Activation Of Neurons Research Articles

Central amygdala NPBWR1 neurons facilitate social novelty seeking and new social interactions.

The formation of new social interactions is vital for social animals, but the underlying neural mechanisms remain poorly understood. We identified CeANpbwr1 neurons, a population in central amygdala expressing neuropeptide B/W receptor-1 (NPBWR1), that play a critical role in these interactions. CeANpbwr1 neurons were activated during encounters with unfamiliar, but not with familiar, mice. Manipulations of CeANpbwr1 neurons showed that their excitation is essential for maintaining physical interactions with novel conspecifics. Activation of CeANpbwr1 neurons alleviated social deficits induced by chronic social defeat stress, suggesting therapeutic potential. Conversely, overexpression of human NPBWR1 in CeANpbwr1 neurons reduced activity of these neurons and impaired social interactions with unfamiliar mice. This effect was absent in a polymorphic variant of the human NPBWR1 gene (404A>T). These findings highlight how CeANpbwr1 neurons promote social novelty seeking and reveal a complex interplay between NPBWR1 genetic variations and social behavior.

Neuronal-ILC2 interactions regulate pancreatic glucagon and glucose homeostasis.

The immune system shapes body metabolism, while interactions between peripheral neurons and immune cells control tissue homeostasis and immunity. However, whether peripheral neuroimmune interactions orchestrate endocrine system functions remains unexplored. After fasting, mice lacking type 2 innate lymphoid cells (ILC2s) displayed disrupted glucose homeostasis, impaired pancreatic glucagon secretion, and inefficient hepatic gluconeogenesis. Additionally, intestinal ILC2s were found in the pancreas, which was dependent on their expression of the adrenergic beta 2 receptor. Targeted activation of catecholaminergic intestinal neurons promoted the accumulation of ILC2s in the pancreas. Our work provides evidence that immune cells can be regulated by neuronal signals in response to fasting, activating an inter-organ communication route that promotes pancreatic endocrine function and regulation of blood glucose levels.

Activation of locus coeruleus noradrenergic neurons rapidly drives homeostatic sleep pressure.

Homeostatic sleep regulation is essential for optimizing the amount and timing of sleep for its revitalizing function, but the mechanism underlying sleep homeostasis remains poorly understood. Here, we show that optogenetic activation of locus coeruleus (LC) noradrenergic neurons immediately increased sleep propensity following a transient wakefulness, contrasting with many other arousal-promoting neurons whose activation induces sustained wakefulness. Fiber photometry showed that repeated optogenetic or sensory stimulation caused a rapid reduction of calcium activity in LC neurons and steep declines in noradrenaline/norepinephrine (NE) release in both the LC and medial prefrontal cortex (mPFC). Knockdown of α2A adrenergic receptors in LC neurons mitigated the decline of NE release induced by repetitive stimulation and extended wakefulness, demonstrating an important role of α2A receptor-mediated auto-suppression of NE release. Together, these results suggest that functional fatigue of LC noradrenergic neurons, which reduces their wake-promoting capacity, contributes to sleep pressure.

Nesfatin-1 Neurons in the Ventral Premammillary Nucleus Integrate Metabolic and Reproductive Signals in Male Rats

