RESUMEN
Upon vascular injury, platelets form a hemostatic plug by binding to the subendothelium and to each other. Platelet-to-matrix binding is initially mediated by von Willebrand factor (VWF) and platelet-to-platelet binding is mediated mainly by fibrinogen and VWF. After binding, the actin cytoskeleton of a platelet drives its contraction, generating traction forces that are important to the cessation of bleeding. Our understanding of the relationship between adhesive environment, F-actin morphology, and traction forces is limited. Here, we examined F-actin morphology of platelets attached to surfaces coated with fibrinogen and VWF. We identified distinct F-actin patterns induced by these protein coatings and found that these patterns were identifiable into three classifications via machine learning: solid, nodular, and hollow. We observed that traction forces for platelets were significantly higher on VWF than on fibrinogen coatings and these forces varied by F-actin pattern. In addition, we analyzed the F-actin orientation in platelets and noted that their filaments were more circumferential when on fibrinogen coatings and having a hollow F-actin pattern, while they were more radial on VWF and having a solid F-actin pattern. Finally, we noted that subcellular localization of traction forces corresponded to protein coating and F-actin pattern: VWF-bound, solid platelets had higher forces at their central region while fibrinogen-bound, hollow platelets had higher forces at their periphery. These distinct F-actin patterns on fibrinogen and VWF and their differences in F-actin orientation, force magnitude, and force localization could have implications in hemostasis, thrombus architecture, and venous versus arterial thrombosis.
Asunto(s)
Hemostáticos , Factor de von Willebrand , Factor de von Willebrand/metabolismo , Fibrinógeno/metabolismo , Plaquetas/metabolismo , Actinas/metabolismo , Tracción , Glicoproteínas de Membrana Plaquetaria/metabolismo , Hemostáticos/metabolismo , Citoesqueleto de Actina/metabolismoRESUMEN
Nitric oxide (NO) and cyclic GMP (cGMP) suppressed glutamatergic synaptic transmission to trigeminal motoneurons in brain stem slices of neonatal rats. Histological studies showed guanylate cyclase (GC) containing fibers in the trigeminal motor pool. Glutamatergic excitatory postsynaptic currents (EPSCs) were recorded from neonatal trigeminal motoneurons in response to stimulation of the supratrigeminal nucleus (SuV). The NO donors DETA/NONOate (DETA/NO), at a concentration which released 275.1 nM of NO, and Spermine/NONOate (Sper/NO) reduced the amplitude of the EPSC to 52.7±0.6% and 60.1±10.8% of control values, respectively. These actions were not blocked by the GC inhibitors, ODQ or NS-2028. However, in the presence of YC-1 or BAY41-2272, modulators of GC that act as NO sensitizers, lower and otherwise ineffective concentrations of DETA/NO induced a reduction of the EPSC to 60.6±5.2%. Moreover, NO effects were mimicked by 8BrcGMP and by Zaprinast, an inhibitor of Phosphodiesterase 5. Glutamatergic currents evoked by exogenous glutamate were not reduced by DETA/NO nor 8BrcGMP. Paired-pulse facilitation was increased by NO donors. Under "minimal stimulation" conditions NO donors and cGMP increased the failure rate of evoked EPSCs. Protein kinase inhibitors antagonized cGMP effects. The results suggest that NO, through the synthesis of cGMP, presynaptically inhibits glutamatergic synaptic transmission on trigeminal motoneurons. We propose that NO has complex actions on motor pools; specific studies are needed to elucidate their physiological significance in the behaving animal.
Asunto(s)
GMP Cíclico/metabolismo , Potenciales Postsinápticos Excitadores/fisiología , Neuronas Motoras/fisiología , Inhibición Neural/fisiología , Óxido Nítrico/metabolismo , Núcleos del Trigémino/fisiología , Factores de Edad , Animales , Animales Recién Nacidos , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Ácido Glutámico/fisiología , Neuronas Motoras/efectos de los fármacos , Inhibición Neural/efectos de los fármacos , Donantes de Óxido Nítrico/farmacología , Técnicas de Cultivo de Órganos , Terminales Presinápticos/fisiología , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Triazenos/farmacología , Núcleos del Trigémino/citologíaRESUMEN
The present retrograde labeling study was designed to determine the presence and pattern of projections from individual subdivisions of the central nucleus of the amygdala (CNA) to the nucleus pontis oralis (NPO), which is a critical brainstem site involved in the generation and maintenance of active (REM) sleep. Projections from the CNA were labeled with the retrograde tracer cholera toxin B-subunit (CTB), which was injected, unilaterally, via microiontophoresis, into the NPO. Sections of the amygdala were immunostained in order to identify CTB-labeled CNA neurons and CNA neurons that contained CTB plus the vesicular glutamate transporter 2 (VGLUT2), which is a marker for glutamatergic neurons. Histological analyses revealed that retrogradely labeled neurons that project to the NPO were localized, ipsilaterally, within the medial, lateral, and capsular subdivisions of the CNA. In addition, a substantial proportion (24%) of all retrogradely labeled CNA neurons also exhibited VGLUT2 immunoreactivity. The present study demonstrates that glutamatergic neurons, which are present within various subdivisions of the CNA, project directly to the NPO. These data lend credence to the hypothesis that NPO neurons that are involved in the control of active sleep are activated by glutamatergic projections from the amygdala.
