Apelin-13 microinjection into the paraventricular nucleus increased sympathetic nerve activity innervating brown adipose tissue in rats

The aim of present study is to clarify the role of apelin in regulating energy homeostasis in brown adipose tissue (BAT). We examined the central effects of apelin-13 on the brain c-fos like immunoreactivity (c-FLI), BAT temperature and the activity of the sympathetic nerve activity innervating BAT in rats. In the hypothalamus, central infusion into the third cerebral ventricle (i3vt) of apelin-13 caused induction of c-FLI in the paraventricular nucleus (PVN) compared with the controls (PBS-treated) group. In addition, microinjection of apelin-13 into the PVN produced significant increases in BAT temperature. Furthermore, microinjection of apelin-13 treatment increased BAT sympathetic nerve activity compared with controls. We conclude that apelin-13 microinjection into PVN increases sympathetic nerve activity innervating BAT.     

Localization of CGRP receptor components and receptor binding sites in rhesus monkey brainstem: A detailed study using in situ hybridization,immunofluorescence, and autoradiography

Functional imaging studies have revealed that certain brainstem areas are activated during migraine attacks. The neuropeptide calcitonin gene–related peptide (CGRP) is associated with activation of the trigeminovascular system and transmission of nociceptive information and plays a key role in migraine pathophysiology. Therefore, to elucidate the role of CGRP, it is critical to identify the regions within the brainstem that process CGRP signaling. In situ hybridization and immunofluorescence were performed to detect mRNA expression and define cellular localization of calcitonin receptor–like receptor (CLR) and receptor activity–modifying protein 1 (RAMP1), respectively. To define CGRP receptor binding sites, in vitro autoradiography was performed with [³H]MK‐3207 (a CGRP receptor antagonist). CLR and RAMP1 mRNA and protein expression were detected in the pineal gland, medial mammillary nucleus, median eminence, infundibular stem, periaqueductal gray, area postrema, pontine raphe nucleus, gracile nucleus, spinal trigeminal nucleus, and spinal cord. RAMP1 mRNA expression was also detected in the posterior hypothalamic area, trochlear nucleus, dorsal raphe nucleus, medial lemniscus, pontine nuclei, vagus nerve, inferior olive, abducens nucleus, and motor trigeminal nucleus; protein coexpression of CLR and RAMP1 was observed in these areas via immunofluorescence. [³H]MK‐3207 showed high binding densities concordant with mRNA and protein expression. The present study suggests that several regions in the brainstem may be involved in CGRP signaling. Interestingly, we found receptor expression and antagonist binding in some areas that are not protected by the blood–brain barrier, which suggests that drugs inhibiting CGRP signaling may not be able to penetrate the central nervous system to antagonize receptors in these brain regions. J. Comp. Neurol. 524:90–118, 2016. © 2015 Wiley Periodicals, Inc.     

Characterization of the parabrachial nucleus input to the hypothalamic paraventricular nucleus in the rat

The brainstem parabrachial nucleus (PBN) is viewed as an increasingly important site for the transfer of autonomic-related information to more rostral structures in the forebrain including the hypothalamus. In this study, we examined electrophysiologically in vivo and anatomically the nature of PBN input to the hypothalamic paraventricular nucleus (PVN) and particularly to the vasopressin-and oxytocin-secreting magnocellular neurosecretory cells within this nucleus. In urethane-anaesthetized rats, extracellular recordings from 108 antidromically identified neurosecretory PVN cells revealed an excitatory (37/43 cells) and less frequently an inhibitory (6/43 cells) response consequent to electrical stimulation in the PBN. Both vasopressin (12/37 cells)-and oxytocin (9/37 cells)-secreting neurons appear to respond to the PBN stimulus. Four cells projecting to the neurohypophysis could also be antidromically activated from PBN, and this observation may be indicative of collateral branching in some PVN neurosecretory neurons. In addition, recordings from 60 non-magnocellular (i.e. non-neurohypophysially-projecting) PVN cells revealed a facilitatory response (43/60 cells) following PBN stimulation, Iontophoretic injections of the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) were made within the rat lateral PBN and brains prepared for immunocytochemical examination of projections to the PVN region. PHA-L-labelled fibres and terminals were visualized within both the parvocellular and magnocellular divisions of the PVN. In addition, labelled fibres were also seen in a region immediately dorsal to the PVN. PHA-L-labelled fibres with axonal varicosities and boutons were visualized over immunocyto-chemically-identified vasopressin and oxytocin neurons within the magnocellular PVN. These convergent electrophysiological and anatomical data provide evidence for a PBN projection to the PVN that is predominantly excitatory to both magnocellular neurosecretory and non-magnocellular cells. Moreover, with respect to vasopressin-and oxytocin-secreting cells, the PBN input appears to be directed at both populations of peptidergic neurons.     

