A master regulator of opioid reward in the ventral prefrontal cortex.

In addition to their intrinsic rewarding properties, opioids can also evoke aversive reactions that protect against misuse. Cellular mechanisms that govern the interplay between opioid reward and aversion are poorly understood. We used whole-brain activity mapping in mice to show that neurons in the dorsal peduncular nucleus (DPn) are highly responsive to the opioid oxycodone. Connectomic profiling revealed that DPn neurons innervate the parabrachial nucleus (PBn). Spatial and single-nuclei transcriptomics resolved a population of PBn-projecting pyramidal neurons in the DPn that express µ-opioid receptors (µORs). Disrupting µOR signaling in the DPn switched oxycodone from rewarding to aversive and exacerbated the severity of opioid withdrawal. These findings identify the DPn as a key substrate for the abuse liability of opioids.

Activation of CGRP neurons in the parabrachial nucleus suppresses addictive behavior.

Punishment such as electric shock or physical discipline employs a mixture of physical pain and emotional distress to induce behavior modification. However, a neural circuit that produces behavior modification by selectively focusing the emotional component, while bypassing the pain typically induced by peripheral nociceptor activation, is not well studied. Here, we show that genetically silencing the activity of neurons expressing calcitonin gene-related peptide (CGRP) in the parabrachial nucleus blocks the suppression of addictive-like behavior induced by footshock. Furthermore, activating CGRP neurons suppresses not only addictive behavior induced by self-stimulating dopamine neurons but also behavior resulting from self-administering cocaine, without eliciting nocifensive reactions. Moreover, among multiple downstream targets of CGRP neurons, terminal activation of CGRP in the central amygdala is effective, mimicking the results of cell body stimulation. Our results indicate that unlike conventional electric footshock, stimulation of CGRP neurons does not activate peripheral nociceptors but effectively curb addictive behavior.

A visual circuit related to the parabrachial nucleus for the antipruritic effects of bright light treatment.

In addition to its role in vision, light also serves non-image-forming visual functions. Despite clinical evidence suggesting the antipruritic effects of bright light treatment, the circuit mechanisms underlying the effects of light on itch-related behaviors remain poorly understood. In this study, we demonstrate that bright light treatment reduces itch-related behaviors in mice through a visual circuit related to the lateral parabrachial nucleus (LPBN). Specifically, a subset of retinal ganglion cells (RGCs) innervates GABAergic neurons in the ventral lateral geniculate nucleus and intergeniculate leaflet (vLGN/IGL), which subsequently inhibit CaMKIIα+ neurons in the LPBN. Activation of both the vLGN/IGL-projecting RGCs and the vLGN/IGL-to-LPBN projections is sufficient to reduce itch-related behaviors induced by various pruritogens. Importantly, we demonstrate that the antipruritic effects of bright light treatment rely on the activation of the retina-vLGN/IGL-LPBN pathway. Collectively, our findings elucidate a visual circuit related to the LPBN that underlies the antipruritic effects of bright light treatment.

Lateral parabrachial nucleus astrocytes control food intake.

Food intake behavior is under the tight control of the central nervous system. Most studies to date focus on the contribution of neurons to this behavior. However, although previously overlooked, astrocytes have recently been implicated to play a key role in feeding control. Most of the recent literature has focused on astrocytic contribution in the hypothalamus or the dorsal vagal complex. The contribution of astrocytes located in the lateral parabrachial nucleus (lPBN) to feeding behavior control remains poorly understood. Thus, here, we first investigated whether activation of lPBN astrocytes affects feeding behavior in male and female rats using chemogenetic activation. Astrocytic activation in the lPBN led to profound anorexia in both sexes, under both ad-libitum feeding schedule and after a fasting challenge. Astrocytes have a key contribution to glutamate homeostasis and can themselves release glutamate. Moreover, lPBN glutamate signaling is a key contributor to potent anorexia, which can be induced by lPBN activation. Thus, here, we determined whether glutamate signaling is necessary for lPBN astrocyte activation-induced anorexia, and found that pharmacological N-methyl D-aspartate (NMDA) receptor blockade attenuated the food intake reduction resulting from lPBN astrocyte activation. Since astrocytes have been shown to contribute to feeding control by modulating the feeding effect of peripheral feeding signals, we further investigated whether lPBN astrocyte activation is capable of modulating the anorexic effect of the gut/brain hormone, glucagon like peptide -1, as well as the orexigenic effect of the stomach hormone - ghrelin, and found that the feeding effect of both signals is modulated by lPBN astrocytic activation. Lastly, we found that lPBN astrocyte activation-induced anorexia is affected by a diet-induced obesity challenge, in a sex-divergent manner. Collectively, current findings uncover a novel role for lPBN astrocytes in feeding behavior control.