Asunto(s)
Amígdala del Cerebelo/citología , Neuronas/fisiología , Puente/fisiología , Amígdala del Cerebelo/fisiología , Animales , Toxina del Cólera/metabolismo , Femenino , Masculino , Vías Nerviosas/fisiología , Porcinos , Proteína 2 de Transporte Vesicular de Glutamato/metabolismoRESUMEN
This report presents the results of a study of the frequency potentiation of inhibitory postsynaptic currents (IPSCs) in hypoglossal motoneurons and its modulation by serotonin. A release-site model of synaptic plasticity was used to characterize the frequency-related potentiation of evoked IPSCs. Data were obtained to determine if the frequency potentiation of IPSCs occurs as a consequence of a low baseline quantal content of evoked IPSCs using whole cell patch-clamp recordings from hypoglossal motoneurons in the neonatal rat brainstem slice preparation. In these motoneurons, EPSCs and GABAergic IPSCs were blocked by the application of CNQX, AP-5 and bicuculline. Glycinergic IPSCs were evoked by threshold stimulation of inhibitory neurons in the nucleus of Roller, which is located ventro-lateral to the hypoglossal nucleus. IPSC responses to trains of stimuli were recorded in control solutions and in solutions containing serotonin, which is known to reduce IPSPs in this preparation. The amplitude of non-potentiated IPSCs was reduced and their frequency potentiation was enhanced when serotonin was added to the bath. These data were examined using a release-site model of synaptic plasticity in which facilitation is attributed to a time-dependent increase in the probability of transmitter release; depression is attributed to a time-dependent decrease in the number of sites available for release. Using this model, the effect of serotonin on frequency potentiation was explained by a combination of a reduction in the baseline probability of transmitter release and an increase in the time constant of decay of the increase in probability of release that follows a stimulus.
Asunto(s)
Glicina/metabolismo , Nervio Hipogloso/fisiología , Potenciales Postsinápticos Inhibidores/fisiología , Neuronas Motoras/fisiología , Serotonina/metabolismo , Animales , Animales Recién Nacidos , Tronco Encefálico/efectos de los fármacos , Tronco Encefálico/fisiología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Nervio Hipogloso/efectos de los fármacos , Técnicas In Vitro , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Modelos Neurológicos , Neuronas Motoras/efectos de los fármacos , Inhibición Neural/efectos de los fármacos , Inhibición Neural/fisiología , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/fisiología , Técnicas de Placa-Clamp , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/fisiología , Ratas , Ratas Wistar , Sinapsis/efectos de los fármacos , Sinapsis/fisiología , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Factores de TiempoRESUMEN
STUDY OBJECTIVE: This study was designed to determine the effects of eszopiclone on apnea-induced excitotoxic synaptic processes and apoptosis in the hippocampus. DESIGN: Recurrent periods of apnea, which consisted of a sequence of apnea (75% SpO2), followed by ventilation with recovery to normoxia (> 95% SpO2), were induced for a period of three hours in anesthetized guinea pigs. The CA3 Schaffer collateral pathway in the hippocampus was stimulated and the field excitatory postsynaptic potential (fEPSP) response was recorded in CA1. Animals in the experimental group received an intravenous injection of eszopiclone (3 mg/kg) 10 min prior to the initiation of the periods of recurrent apnea, and once every 60 min thereafter; control animals received comparable injections of vehicle. At the end of the 3-h period of recurrent apnea, the animals were perfused, and hippocampal sections were immunostained in order to determine the presence of apoptosis, i.e., programmed cell death. ANALYSES AND RESULTS: Apnea resulted in a persistent increase in synaptic responsiveness of CA1 neurons as determined by analyses of the fEPSP. Eszopiclone antagonized the apnea-induced increase in the fEPSP. Morphological analyses revealed significant apoptosis of CA1 neurons in control animals; however, there was no significant apoptosis in eszopiclone-treated animals. CONCLUSIONS: Eszopiclone was determined to suppress the apnea-induced hyperexcitability of hippocampal CA1 neurons, thereby reducing/eliminating neurotoxicity. These data lend credence to our hypothesis that eszopiclone, exclusive of its hypnotic actions, has the capacity to function as a potent neuroprotective agent.