Noradrenaline-induced lipolysis in adipose tissue is suppressed at hibernation temperatures in ground squirrels

Responsiveness of white adipose tissue (WAT) and brown adipose tissue (BAT) from hibernating and nonhibernating golden-mantled ground squirrels (Spermophilus lateralis) to the lipolytic action of the sympathetic neurotransmitter, noradrenaline, was tested in vitro at temperatures characteristic of deep torpor (5 degrees C) and euthermia (37 degrees C). Noradrenaline-stimulated lipolysis, as indicated by WAT glycerol release, of tissue from hibernating ground squirrels was six- to ten-fold greater at 37 degrees C than at 5 degrees C. Noradrenaline was ineffective in increasing lipolysis at 5 degrees C. Noradrenaline-stimulated lipolysis in BAT was similarly suppressed at 5 degrees C. Noradrenaline-stimulated lipolysis was little affected by temperature change below approximately 15 degrees C but strongly correlated with temperature above approximately 15 degrees C. Noradrenaline-induced lipolysis of WAT from nonhibernating and hibernating ground squirrels did not differ at an incubation temperature of 5 degrees C. We conclude that noradrenaline-stimulated WAT lipolytic activity is markedly suppressed at the low temperatures characteristic of deep torpor and that there is no 'hibernation-specific' adaptation of WAT to enhance its responsiveness to noradrenaline at low tissue temperatures. Temperature dependence of noradrenaline-stimulated lipolysis may in part account for the shift from lipid to carbohydrate metabolism during the earliest stage of arousal from deep torpor.     

Intrinsic sensory neurons provide direct input to motor neurons and interneurons in mouse distal colon via varicose baskets

Connections from intrinsic primary afferent neurons (IPANs), to ascending motor and interneurons have been described in guinea pig colon. These mono- and polysynaptic circuits may underlie polarized motor reflexes evoked by local gut stimulation. There is a need to translate findings in guinea pig to mouse, a species increasingly used in enteric neuroscience. Here, mouse distal colon was immunolabeled for CGRP, a marker of putative IPANs. This revealed a combination of large, intensely immunofluorescent axons in myenteric plexus and circular muscle, and thinner varicose axons with less immunofluorescence. The latter formed dense, basket-like varicosity clusters (CGRP+ baskets) that enveloped myenteric nerve cell bodies. Immunolabeling after 4–5 days in organ culture caused loss of large CGRP+ axons, but not varicose CGRP+ fibers and CGRP+ baskets. Baskets were characterized further by triple labeling with CGRP, nitric oxide synthase (NOS) and calretinin (CALR) antibodies. Approximately half (48%) of nerve cell bodies inside CGRP+ baskets lacked both NOS and CALR, while two overlapping populations containing NOS and/or CALR comprised the remainder. Quantitative analysis revealed CGRP+ varicosities were most abundant in baskets, followed by CALR+ varicosities, with a high degree of colocalization between the two markers. Few NOS+ varicosities occurred in baskets. Significantly higher proportions of CALR+ and CGRP+ varicosities colocalized in baskets than in circular muscle. In conclusion, CGRP+ baskets in mouse colon are formed by intrinsic enteric neurons with a neurochemical profile consistent with IPANs and have direct connections to both excitatory and inhibitory neurons.     

