Corticotropin-releasing factor neurons in the hypothalamic paraventricular nucleus are involved in arousal/yawning response of rats

Our previous studies have suggested that activation of the hypothalamic paraventricular (PVN) descending oxytocinergic projections is involved in the induction of yawning accompanied by an arousal response, but the possibility that neural systems other than the oxytocinergic system in the PVN also mediate the arousal/yawning response cannot be ruled out. We assessed the activity of corticotropin-releasing factor (CRF) neurons during yawning induced by the PVN stimulation in anesthetized, spontaneously breathing rats using double-staining for c-Fos and CRF. Yawning response was evaluated by monitoring an intercostals electromyogram as an index of inspiratory activity and a digastric electromyogram as an indicator of mouth opening. We also recorded the electrocorticogram (ECoG) to determine the arousal response during yawning. Microinjection of l-glutamate (2-5 nmol) into the PVN produced a frequent yawning accompanied by an arousal shift in the ECoG, and these behavioral effects were associated with a significant increase of c-Fos positive CRF neurons in the medial parvocellular subdivision of the PVN. In addition, a marked enhancement in the c-Fos expression was found in the both locus coeruleus (LC) and global area in the cortex when the frequency of yawning response was increased by the PVN stimulation, suggesting that the arousal response during yawning might be mediated by the activation of LC neurons. The present study suggests that an activation of CRF neurons in the PVN is responsible for the arousal response accompanied by yawning behavior.     

Contribution of dopamine neurons in the medial zona incerta to the innervation of the central nucleus of the amygdala, horizontal diagonal band of Broca and hypothalamic paraventricular nucleus

Results of previous studies suggested that incertohypothalamic dopamine (IHDA) neurons located in the medial zona incerta (MZI) project to the central nucleus of the amygdala (cAMY), horizontal diagonal band of Broca (HDB), and paraventricular nucleus (PVN). The overall goal of the present study was to determine the relative contribution of IHDA neurons to the DA innervation of these brain regions. A combined fluorescent and in situ hybridization histochemical procedure was employed to localize the retrograde tracer fluoro-gold (FG) in cells expressing tyrosine hydroxylase (TH) mRNA in the MZI following its iontophoretic injection into either the cAMY, HDB or PVN. For comparison, the numbers of dual labeled FG/TH mRNA neurons in the midbrain were also determined. One week after unilateral injection of FG into the cAMY, cells containing FG+TH mRNA were found in the ipsilateral MZI, substantia nigra zona compacta (SNC) and ventral tegmental area (VTA). The total numbers of cells labeled with FG varied with the size of the injection site, but the ratio of dual labeling in the MZI to that of the SNC–VTA remained constant across animals at approximately 1:6. FG injections into the HDB resulted in a ratio of dual labeled cells in the ipsilateral MZI and VTA of approximately 1:2, but no dual labeled cells were found in the SNC. Dual labeled cells were only found in the ipsilateral MZI in animals receiving FG injections in the PVN. Thus, DA terminals in the PVN originate exclusively from IHDA neurons in the MZI, whereas these neurons provide only a portion of the DA innervation of the cAMY and HDB. The similar distribution of dual labeled cells in the MZI following FG injections into the cAMY, HDB and PVN suggests that perikarya of IHDA neurons projecting to these regions are not organized into distinct groups.     

Amygdala enterostatin induces c-Fos expression in regions of hypothalamus that innervate the PVN

Enterostatin selectively inhibits the intake of the dietary fat after both central and peripheral administration. Our previous studies have shown that a central site of action is the central nucleus of amygdala. Serotonergic agonists administered into the paraventricular nucleus (PVN) inhibit fat intake and serotonergic antagonists block the feeding suppression induced by amygdala enterostatin, suggesting that there are functional connections between the PVN and amygdala that affect the feeding response to enterostatin. Our purpose was to identify the anatomic and functional projections from the amygdala to the PVN and hypothalamic area that are responsive to enterostatin, by using a retrograde tracer fluorogold (FG) and c-Fos expression. Rats were injected with fluorogold unilaterally into the PVN and a chronic amygdala cannula was implanted ipsilaterally. After 10 days recovery, rats were injected with either enterostatin (0.1 nmol) or saline vehicle (0.1 microl) into the amygdala and sacrificed 2 h later by cardiac perfusion under anesthesia. The brains were subjected to dual immunohistochemistry to visualize both FG and c-Fos-positive cells. FG/c-Fos double-labeled cells were found in forebrain regions including the PVN, amygdala, lateral hypothalamus (LH), ventral medial hypothalamus (VMH) and arcuate nucleus (ARC). The data provides the first anatomical evidence that enterostatin activates amygdala neurons that have functional and anatomic projections directly to the PVN and also activates neurons in the arcuate, LH and VMH, which innervate the PVN.     