Lateral parabrachial FoxP2 neurons regulate respiratory responses to hypercapnia.

About half of the neurons in the parabrachial nucleus (PB) that are activated by CO2 are located in the external lateral (el) subnucleus, express calcitonin gene-related peptide (CGRP), and cause forebrain arousal. We report here, in male mice, that most of the remaining CO2-responsive neurons in the adjacent central lateral (PBcl) and Kölliker-Fuse (KF) PB subnuclei express the transcription factor FoxP2 and many of these neurons project to respiratory sites in the medulla. PBclFoxP2 neurons show increased intracellular calcium during wakefulness and REM sleep and in response to elevated CO2 during NREM sleep. Photo-activation of the PBclFoxP2 neurons increases respiration, whereas either photo-inhibition of PBclFoxP2 or genetic deletion of PB/KFFoxP2 neurons reduces the respiratory response to CO2 stimulation without preventing awakening. Thus, augmenting the PBcl/KFFoxP2 response to CO2 in patients with sleep apnea in combination with inhibition of the PBelCGRP neurons may avoid hypoventilation and minimize EEG arousals.

Functional dissection of parabrachial substrates in processing nociceptive information.

Painful stimuli elicit first-line reflexive defensive reactions and, in many cases, also evoke second-line recuperative behaviors, the latter of which reflects the sensing of tissue damage and the alleviation of suffering. The lateral parabrachial nucleus (lPBN), composed of external- (elPBN), dorsal- (dlPBN), and central/superior-subnuclei (jointly referred to as slPBN), receives sensory inputs from spinal projection neurons and plays important roles in processing affective information from external threats and body integrity disruption. However, the organizational rules of lPBN neurons that provoke diverse behaviors in response to different painful stimuli from cutaneous and deep tissues remain unclear. In this study, we used region-specific neuronal depletion or silencing approaches combined with a battery of behavioral assays to show that slPBN neurons expressing substance P receptor ( NK1R) (lPBN NK1R) are crucial for driving pain-associated self-care behaviors evoked by sustained noxious thermal and mechanical stimuli applied to skin or bone/muscle, while elPBN neurons are dispensable for driving such reactions. Notably, lPBN NK1R neurons are specifically required for forming sustained somatic pain-induced negative teaching signals and aversive memory but are not necessary for fear-learning or escape behaviors elicited by external threats. Lastly, both lPBN NK1R and elPBN neurons contribute to chemical irritant-induced nocifensive reactions. Our results reveal the functional organization of parabrachial substrates that drive distinct behavioral outcomes in response to sustained pain versus external danger under physiological conditions.

Parabrachial Calca neurons drive nociplasticity.

Pain that persists beyond the time required for tissue healing and pain that arises in the absence of tissue injury, collectively referred to as nociplastic pain, are poorly understood phenomena mediated by plasticity within the central nervous system. The parabrachial nucleus (PBN) is a hub that relays aversive sensory information and appears to play a role in nociplasticity. Here, by preventing PBN Calca neurons from releasing neurotransmitters, we demonstrate that activation of Calca neurons is necessary for the manifestation and maintenance of chronic pain. Additionally, by directly stimulating Calca neurons, we demonstrate that Calca neuron activity is sufficient to drive nociplasticity. Aversive stimuli of multiple sensory modalities, such as exposure to nitroglycerin, cisplatin, or lithium chloride, can drive nociplasticity in a Calca-neuron-dependent manner. Aversive events drive nociplasticity in Calca neurons in the form of increased activity and excitability; however, neuroplasticity also appears to occur in downstream circuitry.