Asunto(s)
Apnea/patología , Compuestos de Azabiciclo/farmacología , Hipocampo/efectos de los fármacos , Hipnóticos y Sedantes/farmacología , Enfermedades Neurodegenerativas/prevención & control , Piperazinas/farmacología , Animales , Apoptosis/efectos de los fármacos , Estimulación Eléctrica/métodos , Eszopiclona , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Femenino , Cobayas , Hipocampo/ultraestructura , Inmunohistoquímica , MasculinoRESUMEN
Patients with obstructive sleep apnea (OSA) exhibit hippocampal damage and cognitive deficits. To determine the effect of apnea on the synaptic transmission in the hippocampus, we performed electrophysiological studies in an in vivo guinea pig model of OSA. Specifically, we determined the cornu ammonis region 1 (CA1) field excitatory postsynaptic potential (fEPSP) response to cornu ammonis region 3 (CA3) stimulation and examined the presynaptic mechanisms underlying the changes in the fEPSP. Single episodes of apnea resulted in a maximal potentiation of the fEPSPs at 1 to 3 min after the termination of each episode of apnea. The mean amplitude and slope of the post-apneic fEPSP was significantly larger compared with the pre-apneic control. These changes were accompanied by a significant decrease in the paired-pulse facilitation ratio during the post-apneic period compared with the pre-apneic control. The N-methyl-D-aspartate (NMDA) glutamate receptor antagonist MK-801, when applied locally to the CA1 recording site by pressure ejection, blocked the apnea-induced potentiation of the fEPSP. In the experimental animals that were subjected to extended periods of recurrent apnea, CA1 neurons exhibited positive immunoreactivity for fragmented DNA strands, which indicates apoptotic cell death. The present results demonstrate that apnea-induced potentiation of the hippocampal CA1 fEPSP is mediated by an NMDA receptor mechanism. We therefore conclude that recurrent apnea produces abnormally high levels of glutamate that results in the apoptosis of CA1 neurons. We hypothesize that this damage is reflected by the cognitive deficits that are commonly observed in patients with breathing disorders such as OSA.
Asunto(s)
Ácido Glutámico/metabolismo , Hipocampo/patología , Neuronas/patología , Apnea Obstructiva del Sueño/patología , Animales , Apoptosis/efectos de los fármacos , Interpretación Estadística de Datos , Maleato de Dizocilpina/farmacología , Estimulación Eléctrica , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/fisiología , Cobayas , Inmunohistoquímica , Apnea Obstructiva del Sueño/metabolismo , Sinapsis/fisiologíaRESUMEN
Neurons that utilize melanin-concentrating hormone (MCH) and others that employ hypocretin as neurotransmitter are located in the hypothalamus and project diffusely throughout the CNS, including areas that participate in the generation and maintenance of the states of sleep and wakefulness. In the present report, immunohistochemical methods were employed to examine the distribution of MCHergic and hypocretinergic neurons. In order to test the hypothesis that the MCHergic system is capable of influencing specific behavioral states, we studied Fos immunoreactivity in MCH-containing neurons during (1) quiet wakefulness, (2) active wakefulness with motor activity, (3) active wakefulness without motor activity, (4) quiet sleep and (5) active sleep induced by carbachol (AS-carbachol). We determined that MCHergic neuronal somata in the cat are intermingled with hypocretinergic neurons in the dorsal and lateral hypothalamus, principally in the tuberal and tuberomammillary regions; however, hypocretinergic neurons extended more in the anterior-posterior axis than MCHergic neurons. Axosomatic and axodendritic contacts were common between these neurons. In contrast to hypocretinergic neurons, which are known to be active during motor activity and AS-carbachol, Fos immunoreactivity was not observed in MCH-containing neurons in conjunction with any of the preceding behavioral conditions. Non-MCHergic, non-hypocretinergic neurons that expressed c-fos during active wakefulness with motor activity were intermingled with MCH and hypocretin-containing neurons, suggesting that these neurons are related to some aspect of motor function. Further studies are required to elucidate the functional sequela of the interactions between MCHergic and hypocretinergic neurons and the phenotype of the other neurons that were active during motor activity.
