New nociceptive circuits to the hypothalamic perifornical area from the spinal cord and spinal trigeminal nucleus via the parabrachial nucleus.

Neurons of the parabrachial nucleus (PB) receive nociceptive input from the dorsal horn (DH) of the spinal cord and caudal part of the spinal trigeminal nucleus (Vc). Previously, we demonstrated that glutamatergic lateral PB neurons innervate orexin (ORX) neurons in the perifornical area (PeF) of the hypothalamus. However, the neural circuit via which ORX neurons receive nociceptive input from the DH and brainstem remains to be determined. In the present study, we aimed to clarify the potential nociceptive circuit from DH/Vc to PeF via lateral PB. We first examined the neuronal activity of fluorogold (FG)-labeled, PeF-projecting lateral PB neurons in Wistar rats following either saline or formalin injection to the forepaw or lips. We clearly detected more abundant c-Fos-positive, FG-labeled neurons in the PB nucleus. To investigate the relay from the DH/Vc to the PeF via the lateral PB, we injected FG into the PeF and biotinylated dextranamine (BDA) into the contralateral DH or ipsilateral Vc. We observed the most prominent overlap between BDA-labeled axon terminals and FG-labeled neurons in the dorsal lateral and central lateral subnuclei. Furthermore, we found that FG-labeled neurons formed close contact sites with BDA-labeled axons with synaptophysin immunoreactivity. Using electron microscopy, we confirmed that these contact sites were truly synapses. Taken together, our results indicate that the DH/Vc transmits nociceptive information to the PeF via the lateral PB, suggesting the involvement of ORX neurons in the pain pathway.

Glutamatergic signaling from the parabrachial nucleus plays a critical role in hypercapnic arousal.

The mechanisms of arousal from apneas during sleep in patients suffering from obstructive sleep apnea are not well understood. However, we know that respiratory chemosensory pathways converge on the parabrachial nucleus (PB), which sends glutamatergic projections to a variety of forebrain structures critical to arousal, including the basal forebrain, lateral hypothalamus, midline thalamus, and cerebral cortex. We tested the role of glutamatergic signaling in this pathway by developing an animal model for repetitive CO2 arousals (RCAs) and investigating the effect of deleting the gene for the vesicular glutamate transporter 2 (Vglut2) from neurons in the PB. We used mice with lox P sequences flanking exon2 of the Vglut2 gene, in which adeno-associated viral vectors containing genes encoding Cre recombinase and green fluorescent protein were microinjected into the PB to permanently and selectively disrupt Vglut2 expression while labeling the affected neurons. We recorded sleep in these mice and then investigated the arousals during RCA. Vglut2 deletions that included the external lateral and lateral crescent subdivisions of the lateral PB more than doubled the latency to arousal and resulted in failure to arouse by 30 s in >30% of trials. By contrast, deletions that involved the medial PB subdivision had minimal effects on arousal during hypercapnia but instead increased non-rapid eye movement (NREM) sleep by ∼43% during the dark period, and increased delta power in the EEG during NREM sleep by ∼50%. Our results suggest that glutamatergic neurons in the lateral PB are necessary for arousals from sleep in response to CO2, while medial PB glutamatergic neurons play an important role in promoting spontaneous waking.

Non-human primate model of amyotrophic lateral sclerosis with cytoplasmic mislocalization of TDP-43.