Centrally administered bombesin activates COX-containing spinally projecting neurons of the PVN in anesthetized rats

The paraventricular nucleus (PVN) of the hypothalamus has a heterogenous structure containing different types of output neurons that project to the median eminence, posterior pituitary, brain stem autonomic centers and sympathetic preganglionic neurons in the spinal cord. Presympathetic neurons in the PVN send mono- and poly-synaptic projections to the spinal cord. In the present study using urethane-anesthetized rats, we examined the effects of centrally administered bombesin (a homologue of the mammalian gastrin-releasing peptide) on the mono-synaptic spinally projecting PVN neurons pre-labeled with a retrograde tracer Fluoro-Gold (FG) injected into T8 level of the spinal cord, with regard to the immunoreactivity for cyclooxygenase (COX) isozymes (COX-1/COX-2) and Fos (a marker of neuronal activation). FG-labeled spinally projecting neurons were abundantly observed in the dorsal cap, ventral part and posterior part of the PVN. The immunoreactivity of each COX-1 and COX-2 was detected in FG-labeled spinally projecting PVN neurons in the vehicle (10 μl of saline/animal, i.c.v.)-treated group, while bombesin (1 nmol/animal, i.c.v.) had no effect on the number of these immunoreactive neurons for each COX isozyme with labeling of FG. On the other hand, the peptide significantly increased the number of double-immunoreactive neurons for Fos and COX-1/COX-2 with FG-labeling in the PVN (except triple-labeled neurons for FG, COX-2 and Fos in the dorsal cap of the PVN), as compared to those of vehicle-treated group. These results suggest that centrally administered bombesin activates spinally projecting PVN neurons containing COX-1 and COX-2 in rats.     

Cholecystokinin activates catecholaminergic neurons in the caudal medulla that innervate the paraventricular nucleus of the hypothalamus in rats

Stimulation of gastric vagal afferents by systemic administration of cholecystokinin octapeptide (CCK) inhibits gastric motility, reduces food intake, and stimulates pituitary secretion of oxytocin and adrenocorticotropic hormone in rats. To characterize further the central neurol circuits responsible for these effects, the present study used triple-labeling immunocytochemical methods to determine whether or not exogenous CCK activates cFos expression in catecholaminergic neurons in the caudal medulla that project to the paraventricular nucleus of the hypothalamus (PVN). To identify these neurons, the retrograde tracer fluorogold (FG) was iontophoresed into the PVN of anesthetized rats under stereotaxic guidance. After 2 weeks, rats were injected with CCK (100 μg/kg, i. p.) and then anesthetized and killed 1 hour later by perfusion fixation. Medullary sections were processed for triple immunocytochemical localization of cFos, retrogradely transported FG, and tyrosine hydroxylase (TH). In rats with FG injections centered in the PVN (n = 10), approximately 70% of the FG-labeled neurons in the caudal nucleus of the solitary tract (NST) and ventrolateral medulla (VLM) expressed cFos. Of these activated PVN-projecting neurons, approximately 78% in the NST and 89% in the VLM were catecholaminergic (TH positive). These results indicate that PVN-projecting catecholaminergic neurons within the caudal medulla are activated by periph eral administration of CCK, further implicating these ascending catecholaminergic path ways in the neuroendocrine, physiological, and behavioral effects produced by gastric vagal stimulation. © 1995 Wiley-Liss, Inc.     

