinética , Masculino , Agonistas Nicotínicos/farmacocinética , Piridinas/farmacocinética , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Receptores Nicotínicos/clasificación , Distribución Tisular , Tritio/farmacocinéticaRESUMEN
The objective of the present work was double. (i) Light microscopic autoradiography was used to determine the distribution of vasopressin and oxytocin binding sites in the spinal cord of rats. (ii) Whole-cell recordings were performed in lumbar spinal cord slices in order to assess whether these receptors are functional, whether they are located pre- or postsynaptically and whether they are present in motoneurons. In newborns, vasopressin binding sites of the V1a type were present in all laminae of the central gray at all segmental levels, whereas oxytocin binding sites were found only in the superficial layers of the dorsal horn. In adults, binding sites for both neuropeptides were also present, but were less dense. The dissociation constants for vasopressin were similar in newborns and adults. Whole-cell recordings showed that in identified motoneurons vasopressin exerted a direct effect, by inducing a membrane depolarization or by generating a sustained inward current, and an indirect effect, by enhancing glycinergic and GABAergic inhibitory transmission. Vasopressin-induced facilitation of inhibitory transmission could also be demonstrated in unidentified ventral horn neurons. All these effects were mediated by V1a but not V1b receptors. In some neurons, glycinergic transmission was also facilitated by a selective oxytocin receptor agonist. Our data, together with data obtained previously in brainstem motor nuclei, suggest that vasopressin of hypothalamic origin could play a role in motricity. The neuropeptide could act as a neuromodulator, because it would not directly activate motoneurons, but rather render them more responsive to incoming excitatory inputs. Vasopressin may thus act as a regulator of muscular force.
Asunto(s)
Células del Asta Anterior/citología , Células del Asta Anterior/fisiología , Receptores de Vasopresinas/fisiología , Animales , Animales Recién Nacidos , Sitios de Unión/efectos de los fármacos , Sitios de Unión/fisiología , Electrofisiología , Técnicas In Vitro , Ratas , Ratas Sprague-Dawley , Receptores de Vasopresinas/agonistas , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Vasopresinas/farmacologíaRESUMEN
The aim of the present work was to determine whether paraventricular neurons possess functional acetylcholine nicotinic receptors. Using infrared videomicroscopy and differential interference contrast optics, we performed whole-cell recordings in hypothalamic slices containing the paraventricular nucleus. Acetylcholine, locally applied by pressure microejection in the presence of the muscarinic antagonist atropine, evoked a rapidly rising inward current in paraventricular magnocellular endocrine neurons. This current persisted in the presence of blockers of synaptic transmission. It could be reversibly suppressed by nanomolar concentrations of methyllycaconitine, a selective antagonist of alpha 7-containing nicotinic receptors, but was insensitive to micromolar concentrations of dihydro-beta-erythroidine, an antagonist acting preferentially on non-alpha 7 nicotinic receptors. In addition, the effect of acetylcholine could be mimicked by exo-2-(2-pyridyl)-7-azabicyclo[2.2.1]heptane, a recently synthesized nicotinic agonist specific for alpha 7 receptors. Acetylcholine also desensitized paraventricular nicotinic receptors. Desensitization was pronounced and recovery from desensitization was rapid, consistent with the notion that paraventricular nicotinic receptors contain the alpha 7 subunit. Nicotinic currents could not be evoked in paraventricular parvocellular neurons, suggesting that these neurons are devoid of functional nicotinic receptors. The electrophysiological data were corroborated by light microscopic autoradiography, showing that [(125)I]alpha-bungarotoxin binding sites are present in all the magnocellular divisions of the paraventricular nucleus but are undetectable in other areas of this nucleus. Immunohistochemistry, performed using antibodies directed against vasopressin and oxytocin, indicated that responsiveness to nicotinic agonists was a property of vasopressin as well as of oxytocin magnocellular endocrine neurons, in both the paraventricular and the supraoptic nucleus. We conclude that nicotinic agonists can influence the magnocellular neurosecretory system by directly increasing the excitability of magnocellular neurons. By contrast, they are probably without direct effects on paraventricular parvocellular neurons.