Amyotrophic lateral sclerosis is a fatal neurodegenerative disease characterized by progressive motoneuron loss. Redistribution of transactive response deoxyribonucleic acid-binding protein 43 from the nucleus to the cytoplasm and the presence of cystatin C-positive Bunina bodies are considered pathological hallmarks of amyotrophic lateral sclerosis, but their significance has not been fully elucidated. Since all reported rodent transgenic models using wild-type transactive response deoxyribonucleic acid-binding protein 43 failed to recapitulate these features, we expected a species difference and aimed to make a non-human primate model of amyotrophic lateral sclerosis. We overexpressed wild-type human transactive response deoxyribonucleic acid-binding protein 43 in spinal cords of cynomolgus monkeys and rats by injecting adeno-associated virus vector into the cervical cord, and examined the phenotype using behavioural, electrophysiological, neuropathological and biochemical analyses. These monkeys developed progressive motor weakness and muscle atrophy with fasciculation in distal hand muscles first. They also showed regional cytoplasmic transactive response deoxyribonucleic acid-binding protein 43 mislocalization with loss of nuclear transactive response deoxyribonucleic acid-binding protein 43 staining in the lateral nuclear group of spinal cord innervating distal hand muscles and cystatin C-positive cytoplasmic aggregates, reminiscent of the spinal cord pathology of patients with amyotrophic lateral sclerosis. Transactive response deoxyribonucleic acid-binding protein 43 mislocalization was an early or presymptomatic event and was later associated with neuron loss. These findings suggest that the transactive response deoxyribonucleic acid-binding protein 43 mislocalization leads to α-motoneuron degeneration. Furthermore, truncation of transactive response deoxyribonucleic acid-binding protein 43 was not a prerequisite for motoneuronal degeneration, and phosphorylation of transactive response deoxyribonucleic acid-binding protein 43 occurred after degeneration had begun. In contrast, similarly prepared rat models expressed transactive response deoxyribonucleic acid-binding protein 43 only in the nucleus of motoneurons. There is thus a species difference in transactive response deoxyribonucleic acid-binding protein 43 pathology, and our monkey model recapitulates amyotrophic lateral sclerosis pathology to a greater extent than rodent models, providing a valuable tool for studying the pathogenesis of sporadic amyotrophic lateral sclerosis.

Coherence analysis of the calcium activity of putative astrocytic and neuronal cells on the L5 ventral horn and neural output in activated lumbar CPG networks.

Astrocytes are thought to play a crucial role in providing structure to the spinal cord and maintaining efficient synaptic function and metabolism because their fine processes envelop the synapses of neurons and form many neuronal networks within the central nervous system (CNS). To investigate whether putative astrocytes and putative neurons distributed on the ventral horn play a role in the modulation of lumbar locomotor central pattern generator (CPG) networks, we used extracellular recording and optical imaging techniques and recorded the neural output from the left L5 ventral root and the calcium activity of putative astrocytes and neurons in the L5 ventral horn at the same time when activating an isolated L1-L5 spinal cord preparation from rats aged 0-2 days. Optical measurements detected cells that showed a fluorescence intensity change under all experimental conditions, namely, (1) 5-HT + NMDA, (2) TTX, and (3) TTX + Low K+. These cells were semiautomatically identified using an in-house MATLAB-based program, as putative astrocytes and neurons according to the cell classification, i.e., increased or decreased fluorescence intensity change (ΔF/F0), and subjective judgment based on their soma size. Coherence and its phase were calculated according to the calcium activity of the putative astrocytes and putative neurons, and neural output was calculated during fictive locomotion with in-house MATLAB-based programs. We found that the number of putative astrocytes activated by applying low K+ tends not to differ from that activated by applying the protease-activated receptor 1 (PAR1) selective agonist TFLLR-NH2 (TFLLR). Moreover, the calcium activity of several putative astrocytes and neurons synchronized with locomotor-like activity at a frequency range below 0.5 Hz and the time lag between peaks of cellular calcium activity and locomotor-like activity ranged from -1000 to + 1000 ms. These findings presumably indicates that these putative astrocytes and neurons in the left L5 ventral horn require -1000 to + 1000 ms to communicate with lumbar CPG networks and maintain efficient synaptic function and metabolism in activated lumbar CPG networks. This finding suggests the possibility that putative astrocytic and neuronal cells in the L5 ventral horn contribute to generating the rhythms and patterns of locomotor-like activity by activated CPG networks in the first to fifth lumbar spinal cord.

Dual orexin receptor blocker suvorexant attenuates hypercapnic ventilatory augmentation in mice.

