Hypocretin/orexin and nociceptin/orphanin FQ coordinately regulate analgesia in a mouse model of stress-induced analgesia.

Stress-induced analgesia (SIA) is a key component of the defensive behavioral "fight-or-flight" response. Although the neural substrates of SIA are incompletely understood, previous studies have implicated the hypocretin/orexin (Hcrt) and nociceptin/orphanin FQ (N/OFQ) peptidergic systems in the regulation of SIA. Using immunohistochemistry in brain tissue from wild-type mice, we identified N/OFQ-containing fibers forming synaptic contacts with Hcrt neurons at both the light and electron microscopic levels. Patch clamp recordings in GFP-tagged mouse Hcrt neurons revealed that N/OFQ hyperpolarized, decreased input resistance, and blocked the firing of action potentials in Hcrt neurons. N/OFQ postsynaptic effects were consistent with opening of a G protein-regulated inwardly rectifying K+ (GIRK) channel. N/OFQ also modulated presynaptic release of GABA and glutamate onto Hcrt neurons in mouse hypothalamic slices. Orexin/ataxin-3 mice, in which the Hcrt neurons degenerate, did not exhibit SIA, although analgesia was induced by i.c.v. administration of Hcrt-1. N/OFQ blocked SIA in wild-type mice, while coadministration of Hcrt-1 overcame N/OFQ inhibition of SIA. These results establish what is, to our knowledge, a novel interaction between the N/OFQ and Hcrt systems in which the corticotropin-releasing factor and N/OFQ systems coordinately modulate the Hcrt neurons to regulate SIA.

Electron microscopic examination of the endomorphin 2-like immunoreactive neurons in the rat hypothalamus.

Endomorphins are endogenous opioid peptides with high affinity and selectivity for the mu-opioid receptor. In the present study, we examined the morphology of the endomorphin 2-like immunoreactive (EM2-LI) neurons in the hypothalamus at the light and electron microscopic levels. At the light microscopic level, EM2-LI neurons were found mostly distributed in the regions between the dorsomedial and ventromedial hypothalamic nuclei and the region near the third ventricle. At the electron microscopic level, EM2-LI perikarya could be divided into two groups. Type I perikarya contained relatively undeveloped endoplasmic reticulum and Golgi apparatus while type II perikarya contained well-developed rough-surfaced endoplasmic reticulum and Golgi apparatus. Both type I and type II neurons contained numerous EM2-LI dense-cored vesicles. Type II perikarya and dendrites received synapses and showed immunoreactivity in the endoplasmic reticulum and Golgi apparatus. EM2-LI axon terminals formed synapses with both immunonegative and immunopositive dendrites. In some cases, the axon terminals contained both immunonegative and immunopositive dense-cored vesicles. EM2-LI neurons often had synaptic relationships with neurons containing immunonegative dense-cored vesicles. Myelinated axon shafts containing EM2-LI were also found. This first demonstration of the ultrastructure and synaptic relationships of EM2-LI neurons in the hypothalamus provides morphological evidence that suggests (1) endomorphin 2-containing neurons modulate physiological function through synaptic relationships; (2) endomorphin 2 may coexist with other neurotransmitters in the same neurons; and (3) endomorphin 2-containing neurons could modulate other endomorphin 2-containing neurons as well as those containing other neurotransmitters.

The distribution of melanin-concentrating hormone in the lamprey brain.

