The hypocretin neuron system: an arousal system in the human brain

Hypocretins are recently discovered neuropeptides produced by a small group of posterior hypothalamic neurons which project widely over the neuroaxis. In this study, we note that hypocretin neuron perikarya in the human brain are localized to the perifornical region of the posterior hypothalamus, extending into the lateral hypothalamus. These neurons lightly innervate all areas of cerebral cortex studied in a variable pattern with denser innervation of association cortex than primary motor or sensory cortex. There is a dense innervation of hypothalamus, locus coeruleus, raphe nuclei, midline thalamus and nucleus of the diagonal band-nucleus basalis complex of the forebrain. This pattern of projections from the hypocretin neurons is compatible with an important role in arousal and the maintenance of the waking state.     

Orexins (hypocretins): novel hypothalamic peptides with divergent functions.

The hypothalamus is the most important region in the control of food intake and body weight. The ventromedial "satiety center" and lateral hypothalamic "feeding center" have been implicated in the regulation of feeding and energy homeostasis by various studies of brain lesions. The discovery of orexin peptides, whose neurons are localized in the lateral hypothalamus and adjacent areas, has given us new insight into the regulation of feeding. Dense fiber projections are found throughout the brain, especially in the raphe nucleus, locus coeruleus, paraventricular thalamic nucleus, arcuate nucleus, and central gray. Orexins mainly stimulate food intake, but by the virtue of wide immunoreactive projections throughout the brain and spinal cord, orexins interact with various neuronal pathways to potentiate divergent functions. In this review, we summarize recent progress in the physiological, neuroanatomical, and molecular studies of the novel neuropeptide orexins (hypocretins).     

Transneuronal tracing of neural pathways that regulate hindlimb muscle blood flow

Despite considerable interest in the neural mechanisms that regulate muscle blood flow, the descending pathways that control sympathetic outflow to skeletal muscles are not adequately understood. The present study mapped these pathways through the transneuronal transport of two recombinant strains of pseudorabies virus (PRV) injected into the gastrocnemius muscles in the left and right hindlimbs of rats: PRV-152 and PRV-BaBlu. To prevent PRV from being transmitted to the brain stem via motor circuitry, a spinal transection was performed just below the L2 level. Infected neurons were observed bilaterally in all of the areas of the brain that have previously been shown to contribute to regulating sympathetic outflow: the medullary raphe nuclei, rostral ventrolateral medulla (RVLM), rostral ventromedial medulla, A5 adrenergic cell group region, locus coeruleus, nucleus subcoeruleus, and the paraventricular nucleus of the hypothalamus. The RVLM, the brain stem region typically considered to play the largest role in regulating muscle blood flow, contained neurons infected following the shortest postinoculation survival times. Approximately half of the infected RVLM neurons were immunopositive for tyrosine hydroxylase, indicating that they were catecholaminergic. Many (47%) of the RVLM neurons were dually infected by the recombinants of PRV injected into the left and right hindlimb, suggesting that the central nervous system has a limited capacity to independently regulate blood flow to left and right hindlimb muscles.     

Neuronal-vascular relationships in the raphe nuclei, locus coeruleus, and substantia nigra in primates.

A fluorescence histochemical and electron microscopic study of the monoaminergic cell groups in the squirrel monkey and Rhesus monkey brains has revealed the direct apposition of blood vessels to perikarya and dendrites of monoaminergic neurons. Capillaries and small arterioles or venules, ranging from 8-50 microns in diameter, showed perikarya and dendrites abutting the basement membrane without evidence of glial interposition. This neuronal-vascular relationship was present in 20% to 30% of the small vessels in the serotonergic nuclei raphe dorsalis and centralis superior and in the noradrenergic locus coeruleus. Such contacts were clearly present but observed less frequently in the dopaminergic substantia nigra pars compacta and in the serotonergic nuclei raphe obscurus, pallidus, magnus, and pontis. We postulate that monoamine-containing neurons apposed to blood vessels in certain regions of the brain may be influenced directly by hormones or other substances in blood.     

