Restricted expression of Neuroglobin in the mouse retina and co-localization with Melanopsin and Tyrosine Hydroxylase.

Neuroglobin (Ngb), a neuronal specific oxygen binding heme-globin, reported to be expressed at high levels in most layers of the murine retina. Ngb's function is presently unknown, but based on its high expression level and oxygen binding capabilities Ngb was proposed to function as an oxygen reservoir facilitating oxygen metabolism in highly active neurons or to function as a neuroprotectant. In the present study, we re-examined the expression pattern of Ngb in the retina using a highly validated antibody. Furthermore, intactness of retino-hypothalamic projections and the retinal expression level of Melanopsin and Tyrosine Hydroxylase were investigated in Ngb-null mice. Ngb-immunoreactivity was found in a few neurons of the ganglion cell and inner nuclear layers co-expressing Melanopsin and Tyrosine Hydroxylase, respectively. Ngb deficiency neither affected the level of Melanopsin and Tyrosine Hydroxylase proteins nor the intactness of PACAP-positive retinohypothalamic projections in the suprachiasmatic nucleus. Based on the present results, it seems unlikely that Ngb could have a major role in retinal oxygen homeostasis and neuronal survival under normal conditions. The present study suggests that a number of previously published reports have relied on antibodies with dubious specificity.

K(ATP) channel openers in the trigeminovascular system.

BACKGROUND: The ATP-sensitive K(+) (K(ATP)) channel openers levcromakalim and pinacidil are vasodilators that induce headache in healthy people. The neuropeptide calcitonin gene-related peptide (CGRP) induces headache in healthy people and migraine in migraineurs, potentially through a mechanism that involves opening of vascular or neuronal K(ATP) channels and mast cell degranulation. Using rat as a model, we studied the molecular presence of K(ATP) channels in the trigeminovascular system. Furthermore, we examined whether K(ATP) channel openers stimulate the in vitro release of CGRP and whether they degranulate dural mast cells. METHODS: mRNA and protein expression of K(ATP) channel subunits were studied in the trigeminal ganglion (TG) and trigeminal nucleus caudalis (TNC) by qPCR and western blotting. In vitro CGRP release was studied after application of levcromakalim (1 µM) and diazoxide (10 µM) to freshly isolated rat dura mater, TG and TNC. Rat dural mast cells were challenged in situ with levcromakalim (10(-5) M) to study its potential degranulation effect. RESULTS: mRNA and protein of K(ATP) channel subunits Kir6.1, Kir6.2, SUR1 and SUR2B were identified in the TG and TNC. K(ATP) channel openers did not release or inhibit capsaicin-induced CGRP release from dura mater, TG or TNC. They did also not induce dural mast cell degranulation. CONCLUSIONS: K(ATP) channel openers do not interact with CGRP release or mast cell degranulation. Activation of these channels in the CNS is antinociceptive and therefore cannot explain the headache induced by K(ATP) channel openers. Thus, they are likely to induce headache by interaction with extracerebral K(ATP) channels, probably the SUR2B isoforms.

The selective alpha7 nicotinic acetylcholine receptor agonist A-582941 activates immediate early genes in limbic regions of the forebrain: Differential effects in the juvenile and adult rat.

Due to the cognitive-enhancing properties of alpha7 nicotinic acetylcholine receptor (alpha7 nAChR) agonists, they have attracted interest for the treatment of cognitive disturbances in schizophrenia. Schizophrenia typically presents in late adolescence or early adulthood. It is therefore important to study whether alpha7 nAChR stimulation activates brain regions involved in cognition in juvenile as well as adult individuals. Here, we compared the effects of the novel and selective alpha7 nAChR agonist 2-methyl-5-(6-phenyl-pyridazin-3-yl)-octahydro-pyrrolo[3,4-c]pyrrole (A-582941) in the juvenile and adult rat forebrain using two markers, activity-regulated cytoskeleton-associated protein (Arc) and c-Fos, to map neuronal activity. Acute administration of A-582941 (1, 3, 10 mg/kg) induced a dose-dependent increase in Arc mRNA expression in the medial prefrontal cortex (mPFC) and the ventral/lateral orbitofrontal (VO/LO) cortex of juvenile, but not adult rats. This effect was mitigated by the alpha7 nAChR antagonist methyllycaconitine. A-582941 also increased c-Fos mRNA expression in the mPFC of juvenile, but not adult rats. Furthermore, A-582941 increased the number of Arc and c-Fos immunopositive cells in the mPFC, VO/LO, and shell of the nucleus accumbens, in both juvenile and adult rats. The A-582941-induced c-Fos protein expression was significantly greater in the mPFC and VO/LO of juvenile compared with adult rats. These data indicate that A-582941-induced alpha7 nAChR stimulation activates brain regions critically involved in working memory and attention. Furthermore, this effect is more pronounced in juvenile than adult rats, indicating that the juvenile forebrain is more responsive to alpha7 nAChR stimulation. This observation may be relevant in the treatment of juvenile-onset schizophrenia.

