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.

Feeding signals inhibit fluid-satiation signals in the mouse lateral parabrachial nucleus to increase intake of highly palatable, caloric solutions.

Chemogenetic activation of oxytocin receptor-expressing neurons in the parabrachial nucleus (OxtrPBN neurons) acts as a satiation signal for water. In this research, we investigated the effect of activating OxtrPBN neurons on satiation for different types of fluids. Chemogenetic activation of OxtrPBN neurons in male and female transgenic OxtrCre mice robustly suppressed the rapid, initial (15-min) intake of several solutions after dehydration: water, sucrose, ethanol and saccharin, but only slightly decreased intake of Ensure®, a highly caloric solution (1 kcal/mL; containing 3.72 g protein, 3.27 g fat, 13.42 g carbohydrates, and 1.01 g dietary fibre per 100 mL). OxtrPBN neuron activation also suppressed cumulative, longer-term (2-h) intake of lower caloric, less palatable solutions, but not highly caloric, palatable solutions. These results suggest that OxtrPBN neurons predominantly control initial fluid-satiation responses after rehydration, but not longer-term intake of highly caloric, palatable solutions. The suppression of fluid intake was not because of anxiogenesis, but because OxtrPBN neuron activation decreased anxiety-like behaviour. To investigate the role of different PBN subdivisions on the intake of different solutions, we examined FOS as a proxy marker of PBN neuron activation. Different PBN subdivisions were activated by different solutions: the dorsolateral PBN similarly by all fluids; the external lateral PBN by caloric but not non-caloric solutions; and the central lateral PBN primarily by highly palatable solutions, suggesting PBN subdivisions regulate different aspects of fluid intake. To explore the possible mechanisms underlying the minimal suppression of Ensure® after OxtrPBN neuron activation, we demonstrated in in vitro slice recordings that the feeding-associated agouti-related peptide (AgRP) inhibited OxtrPBN neuron firing in a concentration-related manner, suggesting possible inhibition by feeding-related neurocircuitry of fluid satiation neurocircuitry. Overall, this research suggests that although palatable beverages like sucrose- and ethanol-containing beverages activate fluid satiation signals encoded by OxtrPBN neurons, these neurons can be inhibited by hunger-related signals (agouti-related peptide, AgRP), which may explain why these fluids are often consumed in excess of what is required for fluid satiation.

Sodium Leak Channel in Glutamatergic Neurons of the Lateral Parabrachial Nucleus Modulates Inflammatory Pain in Mice.

Elevated excitability of glutamatergic neurons in the lateral parabrachial nucleus (PBL) is associated with the pathogenesis of inflammatory pain, but the underlying molecular mechanisms are not fully understood. Sodium leak channel (NALCN) is widely expressed in the central nervous system and regulates neuronal excitability. In this study, chemogenetic manipulation was used to explore the association between the activity of PBL glutamatergic neurons and pain thresholds. Complete Freund's adjuvant (CFA) was used to construct an inflammatory pain model in mice. Pain behaviour was tested using von Frey filaments and Hargreaves tests. Local field potential (LFP) was used to record the activity of PBL glutamatergic neurons. Gene knockdown techniques were used to investigate the role of NALCN in inflammatory pain. We further explored the downstream projections of PBL using cis-trans-synaptic tracer virus. The results showed that chemogenetic inhibition of PBL glutamatergic neurons increased pain thresholds in mice, whereas chemogenetic activation produced the opposite results. CFA plantar modelling increased the number of C-Fos protein and NALCN expression in PBL glutamatergic neurons. Knockdown of NALCN in PBL glutamatergic neurons alleviated CFA-induced pain. CFA injection induced C-Fos protein expression in central nucleus amygdala (CeA) neurons, which was suppressed by NALCN knockdown in PBL glutamatergic neurons. Therefore, elevated expression of NALCN in PBL glutamatergic neurons contributes to the development of inflammatory pain via PBL-CeA projections.