Mating-activated brainstem catecholaminergic neurons in the female rat

Central catecholaminergic systems play an important role in the control of reproductive activities including sexual behavior, luteinizing hormone (LH) and prolactin secretion. It has been reported that catecholaminergic neurons in the locus coeruleus (A6) are activated by mating in rabbits and ferrets, animals known as reflex ovulators. This study used Fos as a marker of neuronal activity to examine whether brainstem catecholaminergic neurons are activated by mating in the spontaneous ovulator, the female rat. Proestrous rats receiving intromissions (mated group) from males or mounts-without-intromission (mounted group) were sacrificed along with rats taken directly from their home cage (control group) 90 min after the beginning of mating or mounting. Double-label immunocytochemistry was used to examine the expression of c-Fos in catecholaminergic neurons labeled by tyrosine hydroxylase (TH) antibody, or adrenergic neurons labeled by phenylethanolamine-N-methyl transferase (PNMT) antibody. Double label immunofluorescent immunohistochemistry was used to determine the number of neurons containing the estrogen receptor (ERalpha) that were activated by mating in these brain areas. The results showed that mating-with-intromissions induced a significant increase in the percentage of TH/Fos colabeled neurons in both A1 and A2 cells compared to mounting-without-intromission or control. In both these areas, over 50% ERalpha-ir neurons were activated after mating while mounting-without-intromission did not affect the percentage of colabeled Fos/ERalpha neurons. In A6 region, neither the expression of Fos nor the percentage of TH/Fos colabeled cells was influenced by either mating or mounting compared to controls. The percentage of PNMT-containing neurons colabeled with Fos was not different in C1 and C2 among the three experimental groups. The results indicate that catecholaminergic neurons were activated by mating in A1 and A2 but not in adjoining adrenergic C1 and C2 cells. In contrast to the findings that catecholaminergic neurons in A6 are activated by mating in induced ovulators, mating did not affect neuronal activity in A6 neurons in the female rat. In A1 and A2 areas, a high percentage of neurons containing ERalpha were activated by mating suggesting both tactile and hormonal information may converge on these populations of neurons. The activated catecholaminergic neurons in A1 and A2 may be an important pathway by which sensory information generated during sexual interaction modulates both behavior and pituitary function.     

Forebrain origins of glutamatergic innervation to the rat paraventricular nucleus of the hypothalamus: differential inputs to the anterior versus posterior subregions

The hypothalamic paraventricular nucleus (PVN) regulates numerous homeostatic systems and functions largely under the influence of forebrain inputs. Glutamate is a major neurotransmitter in forebrain, and glutamate neurosignaling in the PVN is known to mediate many of its functions. Previous work showed that vesicular glutamate transporters (VGluTs; specific markers for glutamatergic neurons) are expressed in forebrain sites that project to the PVN; however, the extent of this presumed glutamatergic innervation to the PVN is not clear. In the present study retrograde FluoroGold (FG) labeling of PVN-projecting neurons was combined with in situ hybridization for VGluT1 and VGluT2 mRNAs to identify forebrain regions that provide glutamatergic innervation to the PVN and its immediate surround in rats, with special consideration for the sources to the anterior versus posterior PVN. VGluT1 mRNA colocalization with retrogradely labeled FG neurons was sparse. VGluT2 mRNA colocalization with FG neurons was most abundant in the ventromedial hypothalamus after anterior PVN FG injections, and in the lateral, posterior, dorsomedial, and ventromedial hypothalamic nuclei after posterior PVN injections. Anterograde tract tracing combined with VGluT2 immunolabeling showed that 1) ventromedial nucleus-derived glutamatergic inputs occur in both the anterior and posterior PVN; 2) posterior nucleus-derived glutamatergic inputs occur predominantly in the posterior PVN; and 3) medial preoptic nucleus-derived inputs to the PVN are not glutamatergic, thereby corroborating the innervation pattern seen with retrograde tracing. The results suggest that PVN subregions are influenced by varying amounts and sources of forebrain glutamatergic regulation, consistent with functional differentiation of glutamate projections.     