The ability to reproduce depends on metabolic status. In rodents, the ventral premammillary nucleus (PMv) integrates metabolic and reproductive signals. While leptin (adiposity-related) signaling in the PMv is critical for female fertility, male reproductive functions are strongly influenced by glucose homeostasis. The anorexigenic peptide nesfatin-1 is a leptin-independent central regulator of blood glucose. Therefore, its integrative role in male rats can be assumed. To investigate this, we mapped the distribution of nesfatin-1 mRNA- and protein-producing cells in the PMv during postnatal development via in situ hybridization and immunohistochemistry, respectively. Fos-nesfatin-1, double immunostaining was used to determine the combined effect of heterosexual pheromone challenge and insulin-induced hypoglycemia on neuronal activation in adults. We found that ~75% of the pheromone-activated neurons were nesfatin-1 cells. Hypoglycemia reduced pheromone-induced cell activation, particularly in nesfatin-1 neurons. Immuno-electron microscopy revealed innervation of PMv nesfatin-1 neurons by urocortin3-immunoreactive terminals, reportedly originating from the medial amygdala. Nesfatin-1 immunopositive neurons expressed GPR10 mRNA, a receptor associated with metabolic signaling, but did not respond with accumulation of phosphorylated STAT3 immunopositivity, a marker of leptin receptor signaling, in response to intracerebroventricular leptin treatment. Our results suggest that PMv nesfatin-1 neurons are primarily responsible for integrating reproductive and metabolic signaling in male rats.

Brain-wide neuronal circuit connectome of human glioblastoma.

Glioblastoma (GBM) infiltrates the brain and can be synaptically innervated by neurons, which drives tumor progression1,2. Synaptic inputs onto GBM cells identified so far are largely short-range and glutamatergic3,4. The extent of GBM integration into the brain-wide neuronal circuitry remains unclear. Here we applied rabies virus- and herpes simplex virus-mediated trans-monosynaptic tracing5,6 to systematically investigate circuit integration of human GBM organoids transplanted into adult mice. We found that GBM cells from multiple patients rapidly integrate into diverse local and long-range neural circuits across the brain. Beyond glutamatergic inputs, we identified various neuromodulatory inputs, including synapses between basal forebrain cholinergic neurons and GBM cells. Acute acetylcholine stimulation induces long-lasting elevation of calcium oscillations and transcriptional reprogramming of GBM cells into a more motile state via the metabotropic CHRM3 receptor. CHRM3 activation promotes GBM cell motility, whereas its downregulation suppresses GBM cell motility and prolongs mouse survival. Together, these results reveal the striking capacity for human GBM cells to rapidly and robustly integrate into anatomically diverse neuronal networks of different neurotransmitter systems. Our findings further support a model wherein rapid connectivity and transient activation of upstream neurons may lead to a long-lasting increase in tumor fitness.

Intranasal delivery of a ghrelin mimetic engages the brain ghrelin signalling system in mice.

Ghrelin, the endogenous ligand of the growth hormone secretagogue receptor (GHSR), promotes food intake, other feeding behaviours and stimulates growth hormone (GH) release from the pituitary. Growth hormone secretagogues (GHS), such as GHRP-6 and MK-0677, are synthetic GHSR ligands that activate orexigenic Neuropeptide Y neurons that co-express Agouti-Related Peptide (AgRP) in the arcuate nucleus of the hypothalamus when administered systemically. Systemic GHRP-6 also stimulates GH release in humans and rats. Thus, GHS and ghrelin have therapeutic relevance in patients who could benefit from its orexigenic and/or GH-releasing effects. This study examined whether intranasal delivery of ghrelin, GHRP-6, or MK-0677 engages the brain ghrelin signalling system. Effective compounds and doses were selected based on increased food intake after intranasal application in mice. Only GHRP-6 (5 mg/kg) increased food intake without adverse effects, prompting detailed analysis of meal patterns, neuronal activation in the arcuate nucleus (via Fos mapping) and neurochemical identification of c-fos mRNA-expressing neurons using RNAscope. We also assessed the impact of intranasal GHRP-6 on serum GH levels. Intranasal GHRP-6 increased food intake by increasing meal frequency and size. Fos expression in the arcuate nucleus was higher in GHRP-6-treated mice than saline controls. When examining the neurochemical identity of c-fos-mRNA-expressing neurons, we found co-expression with 63.5±1.9% Ghsr-mRNA, 79±6.8% Agrp-mRNA and 11.4±2.5% Ghrh-mRNA, demonstrating GHRP-6's ability to engage arcuate nucleus neurons involved in food intake and GH release. Additionally, intranasal GHRP-6 elevated GH serum levels. These findings suggest that intranasal GHRP-6, but not ghrelin or MK-0677, can engage the brain ghrelin signalling system.

