Extended Data Fig. 5: Endogenous MCH increases GABA tone in the dLS.
From: Hypothalamic melanin-concentrating hormone regulates hippocampus-dorsolateral septum activity
a, Pooled data for amplitude. n = 11 cells/9 animals and 14/9 for 5 Hz and 20 Hz, respectively. Paired two-tailed Student’s t-tests were used: 5 Hz, p = 0.2905 and 20 Hz, p = 0.132. AAV-DIO-ChR2-eYFP was injected into the lateral hypothalamic region of MCH-Cre mice, sIPSCs (isolated pharmacologically in the presence of CNQX and APV) were recorded in dLS neurons. MCH fibers were optogenetically activated using two different paradigms (5 Hz 5 second x 5 or 20 Hz 2 min). sIPSCs were recorded before optogenetic stimulation (Control) and 2–3 min after optogenetic stimulation (indicated as 5 Hz or 20 Hz). b, Sample traces showing the impact of optogenetic stimulation of MCH fibers on sIPSCs in dLS neurons, as described above, in the presence of Tc-MCH7c. c, Pooled data show that in the presence of TC-MCH7c, the facilitatory effects induced post-prolonged optogenetic stimulation of MCH fibers were abolished, suggesting the involvement of MCH signaling. n = 14/7 and 13/7 for 5 Hz and 20 Hz, respectively. Paired two-tailed Student’s t-tests were used: 5 Hz, p = 0.4489 and 20 Hz, p = 0.9195. d, Sample traces of sIPSCs in the absence (Control) or presence of MCH. f, Pooled data for e. n = 8/4 neurons/animals. Paired two-tailed Student’s t-tests were used, p = 0.0198. f-h MCH suppresses collateral transmission in the dLS. g, Diagram of experimental setup and sample images showing AAV-ChR2-eYFP infection in the dLS. Whole-cell patch clamp recordings were made from cells without ChR2-eYFP infection in the dLS; fibers and neuronal cell bodies expressing ChR2 were activated by 470 nm LED light (each pulse at 1 ms duration at 1, 5 and 10 Hz); collateral optogenetically-induced IPSCs were recorded. g, Sample traces showing the GABAAR-mediated inhibitory collateral synaptic transmission before (black traces) and after (green traces) administration of exogenous MCH (600 nM) through perfusion chamber. h, Pooled data from b. n = 12 cells/5 animals. Paired two-tailed Student’s t-tests were used, p = 0.0006. i, GABAB receptor-mediated inhibition suppresses spontaneous firing of dLS neurons. Sample traces showing that GABAB receptor-mediated inhibitory synaptic transmission is sufficient to suppress dLS spontaneous action potential firing. AAV-ChR2-eYFP was injected into the dLS. Whole-cell patch clamp recordings under current clamp mode were made from cells without ChR2-eYFP infection in the dLS; fibers and neuronal cell bodies expressing ChR2 were activated by 470 nm LED light (each pulse was 1 ms duration delivered at 1, 5 or 10 Hz). In the presence of GABAAR blocker PTX, optogenetic stimulation delivered at varying frequencies (1 Hz, 5 Hz and 10 Hz) to activate dLS neurons suppressed spontaneous firing. Higher frequencies produce prolonged hyperpolarization in dLS neurons. In the presence of CGP55845, a GABABR blocker, the suppression of firing and hyperpolarization of membrane potential were both blocked, suggesting the involvement of GABAB receptor signaling. Data are mean ± SEM * p < 0.05, ** p < 0.01, ***p < 0.001.