Ethanol depresses neurons in the lateral parabrachial nucleus by potentiating pre- and postsynaptic GABAA receptors.

As a psychoactive substance, ethanol is widely used in people's life. However, the neuronal mechanisms underlying its sedative effect remain unclear. In this study, we investigated the effects of ethanol on the lateral parabrachial nucleus (LPB), which is a novel component related to sedation. Coronal brain slices (280 µm thick) containing the LPB were prepared from C57BL/6J mice. The spontaneous firing and membrane potential of LPB neurons, and GABAergic transmission onto these neurons were recorded using whole-cell patch-clamp recordings. Drugs were applied through superfusion. The LPB neurons exhibited a regular spontaneous discharge at a rate of 1.5-3 Hz without burst firing. Brief superfusion of ethanol (30, 60, and 120 mM) concentration-dependently and reversibly suppressed the spontaneous firing of the neurons in LPB. In addition, when synaptic transmission was blocked by tetrodotoxin (TTX) (1 µM), ethanol (120 mM) caused hyperpolarization of the membrane potential. Furthermore, superfusion of ethanol markedly increased the frequency and amplitude of spontaneous and miniature inhibitory postsynaptic currents, which were abolished in the presence of the GABAA receptor (GABAA-R) antagonist picrotoxin (100 µM). In addition, the inhibitory effect of ethanol on the firing rate of LPB neurons was completely abolished by picrotoxin. Ethanol inhibits the excitability of LPB neurons in mouse slices, possibly via potentiating GABAergic transmission onto the neurons at pre- and postsynaptic sites.

Melanocortin-4 receptors on neurons in the parabrachial nucleus mediate inflammation-induced suppression of food-seeking behavior.

Anorexia is a common symptom during infectious and inflammatory disease. Here we examined the role of melanocortin-4 receptors (MC4Rs) in inflammation-induced anorexia. Mice with transcriptional blockage of the MC4Rs displayed the same reduction of food intake following peripheral injection of lipopolysaccharide as wild type mice but were protected against the anorexic effect of the immune challenge in a test in which fasted animals were to use olfactory cues to find a hidden cookie. By using selective virus-mediated receptor re-expression we demonstrate that the suppression of the food-seeking behavior is subserved by MC4Rs in the brain stem parabrachial nucleus, a central hub for interoceptive information involved in the regulation of food intake. Furthermore, the selective expression of MC4R in the parabrachial nucleus also attenuated the body weight increase that characterizes MC4R KO mice. These data extend on the functions of the MC4Rs and show that MC4Rs in the parabrachial nucleus are critically involved in the anorexic response to peripheral inflammation but also contribute to body weight homeostasis during normal conditions.

A Parabrachial-to-Amygdala Circuit That Determines Hemispheric Lateralization of Somatosensory Processing.

BACKGROUND: The central amygdala (CeA) is a bilateral hub of pain and emotional processing with well-established functional lateralization. We reported that optogenetic manipulation of neural activity in the left and right CeA has opposing effects on bladder pain. METHODS: To determine the influence of calcitonin gene-related peptide (CGRP) signaling from the parabrachial nucleus on this diametrically opposed lateralization, we administered CGRP and evaluated the activity of CeA neurons in acute brain slices as well as the behavioral signs of bladder pain in the mouse. RESULTS: We found that CGRP increased firing in both the right and left CeA neurons. Furthermore, we found that CGRP administration in the right CeA increased behavioral signs of bladder pain and decreased bladder pain-like behavior when administered in the left CeA. CONCLUSIONS: These studies reveal a parabrachial-to-amygdala circuit driven by opposing actions of CGRP that determines hemispheric lateralization of visceral pain.

Parabrachial-to-parasubthalamic nucleus pathway mediates fear-induced suppression of feeding in male mice.