Exposure to an open-field arena increases c-Fos expression in a distributed anxiety-related system projecting to the basolateral amygdaloid complex.

Anxiety states and anxiety-related behaviors appear to be regulated by a distributed and highly interconnected system of brain structures including the basolateral amygdala. Our previous studies demonstrate that exposure of rats to an open-field in high- and low-light conditions results in a marked increase in c-Fos expression in the anterior part of the basolateral amygdaloid nucleus (BLA) compared with controls. The neural mechanisms underlying the anatomically specific effects of open-field exposure on c-Fos expression in the BLA are not clear, however, it is likely that this reflects activation of specific afferent input to this region of the amygdala. In order to identify candidate brain regions mediating anxiety-induced activation of the basolateral amygdaloid complex in rats, we used cholera toxin B subunit (CTb) as a retrograde tracer to identify neurons with direct afferent projections to this region in combination with c-Fos immunostaining to identify cells responding to exposure to an open-field arena in low-light (8-13 lux) conditions (an anxiogenic stimulus in rats). Adult male Wistar rats received a unilateral microinjection of 4% CTb in phosphate-buffered saline into the basolateral amygdaloid complex. Rats were housed individually for 11 days after CTb injections and handled (HA) for 2 min each day. On the test day rats were either, 1) exposed to an open-field in low-light conditions (8-13 lux) for 15 min (OF); 2) briefly HA or 3) left undisturbed (control). We report that dual immunohistochemical staining for c-Fos and CTb revealed an increase in the percentage of c-Fos-immunopositive basolateral amygdaloid complex-projecting neurons in open-field-exposed rats compared with HA and control rats in the ipsilateral CA1 region of the ventral hippocampus, subiculum and lateral entorhinal cortex. These data are consistent with the hypothesis that exposure to the open-field arena activates an anxiety-related neuronal system with convergent input to the basolateral amygdaloid complex.

Exposure to an open-field arena increases c-Fos expression in a subpopulation of neurons in the dorsal raphe nucleus, including neurons projecting to the basolateral amygdaloid complex.

Serotonergic systems in the dorsal raphe nucleus are thought to play an important role in the regulation of anxiety states. To investigate responses of neurons in the dorsal raphe nucleus to a mild anxiety-related stimulus, we exposed rats to an open-field, under low-light or high-light conditions. Treatment effects on c-Fos expression in serotonergic and non-serotonergic cells in the midbrain raphe nuclei were determined 2 h following open-field exposure or home cage control (CO) conditions. Rats tested under both light conditions responded with increases in c-Fos expression in serotonergic neurons within subdivisions of the midbrain raphe nuclei compared with CO rats. However, the total numbers of serotonergic neurons involved were small suggesting that exposure to the open-field may affect a subpopulation of serotonergic neurons. To determine if exposure to the open-field activates a subset of neurons in the midbrain raphe complex that projects to forebrain circuits regulating anxiety states, we used cholera toxin B subunit (CTb) as a retrograde tracer to identify neurons projecting to the basolateral amygdaloid complex (BL) in combination with c-Fos immunostaining to identify cells that responded to open-field exposure. Rats received a unilateral injection of CTb into the BL. Seven to 11 days following CTb injection rats were either, 1) exposed to an open-field in low-light conditions, 2) briefly handled or 3) left undisturbed in home cages. Dual immunostaining for c-Fos and CTb revealed an increase in the percentage of c-Fos-immunoreactive BL-projecting neurons in open-field-exposed rats compared with handled and control rats. Dual immunostaining for tryptophan hydroxylase and CTb revealed that a majority (65%) of BL-projecting neurons were serotonergic, leaving open the possibility that activated neurons were serotonergic, non-serotonergic, or both. These data are consistent with the hypothesis that exposure to anxiogenic stimuli activates a subset of neurons in the midbrain raphe complex projecting to amygdala anxiety circuits.

K(ATP) channel expression and pharmacological in vivo and in vitro studies of the K(ATP) channel blocker PNU-37883A in rat middle meningeal arteries.

