Orexins/hypocretins excite basal forebrain cholinergic neurones.

The orexins (orexin A and B, also known as hypocretin 1 and 2) are two recently identified neuropeptides (de Lecea et al., 1998; Sakurai et al., 1998) which are importantly implicated in the control of wakefulness (for reviews see Hungs and Mignot, 2001; van den Pol, 2000; Willie et al., 2001 ). Indeed, alteration in these peptides' precursor, their receptors or the hypothalamic neurones that produce them leads to the sleep disorder narcolepsy (Chemelli et al., 1999; Lin et al., 1999; Peyron et al., 2000; Thannickal et al., 2000). The mechanisms by which the orexins modulate wakefulness, however, are still unclear. Their presence in fibres coursing from the hypothalamus (Peyron et al., 1998) up to the preoptic area (POA) and basal forebrain (BF) suggests that they might influence the important sleep and waking neural systems situated there (Jones, 2000). The present study, performed in rat brain slices, demonstrates, however, that the orexins have no effect on the GABA sleep-promoting neurones of the POA, whereas they have a strong and direct excitatory effect on the cholinergic neurones of the contiguous BF. In addition, by comparing the effects of orexin A and B we demonstrate here that orexins' action depends upon orexin type 2 receptors (OX(2)), which are those lacking in narcoleptic dogs (Lin et al., 1999). These results suggest that the orexins excite cholinergic neurones that release acetylcholine in the cerebral cortex and thereby contribute to the cortical activation associated with wakefulness.

Orexins (hypocretins) directly excite tuberomammillary neurons.

Wakefulness has recently been shown to depend upon the newly identified orexin (or hypocretin) neuropeptides by the findings that alteration in their precursor protein, their receptors or the neurons that produce them leads to the sleep disorder narcolepsy in both animals and humans. The questions of how and where these brain peptides act to maintain wakefulness remain unresolved. The purpose of the present study was to determine whether the orexins could directly affect hypothalamic histaminergic neurons, which are known to contribute to the state of wakefulness by their diffuse projections through the brain. Using brain slices, we recorded in the ventral tuberomammillary nuclei from neurons identified as histaminergic on the basis of their previously described morphological and electrophysiological characteristics and found that they were depolarized and excited by the orexins through a direct postsynaptic action. We then compared the depolarizing effect of orexin A and B and found that they were equally effective upon these cells. This latter finding suggests that the effect of orexins is mediated by orexin type 2 receptors, which are those lacking in narcoleptic dogs. Our results therefore show that the histaminergic neurons of the tuberomammillary nuclei represent an important target for the orexin system in the maintenance of wakefulness.

Firing properties and electrotonic structure of Xenopus larval spinal neurons.

Whole cell voltage- and current-clamp measurements were done on intact Xenopus laevis larval spinal neurons at developmental stages 42-47. Firing patterns and electrotonic properties of putative interneurons from the dorsal and ventral medial regions of the spinal cord at myotome levels 4-6 were measured in isolated spinal cord preparations. Passive electrotonic parameters were determined with internal cesium sulfate solutions as well as in the presence of active potassium conductances. Step-clamp stimuli were combined with white-noise frequency domain measurements to determine both linear and nonlinear responses at different membrane potential levels. Comparison of analytic and compartmental dendritic models provided a way to determine the number of compartments needed to describe the dendritic structure. The electrotonic structure of putative interneurons was correlated with their firing behavior such that highly accommodating neurons (Type B) had relatively larger dendritic areas and lower electrotonic lengths compared with neurons that showed sustained action potential firing in response to a constant current (Type A). Type A neurons had a wide range of dendritic areas and potassium conductances that were activated at membrane potentials more negative than observed in Type B neurons. The differences in the potassium conductances were in part responsible for a much greater rectification in the steady-state current voltage (I-V curve) of the strongly accommodating neurons compared with repetitively firing cells. The average values of the passive electrotonic parameters found for Rall Type A and B neurons were c(soma) = 3.3 and 2.6 pF, g(soma) = 187 and 38 pS, L = 0.36 and 0.21, and A = 3.3 and 6.5 for soma capacitance, soma conductance, electrotonic length, and the ratio of the dendritic to somatic areas, respectively. Thus these experiments suggest that there is a correlation between the electrotonic structure and the excitability properties elicited from the somatic region.

Active dendritic membrane properties of Xenopus larval spinal neurons analyzed with a whole cell soma voltage clamp.

Voltage- and current-clamp measurements of inwardly directed currents were made from the somatic regions of Xenopus laevis spinal neurons. Current-voltage (I-V) curves determined under voltage clamp, but not current clamp, were able to indicate a negative slope conductance in neurons that showed strong accommodating action potential responses to a constant current stimulation. Voltage-clamp I-V curves from repetitive firing neurons did not have a net negative slope conductance and had identical I-V plots under current clamp. Frequency domain responses indicate negative slope conductances with different properties with or without tetrodotoxin, suggesting that both sodium and calcium currents are present in these spinal neurons. The currents obtained from a voltage clamp of the somatic region were analyzed in terms of spatially controlled soma membrane currents and additional currents from dendritic potential responses. Linearized frequency domain analysis in combination with both voltage- and current-clamp responses over a range of membrane potentials was essential for an accurate determination of consistent neuronal model behavior. In essence, the data obtained at resting or hyperpolarized membrane potentials in the frequency domain were used to determine the electrotonic structure, while both the frequency and time domain data at depolarized potentials were required to characterize the voltage-dependent channels. Finally, the dendritic and somatic membrane properties were used to reconstruct the action potential behavior and quantitatively predict the dependence of neuronal firing properties on electrotonic structure. The reconstructed action potentials reproduced the behavior of two broad distributions of interneurons characterized by their degree of accommodation. These studies suggest that in addition to the ionic conductances, electrotonic structure is correlated with the action potential behavior of larval neurons.