Physiological and Neurochemical Aspects of Corticotropin-Releasing Factor Actions in the Brain: The Role of the Locus Coeruleus

Corticotropin-releasing factor (CRF) is both a major regulator of the hypothalamo-pituitary-adrenal (HPA) axis and the activity of the autonomic nervous system. Besides, it exerts numerous effects on other physiological functions such as appetite control, motor and cognitive behavior and immune function. The basis for these effects is constituted by its distribution in hypothalamic and extrahypothalamic brain areas, the latter being represented by limbic structures such as the central nucleus of the amygdala or by brain stem neurons such as the locus coeruleus (LC) or nucleus of the solitary tract (NTS). The effects of CRF are mediated through recently described CRF-receptor subtypes, whose molecular biology, biochemistry and pharmacological regulation are discussed in detail. In the second part of this review, we will focus on the physiology of CRF-systems in the brain, with a particular emphasis on cardiovascular regulation, respiration, appetite control and stress-related behavior. Finally, the role of the locus coeruleus in the control of CRF-mediated behavioral activities is discussed. The interaction of noradrenergic and CRF-neurons clearly implies that CRF appears to directly activate LC neurons in a stressful situation, thus ultimately coordinating the bodily response to a stressful stimulus.     

Response of central monoaminergic neurons to lisuride: comparison with LSD.

The response of brain serotonergic (dorsal raphe), noradrenergic (locus coeruleus) and dopaminergic (pars compacta, substantia nigra) neurons to lisuride hydrogen maleate, a non-hallucinogenic ergot, was studied in the rat using extracellular single cell recording techniques. As has been previously reported for LSD, minute intravenous infusions of lisuride (1–5 μg/kg) produced a complete but reversible suppression of raphe unit spontaneous firing. A similar depressant response was noted when lisuride was applied to raphe units by microiontophoresis. In contrast, locus coeruleus neurons were accelerated by the drug at somewhat higher doses (25–50 μg/kg). Pars compacta neurons demonstrated a predominately depressant response to lisuride but many of the cells tested were only partially suppressed and a few units were accelerated. It is suggested that the marked alterations in central monoamine turnover which have been observed with lisuride are directly paralled by changes in impulse flow in monoaminergic neurons. The fact that lisuride has powerful suppressant effects on central serotonergic neurons but no psychotomimetic actions in man challenges the “serotonin theory” of hallucinogensis; however, other pharmacological properties may account for lisuride's lack of hallucinogenic effects. Further studies with lisuride may provide insight into those drug characteristics critical to the presence or absence of hallucinogenic action.     

Immunohistochemical mapping of galanin-like neurons in the rat central nervous system

Using an antiserum generated in rabbits against synthetic galanin (GA) and the indirect immunofluorescence method, the distribution of GA-like immunoreactive cell bodies and nerve fibers was studied in the rat central nervous system (CNS) and a detailed stereotaxic atlas of GA-like neurons was prepared. GA-like immunoreactivity was widely distributed in the rat CNS. Appreciable numbers of GA-positive cell bodies were observed in the rostral cingulate and medial prefrontal cortex, the nucleus interstitialis striae terminalis, the caudate, medial preoptic, preoptic periventricular, and preoptic suprachiasmatic nuclei, the medial forebrain bundle, the supraoptic, the hypothalamic periventricular, the paraventricular, the arcuate, dorsomedial, perifornical, thalamic periventricular, anterior dorsal and lateral thalamic nuclei, medial and central amygdaloid nuclei, dorsal and ventral premamillary nuclei, at the base of the hypothalamus, in the central gray matter, the hippocampus, the dorsal and caudoventral raphe nuclei, the interpeduncular nucleus, the locus coeruleus, ventral parabrachial, solitarii and commissuralis nuclei, in the A1, C1 and A4 catechaolamine areas, the posterior area postrema and the trigeminal and dorsal root ganglia. Fibers were generally seen where cell bodies were observed. Very dense fiber bundles were noted in the septohypothalamic tract, the preoptic area, in the hypothalamus, the habenula and the thalamic periventricular nucleus, in the ventral hippocampus, parts of the reticular formation, in the locus coeruleus, the dorsal parabrachial area, the nucleus and tract of the spinal trigeminal area and the substantia gelatinosa, the superficial layers of the spinal cord and the posterior lobe of the pituitary. The localization of the GA-like immunoreactivity in the locus coeruleus suggests a partial coexistence with catecholaminergic neurons as well as a possible involvement of the GA-like peptide in a neuroregulatory role.     