BACKGROUND AND PURPOSE: Dilatation of cerebral and dural arteries causes a throbbing, migraine-like pain, indicating that these structures are involved in migraine. Clinical trials suggest that adenosine 5'-triphosphate-sensitive K(+) (K(ATP)) channel opening may cause migraine by dilatating intracranial arteries, including the middle meningeal artery (MMA). We studied the K(ATP) channel expression profile in rat MMA and examined the potential inhibitory effects of the K(ATP) channel blocker PNU-37883A on K(ATP) channel opener-induced relaxation of the rat MMA, using the three K(ATP) channel openers levcromakalim, pinacidil and P-1075. EXPERIMENTAL APPROACH: mRNA and protein expression of K(ATP) channel subunits in the rat MMA were studied by quantitative real-time PCR and western blotting, respectively. The in vivo and in vitro effects of the K(ATP) channel drugs on rat MMA were studied in the genuine closed cranial window model and in myograph baths, respectively. KEY RESULTS: Expression studies indicate that inwardly rectifying K(+) (Kir)6.1/sulphonylurea receptor (SUR)2B is the major K(ATP) channel complex in rat MMA. PNU-37883A (0.5 mg kg(-1)) significantly inhibited the in vivo dilatory effect of levcromakalim (0.025 mg kg(-1)), pinacidil (0.38 mg kg(-1)) and P-1075 (0.016 mg kg(-1)) in rat MMA. In vitro PNU-37883A significantly inhibited the dilatory responses of the three K(ATP) channel openers in rat MMA at 10(-7) and 3 x 10(-7) M. CONCLUSIONS AND IMPLICATIONS: We suggest that Kir6.1/SUR2B is the major functional K(ATP) channel complex in the rat MMA. Furthermore, we demonstrate the potent in vivo and in vitro blocking potentials of PNU-37883A on K(ATP) channel opener-induced relaxation of the rat MMA.

Serotonergic systems associated with arousal and vigilance behaviors following administration of anxiogenic drugs.

Serotonergic systems play important roles in modulating behavioral arousal, including behavioral arousal and vigilance associated with anxiety states. To further our understanding of the neural systems associated with increases in anxiety states, we investigated the effects of multiple anxiogenic drugs on topographically organized subpopulations of serotonergic neurons using double immunohistochemical staining for c-Fos and tryptophan hydroxylase combined with topographical analysis of the rat dorsal raphe nucleus (DR). Anxiogenic drugs with diverse pharmacological properties including the adenosine receptor antagonist caffeine, the serotonin 5-HT2A/2C receptor agonist m-chlorophenyl piperazine (mCPP), the alpha2-adrenoreceptor antagonist yohimbine, and the benzodiazepine receptor partial inverse agonist N-methyl-beta-carboline-3-carboxamide (FG-7142) induced increases in behavioral arousal and vigilance behaviors consistent with an increase in anxiety state. In addition, these anxiogenic drugs, excluding yohimbine, had convergent actions on an anatomically-defined subset of serotonergic neurons within the middle and caudal, dorsal subdivision of the DR. High resolution topographical analysis revealed that at the mid-rostrocaudal level, caffeine and FG-7142 had convergent effects on c-Fos expression in serotonergic neurons that were restricted to a previously undefined region, which we have named the shell region of the dorsal part of the dorsal raphe nucleus (DRDSh), that overlaps the anatomical border between the dorsal part of the dorsal raphe nucleus, the ventral part of the dorsal raphe nucleus (DRV), and the ventrolateral part of the dorsal raphe nucleus (DRVL). Retrograde tracing methods revealed that DRDSh contains large numbers of neurons projecting to the basolateral amygdaloid nucleus, a forebrain structure important for emotional appraisal and modulation of anxiety-related physiological and behavioral responses. Together these findings support the hypothesis that there is a functional topographical organization in the DR and are consistent with the hypothesis that anxiogenic drugs have selective actions on a subpopulation of serotonergic neurons projecting to a distributed central autonomic and emotional motor control system regulating anxiety states and anxiety-related physiological and behavioral responses.

Regulation of activity-regulated cytoskeleton protein (Arc) mRNA after acute and chronic electroconvulsive stimulation in the rat.

The temporal profile of Arc gene expression after acute and chronic electroconvulsive stimulations (ECS) was studied using semi-quantitative in situ hybridisation in the rat cortex. A single ECS strongly and temporarily increased Arc mRNA levels in dentate granular cells with maximal induction seen up to 4 h after the stimulus, but returned to baseline at 24 h. A single ECS also increased expression of Arc mRNA in the CA1 and the parietal cortex, but the expression peaked within 1 h and returned to baseline levels within 2 h. Repeated or chronic ECS is a model of electroconvulsive therapy and it would be predicted that gene products involved in antidepressant effects accumulate after repeated ECS. However, repeated ECS reduced Arc gene expression in the CA1 24 h after the last stimulus. These results indicate that Arc is an immediate early gene product regulated by an acute excitatory stimulus, but not accumulated by long term repetitive ECS and therefore not a molecular biomarker for antidepressant properties. More likely, Arc is likely a molecular link to the decline in memory consolidation seen in depressive patients subjected to electroconvulsive therapy.