Feeding behavior is adaptively regulated by external and internal environment, such that feeding is suppressed when animals experience pain, sickness, or fear. While the lateral parabrachial nucleus (lPB) plays key roles in nociception and stress, neuronal pathways involved in feeding suppression induced by fear are not fully explored. Here, we investigate the parasubthalamic nucleus (PSTN), located in the lateral hypothalamus and critically involved in feeding behaviors, as a target of lPB projection neurons. Optogenetic activation of lPB-PSTN terminals in male mice promote avoidance behaviors, aversive learning, and suppressed feeding. Inactivation of the PSTN and lPB-PSTN pathway reduces fear-induced feeding suppression. Activation of PSTN neurons expressing pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide enriched in the PSTN, is sufficient for inducing avoidance behaviors and feeding suppression. Blockade of PACAP receptors impaires aversive learning induced by lPB-PSTN photomanipulation. These findings indicate that lPB-PSTN pathway plays a pivotal role in fear-induced feeding suppression.

Masseter muscle contraction and cervical muscle sensitization by nerve growth factor cause mechanical hyperalgesia in masticatory muscle with activation of the trigemino-lateral parabrachial nucleus system in female rats.

OBJECTIVE: To establish a new rat model of craniofacial myalgia, and to clarify which central nervous system pathways are activated in the model. BACKGROUND: Craniofacial myalgia, represented by myogenous temporomandibular disorder and tension-type headache with pericranial tenderness, is more common in female patients. The pain is thought to be a type of multifactorial disorder with several coexisting causes. To our knowledge, there are no models of craniofacial muscle hyperalgesia caused by multiple types of stimuli. METHODS: We injected nerve growth factor into the trapezius muscle of female and male rats and repeatedly stimulated the masseter muscle (MM) electrically for 10 days. We determined the mechanical head-withdrawal threshold of MM and extent of phosphorylated extracellular signal-related kinase 1/2 (pERK) immunoreactivity in various regions of the lower brainstem. We conducted retrograde tract-tracing to determine the projection of mechanosensitive MM-innervating secondary neurons to the lateral parabrachial nucleus. Finally, we administered morphine in rats to determine whether increases of pERK immunoreactivity were dependent on noxious inputs. RESULTS: In female rats, but not male rats, the mechanical head-withdrawal threshold was decreased significantly from days 9 to 12. The number of pERK-immunoreactive neurons in the brainstem was increased significantly in female rats in the group with both stimuli compared to rats in other groups with a single stimulus. Mechanosensitive MM-innervating neurons in the brainstem projected to the parabrachial nucleus. Morphine administration blocked the increase in the number of pERK-immunoreactive neurons in both the brainstem and parabrachial nucleus. CONCLUSIONS: We established a model of craniofacial myalgia by combining trapezius and MM stimuli in female rats. We found mechanical hyperalgesia of the MM and activation of the pain pathway from the brainstem to parabrachial nucleus. The model reflects the characteristics of patients with craniofacial myalgia and might be helpful to clarify the pathogenic mechanisms underlying these disorders.

A central alarm system that gates multi-sensory innate threat cues to the amygdala.

Perception of threats is essential for survival. Previous findings suggest that parallel pathways independently relay innate threat signals from different sensory modalities to multiple brain areas, such as the midbrain and hypothalamus, for immediate avoidance. Yet little is known about whether and how multi-sensory innate threat cues are integrated and conveyed from each sensory modality to the amygdala, a critical brain area for threat perception and learning. Here, we report that neurons expressing calcitonin gene-related peptide (CGRP) in the parvocellular subparafascicular nucleus in the thalamus and external lateral parabrachial nucleus in the brainstem respond to multi-sensory threat cues from various sensory modalities and relay negative valence to the lateral and central amygdala, respectively. Both CGRP populations and their amygdala projections are required for multi-sensory threat perception and aversive memory formation. The identification of unified innate threat pathways may provide insights into developing therapeutic candidates for innate fear-related disorders.

Caffeine excites medial parabrachial nucleus neurons of mice by blocking adenosine A1 receptor.