Asunto(s)
Acetilcolina/metabolismo , Neuronas/metabolismo , Antagonistas Nicotínicos/farmacología , Núcleo Hipotalámico Paraventricular/metabolismo , Receptores Nicotínicos/metabolismo , Transmisión Sináptica/fisiología , Acetilcolina/farmacología , Animales , Tamaño de la Célula/efectos de los fármacos , Tamaño de la Célula/fisiología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Neuronas/citología , Neuronas/efectos de los fármacos , Neurofisinas/metabolismo , Oxitocina/metabolismo , Núcleo Hipotalámico Paraventricular/citología , Núcleo Hipotalámico Paraventricular/efectos de los fármacos , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Receptores Nicotínicos/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Vasopresinas/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
Vasopressin can directly excite facial motoneurons in young rats and mice. It acts by generating a persistent inward current, which is Na(+)-dependent, tetrodotoxin-insensitive and voltage-gated. This peptide-evoked current is unaffected by Ca(++) or K(+) channel blockade and is modulated by extracellular divalent cations. In the present work, we determined how vasopressin alters the input-output properties of facial motoneurons. Whole-cell recordings were obtained from these neurons in the current clamp mode, in brainstem slices of young rats. Repetitive firing was evoked by injecting depolarizing current pulses. Steady-state frequency-current (f-I) relationships were constructed and the effect of vasopressin on these relationships was studied. We found that vasopressin caused a parallel shift to the left of the cell steady-state f-I relationship. This effect persisted in the presence of blockers of K(+) or Ca(++) channels. The peptide effect was distinct from that brought about by Ca(++) channel suppression or by apamin, a blocker of the mAHP. These latter manipulations resulted in an increase in the slope of the steady-state f-I relationship. We conclude that the vasopressin-induced modification of the input-output properties of facial motoneurons is probably exclusively caused by the sodium-dependent, voltage-modulated inward current elicited by the peptide, rather than being due to indirect effects of the peptide on Ca(++) channels, K(+) channels or Ca(++)-dependent K(+) channels. Computer simulation, based on a simple model of facial motoneurons, indicates that the introduction of a conductance having the properties of the vasopressin-dependent conductance can entirely account for the observed peptide-induced shift of the f-I relationship.
Asunto(s)
Músculos Faciales/inervación , Neuronas Motoras/efectos de los fármacos , Vasopresinas/farmacología , Algoritmos , Animales , Bloqueadores de los Canales de Calcio/farmacología , Simulación por Computador , Electrofisiología , Músculos Faciales/efectos de los fármacos , Técnicas In Vitro , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
The properties of nicotinic acetylcholine receptors (nAChRs) were studied following exogenous expression in a host system or using whole-cell recordings in brain slices, autoradiography and immunohistochemistry. When expressed in HEK-293 cells, alpha 4 beta 2 nAChRs displayed both a high and a low affinity component. The ratio of these two states was modified by chronic nicotine exposure, resulting in an enhanced sensitivity and a marked reduction in desensitization. Mutations in the gene coding for the alpha 4 subunit are responsible for a particular form of nocturnal epilepsy. When expressed in Xenopus oocytes, alpha 4 beta 2 nAChRs containing these mutations displayed distinct alterations in agonist affinity, desensitization and calcium permeability. Magnocellular endocrine neurons in the supraoptic (SO) nucleus of the hypothalamus were found to express functional alpha 7-containing nAChRs, which could play a role in regulating neurohypophysial peptide secretion. Facial (VII), hypoglossal (XII) and vagal (X) motoneurons of young rats responded to ACh by a fast inward current. The nAChRs present in VII and XII nuclei were of the non-alpha 7-containing type, whereas those present in the X nucleus contained the alpha 7 subunit. In Bcl-2 transgenic mice, facial nerve axotomy caused nAChRs downregulation by interfering negatively with the expression of the alpha 4 subunit. Binding sites corresponding to alpha 7-containing nAChRs were also detected in spinal motor nuclei and axotomy provoked a reduction of the binding. Together, these data indicate that long-term exposure to nicotine can promote neuroadaptive changes in nAChRs and that genetic alterations of neuronal nAChRs can result in transmissible neurological diseases. They also suggest that these receptors probably play a role in the central regulation of autonomic functions, as well as in motor control.