Tyrosine Hydroxylase Messenger Ribonucleic Acid Levels Increase in A1 but not Locus Ceruleus Noradrenergic Neurons in Proestrous Rats but not in Diestrous or Androgen-Sterilized Animals

Norepinephrine (NE) turnovers (an index of secretion) increase in the hypothalamus of proestrous rats concomitant with luteinizing hormone surges, whereas, neither of these events are observed in diestrous nor in androgen-sterilized rats. Increased hypothalamic NE release may occur as a consequence of the withdrawal of local inhibitory γ-aminobutyric acid and opiate controls on specific presynaptic NE terminals and/or as a result of an increase in activity within noradrenergic neurons. Tyrosine hydroxylase (TH) is the rate-limiting enzyme for the synthesis of NE and our earlier studies revealed that increases in TH mRNA in A1 and locus ceruleus (LC) neurons can serve as an index of increased activity within these cells. In the present study, we evaluated whether TH message levels change in A1 and LC neurons prior to and during the hours when luteinizing hormone surges and increased NE turnovers are observed. As controls, TH mRNA levels in A1 and LC neurons were evaluated at the same hours of day in diestrous day 2 and in androgen-sterilized rats. In situ hybridization histochemistry and quantitative image analysis methods were used to measure changes in TH mRNA levels. Luteinizing hormone surges in proestrous rats began at 1500 h, peaked between 1600 and 1700 h and declined, thereafter, to 2000 h. In contrast, plasma luteinizing hormone remained basal throughout the day in diestrous and androgen-sterilized rats. While A1 neuronal TH mRNA levels did not differ in the three groups of rats during the morning (0930 to 1030 h), these message levels were significantly elevated in proestrous rats during the afternoon (1645 to 1715 h) and remained high at 2000 to 2030 h. In contrast, no changes in TH mRNA levels were observed in A1 neurons throughout the afternoon in diestrous animals or androgen-sterilized rats. TH mRNA levels in the LC did not differ in the three groups of rats and they remained unchanged throughout the afternoon hours we examined. From these observations we conclude that concomitant with afternoon proestrous luteinizing hormone surges and the accompanying increase in hypothalamic NE secretion, there is an increase in activity within A1 but not LC neurons. These data suggest that the proestrous increase in hypothalamic NE turnover we previously observed is not due solely to withdrawal of local inhibitory controls of presynaptic NE release but it also involves an increase in activity within A1 but not LC neurons.     

Regulation of Neuronal Activation by Alpha2A Adrenergic Receptor Agonist

Stress factors induce neuronal activation in brain areas that are related to anxiety and fear. High doses of caffeine induce neuronal activation with Ca2+ influx followed by expression of the immediate early gene c-fos. In the present study, we investigated c-Fos protein expression in stress-responsive brain areas induced by caffeine, as well as the role of alpha2A receptor in the regulation of neuronal activation. Immunohistochemical analysis showed that an acute effect of caffeine induced c-Fos protein expression in the hippocampus, the bed nucleus of stria terminalis (BNST), the lateral septum, the basolateral and central amygdala, the paraventricular hypothalamic nucleus (PVN), the locus coeruleus, and the lateral parabrachial nucleus (LPBN). However, c-Fos expression was attenuated after repeated treatment of caffeine, spaced 24 h apart, compared to a single acute effect. Alpha2A receptor activation with the agonist guanfacine attenuated the acute effect of caffeine in terms of c-Fos expression in neurons in the CA1-CA3 areas of hippocampus, the locus coeruleus and the LPBN as compared with effect of caffeine alone, whereas the number of c-Fos expressing neurons increased in the lateral septum, the dorsal BNST, the central amygdala, and the PVN, areas that are densely innervated by noradrenergic neurons. Guanfacine alone induced c-Fos protein expression in neurons in the central amygdala, the dorsal BNST, the PVN, the LPBN, and the caudal nucleus of the solitary tract. Guanfacine alone also induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in neurons expressing c-Fos in the dorsal BNST, the central amygdala, and the LPBN. These results suggest that alpha2A receptor activation modulates synaptic transmission in neuronal circuits that are correlated with stress in vivo.     

Morphology,axonal projection pattern,and responses to optic nerve stimulation of thalamic neurons in the fire-bellied toad Bombina orientalis