Asunto(s)
Hormonas Hipotalámicas/metabolismo , Hipotálamo/metabolismo , Melaninas/metabolismo , Vías Nerviosas/metabolismo , Neuronas/metabolismo , Hormonas Hipofisarias/metabolismo , Sueño/fisiología , Vigilia/fisiología , Acetilcolina/metabolismo , Animales , Carbacol/farmacología , Gatos , Agonistas Colinérgicos/farmacología , Hipotálamo/citología , Hipotálamo/efectos de los fármacos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Movimiento/efectos de los fármacos , Movimiento/fisiología , Vías Nerviosas/citología , Vías Nerviosas/efectos de los fármacos , Neuronas/citología , Neuronas/efectos de los fármacos , Neuropéptidos/metabolismo , Orexinas , Proteínas Proto-Oncogénicas c-fos/metabolismo , Sueño/efectos de los fármacos , Sueño REM/efectos de los fármacos , Sueño REM/fisiología , Sinapsis/metabolismo , Sinapsis/ultraestructura , Vigilia/efectos de los fármacosRESUMEN
In aged cats, light microscopic studies revealed significant decrease in the soma size of choline acetyltransferase (ChAT)-positive neurons in the laterodorsal and pedunculo-pontine tegmental nuclei (LDT and PPT), compared with adult control animals. In addition, a significant reduction of the total dendritic length and total dendritic segment number of ChAT-positive neurons was detected in both the LDT and PPT of aged cats. However, in contrast to the changes of soma and dendrites, no significant changes in the number of ChAT-positive neurons in aged were found comparing to that in the control cats in both the LDT and PPT; nor were there differences in the staining intensity of the somata of neurons in the adult and aged cats. Electron microscopic analysis highlighted degenerative changes in cholinergic neurons in the LDT and PPT of aged cats which included somata with intracytoplasmic vacuoles, darkened mitochondria, depletion of dendritic microtubules and severe demyelination of axons. These data indicate that profound atrophic changes occur in cholinergic systems of the LDT and PPT as a consequence of the aging process. These alterations likely reflect the cellular bases for the age-related changes in REM sleep that occur in old animals.
Asunto(s)
Envejecimiento/fisiología , Colina O-Acetiltransferasa/metabolismo , Neuronas/metabolismo , Neuronas/ultraestructura , Núcleo Tegmental Pedunculopontino , Factores de Edad , Animales , Gatos , Recuento de Células/métodos , Femenino , Inmunohistoquímica/métodos , Masculino , Microscopía Inmunoelectrónica/métodos , Núcleo Tegmental Pedunculopontino/citología , Núcleo Tegmental Pedunculopontino/metabolismo , Núcleo Tegmental Pedunculopontino/ultraestructuraRESUMEN
Hypocretin-1 (hcrt-1) and hypocretin-2 (hcrt-2) have been implicated in a wide variety of functions including sleep and wakefulness as well as related behaviors. Many of these functions of the hypocretins involve the activation of cholinergic neurons in the basal forebrain (BF). These neurons have been shown to exhibit age-related changes in a variety of species. In the present experiment, in adult and aged guinea pigs, we compared hypocretin immunoreactivity in regions of the BF that include the medial septal nucleus (MS), the vertical and horizontal limbs of the diagonal band of Broca (VDB and HDB) and the magocellular preoptic nucleus (MCPO). In adult guinea pigs (3-5 months of age), all of the preceding BF regions contained dense hypocretin fibers with varicosities. On the contrary, in old guinea pigs (27-28 months), although the MS exhibited a similar intensity of hypocretin immunoreactivity compared with the adult guinea pig, there was a significant decrease in the intensity of immunoreactivity of hypocretinergic fibers in the VDB, HDB and MCPO. These data indicate that the hypocretinergic innervation of specific nuclei of the BF is compromised during the aging process. We suggest that the reduction in hypocretinergic innervation of the BF nuclei may contribute to the age-related changes in the states of sleep and wakefulness as well as deficits in related systems that occur in old age.