Asunto(s)
Sistema Nervioso Central/fisiología , Neuronas/fisiología , Receptores Nicotínicos/fisiología , Animales , Sistema Nervioso Central/efectos de los fármacos , Humanos , Neuronas/efectos de los fármacos , Agonistas Nicotínicos/metabolismo , Agonistas Nicotínicos/farmacología , Antagonistas Nicotínicos/metabolismo , Antagonistas Nicotínicos/farmacología , Receptores Nicotínicos/genéticaRESUMEN
Acetylcholine can stimulate the release of vasopressin. In organ-cultured hypothalamo-neurohypophyseal systems, acetylcholine enhanced vasopressin release by acting in or near the supraoptic nucleus Extracellular recordings suggested that acetylcholine can increase supraoptic neuron excitability. These effects could be mimicked, in part, by nicotine or blocked by nicotinic antagonists, suggesting that they might be mediated by nicotinic acetylcholine receptors. Autoradiography indicated that alpha-bungarotoxin binding sites are present in the supraoptic nucleus; however, neither acetylcholine nor nicotine binding sites could be detected. Thus, the existence, let alone the nature, of nicotinic receptors in the supraoptic nucleus has so far remained elusive. The present work attempts to determine: (i) whether functional nicotinic receptors are present in this nucleus; (ii) whether they are located on neurosecretory magnocellular cells or at presynaptic sites; (iii) what their pharmacological and biophysical properties are; (iv) whether they influence the activity of all or only part of supraoptic neurons. Whole-cell recordings were performed in hypothalamic slices or in acutely dissociated supraoptic neurons and the effect of nicotinic agonists was tested under voltage-clamp conditions. Autoradiography was done in coronal hypothalamic sections, using [3H]epibatidine and [125I]alpha-bungarotoxin as ligands. Our results indicate that supraoptic neurons possess functional nicotinic receptors containing the alpha7 subunit.
Asunto(s)
Neuronas/química , Neuronas/fisiología , Receptores Nicotínicos/fisiología , Núcleo Supraóptico/citología , Acetilcolina/farmacología , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Bungarotoxinas/metabolismo , Bungarotoxinas/farmacología , Dihidro-beta-Eritroidina/farmacología , Relación Dosis-Respuesta a Droga , Electrofisiología , Técnicas In Vitro , Radioisótopos de Yodo , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , Antagonistas Nicotínicos/farmacología , Piridinas/metabolismo , Piridinas/farmacología , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Tritio , Vasodilatadores/farmacología , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
In mammals, nicotinic acetylcholine receptors (nAChRs) play a crucial role in motor control. Muscle-type nAChRs mediate synaptic excitation of skeletal muscle by motoneurons, and nAChRs are present on Renshaw cells, where they produce recurrent inhibition of spinal motoneurons. We asked whether nAChRs are also present in motoneurons. Whole-cell recordings were performed on various motor nuclei in brainstem slices of young rats. Neurons were visualized using infrared (IR) videomicroscopy. Acetylcholine (ACh) or the nicotinic agonist, epibatidine, were delivered by pressure microinjection. Facial (VII), hypoglossal (XII) and vagal (X) motoneurons responded to ACh by generating a fast inward current. In VII motoneurons, the ACh effect was mimicked by epibatidine, and nicotine induced a slow inward current and desensitized the ACh-evoked current. In VII and XII motoneurons, the ACh-evoked current was blocked by the nicotinic antagonist dihydro-beta-erythroidine (DHbetaE), but was unaffected by methyllycaconitine (MLA), an alpha7-specific antagonist. By contrast, the ACh-induced current in X motoneurons was sensitive to MLA. Current-voltage relationships indicated that the currents mediated by either alpha7-containing (X) or non-alpha7-containing (VII, XII) nAChRs displayed inward rectification. In accordance with the electrophysiological data, autoradiography revealed that VII, X and XII nuclei of young rats contained binding sites for [3H]epibatidine; binding sites for [125I]alpha-bungarotoxin, a selective ligand of alpha7-containing nAChRs, were present in X nucleus but were almost undetectable in VII and XII nuclei. Thus, brainstem motoneurons of young rats possess functional nAChRs. They could promote fast synaptic coupling between motoneurons, and thus play a role in somatic and visceral motor functions.