Intracellular recording and biocytin labeling were carried out in the fire-bellied toad Bombina orientalis to study the morphology and axonal projections of thalamic (TH) neurons and their responses to electrical optic nerve stimulation. Labeled neurons (n = 142) were divided into the following groups: TH1 neurons projecting to the dorsal striatum; TH2 neurons projecting to the amygdala, nucleus accumbens, and septal nuclei; TH3 neurons projecting to the medial or dorsal pallium; TH4 neurons with projections ascending to the dorsal striatum or ventral striatum/amygdala and descending to the optic tectum, tegmentum, and rostral medulla oblongata; TH5 neurons with projections to the tegmentum, rostral medulla oblongata, prectectum, or tectum; and TH6 neurons projecting to the hypothalamus. TH1 neurons are found in the central, TH2 neurons in the anterior and central, TH3 neurons in the anterior dorsal nucleus, and TH4 and TH5 neurons in the posterior dorsal or ventral nucleus. Neurons with descending projections arborize in restricted parts of retinal afferents; neurons with ascending projections do not substantially arborize within retinal afferents. At electrical optic nerve stimulation, neurons in the ventral thalamus respond with excitation at latencies of 10.8 msec; one-third of them follow repetitive stimulation and possibly are monosynaptically driven. Neurons in the dorsal thalamus respond mostly with inhibition at latencies of 42.3 msec and are polysynaptically driven. This corroborates the view that neurons in the dorsal thalamus projecting to the telencephalon receive no substantial direct retinal input and that the thalamopallial pathway of amphibians is not homologous to the mammalian retinogeniculocortical pathway.     

Morphology, axonal projection pattern, and responses to optic nerve stimulation of thalamic neurons in the salamander Plethodon jordani

In the salamander Plethodon jordani, the morphology and axonal projections of thalamic (TH) neurons and their responses to electrical optic nerve stimulation were determined by intracellular recording and biocytin labeling under in vitro, whole-brain conditions. Based on their axonal projections, labeled neurons (n = 76) were divided into the following groups: TH1 neurons, with mostly ipsilateral projections to the striatum; TH2 neurons, with ipsilateral or bilateral projections to the medial amygdala and nucleus accumbens; TH3 neurons, with bilateral projections to the medial and dorsal pallium; TH4 neurons, with mostly ipsilateral projections to the striatum and ipsilateral projections to the tectum opticum, tegmentum, and rostral medulla oblongata; and TH5 neurons, with ipsilateral projections to the tegmentum, medulla oblongata, and rostral spinal cord without (TH5.1) or with (TH5.2) additional projections to the optic tectum. TH1-TH4 neurons are found in the dorsal thalamus and around the sulcus medialis, and TH5 neurons are found in the ventral thalamus. Labeled neurons with ascending projections, i.e., the more dorsally situated TH1-TH4 neurons, are mostly inhibited by electrical stimulation of the optic nerve and have significantly longer latencies (mean +/- S.D., 42.1 +/- 11.6 msec) than neurons with exclusively descending projections, i.e., the ventrally located TH5 neurons (8.5 +/- 6.1 msec), which receive the bulk of retinal afferents and show excitation at electrical optic nerve stimulation. Neurons recorded without labeling in the dorsal thalamus likewise exhibit mostly inhibition and have significantly longer latencies (35.7 +/- 18.9 msec) than those recorded in the ventral thalamus (10.9 +/- 7.7 msec), which mostly show excitation. None of the neurons recorded in the dorsal thalamus followed repetitive stimulation of the optic nerve. Thus, neurons situated in the dorsal thalamus and projecting to pallial or subpallial telencephalic targets are unlikely to receive monosynaptic or oligosynaptic, excitatory retinal input. Accordingly, no retino-thalamo-telencephalic pathway homologous to that found in amniotes appears to exist in salamanders.     

Regulatory role of glucocorticoids and glucocorticoid receptor mRNA levels on tyrosine hydroxylase gene expression in the locus coeruleus during repeated immobilization stress