大白鼠脚内核的传入性联系

众所周知,肉食动物和大白鼠的脚内核,相当于灵长类的内侧苍白球(Nagy et al.1978;Fox and Schmitz 1944);它们的细胞形态、传入及传出均相同。早期以及近年来的一些研究工作者,虽然在研究其他核团的投射时,联系到一些本核团的传入,但是尚缺乏对本核团传人的系统研究。本实验即是应用辣根过氧化物酶的逆行传递法来研究大白鼠脚内核的传入性联系。     

Opiocortin and catecholamine projections to raphe nuclei

Afferent projections to the nucleus raphe magnus (NRM) and dorsal raphe nucleus (DRN) were identified using retrograde transport of horseradish peroxidase conjugated wheat germ agglutinin (HRP-WGA). Neurons were labeled in important nociceptive regions including periaqueductal gray (PAG), arcuate nucleus, lateral hypothalamus and medial thalamic nuclei following both injections. We have immunocytochemically identified opiocortin/WGA neurons in the arcuate nucleus following NRM and DRN injections. Dual stained catecholamine/WGA perikarya were found in zona incerta, locus coeruleus, substantia nigra, nucleus tractus solitarius and adjacent A2, C2 and C3, lateral paragigantocellular reticular nucleus/C1 and lateral reticular nucleus/A1 following DRN injections and in zona incerta, substantia nigra, nucleus tractus solitarius/A2 and lateral reticular nucleus/A1 after NRM injections. These results provide further evidence for opiocortin and catecholamine modulation of analgesia.     

Selective spread of herpes simplex virus in the central nervous system after ocular inoculation.

The spread of herpes simplex virus (HSV) was studied in the mouse central nervous system (CNS) after ocular inoculation. Sites of active viral replication in the CNS were identified by autoradiographic localization of neuronal uptake of tritiated thymidine. Labeled neurons were first noted in the CNS at 4 days postinoculation in the Edinger-Westphal nucleus, ipsilateral spinal trigeminal nucleus, pars caudalis, pars interpolaris, and ipsilateral dorsal horn of the rostral cervical spinal cord. By 5 days postinoculation, additional sites of labeling included the seventh nerve nucleus, nucleus locus coeruleus, and the nuclei raphe magnus and raphe pallidus. None of these sites are contiguous to nuclei infected at 4 days, but all are synaptically related to these nuclei. By 7 days postinoculation, no new foci of labeled cells were noted in the brain stem, but labeled neurons were noted in the amygdala, hippocampus, and somatosensory cortex. Neurons in both the amygdala and hippocampus receive axonal projections from the locus coeruleus. On the basis of these findings, we conclude that the spread of HSV in the CNS after intracameral inoculation is not diffuse but is restricted to a small number of noncontiguous foci in the brain stem and cortex which become infected in a sequential fashion. Since these regions are synaptically related, the principal route of the spread of HSV in the CNS after ocular infection appears to be along axons, presumably via axonal transport rather than by local spread.     

