Hypocretin I in the lateral hypothalamus activates key feeding-regulatory brain sites.

Hypocretin I (also referred to as orexin A) administered into the lateral hypothalamus (LH) stimulates feeding in rats. We undertook the present study to determine the brain regions activated by LH administration of hypocretin I. Hypocretin I administered into the LH significantly elevated cFos-immunoreactivity in the lateral septal area, the central nucleus of the amygdala, the shell of the nucleus accumbens, the bed nucleus of the stria terminalis, the LH, the posterior and dorsomedial hypothalamus, the perifornical, arcuate and paraventricular nuclei and the nucleus of the solitary tract. These data indicate that LH hypocretin I communicates with other key energy regulatory sites within the hypothalamus, the limbic region and the hindbrain, and suggest that these areas are important in the feeding-stimulatory actions of hypocretin I.

Increased delayed type hypersensitivity in rats subjected to unilateral mononeuropathy is mediated by neurokinin-1 receptors.

An animal model or peripheral mononeuropathy was utilized in the present study to investigate the potential role of substance P (SP) in modifying immune responses associated with chronic pain conditions. Animals subjected to unilateral sciatic ligation and sham-operated animals were sensitized with keyhole limpet hemocyanin (KLH) and subsequently challenged in the ipsilateral or contralateral hind paw to produce a delayed-type hypersensitivity (DTH) response. Subcutaneous microdialysis and radioimmunoassay were used to measure interstitial fluid SP levels in the challenged tissue prior to and following immune challenge in control and neuropathic animals. Following immune challenge, there was a significant increase in the concentration of SP in tissue dialysate samples from the challenged paw of both sham-operated and neuropathic animals. However, tissue SP levels in neuropathic animals were more than two-fold higher than those obtained from sham-operated controls following challenge. SP concentration remained elevated for 2.5 h following immune challenge in neuropathic animals compared to 90 min in sham-operated animals. Compared with controls, neuropathic animals also exhibited an increased DTH response that was reversed, in a dose-related fashion, by the non-peptide NK-1 receptor blocker L-703,606. The same antagonist had no effect in sham-operated animals. These data suggest that the increased DTH response in animals subjected to unilateral mononeuropathy involves SP and NK-1 receptors present in the challenged tissue.

Distribution of nitric oxide synthase-immunoreactive interneurons in the spinal trigeminal nucleus.

The spinal trigeminal nucleus is involved in the transmission of orofacial sensory information. Neither the distribution of the neuromessenger, nitric oxide, within the trigeminal system nor the possible relationship of this simple gas with trigeminothalamic neurons has been carefully studied. Using immunocytochemical (against nitric oxide synthase) and histochemical (NADPH-diaphorase staining) techniques, we have found that nitric oxide neurons and processes are more prominent in the nucleus caudalis and the dorsomedial aspect of the nucleus oralis than in other spinal trigeminal regions. To study the relationship of nitric oxide to trigeminothalamic neurons and intertrigeminal interneurons of the spinal trigeminal nucleus, spinal trigeminal neurons were retrogradely labeled with fluorogold by thalamic injections or by injections into the junction of the nucleus interpolaris and nucleus caudalis. Medullary sections were subsequently processed with NADPH-diaphorase histochemistry. None of the diaphorase-stained neurons in the spinal trigeminal nucleus was found to contain fluorogold; however, some diaphorase-stained processes were found in close proximity to trigeminothalamic neurons. Following spinal trigeminal nucleus injections, many diaphorase-stained neurons were found to contain fluorogold, especially in the nucleus caudalis, suggesting that nitric oxide-containing neurons in the spinal trigeminal nucleus are intertrigeminal interneurons. Collectively, these data indicate that nitric oxide is most prominent in interneurons located in nucleus caudalis and that these interneurons give rise to processes that appose trigeminothalamic neurons, raising the possibility that they may indirectly influence orofacial nociceptive processing at the level of the spinal trigeminal nucleus via nitric oxide production.

Systemic morphine reduces GABA release in the lateral but not the medial portion of the midbrain periaqueductal gray of the rat.

