Excitation of histaminergic tuberomamillary neurons by thyrotropin-releasing hormone

Abstract

The histaminergic tuberomamillary nucleus (TMN) controls arousal and attention, and the firing of TMN neurons is state-dependent, active during waking, silent during sleep. Thyrotropin-releasing hormone (TRH) promotes arousal and combats sleepiness associated with narcolepsy. Single-cell reverse-transcription-PCR demonstrated variable expression of the two known TRH receptors in the majority of TMN neurons. TRH increased the firing rate of most (ca 70%) TMN neurons. This excitation was abolished by the TRH receptor antagonist chlordiazepoxide (CDZ; 50 mum). In the presence of tetrodotoxin (TTX), TRH depolarized TMN neurons without obvious change of their input resistance. This effect reversed at the potential typical for nonselective cation channels. The potassium channel blockers barium and cesium did not influence the TRH-induced depolarization. TRH effects were antagonized by inhibitors of the Na(+)/Ca(2+) exchanger, KB-R7943 and benzamil. The frequency of GABAergic spontaneous IPSCs was either increased (TTX-insensitive) or decreased [TTX-sensitive spontaneous IPSCs (sIPSCs)] by TRH, indicating a heterogeneous modulation of GABAergic inputs by TRH. Facilitation but not depression of sIPSC frequency by TRH was missing in the presence of the kappa-opioid receptor antagonist nor-binaltorphimine. Montirelin (TRH analog, 1 mg/kg, i.p.) induced waking in wild-type mice but not in histidine decarboxylase knock-out mice lacking histamine. Inhibition of histamine synthesis by (S)-alpha-fluoromethylhistidine blocked the arousal effect of montirelin in wild-type mice. We conclude that direct receptor-mediated excitation of rodent TMN neurons by TRH demands activation of nonselective cation channels as well as electrogenic Na(+)/Ca(2+) exchange. Our findings indicate a key role of the brain histamine system in TRH-induced arousal.

Figure 1.

TRH enhances firing rate of TMN neurons. A, B, Averaged ratemeter recordings of responses to bath applied TRH in mouse (A; 1.5 μm TRH, n = 6) and rat (B; 2–10 μm TRH, n = 10) TMN neurons. Four nonresponding cells are not included. C, Extracellular action potential recordings in rat TMN neuron: regular firing, excitation by TRH 2 μm, and recovery after 15 min. Averaged extracellular triphasic action potentials (80–150 APs for each), reduction of amplitude during TRH indicating depolarization. D, Chlordiazepoxide, a TRH receptor antagonist (CDZ; open bar indicates period of bath application), reduces the TRH response in rat TMN neurons at 10 μm (n = 4) and abolishes it at 50 μm (n = 6). E, TRH applied in the presence of benzamil-enhanced firing rate in a significantly smaller fraction of TMN neurons to a significantly smaller extent (n = 10) compared with the control group (n = 14; top trace).

Figure 2.

Expression of TRH receptors in rat and mouse TMN neurons. A, B, Single-cell RT-PCR analysis of TRH receptor expression in five mouse (A) and five rat (B) TMN neurons (phase-contrast light microscope photographs of corresponding cells are given). Pc, Positive control: posterior hypothalamus; nc, negative control; M-DNA size marker: 100 bp ladder (500 bp, most intense line). C, Summary of single-cell RT-PCR analysis done in 26 mouse and 19 rat TMN neurons positive for the histamine synthesizing enzyme HDC.

Figure 3.

Intracellular sharp-electrode recordings from rat TMN neurons. A, Depolarization and increased firing in the presence of TRH. After washout inhibition by an H3-receptor agonist (R-α-methylhistamine). B, During 1 μm TTX, TRH depolarizes the neuron, indicating a postsynaptic site of action. During maximal depolarization and manual clamping to the resting potential, no obvious change in input resistance is seen. C, Depolarization by TRH is not significantly affected by 500 μm BaCl₂ and 3 mm CsCl but markedly reduced by 80 μm KB-R7943. **p < 0.01, Student's two-tailed t test. Right, Average depolarizations (mean ± SE) under each condition in 1 μm TTX, for responding cells; numbers of cells are near bars. D, The voltage–current plots recorded in the same neuron before and during TRH treatment. The curves intersect at −4.3 ± 6.3 mV, close to the reversal potential predicted for a mixed cationic conductance.

Figure 4.