Distinct Behavioral Profiles and Neuronal Correlates of Heroin Vulnerability Versus Resiliency in a Multi-Symptomatic Model of Heroin Use Disorder in Rats.

The behavioral and diagnostic heterogeneity within the opioid use disorder (OUD) diagnosis is not readily captured in current animal models, limiting the translational relevance of the mechanistic research that is conducted in experimental animals. The authors hypothesized that a nonlinear clustering of OUD-like behavioral traits would capture population heterogeneity and yield subpopulations of OUD vulnerable rats with distinct behavioral and neurocircuit profiles. Over 900 male and female heterogeneous stock rats, a line capturing genetic and behavioral heterogeneity present in humans, were assessed for several measures of heroin use and rewarded and non-rewarded seeking behaviors. A nonlinear stochastic block model clustering analysis was used to assign rats to OUD vulnerable, intermediate, and resilient clusters. Additional behavioral tests and circuit analyses using c-fos protein activation were conducted on the vulnerable and resilient subpopulations. OUD vulnerable rats exhibited greater heroin taking and seeking behaviors relative to those in the intermediate and resilient clusters. Akin to human OUD diagnosis, further vulnerable rat subclustering revealed subpopulations with different combinations of behavioral traits, including sex differences. Lastly, heroin cue-induced neuronal patterns of circuit activation differed between resilient and vulnerable phenotypes. Behavioral sex differences were recapitulated in patterns of circuitry activation, including preferential engagement of extended amygdala stress circuitry in males and cortico-striatal drug cue-seeking circuitry in females. Using a nonlinear clustering approach in rats, the analysis captured behavioral diagnostic heterogeneity reflective of human OUD diagnosis. OUD vulnerability and resiliency were associated with distinct neuronal activation patterns, posing this approach as a translational tool in assessing neurobiological mechanisms underpinning OUD.

Ventral pallidal GABAergic neurons drive consumption in male, but not female rats.

Food intake is controlled by multiple converging signals: hormonal signals that provide information about energy homeostasis, but also hedonic and motivational aspects of food and food cues that can drive non-homeostatic or "hedonic" feeding. The ventral pallidum (VP) is a brain region implicated in the hedonic and motivational impact of food and foods cues, as well as consumption of rewards. Disinhibition of VP neurons has been shown to generate intense hyperphagia, or overconsumption. While VP gamma-Aminobutyric acidergic (GABA) neurons have been implicated in cue-elicited reward seeking and motivation, the role of these neurons in the hyperphagia resulting from VP activation remains unclear. Here, we used Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) to activate VP GABA neurons in non-restricted male and female rats during chow and sucrose consumption. We found that activation of VP GABA neurons increases consumption of chow and sucrose in male rats, but not female rats. Together, these findings suggest that activation of VP GABA neurons can stimulate consumption of routine or highly palatable rewards selectively in male rats.Significance statement The ventral pallidum has been implicated bidirectionally in consumption of both standard food and highly palatable rewards, but the specific neural subpopulations involved have not been identified. Here we chemogenetically excited GABAergic ventral pallidal neurons and tested consumption of standard chow and a sweet sucrose solution. We found that chemogenetic excitation of these neurons stimulated consumption of both rewards but did so specifically in male rats. These results suggest that GABAergic ventral pallidal neurons can drive overconsumption of foods in male rats, but not female rats, raising important questions about the role of ventral pallidum in consumption in females, who have been understudied in this domain.

