Thyrotropin-releasing hormone increases behavioral arousal through modulation of hypocretin/orexin neurons

Abstract

Thyrotropin-releasing hormone (TRH) has previously been shown to promote wakefulness and to induce arousal from hibernation. Expression of TRH-R1 (TRH receptor 1) is enriched in the tuberal and lateral hypothalamic area (LHA), brain regions in which the hypocretin/orexin (Hcrt) cells are located. Because the Hcrt system is implicated in sleep/wake control, we hypothesized that TRH provides modulatory input to the Hcrt cells. In vitro electrophysiological studies showed that bath application of TRH caused concentration-dependent membrane depolarization, decreased input resistance, and increased firing rate of identified Hcrt neurons. In the presence of tetrodotoxin, TRH induced inward currents that were associated with a decrease in frequency, but not amplitude, of miniature postsynaptic currents (PSCs). Ion substitution experiments suggested that the TRH-induced inward current was mediated in part by Ca(2+) influx. Although TRH did not significantly alter either the frequency or amplitude of spontaneous excitatory PSCs, TRH (100 nm) increased the frequency of spontaneous inhibitory PSCs by twofold without affecting the amplitude of these events, indicating increased presynaptic GABA release onto Hcrt neurons. In contrast, TRH significantly reduced the frequency, but not amplitude, of miniature excitatory PSCs without affecting miniature inhibitory PSC frequency or amplitude, indicating that TRH also reduces the probability of glutamate release onto Hcrt neurons. When injected into the LHA, TRH increased locomotor activity in wild-type mice but not in orexin/ataxin-3 mice in which the Hcrt neurons degenerate postnatally. Together, these results are consistent with the hypothesis that TRH modulates behavioral arousal, in part, through the Hcrt system.

Figure 1.

TRH depolarizes hypocretin neurons in the LHA. A, Current-clamp recording showing the excitatory effect of 100 nm TRH on the firing of action potentials of a hypocretin neuron. Membrane potential was adjusted to −60 mV by DC current injection; hyperpolarizing current pulses (−0.3 nA; 800 ms) were delivered every 5 s. B, Concentration dependence of the TRH effect on membrane potential. The IC₅₀ value was estimated at 66 nm (Hill coefficient, 1.0). Data are mean ± SEM (n = 2–15 determinations per concentration). C, The effects of TRH on spike frequency of Hcrt neurons are reversible after washout. Error bars indicate SEM (n = 5).

Figure 2.

TRH depolarizes hypocretin neurons in the presence of TTX. A, Whole-cell current-clamp recording of an Hcrt neuron showing that TRH (100 nm) depolarized Hcrt neurons in the presence of 0.5 μm TTX. B, Representative trace of TRH-induced inward current under voltage-clamp recording at a holding potential of −60 mV. C, I–V relationship under current-clamp recording mode in the presence and absence of TRH when calcium was replaced with strontium in the external medium. D, TRH-induced voltage change produced when calcium was replaced with strontium in the external medium. Data were derived from C. Error bars indicate SEM.

Figure 3.

TRH increases the frequency of sIPSCs, but not sEPSCs, recorded in hypocretin neurons. A, Spontaneous EPSCs were recorded by whole-cell voltage clamp at a holding potential of −60 mV in the presence of bicuculline (40 μm). B, TRH (100 nm) did not affect the frequency or amplitude of sEPSCs (n = 8). C, Spontaneous IPSCs were recorded at a holding potential of −60 mV in the presence of AP-5 (50 μm) and DNQX (20 μm). D, TRH increased the frequency of sIPSCs by twofold (p = 0.027) without affecting sIPSC amplitude (n = 14). Error bars indicate SEM. *p < 0.05.

Figure 4.