Immunocytochemical organization and sour taste activation in the rostral nucleus of the solitary tract of mice

Sensory inputs from the oropharynx terminate in both the trigeminal brainstem complex and the rostral part of the nucleus of the solitary tract (nTS). Taste information is conveyed via the facial and glossopharyngeal nerves, while general mucosal innervation is carried by the trigeminal and glossopharyngeal nerves. In contrast, the caudal nTS receives general visceral information largely from the vagus nerve. Although the caudal nTS shows clear morphological and molecularly delimited subdivisions, the rostral part does not. Thus, linking taste‐induced patterns of activity to morphological subdivisions in the nTS is challenging. To test whether molecularly defined features of the rostral nTS correlate with patterns of taste‐induced activity, we combined immunohistochemistry for markers of various visceral afferent and efferent systems with c‐Fos–based activity maps generated by stimulation with a sour tastant, 30 mM citric acid. We further dissociated taste‐related activity from activity arising from acid‐sensitive general mucosal innervation by comparing acid‐evoked c‐Fos in wild‐type and “taste blind” P2X₂/P2X₃ double knockout (P2X‐dbl KO) mice. In wild‐type mice, citric acid stimulation evoked significant c‐Fos activation in the central part of the rostral nTS—activity that was largely absent in the P2X‐dbl KO mice. P2X‐dbl KO mice, like wild‐type mice, did exhibit acid‐induced c‐Fos activity in the dorsomedial trigeminal brainstem nucleus situated laterally adjacent to the rostral nTS. This dorsomedial nucleus also showed substantial innervation by trigeminal nerve fibers immunoreactive for calcitonin gene‐related peptide (CGRP), a marker for polymodal nociceptors, suggesting that trigeminal general mucosal innervation carries information about acids in the oral cavity. J. Comp. Neurol. 525:271–290, 2017. © 2016 Wiley Periodicals, Inc.     

Brown adipose tissue function is accompanied by cerebral activation in lean but not in obese humans

Brown adipose tissue (BAT) is able to generate heat and dissipate energy in response to cold exposure in mammals. It has recently been acknowledged that adult humans also have functional BAT, whose metabolic activity is reduced in obesity. In healthy humans, the cerebral mechanisms that putatively control BAT function are unclear. By using positron emission tomography (PET), we showed that cold-induced BAT activation is associated with glucose metabolism in the cerebellum, thalamus, and cingulate, temporoparietal, lateral frontal, and occipital cortices in lean participants, whereas no such associations were found under warm control conditions. The cold-induced increase in cerebral glucose metabolism was more robust in lean than obese participants. Cerebral glucose metabolism was not associated with skeletal muscle or white adipose tissue glucose uptake under warm or cold conditions. In conclusion, BAT metabolism was accompanied by the activation of specific cerebral regions, and this shows an uncharacterized role that the brain plays in the regulation of BAT function. In obese participants, the cold-induced response in cerebral activity was attenuated that provides a clue for obesity-induced impairment in BAT metabolism.     

Connections of the laterodorsal tegmental nucleus with the habenular-interpeduncular-raphe system

The laterodorsal tegmental nucleus (LDTg) is a hindbrain cholinergic cell group thought to be involved in mechanisms of arousal and the control of midbrain dopamine cells. Nowadays, there is increasing evidence that LDTg is also engaged in mechanisms of anxiety/fear and promotion of emotional arousal under adverse conditions. Interestingly, LDTg appears to be connected with other regulators of aversive motivational states, including the lateral habenula (LHb), medial habenula (MHb), interpeduncular nucleus (IP), and median raphe nucleus (MnR). However, the circuitry between these structures has hitherto not been systematically investigated. Here, we placed injections of retrograde or anterograde tracers into LDTg, LHb, IP, and MnR. We also examined the transmitter phenotype of LDTg afferents to IP by combining retrograde tracing with immunofluorescence and in situ hybridization techniques. We found LHb inputs to LDTg mainly emerging from the medial division of the LHb (LHbM), which also receives axonal input from LDTg. The bidirectional connections between IP and LDTg displayed a lateralized organization, with LDTg inputs to IP being predominantly GABAergic or cholinergic and mainly directed to the contralateral IP. Moreover, we disclosed reciprocal LDTg connections with structures involved in the modulation of hippocampal theta rhythm including MnR, nucleus incertus, and supramammillary nucleus. Our findings indicate that the habenula is linked with LDTg either by direct reciprocal projections from/to LHbM or indirectly via the MHb-IP axis, supporting a functional role of LDTg in the regulation of aversive behaviors, and further characterizing LHb as a master controller of ascending brainstem state-setting modulatory projection systems.     