In addition to its novel, colour-regulating hormonal role in teleosts, the melanin-concentrating hormone (MCH) serves as a neuromodulatory peptide in all vertebrate brains. In gnathostome vertebrates, it is produced in several neuronal cell groups in the hypothalamus. The present work examines the organisation of the MCH system in the brain of lampreys, which separated from gnathostome vertebrates at an early stage in evolution. In all three lamprey genera examined-Petromyzon, Lampetra, and Geotria spp.-MCH perikarya were found in one major anatomical site, the periventricular dorsal hypothalamic nucleus of the posterior hypothalamus. Axons from these cell bodies projected medially into the ventricular cavity, and laterally to the neuropile of the lateral hypothalamus. From here, they extended anteriorly and posteriorly to the fore- and hindbrain. Other fibres extended dorsomedially to the habenular nucleus. In Lampetra, but not in Petromyzon, MCH fibres were seen in the pituitary neurohypophysis, most prominantly above the proximal pars distalis. The hypothalamic region in which the MCH perikarya are found forms part of the paraventricular organ (PVO), which is rich in monoamines and other neuropeptides. The association of MCH neurones with the PVO, which occurs also in many other nonmammalian vertebrates, may reflect the primary location of the MCH system. These MCH neurones were present in ammocoetes, postmetamorphic juveniles, and adults. They were more heavily granulated in adults than in young lampreys but showed no marked change in secretory appearance associated with metamorphosis or experimental osmotic challenge to indicate a role in feeding or osmoregulation. In sexually maturing Lampetra fluviatilis, however, a second group of small MCH neurones became detectable in the telencephalon, suggesting a potential role in reproduction and/or behaviour.

Histaminergic system in co-cultures of hippocampus and posterior hypothalamus: a morphological and electrophysiological study in the rat.

Neurons of the tuberomammillary nucleus in the posterior hypothalamus diffusely project to most parts of the central nervous system, where their main transmitter, histamine, modulates the excitability of the target neurons. The development of a histaminergic hypothalamo-hippocampal pathway and its function were studied in organotypic co-cultures. Immunocytochemistry for histidine decarboxylase, the specific synthesizing enzyme, stained clusters of neurons in the hypothalamic tuberomammillary area. Immunolabelled varicose processes innervated the co-cultured hippocampus and established a few synaptic contacts on dendrites. Cultured tuberomammillary neurons displayed their typical membrane properties and were spontaneously active. In hippocampal pyramidal cells of the CA3 region the long-lasting afterhyperpolarization was reduced by histamine or impromidine and increased by the H2 antagonist cimetidine, but not by the H1 antagonist mepyramine. The membrane potential was depolarized in presence of an H2 agonist and hyperpolarized by an H2 antagonist. In single hippocampal cultures histamine antagonists did not affect afterhyperpolarization and membrane potential. Histaminergic neurons retain their main morphological and physiological characteristics in slice cultures and establish a functional connection with co-cultured target cells.

Ultrastructural immunolocalization of adenosine deaminase in histaminergic neurons of the tuberomammillary nucleus of rat.

Neurons in the tuberomammillary nucleus (TM) of the rat hypothalamus were immunolabelled for the enzyme adenosine deaminase (ADA) and investigated by electron microscopic immunohistochemical techniques. ADA-immunoreactivity was distributed throughout the somal and dendritic cytoplasm of TM neurons and in the karyoplasm of most, but not all of these neurons. Immunoreactive axons were rarely observed within the tightly packed cell clusters of the TM subdivisions examined. Dense deposition of immunoreaction product together with reasonable preservation of morphological detail facilitated identification of immunoreaction product together with reasonable preservation of morphological detail facilitated identification of immunoreactive profiles and allowed characterization of the ultrastructural features of labelled neurons and the relationships of these with each other and with surrounding unlabelled neuronal and glial elements. Immunolocalization of ADA therefore represents a reliable and convenient method for the identification of TM neurons in EM studies of their ultrastructure and synaptic interactions.

A freeze-fracture study of the soma membranes of monoaminergic neurons in the hypothalamic nucleus recessi posterioris of the rainbow trout (Salmo gairdneri Rich.).

