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Inhibition of vGlut2⁺ PSTh neurons attenuates 2MT-evoked hypothermia and tail temperature increase a Schematic of retrograde tracing from NTS neurons by CTB injection. b Representative image showing double immunostaining of CTB and c-fos. The percentage of double-positive neurons (CTB⁺, c-fos⁺/CTB⁺%) in PSTh is in parenthesis (n = 3). c Representative images and quantitative analysis showing the percentage of c-fos⁺, vGlut2⁺ or c-fos⁺, vGAT⁺ double-positive neurons among c-fos⁺ neurons in PSTh by two-color in situ hybridization (n = 3). d Schematic of chemogenetic inhibition experiment of vGlut2⁺ PSTh neurons in vGlut2-IRES-Cre mice. e Representative image of hMD4i-labeled vGlut2⁺ PSTh neurons (n = 7). f Time-lapsed thermal images of mCherry-expressing mice and hM4Di-expressing mice during 2MT treatment following administration of C21. Tail (g), skin (i), and core (k) temperature curves of mice with (hM4Di, n = 7) or without (mCherry, n = 8) inactivation of vGlut2⁺ PSTh neurons before and during 2MT treatment. Average tail (h), skin (j), and core (l) temperature changes of mice with (hM4Di, n = 7) or without (mCherry, n = 8) inactivation of vGlut2⁺ PSTh neurons during 2MT treatment. A few mice were not included in the analysis of tail temperature (e) because their tails were frequently obscured in the thermal images. g–l Data are mean ± SEM; two-side Student’s t test. Scale bars, 100 µm.
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The neural mechanisms of fear-associated thermoregulation remain unclear. Innate fear odor 2-methyl-2-thiazoline (2MT) elicits rapid hypothermia and elevated tail temperature, indicative of vasodilation-induced heat dissipation, in wild-type mice, but not in mice lacking Trpa1–the chemosensor for 2MT. Here we report that Trpa1 −/− mice show diminis...
Citations
... Our study also showed that PSTh Tac1+ neurons contribute to odor-driven innate fear and may further uncover the cellular mechanism involved in PSTh-mediated cardiovascular, temperature, appetite and autonomic regulation under fear conditions [40][41][42][43][44][45] . ...
Fear-related disorders (for example, phobias and anxiety) cause a substantial public health problem. To date, studies of the neural basis of fear have mostly focused on the amygdala. Here we identify a molecularly defined amygdala-independent tetra-synaptic pathway for olfaction-evoked innate fear and anxiety in male mice. This pathway starts with inputs from the olfactory bulb mitral and tufted cells to pyramidal neurons in the dorsal peduncular cortex that in turn connect to cholecystokinin-expressing (Cck⁺) neurons in the superior part of lateral parabrachial nucleus, which project to tachykinin 1-expressing (Tac1⁺) neurons in the parasubthalamic nucleus. Notably, the identified pathway is specifically involved in odor-driven innate fear. Selective activation of this pathway induces innate fear, while its inhibition suppresses odor-driven innate fear. In addition, the pathway is both necessary and sufficient for stress-induced anxiety-like behaviors. These findings reveal a forebrain-to-hindbrain neural substrate for sensory-triggered fear and anxiety that bypasses the amygdala.
... Optogenetic and chemogenetic techniques have been used to discover that 2MT stimulation activates a large number of neurons in the posterior subthalamic nucleus (PSTh), the external lateral parabrachial subnucleus (PBel), and NST in mice (Fig. 1). Deleting TRPA1 from neurons in these brain regions reduces their activation, revealing the mechanism of the PBel-PSTh neural circuitry in 2MT-induced hypothermia [34]. As a central hub connected to both PBel and NST, PSTh mediates 2MT-induced body temperature reduction and tail vasodilation. ...
... As a central hub connected to both PBel and NST, PSTh mediates 2MT-induced body temperature reduction and tail vasodilation. Specifically, the PSTh-NST pathway plays a major role in mediating the low temperature induced by 2MT in mice, while the PBel-PSTh pathway plays a major role in tail vasodilation [34]. Contrary to the decrease in tail temperature caused by electric shock in mice, 2MT induced an increase in tail temperature. ...
... Contrary to the decrease in tail temperature caused by electric shock in mice, 2MT induced an increase in tail temperature. This may be due to 2MT being an indirect fear message that lowers core body temperature by relaxing the blood vessels in the tail of mice [15,34]. Vascular movements are controlled by sympathetic preganglionic neurons in the spinal cord through sympathetic ganglia in the brainstem, which may also be related to the low body temperature and low metabolic state induced by 2MT [35]. ...
