Low-voltage-activated A-current controls the firing dynamics of mouse hypothalamic orexin neurons

Abstract

The activity of hypothalamic neurons that release the neuropeptides orexin-A and orexin-B is essential for normal wakefulness. Orexin neurons fire spontaneously and are hyperpolarized and inhibited by physiological neuromodulators, but the intrinsic determinants of their electrical activity are poorly understood. We show that mouse orexin neurons coexpress orexin-A and orexin-B, and possess a low-voltage-activated A-type K(+) current (A-current) likely to be composed of Kv4.3 subunits. The A-current enhances the inhibitory influence of hyperpolarizing currents via two mechanisms: by delaying the resumption of spiking after hyperpolarization and by increasing the slope of the relation between the firing frequency and injected current. These results identify an important determinant of the firing dynamics of orexin neurons, and support the idea that the A-current can control neuronal gain.

Fig. 1. Neurochemical and electrical properties of orexin neurons in the mouse LH.

(A) LH brain slice double-stained with orexin-A (orx-A, blue) and orexin-B (orx-B, green) antibodies. In simultaneous confocal imaging (orx-A/orx-B), neurons that contain both peptides appear in cyan. All neurons imaged contained both peptides. (B) Electrical properties of orexin cells: nonadapting firing response to depolarization (i), depolarizing sag in response to hyperpolarization (ii), and low-threshold spike on depolarization from a hyperpolarized potential (iii). The current-clamp protocol is shown under the trace. Action potentials are truncated at 0 mV. (C) Immunocytochemical identification of an LH neuron (arrow) filled with neurobiotin (red) and expressing orexin-A (orx-A, blue).

Fig. 2. Biophysical and molecular properties of A-currents in LH orexin neurons. Currents were recorded in 1 μm TTX.

(A) Transient outward current elicited by a step to −40 mV from −90 mV is blocked by 10 mm 4-AP (n = 7). (B) Immunolabelling of an LH neuron for orexin-B (orx-B, green) and Kv4.3 (blue). Simultaneous confocal imaging (orx-B/Kv4.3) shows that these proteins are found in the same cell (n = 50). Scale bar, 5 μm. (C) Left, A-currents evoked by steps to −60 mV (blue), −50 mV (green) and −40 mV (red), from −90 mV. Right, voltage-dependence of A-type conductance (g_A, see Materials and methods) expressed as a fraction of maximal conductance (g_Amax) (n = 6). The curve is the best fit of Eqn 1 (see Materials and methods) to the data. (D) Voltage-dependence of the extent of A-current inactivation. Left, A-currents induced by a test pulse to 0 mV, after an 800 ms prepulse to −40 mV (red), −50 mV (green) and −60 mV (blue). Right, peak A-current (I_A) is expressed as a fraction of that elicited following a prepulse to −120 mV (I_Amax), and plotted against the prepulse potential (n = 6). The curve is the best fit of Eqn 2 (see Materials and methods) to the data. (E) Time-dependence of I_A recovery from inactivation. Left, the neuron was held at −40 mV for 1 s to inactivate I_A, then stepped to −90 mV for varying durations to remove inactivation, following which I_A was elicited by a test pulse to −40 mV. The protocol is illustrated above the data traces. Right, mean peak I_A (n = 7) during the test pulse plotted against the duration of the de-inactivating −90 mV interpulse. The curve is the best fit of a single exponential function with a time constant of 140 ± 20 ms.

Fig. 3. Effects of A-currents on the firing dynamics of LH orexin neurons.

(A) Rebound hyperpolarization (arrowed) on recovery from hyperpolarizing current pulses is reversibly abolished by 10 mm 4-AP (n = 5). (B) Firing responses to sustained hyperpolarizing current injections of two neurons, one with a large (blue) and one with a small (red) A-current (A-currents elicited as in Fig. 2A). The magnitudes of A-currents and the injected current were divided by cell capacitance (pF) to correct for differences in cell size. Action potentials are truncated at 0 mV. (C) Summary of the data shown in B, for seven orexin cells. There is a strong positive correlation (r = 0.91, P < 0.005, slope = 11, n = 7) between A-type conductance density and the sensitivity of firing to current injection (slope of the firing-current relations). (D) Blocking the A-current with 4-AP (10 mm) decreases the slope of the firing-current relation in an orexin neuron (n = 3).