The dorsal raphe nucleus (DRN) is an important nucleus in pain regulation. However, the underlying neural pathway and the function of specific cell types remain unclear. Here, we report a previously unrecognized ascending facilitation pathway, the DRN to the mesoaccumbal dopamine (DA) circuit, for regulating pain. Chronic pain increased the activity of DRN glutamatergic, but not serotonergic, neurons projecting to the ventral tegmental area (VTA) (DRNGlu-VTA) in male mice. The optogenetic activation of DRNGlu-VTA circuit induced a pain-like response in naive male mice, and its inhibition produced an analgesic effect in male mice with neuropathic pain. Furthermore, we discovered that DRN ascending pathway regulated pain through strengthened excitatory transmission onto the VTA DA neurons projecting to the ventral part of nucleus accumbens medial shell (vNAcMed), thereby activated the mesoaccumbal DA neurons. Correspondingly, optogenetic manipulation of this three-node pathway bilaterally regulated pain behaviors. These findings identified a DRN ascending excitatory pathway that is crucial for pain sensory processing, which can potentially be exploited toward targeting pain disorders.

Feeding requires sophisticated orchestration of neural processes to satiate appetite in natural, capricious settings. However, the complementary roles of discrete neural populations in orchestrating distinct behaviors and motivations throughout the feeding process are largely unknown. Here, we delineate the behavioral repertoire of mice by developing a machine-learning-assisted behavior tracking system and show that feeding is fragmented and divergent motivations for food consumption or environment exploration compete throughout the feeding process. An iterative activation sequence of agouti-related peptide (AgRP)-expressing neurons in arcuate (ARC) nucleus, GABAergic neurons in the lateral hypothalamus (LH), and in dorsal raphe (DR) orchestrate the preparation, initiation, and maintenance of feeding segments, respectively, via the resolution of motivational conflicts. The iterative neural processing sequence underlying the competition of divergent motivations further suggests a general rule for optimizing goal-directed behaviors.

Serotonin (5HT) is a well-known anorexigenic molecule, and 5HT neurons of dorsal raphe nucleus (DRN) have been implicated in suppression of feeding; however, the downstream circuitry is poorly understood. Here we explored major projections of DRN5HT neurons for their capacity to modulate feeding.
We used optogenetics to selectively activate DRN5HT axonal projections in hypothalamic and extrahypothalamic areas and monitored food intake. We next used fiber photometry to image the activity dynamics of DRN5HT axons and 5HT levels in projection areas in response feeding and metabolic hormones. Finally, we used electrophysiology to determine how DRN5HT axons affect downstream neuron activity.
We found that selective activation of DRN5HT axons in (DRN5HT → LH) and (DRN5HT → BNST) suppresses feeding whereas activating medial hypothalamic projections has no effect. Using in vivo imaging, we found that food access and satiety hormones activate DRN5HT projections to LH where they also rapidly increase extracellular 5HT levels. Optogenetic mapping revealed that DRN5HT → LHvGAT and DRN5HT → LHvGlut2 connections are primarily inhibitory and excitatory respectively. Further, in addition to its direct action on LH neurons, we found that 5HT suppresses GABA release from presynaptic terminals arriving from AgRP neurons.
These findings define functionally redundant forebrain circuits through which DRN5HT neurons suppress feeding and reveal that these projections can be modulated by metabolic hormones.