Asunto(s)
Envejecimiento/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neuropéptidos/metabolismo , Telencéfalo/metabolismo , Animales , Femenino , Cobayas , Neuronas/citología , Neuronas/metabolismo , Orexinas , Sueño/fisiología , Telencéfalo/citología , Vigilia/fisiologíaRESUMEN
The present study was undertaken to determine the location of trigeminal and hypoglossal premotor neurons that express neuronal nitric oxide synthase (nNOS) in the cat. Cholera toxin subunit b (CTb) was injected into the trigeminal (mV) or the hypoglossal (mXII) motor nuclei in order to label the corresponding premotor neurons. CTb immunocytochemistry was combined with NADPH-d histochemistry or nNOS immunocytochemistry to identify premotor nitrergic (NADPH-d(+)/CTb(+) or nNOS(+)/ CTb(+) double-labeled) neurons. Premotor trigeminal as well as premotor hypoglossal neurons were located in the ventro-medial medullary reticular formation in a region corresponding to the nucleus magnocellularis (Mc) and the ventral aspect of the nucleus reticularis gigantocellularis (NRGc). Following the injection of CTb into the mV, this region was found to contain a total of 60 +/- 15 double-labeled neurons on the ipsilateral side and 33 +/- 14 on the contralateral side. CTb injections into the mXII resulted in 40 +/- 17 double-labeled neurons in this region on the ipsilateral side and 16 +/- 5 on the contralateral side. Thus, we conclude that premotor trigeminal and premotor hypoglossal nitrergic cells coexist in the same medullary region. They are colocalized with a larger population of nitrergic cells (7200 +/- 23). Premotor neurons in other locations did not express nNOS. The present data demonstrate that a population of neurons within the Mc and the NRGc are the source of the nitrergic innervation of trigeminal and hypoglossal motoneurons. Based on the characteristics of nitric oxide actions and its diffusibility, we postulate that these neurons may serve to synchronize the activity of mV and mXII motoneurons.
Asunto(s)
Bulbo Raquídeo/enzimología , Neuronas Motoras/enzimología , Proteínas del Tejido Nervioso/metabolismo , Óxido Nítrico Sintasa/metabolismo , Formación Reticular/enzimología , Núcleos del Trigémino/enzimología , Animales , Gatos , Femenino , Nervio Hipogloso/citología , Nervio Hipogloso/enzimología , Masculino , Bulbo Raquídeo/citología , Vías Nerviosas/citología , Vías Nerviosas/enzimología , Óxido Nítrico Sintasa de Tipo I , Formación Reticular/citología , Núcleos del Trigémino/citologíaRESUMEN
In a previous study, we noted the presence of enlarged "spot-like" structures that are immunoreactive to hypocretin in specific regions of brainstem of aged cats; similar structures were not seen in the same regions of adult cats. In the present study, electron microscopy was combined with hypocretin immunohistochemistry to examine the ultrastructure of these enlarged "spot-like" structures, which were found to consist of enlarged axonal terminals. The terminals were comprised of a large pale core with a dark peripheral rim. Many granules were present within the core; most of these granular structures were not immunostained. On the other hand, the rim contained a high density of hypocretin immunoreactivity; in this peripheral region, large dense-core vesicles and small synaptic vesicles without dense cores were observed. In addition, mitochondria in the peripheral rim region exhibited high electron density, which is indicative of the presence of age-related degenerative changes. Synaptic contacts were observed between the enlarged terminals and adjacent dendrites. Most of these synapses were asymmetric (Gray type I), although a few of them were symmetric (Gray type II). These data suggest that hypocretin transmission is altered during aging, which would be expected to result in age-related changes in the functioning of the hypocretinergic system.
Asunto(s)
Envejecimiento/patología , Tronco Encefálico/patología , Péptidos y Proteínas de Señalización Intracelular/análisis , Neuropéptidos/análisis , Médula Espinal/patología , Animales , Tronco Encefálico/química , Gatos , Femenino , Masculino , Orexinas , Terminales Presinápticos/química , Terminales Presinápticos/patología , Médula Espinal/químicaRESUMEN
The cholinergic system within the nucleus pontis oralis (NPO) of the pontine tegmentum is critically involved in the generation of active (rapid eye movement) sleep. Previously, we demonstrated that a GABAergic system in the NPO also plays an important role in the control of the behavioral states of wakefulness as well as active sleep. The present study examined interactions between these two neuronal systems vis-a-vis the occurrence of these behavioral states. Accordingly, cholinergic and GABAergic agonists and antagonists were injected into the NPO, and their combined effects on sleep and waking states of chronic, unanesthetized cats were examined. Microinjections of carbachol into the NPO elicited active sleep with a short latency. However, a preinjection of muscimol (a GABA(A) agonist) completely blocked the active sleep-inducing effects of carbachol. The induction of active sleep by carbachol was also suppressed by a subsequent injection of muscimol. On the other hand, the microinjection of scopolamine (a muscarinic receptor antagonist) did not block the induction of active sleep by bicuculline (a GABA(A) antagonist). We conclude that the excitatory cholinergic control of NPO neurons that are involved in the generation of active sleep is gated by a pontine GABAergic system that exerts its effects postsynaptically by inhibiting NPO neurons, resulting in the suppression of active sleep and the generation of wakefulness. In the absence of the activation of this GABAergic gating mechanism, active sleep occurs. These results reveal that specific interactions between cholinergic and GABAergic processes in the NPO play a critical role in the generation of active sleep and wakefulness.
