Regulation of synaptic efficacy in hypocretin/orexin-containing neurons by melanin concentrating hormone in the lateral hypothalamus

Abstract

The lateral hypothalamus (LH) is a central hub that integrates inputs from, and sends outputs to, many other brain areas. Two groups of neurons in the LH, expressing hypocretin/orexin or melanin concentrating hormone (MCH), have been shown to participate in sleep regulation, energy homeostasis, drug addiction, motor regulation, stress response, and social behaviors. The elucidation of crosstalk between these two systems is essential to understand these behaviors and functions because there is evidence that there are reciprocal innervations between hypocretin/orexin and MCH neurons. In this study, we used MCH receptor-1 knock-out (MCHR1 KO) and wild-type (WT) mice expressing green fluorescent protein in hypocretin/orexin-containing neurons to examine the hypothesis that MCH modulates hypocretin/orexin-mediated effects on behavioral state and synaptic transmission in the LH. In MCHR1 KO mice, the efficacy of glutamatergic synapses on hypocretin/orexin neurons is potentiated and hypocretin-1-induced action potential firing is facilitated, potentially explaining an increased effect of modafinil observed in MCHR1 KO mice. In wild-type mice with intact MCHR1 signaling, MCH significantly attenuated the hypocretin-1-induced enhancement of spike frequency in hypocretin/orexin neurons. The MCH effect was dose dependent, pertussis toxin sensitive, and was abolished in MCHR1 KO mice. Consistent with this effect, MCH attenuated hypocretin-1-induced enhancement of the frequency of miniature EPSCs in hypocretin/orexin neurons. These data from MCHR1 KO and WT mice demonstrate a novel interaction between these two systems, implying that MCH may exert a unique inhibitory influence on hypocretin/orexin signaling as a way to fine-tune the output of the LH.

Figure 1.

Sensitization to modafinil-induced increase in locomotor activity during the light phase in MCHR1 ^−/−/hypocretin–GFP mice. A, DNA bands demonstrate the genotyping of MCHR1 ^−/−/hypocretin–GFP (KO), MCHR1 ^+/+/hypocretin–GFP (WT), and MCHR1 ^+/−/hypocretin–GFP (HT) mice. B, Pooled data of beam breaks recorded 30 min after administration of saline or modafinil (10 or 20 mg/kg) from all tested WT and MCHR1 KO mice. *p < 0.05. C, Photomicrographs demonstrate double labeling (indicated by arrows) of hypocretin/orexin (green) and c-Fos (red) in hypocretin/orexin neurons in saline-treated (left) and modafinil-treated (right) MCHR1 KO mice. Scale bar, 15 μm. D, Pooled data showing the percentage of c-Fos-positive hypocretin/orexin neurons from WT and MCHR1 KO mice treated with modafinil (10 mg/kg). *p < 0.05; **p < 0.01.

Figure 2.

Potentiation of hypocretin-1-induced enhancement of the frequency of spikes in hypocretin/orexin neurons in MCHR1 KO mice. A, Sample traces of spikes (action currents) recorded in hypocretin/orexin neurons before, during, and after the application of hypocretin-1 in MCHR1 WT and KO mice. B, The time course of a typical experiment shows that the frequency of spikes recorded in hypocretin/orexin neurons before, during, and after the application of hypocretin-1 in WT (open circles) and KO (filled circles) mice. Time points at which raw traces were taken are indicated by 1–3. The bar below the time course trace indicates the application of hypocretin-1 (300 nm). C, Pooled data from all recorded hypocretin/orexin neurons from MCHR1 KO and WT mice show that the hypocretin-1-mediated effect on the frequency of spikes is significantly higher in KO mice than in WT mice. *p < 0.05.

Figure 3.