Asunto(s)
Tronco Encefálico/metabolismo , Neuronas Motoras/metabolismo , Receptores Nicotínicos/metabolismo , Animales , Autorradiografía , Tronco Encefálico/citología , Tronco Encefálico/fisiología , Electrofisiología , Nervio Facial/citología , Nervio Facial/fisiología , Nervio Hipogloso/citología , Nervio Hipogloso/fisiología , Técnicas In Vitro , Neuronas Motoras/fisiología , Ratas , Ratas Sprague-Dawley , Receptores Nicotínicos/fisiología , Nervio Vago/citología , Nervio Vago/fisiologíaRESUMEN
We have reported previously that axotomy induced a marked increase of vasopressin receptor binding in the adult rat facial nucleus, suggesting an increased number of vasopressin receptors. These receptors were pharmacologically undistinguishable from peripheral V(1a) vasopressin receptors. In the present study, we show, using in situ hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR), that axotomy regulates the expression of the vasopressin V(1a) receptor mRNA in the facial nucleus. Results were obtained from adult male rats killed 1 week following crush of the right facial nerve. In situ hybridization was performed with a (35)S-labelled riboprobe. A specific hybridization signal was detected in both left and right facial nuclei, with a significantly higher intensity in the nucleus ipsilateral to the lesion. V(1a) receptor transcripts were found associated with large facial motoneuronal cell bodies, not with other cells present in the nucleus, i.e., glial or epithelial cells. RT-PCR analysis of unlesioned facial tissue revealed the presence of mRNAs encoding vasopressin V(1a), vasopressin V(1b) and oxytocin receptors, whereas only the V(1a) receptor mRNA was found to be increased following axotomy in the lesioned facial tissue. These data suggest that the axotomy-induced expression of vasopressin receptors in the rat facial nucleus is due, at least to a large extent, to an increase of the V(1a) vasopressin receptor mRNA in facial motoneurons.