A spatially-resolved transcriptional atlas of the murine dorsal pons at single-cell resolution.

The "dorsal pons", or "dorsal pontine tegmentum" (dPnTg), is part of the brainstem. It is a complex, densely packed region whose nuclei are involved in regulating many vital functions. Notable among them are the parabrachial nucleus, the Kölliker Fuse, the Barrington nucleus, the locus coeruleus, and the dorsal, laterodorsal, and ventral tegmental nuclei. In this study, we applied single-nucleus RNA-seq (snRNA-seq) to resolve neuronal subtypes based on their unique transcriptional profiles and then used multiplexed error robust fluorescence in situ hybridization (MERFISH) to map them spatially. We sampled ~1 million cells across the dPnTg and defined the spatial distribution of over 120 neuronal subtypes. Our analysis identified an unpredicted high transcriptional diversity in this region and pinpointed the unique marker genes of many neuronal subtypes. We also demonstrated that many neuronal subtypes are transcriptionally similar between humans and mice, enhancing this study's translational value. Finally, we developed a freely accessible, GPU and CPU-powered dashboard ( http://harvard.heavy.ai:6273/ ) that combines interactive visual analytics and hardware-accelerated SQL into a data science framework to allow the scientific community to query and gain insights into the data.

A brainstem-hypothalamus neuronal circuit reduces feeding upon heat exposure.

Empirical evidence suggests that heat exposure reduces food intake. However, the neurocircuit architecture and the signalling mechanisms that form an associative interface between sensory and metabolic modalities remain unknown, despite primary thermoceptive neurons in the pontine parabrachial nucleus becoming well characterized1. Tanycytes are a specialized cell type along the wall of the third ventricle2 that bidirectionally transport hormones and signalling molecules between the brain's parenchyma and ventricular system3-8. Here we show that tanycytes are activated upon acute thermal challenge and are necessary to reduce food intake afterwards. Virus-mediated gene manipulation and circuit mapping showed that thermosensing glutamatergic neurons of the parabrachial nucleus innervate tanycytes either directly or through second-order hypothalamic neurons. Heat-dependent Fos expression in tanycytes suggested their ability to produce signalling molecules, including vascular endothelial growth factor A (VEGFA). Instead of discharging VEGFA into the cerebrospinal fluid for a systemic effect, VEGFA was released along the parenchymal processes of tanycytes in the arcuate nucleus. VEGFA then increased the spike threshold of Flt1-expressing dopamine and agouti-related peptide (Agrp)-containing neurons, thus priming net anorexigenic output. Indeed, both acute heat and the chemogenetic activation of glutamatergic parabrachial neurons at thermoneutrality reduced food intake for hours, in a manner that is sensitive to both Vegfa loss-of-function and blockage of vesicle-associated membrane protein 2 (VAMP2)-dependent exocytosis from tanycytes. Overall, we define a multimodal neurocircuit in which tanycytes link parabrachial sensory relay to the long-term enforcement of a metabolic code.

Oxytocin-receptor-expressing neurons in the lateral parabrachial nucleus activate widespread brain regions predominantly involved in fluid satiation.

Fluid satiation is an important signal and aspect of body fluid homeostasis. Oxytocin-receptor-expressing neurons (OxtrPBN) in the dorsolateral subdivision of the lateral parabrachial nucleus (dl LPBN) are key neurons which regulate fluid satiation. In the present study, we investigated brain regions activated by stimulation of OxtrPBN neurons in order to better characterise the fluid satiation neurocircuitry in mice. Chemogenetic activation of OxtrPBN neurons increased Fos expression (a proxy marker for neuronal activation) in known fluid-regulating brain nuclei, as well as other regions that have unclear links to fluid regulation and which are likely involved in regulating other functions such as arousal and stress relief. In addition, we analysed and compared Fos expression patterns between chemogenetically-activated fluid satiation and physiological-induced fluid satiation. Both models of fluid satiation activated similar brain regions, suggesting that the chemogenetic model of stimulating OxtrPBN neurons is a relevant model of physiological fluid satiation. A deeper understanding of this neural circuit may lead to novel molecular targets and creation of therapeutic agents to treat fluid-related disorders.

