Histaminergic modulation of GABAergic transmission in rat ventromedial hypothalamic neurones

Abstract

1. The ventromedial nucleus of the hypothalamus (VMH) is a key nucleus in the homeostatic regulation of neuroendocrine and behavioural functions. In mechanically dissociated rat VMH neurones with attached native presynaptic nerve endings, GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded using the nystatin perforated patch recording mode under voltage-clamp conditions. 2. Histamine reversibly inhibited the sIPSC frequency in a concentration-dependent manner without affecting the mean current amplitude. The selective histamine receptor type 3 (H(3)) agonist imetit (100 nM) mimicked this effect and it was completely abolished by the selective H(3) receptor antagonists clobenpropit (3 microM) and thioperamide (10 microM). 3. The GTP-binding protein inhibitor N-ethylmaleimide (10 microM) removed the histaminergic inhibition of GABAergic sIPSCs. 4. Elimination of external Ca(2+) reduced the GABAergic sIPSC frequency without affecting the distribution of current amplitudes. In this condition, the inhibitory effect of imetit on the sIPSC frequency completely disappeared, suggesting that the histaminergic inhibition requires extracellular Ca(2+). 5. The P/Q-type Ca(2+) channel blocker omega-agatoxin IVA (300 nM) attenuated the histaminergic inhibition of the GABAergic sIPSC frequency, but neither the N-type Ca(2+) channel blocker omega-conotoxin GVIA (3 microM) nor the L-type Ca(2+) channel blocker nicardipine (3 microM) was effective. 6. Activation of adenylyl cyclase with forskolin (10 microM) had no effect on histaminergic inhibition of the sIPSCs. 7. In conclusion, histamine inhibits spontaneous GABA release from presynaptic nerve terminals projecting to VMH neurones by inhibiting presynaptic P/Q-type Ca(2+) channels via a G-protein coupled to H(3) receptors and this may modulate the excitability of VMH neurones.

Figure 1. GABAergic sIPSCs

Application of 3 μm bicuculline rapidly and completely blocked sIPSCs in responsive, dissociated VMH neurones. Aa and b, typical traces of sIPSCs. B, traces of sIPSCs recorded at various V_H(a) and their mean amplitude I-V curve (b). In b, each point is the mean of 4 neurones. [Cl⁻]_i and [Cl⁻]_o show the intra- and extracellular Cl⁻ concentrations, respectively. The continuous line is the least-squares linear fit to the mean sIPSC values at each V_H.

Figure 2. Histaminergic depression of GABAergic sIPSCs

A, application of 10 μm histamine (a) in a typical sIPSC example decreased the number of events observed per 10 s (b). Each point is the mean of 6 neurones. Dotted line is the mean of the 10 min control. B, cumulative distributions for inter-event interval (a) and current amplitude (b) of sIPSCs recorded from the same neurone. P values indicate the results of K-S tests for frequency and amplitude (341 events for control and 116 events for histamine). Inset, all amplitudes and frequencies of sIPSCs are normalized to their control (n = 6). His, histamine; Cont, control. *P < 0.05. C, concentration-response curves for the normalized sIPSC frequency of histamine and imetit. The continuous lines represent the least-squares fit. Each point is the mean of 4-8 neurones. EC₅₀ values of histamine and imetit are about 440 and 4.7 nm, respectively. The vertical error bars represent ±s.e.m.

Figure 3. Presynaptic inhibition of GABAergic sIPSCs by histamine

A, typical traces of sIPSCs observed before, during and after the application of 10 μm histamine or 100 nm imetit in the standard solution with or without 3 μm clobenpropit. The lower two traces were obtained from the same neurone. B, cumulative distributions for inter-event interval (a) and amplitude (b) of sIPSCs in the same neurone. P values indicate the results of K-S tests for frequency and amplitude (256 events for clobenpropit and 94 events for imetit in the presence of clobenpropit). C, each column is the mean of 6 neurones. All frequencies and amplitudes of sIPSCs are normalized to the control. *P < 0.05.

Figure 4. H₃ receptor-mediated presynaptic inhibition of GABAergic sIPSCs is coupled to G-protein

A, a typical trace of sIPSCs observed before, during and after the application of 100 nm imetit in solution containing 10 μm NEM. B, cumulative distributions for inter-event interval (a) and amplitude (b) of sIPSCs in the same neurone. P values indicate the results of K-S tests for frequency and amplitude (698 events for NEM and 364 events for imetit in the presence of NEM). C, each column is the mean of 4 neurones. All amplitudes and frequencies are normalized to the control. *P < 0.05.

Figure 5. Effect of external Ca²⁺ on H₃ receptor-mediated presynaptic inhibition

A, a typical trace of sIPSCs observed before, during and after the application of 100 nm imetit in Ca²⁺-free external solution. B, cumulative distributions for inter-event interval (a) and amplitude (b) of sIPSCs in the same neurone. P values indicate the results of K-S tests for frequency and amplitude (104 and 197 events for Ca²⁺-free solution with and without imetit, respectively). C, each column is the mean of 5 neurones. All amplitudes and frequencies are normalized to the control. *P < 0.05, **P < 0.01; n.s., not significant.

Figure 6. Effect of VDCC antagonists on the H₃ receptor-mediated inhibitory effect

Aa, effect of 100 nm imetit on a typical time course of event frequency of sIPSCs under the cumulative application of VDCC antagonists in a neurone. The number of events per 5 s is plotted. Ab, cumulative distributions for inter-event interval in the same neurone. P values indicate the results of K-S tests. B, the effects of VDCC antagonists on the sIPSC frequency (a) and the inhibition ratio of imetit in the presence of VDCC antagonists (b). Each column is the mean of 6-9 neurones and is normalized to the control. * P < 0.05.

Figure 7. Inhibitory effect of H₃ receptors on GABAergic sIPSCs mediated by P/Q-type Ca²⁺ channels

A, typical traces of sIPSCs observed before, during and after the application of 100 nm imetit in the absence (upper trace) or presence (lower trace) of 300 nmω-AgTx IVA. B, each column is the mean of 5 neurones. All frequencies (a) and amplitudes (b) are normalized to control. * P < 0.05.

Figure 8. Effect of presynaptic depolarization on the H₃ receptor-mediated inhibitory effect

Aa, a typical time course of event frequency of sIPSCs observed before, during and after the application of 100 nm imetit in the presence of 100 μm 4-AP and various VDCC antagonists in a neurone. VDCC antagonists were applied cumulatively in the presence of 4-AP. The number of events per 5 s is plotted. Ab, cumulative distributions for inter-event intervals in the same neurone. P values indicate the results of K-S tests. B, the effects of VDCC antagonists on the sIPSC frequency in the presence of 4-AP (a) and inhibition ratio of imetit in the presence of VDCC antagonists (b). Each column is the mean of 6-7 neurones and is normalized to the control. * P < 0.05, ** P < 0.01.

Figure 9. H₃ receptor-mediated presynaptic inhibition of sIPSC is not related to the adenylyl cyclase-cAMP signal transduction pathway

A, effect of 100 nm imetit on a typical trace of sIPSCs in the presence of 10 μm forskolin. B, cumulative distributions for inter-event intervals (a) and amplitude (b) of sIPSCs in the same neurone. P values indicate the results of K-S tests for frequency and amplitude (231 and 524 events for forskolin with or without imetit, respectively). C, each column is the mean of 4 neurones. All amplitudes and frequencies are normalized to the control. * P < 0.05.