The dorsal raphe (DR) nucleus contains many tyrosine hydroxylase (TH)-positive neurons which are regarded as dopaminergic (DA) neurons. These DA neurons in the DR and periaqueductal gray (PAG) region (DADR-PAG neurons) are a subgroup of the A10 cluster, which is known to be heterogeneous. This DA population projects to the central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BNST) and has been reported to modulate various affective behaviors. To characterize, the histochemical features of DADR-PAG neurons projecting to the CeA and BNST in mice, the current study combined retrograde labeling with Fluoro-Gold (FG) and histological techniques, focusing on TH, dopamine transporter (DAT), vasoactive intestinal peptide (VIP), and vesicular glutamate transporter 2 (VGlut2). To identify putative DA neurons, DAT-Cre::Ai14 mice were used. It was observed that DATDR-PAG neurons consisted of the following two subpopulations: TH+/VIP- and TH-/VIP+ neurons. The DAT+/TH-/VIP+ subpopulation would be non-DA noncanonical DAT neurons. Anterograde labeling of DATDR-PAG neurons with AAV in DAT-Cre mice revealed that the fibers exclusively innervated the lateral part of the CeA and the oval nucleus of the BNST. Retrograde labeling with FG injections into the CeA or BNST revealed that the two subpopulations similarly innervated these regions. Furthermore, using VGlut2-Cre::Ai14 mice, it was turned out that the TH-/VIP+ subpopulations innervating both CeA and BNST were VGlut2-positive neurons. These two subpopulations of DATDR-PAG neurons, TH+/VIP- and TH-/VIP+, might differentially interfere with the extended amygdala, thereby modulating affective behaviors.

Social contacts play an important role in the development and survival of social animals. Social isolation (SI) at adolescence often induces abnormalities in many kinds of behaviors. This study assessed whether five weeks of continuous SI at adulthood could alter social behaviors and whether dorsal raphe nucleus (DR) to medial prefrontal cortex (mPFC) 5-HT neural projections were involved in this alteration in C57BL/6J adult male mice. The present study found that five weeks chronic social isolation (CSI) at adulthood increased mounting and sniffing behaviors in resident-intruder test, and lengthened duration staying in interaction zone of stranger cage in the three-chamber social preference test. CSI also reduced the release of 5-HT in the mPFC detected by 5-HT 1.0 sensor and measured by in vivo fiber photometry test. Meanwhile, the c-Fos expression indicated that CSI reduced the activity of serotonergic neurons. Chemogenetic activation of DR-mPFC 5-HTergic projection reduced sniffing of CSI mice in the resident-intruder test, but didn\'t significantly affect mounting behavior. It also decreased the interaction time during the three-chamber social preference test. Thus, 5-HT neural projections from the DR to the mPFC are involved in changes of social exploration behaviors induced by CSI at adulthood.

General anesthesia has been widely applied in surgical or nonsurgical medical procedures, but the mechanism behind remains elusive. Because of shared neural circuits of sleep and anesthesia, whether serotonergic system, which is highly implicated in modulation of sleep and wakefulness, regulates general anesthesia as well is worth investigating.
Immunostaining and fiber photometry were used to assess the neuronal activities. Electroencephalography spectra and burst-suppression ratio (BSR) were used to measure anesthetic depth and loss or recovery of righting reflex to indicate the induction or emergence time of general anesthesia. Regulation of serotonergic system was achieved through optogenetic, chemogenetic, or pharmacological methods.
We found that both Fos expression and calcium activity were significantly decreased during general anesthesia. Activation of 5-HT neurons in the dorsal raphe nucleus (DRN) decreased the depth of anesthesia and facilitated the emergence from anesthesia, and inhibition deepened the anesthesia and prolonged the emergence time. Furthermore, agonism or antagonism of 5-HT 1A or 2C receptors mimicked the effect of manipulating DRN serotonergic neurons.
Our results demonstrate that 5-HT neurons in the DRN play a regulative role of general anesthesia, and activation of serotonergic neurons could facilitate emergence from general anesthesia partly through 5-HT 1A and 2C receptors.

