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.

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.

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.

Orexinergic fibers are in contact with Kölliker-Fuse nucleus neurons projecting to the respiration-related nuclei in the medulla oblongata and spinal cord of the rat.

The neural pathways underlying the respiratory variation dependent on vigilance states remain unsettled. In the present study, we examined the orexinergic innervation of Kölliker-Fuse nucleus (KFN) neurons sending their axons to the rostral ventral respiratory group (rVRG) and phrenic nucleus (PhN) as well as to the hypoglossal nucleus (HGN) by using a combined retrograde tracing and immunohistochemistry. After injection of cholera toxin B subunit (CTb) into the KFN, CTb-labeled neurons that are also immunoreactive for orexin (ORX) were found prominently in the perifornical and medial regions and additionally in the lateral region of the hypothalamic ORX field. After injection of fluorogold (FG) into the rVRG, PhN or HGN, we found an overlapping distribution of ORX-immunoreactive axon terminals and FG-labeled neurons in the KFN. Within the neuropil of the KFN, asymmetrical synaptic contacts were made between these terminals and neurons. We further demonstrated that many neurons labeled with FG injected into the rVRG, PhN, or HGN are immunoreactive for ORX receptor 2. Present data suggest that rVRG-, PhN- and HGN-projecting KFN neurons may be under the excitatory influence of the ORXergic neurons for the state-dependent regulation of respiration.

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.

Glutamatergic lateral parabrachial neurons innervate orexin-containing hypothalamic neurons in the rat.

We performed this study to understand the anatomical substrates of parabrachial nucleus (PBN) modulation of orexin (ORX)-containing neurons in the hypothalamus. After biotinylated dextranamine (BDA) injection into the lateral PBN and immunostaining of ORX-containing neurons in the rat, the prominent overlap of the distribution field of the BDA-labeled fibers and that of the ORX-immunoreactive (ir) neurons was found in the lateralmost part of the dorsomedial nucleus and adjacent dorsal perifornical area (this overlapping field was referred to as "suprafornical area" in the present study), and the labeled axon terminals made asymmetrical synaptic contacts with somata and dendrites of the ORX-ir neurons. We further revealed that almost all the "suprafornical area"-projecting lateral PBN neurons were positive for vesicular glutamate transporter 2 mRNA and very few of them were positive for glutamic acid decarboxylase 67 mRNA. The present data suggest that ORX-containing neurons in the "suprafornical area" may be under the excitatory influence of the glutamatergic lateral PBN neurons probably for the regulation of arousal and waking.

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.

Amygdaloid axons innervate melanin-concentrating hormone- and orexin-containing neurons in the mouse lateral hypothalamus.

This study was performed to understand the anatomical substrates of amygdaloid modulation of feeding-related peptides-containing neurons in the lateral hypothalamic area (LHA). After biotinylated dextranamine (BDA) injection into the central amygdaloid nucleus (CeA) and immunostaining of melanin-concentrating hormone (MCH)- or orexin (ORX)-containing hypothalamic neurons in the mouse, the prominent overlap of the distribution field of the BDA-labeled fibers and that of the MCH-immunoreactive (ir) or ORX-ir neurons was found in the dorsolateral part of the LHA, and the labeled axon terminals made symmetrical synaptic contacts with somata and dendrites of the MCH-ir or ORX-ir neurons. It was further revealed that nearly all the BDA-labeled axon terminals in the dorsolateral part of LHA were immunoreactive for glutamic acid decarboxylase, an enzyme for conversion of glutamic acid to gamma-aminobutyric acid (GABA). The present data suggest that the CeA is involved in the regulation of feeding behavior by exerting its GABAergic inhibitory action upon the MCH- and ORX-containing LHA neurons.

Posterior lateral hypothalamic axon terminals are in contact with trigeminal premotor neurons in the parvicellular reticular formation of the rat medulla oblongata.

This study was performed to understand the anatomical substrates of hypothalamic modulation of jaw movements. After cholera toxin B subunit (CTb) injection into the parvicellular reticular formation (RFp) of the rat medulla oblongata, where many trigeminal premotor neurons have been known to exist, numerous CTb-labeled neurons were found in the posterior lateral hypothalamus (PLH) bilaterally with a clear-cut ipsilateral dominance. After ipsilateral injections of biotinylated dextran amine (BDA) into the PLH and CTb into the motor trigeminal nucleus (Vm), the prominent distribution of BDA-labeled axon terminals around CTb-labeled neurons was found in the RFp region just ventral to the nucleus of the solitary tract and medial to the spinal trigeminal nucleus ipsilateral to the injection sites. Within the neuropil of the RFp, BDA-labeled axon terminals made an asymmetrical synaptic contact predominantly with dendrites and additionally with somata of the RFp neurons, some of which were labeled with CTb. It was further revealed that these BDA-labeled axon terminals were immunoreactive for vesicular glutamate transporter 2. The present data suggest that the PLH plays an important role in the control of jaw movements by exerting its glutamatergic excitatory action upon RFp neurons presynaptic to trigeminal motoneurons.

Synaptic organization of GABAergic projections from the substantia nigra pars reticulata and the reticular thalamic nucleus to the parafascicular thalamic nucleus in the rat.

The ventrolateral part of the parafascicular thalamic nucleus (PF), which is considered to take part in the control mechanism of orofacial motor functions, receives projection fibers not only from the dorsolateral part of the substantia nigra pars reticulata (SNr) but also from the ventral part of the reticular thalamic nucleus (RT) [Tsumori et al., Brain Res. 858 (2000) 429]. In order to better understand the influence of these fibers upon the PF projection neurons, the morphology, synaptology and chemical nature of them were examined in the present study. After ipsilateral injections of Phaseolus vulgaris-leucoagglutinin (PHA-L) into the dorsolateral part of the SNr and biotinylated dextran amine (BDA) into the ventral part of the RT, overlapping distributions of PHA-L-labeled SNr fibers and BDA-labeled RT fibers were seen in the ventrolateral part of the PF. At the electron microscopic level, the SNr terminals made synapses predominantly with the medium to small dendrites and far less frequently with the somata and large dendrites, whereas approximately half of the RT terminals made synapses with the somata and large dendrites and the rest did with the medium to small dendrites of PF neurons. Some of single dendritic as well as single somatic profiles received convergent synaptic inputs from both sets of terminals. These terminals were packed with pleomorphic synaptic vesicles and formed symmetrical synapses. After combined injections of PHA-L into the dorsolateral part of the SNr, BDA into the ventral part of the RT and wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the ventrolateral part of the striatum or into the rostroventral part of the lateral agranular cortex, WGA-HRP-labeled neurons were embedded in the plexus of PHA-L- and BDA-labeled axon terminals within the ventrolateral part of the PF, where the PHA-L- and/or BDA-labeled terminals were in synaptic contact with single somatic and dendritic profiles of the WGA-HRP-labeled neurons. Furthermore, the SNr and RT axon terminals were revealed to be immunoreactive for gamma-aminobutyric acid (GABA), by using the anterograde BDA tracing technique combined with immunohistochemistry for GABA. The present data suggest that GABAergic SNr and RT fibers may exert different inhibitory influences on the PF neurons for regulating the thalamic outflow from the PF to the cerebral cortex and/or striatum in the control of orofacial movements.