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Injections of rhodamine-B into the dorsal raphe nucleus (DRN) and Fluoro-Gold into the lateral geniculate nucleus (LGN) revealed double-labeled retinal ganglion cells (DL RGCs) projecting to both nuclei. The soma-size distribution of DL RGCs was compared with three other distributions: DRN-projecting RGCs, LGN-projecting RGCs, and a large sample of RGCs labeled via the optic nerve with DiI. DL RGC soma diameters fell primarily within the mid-to-upper size range of all three distributions. DL RGCs may provide information to both nuclei concerning comparable aspects of light and visual stimulation via collateralized axons.

The alpha 7 nicotinic acetylcholine receptor (nAChR) subunit can be assembled to form a homomeric-pentamer with high permeability to calcium. Although the expression of the alpha 7-nAChR has been demonstrated throughout the CNS, the neurochemical phenotype of neurons expressing alpha 7 remains to a large extent unknown. Using an antibody against the alpha 7 nAChR subunit, immunohistochemical staining was observed in rat dorsal raphe nucleus (DRN) and locus coeruleus (LC), serotonergic and noradrenergic brainstem nuclei, respectively. In both the DRN and LC, there appeared to be two histologically distinct alpha 7-expressing cell types as distinguished by size, i.e. large versus small diameter. In rats treated with either a serotonergic (5,7-dihydroxytryptamine) or noradrenergic (anti-dopamine-beta-hydroxylase saporin) neurotoxin, tryptophan hydroxylase and tyrosine hydroxylase immunostaining was abolished, respectively. Similarly, the alpha 7-positive large-diameter cells were no longer detectable, suggesting that these cells were serotonergic DRN and noradrenergic LC neurons. Indeed, double-labeling experiments revealed in the large cell types coexpression of alpha 7 with tryptophan hydroxylase in the DRN and with tyrosine hydroxylase in the LC of saline-treated rats. In contrast to the large-diameter cells, the alpha 7-positive small-diameter cells were neither serotonergic nor adrenergic, and were still detected in both the DRN and LC of lesioned rats. Moreover, cell counts revealed an increase number of these cells in lesioned rats with expression of alpha 7 in somal processes not seen in non-lesioned controls. Double labeling revealed coexpression of alpha 7 and GABA within the majority, but not all, of the toxin-resistant cells. The results of these studies suggest that both serotonergic and noradrenergic neurons express alpha 7 nAChRs. In addition, there appears to be a small-diameter cell-type in both the DRN and LC, possibly a GABAergic interneuron, expressing alpha 7 that may be regulated by neurotoxic injury.

The morphology and electrophysiological properties of serotonergic and non-serotonergic projection neurons in the dorsal raphe nucleus (DRN) of the rat were examined in frontal brain slices. Biocytin was injected intracellularly into the intracellularly recorded neurons. Then the morphology of the recorded neurons was observed after histochemical visualization of biocytin. The recorded neurons extending their main axons outside the DRN were considered as projection neurons. Subsequently, serotonergic nature of the neurons was examined by serotonin (5-HT) immunohistochemistry. The general form of the dendritic trees is radiant and poorly branching in both 5-HT- and non-5-HT neurons. However, the dendrites of the 5-HT neurons were spiny, whereas those of the non-5-HT neurons were aspiny. The main axons of both 5-HT- and non-5-HT neurons were observed to send richly branching axon collaterals to the DRN, ventrolateral part of the periaqueductal gray and the midbrain tegmentum. In response to weak, long depolarizing current pulses, the 5-HT neurons displayed a slow and regular firing activity. The non-5-HT neurons fired at higher frequencies even when stronger current was injected. Some other differences in electrophysiological properties were also observed between the 5-HT-immunoreactive spiny projection neurons and the 5-HT-immunonegative aspiny projection neurons.