The functional and anatomical characterization of three spinal output pathways of the anterolateral tract.

The nature of spinal output pathways that convey nociceptive information to the brain has been the subject of controversy. Here, we provide anatomical, molecular, and functional characterizations of two distinct anterolateral pathways: one, ascending in the lateral spinal cord, triggers nociceptive behaviors, and the other one, ascending in the ventral spinal cord, when inhibited, leads to sensorimotor deficits. Moreover, the lateral pathway consists of at least two subtypes. The first is a contralateral pathway that extends to the periaqueductal gray (PAG) and thalamus; the second is a bilateral pathway that projects to the bilateral parabrachial nucleus (PBN). Finally, we present evidence showing that activation of the contralateral pathway is sufficient for defensive behaviors such as running and freezing, whereas the bilateral pathway is sufficient for attending behaviors such as licking and guarding. This work offers insight into the complex organizational logic of the anterolateral system in the mouse.

Transient cAMP production drives rapid and sustained spiking in brainstem parabrachial neurons to suppress feeding.

Brief stimuli can trigger longer-lasting brain states. G-protein-coupled receptors (GPCRs) could help sustain such states by coupling slow-timescale molecular signals to neuronal excitability. Brainstem parabrachial nucleus glutamatergic (PBNGlut) neurons regulate sustained brain states such as pain and express Gs-coupled GPCRs that increase cAMP signaling. We asked whether cAMP in PBNGlut neurons directly influences their excitability and effects on behavior. Both brief tail shocks and brief optogenetic stimulation of cAMP production in PBNGlut neurons drove minutes-long suppression of feeding. This suppression matched the duration of prolonged elevations in cAMP, protein kinase A (PKA) activity, and calcium activity in vivo and ex vivo, as well as sustained, PKA-dependent increases in action potential firing ex vivo. Shortening this elevation in cAMP reduced the duration of feeding suppression following tail shocks. Thus, molecular signaling in PBNGlut neurons helps prolong neural activity and behavioral states evoked by brief, salient bodily stimuli.

Sex-specific negative affect-like behaviour and parabrachial nucleus activation induced by BNST stimulation in adult mice with adolescent alcohol history.

Adolescent alcohol use is a strong predictor for the subsequent development of alcohol use disorders later in life. Additionally, adolescence is a critical period for the onset of affective disorders, which can contribute to problematic drinking behaviours and relapse, particularly in females. Previous studies from our laboratory have shown that exposure to adolescent intermittent ethanol (AIE) vapour alters glutamatergic transmission in the bed nucleus of the stria terminalis (BNST) and, when combined with adult stress, elicits sex-specific changes in glutamatergic plasticity and negative affect-like behaviours in mice. Building on these findings, the current work investigated whether BNST stimulation could substitute for stress exposure to increase the latency to consume a palatable food in a novel context (hyponeophagia) and promote social avoidance in adult mice with AIE history. Given the dense connections between the BNST and the parabrachial nucleus (PBN), a region involved in mediating threat assessment and feeding behaviours, we hypothesized that increased negative affect-like behaviours would be associated with PBN activation. Our results revealed that the chemogenetic stimulation of the dorsolateral BNST induced hyponeophagia in females with AIE history, but not in female controls or males of either group. Social interaction remained unaffected in both sexes. Notably, this behavioural phenotype was associated with higher activation of calcitonin gene-related peptide and dynorphin cells in the PBN. These findings provide new insights into the neurobiological mechanisms underlying the development of negative affect in females and highlight the potential involvement of the BNST-PBN circuitry in regulating emotional responses to alcohol-related stimuli.

Pontine parabrachial nucleus-basal forebrain circuitry regulating cortical and hippocampal arousal.