Sustained responsiveness of the hypothalamic-pituitary-adrenal (HPA) axis during chronic or repeated stress is associated with continuous activation of ascending noradrenergic neurons from the brainstem to the hypothalamic paraventricular nucleus (PVN). The fact that glucocorticoid receptor (GR) exists in the brainstem noradrenergic neurons including locus coeruleus (LC) suggests that glucocorticoids play a modulatory role in maintaining the activity of these neurons during chronic stress. To determine whether alterations in the sensitivity of noradrenergic neuronal activity to endogenous CORT occur during chronic or repeated stress, tyrosine hydroxylase (TH) and GR mRNA expressions in the LC were examined in acute (2 h) and repeated (2 h daily, 14 days) immobilization stress, using sham-operated rats and adrenalectomized rats with a moderate dose of CORT replacement (ADX+CORT group). In acute stress, TH mRNA in the LC increased in the ADX+CORT rats, but not in sham operated rats. In repeated stress, however, elevated endogenous CORT failed to inhibit TH mRNA responses in sham rats; LC TH mRNA in sham rats responded to the same extent as in ADX+CORT rats. A reduction of GR mRNA in the LC was observed in the acutely stressed and repeatedly stressed sham group, but not in the ADX+CORT groups. The decrease in LC GR mRNA levels in sham rats tended to be greater after repeated than after acute stress. LC GR mRNA levels decreased in response to systemic CORT treatment (200 mg pellet sc, for 14 days) and increased in response to adrenalectomy; neither CORT treatment nor adrenalectomy influenced TH mRNA levels in the LC. These results suggest that glucocorticoid responses to acute immobilization prevent LC TH mRNA levels from rising significantly, while glucocorticoids appear to decrease their capacity to restrain LC TH mRNA during repeated immobilization. Although the results clearly show glucocorticoid-dependent alterations in LC GR mRNA expression, the association between increased TH mRNA and decreased GR mRNA in the LC remains to be elucidated.     

Branching projections of catecholaminergic brainstem neurons to the paraventricular hypothalamic nucleus and the central nucleus of the amygdala in the rat

In this study, we have employed triple fluorescent-labelling to reveal the distribution of catecholaminergic neurons within three brainstem areas which supply branching collateral input to the central nucleus of the amygdala (CNA) and the hypothalamic paraventricular nucleus (PVN): the ventrolateral medulla (VLM), the nucleus of the solitary tract (NTS) and the locus coeruleus (LC). The catecholaminergic identity of the neurons was revealed by immunocytochemical detection of the biosynthetic enzyme, tyrosine hydroxylase. The projections were defined by injections of two retrograde tracers, rhodamine- and fluorescein-labelled latex microspheres, in the CNA and PVN, respectively. In the VLM and NTS, the greatest incidence of neurons which contained both retrograde tracers was found at the level of the area postrema. These neurons were mainly located within the confines of the A1/C1 (VLM) and A2 (NTS) catecholaminergic neuronal groups. Double-projecting neurons in the LC (A6) were distributed randomly within the nucleus. It was found that 15% in the VLM, 10% in the NTS and 5% in the LC of the retrogradely labelled cells projected via branching collaterals to the PVN and CNA. One half of these neurons in the VLM and NTS were catecholaminergic, in contrast to the LC where virtually all double-retrogradely labelled neurons revealed tyrosine hydroxylase immunoreactivity. In the other brainstem catecholaminergic cell groups (A5, A7, C3), no catecholaminergic neurons were found that supplied branching collaterals to the CNA and PVN. Our results indicate that brainstem neurons may be involved in the simultaneous transmission of autonomic-related signals to the CNA and the PVN. Catecholamines are involved in these pathways as chemical messengers. Brainstem catecholaminergic and non-catecholaminergic neurons, through collateral branching inputs may provide coordinated signalling of visceral input to rostral forebrain sites. This may lead to a synchronized response of the CNA and PVN for the maintenance of homeostasis.     

Hypocretin1/OrexinA-containing axons innervate locus coeruleus neurons that project to the Rat medial prefrontal cortex. Implication in the sleep-wakefulness cycle and cortical activation

The Hypocretin1/OrexinA (Hcrt1/OxA) neuropeptides are found in a group of posterolateral hypothalamus neurons and are involved in sleep-wakefulness cycle regulation. Hcrt1/OxA neurons project widely to brainstem aminergic structures, such as the locus coeruleus (LC), which are involved in maintenance of wakefulness and EEG activation through intense projections to the medial prefrontal cortex (mPFC). Moreover, defects of the Hcrt1/OxA system are linked to narcolepsy, a disorder characterized by excessive diurnal hypersomnia and REM state disturbance. We aimed to determine whether Hcrt1/OxA neurons innervate LC neurons (noradrenergic and nonnoradrenergic) that project to the mPFC, thereby sustaining behavioral wakefulness. To assess this, we used retrograde tracing from mPFC injections and either Hcrt1/OxA or tyrosine hydroxylase (TH) immunohistochemical labeling in single sections of rat LC. The retrograde tracer Fluorogold (FG) was microinjected into mPFC and, at optimal survival periods, sections through the LC were processed for dual immunolabeling of anti-FG and either anti-Hcrt1/OxA or anti-TH antisera. Many LC neurons projecting to mPFC were nonnoradrenergic. Electron microscopy revealed a prominent localization of Hcrt1/OxA in unmyelinated axons and axon boutons (varicosities and axon terminals) within the LC. Hcrt1/OxA-immunoreactive axon boutons frequently apposed (104/1907) or made asymmetric excitatory-type synapses (60/1907) with FG-immunolabeled dendrites, indicating that Hcrt1/OxA can modulate the activity of LC neurons with cortical projections. Our results show that Hcrt1/OxA hypothalamic neurons likely excite LC neurons that project to the mPFC, and thus activate EEG and facilitate wakefulness. In narcoleptics, who are deficient in Hcrt1/OxA, impairment of this Hcrt1/OxA hypothalamic input to LC might contribute to the appearance of excessive daytime sleepiness.     