Asunto(s)
Puente/fisiología , Receptores Colinérgicos/fisiología , Receptores de GABA/fisiología , Sueño REM/fisiología , Vigilia/fisiología , Animales , Bicuculina/farmacología , Carbacol/farmacología , Gatos , Agonistas Colinérgicos/farmacología , Antagonistas Colinérgicos/farmacología , Agonistas del GABA/farmacología , Antagonistas del GABA/farmacología , Muscimol/farmacología , Neuronas/fisiología , Puente/citología , Puente/efectos de los fármacos , Escopolamina/farmacología , Sueño REM/efectos de los fármacos , Técnicas Estereotáxicas , Vigilia/efectos de los fármacosRESUMEN
Loss of muscle tone during active (rapid-eye-movement, REM) sleep is due to the inhibition of motoneurons. This inhibition is manifest in high-gain intracellular electrophysiological records as hyperpolarizing synaptic noise, which includes large amplitude active sleep-specific inhibitory postsynaptic potentials (IPSPs). We report here evidence that the large active sleep-specific IPSPs are comprised of a small number of minimal unitary potentials that are characterized by fast rise-times (10-90% rise-times < or = 0.75 ms); they are present in high-gain records during quiet sleep or during active sleep where they are intermingled with larger active sleep-specific IPSPs with 10-90% rise-times > or = 1.00 ms and amplitudes that are integer multiples of the minimal unitary potentials. In hypoglossal motoneurons, the amplitude of these minimal unitary potentials averaged 0.33 +/- 0.04 mV (mean +/- S.D., n = 6). It is concluded that the large IPSPs with slow rise-times that are observed in motoneurons during active sleep are due to the nearly simultaneous arrival of multiple (< or = 5) minimal unitary potentials. We hypothesize that the same inhibitory interneurons that produce small IPSPs with fast rise-times during quiet sleep are also responsible for the large amplitude active sleep-specific IPSPs.
Asunto(s)
Nervio Hipogloso/fisiología , Bulbo Raquídeo/fisiología , Neuronas Motoras/fisiología , Inhibición Neural/fisiología , Sueño REM/fisiología , Transmisión Sináptica/fisiología , Vías Aferentes/fisiología , Vías Aferentes/ultraestructura , Animales , Gatos , Interneuronas/citología , Interneuronas/fisiología , Bulbo Raquídeo/citología , Potenciales de la Membrana/fisiología , Neuronas Motoras/citología , Terminales Presinápticos/fisiología , Terminales Presinápticos/ultraestructuraRESUMEN
The distribution of hypocretin-1 (hcrt-1) and hypocretin-2 (hcrt-2) immunoreactivities in the cat brainstem was examined using immunohistochemical techniques. Hcrt-1- and hcrt-2-positive fibers with varicosities were detected in almost all brainstem regions. However, no hcrt-1- or hcrt-2-immunoreactive neuronal somata were observed in the cat brainstem. Both hcrt-1- and hcrt-2-labeled fibers exhibited different densities in distinct regions of the brainstem. In most brainstem regions, the intensity of hcrt-1 immunoreactivity was higher than that of hcrt-2 immunoreactivity. The highest densities of hcrt-1- and hcrt-2-positive fibers were found in the nucleus raphe dorsalis (RD), the laterodorsal tegmental nucleus (LDT) and the locus coeruleus (LC), suggesting an important role for these peptides in functions related to sleep-wake behavior.