Monoaminergic neurons in the hypothalamic nucleus recessi posterioris of the rainbow trout were studied by freeze-fracturing. The size and distribution of intramembrane particles in the nuclear and plasma membranes and the density of nuclear pores in the nuclear envelope were determined. The density of nuclear pores in these cells was significantly greater than in the non-granulated neurons of this nucleus. The density of IMPs in the P face of the inner nuclear membrane was significantly greater than in the P face of the outer nuclear membrane; however in the E faces the density is greater in the outer nuclear membrane but the differences are not statistically significant. In both membranes, the proportion of large particles (greater than 9.6 nm) was greater in the E than in the P faces. The coefficient of partition between the plasma membrane faces was 1.97 +/- 0.24 while in non-granulated neurons was 4.45 +/- 0.51. There are significant differences among these values. Interestingly, the coefficient of partition of the plasma membrane in monoaminergic neurons was lesser than in some other types of neurons. Postsynaptic areas or other membrane specializations were not found on the cell body plasma membrane, which corroborates the results of conventional transmission electron microscopy.

Brainstem afferents to the tuberomammillary nucleus in the rat brain with special reference to monoaminergic innervation.

Monoaminergic innervation of a histamine-producing cell group, the tuberomammillary nucleus in the posterior hypothalamus, was investigated in the rat by light and electron microscopic immunohistochemical techniques. Immunohistochemical staining of sections of the posterior hypothalamus was demonstrated afferent fibers immunoreactive to tyrosine hydroxylase in ventral and medial subgroups of the tuberomammillary nucleus afferent fibers immunoreactive to tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), phenyletanolamine-N-methyltransferase (PNMT), and serotonin (5-HT). TH- and DBH-immunoreactive fibers were similar and were evenly and densely distributed throughout the tuberomammillary nucleus. Fibers stained with 5-HT antibodies were also present throughout the tuberomammillary nucleus but exhibited the densest labeling in the dendritic layer adjacent to the glia limitans in the ventral subgroup. Innervation by PNMT-immunoreactive axons was sparse. Electron microscopic analysis of TH-, DBH-, and 5-HT-immunoreactive fibers in the tuberomammillary nucleus revealed vesicle-containing terminal boutons, which formed synapses with dendrites of varying size. Synaptic contacts with nerve cell bodies were not found. Retrograde transport of the fluorescent dye Fast Blue injected into the tuberomammillary nucleus, combined with immunofluorescent staining with anti-TH, anti-DBH, anti-PNMT, and anti-5-HT antibodies, showed that monoaminergic input to the tuberomammillary nucleus originated mainly from the adrenergic and noradrenergic cell groups C1-C3 and A1-A2, respectively, and from the serotoninergic cell groups B5-B9 as designated by Dahlström and Fuxe ('65). Few double-labeled neurons were found in the nucleus locus coeruleus and the dopaminergic cell groups of the rostral brain stem. The present findings suggest that the activity of the histamine-producing neurons of the tuberomammillary nucleus is influenced by monoaminergic neurons in the ventrolateral and dorsomedial medulla oblongata and the raphe nuclei of the rostral brainstem.

[Ultrastructural analysis of changes in the synapses in the rabbit brain after experimental concussion of the brain]. / Ul'trastrukturnyi analiz izmeneniie sinapsov v golovnom mozge krolikov v dinamike éksperimental'nogo sotriaseniia mozga.

The authors discuss the results of electron-microscopic study of the synaptic contacts in the brain of rabbits during 4 months after experimental brain concussion. A complex of ultrastructural changes of the cerebral synaptic apparatus with a characteristic dynamics of development in time and space is described, and its peculiarities and features distinguishing it from other types of pathology as well as its role in the pathogenesis of craniocerebral injury are discussed.

Galanin immunoreactivity in hypothalamic neurons: further evidence for multiple chemical messengers in the tuberomammillary nucleus.