Regulation of tyrosine hydroxylase levels and activity and Fos expression during opioid withdrawal in the hypothalamic PVN and medulla oblongata catecholaminergic cell groups innervating the PVN

Morphine withdrawal increases the hypothalamic-pituitary-adrenocortical (HPA) axis activity, which is dependent on an hyperactivity of noradrenergic pathways innervating the hypothalamic paraventricular nucleus (PVN). However, the possible adaptive changes that can occur in these pathways during morphine dependence are not known. We studied the alterations in tyrosine hydroxylase (TH; the rate-limiting enzyme in catecholamines biosynthesis) immunoreactivity levels and TH enzyme activity in the rat NTS-A2/VLM-A1 noradrenergic cell groups and in the PVN during morphine withdrawal. In the same paradigm, we measured Fos expression as a marker of neuronal activation. TH and Fos immunoreactivity was determined by quantitative Western blot analysis, combined with immunostaining for TH and Fos for immunohistochemical identification of active neurons during morphine withdrawal. Dependence on morphine was induced by a 7-day s.c. implantation of morphine pellets. Morphine withdrawal was precipitated on day 8 by an injection of naloxone (5 mg/kg s.c.). Morphine withdrawal induced the expression of Fos in the PVN and NTS/VLM, which indicates an activation of neurons in these nuclei. TH immunoreactivity in the NTS/VLM was increased 90 min after morphine withdrawal, whereas there was a decrease in TH levels in the PVN at the same time point. Following withdrawal, Fos immunoreactivity was present in most of the TH-positive neurons of the A2 and A1 neurons. TH activity was measured in the PVN, a projection area of noradrenergic neurons arising from NTS-A2/VLM-A1. Morphine withdrawal was associated with an increase in the enzyme activity at different time points after naloxone-precipitated morphine withdrawal. The present results suggest that an increase in TH protein levels and TH enzyme activity might contribute to the enhanced noradrenergic activity in the PVN in response to morphine withdrawal.     

Origin of noradrenergic afferents to the shell subregion of the nucleus accumbens: anterograde and retrograde tract-tracing studies in the rat

The nucleus accumbens (NAcc) can be subdivided into `core' and `shell' based on anatomical connections and histochemical markers. Previous studies have demonstrated dopamine-β-hydroxylase immunoreactive (DBH-ir) fibers in the NAcc shell, but the source of these noradrenergic (NE) afferents has not been determined. Therefore, we have investigated in detail the anatomy of NE afferents to this subregion. Dual immunohistochemistry for DBH and substance P demonstrated numerous DBH-ir fibers in the caudal NAcc shell. Neurons projecting to the NAcc were identified with Fluoro-Gold (FG) or cholera toxin B (CTb) retrograde tracing and tyrosine hydroxylase (TH) immunohistochemistry. Single- and double-labeled neurons were observed in the A2 and A1 NE cell groups following FG injections into the caudal NAcc shell. Numerous FG and CTb single-labeled neurons were found in the rostral locus coeruleus (LC), subcoeruleus and pericoerulear dendritic region, with an occasional double-labeled neuron in the LC. Few labeled neurons were seen in the brainstem after FG injections into the NAcc core, consistent with the lack of DBH-ir in this subterritory. To confirm these results, injections of Phaseolus vulgaris leucoagglutinin or biotinylated dextran amine were made into the LC or nucleus tractus solitarius (NTS). Virtually no labeled fibers were observed in the NAcc following injections into central LC. However, fibers were observed in the NAcc shell after injections in the NTS. These results indicate that the primary source(s) of NE afferents to the NAcc shell is the A2 region of the NTS, with lesser contributions from A1 and LC.     

