Development of a genetically encoded sensor for probing endogenous nociceptin opioid peptide release.

Nociceptin/orphanin-FQ (N/OFQ) is a recently appreciated critical opioid peptide with key regulatory functions in several central behavioral processes including motivation, stress, feeding, and sleep. The functional relevance of N/OFQ action in the mammalian brain remains unclear due to a lack of high-resolution approaches to detect this neuropeptide with appropriate spatial and temporal resolution. Here we develop and characterize NOPLight, a genetically encoded sensor that sensitively reports changes in endogenous N/OFQ release. We characterized the affinity, pharmacological profile, spectral properties, kinetics, ligand selectivity, and potential interaction with intracellular signal transducers of NOPLight in vitro. Its functionality was established in acute brain slices by exogeneous N/OFQ application and chemogenetic induction of endogenous N/OFQ release from PNOC neurons. In vivo studies with fibre photometry enabled direct recording of NOPLight binding to exogenous N/OFQ receptor ligands, as well as detection of endogenous N/OFQ release within the paranigral ventral tegmental area (pnVTA) during natural behaviors and chemogenetic activation of PNOC neurons. In summary, we show here that NOPLight can be used to detect N/OFQ opioid peptide signal dynamics in tissue and freely behaving animals.

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 cluster of neuropeptide S neurons regulates breathing and arousal.

Neuropeptide S (NPS) is a highly conserved peptide found in all tetrapods that functions in the brain to promote heightened arousal; however, the subpopulations mediating these phenomena remain unknown. We generated mice expressing Cre recombinase from the Nps gene locus (NpsCre) and examined populations of NPS+ neurons in the lateral parabrachial area (LPBA), the peri-locus coeruleus (peri-LC) region of the pons, and the dorsomedial thalamus (DMT). We performed brain-wide mapping of input and output regions of NPS+ clusters and characterized expression patterns of the NPS receptor 1 (NPSR1). While the activity of all three NPS+ subpopulations tracked with vigilance state, only NPS+ neurons of the LPBA exhibited both increased activity prior to wakefulness and decreased activity during REM sleep, similar to the behavioral phenotype observed upon NPSR1 activation. Accordingly, we found that activation of the LPBA but not the peri-LC NPS+ neurons increased wake and reduced REM sleep. Furthermore, given the extended role of the LPBA in respiration and the link between behavioral arousal and breathing rate, we demonstrated that the LPBA but not the peri-LC NPS+ neuronal activation increased respiratory rate. Together, our data suggest that NPS+ neurons of the LPBA represent an unexplored subpopulation regulating breathing, and they are sufficient to recapitulate the sleep/wake phenotypes observed with broad NPS system activation.

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 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 neurotransmitter or directly stimulating them we demonstrate that activation of Calca neurons is both necessary for the manifestation of chronic pain after nerve ligation and is sufficient to drive nociplasticity in wild-type mice. Aversive stimuli such as exposure to nitroglycerin, cisplatin, or LiCl can drive nociplasticity in a Calca-neuron-dependent manner. Calcium fluorescence imaging reveals that nitroglycerin activates PBN Calca neurons and potentiates their responses to mechanical stimulation. The activity and excitability of Calca neurons increased for several days after aversive events, but prolonged nociplasticity likely occurs in downstream circuitry.

Parallel neural pathways control sodium consumption and taste valence.

The hedonic value of salt fundamentally changes depending on the internal state. High concentrations of salt induce innate aversion under sated states, whereas such aversive stimuli transform into appetitive ones under sodium depletion. Neural mechanisms underlying this state-dependent salt valence switch are poorly understood. Using transcriptomics state-to-cell-type mapping and neural manipulations, we show that positive and negative valences of salt are controlled by anatomically distinct neural circuits in the mammalian brain. The hindbrain interoceptive circuit regulates sodium-specific appetitive drive , whereas behavioral tolerance of aversive salts is encoded by a dedicated class of neurons in the forebrain lamina terminalis (LT) expressing prostaglandin E2 (PGE2) receptor, Ptger3. We show that these LT neurons regulate salt tolerance by selectively modulating aversive taste sensitivity, partly through a PGE2-Ptger3 axis. These results reveal the bimodal regulation of appetitive and tolerance signals toward salt, which together dictate the amount of sodium consumption under different internal states.

Vestibular CCK signaling drives motion sickness-like behavior in mice.