Neuroanatomical, electrophysiological and pharmacological studies have provided indirect evidence indicating that GABAergic neurons play a key role in opiate analgesia mediated by the midbrain periaqueductal gray (PAG) and ventromedial medulla. Although these studies suggest that systemic administration of opiates inhibits GABA release in the PAG, there have been no investigations to date that have directly examined this issue. The present study was thus designed to determine whether systemic morphine injection inhibits GABA release in the PAG of awake, freely moving rats using in vivo microdialysis and subsequent HPLC analysis. Extracellular levels of GABA, glutamate, aspartate, glycine, homocysteic acid and taurine were monitored with the microdialysis technique in either the lateral or medial portion of the ventrocaudal PAG in unanesthetized, unrestrained rats. Amino acid release was induced by infusing veratridine (75 microM, a sodium channel activator) directly through the dialysis probe. The effect of veratridine alone and the effect of veratridine in the presence of systemic morphine on the concentrations of amino acids in the PAG dialysate were determined. There were no significant differences in the basal concentrations of GABA, taurine, aspartate, glutamate, homocysteic acid and glycine between dialysates collected from the medial versus the lateral ventrocaudal PAG. Glycine, taurine and glutamate were present in the highest concentrations in dialysis samples both before and after treatment with veratridine, whereas GABA, homocysteic acid and aspartate were present in the lowest concentrations. Perfusion of veratridine into the ventrocaudal PAG resulted in significant elevation of all amino acids investigated. Except for taurine, no significant difference in veratridine-induced release between the lateral and medial PAG was observed. Tetrodotoxin (TTX) significantly blocked veratridine-induced release of GABA, aspartate, glutamate, glycine and taurine but not homocysteic acid. When rats were injected with morphine (10 mg/kg i.p.), veratridine-induced release of GABA was selectively and significantly decreased in the lateral but not the medial PAG as compared to control rats injected with saline followed by veratridine perfusion. Systemic injection of morphine or saline caused no significant change in the basal concentration of amino acids in PAG dialysate samples. These findings are consistent with the proposed mechanism of action of morphine in the lateral ventrocaudal PAG and offer the first direct evidence that systemic opiates decrease GABA release in this midbrain region.

Fluorogold administration via microdialysis labels neurons terminating within the dialysis region.

The use of microdialysis to monitor the release of neurotransmitters in selected regions of the CNS has increased substantially in the last several years. We describe here a method for retrogradely labeling neurons that terminate within the effective sampling region surrounding the dialysis cannula. This is accomplished by direct infusion of fluorogold through the dialysis cannula. By combining this technique with immunohistochemistry, it is possible to identify neurons that could contribute to the neurotransmitter release measured by microdialysis.

Differential origin of brainstem serotoninergic projections to the midbrain periaqueductal gray and superior colliculus of the rat.

Previous studies have shown that both the midbrain periaqueductal gray (PAG) and the superior colliculus receive a significant serotoninergic (5-HT) innervation. In the present study the origins of these 5-HT projections to the rodent PAG and superior colliculus were analyzed by using a combined immunohistochemical-retrograde transport technique. Thirteen brainstem regions were found to contain double-labelled 5-HT-like immunoreactive neurons following HRP injections into the PAG while only four brainstem nuclei contained double-labelled neurons following superior collicular injections. After HRP deposits into the ventral PAG, the largest percentage of double-labelled neurons was identified in nucleus raphe magnus, pars alpha of the nucleus gigantocellularis, and the paragigantocellular nucleus. The dorsal PAG, on the other hand, received the largest percentage of its 5-HT projections from nuclei raphe dorsalis, raphe obscurus, raphe pontis, and raphe medianis. The 5-HT input to the superior colliculus was found to arise exclusively from nuclei raphe dorsalis, raphe medianis, and raphe pontis and from the contralateral periaqueductal gray. Raphe nuclei were found to contribute serotoninergic projections to both the PAG and the superior colliculus while reticular nuclei contributed 5-HT projections only to the PAG. Injections of the fluorescent retrograde tracers true blue and nuclear yellow were then made into the PAG and superior colliculus to ascertain if neurons located in raphe nuclei that projected to both structures provided axon collaterals to both areas. Generally, less than 10% of raphe neurons projecting to the superior colliculus were identified as providing axon collaterals to the PAG. The present results demonstrate major quantitative and qualitative differences in the origin of 5-HT projections to the ventral PAG and superior colliculus. The origin of 5-HT input to the dorsal PAG, on the other hand, showed many similarities to the origin of 5-HT innervation of the superior colliculus. These data also indicate that approximately 35% of raphe neurons provide nonserotoninergic projections to the PAG and superior colliculus.