Suvorexant (Belsomra(R)), a dual orexin receptor antagonist widely used in the treatment of insomnia, inhibits the arousal system in the brain. However, the drug's ventilatory effects have not been fully explored. This study aims to investigate the expression of orexin receptors in respiratory neurons and the effects of suvorexant on ventilation. Immunohistology of brainstem orexin receptor OX2R expression was performed in adult mice (n = 4) in (1) rostral ventral respiratory group (rVRG) neurons projecting to the phrenic nucleus (PhN) retrogradely labeled by Fluoro-Gold (FG) tracer, (2) neurons immunoreactive for paired like homeobox 2b (Phox2b) in the parafacial respiratory group/retrotrapezoid nucleus (pFRG/RTN), and (3) neurons immunoreactive for neurokinin 1 receptor (NK1R) and somatostatin (SST) in the preBötzinger complex (preBötC). Additionally, we measured in vivo ventilatory responses to hyperoxic hypercapnia (5% CO2) and hypoxia (10% O2) before and after suvorexant pretreatment (10 and cumulative 100 mg/kg) in unrestrained mice (n = 10) in a body plethysmograph. We found the OX2R immunoreactive materials in pFRG/RTN Phox2b and preBötC NK1R/SST immunoreactive neurons but not in FG-labeled rVRG neurons, which suggests the involvement of orexin in respiratory control. Further, suvorexant expressly suppressed the hypercapnic ventilatory augmentation, otherwise unaffecting ventilation. Central orexin is involved in shaping the hypercapnic ventilatory chemosensitivity. Suppression of hypercapnic ventilatory augmentation by the orexin receptor antagonist suvorexant calls for caution in its use in pathologies that may progress to hypercapnic respiratory failure, or sleep-disordered breathing. Clinical trials are required to explore the role of targeted pharmacological inhibition of orexin in ventilatory pathologies.

Depletion of vitamin E increases amyloid beta accumulation by decreasing its clearances from brain and blood in a mouse model of Alzheimer disease.

Increased oxidative damage is a prominent and early feature in Alzheimer disease. We previously crossed Alzheimer disease transgenic (APPsw) model mice with alpha-tocopherol transfer protein knock-out (Ttpa(-/-)) mice in which lipid peroxidation in the brain was significantly increased. The resulting double-mutant (Ttpa(-/-)APPsw) mice showed increased amyloid beta (Abeta) deposits in the brain, which was ameliorated with alpha-tocopherol supplementation. To investigate the mechanism of the increased Abeta accumulation, we here studied generation, degradation, aggregation, and efflux of Abeta in the mice. The clearance of intracerebral-microinjected (125)I-Abeta(1-40) from brain was decreased in Ttpa(-/-) mice to be compared with wild-type mice, whereas the generation of Abeta was not increased in Ttpa(-/-)APPsw mice. The activity of an Abeta-degrading enzyme, neprilysin, did not decrease, but the expression level of insulin-degrading enzyme was markedly decreased in Ttpa(-/-) mouse brain. In contrast, Abeta aggregation was accelerated in Ttpa(-/-) mouse brains compared with wild-type brains, and well known molecules involved in Abeta transport from brain to blood, low density lipoprotein receptor-related protein-1 (LRP-1) and p-glycoprotein, were up-regulated in the small vascular fraction of Ttpa(-/-) mouse brains. Moreover, the disappearance of intravenously administered (125)I-Abeta(1-40) was decreased in Ttpa(-/-) mice with reduced translocation of LRP-1 in the hepatocytes. These results suggest that lipid peroxidation due to depletion of alpha-tocopherol impairs Abeta clearances from the brain and from the blood, possibly causing increased Abeta accumulation in Ttpa(-/-)APPsw mouse brain and plasma.

Vesicular glutamate transporter 2-immunoreactive synapses onto phrenic motoneurons in the neonatal rat.