Asunto(s)
Encéfalo/metabolismo , Nervio Facial/fisiología , Hígado/metabolismo , Neuronas/metabolismo , Receptores de Vasopresinas/genética , Transcripción Genética , Regulación hacia Arriba , Animales , Arginina Vasopresina/metabolismo , Axotomía , Cartilla de ADN , Traumatismos del Nervio Facial , Masculino , Compresión Nerviosa , Oxitocina/genética , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Receptores de Oxitocina/genética , Valores de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vasopresinas/genéticaRESUMEN
A potent non-peptide vasopressin (AVP) antagonist, SR-49059, displaying high stability and selective affinity for the V1a AVP receptor subtype, has recently been described. The objective of this study was to assess the binding properties and the penetrability of this compound in the rat brain. Both in vitro and in vivo binding autoradiography experiments were performed. In all studies, the liver was used as a reference V1a tissue. In vitro labelling of rat brain sections with [3H]SR-49059 was similar to that previously detected with [3H]AVP, which confirms that the majority of central AVP binding sites are V1a sites similar to peripheral V1a receptors. As expected, intense specific labelling occurred mainly in the lateral septum, the fundus striatum, the hypothalamic stigmoid nucleus and the area postrema-nucleus of the solitary tract complex. In vivo binding autoradiography showed that [3H]SR-49059 injected intravenously did not enter the brain parenchyma. Specific labelling was however clearly detectable in brain regions with permeable hematoencephalic barrier, the choroid plexus and other circumventricular organs expressing V1a receptors, namely the subfornical organ, the pineal gland and the area postrema. The specificity of [3H]SR-49059 binding in the latter structures was confirmed by the fact that labelling was prevented by pretreatment of animals with high doses of nonradioactive SR-49059. In conclusion, our study shows that [3H]SR-49059 is a suitable probe to investigate V1a receptors in the rat brain. We also demonstrate that although this compound is not able to enter the brain tissue from the peripheral circulation, it does bind specifically to regions devoid of blood-brain barrier and known to be involved in autonomic regulations.
Asunto(s)
Autorradiografía , Encéfalo/metabolismo , Antagonistas de Hormonas/metabolismo , Indoles/metabolismo , Pirrolidinas/metabolismo , Receptores de Vasopresinas/metabolismo , Animales , Antagonistas de los Receptores de Hormonas Antidiuréticas , Cuerpo Estriado/metabolismo , Hipotálamo/metabolismo , Indoles/administración & dosificación , Inyecciones Intravenosas , Hígado/metabolismo , Masculino , Glándula Pineal/metabolismo , Pirrolidinas/administración & dosificación , Ratas , Ratas Sprague-Dawley , Tabique Pelúcido/metabolismo , Núcleo Solitario/metabolismo , Órgano Subfornical/metabolismo , TritioRESUMEN
We reported previously a significant reduction of oxytocin (OT) receptor binding in the brain of 20-month old rats relative to 3-month old ones. The present study shows that testosterone treatment of aging rats restores normal adult levels of OT receptor binding in the olfactory tubercle and in the hypothalamic ventromedial nucleus, but not in the caudate-putamen. These data indicate that the reduced plasma testosterone found in 20-month old rats is responsible for the loss of OT receptors in the olfactory tubercle and hypothalamic ventromedial nucleus, whereas other aging-related mechanisms may account for the decrease of OT receptor binding in the caudate-putamen.
Asunto(s)
Envejecimiento/metabolismo , Encéfalo/metabolismo , Receptores de Oxitocina/metabolismo , Testosterona/farmacología , Animales , Autorradiografía , Encéfalo/efectos de los fármacos , Encéfalo/crecimiento & desarrollo , Núcleo Caudado/metabolismo , Radioisótopos de Yodo , Masculino , Bulbo Olfatorio/metabolismo , Especificidad de Órganos , Putamen/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Oxitocina/efectos de los fármacos , Núcleo Hipotalámico Ventromedial/metabolismoRESUMEN
High affinity binding sites for vasopressin (VP) are widely distributed within the rat brain and spinal cord. Since their presence is associated with neuronal sensitivity to VP application, their anatomical distribution maps structures which could be activated by endogenous VP. Interestingly, marked species-related differences of the VP receptor distribution have been revealed. Some evidence has also been provided that mechanisms of receptor regulation may vary among species. In the rat, the expression of VP binding sites in some motor nuclei shows remarkable plasticity, in particular up-regulation after axotomy. These data suggest that VP may, in addition to affecting motoneuronal excitability, act as a trophic factor onto motoneurones.