Transgenic mice bearing a transgene coding for a glucocorticoid receptor antisense mRNA, which partially blocks glucocorticoid receptor expression, were used in order to clarify the role of glucocorticoid receptors in the regulation of 5-HT(1A), 5-HT(1nonA) and 5-HT(2) binding sites labelled by quantitative autoradiography in the frontal and prefrontal cortex, striatum, hypothalamus, amygdala and raphe nuclei. We found that 1 nM [3H]8-hydroxy-2-[di-N-propylamino]tetralin ([3H]8-OH-DPAT) binding to 5-HT(1A) sites was decreased in strata oriens (-15.1+/-3.5%) and radiatum-lacunosum-moleculare (-13.3+/-4.3%) of the hippocampal CA(3) area, and 2 nM [3H]5-hydroxytryptamine binding to 5-HT(1nonA) sites in the presence of 100 nM 8-OH-DPAT and mesulergine was decreased in the dorsal subiculum (-17.8+/-6.9%). By contrast, 5-HT(2) sites labelled by 0.5 nM of (+/-)-1-(2, 5-dimethoxy-4-[125I]iodophenyl)-2-aminopropane was increased in the dorsal subiculum (+35.2+/-11.5%) and CA(2) area (+29.2+/-11.3%). The observed differences in binding to 5-HT(1) and 5-HT(2) sites were all located in areas of the hippocampus that contain both gluco- and mineralo-corticoid receptors, and no difference was observed in anatomical structures which contain only glucocorticoid receptors. Therefore, it seems that the important factor for the regulation of these 5-HT receptors is the interaction between gluco- and mineralo-corticoid receptors rather than the absolute density of glucocorticoid receptors. These results suggest that some of the alterations of the serotonergic neurotransmission observed in depressed patients might be secondary to an altered glucocorticoid receptor function.

CRF and urocortin, administrated systemically, exert peripheral biological actions which may be mediated by brain pathways. We identified brain neuronal activation induced by intravenous (i.v.) injection of CRF and urocortin in conscious rats by monitoring Fos expression 60 min later. Both peptides (850 pmol/kg, i.v.) increased the number of Fos immunoreactive cells in the paraventricular nucleus of the hypothalamus, supraoptic nucleus, central amygdala, nucleus tractus solitarius and area postrema compared with vehicle injection. Urocortin induced a 4-fold increase in the number of Fos-positive cells in the supraoptic nucleus and a 3.4-fold increase in the lateral magnocellular part of the paraventricular nucleus compared with CRF. Urocortin also elicited Fos expression in the accessory hypothalamic neurosecretory nuclei, ependyma lining the ventricles and choroid plexus which was not observed after CRF. The intensity and pattern of the Fos response were dose-related (85, 255 and 850 pmol/kg, i.v.) and urocortin was more potent than CRF. Neither CRF nor urocortin induced Fos expression in the lateral septal nucleus, Edinger-Westphal nucleus, dorsal raphe nucleus, locus coeruleus, or hypoglossal nucleus. These results show that urocortin, and less potently CRF, injected into the circulation at picomolar doses activate selective brain nuclei involved in the modulation of autonomic/endocrine function; in addition, urocortin induces a distinct activation of hypothalamic neuroendocrine neurons.

Activation of neurons in the ventromedial medulla by electrical stimulation or by microinjection of opioid or cholinergic agonists produces antinociception that is mediated in part by spinally-projecting noradrenergic neurons. Several lines of evidence indicate that these noradrenergic neurons are located in the pontine A7 catecholamine cell group. For example, anatomical studies have demonstrated that neurons in the ventromedial medulla project to the noradrenergic neurons in the A7 catecholamine cell group that provide the major noradrenergic innervation of the spinal cord dorsal horn. In addition, electrical and chemical stimulation of A7 neurons produces antinociception that can be reduced by intrathecal injection of alpha2-adrenoceptor antagonists. The present studies provide more direct evidence that activation of neurons in the ventromedial medulla produces antinociception by activating noradrenergic neurons in the A7 cell group. Neurons in the ventromedial medulla were stimulated by microinjecting the cholinergic agonist carbachol (5 microg) into sites in the nucleus raphe magnus or the nucleus gigantocellularis pars alpha of pentobarbital anesthetized Sprague-Dawley rats. In some experiments, the local anesthetic tetracaine (10 microg) was then microinjected near the A7 cell group to inactivate the spinally-projecting noradrenergic neurons. In other experiments, cobalt chloride (100 mM) was microinjected near the A7 cell group to block synaptic activation of spinally-projecting noradrenergic neurons. Microinjection of carbachol into sites in the ventromedial medulla produced antinociception, assessed using the tail flick test, that lasted more than 60 min. However, the effects of carbachol were attenuated by microinjection of either tetracaine or cobalt into sites near the A7 cell group neurons identified by tyrosine hydroxylase-immunoreactivity. Similar injections of tetracaine or cobalt more than 500 microm from the A7 neurons did not alter the antinociceptive effect of carbachol. These results support the conclusion that the antinociception produced by activating neurons in the ventromedial medulla is mediated in part by the subsequent activation of spinally-projecting noradrenergic neurons in the A7 cell group., (Copyright 1999 Elsevier Science B.V.)

