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Cited by 112Regulation of Appetite and ObesityNeuroscience and Neuropharmacology ResearchSleep and Wakefulness Research

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Melanin-Concentrating Hormone Neurons Release Glutamate for Feedforward Inhibition of the Lateral Septum

Open access
Chee, Melissa J.S., Arrigoni, Elda, Maratos-Flier, Eleftheria·2015·The Journal of Neuroscience
DOI 10.1523/jneurosci.4187-14.2015PMC PMC6605558
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Abstract

Melanin-concentrating hormone (MCH) regulates vital physiological functions, including energy balance and sleep. MCH cells are thought to be GABAergic, releasing GABA to inhibit downstream targets. However, there is little experimental support for this paradigm. To better understand the synaptic mechanisms of mouse MCH neurons, we performed neuroanatomical mapping and characterization followed by optogenetics to test their functional connectivity at downstream targets. Synaptophysin-mediated projection mapping showed that the lateral septal nucleus (LS) contained the densest accumulation of MCH nerve terminals. We then expressed channelrhodopsin-2 in MCH neurons and photostimulated MCH projections to determine their effect on LS activity. Photostimulation of MCH projections evoked a monosynaptic glutamate release in the LS. Interestingly, this led to a feedforward inhibition that depressed LS firing by a robust secondary GABA release. This study presents a circuit analysis between MCH and LS neurons and confirms their functional connection via monosynaptic and polysynaptic pathways. Our findings indicate that MCH neurons are not exclusively GABAergic and reveal a glutamate-mediated, feedforward mechanism that inhibits LS cells.