Distribution of B/K protein in rat brain

B/K protein is a recently isolated member of the double C2-like-domain protein family, which is highly abundant in rat brain. We generated high-titer rabbit polyclonal antibodies with specificity to the 55-kDa rat B/K protein, and examined the expression pattern of B/K protein in rat brain using an immunohistochemical staining method. Immunoreactivity to B/K protein was widely found in distinct regions of rat brain: strongly in the hypothalamus, most of the circumventricular organs, the locus coeruleus, the A5 neurons of the pons, and the anterior pituitary; moderately in the anterior olfactory nucleus, the raphe nucleus, the subfornical organ, and the median eminence; and faintly in the olfactory bulb, the telencephalon, the substantia nigra pars compacta, and the ventral tegmental area. In contrast, immunoreactivity to B/K protein was not observed in the thalamus, the cerebellum, the posterior pituitary, or the spinal cord. In most of the B/K-expressing neurons, immunoreactivity was expressed mainly in soma but not in nerve fibers. B/K was also expressed in nonneuronal cells such as the tanycytes and the subcommissural organ. In the vasopressin-secreting supraoptic and paraventricular nuclei of the hypothalamus, the site where B/K cDNA was originally isolated from, all of the neurons showing vasopressin immunoreactivity also expressed B/K protein, suggesting an overlap of their expression patterns.     

Visualization of monoaminergic neurons and neurotoxicity of MPTP in live transgenic zebrafish

We describe an enhancer trap transgenic zebrafish line, ETvmat2:GFP, in which most monoaminergic neurons are labeled by green fluorescent protein (GFP) during embryonic development. The reporter gene of ETvmat2:GFP was inserted into the second intron of vesicular monoamine transporter 2 (vmat2) gene, and the GFP expression pattern recapitulates that of the vmat2 gene. The GFP positive neurons include the large and pear-shaped tyrosine hydroxylase positive neurons (TH populations 2 and 4) in the posterior tuberculum of ventral diencephalon (PT neurons), which are thought to be equivalent to the midbrain dopamine neurons in mammals. We found that these PT neurons and two other GFP labeled non-TH type neuronal groups, one in the paraventricular organ of the posterior tuberculum and the other in the hypothalamus, were significantly reduced after exposure to MPTP, while the rest of GFP-positive neuronal clusters, including those in telencephalon, pretectum, raphe nuclei and locus coeruleus, remain largely unchanged. Furthermore, we showed that the effects of hedgehog signaling pathway inhibition on the development of monoaminergic neurons can be easily visualized in individual living ETvmat2:GFP embryos. This enhancer trap line should be useful for genetic and pharmacological analyses of monoaminergic neuron development and processes underlying Parkinson's disease.     

蓝斑核和中缝背核参与电剌激弓状核引起的大鼠...

The effect of electrical stimulation of hypothalamic arcuate nucleus (ARC) on intragastric pressure (IGP) was observed on 80 Wistar rats anaesthetized with urethan. The main results are as follows: (1) Electrical stimulation of ARC could cause an obvious decrease of IGP. (2) The reduction of IGP induced by electrical stimulation of ARC was not affected by intracerebroventricular injection of naloxone. (3) After lesioning of locus coeruleus or dorsal raphe, the effect of ARC stimulation was depressed. The results suggest that the locus coeruleus and dorsal raphe nucleus may be involved in the reduction of IGP induced by ARC stimulation, but without the involvement of beta-endorphinergic neurons.     

Effects of coagulation of thelocus coeruleus and pontine raphe nuclei on the background activity recorded from the rat cerebellar fastigial nucleus neurons

Simultaneous or separate coagulation of thelocus coeruleus (LC) and the pontine raphe nucleus (PRN) results in a significant increase of irregular-type background activity in the cerebellar fastigial nucleus neurons. There are also considerable changes in the dynamics of impulse sequences, in particular, the number of neurons with random interpulse intervals markedly increases. Destruction of theLC and/or PRN is followed by a marked drop in the mean frequency of discharges in the neurons of the fastigial nucleus.Neirofiziologiya/Neurophysiology, Vol. 26, No. 6, pp. 437–442, November–December, 1994.     