The effects of infusing N-methyl-d-aspartate (NMDA) into the raphe nuclei on release of 5-HT in this brain region and also the frontal cortex of the same animal were studied using in vivo microdialysis in freely moving rats. Infusion of 25 microM NMDA into the raphe led to a substantial decrease in dialysate 5-HT in this region and a prolonged increase in terminal 5-HT release in the frontal cortex. These effects were blocked by the specific NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (D-AP5; 100 microM). When 25 microM NMDA was co-infused into the raphe with the selective 5-HT1A receptor antagonist (N-¿2-¿4-(2-methoxyphenyl)-1-piperazinyl¿ethyl-N-(2-pyridinyl) cyclohexanecarboxamide) (WAY-100635; 1.0 microM) the effect of NMDA infusion was unaltered. WAY-100635 infused alone into the raphe did not alter local 5-HT or extracellular 5-HT in the cortex. Infusion of 100 microM NMDA into the raphe was followed by an increase in local dialysate 5-HT and a decrease in 5-HT release in the cortex. These changes were reversed by D-AP5. Following infusion of 100 microM NMDA with 1.0 microM WAY-100635 into the raphe local 5-HT release was still increased, however, the decrease in 5-HT observed in the frontal cortex was abolished. These data suggest that the degree of NMDA receptor activation leads to dramatically different outcomes with regard to serotonergic transmission to the frontal cortex. Furthermore, there appears to be a differential role of the 5-HT1A autoreceptor in regulating these effects. These data are discussed in relation to other studies on the regulation of serotonergic transmission in ascending pathways.

The high-affinity serotonin (5-HT) transporter (5-HTT) plays an important role in the removal of extracellular serotonin, thereby modulating and terminating the action of this neurotransmitter at various pre- and post-synaptic serotonergic receptors and heteroreceptors. In order to characterize the anatomical distribution of the 5-HTT in mouse brain, in situ hybridization histochemistry using 35S-labeled riboprobes was performed. These results were compared with 5-HTT binding site distribution as evaluated by [125I]RTI-55 autoradiography. High levels of 5-HTT mRNA were detected in all brain stem raphe nuclei, with variations in labeling among the various subnuclei. Those brain areas known to possess serotonergic cell bodies stained intensely for both 5-HTT mRNA and 5-HTT binding sites. In contrast to previous findings in rat brain, the highest densities of 5-HTT sites were found in areas outside the raphe complex, particularly in the substantia nigra, globus pallidus, and superior colliculi.

GTPCH-I immunoreactive structures in the rat brain were studied using a polyclonal antibody raised in the chick. General mapping was made using the avidin-biotin-peroxidase technique and compared with the distribution of tyrosine hydroxylase and serotonin immunoreactivities. Double immunofluorescence was performed in order to establish real intracellular colocalization. GTPCH-I immunoreactivity was generally found to be low. Immunostained neurons were present in all the serotonin cell groups. In catecholaminergic neurons, although tyrosine hydroxylase immunoreactivity was always very high, GTPCH-I immunoreactivity was extremely variable, from relatively strong (substantia nigra, ventral tegmental area) to low (locus coeruleus, caudal part of the hypothalamus), extremely low (rostral hypothalamus, ventral brainstem) or almost absent (dorsal brainstem, some hypothalamic nuclei). When feasible, double immunolabeling revealed that all the serotonin cells and most of the tyrosine hydroxylase cells were also expressing GTPCH-I. Our results argue in favor of a regulation of tyrosine hydroxylase activity by the intracellular synthesis of BH4.

Sleep/waking stages and behavior were studied following the selective 5-HT1A agonist 8-OH-DPAT given subcutaneously (s.c.) (0.010-0.375 mg/kg) as well as perfused continuously (10 microM) for 6 h into the dorsal raphe nucleus (DRN) using microdialysis. Given systemically, 8-OH-DPAT at 0.375 mg/kg s.c. induced 5-HT behavioral syndrome, increased waking to 149% and reduced slow wave sleep (SWS) to 86%, transition to 76% and rapid eye movement (REM) sleep to 73%. The effect on deep SWS (SWS-2) was biphasic, with an increase after 2 h. 8-OH-DPAT at 0.010 mg/kg did not have any vigilance effects. 8-OH-DPAT perfusion in DRN produced a fourfold increase in REM sleep compared to perfusion of artificial cerebrospinal fluid. This is consistent with the hypothesis that reduced 5-HT neurotransmission following 5-HT1A autoreceptor stimulation will disinhibit cholinergic REM-promoting mesopontine neurons and thereby lead to a REM sleep increase. The other sleep/waking stages were not significantly affected by 8-OH-DPAT perfusion in DRN.