The synaptic organization between vesicular glutamate transporter 2 (VGLUT2)-immunoreactive (ir) axon terminals and phrenic motoneurons in the neonatal rat was examined using a combined retrograde tracing and immunohistochemistry for VGLUT2. The phrenic nucleus (PhN) contained large numbers of VGLUT2-ir axon terminals, some of which made axosomatic and axodendritic synapses with PhN motoneurons. These terminals were of asymmetrical type and contained spherical clear synaptic vesicles. The results suggest that in the neonatal rat glutamatergic synapses onto PhN motoneurons exist and mediate excitatory transmission to drive PhN motoneurons.

Anatomical changes of phrenic motoneurons during development.

Although the phrenic motoneurons are relatively well-developed at the time of birth as compared to non-respiratory motoneurons, they show distinct anatomical changes during postnatal development. In the present review we summarize anatomical changes of phrenic motoneurons during pre- and postnatal development. Cell bodies of phrenic motoneurons migrate into the ventromedial region of the ventral horn of C3-C6 by E13-E14 in the rat. During development the sizes and surface areas of phrenic motoneurons are increased with changes in dendritic morphology.

VGLUT2-expressing neurons in the vestibular nuclear complex mediate gravitational stress-induced hypothermia in mice.

The vestibular system, which is essential for maintaining balance, contributes to the sympathetic response. Although this response is involved in hypergravity load-induced hypothermia in mice, the underlying mechanism remains unknown. This study showed that hypergravity (2g) decreased plasma catecholamines, which resulted in hypoactivity of the interscapular brown adipose tissue (iBAT). Hypothermia induced by 2g load was significantly suppressed by administration of beta-adrenergic receptor agonists, suggesting the involvement of decrease in iBAT activity through sympathoinhibition. Bilateral chemogenetic activation of vesicular glutamate transporter 2 (VGLUT2)-expressing neurons in the vestibular nuclear complex (VNC) induced hypothermia. The VGLUT2-expressing neurons contributed to 2g load-induced hypothermia, since their deletion suppressed hypothermia. Although activation of vesicular gamma-aminobutyric acid transporter-expressing neurons in the VNC induced slight hypothermia instead of hyperthermia, their deletion did not affect 2g load-induced hypothermia. Thus, we concluded that 2g load-induced hypothermia resulted from sympathoinhibition via the activation of VGLUT2-expressing neurons in the VNC.

Blockade of astrocytic activation delays the occurrence of severe hypoxia-induced seizure and respiratory arrest in mice.

Seizures are induced when subjects are exposed to severe hypoxia. It is followed by ventilatory fall-off and eventual respiratory arrest, which may underlie the pathophysiology of death in patients with epilepsy and severe respiratory disorders. However, the mechanisms of hypoxia-induced seizures have not been fully understood. Because astrocytes are involved in various neurological disorders, we aimed to investigate whether astrocytes are operational in seizure generation and respiratory arrest in a severe hypoxic condition. We examined the effects of astrocytic activation blockade on responses of EEG and ventilation to severe hypoxia. Adult mice were divided into two groups; in one group (n = 24) only vehicle was injected, and in the other group (n = 24) arundic acid, an inhibitory modulator of astrocytic activation, was administered before initiation of recording. After recording EEG and ventilation by whole body plethysmography in room air, the gas in the recording chamber was switched to 5% oxygen (nitrogen balanced) until a seizure and ventilatory depression occurred, followed by prompt switch back to room air. Severe hypoxia initially increased ventilation, followed by a seizure and ventilatory suppression in all mice examined. Fourteen mice without arundic acid showed respiratory arrest during loading of hypoxia. However, 22 mice pretreated with arundic acid did not suffer from respiratory arrest. Time from the onset of hypoxia to the occurrence of seizures was significantly longer in the group with arundic acid than that in the group without arundic acid. We suggest that blockade of astrocytic activation delays the occurrence of seizures and prevents respiratory arrest.

Structural and functional connectivity from the dorsomedial hypothalamus to the ventral medulla as a chronological amplifier of sympathetic outflow.