Travel can induce motion sickness (MS) in susceptible individuals. MS is an evolutionary conserved mechanism caused by mismatches between motion-related sensory information and past visual and motion memory, triggering a malaise accompanied by hypolocomotion, hypothermia, hypophagia, and nausea. Vestibular nuclei (VN) are critical for the processing of movement input from the inner ear. Motion-induced activation of VN neurons recapitulates MS-related signs. However, the genetic identity of VN neurons mediating MS-related autonomic and aversive responses remains unknown. Here, we identify a central role of cholecystokinin (CCK)-expressing VN neurons in motion-induced malaise. Moreover, we show that CCK VN inputs onto the parabrachial nucleus activate Calca-expressing neurons and are sufficient to establish avoidance to novel food, which is prevented by CCK-A receptor antagonism. These observations provide greater insight into the neurobiological regulation of MS by identifying the neural substrates of MS and providing potential targets for treatment.

PD-L1/PD-1 checkpoint pathway regulates hippocampal neuronal excitability and learning and memory behavior.

Programmed death protein 1 (PD-1) and its ligand PD-L1 constitute an immune checkpoint pathway. We report that neuronal PD-1 signaling regulates learning/memory in health and disease. Mice lacking PD-1 (encoded by Pdcd1) exhibit enhanced long-term potentiation (LTP) and memory. Intraventricular administration of anti-mouse PD-1 monoclonal antibody (RMP1-14) potentiated learning and memory. Selective deletion of PD-1 in excitatory neurons (but not microglia) also enhances LTP and memory. Traumatic brain injury (TBI) impairs learning and memory, which is rescued by Pdcd1 deletion or intraventricular PD-1 blockade. Conversely, re-expression of Pdcd1 in PD-1-deficient hippocampal neurons suppresses memory and LTP. Exogenous PD-L1 suppresses learning/memory in mice and the excitability of mouse and NHP hippocampal neurons through PD-1. Notably, neuronal activation suppresses PD-L1 secretion, and PD-L1/PD-1 signaling is distinctly regulated by learning and TBI. Thus, conditions that reduce PD-L1 levels or PD-1 signaling could promote memory in both physiological and pathological conditions.

Prolonged activation of EP3 receptor-expressing preoptic neurons underlies torpor responses.

Many species use a temporary drop in body temperature and metabolic rate (torpor) as a strategy to survive food scarcity. A similar profound hypothermia is observed with activation of preoptic neurons that express the neuropeptides Pituitary Adenylate-Cyclase-Activating Polypeptide (PACAP)1, Brain Derived Neurotrophic Factor (BDNF)2, or Pyroglutamylated RFamide Peptide (QRFP)3, the vesicular glutamate transporter, Vglut24,5 or the leptin receptor6 (LepR), estrogen 1 receptor (Esr1)7 or prostaglandin E receptor 3 (EP3R) in mice8. However, most of these genetic markers are found on multiple populations of preoptic neurons and only partially overlap with one another. We report here that expression of the EP3R marks a unique population of median preoptic (MnPO) neurons that are required both for lipopolysaccharide (LPS)-induced fever9 and for torpor. These MnPOEP3R neurons produce persistent fever responses when inhibited and prolonged hypothermic responses when activated either chemo- or opto-genetically even for brief periods of time. The mechanism for these prolonged responses appears to involve increases in intracellular calcium in individual EP3R-expressing preoptic neurons that persist for many minutes up to hours beyond the termination of a brief stimulus. These properties endow MnPOEP3R neurons with the ability to act as a two-way master switch for thermoregulation.

CPT1A in AgRP neurons is required for sex-dependent regulation of feeding and thirst.