Caffeine has been used as a first-line drug for treatment of apnea neonatorum for decades due to its high safety and effectiveness. Studies report that caffeine mainly acts as a blocker of Adenosine Receptors (ARs). However, the mechanism of caffeine in reducing apnea neonatorum in the central nervous system has not been fully explored. Medial parabrachial nucleus (MPB) is part of the respiratory center of the pons that may be related to the activity of caffeine. Previous studies have not explored the effect and mechanism of caffeine on MPB neurons. To elucidate this, the current study used antagonists of A1 and A2a receptors to mimic the effect of caffeine in MPB of mice in vitro using the patch-clamp technique. The firing rates and spontaneous post-synaptic currents were recorded. The findings of the study showed that caffeine excited MPB neurons. Notably, the adenosine A1R antagonist 8-cyclopentyl-1,3-dimethyl-xanthine (CPT) but not the adenosine A2aR antagonist Istradefylline (KW6002) mimicked the exciting effect of caffeine, implying that caffeine excited MPB neurons in mice by blocking A1Rs. Further, the results indicated that caffeine could increase efficiency of synaptic transmission to excite MPB neurons. These findings suggest that A1Rs in MPB may be potential targets for caffeine in reducing apnea neonatorum.

Divergent brainstem opioidergic pathways that coordinate breathing with pain and emotions.

Breathing can be heavily influenced by pain or internal emotional states, but the neural circuitry underlying this tight coordination is unknown. Here we report that Oprm1 (µ-opioid receptor)-expressing neurons in the lateral parabrachial nucleus (PBL) are crucial for coordinating breathing with affective pain in mice. Individual PBLOprm1 neuronal activity synchronizes with breathing rhythm and responds to noxious stimuli. Manipulating PBLOprm1 activity directly changes breathing rate, affective pain perception, and anxiety. Furthermore, PBLOprm1 neurons constitute two distinct subpopulations in a "core-shell" configuration that divergently projects to the forebrain and hindbrain. Through non-overlapping projections to the central amygdala and pre-Bötzinger complex, these two subpopulations differentially regulate breathing, affective pain, and negative emotions. Moreover, these subsets form recurrent excitatory networks through reciprocal glutamatergic projections. Together, our data define the divergent parabrachial opioidergic circuits as a common neural substrate that coordinates breathing with various sensations and behaviors such as pain and emotional processing.

Activation of Lateral Parabrachial Nucleus (LPBn) PACAP-Expressing Projection Neurons to the Bed Nucleus of the Stria Terminalis (BNST) Enhances Anxiety-like Behavior.

Anxiety disorders are among the most common psychiatric disorders, and understanding the underlying neurocircuitry of anxiety- and stress-related behaviors may be important for treatment. The bed nucleus of the stria terminalis (BNST) has been studied for its role in many stress-related pathologies, such as anxiety, pain, depression, and addiction. Our prior work has demonstrated that pituitary adenylate cyclase-activating polypeptide (PACAP) receptor activation in the BNST mediates many of the behavioral consequences of chronic stress. While the BNST contains local PACAP-expressing neurons, a major source of afferent PACAP is the lateral parabrachial nucleus (LPBn), and excitotoxic lesions of the LPBn substantially decreasess PACAP immunostaining in the BNST. Here, we first assessed Cre-dependent reporter expression by injecting AAV2-hSyn-DIO-mCherry into the LPBn of PACAP-IRES-Cre mice for circuit mapping studies and identified PACAP projections to the BNST, lateral capsular central nucleus of the amygdala (CeLC), and ventromedial hypothalamus (VMH). In a second study, we assessed the effects of chemogenetically activating LPBn PACAP afferents in the BNST by injecting AAV2-hSyn-DIO-hM3D(Gq)-mCherry into the LPBn of PACAP-IRES-Cre mice for Cre-dependent expression of excitatory designer receptors exclusively activated by designer drugs (DREADDs). Before behavioral testing, clozapine-N-oxide (CNO), the selective agonist of our DREADD, was infused directly into the BNST. We found that after specific activation of LPBn PACAP afferents in the BNST, mice had increased anxiety-like behavior compared with controls, while total locomotor activity was unaffected. These results indicate that activation of PACAPergic LPBn projections to the BNST may play an important role in producing anxiety-like behavior.

