The wake-promoting hypocretin-orexin neurons are in an intrinsic state of membrane depolarization

Abstract

Wakefulness depends on the activity of hypocretin-orexin neurons because their lesion results in narcolepsy. How these neurons maintain their activity to promote wakefulness is not known. Here, by recording for the first time from hypocretin-orexin neurons and comparing their properties with those of neurons expressing melanin-concentrating hormone, we show that hypocretin-orexin neurons are in an intrinsic state of membrane depolarization that promotes their spontaneous activity. We propose that wakefulness and associated energy expenditure thus depend on that property, which allows the hypocretin-orexin neurons to maintain a tonic excitatory influence on the central arousal and peripheral sympathetic systems.

Fig. 1.

Characterization of neurons expressing Hcrt/Orx or MCH. A₁, Tonic firing in response to a depolarizing current pulse delivered from the level of resting potential (arrowhead).A₂,A₃, LTS (arrow) and ADP (asterisk) triggered by a depolarizing current pulse delivered from an hyperpolarized level. Additional hyperpolarization eliminates the LTS and the ADP (bottom trace in A₃).B, Superimposed responses to hyperpolarizing pulses suggesting the presence of an I_h current (dot). Note that only the trace with the deepest hyperpolarization is shown in full. C, Tonic firing at rest and its elimination by TTX (1.0 μm) to determine resting potentials. D, F, Immunohistochemical identification of an Hcrt/Orx neuron injected with neurobiotin (arrowhead in D) and expressing immunoreactivity for Hcrt/Orx (E) but not for the MCH (F). G₁, Firing with accommodation triggered by a depolarizing current pulse delivered from the resting potential level.G₂,G₃, Absence of either LTS or ADP in response to depolarizing pulses applied from more hyperpolarized levels. H, Responses to hyperpolarizing current pulses demonstrating the absence (dot) of any sag that could have indicated the presence of an I_hcurrent. I, Absence of spontaneous firing in such neurons and their mean resting potential. J,L, Immunohistochemical identification of an MCH neuron injected with neurobiotin (arrowhead inJ) and expressing immunoreactivity for MCH (L) but not for Hcrt/Orx (K). Membrane potentials are as follows (arrowheads): −47 mV (A), −44 mV (B), −48 mV (C), −61 mV (G), and −61 mV (H,I). Fx, Fornix.

Fig. 2.

Properties of hypocretin–orexin neurons.A, Persistence of the LTS and ADP in the presence of TTX (1.0 μm) and cesium chloride (2 mm).Inset illustrates elimination of the time- and voltage-dependent sag by cesium chloride. B,D, Elimination of the ADP but persistence of the LTS in presence of NMDG (B), BAPTA (20 mm;C), or FFA (100 μm; D).Inset in D shows that further addition of Ni (200 μm) eliminates the LTS.

Fig. 3.

Persistence of the membrane depolarization and spontaneous activity of hypocretin–orexin neurons in conditions of synaptic blockade and their inhibition by GABA. A, Persistence of the membrane depolarization in TTX (1.0 μm), ionotropic blockers (MK801 at 20 μm, NBQX at 10 μm, and bicuculline at 10 μm), and an ACSF with 0.1 mm Ca²⁺ and 10 mm Mg²⁺. B, Persistence of the spontaneous activity in the presence of synaptic blockade (right is an enlargement of the area identified by anasterisk in the left). C, Inhibition by a brief application of muscimol (5 sec at 100 μm). Membrane potentials are as follows (arrowheads): −42 mV (A), −43 mV (B), and −44 mV (C).