A 1 h immobilization stress (IS) was imposed to rats at the beginning of the dark period, i.e., when the animals start to be active. The IS was accompanied by an intense polygraphic waking and followed, over 12 h of the dark period, by a significant rebound of slow-wave sleep (SWS, +17%) and paradoxical sleep (PS, +57%). In order to estimate the IS-related changes in the endogenous concentrations of corticotropin-like intermediate lobe peptide (CLIP, ACTH18-39) and related compounds, a specific radioimmunoassay (RIA) was used. Assays performed in cerebral biopsies taken from arcuate (AN) and raphe dorsalis (nRD) nuclei led to the obtention of 2 main immunoreactive peaks, corresponding to CLIP and its phosphorylated form Ph-CLIP. Just after end of the IS and within the nRD. Ph-CLIP immunoreactivity increased by about 95%. Four hours later, i.e., when PS rebound was maximal, a 37% increase in Ph-CLIP immunoreactivity was measured in the AN. These observations have never been described before. In the blood, at the end of the restraint, CLIP/ACTH1-39 total immunoreactivity was increased by 330%. It returned to baseline level 4 h later. Blood concentration of corticosterone was also increased by 56% at the end of the IS and was close to baseline level 4 h later. Data reported here indicate that the IS first triggers an increase in Ph-CLIP within the nRD. Since the nRD contains sleep permissive components, this increase might be determinant for the SWS and PS rebound induction. The changes observed in the blood as regards CLIP/ACTH1-39 total immunoreactivity and corticosterone concentration testify to the efficacy of the IS and are part of the conventional picture accompanying such a situation. Finally, the increase in Ph-CLIP, occurring in the AN 4 h after the end of the restraint, might be part of the restorative processes necessary to compensate the stress overshoot.

After trigeminal rhizotomy, some substance P-like immunoreactive (SP-LI) fibers and terminals in the spinal trigeminal caudal subnucleus (Vc), specially in its superficial laminae (laminae I and II), still remained in the rat. Employing a combination of Fluoro-Gold retrograde tracing and immunofluorescence histochemical staining for SP, we found that the main central origins of these SP-LI fibers and terminals were midbrain periaqueductal gray (PAG), nucleus raphe magnus (NRM) and other raphe nuclei, and nucleus reticularis gigantocellularis pars alpha; all of them are important structures of the endogenous pain control system. The present results provided morphological evidence for PAG or NRM stimulation could inhibit neuronal activities in the Vc evoked by orofacial nociceptive stimulation and also suggested that SP might be an important neurotransmitter or neuromodulator for endogenous pain control system.

Retrograde axonal transport of the cholera toxin B subunit (CTb) was combined with 5-HT immunohistochemistry to determine the origin of the serotonergic innervation of the piriform cortex (PC) in the rat. After iontophoretic CTb injections in the PC, a substantial number of retrogradely labeled cells were found in the middle and medio-ventral part of the dorsal raphe nucleus (RD). A few retrogradely labeled cells were also observed in the median raphe nucleus (MnR) and the B9 serotonergic cell groups. Following CTb and 5-HT immunohistochemistry on the same sections, double-labeled cells were observed in the RD, MnR and B9 groups. In the RD, 30% of CTb stained cells were immunoreactive to 5-HT. After colchicine or nialamide (a monoamine oxidase inhibitor) pretreatment the percentage of these double-labeled cells reached 70%. These results indicate that both 5-HT and non-5-HT neurons in the RD innervate the PC and that the percentage of double-labeled cells is influenced by drug pretreatment. To determine the terminal fields of the RD efferent fibers in the PC, injections of the anterograde tracer PHA-L were also performed. Analysis of the fiber distribution in the PC further revealed some medio-lateral and antero-posterior differences.

Proestrous rats were infused unilaterally into the median raphe nucleus with 200-2,000 ng of the 5-HT1A agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). Inhibition of the lordosis to mount ratio occurred within 15 and 10 min, respectively, following infusion with 1,000 or 2,000 ng of the drug. Infusion of 2,000 ng of the 5-HT2 agonist, (+/-)-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl (DOI), or 4,000 ng 5-HT (creatinine sulfate) failed to substantially reduce lordosis behavior.