The Role of Prostaglandin E2 Synthesized in Rat Lateral Parabrachial Nucleus in LPS-Induced Fever.

INTRODUCTION: The lateral parabrachial nucleus (LPBN) is considered to be a brain site of the pyrogenic action of prostaglandin (PG) E2 outside of the preoptic area. Yet, the role of the LPBN in fever following a systemic immune challenge remains poorly understood. METHODS: We examined the expression of cyclooxygenase-2 (COX-2) and microsomal PGE synthase-1 (mPGES-1) in the LPBN after the intraperitoneal injection of lipopolysaccharide (LPS). We investigated the effects of LPBN NS-398 (COX-2 inhibitor) on LPS-induced fever, the effects of direct LPBN PGE2 administration on the energy expenditure (EE), brown adipose tissue (BAT) thermogenesis, neck muscle electromyographic activity and tail temperature, and the effects of PGE2 on the spontaneous firing activity and thermosensitivity of in vitro LPBN neurons in a brain slice. RESULTS: The COX-2 and mPGES-1 enzymes were upregulated at both mRNA and protein levels. The microinjection of NS-398 in the LPBN attenuated the LPS-induced fever. Direct PGE2 administration in the LPBN resulted in a febrile response by a coordinated response of increased EE, BAT thermogenesis, shivering, and possibly decreased heat loss through the tail. The LPBN neurons showed a clear anatomical distinction in the firing rate response to PGE2, with the majority of PGE2-excited or -inhibited neurons being located in the external lateral or dorsal subnucleus of the LPBN, respectively. However, neither the firing rate nor the thermal coefficient response to PGE2 showed any difference between warm-sensitive, cold-sensitive, and temperature-insensitive neurons in the LPBN. CONCLUSIONS: PGE2 synthesized in the LPBN was at least partially involved in LPS-induced fever via its different modulations of the firing rate of neurons in different LPBN subnuclei.

Activation of parabrachial nucleus - ventral tegmental area pathway underlies the comorbid depression in chronic neuropathic pain in mice.

Depression symptoms are often found in patients suffering from chronic pain, a phenomenon that is yet to be understood mechanistically. Here, we systematically investigate the cellular mechanisms and circuits underlying the chronic-pain-induced depression behavior. We show that the development of chronic pain is accompanied by depressive-like behaviors in a mouse model of trigeminal neuralgia. In parallel, we observe increased activity of the dopaminergic (DA) neuron in the midbrain ventral tegmental area (VTA), and inhibition of this elevated VTA DA neuron activity reverses the behavioral manifestations of depression. Further studies establish a pathway of glutamatergic projections from the spinal trigeminal subnucleus caudalis (Sp5C) to the lateral parabrachial nucleus (LPBN) and then to the VTA. These glutamatergic projections form a direct circuit that controls the development of the depression-like behavior under the state of the chronic neuropathic pain.

Ultra-Low Frequency Transcutaneous Electrical Nerve Stimulation on Pain Modulation in a Rat Model with Myogenous Temporomandibular Dysfunction.

Masticatory myofascial pain (MMP) is one of the most common causes of chronic orofacial pain in patients with temporomandibular disorders. To explore the antinociceptive effects of ultra-low frequency transcutaneous electrical nerve stimulation (ULF-TENS) on alterations of pain-related biochemicals, electrophysiology and jaw-opening movement in an animal model with MMP, a total of 40 rats were randomly and equally assigned to four groups; i.e., animals with MMP receiving either ULF-TENS or sham treatment, as well as those with sham-MMP receiving either ULF-TENS or sham treatment. MMP was induced by electrically stimulated repetitive tetanic contraction of masticatory muscle for 14 days. ULF-TENS was then performed at myofascial trigger points of masticatory muscles for seven days. Measurable outcomes included maximum jaw-opening distance, prevalence of endplate noise (EPN), and immunohistochemistry for substance P (SP) and µ-opiate receptors (MOR) in parabrachial nucleus and c-Fos in rostral ventromedial medulla. There were significant improvements in maximum jaw-opening distance and EPN prevalence after ULF-TENS in animals with MMP. ULF-TENS also significantly reduced SP overexpression, increased MOR expression in parabrachial nucleus, and increased c-Fos expression in rostral ventromedial medulla. ULF-TENS may represent a novel and applicable therapeutic approach for improvement of orofacial pain induced by MMP.