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Figures

Figure 1 The LS receives dense MCH axon terminals. A , Merged low-magnification ( i ) and high-magnification ( ii , outlined from i ) confocal photomicrographs showing native synaptophysinmCherry fluorescence (red) and MCH immunoreactivity (green) in the lateral hypothalamus of Pmch-cre mice injected with AAV8 -DIO-Ef1 𝛂 -synaptophysin-mCherry. White arrowheads mark some representative double-labeled neurons (yellow). B , Epifluorescence photomicrograph of the LS showing native synaptophysin-mCherry-labeled fluorescent ( Figure legend continues. )
Figure 1 The LS receives dense MCH axon terminals. A , Merged low-magnification ( i ) and high-magnification ( ii , outlined from i ) confocal photomicrographs showing native synaptophysinmCherry fluorescence (red) and MCH immunoreactivity (green) in the lateral hypothalamus of Pmch-cre mice injected with AAV8 -DIO-Ef1 𝛂 -synaptophysin-mCherry. White arrowheads mark some representative double-labeled neurons (yellow). B , Epifluorescence photomicrograph of the LS showing native synaptophysin-mCherry-labeled fluorescent ( Figure legend continues. )CC-BY-NC-SA · From the PDF
Figure 2 MCHneurons express vGLUT2 but not vGAT. A , B , Merged low-magnification ( i ) and high-magnification ( ii , outlined in i ) confocal photomicrographs showing MCH immunoreactivity (red) in GFP-positive (green) vGAT ( A ) and vGLUT2 ( B ) neurons. Insets (outlined in Aii and Bii ) showing the colocalization of MCH immunoreactivity with vGLUT2 but not vGAT expression. White arrowheads indicate double-labeled neurons (yellow). Asterisks mark non-colocalized MCH neurons. Scale bars: i , 200 𝛍 m; ii , 100 𝛍 m. C , Unilateral cell count of MCH-IR neurons expressing vGAT or vGLUT2. cp, Cerebral peduncle; DMH, dorsomedial hypothalamic nucleus; opt, optic tract; VMH, ventromedial hypothalamic nucleus.
Figure 2 MCHneurons express vGLUT2 but not vGAT. A , B , Merged low-magnification ( i ) and high-magnification ( ii , outlined in i ) confocal photomicrographs showing MCH immunoreactivity (red) in GFP-positive (green) vGAT ( A ) and vGLUT2 ( B ) neurons. Insets (outlined in Aii and Bii ) showing the colocalization of MCH immunoreactivity with vGLUT2 but not vGAT expression. White arrowheads indicate double-labeled neurons (yellow). Asterisks mark non-colocalized MCH neurons. Scale bars: i , 200 𝛍 m; ii , 100 𝛍 m. C , Unilateral cell count of MCH-IR neurons expressing vGAT or vGLUT2. cp, Cerebral peduncle; DMH, dorsomedial hypothalamic nucleus; opt, optic tract; VMH, ventromedial hypothalamic nucleus.CC-BY-NC-SA · From the PDF
Figure 3 Photostimulation of ChR2-expressing MCH terminals evoked glutamate release in the LS. A , Experimental design schematic showing patch-clamp recording while photostimulating ChR2-expressing MCH neurons. B , Merged epifluorescence and IR-DIC photomicrographs of a coronal Pmch-cre brain slice injected with AAV8 -EF1 𝛂 -DIO-hChR2(H134R)-mCherry showing whole-cell recording from a mCherry-labeled MCH neuron (inset, outlined region). Scale bars: 200 𝛍 m;inset, 50 𝛍 m. C , Sample traces of action potential firing from a ChR2-expressing MCH neuron evoked by 5 s light trains (5 ms pulses) at 1, 5, 20, and 100 Hz light frequency (dashed line, 0 mV; left). Percentage change in spike fidelity (black, left axis) and MCH neuronal firing (gray, right axis) showing effective entrainment up to 20 Hz photostimulation ( n = 12). D , Experimental design schematic showing whole-cell recordings from LS neurons while photostimulating ChR2-expressing MCHprojections. E , Merged low-magnification ( i ) and high-magnification ( ii ) epifluorescence and IR-DIC photomicrographs of a coronal brain slice showing ChR2-expressing MCH axon terminals in the LS ( i ) and mCherry-fluorescent puncta outlining LS cells ( ii ). Scale bars: i , 200 𝛍 m; ii , 100 𝛍 m. F -I , Overlay of individual (gray) and averaged (black) oIPSCs ( V h of -5 mV; F ) and oEPSCs ( V h of -60 mV; H ) after photostimulation (3 5-ms light pulses in 1 s, repeated every 5 s for 20 trials). Averaged responses ( F , H ) magnified in insets (top) and raster plots (bottom; 50 ms bins) showing event synchronization during photostimulation. Probability plots (50 ms bins) showing that photostimulation increased IPSC and EPSC probability in control (con; n = 14), which is abolished by 20 𝛍 M BIC ( n = 4; G ) and 1 mM KYN ( n = 6; I ), respectively. J , K , Range (line, median; box, 25th to 75th percentiles; whisker, minimum to maximum) of absolute amplitudes ( J ) and delay latency ( K ) of oIPSCs and oEPSCs averaged from 20 trials. *** p < 0.001. L , Overlay of individual (gray) and averaged (black) oIPSCs (top) and oEPSCs (bottom) in control ( i ), BIC ( ii ), KYN ( iii ), 500 nM TTX ( iv ), TTX and KYN ( v ), and washout ( vi ). M , Effect of KYN and BIC on oIPSC and oEPSC amplitude. 3V, Third ventricle; cc, corpus callosum; con, control; CPu, caudate putamen; f, fornix; LHA, lateral hypothalamic area; LV, lateral ventricle.
Figure 3 Photostimulation of ChR2-expressing MCH terminals evoked glutamate release in the LS. A , Experimental design schematic showing patch-clamp recording while photostimulating ChR2-expressing MCH neurons. B , Merged epifluorescence and IR-DIC photomicrographs of a coronal Pmch-cre brain slice injected with AAV8 -EF1 𝛂 -DIO-hChR2(H134R)-mCherry showing whole-cell recording from a mCherry-labeled MCH neuron (inset, outlined region). Scale bars: 200 𝛍 m;inset, 50 𝛍 m. C , Sample traces of action potential firing from a ChR2-expressing MCH neuron evoked by 5 s light trains (5 ms pulses) at 1, 5, 20, and 100 Hz light frequency (dashed line, 0 mV; left). Percentage change in spike fidelity (black, left axis) and MCH neuronal firing (gray, right axis) showing effective entrainment up to 20 Hz photostimulation ( n = 12). D , Experimental design schematic showing whole-cell recordings from LS neurons while photostimulating ChR2-expressing MCHprojections. E , Merged low-magnification ( i ) and high-magnification ( ii ) epifluorescence and IR-DIC photomicrographs of a coronal brain slice showing ChR2-expressing MCH axon terminals in the LS ( i ) and mCherry-fluorescent puncta outlining LS cells ( ii ). Scale bars: i , 200 𝛍 m; ii , 100 𝛍 m. F -I , Overlay of individual (gray) and averaged (black) oIPSCs ( V h of -5 mV; F ) and oEPSCs ( V h of -60 mV; H ) after photostimulation (3 5-ms light pulses in 1 s, repeated every 5 s for 20 trials). Averaged responses ( F , H ) magnified in insets (top) and raster plots (bottom; 50 ms bins) showing event synchronization during photostimulation. Probability plots (50 ms bins) showing that photostimulation increased IPSC and EPSC probability in control (con; n = 14), which is abolished by 20 𝛍 M BIC ( n = 4; G ) and 1 mM KYN ( n = 6; I ), respectively. J , K , Range (line, median; box, 25th to 75th percentiles; whisker, minimum to maximum) of absolute amplitudes ( J ) and delay latency ( K ) of oIPSCs and oEPSCs averaged from 20 trials. *** p < 0.001. L , Overlay of individual (gray) and averaged (black) oIPSCs (top) and oEPSCs (bottom) in control ( i ), BIC ( ii ), KYN ( iii ), 500 nM TTX ( iv ), TTX and KYN ( v ), and washout ( vi ). M , Effect of KYN and BIC on oIPSC and oEPSC amplitude. 3V, Third ventricle; cc, corpus callosum; con, control; CPu, caudate putamen; f, fornix; LHA, lateral hypothalamic area; LV, lateral ventricle.CC-BY-NC-SA · From the PDF
Figure4 PhotostimulationofMCHterminalsinhibitedLScellsbyglutamate-mediatedGABArelease. A , B , Sample trace ( i ) and meanpercentage change ( ii ) in spontaneous LS firing frequency in response to photostimulation (5 ms light pulses, 10 Hz train for 5 s) before (control), during 1 mM KYN ( A ) or 20 𝛍 M BIC ( B ), and after washout (wash). * p < 0.05. C , Model of monosynaptic and disynaptic pathways betweenMCHandLSneurons.MCHneuronsinthelateralhypothalamicarea(LHA)directlyreleaseglutamate (Glu) onto LS neurons and GABAergic interneurons or afferents. Glutamate release at GABAergic intermediates elicits robust feedforward inhibition of LS neuronal activity.
Figure4 PhotostimulationofMCHterminalsinhibitedLScellsbyglutamate-mediatedGABArelease. A , B , Sample trace ( i ) and meanpercentage change ( ii ) in spontaneous LS firing frequency in response to photostimulation (5 ms light pulses, 10 Hz train for 5 s) before (control), during 1 mM KYN ( A ) or 20 𝛍 M BIC ( B ), and after washout (wash). * p < 0.05. C , Model of monosynaptic and disynaptic pathways betweenMCHandLSneurons.MCHneuronsinthelateralhypothalamicarea(LHA)directlyreleaseglutamate (Glu) onto LS neurons and GABAergic interneurons or afferents. Glutamate release at GABAergic intermediates elicits robust feedforward inhibition of LS neuronal activity.CC-BY-NC-SA · From the PDF