TRH reduces the frequency of miniature EPSCs but not miniature IPSCs recorded in Hcrt neurons. A, mEPSCs were recorded by whole-cell voltage clamp at a holding potential of −60 mV in the presence of TTX (0.5 μm) and bicuculline (40 μm). B, TRH (100 nm) reduced the frequency (p < 0.0001) but not the amplitude of mEPSCs (n = 13). *p < 0.05. C, mIPSCs were recorded by whole-cell voltage clamp at a holding potential of −60 mV in the presence of TTX (0.5 μm), AP-5 (50 μm), and DNQX (20 μm). D, TRH (100 nm) did not significantly affect either mIPSC frequency or amplitude (n = 4). Error bars indicate SEM.

Figure 5.

Identification of injection sites in wild-type and orexin/ataxin-3 mice. A, Distribution of hypocretin-immunoreactive cells in a representative brain section of a wild-type mouse. The arrows show location of the guide cannula, and the triangles show the tracks of the injection cannula. B, Location of the injection site in the same brain section as A is indicated by FluoroGold fluorescence. Note that the location of the injection site is dorsal to the distribution of hypocretin neurons. C, In orexin/ataxin-3 mice, there is a dramatic reduction in the number of hypocretin-immunoreactive cells (compare C, A). D, The location of FluoroGold injection site in the same brain section from the orexin/ataxin-3 mouse immunostained in C.

Figure 6.

Effect of unilateral TRH injections into the lateral hypothalamus on LMA in wild-type and orexin/ataxin-3 mice. LMA counts recorded by the transmitters were averaged over 5 min intervals. The zero time point represents the LMA calculated during the first 5 min after the injection. A–C, Saline (A), 0.1 μg of TRH (B), or 1 μg of TRH (C) was injected in a balanced order through a guide cannula implanted in the lateral hypothalamus in both wild-type (n = 6) and orexin/ataxin-3 mice (n = 6). D, Average LMA counts in the 10 min interval from 5 to 15 min after injection of TRH or saline. TRH at the dose of 1 μg produces significantly more LMA counts than saline injection in wild-type mice but not in orexin/ataxin-3 mice. *p < 0.05 compared with the saline group of the same genotype. Error bars indicate SEM.

Figure 7.

Effects of 2 min versus 2 h exposure to isoflurane on LMA in orexin/ataxin-3 mice compared with litter-matched controls. A, Mice were anesthetized with 2% isoflurane for 2 min and then allowed to recover while LMA was recorded. B, The same mice were anesthetized with 2% isoflurane for induction followed by 1.5% isoflurane for 2 h and then allowed to recover while LMA was recorded. One week was allowed for the mice to recover between 2 min and 2 h isoflurane treatments. Error bars indicate SEM.

Figure 8.

Relationship between injection site location and change in LMA induced in mice injected with 1 μg of TRH. The symbols overlaid on a mouse brain atlas image indicate the location of the injection sites and are color-coded to indicate the magnitude of the effect of 1 μg of TRH on average LMA counts. The greatest increase in LMA was seen in the wild-type mice in which the tip of injection cannula was located near the core of hypocretin neurons. In the orexin/ataxin-3 mice, the increase of LMA was not significant (compare Fig. 6D), although some injections were located at the brain sites in which TRH effectively increased LMA in wild-type mice.

Figure 9.

Effect of unilateral TRH injections into the lateral hypothalamus on body temperature. T_b data were averaged over 5 min intervals; the zero time point represents the T_b observed within the first 5 min after the injection. A–C, Saline (A), 0.1 μg of TRH (B), or 1 μg of TRH (C) was injected as described in Figure 6. D, Average T_b for the 60 min after injection of TRH or saline in wild-type and orexin/ataxin-3 mice. TRH at the dose of 1 μg produces significant increase in T_b compared with saline in both wild-type and orexin/ataxin-3 mice. *p < 0.05 compared with the saline group of the same genotype. Error bars indicate SEM.

Figure 10.

Schematic summarizing the direct and indirect effects of TRH on hypocretin neurons. In addition to direct postsynaptic excitatory effects (presumably mediated by TRH-R1), TRH increases GABA release but reduces the probability of glutamate release at presynaptic terminals.