Ventrolateral TMN neurons respond to TRH and montirelin in a dose-dependent manner. A, TMN neuron stained with both biocytin (red) and HDC (green). Scale bar, 25 μm. B, Photograph of TMN neuron in slice approached with a recording patch pipette (right) and puff pipette filled with TRH (left); scale bar, 20 μm. C, Examples of TMN neuronal firing in control and in the presence of TRH or montirelin. D, Time course diagram illustrating change in averaged (±SE) firing frequency of TMN neurons in the presence of different concentrations of montirelin: n = 5, 4, and 4 cells tested for montirelin 2.5, 10, and 40 μm, respectively. E, Averaged time course diagrams for TRH 1 μm (n = 5) and 10 μm (n = 6).

Figure 5.

Whole-cell voltage-clamp sIPSC recordings from TMN neurons in rat thin slices. A, TMN neuron approached with a patch pipette (scale bar, 20 μm) and identified with the stimulation protocol shown at right. Hyperpolarization-activated inward current (I_h) becomes obvious after voltage jumps from −50 to −90 mV and to more negative values. Maximal outward I_A current is seen after return to the holding membrane potential from −120 mV. B, TRH (2.5 μm)-induced depression of sIPSC frequency (at room temperature). The GABA_A receptor antagonist gabazine (10 μm) blocks spontaneous synaptic activity. C, AMPA and NMDA receptor antagonists (DNQX and d-AP5, respectively) reduce frequency of sIPSCs without affecting their kinetics or amplitude.

Figure 6.

TRH induces inward current and modulates frequency of sIPSCs in TMN neurons recorded in whole-cell voltage-clamp at 32°C. A, TRH-mediated inward current in TMN neuron where frequency of sIPSCs was increased. Summary diagram at right shows three neuronal groups: no change, decrease, and increase in sIPSC frequency under TRH (control 100%). Dark gray bars show percentage frequency changes in neurons, where no direct postsynaptic currents in response to TRH were recorded. Light gray bars show percentage frequency changes in cells where TRH-evoked currents measured; averages of these currents are given as black bars at the right side of the box. B, Neuron displaying an increased sIPSC frequency in response to TRH and summary of frequency changes in all cells belonging to the same group at right. co, Control; wa, washout. C, Neuron displaying decreased sIPSC frequency under TRH. As no difference in sIPSC occurrence (suppression or enhancement) by TRH was obtained with Fisher's exact probability test (p = 0.36) between neurons recorded at room or more physiological temperature, all recordings were pooled. Cells with control sIPSC frequency <0.1 Hz were not included in summary diagrams.

Figure 7.

Unimodal presynaptic modulation of spontaneous IPSCs by TRH is seen either in the presence of TTX or the antagonist at the κ-opioid receptor, nor-binaltorphimine (1 μm each). A, Neuron with an increased miniature IPSC frequency in response to TRH and summary of frequency changes in all cells recorded in the presence of TTX (at right). Kolmogorov–Smirnov Z (K–S Z) and p values are given on corresponding cumulative fraction histograms, illustrating no change in amplitude but increase of frequency (decrease in interevent intervals) in the presence of TRH. B, Neuron with a decreased sIPSC frequency in response to TRH in the presence of nor-binaltorphimine and summary of frequency changes in all cells belonging to the same group (at right). co, Control; wa, washout.

Figure 8.

The TRH analog montirelin lacks waking effect in histamine deficient mice (HDC^−/−). A, Waking effect of montirelin (1 mg/kg, i.p.) in wild-type mice (HDC^+/+) and its absence in HDC^−/− mice (EEG, EMG, hypnogram). B, Hourly cumulative values of waking during 1 h before and 4 h after the injection of vehicle (NaCl; no symbols), montirelin at 1 mg/kg (open symbols), or 3 mg/kg (filled symbols) in HDC^+/+ and HDC^−/− mice. C, Latencies to SWS and PS after compound injection (n = 8 and 6 in 4 and 3 animals for HDC^+/+ and HDC^−/− mice, respectively). Note the significant effect induced by montirelin in HDC^+/+ mice and its absence in HDC^−/− mice [a, b: montirelin vs NaCl in the same group, p < 0.01, 0.05, Dunnett's t test; c, d: p < 0.05 HDC^+/+ vs HDC^−/−, montirelin 1 mg/kg plus α-FMH ((S)-α-fluoromethylhistidine) 60 mg/kg vs montirelin 1 mg/kg, two-tailed t test after ANOVA for repeated measures].