Stimulation of orexin/hypocretin neurones by thyrotropin-releasing hormone

Abstract

Central orexin/hypocretin neurones are critical for sustaining consciousness: their firing stimulates wakefulness and their destruction causes narcolepsy. We explored whether the activity of orexin cells is modulated by thyrotropin-releasing hormone (TRH), an endogenous stimulant of wakefulness and locomotor activity whose mechanism of action is not fully understood. Living orexin neurones were identified by targeted expression of green fluorescent protein (GFP) in acute brain slices of transgenic mice. Using whole-cell patch-clamp recordings, we found that TRH robustly increased the action potential firing rate of these neurones. TRH-induced excitation persisted under conditions of synaptic isolation, and involved a Na(+)-dependent depolarization and activation of a mixed cation current in the orexin cell membrane. By double-label immunohistochemistry, we found close appositions between TRH-immunoreactive nerve terminals and orexin-A-immunoreactive cell bodies. These results identify a new physiological modulator of orexin cell firing, and suggest that orexin cell excitation may contribute to the arousal-enhancing actions of TRH.

Figure 1. Effects of thyrotropin-releasing hormone (TRH) on the membrane potential of orexin neurones

A, an eGFP-expressing orexin neurone during a whole-cell recording (left; scale bar, 30 μm). The cell was identified in a brain slice by epifluorescence (right). B, effect of 250 nm TRH on an orexin cell recorded using a KCl pipette solution. Breaks in this and subsequent current-clamp traces correspond to intervals (< 30 s) where the recording was paused to perform voltage-clamp analysis. C, effect of 250 nm TRH on an orexin cell recorded using a potassium gluconate pipette solution. D, effect of 250 nm TRH in the presence of 1 μm bath tetrodotoxin. E, lack of effect of 250 nm TRH free acid, a biologically inactive TRH analogue. F, mean firing rate of orexin neurones (n= 11) in the absence and presence of 250 nm TRH, **P < 0.001. G, TRH dose–response curve (EC₅₀= 6.2 nm, see Methods). Each point corresponds to ≥ 3 cells.

Figure 2. Effects of ion substitution and drugs on membrane potential responses to TRH

A, effect of 250 nm TRH on an orexin cell in ‘low Ca²⁺’ extracellular solution (see Methods). B, effect of 250 nm TRH on an orexin cell in ‘low Na⁺’ extracellular solution (see Methods). C, lack of effect of the Na⁺/Ca²⁺ exchange blocker KB−R7943 (70 μm) on depolarization induced by 250 nm TRH. D, summary of mean depolarization elicited by 250 nm TRH in the presence of different solutions and drugs. **P < 0.005, each bar corresponds to at least 4 cells. n.s. = no significant difference (P > 0.2).

Figure 3. Effects of TRH on membrane current–voltage relationship of orexin neurones

A, the voltage-clamp protocol used to obtain data in B. B, currents obtained in response to voltage steps using a ‘low Ca²⁺’ extracellular solution (see Methods). Grey bars (a and b) show where the steady-state values were measured to produce the plot in C. C, net current activated by TRH (b minus a), n= 4 cells.

Figure 4. Appositions between TRH terminals and orexin cell bodies

Confocal micrographs from coronal sections of the mouse hypothalamus double-labelled with immunofluorescence for orexin (green) and TRH (red). A–C show low-magnification overview of the lateral hypothalamic area (LHA); panels A and B are merged in C. Note orexin-immunoreactive cell bodies clustered in the LHA, while a dense plexus of TRH-immunoreactive fibres is seen in the dorsomedial hypothalamus (DMH). An area of overlap is especially evident in the border zone. At high resolution (D–F), TRH-immunoreactive terminals can be seen forming close appositions (arrowheads) on orexin-immunoreactive cell somata (asterisk) and proximal dendrites (arrows). Scale bars are: 100 μm in A–C, 10 μm in D, 5 μm in E and F. V3−, third ventricle; fx, fornix.