RESUMEN
Prolactin-releasing peptide (PrRP) is a recently discovered neuropeptide implicated in the central control of feeding behaviour and autonomic homeostasis. PrRP-containing neurones and PrRP receptor mRNA are found in abundance in the caudal portion of the nucleus tractus solitarius (NTS), an area which together with the dorsal motor nucleus of the vagus (DMV) comprises an integrated structure, the dorsal vagal complex (DVC) that processes visceral afferent signals from and provides parasympathetic motor innervation to the gastrointestinal tract. In this study, microinjection experiments were conducted in vivo in combination with whole-cell recording from neurones in rat medullary slices to test the hypothesis that PrRP plays a role in the central control of gastric motor function, acting within the DVC to modulate the activity of preganglionic vagal motor neurones that supply the stomach. Microinjection of PrRP (0.2 pmol (20 nl)(-1)) into the DMV at the level of the area postrema (+0.2 to +0.6 mm from the calamus scriptorius, CS) markedly stimulated gastric contractions and increased intragastric pressure (IGP). Conversely, administration of peptide into the DMV at sites caudal to the obex (0.0 to -0.3 mm from the CS) decreased IGP and reduced phasic contractions. These effects occurred without change in mean arterial pressure and were abolished by ipsilateral vagotomy, indicating mediation via a vagal-dependent mechanism(s). The pattern of gastric motor responses evoked by PrRP mimicked that produced by administration of L-glutamate at the same sites, and both the effects of L-glutamate and PrRP were abolished following local administration of NMDA and non-NMDA-type glutamate receptor antagonists. On the other hand, microinjection of PrRP into the medial or comissural nucleus of the solitary tract (mNTS and comNTS, respectively) resulted in less robust changes in IGP in a smaller percentage of animals, accompanied by marked alterations in arterial pressure. Superfusion of brain slices with PrRP (100-300 nm) produced a small depolarization and increased spontaneous firing in 10 of 30 retrogradely labelled gastric-projecting DMV neurones. The excitatory effects were blocked by administration of TTX (2 mum) or specific glutamate receptor antagonists, indicating that they resulted from interactions of PrRP at a presynaptic site. Congruent with this, PrRP increased the amplitude of excitatory postsynaptic currents (EPSCs, 154 +/- 33%, 12 of 25 neurones) evoked by electrical stimulation in mNTS or comNTS. In addition, administration of PrRP decreased the paired-pulse ratio of EPSCs evoked by two identical stimuli delivered 100 ms apart (from 0.95 +/- 0.08 to 0.71 +/- 0.11, P < 0.05), whereas it did not affect the amplitude of inward currents evoked by exogenous application of L-glutamate to the slice. The frequency, but not amplitude of spontaneous EPSCs and action potential-independent miniature EPSCs was also increased by administration of PrRP, suggesting that the peptide was acting at least in part at receptors on presynaptic nerve terminals to enhance glutamatergic transmission. In recordings obtained from a separate group of slices, we did not observe any direct effects of PrRP on spontaneous discharge or postsynaptic excitability in either mNTS or comNTS neurones (n = 31). These data indicate that PrRP may act within the DVC to regulate gastric motor function by modulating the efficacy of conventional excitatory synaptic inputs from the NTS onto gastric-projecting vagal motor neurones.