Epilepsy, frequently comorbid with depression, easily develops drug resistance. Here, we investigated how dorsal raphe (DR) and its 5-HTergic neurons are implicated in epilepsy.
In mouse hippocampal kindling model, using immunochemistry, calcium fiber photometry, and optogenetics, we investigated the causal role of DR 5-HTergic neurons in seizure of temporal lobe epilepsy (TLE). Further, deep brain stimulation (DBS) of the DR with different frequencies was applied to test its effect on hippocampal seizure and depressive-like behavior.
Number of c-fos+ neurons in the DR and calcium activities of DR 5-HTergic neurons were both increased during kindling-induced hippocampal seizures. Optogenetic inhibition, but not activation, of DR 5-HTergic neurons conspicuously retarded seizure acquisition specially during the late period. For clinical translation, 1-Hz-specific, but not 20-Hz or 100-Hz, DBS of the DR retarded the acquisition of hippocampal seizure. This therapeutic effect may be mediated by the inhibition of DR 5-HTergic neurons, as optogenetic activation of DR 5-HTergic neurons reversed the anti-seizure effects of 1-Hz DR DBS. However, DBS treatment had no effect on depressive-like behavior.
Inhibition of hyperactivity of DR 5-HTergic neuron may present promising anti-seizure effect and the DR may be a potential DBS target for the therapy of TLE.

The brain dopamine (DA) system participates in forming and expressing memory. Despite a well-established role of DA neurons in the ventral tegmental area in memory formation, the exact DA circuits that control memory expression remain unclear. Here, we show that DA neurons in the dorsal raphe nucleus (DRN) and their medulla input control the expression of incentive memory. DRN DA neurons are activated by both rewarding and aversive stimuli in a learning-dependent manner and exhibit elevated activity during memory recall. Disrupting their physiological activity or DA synthesis blocks the expression of natural appetitive and aversive memories as well as drug memories associated with opioids. Moreover, a glutamatergic pathway from the lateral parabrachial nucleus to the DRN selectively regulates the expression of reward memories associated with opioids or foods. Our study reveals a specialized DA subsystem important for memory expression and suggests new targets for interventions against opioid addiction.

Serotonin neurons of the dorsal and median raphe nuclei (DR, MR) collectively innervate the entire forebrain and midbrain, modulating diverse physiology and behavior. To gain a fundamental understanding of their molecular heterogeneity, we used plate-based single-cell RNA-sequencing to generate a comprehensive dataset comprising eleven transcriptomically distinct serotonin neuron clusters. Systematic in situ hybridization mapped specific clusters to the principal DR, caudal DR, or MR. These transcriptomic clusters differentially express a rich repertoire of neuropeptides, receptors, ion channels, and transcription factors. We generated novel intersectional viral-genetic tools to access specific subpopulations. Whole-brain axonal projection mapping revealed that DR serotonin neurons co-expressing vesicular glutamate transporter-3 preferentially innervate the cortex, whereas those co-expressing thyrotropin-releasing hormone innervate subcortical regions in particular the hypothalamus. Reconstruction of 50 individual DR serotonin neurons revealed diverse and segregated axonal projection patterns at the single-cell level. Together, these results provide a molecular foundation of the heterogenous serotonin neuronal phenotypes.

Both the ventral tegmental area (VTA) and dorsal raphe nucleus (DRN) are involved in affective control and reward-related behaviors. Moreover, the neuronal activities of the VTA and DRN are modulated by opioids. However, the precise circuits from the VTA to DRN and how opioids modulate these circuits remain unknown. Here, we found that neurons projecting from the VTA to DRN are primarily GABAergic. Rostral VTA (rVTA) GABAergic neurons preferentially innervate DRN GABAergic neurons, thus disinhibiting DRN serotonergic neurons. Optogenetic activation of this circuit induces aversion. In contrast, caudal VTA (cVTA) GABAergic neurons mainly target DRN serotonergic neurons, and activation of this circuit promotes reward. Importantly, μ-opioid receptors (MOPs) are selectively expressed at rVTA→DRN GABAergic synapses, and morphine depresses the synaptic transmission. Chronically elevating the activity of the rVTA→DRN pathway specifically interrupts morphine-induced conditioned place preference. This opioid-modulated inhibitory circuit may yield insights into morphine reward and dependence pathogenesis.