Glutamatergic Neurons in the Dorsal Hypothalamic Area Dissociate Thermogenesis from Heat Conserving Pathways During Stress Responses

Abstract

Stress elicits a variety of autonomic responses, including thermal changes in humans and animals. In this present study we investigate the circuit basis for stress‐induced hyperthermia with focus on the hypothalamic‐medulary pathway in adaptive thermoregulatory responses.We injected retrograde tracer (CTb) in the raphe pallidus (RPa) of Vgat and Vglut2‐IRES‐cre L10‐GFP reporter mice to identify the phenotype of neurons in the dorsal hypothalamic area (DHA) projecting to RPa.In a separate series of experiment, we bilaterally injected AAV‐Flex‐hGlyR‐mCherry in the DHA of Vglut2‐IRES‐cre mice to promote inhibition of DHAVglut2 neurons. hGlyR is a mutated glycine receptor in which ivermectin (hGlyR ligand) gates a chloride channel. Using AAV8‐hSyn‐DIO‐hM3D(Gq)‐mCherry (DREADD) we induced activation of DHAVglut2 neurons and measured changes in brown adipose tissue (BAT) and tail temperature. An optogenetic approach in freely‐behaving mice was used to promote inhibition of the synaptic terminal of the DHAVglut2 neurons in the RPa.We identified that glutamatergic neurons in the DHA (DHAVglut2) sending direct inputs to RPa are activated during cage switch stress (singly housed male was switched to a cage previously occupied by another male) causing 39 + 6% of Vglut2/CTb neurons to express cFos, whereas only 13 + 2% of Vglut2/CTb neurons expressed cFos in the control animals (mean + SEM, n=4,3 respectively, *p=0.016).The long‐lasting inhibition induced by hGlyR‐IVM reduced cFOS expression in our stress protocol (after IVM 6 + 1% cFOS+/hGlyR Vglut2 labeled neurons vs vehicle 40 + 8% cFOS+/hGlyR Vglut2 cFos doubly labeled neurons in the DHA; n=3 each group, *p=0.013) and also decreased stress‐induced hyperthermia (2.8 + 0.1°C after vehicle vs. 2.0 +0.1°C after IVM, n=11, FTc(interaction) (164,1278)=1.556).During the stress protocol we found a large increase in BAT thermogenesis (rise of 1.2 + 0.2°C), and tail artery vasoconstriction (fall in tail temperature by −2.3 + 0.4°C). Surprisingly, activation of DHAVglut2 neurons using hM3D excitatory receptor caused profound BAT thermogenesis (1.0 + 0.14°C), but it did not cause vasoconstriction, instead resulting in tail artery vasodilation (2.5 + 0.6°C) as a delayed reflex response to central hyperthermia.Finally, optogenetic experiments identified that DHAVglut2—>RPa pathway is a mediator of stress‐induced hyperthermia, as photoinhibition of terminals in the RPa reduced the peak of stress‐induced hyperthermia by 0.4+0.06°C, (comparing same group before vs. during photoinhibitoin). We propose that the DHAVglut2—‐>RPa is a thermogenic pathway regulating BAT thermogenesis but not thermoregulatory vasoconstriction during stress.Support or Funding InformationGrant support: NIH Grant NS085477 (C.B.S.), NS073613 (P.M.F.), CAPES and CNPq