Neurotransmitter diversity in pre‐synaptic terminals located in the parvicellular neuroendocrine paraventricular nucleus of the rat and mouse hypothalamus

Virtually all rodent neuroendocrine corticotropin‐releasing‐hormone (CRH) neurons are in the dorsal medial parvicellular (mpd) part of the paraventricular nucleus of the hypothalamus (PVH). They form the final common pathway for adrenocortical stress responses. Their activity is controlled by sets of GABA‐, glutamate‐, and catecholamine‐containing inputs arranged in an interactive pre‐motor network. Defining the nature and arrangement of these inputs can help clarify how stressor type and intensity information is conveyed to neuroendocrine neurons. Here we use immunohistochemistry with high‐resolution 3‐dimensional image analyses to examine the arrangement of single‐ and co‐occurring GABA, glutamate, and catecholamine markers in synaptophysin‐defined pre‐synaptic terminals in the PVHmpd of unstressed rats and Crh‐IRES‐Cre;Ai14 transgenic mice: respectively, vesicular glutamate transporter 2 (VGluT2), vesicular GABA transporter (VGAT), dopamine β‐hydroxylase (DBH), and phenylethanolamine n ‐methyltransferase (PNMT). Just over half of all PVHmpd pre‐synaptic terminals contain VGAT, with slightly less containing VGluT2. The vast majority of terminal appositions with mouse CRH neurons occur non‐somatically. However, there are significantly more somatic VGAT than VGluT2 appositions. In the rat PVHmpd, about five times as many pre‐synaptic terminals contain PNMT than DBH only. However, because epinephrine release has never been detected in the PVH, PNMT terminals may functionally be noradrenergic not adrenergic. PNMT and VGluT2 co‐occur in some pre‐synaptic terminals indicating the potential for co‐transmission of glutamate and norepinephrine. Collectively, these results provide a structural basis for how GABA/glutamate/catecholamine interactions enable adrenocortical responses to fast‐onset interosensory stimuli, and more broadly, how combinations of PVH neurotransmitters and neuromodulators interact dynamically to control adrenocortical activity.     

Peripheral and central anatomical organization of cutaneous afferent subtypes in a rat nociceptive intersegmental spinal reflex

Stimulation of rat segmental dorsal cutaneous nerves (DCNs) evokes the nociceptive intersegmental cutaneus trunci muscle (CTM) reflex. The reflex consists of early and late responses, mediated by Aδ and C fibers, respectively, based on required stimulation strengths, and shows segmental differences in terms of amplitude and duration. We have now investigated whether the peripheral or central anatomy of nociceptive afferent subtypes in different DCNs also vary in a segmental manner. The numbers of different axon subtypes, determined by axon diameter, were analyzed across peripheral DCNs from T6 to L1. The central projections of T7 and T13 DCN afferents were traced using DCN injections of cholera toxin subunit B (CTB) for myelinated A fibers and isolectin B4 (IB4) for unmyelinated C fibers and both labels were quantified in the dorsal horns. Peripheral axon subtype numbers did not differ significantly across DCNs. Centrally, IB4⁺, but not CTB⁺, projection areas were different between T7 and T13, consistent with different segmental CTM neurogram responses. At both levels, A fibers projected to deeper layers of the dorsal horn than did C fibers. These termination sites are consistent with both mono‐ and polysynaptic connections between these afferents and the ascending propriospinal interneurons of the reflex. Also analyzed were the spatial distribution, the synaptic termination, and the glutamatergic transporter profiles of DCN A and C fibers and their relationship to calcitonin gene‐related peptide (CGRP) staining in the dorsal horn.     

Characterization of the relaxin family peptide receptor 3 system in the mouse bed nucleus of the stria terminalis

The bed nucleus of the stria terminalis (BNST) is a critical node involved in stress and reward-related behaviors. Relaxin family peptide receptor 3 (RXFP3) signaling in the BNST has been implicated in stress-induced alcohol seeking behavior. However, the neurochemical phenotype and connectivity of BNST RXFP3-expressing (RXFP3+) cells have yet to be elucidated. We interrogated the molecular signature and electrophysiological properties of BNST RXFP3+ neurons using a RXFP3-Cre reporter mouse line. BNST RXFP3+ cells are circumscribed to the dorsal BNST (dBNST) and are neurochemically heterogeneous, comprising a mix of inhibitory and excitatory neurons. Immunohistochemistry revealed that ~48% of BNST RXFP3+ neurons are GABAergic, and a quarter of these co-express the calcium-binding protein, calbindin. A subset of BNST RXFP3+ cells (~41%) co-express CaMKIIα, suggesting this subpopulation of BNST RXFP3+ neurons are excitatory. Corroborating this, RNAscope® revealed that ~35% of BNST RXFP3+ cells express vVGluT2 mRNA, indicating a subpopulation of RXFP3+ neurons are glutamatergic. RXFP3+ neurons show direct hyperpolarization to bath application of a selective RXFP3 agonist, RXFP3-A2, while around 50% of cells were depolarised by exogenous corticotrophin releasing factor. In behaviorally naive mice the majority of RXFP3+ neurons were Type II cells exhibiting I_h and T type calcium mediated currents. However, chronic swim stress caused persistent plasticity, decreasing the proportion of neurons that express these channels. These studies are the first to characterize the BNST RXFP3 system in mouse and lay the foundation for future functional studies appraising the role of the murine BNST RXFP3 system in more complex behaviors.     