Asunto(s)
Motilidad Gastrointestinal/fisiología , Hormonas Hipotalámicas/fisiología , Neuropéptidos/fisiología , Transmisión Sináptica/fisiología , Nervio Vago/fisiología , Animales , Tronco Encefálico/efectos de los fármacos , Tronco Encefálico/fisiología , Potenciales Evocados , Potenciales Postsinápticos Excitadores , Femenino , Motilidad Gastrointestinal/efectos de los fármacos , Ácido Glutámico , Hormonas Hipotalámicas/farmacología , Masculino , Neuronas/efectos de los fármacos , Neuronas/fisiología , Neuropéptidos/farmacología , Hormona Liberadora de Prolactina , Ratas , Ratas Sprague-Dawley , Estómago/inervación , Transmisión Sináptica/efectos de los fármacos , Nervio Vago/efectos de los fármacosRESUMEN
In addition to action potential-evoked exocytotic release at neurohypophysial nerve terminals, the neurohormones arginine vasopressin (aVP) and oxytocin (OT) undergo Ca(2+)-dependent somatodendritic release within the supraoptic and paraventricular hypothalamic nuclei. However, the cellular and molecular mechanisms that underlie this release have not been elucidated. In the present study, the whole-cell patch-clamp technique was utilized in combination with high-time-resolved measurements of membrane capacitance (C(m)) and microfluorometric measurements of cytosolic free Ca(2+) concentration ([Ca(2+)](i)) to examine the Ca(2+) and stimulus dependence of exocytosis in the somata of magnocellular neurosecretory cells (MNCs) isolated from rat supraoptic nucleus (SON). Single depolarizing steps (> or =20 ms) that evoked high-voltage-activated (HVA) Ca(2+) currents (I(Ca)) and elevations in intracellular Ca(2+) concentration were accompanied by an increase in C(m) in a majority (40/47) of SON neurones. The C(m) responses were composed of an initial Ca(2+)-independent, transient component and a subsequent, sustained phase of increased C(m) (termed DeltaC(m)) mediated by an influx of Ca(2+), and increased with corresponding prolongation of depolarizing step durations (20-200 ms). From this relationship we estimated the rate of vesicular release to be 1533 vesicles s(-1). Delivery of neurone-derived action potential waveforms (APWs) as stimulus templates elicited I(Ca) and also induced a DeltaC(m), provided APWs were applied in trains of greater than 13 Hz. A train of APWs modelled after the bursting pattern recorded from an OT-containing neurone during the milk ejection reflex was effective in supporting an exocytotic DeltaC(m) in isolated MNCs, indicating that the somata of SON neurones respond to physiological patterns of neuronal activity with Ca(2+)-dependent exocytotic activity.
Asunto(s)
Calcio/metabolismo , Exocitosis/fisiología , Neuronas/fisiología , Núcleo Supraóptico/fisiología , Potenciales de Acción , Animales , Señalización del Calcio , Citosol/metabolismo , Capacidad Eléctrica , Electrofisiología , Fluorometría , Lactancia/fisiología , Neuronas/metabolismo , Concentración Osmolar , Oxitocina/metabolismo , Ratas , Ratas Sprague-Dawley , Tiempo de Reacción , Núcleo Supraóptico/metabolismoRESUMEN
Orexin (hypocretin)-containing projections from lateral hypothalamus (LH) are thought to play an important role in the regulation of feeding behaviour and energy balance. In rodent studies, central administration of orexin peptides increases food intake, and orexin neurones in the LH are activated by hypoglycaemia during fasting. In addition, administration of orexins into the fourth ventricle or the dorsal motor nucleus of the vagus (DMV) has been shown to stimulate gastric acid secretion and motility, respectively, via vagal efferent pathways. In this study, whole-cell recordings were obtained from DMV neurones in rat brainstem slices to investigate the cellular mechanism(s) by which orexins produce their gastrostimulatory effects. To determine whether responsiveness to orexins might be differentially expressed among distinct populations of preganglionic vagal motor neurones, recordings were made from neurones whose projections to the gastrointestinal tract had been identified by retrograde labelling following apposition of the fluorescent tracer DiI to the gastric fundus, corpus or antrum/pylorus, the duodenum or caecum. Additionally, the responses of neurones to orexins were compared with those produced by oxytocin, which acts within the DMV to stimulate gastric acid secretion, but inhibits gastric motor function. Bath application of orexin-A or orexin-B (30-300 nM) produced a slow depolarization, accompanied by increased firing in 47 of 102 DMV neurones tested, including 70 % (30/43) of those that projected to the gastric fundus or corpus. In contrast, few DMV neurones that supplied the antrum/pylorus (3/13), duodenum (4/18) or caecum (1/13) were responsive to these peptides. The depolarizing responses were concentration dependent and persisted during synaptic isolation of neurones with TTX or Cd2+, indicating they resulted from activation of postsynaptic orexin receptors. They were also associated with a small increase in membrane resistance, and in voltage-clamp recordings orexin-A induced an inward current that reversed near the estimated equilibrium potential for K+, indicating the depolarization was due in large part to a reduction in K+ conductance. Orexins did not affect synaptically evoked excitation, but did reduce membrane excitability in a subset of gastric-projecting DMV neurones by enhancing GABA-mediated synaptic input. Lastly, although many DMV neurones responded to orexins and oxytocin with excitation, for the most part these peptides modulated excitability of distinct populations of gastric-projecting vagal motor neurones. These results indicate that orexins act preferentially within the DMV to directly excite vagal motor neurones that project to gastric fundus and corpus. In this way, release of endogenous orexins from descending hypothalamic projections into the DMV may mediate the increase in gastric acid secretion and motor activity associated with the cephalic phase of feeding.