BACKGROUND: Fatty acid metabolism in the hypothalamus has an important role in food intake, but its specific role in AgRP neurons is poorly understood. Here, we examined whether carnitinea palmitoyltransferase 1A (CPT1A), a key enzyme in mitochondrial fatty acid oxidation, affects energy balance. METHODS: To obtain Cpt1aKO mice and their control littermates, Cpt1a(flox/flox) mice were crossed with tamoxifen-inducible AgRPCreERT2 mice. Food intake and body weight were analyzed weekly in both males and females. At 12 weeks of age, metabolic flexibility was determined by ghrelin-induced food intake and fasting-refeeding satiety tests. Energy expenditure was analyzed by calorimetric system and thermogenic activity of brown adipose tissue. To study fluid balance the analysis of urine and water intake volumes; osmolality of urine and plasma; as well as serum levels of angiotensin and components of RAAS (renin-angiotensin-aldosterone system) were measured. At the central level, changes in AgRP neurons were determined by: (1) analyzing specific AgRP gene expression in RiboTag-Cpt1aKO mice obtained by crossing Cpt1aKO mice with RiboTag mice; (2) measuring presynaptic terminal formation in the AgRP neurons with the injection of the AAV1-EF1a-DIO-synaptophysin-GFP in the arcuate nucleus of the hypothalamus; (3) analyzing AgRP neuronal viability and spine formations by the injection AAV9-EF1a-DIO-mCherry in the arcuate nucleus of the hypothalamus; (4) analyzing in situ the specific AgRP mitochondria in the ZsGreen-Cpt1aKO obtained by breeding ZsGreen mice with Cpt1aKO mice. Two-way ANOVA analyses were performed to determine the contributions of the effect of lack of CPT1A in AgRP neurons in the sex. RESULTS: Changes in food intake were just seen in male Cpt1aKO mice while only female Cpt1aKO mice increased energy expenditure. The lack of Cpt1a in the AgRP neurons enhanced brown adipose tissue activity, mainly in females, and induced a substantial reduction in fat deposits and body weight. Strikingly, both male and female Cpt1aKO mice showed polydipsia and polyuria, with more reduced serum vasopressin levels in females and without osmolality alterations, indicating a direct involvement of Cpt1a in AgRP neurons in fluid balance. AgRP neurons from Cpt1aKO mice showed a sex-dependent gene expression pattern, reduced mitochondria and decreased presynaptic innervation to the paraventricular nucleus, without neuronal viability alterations. CONCLUSIONS: Our results highlight that fatty acid metabolism and CPT1A in AgRP neurons show marked sex differences and play a relevant role in the neuronal processes necessary for the maintenance of whole-body fluid and energy balance.

Activation of Parabrachial Tachykinin 1 Neurons Counteracts Some Behaviors Mediated by Parabrachial Calcitonin Gene-related Peptide Neurons.

Many threats activate parabrachial neurons expressing calcitonin gene-related peptide (CGRPPBN) which transmit alarm signals to forebrain regions. Most CGRPPBN neurons also express tachykinin 1 (Tac1), but there are also Tac1-expressing neurons in the PBN that do not express CGRP (Tac1+;CGRP- neurons). Chemogenetic or optogenetic activation of all Tac1PBN neurons in mice elicited many physiological/behavioral responses resembling the activation of CGRPPBN neurons, e.g., anorexia, jumping on a hot plate, avoidance of photostimulation; however, two key responses opposed activation of CGRPPBN neurons. Activating Tac1PBN neurons did not produce conditioned taste aversion and it elicited dynamic escape behaviors rather than freezing. Activating Tac1+;CGRP- neurons, using an intersectional genetic targeting approach, resembles activating all Tac1PBN neurons. These results reveal that activation of Tac1+;CGRP- neurons can suppress some functions attributed to the CGRPPBN neurons, which provides a mechanism to bias behavioral responses to threats.

Parabrachial tachykinin1-expressing neurons involved in state-dependent breathing control.

Breathing is regulated automatically by neural circuits in the medulla to maintain homeostasis, but breathing is also modified by behavior and emotion. Mice have rapid breathing patterns that are unique to the awake state and distinct from those driven by automatic reflexes. Activation of medullary neurons that control automatic breathing does not reproduce these rapid breathing patterns. By manipulating transcriptionally defined neurons in the parabrachial nucleus, we identify a subset of neurons that express the Tac1, but not Calca, gene that exerts potent and precise conditional control of breathing in the awake, but not anesthetized, state via projections to the ventral intermediate reticular zone of the medulla. Activating these neurons drives breathing to frequencies that match the physiological maximum through mechanisms that differ from those that underlie the automatic control of breathing. We postulate that this circuit is important for the integration of breathing with state-dependent behaviors and emotions.

Novel genetically encoded tools for imaging or silencing neuropeptide release from presynaptic terminals in vivo.