Psychological stress activates the hypothalamus, augments the sympathetic nervous output, and elevates blood pressure via excitation of the ventral medullary cardiovascular regions. However, anatomical and functional connectivity from the hypothalamus to the ventral medullary cardiovascular regions has not been fully elucidated. We investigated this issue by tract-tracing and functional imaging in rats. Retrograde tracing revealed the rostral ventrolateral medulla was innervated by neurons in the ipsilateral dorsomedial hypothalamus (DMH). Anterograde tracing showed DMH neurons projected to the ventral medullary cardiovascular regions with axon terminals in contiguity with tyrosine hydroxylase-immunoreactive neurons. By voltage-sensitive dye imaging, dynamics of ventral medullary activation evoked by electrical stimulation of the DMH were analyzed in the diencephalon-lower brainstem-spinal cord preparation of rats. Although the activation of the ventral medulla induced by single pulse stimulation of the DMH was brief, tetanic stimulation caused activation of the DMH sustained into the post-stimulus phase, resulting in delayed recovery. We suggest that prolonged excitation of the DMH, which is triggered by tetanic electrical stimulation and could also be triggered by psychological stress in a real life, induces further prolonged excitation of the medullary cardiovascular networks, and could contribute to the pathological elevation of blood pressure. The connectivity from the DMH to the medullary cardiovascular networks serves as a chronological amplifier of stress-induced sympathetic excitation. This notion will be the anatomical and pathophysiological basis to understand the mechanisms of stress-induced sustained augmentation of sympathetic activity.

Differential effects of sodium-glucose cotransporter 2 inhibitor and low-carbohydrate diet on body composition and metabolic profile in obese diabetic db/db mice.

INTRODUCTION: Treatment using sodium-glucose cotransporter (SGLT) 2 inhibitor and low-carbohydrate diet (LCD) for obesity and type 2 diabetes are similar in terms of carbohydrate limitation. However, their mechanisms of action differ, and the effects on the body remain unclear. We investigated the effects of SGLT2 inhibitor and LCD on body composition and metabolic profile using the db/db mouse model for obesity and type 2 diabetes. RESEARCH DESIGN AND METHODS: Eight-week-old male db/db mice were divided into four groups: mice receiving normal diet and vehicle or canagliflozin (Cana) administration and mice receiving LCD and vehicle or Cana administration for 8 weeks. Consumed calories were adjusted to be equal among the groups. RESULTS: Both Cana administration and LCD feeding resulted in significant weight gain. Cana administration significantly decreased plasma glucose levels and increased plasma insulin levels with preservation of pancreatic ß cells. However, LCD feeding did not improve plasma glucose levels but deteriorated insulin sensitivity. LCD feeding significantly reduced liver weight and hepatic triglyceride content; these effects were not observed with Cana administration. Combined treatment with LCD did not lead to an additive increase in blood ß-ketone levels. CONCLUSIONS: SGLT2 inhibitors and LCD exert differential effects on the body. Their combined use may achieve better metabolic improvements in obesity and type 2 diabetes.

The role of the hypothalamus in modulation of respiration.

The hypothalamus is a higher center of the autonomic nervous system and maintains essential body homeostasis including respiration. The paraventricular nucleus, perifornical area, dorsomedial hypothalamus, and lateral and posterior hypothalamus are the primary nuclei of the hypothalamus critically involved in respiratory control. These hypothalamic nuclei are interconnected with respiratory nuclei located in the midbrain, pons, medulla and spinal cord. We provide an extensive review of the role of the above hypothalamic nuclei in the maintenance of basal ventilation, and modulation of respiration in hypoxic and hypercapnic conditions, during dynamic exercise, in awake and sleep states, and under stress. Dysfunction of the hypothalamus causes abnormal breathing and hypoventilation. However, the cellular and molecular mechanisms how the hypothalamus integrates and modulates autonomic and respiratory functions remain to be elucidated.

Lateral parabrachial neurons innervate orexin neurons projecting to brainstem arousal areas in the rat.