INTRODUCTION: The basal forebrain (BF) and the medial septum (MS) respectively drive neuronal activity of cerebral cortex and hippocampus (HPC) in sleep-wake cycle. Our previous studies of lesions and neuronal circuit tracing have shown that the pontine parabrachial nucleus (PB) projections to the BF and MS may be a key circuit for cortical and HPC arousal. AIMS: This study aims to demonstrate that PB projections to the BF and MS activate the cerebral cortex and HPC. RESULTS: By using chemogenetic stimulation of the BF, the PB-BF and the PB-MS pathway combined with electroencephalogram (EEG) Fast Fourier Transformation (FFT) analysis in rats, we demonstrated that chemogenetic stimulation of the BF or PB neurons projecting to the BF activated the cerebral cortex while chemogenetic stimulation of the MS or PB neurons projecting to the MS activated HPC activity, in sleep and wake state. These stimulations did not significantly alter sleep-wake amounts. CONCLUSIONS: Our results support that PB projections to the BF and MS specifically regulating cortical and HPC activity.

The Lateral Parabrachial Nucleus Inputs to the Lateral Hypothalamus Trigger Nocifensive Behaviors.

The lateral parabrachial nucleus (LPBN) is known to play a key role in relaying noxious information from the spinal cord to the brain. Different LPBN efferent mediate different aspects of the nocifensive response. However, the function of the LPBN â†’ lateral hypothalamus (LH) circuit in response to noxious stimuli has remained unknown. Here, we show that LPBN â†’ LH circuit is activated by noxious stimuli. Interestingly, either activation or inhibition of this circuit induced analgesia. Optogenetic activation of LPBN afferents in the LH elicited spontaneous jumping and induced place aversion. Optogenetic inhibition inhibited jumping behavior to noxious heat. Ablation of LH glutamatergic neurons could abolish light-evoked analgesia and jumping behavior. Our study revealed a role for the LPBN â†’ LH pathway in nocifensive behaviors.

The parabrachial to central amygdala pathway is critical to injury-induced pain sensitization in mice.

The spino-ponto-amygdaloid pathway is a major ascending circuit relaying nociceptive information from the spinal cord to the brain. Potentiation of excitatory synaptic transmission in the parabrachial nucleus (PBN) to central amygdala (CeA) pathway has been reported in rodent models of persistent pain. However, the functional significance of this pathway in the modulation of the somatosensory component of pain was recently challenged by studies showing that spinal nociceptive neurons do not target CeA-projecting PBN cells and that manipulations of this pathway have no effect on reflexive-defensive somatosensory responses to peripheral noxious stimulation. Here, we showed that activation of CeA-projecting PBN neurons is critical to increase both stimulus-evoked and spontaneous nociceptive responses following an injury in male and female mice. Using optogenetic-assisted circuit mapping, we confirmed a functional excitatory projection from PBN→CeA that is independent of the genetic or firing identity of CeA cells. We then showed that peripheral noxious stimulation increased the expression of the neuronal activity marker Fos in CeA-projecting PBN neurons and that chemogenetic inactivation of these cells decreased behavioral hypersensitivity in models of neuropathic and inflammatory pain without affecting baseline nociception. Lastly, we showed that chemogenetic activation of CeA-projecting PBN neurons is sufficient to induced bilateral hypersensitivity without injury. Together, our results indicate that the PBN→CeA pathway is a key modulator of pain-related behaviors that can increase reflexive-defensive and affective-motivational responses to somatosensory stimulation in injured states without affecting nociception under normal physiological conditions.

Excitatory neurons in the lateral parabrachial nucleus mediate the interruptive effect of inflammatory pain on a sustained attention task.

BACKGROUND: Attentional deficits are among the most common pain-induced cognitive disorders. Pain disrupts attention and may excessively occupy attentional resources in pathological states, leading to daily function impairment and increased disability. However, the neural circuit mechanisms by which pain disrupts attention are incompletely understood. METHODS: We used a three-choice serial reaction time task (3CSRTT) to construct a sustained-attention task model in male C57BL/6J mice. Formalin or complete Freund's adjuvant was injected into a paw to establish an inflammatory pain model. We measured changes in 3CSRTT performance in the two inflammatory pain models, and investigated the neural circuit mechanisms of pain-induced attentional deficits. RESULTS: Acute inflammatory pain impaired 3CSRTT performance, while chronic inflammatory pain had no effect. Either inhibition of the ascending pain pathway by blockade of the conduction of nociceptive signals in the sciatic nerve using the local anesthetic lidocaine or chemogenetic inhibition of Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) neurons in the lateral parabrachial nucleus (LPBN) attenuated the acute inflammatory pain-induced impairment of 3CSRTT performance, while chemogenetic activation of CaMKIIα neurons in the LPBN disrupted the 3CSRTT. Furthermore, the activity of CaMKIIα neurons in the LPBN was significantly lower on Day 2 after complete Freund's adjuvant injection than on the day of injection, which correlated with the recovery of 3CSRTT performance during chronic inflammatory pain. CONCLUSIONS: Activation of excitatory neurons in the LPBN is a mechanism by which acute inflammatory pain disrupts sustained attention. This finding has implications for the treatment of pain and its cognitive comorbidities.