Neurons produce and release neuropeptides to communicate with one another. Despite their profound impact on critical brain functions, circuit-based mechanisms of peptidergic transmission are poorly understood, primarily due to the lack of tools for monitoring and manipulating neuropeptide release in vivo. Here, we report the development of two genetically encoded tools for investigating peptidergic transmission in behaving mice: a genetically encoded large dense core vesicle (LDCV) sensor that detects the neuropeptides release presynaptically, and a genetically encoded silencer that specifically degrades neuropeptides inside the LDCV. Monitoring and silencing peptidergic and glutamatergic transmissions from presynaptic terminals using our newly developed tools and existing genetic tools, respectively, reveal that neuropeptides, not glutamate, are the primary transmitter in encoding unconditioned stimulus during Pavlovian threat learning. These results show that our sensor and silencer for peptidergic transmission are reliable tools to investigate neuropeptidergic systems in awake behaving animals.

Topographic representation of current and future threats in the mouse nociceptive amygdala.

Adaptive behaviors arise from an integration of current sensory context and internal representations of past experiences. The central amygdala (CeA) is positioned as a key integrator of cognitive and affective signals, yet it remains unknown whether individual populations simultaneously carry current- and future-state representations. We find that a primary nociceptive population within the CeA of mice, defined by CGRP-receptor (Calcrl) expression, receives topographic sensory information, with spatially defined representations of internal and external stimuli. While Calcrl+ neurons in both the rostral and caudal CeA respond to noxious stimuli, rostral neurons promote locomotor responses to externally sourced threats, while caudal CeA Calcrl+ neurons are activated by internal threats and promote passive coping behaviors and associative valence coding. During associative fear learning, rostral CeA Calcrl+ neurons stably encode noxious stimulus occurrence, while caudal CeA Calcrl+ neurons acquire predictive responses. This arrangement supports valence-aligned representations of current and future threats for the generation of adaptive behaviors.

A gut-retching discovery.

In this issue of Cell, Xie et al. identify a gut-to-brain pathway that triggers retching after toxic food ingestion or emetic agent administration. Their results shed light on how peripheral signals reach the brain to orchestrate appropriate behavioral responses and facilitate learning to prevent repeated ingestion of harmful substances.

Polony gels enable amplifiable DNA stamping and spatial transcriptomics of chronic pain.

Methods for acquiring spatially resolved omics data from complex tissues use barcoded DNA arrays of low- to sub-micrometer features to achieve single-cell resolution. However, fabricating such arrays (randomly assembled beads, DNA nanoballs, or clusters) requires sequencing barcodes in each array, limiting cost-effectiveness and throughput. Here, we describe a vastly scalable stamping method to fabricate polony gels, arrays of ∼1-micrometer clonal DNA clusters bearing unique barcodes. By enabling repeatable enzymatic replication of barcode-patterned gels, this method, compared with the sequencing-dependent array fabrication, reduced cost by at least 35-fold and time to approximately 7 h. The gel stamping was implemented with a simple robotic arm and off-the-shelf reagents. We leveraged the resolution and RNA capture efficiency of polony gels to develop Pixel-seq, a single-cell spatial transcriptomic assay, and applied it to map the mouse parabrachial nucleus and analyze changes in neuropathic pain-regulated transcriptomes and cell-cell communication after nerve ligation.

Molecular and anatomical characterization of parabrachial neurons and their axonal projections.

The parabrachial nucleus (PBN) is a major hub that receives sensory information from both internal and external environments. Specific populations of PBN neurons are involved in behaviors including food and water intake, nociceptive responses, breathing regulation, as well as learning and responding appropriately to threatening stimuli. However, it is unclear how many PBN neuron populations exist and how different behaviors may be encoded by unique signaling molecules or receptors. Here we provide a repository of data on the molecular identity, spatial location, and projection patterns of dozens of PBN neuron subclusters. Using single-cell RNA sequencing, we identified 21 subclusters of neurons in the PBN and neighboring regions. Multiplexed in situ hybridization showed many of these subclusters are enriched within specific PBN subregions with scattered cells in several other regions. We also provide detailed visualization of the axonal projections from 21 Cre-driver lines of mice. These results are all publicly available for download and provide a foundation for further interrogation of PBN functions and connections.

Control of exploration, motor coordination and amphetamine sensitization by cannabinoid CB1 receptors expressed in medium spiny neurons.