Calcitonin gene related peptide α is dispensable for many danger-related motivational responses.

Calcitonin gene related peptide (CGRP) expressing neurons in the parabrachial nucleus have been shown to encode danger. Through projections to the amygdala and other forebrain structures, they regulate food intake and trigger adaptive behaviors in response to threats like inflammation, intoxication, tumors and pain. Despite the fact that this danger-encoding neuronal population has been defined based on its CGRP expression, it is not clear if CGRP is critical for its function. It is also not clear if CGRP in other neuronal structures is involved in danger-encoding. To examine the role of CGRP in danger-related motivational responses, we used male and female mice lacking αCGRP, which is the main form of CGRP in the brain. These mice had no, or only very weak, CGRP expression. Despite this, they did not behave differently compared to wildtype mice when they were tested for a battery of danger-related responses known to be mediated by CGRP neurons in the parabrachial nucleus. Mice lacking αCGRP and wildtype mice showed similar inflammation-induced anorexia, conditioned taste aversion, aversion to thermal pain and pain-induced escape behavior, although it should be pointed out that the study was not powered to detect any possible differences that were minor or sex-specific. Collectively, our findings suggest that αCGRP is not necessary for many threat-related responses, including some that are known to be mediated by CGRP neurons in the parabrachial nucleus.

Reward signalling in brainstem nuclei under fluctuating blood glucose.

Phasic dopamine release from mid-brain dopaminergic neurons is thought to signal errors of reward prediction (RPE). If reward maximisation is to maintain homeostasis, then the value of primary rewards should be coupled to the homeostatic errors they remediate. This leads to the prediction that RPE signals should be configured as a function of homeostatic state and thus diminish with the attenuation of homeostatic error. To test this hypothesis, we collected a large volume of functional MRI data from five human volunteers on four separate days. After fasting for 12 hours, subjects consumed preloads that differed in glucose concentration. Participants then underwent a Pavlovian cue-conditioning paradigm in which the colour of a fixation-cross was stochastically associated with the delivery of water or glucose via a gustometer. This design afforded computation of RPE separately for better- and worse-than expected outcomes during ascending and descending trajectories of serum glucose fluctuations. In the parabrachial nuclei, regional activity coding positive RPEs scaled positively with serum glucose for both ascending and descending glucose levels. The ventral tegmental area and substantia nigra became more sensitive to negative RPEs when glucose levels were ascending. Together, the results suggest that RPE signals in key brainstem structures are modulated by homeostatic trajectories of naturally occurring glycaemic flux, revealing a tight interplay between homeostatic state and the neural encoding of primary reward in the human brain.

Efferent projections of CGRP/Calca-expressing parabrachial neurons in mice.