迷走神经参与胃伤害性信息向下丘脑的传递

目的　 研究迷走神经是否参与胃伤害性信息向下丘脑室旁核的传递。 方法　 检测下列条件下c Fos蛋白在孤束核及下丘脑室旁核的表达 :①胃内注入福尔马林引起伤害性刺激 ;②福尔马林刺激结合双侧膈下迷走神经切断术。结果　胃内注入福尔马林引起的伤害性刺激可以诱导c Fos蛋白在孤束核和下丘脑室旁核等脑区的表达 ,但在胸段脊髓的I,V ,VII和X层无明显表达。胃内注入生理盐水的对照组则仅有极少量的表达 ,双侧膈下迷走神经切断术可以减少c Fos蛋白在这些部位的表达。 结论　 该研究结果表明 ,迷走神经参与了胃内脏伤害性信息向孤束核及下丘脑室旁核的传递     

Light-induced c-Fos expression in suprachiasmatic nuclei neurons targeting the paraventricular nucleus of the hamster hypothalamus: phase dependence and immunochemical identification.

The suprachiasmatic nuclei (SCN) contain a master clock driving the majority of circadian rhythms in mammals. It is believed that the SCN confers circadian rhythmicity as well as light responsiveness to pineal melatonin secretion via a direct projection to the paraventricular nucleus of the hypothalamus (PVN). Neurons in the SCN respond to light during subjective night with an expression of the immediate early gene c-fos. The number and distribution of c-Fos protein-containing neurons depend on the zeitgeber time (ZT) at which the light stimulus is presented. To investigate whether this phase-dependent activity is present in the SCN output neurons targeting the PVN, we combined retrograde cholera toxin subunit B (ChB) tracing from the PVN with c-Fos immunohistochemistry. Male golden hamsters were injected iontophoretically with ChB into the PVN area and 7 days later given a 1.5-hr light stimulus at either ZT 14 or ZT 19 followed by vascular fixation. Light stimulation at ZT 19 gave rise to more c-Fos containing neurons in the SCN than light presented at ZT 14. Double immunostaining for ChB and c-Fos revealed that light stimulation at ZT 14 induced c-Fos expression in 26.6% +/- 2.8% of the retrogradely filled perikarya, whereas light-stimulation at ZT 19 increased this fraction to 40.7% +/- 1.9%. This demonstrates the presence of a phase-dependent c-Fos induction in the suprachiasmatic-paraventricular projection system. Triple immunohistochemistry showed that light-activated output neurons contained both gastrin-releasing peptide and vasoactive intestinal polypeptide and to a lesser extent vasopressin. The present findings provide functional evidence of light activation of central pathways involved in the regulation of circadian output rhythms.     

Role of the locus coeruleus in the prolactin secretion of female rats

Since locus coeruleus (LC) lesion blocks preovulatory prolactin surge, the aim of this study was to determine if this lesion would also block prolactin surges induced by steroids in ovariectomized rats and would modify basal prolactin secretion. To determine the time of the steroid-induced prolactin surges, ovariectomized rats treated with estradiol (OVE) or estradiol and progesterone (OVEP) were cannulated at 08:00 h and blood samples were collected hourly between 14:00 and 18:00 h. Ovariectomized rats treated with oil (OV-Oil) were used as control. Prolactin peaked at 16:00 h in OVE rats and at 15:00 h in OVEP. In a second experiment, male rats, cycling rats, OVE, OVEP, and OV-Oil groups were cannulated at 08:00 h, followed by LC lesion or sham-surgery. Blood samples were withdrawn at times of basal and peak prolactin levels. LC lesion blocked afternoon prolactin surges of OVE, OVEP and proestrus rats. However, the low levels observed at 16:00 h in OV-Oil, diestrus and male rats as well as at 11:00 h in OVE, OVEP, estrus, and proestrus rats were not modified by LC lesion. The high prolactin levels observed on estrus afternoon were dramatically reduced by LC lesion. Data suggest that LC neurons are important for steroid-induced prolactin surge genesis, but not for prolactin basal secretion.