Activation of cannabinoid 1 receptors (CB1 R) modulates multiple behaviours, including exploration, motor coordination and response to psychostimulants. It is known that CB1 R expressed by either excitatory or inhibitory neurons mediates different behavioural responses to CB1 R activation, yet the involvement of CB1 R expressed by medium spiny neurons (MSNs), the neuronal subpopulation that expresses the highest level of CB1 R in the CNS, remains unknown. We report a new genetically modified mouse line that expresses functional CB1 R in MSN on a CB1 R knockout (KO) background (CB1 R(MSN) mice). The absence of cannabimimetic responses measured in CB1 R KO mice was not rescued in CB1 R(MSN) mice, nor was decreased spontaneous locomotion, impaired instrumental behaviour or reduced amphetamine-triggered hyperlocomotion measured in CB1 R KO mice. Significantly, reduced novel environment exploration of an open field and absence of amphetamine sensitization (AS) measured in CB1 R KO mice were fully rescued in CB1 R(MSN) mice. Impaired motor coordination in CB1 R KO mice measured on the Rotarod was partially rescued in CB1 R(MSN) mice. Thus, CB1 R expressed by MSN control exploration, motor coordination, and AS. Our study demonstrates a new functional roles for cell specific CB1 R expression and their causal link in the control of specific behaviors.

Cre Recombinase Driver Mice Reveal Lineage-Dependent and -Independent Expression of Brs3 in the Mouse Brain.

Bombesin receptor subtype-3 (BRS3) is an orphan receptor that regulates energy homeostasis. We compared Brs3 driver mice with constitutive or inducible Cre recombinase activity. The constitutive BRS3-Cre mice show a reporter signal (Cre-dependent tdTomato) in the adult brain because of lineage tracing in the dentate gyrus, striatal patches, and indusium griseum, in addition to sites previously identified in the inducible BRS3-Cre mice (including hypothalamic and amygdala subregions, and parabrachial nucleus). We detected Brs3 reporter expression in the dentate gyrus at day 23 but not at postnatal day 1 or 5 months of age. Hypothalamic sites expressed reporter at all three time points, and striatal patches expressed Brs3 reporter at 1 day but not 5 months. Parabrachial nucleus Brs3 neurons project to the preoptic area, hypothalamus, amygdala, and thalamus. Both Cre recombinase insertions reduced Brs3 mRNA levels and BRS3 function, causing obesity phenotypes of different severity. These results demonstrate that driver mice should be characterized phenotypically and illustrate the need for knock-in strategies with less effect on the endogenous gene.

Neural basis of opioid-induced respiratory depression and its rescue.

Opioid-induced respiratory depression (OIRD) causes death following an opioid overdose, yet the neurobiological mechanisms of this process are not well understood. Here, we show that neurons within the lateral parabrachial nucleus that express the µ-opioid receptor (PBL Oprm1 neurons) are involved in OIRD pathogenesis. PBL Oprm1 neuronal activity is tightly correlated with respiratory rate, and this correlation is abolished following morphine injection. Chemogenetic inactivation of PBL Oprm1 neurons mimics OIRD in mice, whereas their chemogenetic activation following morphine injection rescues respiratory rhythms to baseline levels. We identified several excitatory G protein-coupled receptors expressed by PBL Oprm1 neurons and show that agonists for these receptors restore breathing rates in mice experiencing OIRD. Thus, PBL Oprm1 neurons are critical for OIRD pathogenesis, providing a promising therapeutic target for treating OIRD in patients.

Intercalated amygdala clusters orchestrate a switch in fear state.

Adaptive behaviour necessitates the formation of memories for fearful events, but also that these memories can be extinguished. Effective extinction prevents excessive and persistent reactions to perceived threat, as can occur in anxiety and 'trauma- and stressor-related' disorders1. However, although there is evidence that fear learning and extinction are mediated by distinct neural circuits, the nature of the interaction between these circuits remains poorly understood2-6. Here, through a combination of in vivo calcium imaging, functional manipulations, and slice physiology, we show that distinct inhibitory clusters of intercalated neurons (ITCs) in the mouse amygdala exert diametrically opposed roles during the acquisition and retrieval of fear extinction memory. Furthermore, we find that the ITC clusters antagonize one another through mutual synaptic inhibition and differentially access functionally distinct cortical- and midbrain-projecting amygdala output pathways. Our findings show that the balance of activity between ITC clusters represents a unique regulatory motif that orchestrates a distributed neural circuitry, which in turn regulates the switch between high- and low-fear states. These findings suggest that the ITCs have a broader role in a range of amygdala functions and associated brain states that underpins the capacity to adapt to salient environmental demands.