From pain to meningitis: bacteria hijack nociceptors to promote meningitis

Bacterial meningitis is a severe and life-threatening infection of the central nervous system (CNS), primarily caused by Streptococcus pneumoniae and Neisseria meningitidis. This condition carries a high risk of mortality and severe neurological sequelae, such as cognitive impairment and epilepsy. Pain, a central feature of meningitis, results from the activation of nociceptor sensory neurons by inflammatory mediators or bacterial toxins. These nociceptors, abundantly present in the meninges, trigger neuroimmune signaling pathways that influence the host immune response. However, the mechanisms by which bacteria hijack these nociceptors to promote CNS invasion and exacerbate the disease remain poorly understood. This review examines the interactions between bacteria and meningeal nociceptors, focusing on their direct and indirect activation via ion channels, such as transient receptor potential vanilloid-1 (TRPV1) and transient receptor potential ankyrin 1 (TRPA1), or through the release of neuropeptides like calcitonin gene-related peptide (CGRP). These interactions suppress immune defenses by inhibiting macrophage activity and neutrophil recruitment, thus facilitating bacterial survival and invasion of the CNS. Understanding this neuroimmune axis may open potential therapeutic targets for treating bacterial meningitis by enhancing host defenses and mitigating pain. Further research using advanced methodologies is essential to clarify the role of nociceptor-mediated immune modulation in this disease.

A membrane-targeted photoswitch restores physiological ON/OFF responses to light in the degenerate retina

The lack of effective therapies for visual restoration in Retinitis pigmentosa and macular degeneration has led to the development of new strategies, such as optogenetics and retinal prostheses. However, visual restoration is poor due to the massive light-evoked activation of retinal neurons, regardless of the segregation of visual information in ON and OFF channels, which is essential for contrast sensitivity and spatial resolution. Here, we show that Ziapin2, a membrane photoswitch that modulates neuronal capacitance and excitability in a light-dependent manner, is capable of reinstating, in mouse and rat genetic models of photoreceptor degeneration, brisk and sluggish ON, OFF, and ON-OFF responses in retinal ganglion cells evoked by full-field stimuli, with reactivation of their excitatory and inhibitory conductances. Intravitreally injected Ziapin2 in fully blind rd10 mice restores light-driven behavior and optomotor reflexes. The results indicate that Ziapin2 is a promising molecule for reinstating physiological visual responses in the late stages of retinal degeneration.

Parabrachial CGRP neurons modulate active defensive behavior under a naturalistic threat.

Recent studies suggest that calcitonin gene-related peptide (CGRP) neurons in the parabrachial nucleus (PBN) represent aversive information and signal a general alarm to the forebrain. If CGRP neurons serve as a true general alarm, their activation would modulate both passive nad active defensive behaviors depending on the magnitude and context of the threat. However, most prior research has focused on the role of CGRP neurons in passive freezing responses, with limited exploration of their involvement in active defensive behaviors. To address this, we examined the role of CGRP neurons in active defensive behavior using a predator-like robot programmed to chase mice. Our electrophysiological results revealed that CGRP neurons encode the intensity of aversive stimuli through variations in firing durations and amplitudes. Optogenetic activation of CGRP neuron during robot chasing elevated flight responses in both conditioning and retention tests, presumably by amyplifying the perception of the threat as more imminent and dangerous. In contrast, animals with inactivated CGRP neurons exhibited reduced flight responses, even when the robot was programmed to appear highly threatening during conditioning. These findings expand the understanding of CGRP neurons in the PBN as a critical alarm system, capable of dynamically regulating active defensive behaviors by amplifying threat perception, ensuring adaptive responses to varying levels of danger.

Neurogenic inflammation and itch in barrier tissues.

Once regarded as distinct systems, the nervous system and the immune system are now recognized for their complex interactions within the barrier tissues. The neuroimmune circuitry comprises a dual-network system that detects external and internal disturbances, providing critical information to tailor a context-specific response to various threats to tissue integrity, such as wounding or exposure to noxious and harmful stimuli like pathogens, toxins, or allergens. Using the skin as an example of a barrier tissue with the polarized sensory neuronal responses of itch and pain, we explore the molecular pathways driving neuronal activation and the effects of this activation on the immune response. We then apply these findings to other barrier tissues, to find common pathways controlling neuroimmune responses in the barriers.