Upregulation of glutamatergic synaptic transmission in hypocretin/orexin neurons from MCHR1 KO mice. mEPSCs were recorded in hypocretin/orexin neurons under voltage clamp in the presence of TTX and bicuculline. A, Sample traces of mEPSCs recorded in hypocretin/orexin neurons from MCHR1 WT and KO mice. B, Pooled data for the frequency of mEPSCs recorded in hypocretin/orexin neurons from MCHR1 WT and KO mice show no significant difference between WT and KO mice. C, Cumulative probability of the amplitude of mEPSC events recorded in hypocretin/orexin neurons from MCHR1 WT and KO mice indicates a significant right shift in KO mice, suggesting enhancement of the amplitude of mEPSCs in these mice (p < 0.05, Kolmogorov–Smirnov test). D, Sample traces of evoked EPSCs carried by AMPA and NMDA receptors in hypocretin/orexin neurons from MCHR1 WT (left) and KO (right) mice. E, Pooled data for AMPAR/NMDAR ratio in all tested hypocretin/orexin neurons from MCHR1 WT and KO mice. *p < 0.05, t test. F, Sample traces of mIPSCs recorded in hypocretin/orexin neurons from MCHR1 WT and KO mice. G, Pooled data for the frequency of mIPSCs recorded in hypocretin/orexin neurons from MCHR1 WT and KO mice show no significant difference between WT and KO mice. H, Cumulative probability of the amplitude of mIPSC events recorded in hypocretin/orexin neurons from MCHR1 WT and KO mice indicates no changes in the amplitude of mIPSCs in KO mice (p > 0.05, Kolmogorov–Smirnov test).

Figure 4.

Upregulation of D₁ DA receptor-triggered glutamatergic transmission in hypocretin/orexin neurons in MCHR1 KO mice. A, Sample traces of mEPSCs recorded in hypocretin/orexin neurons from MCHR1 WT and KO mice before, during, and after the application of SKF81297 (SKF), a selective D₁ DA receptor agonist. B, A typical time course shows the frequency of mEPSCs before, during, and after the application of SKF81297 in hypocretin/orexin neurons from MCHR1 WT (open circles) and KO (filled circles) mice. Time points at which raw traces were taken are indicated by 1–3. The bar below the trace indicates the application of SKF81297. C, Pooled data for normalized frequency of mEPSCs recorded during the application of SKF81297 in hypocretin/orexin neurons from WT and KO mice indicates a significant increase in the SKF81297-triggered effect in MCHR1 KO mice compared with WT littermates. *p < 0.05.

Figure 5.

MCH reverses hypocretin-1-induced enhancement of action potentials in hypocretin/orexin neurons. A, B, The time course of a typical experiment and pooled data show that MCH (1 μm) applied to the recorded neurons did not attenuate the basal frequency of spikes in hypocretin neurons. C, D, MCH (1 μm) applied to the recorded neurons attenuated hypocretin-1-induced enhancement of spike frequency in hypocretin/orexin neurons. This attenuation was reversible during the removal of MCH. Sample traces recorded in our experiments are presented in C and the time course of a typical experiment is shown in D. Time points at which raw traces were taken are indicated by 1–5. E, Pooled data for the frequency of spikes show the dose dependency of MCH-mediated inhibition of hypocretin-1-induced firing of spikes in hypocretin/orexin neurons. *p < 0.05. F, Pooled data from experiments with MCHR1 KO mice indicate that the MCH-mediated inhibitory effect was abolished in KO mice.

Figure 6.

PTX abolishes the effect mediated by MCH in hypocretin/orexin neurons. A, The time course of a typical experiment showing MCH does not decrease the enhancement of the frequency of spikes induced by hypocretin-1 in hypocretin/orexin neurons in slices pretreated with PTX for 3 h. Time points at which raw traces were taken are indicated by 1–4. The dotted line represents the baseline. B, C, Sample traces of spikes recorded and pooled data from all tested neurons are presented. *p < 0.01, compared with control.

Figure 7.

MCH depresses hypocretin-1-induced enhancement of the frequency of mEPSCs in hypocretin/orexin neurons. After the increment in the frequency of mEPSCs induced by hypocretin-1 reaches a plateau, MCH was applied to the recorded neurons in the presence of hypocretin-1. A, The time course of a typical experiment is presented. Inset, A bar graph indicates that MCH does not inhibit the basal frequency of mEPSCs in hypocretin/orexin neurons. Time points at which raw traces were taken are indicated by 1–4. B, Sample traces of mEPSCs recorded in different phases of our experiments are shown. C, Pooled data from all seven hypocretin/orexin neurons show that the frequency of mEPSCs in the presence of MCH plus hypocretin-1 is significantly lower than that in the presence of hypocretin-1 alone. *p < 0.05.