The parabrachial nucleus (PB) is composed of glutamatergic neurons at the midbrain-hindbrain junction. These neurons form many subpopulations, one of which expresses Calca, which encodes the neuropeptide calcitonin gene-related peptide (CGRP). This Calca-expressing subpopulation has been implicated in a variety of homeostatic functions, but the overall distribution of Calca-expressing neurons in this region remains unclear. Also, while previous studies in rats and mice have identified output projections from CGRP-immunoreactive or Calca-expressing neurons, we lack a comprehensive understanding of their efferent projections. We began by identifying neurons with Calca mRNA and CGRP immunoreactivity in and around the PB, including populations in the locus coeruleus and motor trigeminal nucleus. Calca-expressing neurons in the PB prominently express the mu opioid receptor (Oprm1) and are distinct from neighboring neurons that express Foxp2 and Pdyn. Next, we used Cre-dependent anterograde tracing with synaptophysin-mCherry to map the efferent projections of these neurons. Calca-expressing PB neurons heavily target subregions of the amygdala, bed nucleus of the stria terminalis, basal forebrain, thalamic intralaminar and ventral posterior parvicellular nuclei, and hindbrain, in different patterns depending on the injection site location within the PB region. Retrograde axonal tracing revealed that the previously unreported hindbrain projections arise from a rostral-ventral subset of CGRP/Calca neurons. Finally, we show that these efferent projections of Calca-expressing neurons are distinct from those of neighboring PB neurons that express Pdyn. This information provides a detailed neuroanatomical framework for interpreting experimental work involving CGRP/Calca-expressing neurons and opioid action in the PB region.

Role of calcitonin gene-related peptide in pain regulation in the parabrachial nucleus of naive rats and rats with neuropathic pain.

Researches have shown that calcitonin gene-related peptide (CGRP) plays a pivotal role in pain modulation. Nociceptive information from the periphery is relayed from parabrachial nucleus (PBN) to brain regions implicated involved in pain. This study investigated the effects and mechanisms of CGRP and CGRP receptors in pain regulation in the PBN of naive and neuropathic pain rats. Chronic sciatic nerve ligation was used to model neuropathic pain, CGRP and CGRP 8-37 were injected into the PBN of the rats, and calcitonin receptor-like receptor (CLR), a main structure of CGRP receptor, was knocked down by lentivirus-coated CLR siRNA. The hot plate test (HPT) and the Randall Selitto Test (RST) was used to determine the latency of the rat hindpaw response. The expression of CLR was detected with RT-PCR and western blotting. We found that intra-PBN injecting of CGRP induced an obvious anti-nociceptive effect in naive and neuropathic pain rats in a dose-dependent manner, the CGRP-induced antinociception was significantly reduced after injection of CGRP 8-37, Moreover, the mRNA and protein levels of CLR, in PBN decreased significantly and the antinociception CGRP-induced was also significantly lower in neuropathic pain rats than that in naive rats. Knockdown CLR in PBN decreased the expression of CLR and the antinociception induced by CGRP was observably decreased. Our results demonstrate that CGRP induced antinociception in PBN of naive or neuropathic pain rats, CGRP receptor mediates this effect. Neuropathic pain induced decreases in the expression of CGRP receptor, as well as in CGRP-induced antinociception in PBN.

Projecting neurons in spinal dorsal horn send collateral projections to dorsal midline/intralaminar thalamic complex and parabrachial nucleus.

Itch is an annoying sensation that always triggers scratching behavior, yet little is known about its transmission pathway in the central nervous system. Parabrachial nucleus (PBN), an essential transmission nucleus in the brainstem, has been proved to be the first relay station in itch sensation. Meanwhile, dorsal midline/intralaminar thalamic complex (dMITC) is proved to be activated with nociceptive stimuli. However, whether the PBN-projecting neurons in spinal dorsal horn (SDH) send collateral projections to dMITC, and whether these projections involve in itch remain unknown. In the present study, a double retrograde tracing method was applied when the tetramethylrhodamine-dextran (TMR) was injected into the dMITC and Fluoro-gold (FG) was injected into the PBN, respectively. Immunofluorescent staining for NeuN, substance P receptor (SPR), substance P (SP), or FOS induced by itch or pain stimulations with TMR and FG were conducted to provide morphological evidence. The results revealed that TMR/FG double-labeled neurons could be predominately observed in superficial laminae and lateral spinal nucleus (LSN) of SDH; Meanwhile, most of the collateral projection neurons expressed SPR and some of them expressed FOS in acute itch model induced by histamine. The present results implicated that some of the SPR-expressing neurons in SDH send collateral projections to the dMITC and PBN in itch transmission, which might be involved in itch related complex affective/emotional processing to the higher brain centers.