Enhancing Neuron Activity Promotes Functional Recovery by Inhibiting Microglia-Mediated Synapse Elimination After Stroke.

Activating glutamatergic neurons in the ipsilesional motor cortex can promote functional recovery after stroke. However, the underlying molecular mechanisms remain unclear. Clarifying key molecular mechanisms involved in recovery could help understand the development of neuromodulation strategies after stroke. Adeno-associated virus 2/9-CamKIIa-hM3Dq-mCherry was injected into ipsilesional motor cortex by stereotaxic in the photothrombotic stroke model. Starting from the third day after the stroke, male mice were injected intraperitoneally with clozapine-N-oxide every day to activate excitatory neurons. C1q-blocking antibody and annexin V were used to inhibit C1q and exposed phosphatidylserine (EPS), respectively. The cylinder test and grid-walking test were performed to evaluate functional recovery. The potential molecular mechanisms of excitatory neuronal activation on microglia-mediated synaptic pruning after stroke by immunofluorescence, real-time polymerase chain reaction, Western blotting, and RNA sequencing. Activating excitatory neurons significantly promoted functional recovery and inhibited microglia-mediated synaptic pruning after stroke. Furthermore, it decreased EPS and C1q levels in synapses. On the contrary, inhibiting excitatory neurons aggravated functional defects, promoted microglia-mediated synaptic pruning, and increased EPS and C1q levels in synapses. Selective blocking of EPS repressed C1q tagging of synapses and microglia-mediated synaptic pruning and improved functional recovery. Meanwhile, blocking EPS markedly rescued synaptic density, and motor function deteriorated by chemogenetic inhibition. In addition, C1q-blocking antibody prevented phosphatidylserine engulfment by microglia. Together, these data provide mechanistic insight into microglia-mediated synapse pruning after neuronal activation after stroke and identify the role of C1q binding to EPS in stroke treatment during the repair phase.

Maternally activated connections of the ventral lateral septum reveal input from the posterior intralaminar thalamus.

The lateral septum (LS) demonstrates activation in response to pup exposure in mothers, and its lesions eliminate maternal behaviors suggesting it is part of the maternal brain circuitry. This study shows that the density of pup-activated neurons in the ventral subdivision of the LS (LSv) is nearly equivalent to that in the medial preoptic area (MPOA), the major regulatory site of maternal behavior in rat dams. However, when somatosensory inputs including suckling were not allowed, pup-activation was markedly reduced in the LSv. Retrograde tract tracing identified various brain regions potentially influencing LSv neuronal activation through their projections. Among all, anterograde tract tracing confirmed that the posterior intralaminar thalamic nucleus (PIL), implicated in processing touch-related stimuli, targets the pup-activated region of the LSv. Moreover, nerve terminals containing the maternally induced PIL neuropeptide parathyroid hormone 2 (PTH2), were found to form synaptic connections with c-Fos activated LSv neurons using electron microscopy. Confirmation of PTH2 + PIL fibers projecting to LSv was achieved by retrograde tract tracing methods. Furthermore, double retrograde injections revealed that neurons within the PIL can project to both LSv and MPOA, suggesting their simultaneous regulation by PIL input. We also established that septal neurons activated by the pups in the mother are GABAergic and send inhibitory projections to the MPOA and other components of the maternal brain circuitry. This implies that the LSv and MPOA form an interconnected subcircuit in the maternal brain network, which is primarily driven by somatosensory input from the pups via the PIL PTH2 + neurons.

Dopaminergic modulation of propofol-induced activation in VLPO neurons: the role of D1 receptors in sleep-promoting neural circuits

Dopaminergic modulation of propofol-induced activation in VLPO neurons: the role of D1 receptors in sleep-promoting neural circuits

Temperature Regulates Astroglia Morphogenesis Through Thermosensory Circuitry in Caenorhabditis elegans.