Immunocytochemical localization of glutamate-, glutaminase- and aspartate aminotransferase-like immunoreactivity in the rat deep cerebellar nuclei.

Although the anatomy and the connectivity of the deep cerebellar nuclei have been well documented, little is known about the neurotransmitter systems mediating cerebellar efferent pathways. The present study utilizes immunohistochemical procedures in conjunction with a novel monoclonal antibody specific for carbodiimide-fixed glutamate and polyclonal antisera against glutaminase (GLNase) and aspartate aminotransferase (AATase) to examine the presence of putative excitatory amino acid transmitters in neurons of the deep cerebellar nuclei. Carbodiimide-fixed glutamate-like, GLNase-like and AATase-like immunoreactivities were observed in neurons of the lateral, posterior interpositus, anterior interpositus and medial deep cerebellar nuclei. More neurons were stained with AATase antiserum than with the GLNase antiserum or the monoclonal antibody. These results suggest glutamate, GLNase and AATase are present in neurons of the deep cerebellar nuclei and raise the possibility that glutamate may be an excitatory transmitter in these structures.

Immunocytochemical localization of serotonin in the rat periaqueductal gray: a quantitative light and electron microscopic study.

The distribution of serotonin-like immunoreactivity in five regions of the rodent midbrain periaqueductal gray (PAG) was studied by using light and electron microscopic immunohistochemistry in combination with quantitative analysis. Light microscopic analysis revealed the presence of serotonin-like immunoreactive cell bodies located in the ventrolateral and ventromedial regions of the caudal PAG and serotonin-like immunoreactive processes throughout the PAG. Ultrastructural analysis showed dendritic profiles that stained positively for serotonin primarily in ventral regions, although an occasional profile was seen dorsally. Numerous synaptic contacts between unstained axon terminals and ventral dendritic profiles were seen. Axonal profiles that contained reaction product were identified throughout the PAG, but were rarely observed to make any type of specialized contact. Ultrastructural quantification of serotonin-like immunoreactive processes indicated that the highest volume fraction of serotonin immunoreactivity occurred caudoventrally where stained processes constituted 2.6% of the neuropil volume. Rostroventrally stained processes constituted only 0.14% of the neuropil volume at the level of the posterior commissure. By contrast the amount of serotonin-like immunoreactivity found dorsally remained relatively constant at all rostrocaudal levels. Analysis of serotonin staining among PAG regions demonstrated the lowest overall volume fraction in the dorsal region and the highest overall volume fraction in the ventromedial region. No significant differences were observed between medial and lateral regions. A comparison of the results of light microscopic quantitative analysis of serotoninergic processes with electron microscopic quantitative analysis indicated that both techniques produce comparable results.

The periaqueductal gray projections to the rat spinal trigeminal, raphe magnus, gigantocellular pars alpha and paragigantocellular nuclei arise from separate neurons.

Possible collateral branches of periaqueductal gray axons which distribute to the nucleus raphe magnus, nucleus reticularis paragigantocellularis, nucleus reticularis gigantocellularis pars alpha and the spinal trigeminal nucleus were analyzed with the double fluorescent retrograde tracer technique. With the exception of a small number of double-labeled neurons observed in the periaqueductal gray following injections of fluorescent dyes into the nuclei reticularis paragigantocellularis and gigantocellularis pars alpha, no double-labeled cells were found in this midbrain region following injections of tracers into various combinations of the above 4 nuclear groups. The results of this investigation indicate that these 4 brainstem nuclei are innervated predominantly by separate neuronal populations within the periaqueductal gray.

Effects of progesterone treatment on basal and LH-RH-induced plasma LH concentrations in anoestrous and ovariectomized sheep.

Anoestrous and ovariectomized sheep were treated with progesterone-containing implants or left as controls. Plasma LH concentrations were decreased by progesterone treatment (P less than 0.025) and increased by ovariectomy (P less than 0.001). When the ewes were treated with 0.25 micrograms LH-RH, the LH response was reduced (P less than 0.025) in the progesterone-treated anoestrous ewes but not in the progesterone-treated ovariectomized sheep. These data demonstrate that low concentrations of progesterone, acting together with another ovarian hormone, can markedly reduce the pituitary response to a small dose of LH-RH.