Asunto(s)
Proteínas Portadoras/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Bulbo Raquídeo/metabolismo , Neuronas/metabolismo , Neuropéptidos/metabolismo , Puente/metabolismo , Animales , Gatos , Inmunohistoquímica , Orexinas , Sueño/fisiología , Vigilia/fisiologíaRESUMEN
Cholinergic and gamma-aminobutyric acid (GABA) mechanisms in the dorsolateral pontomesencephalic tegmentum have been implicated in the control of active (REM) sleep and wakefulness. To determine the relationships between neurons that contain these neurotransmitters in this region of the brainstem in adult cats, combined light and electron microscopic immunocytochemical procedures were employed. Light microscopic analyses revealed that choline acetyltransferase (ChAT) and GABA immunoreactive neurons were distributed throughout the laterodorsal and pedunculopontine tegmental nuclei (LDT and PPT). Surprisingly, approximately 50% of the ChAT immunoreactive neurons in these nuclei also contained GABA. Using electron microscopic pre-embedding immunocytochemistry, GABA immunoreactivity was observed in somas, dendrites and axon terminals in both the LDT and PPT. Most of the GABA immunoreactive terminals formed symmetrical synapses with non-immunolabeled dendrites. Electron microscopic double-immunolabeling techniques revealed that ChAT and GABA were colocalized in axon terminals in the LDT/PPT. Approximately 30% of the ChAT immunoreactive terminals were also GABA immunoreactive, whereas only 6-8% of the GABA immunoreactive terminals were ChAT immunoreactive. Most of the ChAT/GABA immunoreactive terminals formed symmetrical synapses with non-immunolabeled dendrites; however, ChAT/GABA immunoreactive terminals were also observed that contacted ChAT immunoreactive dendrites. With respect to ChAT immunoreactive postsynaptic profiles, approximately 40% of the somas and 50% of the dendrites received synaptic contact from GABA immunoreactive terminals in both the LDT and PPT. These findings (a) indicate that there are fundamental interactions between cholinergic and GABAergic neurons within the LDT/PPT that play an important role in the control of active sleep and wakefulness and (b) provide an anatomical basis for the intriguing possibility that a mechanism of acetylcholine and GABA co-release from the terminals of LDT/PPT neurons is involved in the regulation of behavioral states.
Asunto(s)
Acetilcolina/metabolismo , Neuronas/metabolismo , Núcleo Tegmental Pedunculopontino/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Gatos , Colina O-Acetiltransferasa/metabolismo , Dendritas/metabolismo , Dendritas/ultraestructura , Inmunohistoquímica , Microscopía Electrónica , Neuronas/ultraestructura , Núcleo Tegmental Pedunculopontino/ultraestructura , Terminales Presinápticos/metabolismo , Terminales Presinápticos/ultraestructura , Sueño/fisiología , Transmisión Sináptica/fisiología , Vigilia/fisiologíaRESUMEN
The present study was undertaken to explore the neuronal mechanisms of hypocretin actions on neurons in the nucleus pontis oralis (NPO), a nucleus which plays a key role in the generation of active (REM) sleep. Specifically, we sought to determine whether excitatory postsynaptic potentials (EPSPs) evoked by stimulation of the laterodorsal tegmental nucleus (LDT) and spontaneous EPSPs in NPO neurons are modulated by hypocretin. Accordingly, recordings were obtained from NPO neurons in the cat in conjunction with the juxtacellular microinjection of hypocretin-1 onto intracellularly recorded cells. The application of hypocretin-1 significantly increased the mean amplitude of LDT-evoked EPSPs of NPO neurons. In addition, the frequency and the amplitude of spontaneous EPSPs in NPO neurons increased following hypocretin-1 administration. These data suggest that hypocretinergic processes in the NPO are capable of modulating the activity of NPO neurons that receive excitatory cholinergic inputs from neurons in the LDT.
Asunto(s)
Proteínas Portadoras/farmacología , Péptidos y Proteínas de Señalización Intracelular , Neuronas/efectos de los fármacos , Neuropéptidos/farmacología , Puente/efectos de los fármacos , Simpatomiméticos/farmacología , Sinapsis/efectos de los fármacos , Acetilcolina/fisiología , Animales , Gatos , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Neuronas/fisiología , Orexinas , Puente/citología , Sueño REM/efectos de los fármacos , Sinapsis/fisiología , Tegmento Mesencefálico/fisiologíaRESUMEN
The hypocretinergic system has been implicated in the generation and/or maintenance of wakefulness. Our results challenge this hypothesis. Utilizing cats as an animal model and immunocytochemical procedures for the simultaneous detection of hypocretin and Fos, we determined that hypocretinergic neurons are activated during wakefulness but only when somatomotor activity is present. These neurons are not activated during alert or quiet wakefulness in the absence of motor activity or during quiet sleep. We conclude that the hypocretinergic system is not responsible for the generation and/or maintenance of wakefulness, per se; on the contrary, we suggest that hypocretinergic neurons are primarily involved in motor functions irrespective of the animal's behavioral state.