Orexin (ORX) neurons in the hypothalamus send their axons to arousal-promoting areas. We have previously shown that glutamatergic neurons in the lateral parabrachial nucleus (LPB) innervate ORX neurons. In this study, we examined potential pathways from the LPB to ORX neurons projecting to arousal-promoting areas in the brainstem by a combination of tract-tracing techniques in male Wistar rats. We injected the anterograde tracer biotinylated dextranamine (BDA) into the LPB and the retrograde tracer cholera toxin B subunit (CTb) into the ventral tegmental area, dorsal raphe nucleus, pedunculopontine tegmental nucleus, laterodorsal tegmental area, or locus coeruleus (LC). We then analyzed the BDA-labeled fibers and ORX-immunoreactive neurons in the hypothalamus. We found that double-labeled ORX and CTb neurons were the most abundant after CTb was injected into the LC. We also observed prominently overlapping distribution of BDA-labeled fibers, arising from neurons located in the lateral-most part of the dorsomedial nucleus and adjacent dorsal perifornical area. In these areas, we confirmed by confocal microscopy that BDA-labeled synaptophysin-immunoreactive axon terminals were in contiguity with cell bodies and dendrites of CTb-labeled ORX-immunoreactive neurons. These results suggest that the LPB innervates arousal-promoting areas via ORX neurons and is likely to promote arousal responses to stimuli.

Role of the α2 subunit of AMP-activated protein kinase and its nuclear localization in mitochondria and energy metabolism-related gene expressions in C2C12 cells.

BACKGROUND: AMP-activated protein kinase (AMPK), a heterotrimer with α1 or α2 catalytic subunits, acts as an energy sensor and regulates cellular homeostasis. Whereas AMPKα1 is necessary for myogenesis in skeletal muscle, the role of AMPKα2 in myogenic differentiation and energy metabolism-related gene expressions has remained unclear. We here examined the specific roles of AMPKα1 and AMPKα2 in the myogenic differentiation and mitochondria and energy metabolism-related gene expressions in C2C12 cells. MATERIALS AND METHODS: Stable C2C12 cell lines expressing a scramble short hairpin RNA (shRNA) or shRNAs specific for AMPKα1 (shAMPKα1), AMPKα2 (shAMPKα2), or both AMPKα1 and AMPKα2 (shPanAMPK) were generated by lentivirus infection. Lentiviruses encoding wild-type AMPKα2 (WT-AMPKα2) or AMPKα2 with a mutated nuclear localization signal (ΔNLS-AMPKα2) were also constructed for introduction into myoblasts. Myogenesis was induced by culture of C2C12 myoblasts for 6 days in differentiation medium. RESULTS: The amount of AMPKα2 increased progressively, whereas that of AMPKα1 remained constant, during the differentiation of myoblasts into myotubes. Expression of shPanAMPK or shAMPKα1, but not that of shAMPKα2, attenuated the proliferation of myoblasts as well as the phosphorylation of both acetyl-CoA carboxylase and the autophagy-initiating kinase ULK1 in myotubes. Up-regulation of myogenin mRNA, a marker for the middle stage of myogenesis, was attenuated in differentiating myotubes expressing shPanAMPK or shAMPKα1. In contrast, up-regulation of gene expression for muscle creatine kinase (MCK), a late-stage differentiation marker, as well as for genes related to mitochondrial biogenesis including the transcriptional coactivator peroxisome proliferator-activated receptor-γ coactivator-1α1 and α4 (PGC-1α1 and PGC-1α4) and mitochondria-specific genes such as cytochrome c were attenuated in myotubes expressing shAMPKα2 or shPanAMPK. The diameter of myotubes expressing shPanAMPK or shAMPKα2 was reduced, whereas that of those expressing shAMPKα1 was increased, compared with myotubes expressing scramble shRNA. A portion of AMPKα2 became localized to the nucleus during myogenic differentiation. The AMPK activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) and 2-deoxyglucose (2DG) each induced the nuclear translocation of WT-AMPKα2, but not that of ΔNLS-AMPKα2. Finally, expression of WT-AMPKα2 increased the mRNA abundance of PGC-1α1 and MCK mRNAs as well as cell diameter and tended to increase that of PGC-1α4, whereas that of ΔNLS-AMPKα2 increased only the abundance of MCK mRNA, in myotubes depleted of endogenous AMPKα2. CONCLUSION: TAMPKα1 and AMPKα2 have distinct roles in myogenic differentiation of C2C12 cells, with AMPKα1 contributing to the middle stage of myogenesis and AMPKα2 to the late stage. AMPKα2 regulates gene expressions including MCK, PGC-1α1 and PGC-1α4 and mitochondria-specific genes such as cytochrome c during the late stage of differentiation. Furthermore, the nuclear translocation of AMPKα2 is necessary for maintenance of PGC-1α1 mRNA during myogenesis.