Hibernation is a prolonged state of low metabolism that animals enter in response to extreme environmental conditions to enhance their survival in harsh environments. Recent studies have shown that non-hibernating species can also be induced to enter a hibernation-like state. 2-methyl-2-thiazoline (2MT), a potent analog of fox odor, can induce fear-related behavior in mice with low body temperature and low metabolism, and has specific organ-protective effects. A systematic understanding of 2MT-induced hibernation and its underlying mechanisms may aid in expanding its applications in medicine and other fields.
... In the staining process, permeabilization with a detergent is critical for obtaining good results. As previously reported, a 3-h incubation with 1% Triton-X in phosphate buffered saline enables permeabilization and staining with anti-c-Fos antibody (14,15). To reveal drug treatment-activated neurons throughout the brain (e.g., visualizing GPCR agonist/antagonistactivated neurons), the availability of approximately 100 coronal slices is sufficient (80 μm each, not containing olfactory bulb or spinal cord tissue) to perform screening of c-Fos expression in the whole brains of adult mice. ...
Understanding the mechanisms of drug action in the brain, from the genetic to the neural circuit level, is crucial for the development of new agents that act upon the central nervous system. Determining the brain regions and neurons affected by a drug is essential for revealing its mechanism of action in the brain. c-Fos, a marker of neuronal activation, has been widely used to detect neurons activated by stimuli with high spatial resolution. In this review, the use of c-Fos for the visualization and manipulation of activated neurons is introduced. I also explain that a higher temporal resolution can be achieved by changing the staining method for visualization of c-Fos. Moreover, a new method that allows labeling and manipulating commonly activated neurons using two different stimuli is proposed.
... Whether LPB neurons simply relay these afferent signals or actively process them remains an interesting question. Considering that the LPB receives diverse inputs from brain regions with functions related to internal state sensing and fear, including the nucleus of the solitary tract (NTS) 25,54 , the rostral ventrolateral medulla 55 , and the paraventricular hypothalamus 56 , we propose that the LPB may also incorporate other types of signals to modify thermal afferent signals. In support of this hypothesis, it has recently been suggested that the LPBel receives input from the NTS and sends projections to the posterior subthalamic nucleus to mediate innate fear-associated hypothermia 54 . ...
... Considering that the LPB receives diverse inputs from brain regions with functions related to internal state sensing and fear, including the nucleus of the solitary tract (NTS) 25,54 , the rostral ventrolateral medulla 55 , and the paraventricular hypothalamus 56 , we propose that the LPB may also incorporate other types of signals to modify thermal afferent signals. In support of this hypothesis, it has recently been suggested that the LPBel receives input from the NTS and sends projections to the posterior subthalamic nucleus to mediate innate fear-associated hypothermia 54 . ...
Thermal homeostasis is vital for mammals and is controlled by brain neurocircuits. Yet, the neural pathways responsible for cold defense regulation are still unclear. Here, we found that a pathway from the lateral parabrachial nucleus (LPB) to the dorsomedial hypothalamus (DMH), which runs parallel to the canonical LPB to preoptic area (POA) pathway, is also crucial for cold defense. Together, these pathways make an equivalent and cumulative contribution, forming a parallel circuit. Specifically, activation of the LPB → DMH pathway induced strong cold-defense responses, including increases in thermogenesis of brown adipose tissue (BAT), muscle shivering, heart rate, and locomotion. Further, we identified somatostatin neurons in the LPB that target DMH to promote BAT thermogenesis. Therefore, we reveal a parallel circuit governing cold defense in mice, which enables resilience to hypothermia and provides a scalable and robust network in heat production, reshaping our understanding of neural circuit regulation of homeostatic behaviors.
... The PSTN is a distinct component located at the caudal part of the lateral hypothalamic area 13 . Functional manipulation of defined pathways has demonstrated that the PSTN is involved in motivation, the cardiovascular system, fear-induced thermoregulation, and metabolic homeostasis (by control of feeding behavior) [14][15][16][17] . Most recently, the PSTN is reported to play a key role in appetite suppression 18,19 . ...