Asunto(s)
Sistema Nervioso Central/química , Sistema Nervioso Central/metabolismo , Neuronas Motoras/química , Vasopresinas/análisis , Vasopresinas/metabolismo , Animales , Sitios de Unión/fisiología , Sistema Nervioso Central/citología , Neuronas Motoras/metabolismo , RatasRESUMEN
A vasopressin receptor antagonist, [1-(beta-mercapto-beta,beta-pentamethylenepropionic acid), 2-o-ethyl-D-tyrosine, 4-valine, 9-tyrosylamide] arginine vasopressin (d(CH2)5[o-ethyl-D-Tyr2,Val4,Tyr-NH9(2)]AVP), has been prepared. This antagonist is a potent antiantidiuretic, antivasopressor and antioxytocic peptide with pA2 values of 7.69-7.94 and affinities of 1.12-11.0 nM. When radioiodinated at the phenyl moiety of the tyrosylamide residue at position 9, this peptide was demonstrated to bind to vasopressin V2 and V1a receptors with a dissociation constant of 0.22-0.75 nM. This ligand is a good tool for further studies on human vasopressin V2 receptor localization and characterization, when used in combination with a selective vasopressin V1a ligand.
Asunto(s)
Antagonistas de los Receptores de Hormonas Antidiuréticas , Arginina Vasopresina/análogos & derivados , Animales , Arginina Vasopresina/síntesis química , Arginina Vasopresina/química , Arginina Vasopresina/farmacología , Autorradiografía , ADN/biosíntesis , Diuresis/efectos de los fármacos , Humanos , Inosina Trifosfato/metabolismo , Radioisótopos de Yodo , Ratas , Receptores de Oxitocina/efectos de los fármacos , Receptores de Oxitocina/metabolismo , Receptores de Vasopresinas/biosíntesis , Receptores de Vasopresinas/metabolismoRESUMEN
The distribution of vasopressin and oxytocin receptors was established by in vitro autoradiography in the spinal cord of adult rats of either sex, as well as in male castrates. In both males and females, high concentrations of vasopressin binding sites were found in a few groups of somatic motoneurons: the large lateral group at the cervicothoracic junction in segments C8 and Th1; the small medial group in segments L3-L5; and the pudendal and retrodorsolateral nuclei in segments L5-L6. The extension and intensity of labelling in pudendal nuclei were markedly lower in females than in males, in particular in the dorsomedial nucleus, where binding was either not or hardly detectable. Gonadectomy in males resulted in a significant reduction of binding in pudendal nuclei, but not in other labelled motor nuclei. Moderate amounts of vasopressin binding sites were also found evenly distributed throughout the central gray at all segmental levels. Oxytocin binding sites were detectable in all spinal segments, but in low amounts and restricted to the superficial layers of the dorsal horn. The abundance of vasopressin binding sites in the central gray suggests that vasopressin may be involved in most spinal functions. The permanent expression of vasopressin binding sites in pudendal motor nuclei of is particular interest with regard to the known plasticity of pudendal motoneurons.
Asunto(s)
Orquiectomía , Ovariectomía , Receptores de Oxitocina/metabolismo , Receptores de Vasopresinas/metabolismo , Médula Espinal/anatomía & histología , Médula Espinal/metabolismo , Nervios Espinales/fisiología , Animales , Autorradiografía , Femenino , Radioisótopos de Yodo , Ligandos , Masculino , Ratas , Ratas Sprague-Dawley , Caracteres Sexuales , Nervios Espinales/metabolismo , Vasopresinas/metabolismoRESUMEN
This review concentrates on the pharmacological properties and the regional distribution of the arginine vasopressin (AVP) and oxytocin (OT) receptors that are present in the central nervous system. Of particular interest are the kinetics and the pharmacological profiles of these receptors that resemble those present at the periphery. However, their transduction mechanisms need to be further investigated. This should be rendered easier by molecular biology technology. The current knowledge of the anatomical distribution of AVP and OT receptors in the brain is reviewed. Also of great interest for double labeling studies will be the receptor antibodies, now being developed. The existence of additional receptors (AVP4-9) is examined, and aspects of the regulation of the receptors' expression in relation to the age, the species, and the hormonal status or following injury are also discussed.