Astrocytes are the most abundant type of macroglia in the brain and play crucial roles in regulating neural development and functions. The diverse functions of astrocytes are largely determined by their morphology, which is regulated by genetic and environmental factors. However, whether and how the astrocyte morphology is affected by temperature remains largely unknown. Here we discovered that elevated cultivation temperature (26°C) stimulates Caenorhabditis elegans ventral CEPsh glia endfoot extension during early developmental stages. This extension depends on the activation of glutamate AWC neurons, which inhibit the postsynaptic cholinergic AIY interneurons through glutamate-gated chloride channels, GLC-3 and GLC-4. In responding to the thermosensory signal, the guanyl-nucleotide exchange factor EPHX-1 and Rho GTPase CDC-42/Cdc42 in the glia facilitate the endfoot extension via F-actin assembly. This study elucidates the significant role of thermosensory circuitry in glia morphogenesis and the underlying molecular mechanism.

A novel SIRT1 activator attenuates neuropathic pain by inhibiting spinal neuronal activation via the SIRT1-mGluR1/5 pathway

Neuropathic pain is a type of pain caused by an injury or disease of the somatosensory nervous system. Currently, there is still absence of effective therapeutic drugs for neuropathic pain, so developing new therapeutic drugs is urgently needed. In the present study, we observed the effect of Comp 6d, a novel silent information regulator 1 (SIRT1) activator synthesized in our laboratory, on neuropathic pain and investigated the mechanisms involved. We found that both intrathecal and intraperitoneal injections of Comp 6d effectively alleviated neuropathic pain induced by chronic constriction injury (CCI) or spared nerve injury (SNI). However, the effect of Comp 6d on neuropathic pain was abolished in SIRT1 knockout mice. These results demonstrated that Comp 6d alleviated neuropathic pain by specifically activating SIRT1 in the spinal cord. Moreover, long-term intraperitoneal injection of Comp 6d had no significant side effects on heart, liver and kidney in SNI mice. Further study showed that the improvement of neuropathic pain by Comp 6d was mediated by the downregulation of mGluR1/5 levels and the subsequent inhibition of spinal neuronal activation. Taken together, the present findings suggest that the novel SIRT1 activator Comp 6d inhibits the activation of spinal cord neurons via the SIRT1-mGluR1/5 pathway, thereby attenuating neuropathic pain. Comp 6d is expected to be an effective therapeutic agent for neuropathic pain.

The cerebral blood flow response to neuroactivation is reduced in cognitively normal men with β-amyloid accumulation

BackgroundAccumulation of β-amyloid (Aβ) in the brain is a hallmark of Alzheimer’s Disease (AD). Cerebral deposition of Aβ initiates deteriorating pathways which eventually can lead to AD. However, the exact mechanisms are not known. A possible pathway could be that Aβ affects the cerebral vessels, causing inadequate cerebrovascular function. In the present study, we examined if Aβ accumulation is associated with a reduced cerebral blood flow response (CBF) to neuronal activation by visual stimulation (ΔCBFVis.Act.) in cognitively normal subjects from the Metropolit Danish Male Birth Cohort.Methods64 subjects participated in the present study. ΔCBFVis.Act. was measured using arterial spin labelling (ASL) combined with blood-oxygen-level-dependent (BOLD) MRI. Neuronal activation was obtained by visual stimulation by a flickering checkerboard presented on a screen in the MRI-scanner. Brain Aβ accumulation and cerebral glucose metabolism were assessed by PET imaging using the radiotracers [11C]Pittsburgh Compound-B (PiB) and [18F]Fluorodeoxyglucose (FDG), respectively. Cortical thickness was measured from structural MRI.ResultsΔCBFVis.Act. correlated negatively (β = -32.1 [95% confidence interval (CI): -60.2; -4.1], r = -0.30, p = 0.025) with PiB standardized uptake value ratio (SUVr) in the brain regions activated by visual stimulation. ΔCBFVis.Act. did not correlate with FDG SUVr (β = 1.9 [CI: -23.8; 27.6], r = 0.02, p = 0.88) or cortical thickness (β = 10.3 [CI: -8.4; 29.0], r = 0.15, p = 0.27) in the activated brain regions. Resting CBF did not correlate with PiB SUVr neither in the regions activated by visual stimulation (β = -17.8 [CI:-71.9; 36.2], r =- 0.09, p = 0.51) nor in the remaining cortex (β = 5.2 [CI:-3.9; 14.2], r = 0.15, p = 0.26).ConclusionWe found a correlation between high PiB SUVr and reduced CBF response to neuronal activation, indicating a link between Aβ accumulation and impaired cerebrovascular function. The impairment was not associated with cortical thinning or hypometabolism, suggesting that Aβ accumulation affecting brain vessel function could be a very early pathology leading to neurodegenerative disease.