State-dependent modulation of positive and negative affective valences by a parabrachial nucleus-to-ventral tegmental area pathway in mice.

Appropriately responding to various sensory signals in the environment is essential for animal survival. Accordingly, animal behaviors are closely related to external and internal states, which include the positive and negative emotional values of sensory signals triggered by environmental factors. While the lateral parabrachial nucleus (LPB) plays a key role in nociception and supports negative valences, it also transmits signals including positive valences. However, the downstream neuronal mechanisms of positive and negative valences have not been fully explored. In the present study, we investigated the ventral tegmental area (VTA) as a projection target for LPB neurons. Optogenetic activation of LPB-VTA terminals in male mice elicits positive reinforcement in an operant task and induces both avoidance and attraction in a place-conditioning task. Inhibition of glutamic acid decarboxylase (GAD) 65-expressing cells in the VTA promotes avoidance behavior induced by photoactivation of the LPB-VTA pathway. These findings indicate that the LPB-VTA pathway is one of the LPB outputs for the transmission of positive and negative valence signals, at least in part, with GABAergic modification in VTA.

A Purkinje cell to parabrachial nucleus pathway enables broad cerebellar influence over the forebrain.

In addition to its motor functions, the cerebellum is involved in emotional regulation, anxiety and affect. We found that suppressing the firing of cerebellar Purkinje cells (PCs) rapidly excites forebrain areas that contribute to such functions (including the amygdala, basal forebrain and septum), but that the classic cerebellar outputs, the deep cerebellar nuclei, do not directly project there. We show that PCs directly inhibit parabrachial nuclei (PBN) neurons that project to numerous forebrain regions. Suppressing the PC-PBN pathway influences many regions in the forebrain and is aversive. Molecular profiling shows that PCs directly inhibit numerous types of PBN neurons that control diverse behaviors that are not involved in motor control. Therefore, the PC-PBN pathway allows the cerebellum to directly regulate activity in the forebrain, and may be an important substrate for cerebellar disorders arising from damage to the posterior vermis.

Selective optogenetic modulation of the PBN terminals in the lateral hypothalamic area and basal forebrain regulates emergence from isoflurane anesthesia in mice.

While the mechanism of general anesthesia has been extensively studied, the underlying neural circuitry has yet to be fully understood. The parabrachial nucleus (PBN) plays a crucial role in modulating wakefulness and promoting arousal from general anesthesia. However, the specific role of PBN projections in the process of general anesthesia remains unclear. In this study, we bilaterally injected AAV-associated viruses encoding excitatory or inhibitory optogenetic probes into the PBN and implanted optical fibers in the LH or BF area. After four weeks, we optogenetically activated or inhibited the PBN-LH and PBN-BF pathways under 1.5 vol% isoflurane. We calculated the time it took for anesthesia induction and emergence, simultaneously monitoring changes in the burst-suppression ratio using electroencephalogram recording. Our findings indicate that optogenetic activation of the PBN-LH and PBN-BF projections plays a significant role in promoting both cortical and behavioral emergence from isoflurane inhalation, without significantly affecting the induction time. Conversely, photoinhibition of these pathways prolonged the recovery time, with no notable difference observed during the induction phase.In summary, our results demonstrate that the PBN-LH and PBN-BF pathways are crucial for promoting arousal from isoflurane general anesthesia, but do not have a pronounced impact on the induction phase.