Electrophysiological Properties of Melanin-Concentrating Hormone and Orexin Neurons in Adolescent Rats

Abstract

Orexin and melanin-concentrating hormone (MCH) neurons have complementary roles in various physiological functions including energy balance and the sleep/wake cycle. in vitro electrophysiological studies investigating these cells typically use post-weaning rodents, corresponding to adolescence. However, it is unclear whether these neurons are functionally mature at this period and whether these studies can be generalized to adult cells. Therefore, we examined the electrophysiological properties of orexin and MCH neurons in brain slices from post-weaning rats and found that MCH neurons undergo an age-dependent reduction in excitability, but not orexin neurons. Specifically, MCH neurons displayed an age-dependent hyperpolarization of the resting membrane potential (RMP), depolarizing shift of the threshold, and decrease in excitatory transmission, which reach the adult level by 7 weeks of age. In contrast, basic properties of orexin neurons were stable from 4 weeks to 14 weeks of age. Furthermore, a robust short-term facilitation of excitatory synapses was found in MCH neurons, which showed age-dependent changes during the post-weaning period. On the other hand, a strong short-term depression was observed in orexin neurons, which was similar throughout the same period. These differences in synaptic responses and age dependence likely differentially affect the network activity within the lateral hypothalamus where these cells co-exist. In summary, our study suggests that orexin neurons are electrophysiologically mature before adolescence whereas MCH neurons continue to develop until late adolescence. These changes in MCH neurons may contribute to growth spurts or consolidation of adult sleep patterns associated with adolescence. Furthermore, these results highlight the importance of considering the age of animals in studies involving MCH neurons.

Keywords: adolescence; development; hypocretin; lateral hypothalamus; melanin-concentrating hormone; orexin; patch clamp; short-term synaptic plasticity.

Figure 1

Post hoc immunohistochemical identification of melanin-concentrating hormone (MCH) and orexin neurons. (A) Sample images of a confirmed MCH neuron. Post hoc immunohistochemical staining of the biocytin labeled cell (A1, blue) shows co-localization with MCH (A2, red) but not orexin A (A3, green), shown by overlap (A4). (B) Sample images of a confirmed orexin neuron. The biocytin stained cell (B1) is co-localized with orexin A (B3) but not MCH (B2), shown by overlap (B4). Scale bar: 20 μm.

Figure 2

MCH neurons undergo an age-dependent decrease in excitability. (A) Sample traces during a 200-pA current injection (left) and averaged action potential (AP) waveform (right) recorded from MCH neurons of 4, 7 and 14-week old rats. Dotted reference line in the left panel is −80 mV. (B) Resting membrane potential (RMP) of MCH neurons from different age groups. (C,D) AP frequency (C) and first spike latency (D) of MCH neurons during positive current injections. (E–J) Various parameters of AP waveform as indicated on the Y-axis. *p < 0.05, **p < 0.01, one-way analysis of variance (ANOVA) with Holm–Sidak post-test. 4 weeks vs. 7 weeks: ^$$p < 0.01, ^$$$p < 0.001, ^$$$$p < 0.0001; 4 weeks vs. 14 weeks: ^#p < 0.05, ^##p < 0.01, ^####p < 0.0001, two-way repeated measures (RM) ANOVA with Holm-Sidak post-test.

Figure 3

Age-dependent changes in excitatory synaptic transmission to MCH neurons. (A) Sample traces of miniature excitatory postsynaptic currents (mEPSCs) recorded from MCH neurons of 4, 7 and 14-week old rats. (B,C) Frequency (B) and amplitude (C) of mEPSCs in MCH neurons. (D) Pairs of EPSCs evoked at 50 Hz recorded from MCH neurons. Traces are scaled to the first EPSC. (E) Paired pulse ratio (PPR) at 10 and 50 Hz. *p < 0.05, **p < 0.01, one-way ANOVA (B,C) or two-way RM ANOVA (E) with Holm-Sidak post-test.

Figure 4

Activity-dependent short-term plasticity in MCH neurons of 4-week old rats. (A) Sample recordings of evoked EPSCs during 10 and 50 Hz trains. Expanded traces below show the 1st, 5th and 50th EPSC of the train. (B) Normalized amplitude of 50 EPSCs during 10 and 50 Hz train stimulation. (C) Plateau of EPSC amplitude during 10 and 50 Hz train stimulation. ****p < 0.0001, paired t-test.

Figure 5

Activity-dependent short-term plasticity in MCH neurons is modulated by age. (A) Sample traces of EPSCs evoked at 50 Hz in MCH neurons (first five EPSCs are shown). (B,C) Normalized EPSC amplitude during 10 Hz (B) and 50 Hz trains (C) from different age groups as indicated. Note that the 4-week data is also shown in Figure 4B. (D) Synaptic facilitation at the beginning of 10 Hz and 50 Hz trains (average of EPSC2–5, normalized to EPSC1). (E) EPSC plateau at the end of 10 Hz and 50 Hz trains (average of EPSC26–30, normalized to EPSC1). *p < 0.05, **p < 0.01, ****p < 0.0001, two-way RM ANOVA with Holm-Sidak post-test.

Figure 6

Excitability and AP waveform of orexin neurons after weaning. (A) Sample traces of spontaneous firing (left) and averaged AP waveform (right) recorded from orexin neurons of 4, 7 and 14-week old rats. Dotted line in the left panel is −59 mV. (B) RMP of orexin neurons from different age groups. (C,D) AP frequency (C) and first spike latency (D) of orexin neurons during positive current injections. (E–J) Various parameters of AP waveform as indicated on the Y-axis. *p < 0.05, **p < 0.01, one-way ANOVA with Holm-Sidak post-test.

Figure 7

Basal properties of excitatory transmission to orexin neurons do not change after weaning. (A) Sample traces of mEPSCs in orexin neurons of 4, 7 and 14-week old rats. (B,C) The frequency (B) and amplitude (C) of mEPSCs in orexin neurons. (D) Paired EPSCs recorded from orexin neurons. Traces are scaled to the first EPSC. (E) Paired pulse ratio (PPR) at 10 Hz and 50 Hz stimulation.

Figure 8

Activity-dependent short-term plasticity in orexin neurons of 4-week old rats. (A) Sample recordings of evoked EPSCs during 10 and 50 Hz train stimulation. Expanded traces below show the 1st, 5th and 50th EPSC of the train. (B) Normalized amplitude of EPSCs in response to10 and 50 Hz train stimulation. (C) Plateau of EPSC amplitude (average of last five EPSCs normalized to EPSC1) during 10 Hz and 50 Hz train stimulation. ****p < 0.0001, paired t-test.

Figure 9

Activity-dependent short-term plasticity does not change in orexin neurons after weaning. (A) Sample traces of five EPSCs evoked at 50 Hz in orexin neurons from different age groups. (B,C) Normalized EPSC amplitude during 10 Hz (B) or 50 Hz train stimulation (C). Note that the 4-week data is also shown in Figure 8B. (D) Synaptic depression at the beginning of 10 and 50 Hz trains (average of EPSC2–5, normalized to EPSC1). (E) Plateau at the end of 10 Hz and 50 Hz trains (average of EPSC26–30, normalized to EPSC1).