Hydrogen-bonded organic framework nanotransducers enabled sono-optogenetics for Parkinsonian rats.

Cell-type-specific activation of parvalbumin (PV)-expressing neurons in the external globus pallidus (GPe) through optogenetics has shown promise in facilitating long-lasting movement dysfunction recovery in mice with Parkinson's disease. However, its translational potential is hindered by adverse effects stemming from the invasive implantation of optical fibers into the brain. In this study, we have developed a non-invasive optogenetics approach, utilizing focused ultrasound-triggered mechanoluminescent nanotransducers to enable remote photon delivery deep in the brain for genetically targeted neuromodulation. These mechanoluminescent nanotransducers consist of sonosensitized hydrogen-bonded frameworks and chemiluminescent L012, serving as a nanoscale light source through ultrasound-induced cascade reactions. This system offers high ultrasound-triggered brightness and long-lasting light emission, facilitating repeatable deep brain stimulation. Our sono-optogenetics technology demonstrated effective modulation in the mouse motor cortex for limb motion control and activation of PV-GPe neurons for rescuing movement dysfunction over time in dopamine-depleted Parkinson's disease rats. This approach demonstrates the pathway for achieving genetically targeted and non-invasive neuromodulation for long-lasting treatment of Parkinson's disease, towards non-human primate models and clinical applications.

Convergent state-control of endogenous opioid analgesia.

Pain is a dynamic and nonlinear experience shaped by injury and contextual factors, including expectations of future pain or relief 1 . While µ opioid receptors are central to the analgesic effects of opioid drugs, the endogenous opioid neurocircuitry underlying pain and placebo analgesia remains poorly understood. The ventrolateral column of the posterior periaqueductal gray is a critical hub for nociception and endogenous analgesia mediated by opioid signaling 2 . However, significant gaps remain in understanding the cell-type identities, the sub-second neural dynamics involved in pain modulation, the role of endogenous peptide neuromodulators, and the contextual factors influencing these processes. Using spatial mapping with single-nuclei RNA sequencing of pain-active neurons projecting to distinct long-range brain targets, alongside cell type-specific and activity-dependent genetic tools for in vivo optical recordings and modulation of neural activity and opioid peptide release, we identified a functional dichotomy in the ventrolateral periaqueductal gray. Neurons expressing µ opioid receptors encode active nociceptive states, whereas enkephalin-releasing neurons drive pain relief during recovery from injury, in response to learned fear predictions, and during placebo analgesia. Finally, by leveraging the functional effects of placebo analgesia, we used direct optogenetic activation of vlPAG enkephalin neurons to drive opioid peptide release, resulting in a robust reduction in pain. These findings show that diverse need states converge on a shared midbrain circuit that releases endogenous opioids with high spatiotemporal precision to suppress nociceptive activity and promote analgesia.