... Most recently, the PSTN is reported to play a key role in appetite suppression 18,19 . Meanwhile, the PSTN is implicated in conditioned taste aversion, place avoidance, and impulsive action 13,16,20 . Interestingly, PSTN neurons notably respond to arousing stimuli, especially conspicuous stimuli, mobilized by predation, and novelty reliant on the exploration, which operates on the basis of wakefulness 13,21 . ...
... In contrast, our findings are consistent with a previous observation that activation of serotonergic terminals in the STN/PSTN from the dorsal raphe nucleus does not affect anxiety-or antidepressant-like behavior in the elevated plus maze or forced swim test, respectively 20 . Recently, the posterior subthalamic nucleus, including the PSTN, was reported to mediate innate fear-associated hypothermia in mice 16 . Chemogenetic inhibition of glutamatergic neurons in the posterior subthalamic nucleus attenuates 2-methyl-2-thiazoline-evoked hypothermia 16 , indicating that the thermoregulation activity of the PSTN is consistent with its ability to induce wakefulness. ...
The parasubthalamic nucleus (PSTN) is considered to be involved in motivation, feeding and hunting, all of which are highly depending on wakefulness. However, the roles and underlying neural circuits of the PSTN in wakefulness remain unclear. Neurons expressing calretinin (CR) account for the majority of PSTN neurons. In this study in male mice, fiber photometry recordings showed that the activity of PSTNCR neurons increased at the transitions from non-rapid eye movement (non-REM, NREM) sleep to either wakefulness or REM sleep, as well as exploratory behavior. Chemogenetic and optogenetic experiments demonstrated that PSTNCR neurons were necessary for initiating and/or maintaining arousal associated with exploration. Photoactivation of projections of PSTNCR neurons revealed that they regulated exploration-related wakefulness by innervating the ventral tegmental area. Collectively, our findings indicate that PSTNCR circuitry is essential for the induction and maintenance of the awake state associated with exploration.
... Whether LPB neurons simply relay these afferent signals or actively process them remains an interesting question. Considering that the LPB receives diverse inputs from brain regions with functions related to internal state sensing and fear, including the nucleus of the solitary tract (NTS) 25,54 , the rostral ventrolateral medulla 55 , and the paraventricular hypothalamus 56 , we propose that the LPB may also incorporate other types of signals to modify thermal afferent signals. In support of this hypothesis, it has recently been suggested that the LPBel receives input from the NTS and sends projections to the posterior subthalamic nucleus to mediate innate fear-associated hypothermia 54 . ...
... Considering that the LPB receives diverse inputs from brain regions with functions related to internal state sensing and fear, including the nucleus of the solitary tract (NTS) 25,54 , the rostral ventrolateral medulla 55 , and the paraventricular hypothalamus 56 , we propose that the LPB may also incorporate other types of signals to modify thermal afferent signals. In support of this hypothesis, it has recently been suggested that the LPBel receives input from the NTS and sends projections to the posterior subthalamic nucleus to mediate innate fear-associated hypothermia 54 . ...
Thermal homeostasis is vital for mammals and is controlled by brain neurocircuits. Remarkable advances have been made in understanding how neurocircuits centered in the hypothalamic preoptic area (POA), the brain's thermoregulation center, control warm defense, whereas mechanisms by which the POA regulates cold defense remain unclear. Here, we confirmed that the pathway from the lateral parabrachial nucleus (LPB) to the POA, is critical for cold defense. Parallel to this pathway, we uncovered that a pathway from the LPB to the dorsomedial hypothalamus (DMH), namely the LPB-DMH pathway, is also essential for cold defense. Projection-specific blockings revealed that both pathways provide an equivalent and cumulative contribution to cold defense, forming a parallel circuit. Specifically, activation of the LPB-DMH pathway induced strong cold-defense responses, including increases in thermogenesis of brown adipose tissue (BAT), muscle shivering, heart rate, and physical activity. Further, we identified a subpopulation of somatostatin+ neurons in the LPB that target the DMH to promote BAT thermogenesis. Therefore, we reveal a parabrachial-hypothalamic parallel circuit in governing cold defense in mice. This not only enables resilience to hypothermia but also provides a scalable and robust network in heat production, reshaping our understanding of how neural circuits regulate essential homeostatic behaviors.