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Metadata

Authors
Chee, M. J. S., Arrigoni, E., & Maratos-Flier, E.
Affiliations
Beth Israel Deaconess Medical Center (US)
ORCID
Venue
The Journal of Neuroscience
Publisher
Society for Neuroscience
Published
2015-02-25
Volume
35
Issue
8
Pages
3644-3651
ISSN
  • 0270-6474 (ISSN-L)
  • 1529-2401
DOI
10.1523/jneurosci.4187-14.2015
PMC
PMC6605558
OpenAlex
W1970610262
Cited by
112
References
48
Citations by year
2026: 11 · 2025: 7 · 2024: 11 · 2023: 11 · 2022: 8 · 2021: 11 · 2020: 11 · 2019: 5 · 2018: 13 · 2017: 10 · 2016: 11 · 2015: 3
FWCI
6.84
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Neuroscience (publishedVersion, cc-by-nc-sa)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Topics
  • Life Sciences › Neuroscience › Endocrine and Autonomic Systems › Regulation of Appetite and Obesity
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
  • Life Sciences › Neuroscience › Cognitive Neuroscience › Sleep and Wakefulness Research
Keywords
melanin-concentrating hormone, lateral septal nucleus, glutamate release, feedforward inhibition, GABAergic neurons, MCH neurons, synaptic connectivity, monosynaptic pathway, polysynaptic pathways, optogenetic stimulation, channelrhodopsin-2, neural circuits
MeSH terms
Animals, Hypothalamic Hormones, Melanins, Pituitary Hormones, Septal Nuclei, Presynaptic Terminals, Glutamic Acid, Excitatory Postsynaptic Potentials, Feedback, Physiological, Mice, Inhibitory Postsynaptic Potentials, GABAergic Neurons, Optogenetics

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