By using a specific antibody against the 29 amino-acid peptide galanin (Gal) with light and electron microscopic immunocytochemistry, we have studied the distribution of Gal immunoreactivity in the posterior hypothalamic magnocellular neurons of the rat. In colchicine-treated rats, a large number of Gal-immunoreactive cells were identified within all subdivisions of the tuberomammillary nucleus. The majority of these cells are large multipolar or fusiform neurons, with long, sparsely branching dendrites. A small number project to the ventral hippocampus, as shown by experiments with the retrograde tracing of Fast Blue. Ultrastructural examination of the Gal-immunoreactive cells confirms their indentity as magnocellular neurons, with dense deposits of immunoreaction product, particularly in small ribosomal arrays and in large, dense-cored vesicles. Axosomatic synapses occur on these neurons. The axonal boutons synapse with asymmetric and symmetric junctions and contain small synaptic vesicles as well as numerous large, dense-cored vesicles, which display Gal immunoreactivity. Sequential staining of thin, alternate sections with antibodies against Gal and L-histidine decarboxylase (HDCase; EC 4.1.1.22) showed colocalization of Galand HDCase-immunoreactivities in a majority of tuberomammilary neurons. The finding of Gal immunoreactivity within histamine-producing neurons of the tuberomammillary nucleus adds to the multiplicity of potential neuronal messengers utilized by these cells.

An EM autoradiographic study of the hypothalamo-hippocampal projection.

Previous studies have shown that in many different mammals there is a small but distinct projection from the supramammillary region in the caudal hypothalamus to the junctional region between the regio superior and regio inferior of the hippocampus. We have analyzed the mode of termination of this hypothalamo-hippocampal projection in the rat by electron microscopic (EM) autoradiography following injections of [3H]proline into the caudal hypothalamus. The projection is confined to the regio inferior where it is centered over the subicular end of field CA3, but also spans the adjoining region, field CA2. In our material the highest densities of labeling have been seen over the deeper part of the pyramidal cell layer and in the adjacent stratum oriens but, in addition, above background levels of labeling have been found superficial to the pyramidal cell layer in the stratum lucidum and the deeper part of the stratum radiatum. Most of the labeled synapses appear to be on the perikarya and primary dendrites of the hippocampal pyramidal cells, but some axo-spinous contacts have also been seen. All the labeled boutons contained clear, spheroidal synaptic vesicles and made asymmetric, Type I, contacts with their targets.

[Ultrastructure of the neurons and synapses of the posterior hypothalamic field in immune reactions]. / Ul'trastruktura neironov i sinapsov zadnego gipotalamicheskogo polia pri immunnykh reaktsiiakh.

The antigenic stimulation induces alteration of the ultrastructure of neurons, synapses and glial cells in area hypothalamic posterior of the brain of experimental animals. Swelling of mitochondria, partial or total destruction of cristae points out to the involvement of the energy apparatus of neurons and synapses. An intensive formation of antibodies is accompanied by an increase in the structural functional activity of neurons. Ultrastructural alterations of some organelles of neurones, synapses and glial cells are connected with adaptive reactions of intracellular structures and have a non-specific nature.

[Morphometric study of the effect of 5-hydroxytryptophan on the activity of various hypothalamic centers in rats depending on the state of their sexual system]. / Morfometricheskoe izuchenie vliianiia 5-oksitriptofana na aktivost' nekotorykh gipotalamicheskikh tsentrov u krys v zavisimosti ot sostoianiia ikh polovoi sistemy.

The effect of 5-oxytryptophan (serotonin metabolic precursor), injected intraperitoneally to female rats (intact and with deafferented mediobasal hypothalamus) on the activity of a number of hypothalamic centres was studied. The activity of cells, both outside and inside the isolated area, proved to alter under the effect of 5-oxytryptophan. The direction of these effects depends on the initial state of the hypothalamic centres and on the hormone in whose presence serotonin acts.

Astrocytic gap junctions in the rat lateral hypothalamic area.

An extensive system of gap or nexus junctions has been found between astrocytic processes in the neuropil of the lateral hypothalamic area in the albino rat. These specialized interastrocytic junctions occur in regions of high synaptic density where neural processes are separated by the interconnected glial system. In this study, 90% of the gap junctions observed in the lateral hypothalamic neuropil are in the immediate proximity of synaptic terminals. The close morphological relationship of these glial gap junctions with synaptic contacts suggests that they may play a significant role in the process of synaptic transmission.