... Direct or indirect innervation of the DMH from other brain regions and neurons, such as the dlPAG, LHb and orexin neurons, likely contribute to sympathetic and behavioral stress responses, although more studies are needed to understand the roles of these structures and neurons in the central network of stress signaling. Moreover, some types of fear stress induce hypothermia as opposed to sympathoexcitation, and a neural pathway involving the parabrachial nucleus, posterior subthalamic nucleus and nucleus of the solitary tract has recently been proposed to mediate skin vasodilation and hypothermia evoked by a fear odor (Liu et al., 2021). However, the functional neural connection of this pathway to the autonomic nervous system for the hypothermic response remains to be determined. ...
In mammals, many types of psychological stressors elicit a variety of sympathoexcitatory responses paralleling the classic fight-or-flight response to a threat to survival, including increased body temperature via brown adipose tissue thermogenesis and cutaneous vasoconstriction, and increased skeletal muscle blood flow via tachycardia and visceral vasoconstriction. Although these responses are usually supportive for stress coping, aberrant sympathetic responses to stress can lead to clinical issues in psychosomatic medicine. Sympathetic stress responses are mediated mostly by sympathetic premotor drives from the rostral medullary raphe region (rMR) and partly by those from the rostral ventrolateral medulla (RVLM). Hypothalamomedullary descending pathways from the dorsomedial hypothalamus (DMH) to the rMR and RVLM mediate important, stress-driven sympathoexcitatory transmission to the premotor neurons to drive the thermal and cardiovascular responses. The DMH also likely sends an excitatory input to the paraventricular hypothalamic nucleus to stimulate stress hormone release. Neurons in the DMH receive a stress-related excitation from the dorsal peduncular cortex and dorsal tenia tecta (DP/DTT) in the ventromedial prefrontal cortex. By connecting the corticolimbic emotion circuit to the central sympathetic and somatic motor systems, the DP/DTT → DMH pathway plays as the primary mediator of the psychosomatic signaling that drives a variety of sympathetic and behavioral stress responses. These brain regions together with other stress-related regions constitute a central neural network for physiological stress responses. This network model is relevant to understanding the central mechanisms by which stress and emotions affect autonomic regulations of homeostasis and to developing new therapeutic strategies for various stress-related disorders.
In mammals, males execute a stereotypical and organized sequence of sexual behaviours, such as mounting, intromission, and ejaculation, to successfully complete copulation. However, the neural mechanisms that govern the sequential transitions of male copulatory behaviours remain unclear. Here, we report that dopamine (DA) and acetylcholine (ACh) dynamics in the ventral shell of the nucleus accumbens (vsNAc) closely align with serial transitions of sexual behaviours in male mice. In particular, the vsNAc exhibits a unique pattern of 1.5—2.2 Hz dual ACh/DA rhythms that correspond to the pelvic thrust rhythm during intromission. The dual ACh/DA rhythms are generated locally by reciprocal regulation between ACh and DA signalling via nicotinic acetylcholine (nAChR) and dopamine D2 (D2R) receptors, respectively. Knockdown of choline acetyltransferase (ChAT) and D2R expression in the vsNAc diminishes intromission and ejaculation. We showed that ACh signalling promotes the initiation of intromission, whereas DA signalling sustains intromission by inhibiting the activities of D2R—expressing neurons in the vsNAc. Moreover, optogenetic activation of ChATvsNAc neurons during intromission slows down the DA rhythm, a specific activity signature that precedes ejaculation, and leads to immediate ejaculation. Taken together, dual ACh/DA dynamics in the vsNAc coordinate sequential transitions of male copulatory behaviours from intromission to ejaculation.
In terrestrial vertebrates, the olfactory system is divided into main (MOS) and accessory (AOS) components that process both volatile and nonvolatile cues to generate appropriate behavioral responses. While much is known regarding the molecular diversity of neurons that comprise the MOS, less is known about the AOS. Here, focusing on the vomeronasal organ (VNO), the accessory olfactory bulb (AOB), and the medial amygdala (MeA), we reveal that populations of neurons in the AOS can be molecularly subdivided based on their ongoing or prior expression of the transcription factors Foxp2 or Dbx1, which delineate separate populations of GABAergic output neurons in the MeA. We show that a majority of AOB neurons that project directly to the MeA are of the Foxp2 lineage. Using single-neuron patch-clamp electrophysiology, we further reveal that in addition to sex-specific differences across lineage, the frequency of excitatory input to MeA Dbx1- and Foxp2-lineage neurons differs between sexes. Together, this work uncovers a novel molecular diversity of AOS neurons, and lineage and sex differences in patterns of connectivity.