侧脑室注射肾上腺髓质素增加大鼠心血管相关核团中c-fos的表达并激活脑内一氧化氮神经元

本研究利用Fos蛋白和一氧化氮合酶(nNOS)双重免疫组化方法,观察侧腑脑室注射肾上腺髓质素(adrenomedullin,ADM)对大鼠心血管相关核中c-fos表达及一氧化氮神经元的影响,以探讨ADM在中枢的作用部位并研究其在中枢的作用是否有NO神经元参与。侧脑室注射ADM(1nmol／kg,3nmol／kg)诱发脑干的孤束核、最后区、蓝斑核、臂旁核和外侧巨细胞旁核,下丘脑的室旁核、视上核才腹内侧核以及前脑的中央杏仁核和外侧缰核等多个部位的心血管中枢出现大量Fos样免疫反应神经元。侧脑室注射ADM(3nmol／kg),引起脑干的孤束核、外侧巨细胞旁核,下丘脑的室旁核、视上核内的Fos-nNOS双标神经元增加;ADM(1nmol／kg)亦可引起室旁核、视上核内的Fos-nNOS双标神经元增加,而对孤束核、外侧巨细胞旁核内的Fos-nNOS双标神经元无影响。降钙素基因相关肽(calcitonin gene—related peptide,CGRP)受体拈抗剂CGRP8-37(30nmol／kg)可明显减弱此效应。以上结果表明,ADM可兴奋脑内多个心血管相关核闭的神经元并激活室旁核、视上核、孤束核及外侧巨细胞核内一氧化氮神经元,此效应可能部分山CGRP受体介导。     

Interaction of opiate peptide and noradrenalin systems: Light microscopic studies

In this light microscopic immunocyto-chemical study β-Endorphin (β-END), leu-enkephalin and dopamine-βhydroxylase (DBH) antisera are used to obtain an overview of the interaction of the noradrenergic and opiate peptide systems in brain. Serial brain areas were analyzed for DBH and then for β-END or leu-enkephalin. Several areas were evaluated for cell and fiber interactions between these systems. The areas of richest possible contact between β-END and DBH positive systems include the rostral locus coeruleus region, the periaqueductal grey, possibly the dorsal thalamus, the paraventricular hypothalamus and the arcuate nucleus. Enkephalin cells and fibers were seen surrounding the locus coeruleus throughout its length with a few fibers in the nucleus itself.     

Substance P-immunoreactive neurons in the brainstem of the cat related to cardiovascular centers

Summary The distribution of substance P-immunoreactivity (SP-IR) in the brainstem and spinal cord of normal and colchicine-pretreated cats was analysed using the peroxidase-antiperoxidase (PAP) technique. Numerous SP-IR fibers are present in the nucleus solitarius, nucleus dorsalis nervi vagi and nucleus spinalis nervi trigemini, various parts of the formatio reticularis, substantia grisea centralis mesencephali, locus coeruleus and nucleus parabrachialis. SP-IR perikarya occur in the substantiae gelatinosa and intermedia of the spinal cord, the nucleus spinalis nervi trigemini-pars caudalis, the nucleus dorsalis nervi vagi, and the nucleus solitarius, as well as in the adjacent formatio reticularis and the medullary nuclei of the raphe. In addition, SP-IR cell bodies are located in the nuclei raphe magnus and incertus, ventral and dorsal to the nucleus tegmentalis dorsalis (Gudden), nucleus raphe dorsalis, substantia grisea centralis mensencephali, locus coeruleus, nucleus parabrachialis and colliculus superior.The results indicate that SP-IR neurons may be involved in the regulation of cardiovascular functions both at the central and peripheral level. A peripheral afferent portion seems to terminate in the nucleus solitarius and an efferent part is postulated to originate from the nucleus dorsalis nervi vagi and from the area of the nuclei retroambiguus, ambiguus and retrofacialis.     

Dorsomedial hypothalamic and brainstem pathways controlling thermogenesis in brown adipose tissue

1. The rostral medullary raphe pallidus contains sympathetic premotor neurons controlling thermogenesis in brown adipose tissue (BAT).

2. Disinhibition of neurons in the dorsomedial hypothalamus (DMH) stimulates BAT thermogenesis through activation of neurons in raphe pallidus.

3. An increase in BAT sympathetic outflow and BAT thermogenesis following microinjection of prostaglandin E2 into the preoptic area requires activation of both DMH neurons and raphe pallidus neurons.

4. DMH contains a population of neurons receiving a tonically- active GABAergic inhibition which mediate increases in BAT thermogenesis through stimulation of BAT sympathetic premotor neurons in raphe pallidus.