New viral-genetic mapping uncovers an enrichment of corticotropin-releasing hormone-expressing neuronal inputs to the nucleus accumbens from stress-related brain regions

Corticotropin-releasing hormone (CRH) is an essential, evolutionarily-conserved stress neuropeptide. In addition to hypothalamus, CRH is expressed in brain regions including amygdala and hippocampus where it plays crucial roles in modulating the function of circuits underlying emotion and cognition. CRH⁺ fibers are found in nucleus accumbens (NAc), where CRH modulates reward/motivation behaviors. CRH actions in NAc may vary by the individual's stress history, suggesting roles for CRH in neuroplasticity and adaptation of the reward circuitry. However, the origin and extent of CRH⁺ inputs to NAc are incompletely understood. We employed viral genetic approaches to map both global and CRH⁺ projection sources to NAc in mice. We injected into NAc variants of a new designer adeno-associated virus that permits robust retrograde access to NAc-afferent projection neurons. Cre-dependent viruses injected into CRH-Cre mice enabled selective mapping of CRH⁺ afferents. We employed anterograde AAV1-directed axonal tracing to verify NAc CRH⁺ fiber projections and established the identity of genetic reporter-labeled cells via validated antisera against native CRH. We quantified the relative contribution of CRH⁺ neurons to total NAc-directed projections. Combined retrograde and anterograde tracing identified the paraventricular nucleus of the thalamus, bed nucleus of stria terminalis, basolateral amygdala, and medial prefrontal cortex as principal sources of CRH⁺ projections to NAc. CRH⁺ NAc afferents were selectively enriched in NAc-projecting brain regions involved in diverse aspects of the sensing, processing and memory of emotionally salient events. These findings suggest multiple, complex potential roles for the molecularly-defined, CRH-dependent circuit in modulation of reward and motivation behaviors.     

The dromedary camel displays annual variation in hypothalamic kisspeptin and Arg–Phe-amide-related peptide-3 according to sex,season, and breeding activity

The dromedary camel (Camelus dromedarius) is a desert mammal whose cycles in reproductive activity ensure that the offspring's birth and weaning coincide with periods of abundant food resources and favorable climate conditions. In this study, we assessed whether kisspeptin (Kp) and arginine–phenylalanine (RF)-amide related peptide-3 (RFRP-3), two hypothalamic peptides known to regulate the mammalian hypothalamo-pituitary gonadal axis, may be involved in the seasonal control of camel's reproduction. Using specific antibodies and riboprobes, we found that Kp neurons are present in the preoptic area (POA), suprachiasmatic (SCN), and arcuate (ARC) nuclei, and that RFRP-3 neurons are present in the paraventricular (PVN), dorsomedial (DMH), and ventromedial (VMH) hypothalamic nuclei. Kp fibers are found in various hypothalamic areas, notably the POA, SCN, PVN, DMH, VMH, supraoptic nucleus, and the ventral and dorsal premammillary nucleus. RFRP-3 fibers are found in the POA, SCN, PVN, DMH, VMH, and ARC. POA and ARC Kp neurons and DMH RFRP-3 neurons display sexual dimorphism with more neurons in female than in male. Both neuronal populations display opposed seasonal variations with more Kp neurons and less RFRP-3 neurons during the breeding (December–January) than the nonbreeding (July–August) season. This study is the first describing Kp and RFRP-3 in the camel's brain with, during the winter period lower RFRP-3 expression and higher Kp expression possibly responsible for the HPG axis activation. Altogether, our data indicate the involvement of both Kp and RFRP-3 in the seasonal control of the dromedary camel's breeding activity.