Structural and functional identification of two distinct inspiratory neuronal populations at the level of the phrenic nucleus in the rat cervical spinal cord.

The diaphragm is driven by phrenic motoneurons that are located in the cervical spinal cord. Although the anatomical location of the phrenic nucleus and the function of phrenic motoneurons at a single cellular level have been extensively analyzed, the spatiotemporal dynamics of phrenic motoneuron group activity have not been fully elucidated. In the present study, we analyzed the functional and structural characteristics of respiratory neuron population in the cervical spinal cord at the level of the phrenic nucleus by voltage imaging, together with histological analysis of neuronal and astrocytic distribution in the cervical spinal cord. We found spatially distinct two cellular populations that exhibited synchronized inspiratory activity on the transversely cut plane at C4-C5 levels and on the ventral surface of the mid cervical spinal cord in the isolated brainstem-spinal cord preparation of the neonatal rat. Inspiratory activity of one group emerged in the central portion of the ventral horn that corresponded to the central motor column, and the other appeared in the medial portion of the ventral horn that corresponded to the medial motor column. We identified by retrogradely labeling study that the anatomical distributions of phrenic and scalene motoneurons coincided with optically detected central and medial motor regions, respectively. Furthermore, we anatomically demonstrated closely located features of putative motoneurons, interneurons and astrocytes in these regions. Collectively, we report that phrenic and scalene motoneuron populations show synchronized inspiratory activities with distinct anatomical locations in the mid cervical spinal cord.

Neuroanatomical and neurochemical organization of projections from the central amygdaloid nucleus to the nucleus retroambiguus via the periaqueductal gray in the rat.

The periaqueductal gray (PAG)-nucleus retroambiguus (NRA) pathway has been known to be involved in the control of vocalization and sexual behavior. To know how the amygdaloid complex influences the PAG-NRA pathway, here we first examined the synaptic organization between the central amygdaloid nucleus (CeA) fibers and the PAG neurons that project to the NRA by using anterograde and retrograde tract-tracing techniques in the rat. After ipsilateral injections of biotinylated dextran amine (BDA) into the CeA and cholera toxin B subunit (CTb) into the NRA, the prominent overlapping distribution of BDA-labeled axon terminals and CTb-labeled neurons was found ipsilaterally in the lateral/ventrolateral PAG, where some of the BDA-labeled terminals made symmetrical synaptic contacts with somata and dendrites of the CTb-labeled neurons. After CTb injection into the lateral/ventrolateral PAG, CTb-labeled neurons were distributed mainly in the medial division of the CeA. After BDA injection into the lateral/ventrolateral PAG, BDA-labeled fibers were distributed mainly in and around the NRA within the medulla oblongata. Using a combined retrograde tracing and in situ hybridization technique, we further demonstrated that more than half of the CeA neurons labeled with Fluoro-Gold (FG) injected into the lateral/ventrolateral PAG were positive for glutamic acid decarboxylase 67 mRNA and that the vast majority of PAG neurons labeled with FG injected into the NRA expressed vesicular glutamate transporter 2 mRNA. The present results suggest that the glutamatergic PAG-NRA pathway is under the inhibitory influence of the GABAergic CeA neurons.