Asunto(s)
Proteínas Portadoras/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Locomoción/fisiología , Neuronas/metabolismo , Neuropéptidos/metabolismo , Vigilia/fisiología , Animales , Gatos , Electroencefalografía , Electromiografía , Electrooculografía , Hipotálamo/metabolismo , Masculino , Orexinas , Sueño REM/fisiologíaRESUMEN
In the present study we found that mesencephalic trigeminal (Mes-V) neurons of the rat are innervated by nitrergic fibers and that nitric oxide (NO) modifies the electrophysiological properties of these cells. Mes-V neurons were surrounded by a network of fibers that contained neuronal nitric oxide synthase (nNOS); these fibers gave rise to terminal-, bouton-like structures which ended in Mes-V cells bodies. These cells, which did not display nNOS-like immunoreactivity were immunoreactive to a cGMP antibody. By performing intracellular recordings in the adult rat brain slice preparation, the effects of diethylenetriamine/NO adduct (DETA/NO) applications were examined. DETA/NO induced a depolarization that averaged 2.2 mV (range: 1-6 mV) in nine of 22 neurons. In 15 of 22 neurons (68% of the cells), there was a decrease in current threshold from 0.74 to 0.60 nA (19%; P<0.001). The excitatory effects of DETA/NO were abolished by ODQ, a blocker of soluble guanylate cyclase. Input resistance (R(in)) decreased in 80% of the cells from a mean of 24.8 to 20.6 Momega (17%; P<0.001) and the membrane time constant (tau(m)) decreased from 7.5 to 5.6 ms (25%; P<0.05). The 'sag' seen in the membrane response of these cells to current pulses was augmented during DETA/NO application. These findings indicate that there is a nitrergic innervation of Mes-V neurons and that these sensory cells are target for NO that may act on them as an excitatory neuromodulator promoting the synthesis of intracellular cGMP.
Asunto(s)
Mesencéfalo/fisiología , Fibras Nerviosas/enzimología , Óxido Nítrico Sintasa/fisiología , Óxido Nítrico/farmacología , Nervio Trigémino/fisiología , Animales , Toxina del Cólera , GMP Cíclico/metabolismo , Electrofisiología , Inhibidores Enzimáticos/farmacología , Guanilato Ciclasa/antagonistas & inhibidores , Peroxidasa de Rábano Silvestre , Inmunohistoquímica , Técnicas In Vitro , Músculo Masetero/inervación , Músculo Masetero/fisiología , Potenciales de la Membrana/efectos de los fármacos , Mesencéfalo/citología , Mesencéfalo/efectos de los fármacos , NADPH Deshidrogenasa/metabolismo , Fibras Nerviosas/efectos de los fármacos , Donantes de Óxido Nítrico/antagonistas & inhibidores , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa de Tipo I , Poliaminas/antagonistas & inhibidores , Poliaminas/farmacología , Ratas , Ratas Wistar , Nervio Trigémino/efectos de los fármacos , Nervio Trigémino/enzimologíaRESUMEN
Hypocretin-1 (hcrt-1) and hypocretin-2 (hcrt-2) are two recently discovered hypothalamic neuropeptides. In the present study, using double immunofluorescent techniques, the co-localization of hcrt-1 and hcrt-2 was examined in neuronal soma and fibers/terminals located, respectively, in the cat hypothalamus and brainstem. In the hypothalamus, all hcrt-1 positive neuronal soma also displayed hcrt-2 immunoreactivity. In the brainstem, both hcrt-1 and hcrt-2 antibodies labeled the same fibers/terminals, indicating that hcrt-1 and hcrt-2 co-localize not only in the neuronal soma (hypothalamus) but also in their fibers/terminals (brainstem). If both peptides are released following neuronal activity, then the distinct effects of these peptides in the brain are likely to depend on the types of postsynaptic receptors that are activated.
Asunto(s)
Tronco Encefálico/metabolismo , Proteínas Portadoras/metabolismo , Hipotálamo/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Neuropéptidos/metabolismo , Animales , Gatos , Técnica del Anticuerpo Fluorescente , Orexinas , Terminales Presinápticos/metabolismoRESUMEN
We demonstrate the presence of nitric oxide synthase containing fibers within the guinea pig trigeminal motor nucleus and describe the effects of nitric oxide (NO) on trigeminal motoneurons. Using immunohistochemical techniques, we observed nitrergic fibers displaying varicosities and giving rise to bouton-like structures in apposition to retrogradely labeled motoneuron processes, most of which were dendrites. NO-donors evoked a membrane depolarization (mean 7.5 mV) and a decrease in rheobase (mean 38%). These substances also evoked an apparent increase in an hyperpolarization-activated cationic current (I(H)). These changes were not accompanied by any modification of the motoneurons' input resistance or time constant. The effects were suppressed by blocking the cytosolic guanlyate cyclase. A membrane-permeant cyclic guanosine 3,5'-monophosphate (cGMP) analogue mimicked the effects of NO. There was a considerable increase in synaptic activity following NO-donors or db-cGMP application. Tetrodotoxin supressed the increase in synaptic activity evoked by NO-donors. The histological and electrophysiological evidence, taken together, indicates the existence of a nitrergic system able to modulate trigeminal motoneurons under yet unknown physiological conditions.