GABAergic neurons in the ventrolateral subnucleus of the nucleus tractus solitarius are in contact with Kölliker-Fuse nucleus neurons projecting to the rostral ventral respiratory group and phrenic nucleus in the rat.

After ipsilateral injections of biotinylated dextran amine (BDA) into the ventrolateral subnucleus of the nucleus tractus solitarius (vlNTS) and Fluoro-gold (FG) into the rostral ventral respiratory group (rVRG) region or into the phrenic nucleus (PhN) region in the rat, an overlapping distribution of BDA-labeled axon terminals and FG-labeled neurons was found in the Kölliker-Fuse (KF) nucleus ipsilateral to the injection sites. Using retrograde tracing combined with immunohistochemistry for glutamic acid decarboxylase isoform 67 (GAD67), we indicated that as many as 40% of the vlNTS neurons projecting to the KF were immunoreactive for GAD67. Using a combination of anterograde and retrograde tracing techniques, and immunohistochemistry for GAD67, we further demonstrated that the vlNTS axon terminals with GAD67 immunoreactivity established close contact to the rVRG- or PhN-projecting KF neurons. The present results suggest that GABAergic vlNTS fibers may exert inhibitory influences on the rVRG- as well as PhN-projecting KF neurons and these circuits may be involved in the respiratory reflexes such as the Hering-Breuer reflex.

Role of serum myostatin in the association between hyperinsulinemia and muscle atrophy in Japanese obese patients.

AIMS: The protein myostatin is a member of the transforming growth factor ß superfamily. This is mainly expressed in skeletal muscle and negatively regulates skeletal muscle growth. The present study aimed to elucidate the associations among circulating myostatin level, skeletal muscle mass, and metabolic profiles in Japanese obese patients. METHODS: Japanese obese outpatients (n = 74) were enrolled. We measured clinical parameters, quantified serum myostatin levels, and examined their associations in a cross-sectional manner. RESULTS: Both total skeletal muscle mass and serum myostatin level were higher in males than in females. Among 74 patients, serum myostatin level was positively correlated with skeletal muscle mass and serum immunoreactive insulin (IRI) level [correlation coefficient (r) = 0.294, P = 0.011; r = 0.262, P = 0.024, respectively]. Furthermore, multivariate linear regression analysis revealed that serum myostatin level was positively correlated with IRI after adjusting for gender and skeletal muscle mass (ß-coefficient = 0.230, P = 0.029, R2 = 0.236). CONCLUSIONS: In obese patients, serum myostatin level was elevated in conjunction with an increase in IRI level independent of skeletal muscle mass. This may imply possible novel pathological implications of serum myostatin in muscle mass and metabolism in obese patients with hyperinsulinemia.

Activation of AMPK-Regulated CRH Neurons in the PVH is Sufficient and Necessary to Induce Dietary Preference for Carbohydrate over Fat.

Food selection is essential for metabolic homeostasis and is influenced by nutritional state, food palatability, and social factors such as stress. However, the mechanism responsible for selection between a high-carbohydrate diet (HCD) and a high-fat diet (HFD) remains unknown. Here, we show that activation of a subset of corticotropin-releasing hormone (CRH)-positive neurons in the rostral region of the paraventricular hypothalamus (PVH) induces selection of an HCD over an HFD in mice during refeeding after fasting, resulting in a rapid recovery from the change in ketone metabolism. These neurons manifest activation of AMP-activated protein kinase (AMPK) during food deprivation, and this activation is necessary and sufficient for selection of an HCD over an HFD. Furthermore, this effect is mediated by carnitine palmitoyltransferase 1c (CPT1c). Thus, our results identify the specific neurons and intracellular signaling pathway responsible for regulation of the complex behavior of selection between an HCD and an HFD. VIDEO ABSTRACT.