Cloning and analysis of an HMG gene from the lamprey Lampetra fluviatilis: gene duplication in vertebrate evolution.

Evolution has shaped the organisation of vertebrate genomes, including the human genome. To shed further light on genome history, we have cloned and analysed an HMG gene from lamprey, representing one of the earliest vertebrate lineages. Genes of the HMG1/2 family encode chromosomal proteins that bind DNA in a non-sequence-specific manner, and have been implicated in a variety of cellular processes dependent on chromatin structure. They are characterised by two copies of a conserved motif, the HMG box, followed by an acidic C-terminal region. We report here the cloning of a cDNA clone from the river lamprey Lampetra fluviatilis containing a gene with two HMG boxes and an acidic tail; we designate this gene LfHMG1. Molecular phylogenetic analysis shows that LfHMG1 is descended from a gene ancestral to mammalian HMG1 and HMG2. This implies that there was a duplication event in the HMG1/2 gene family, that occurred after the divergence of the jawed and jawless fishes, 450 million years ago. This conclusion supports and refines the hypothesis that there was a period of extensive gene duplication early in vertebrate evolution. We also show that the HMG1/2 family originated before the protostomes and deuterostomes diverged, over 525 million years ago.

Efferent connections from the lateral hypothalamic region and the lateral preoptic area to the hypothalamic paraventricular nucleus of the rat.

The lateral preoptic and lateral hypothalamic regions contain the majority of the cell groups embedded in the fibre trajectories of the medial forebrain bundle on its course through the hypothalamus. Recent studies have extended considerably the parcellation of the lateral hypothalamic region, and therefore, the need to emphasize new insights into the anatomical organisation of projections from the neurons of the lateral hypothalamic region. In the present study we describe the anatomical organisation of efferent projections from the lateral preoptic and lateral hypothalamic regions to the hypothalamic paraventricular nucleus (PVN) on the basis of retrograde- and anterograde-tracing techniques. Iontophoretic injections of the retrograde tracer, cholera toxin subunit B, into the PVN revealed that most hypothalamic nuclei project to the PVN. Within the lateral hypothalamic region, retrogradely labelled cells were concentrated in the intermediate hypothalamic area, the lateral hypothalamic area, and the perifornical nucleus, whereas fewer retrogradely labelled cells were found in the lateral preoptic area. To determine the distribution of terminating fibres in subnuclei of the heterogeneous PVN, iontophoretic injections of the anterograde tracer Phaseolus vulgaris-leucoagglutinin were delivered into distinct areas of the lateral hypothalamic region. Neurons of the intermediate hypothalamic area projected mainly to the PVN subnuclei, which contained parvicellular neuroendocrine cells. In contrast, neurons of the rostral and tuberal parts of the lateral hypothalamic area and the perifornical nucleus projected to the PVN subnuclei, which contained parvicellular neurons that send descending projections to preganglionic cell groups in the medulla and spinal cord. The perifornical nucleus was the only area within the lateral hypothalamic region that consistently innervated magnocellular perikarya of the PVN. Finally, all areas of the lateral hypothalamic region contributed substantially to fibres terminating in the perinuclear shell of the PVN. These results demonstrate that anatomically distinct areas of the lateral hypothalamic region have distinct projections to subnuclei of the PVN and further substantiate the view that the lateral hypothalamic region as well as the PVN constitute anatomically and functionally heterogeneous structures.

Central innervation of the rat ependyma and subcommissural organ with special reference to ascending serotoninergic projections from the raphe nuclei.

The subcommissural organ (SCO) and the cerebral ependyma receive serotoninergic innervation, but little is known about their origin in the raphe nuclei. Application of the retrograde tracer cholera toxin subunit B (ChB) in the third ventricle resulted in uptake in ependymal axons and backfilling of perikarya in the dorsomedian part of the dorsal raphe nucleus, immediately under the caudal aqueduct. By using dual staining with antisera against serotonin and ChB, a portion of the retrogradely labeled neurons was observed to co-store serotonin. Phaseolus vulgaris-leucoagglutinin (PHA-L) was injected into different raphe nuclei to fill the neurons in the same areas where the retrogradely labeled neurons were found. PHA-L injection in the midline of the dorsal raphe nucleus gave rise to ascending axonal processes in the mesencephalic central gray, from where they entered the periventricular strata and the third ventricular ependyma. In the cerebral ependyma, large numbers of positive fibers were consistently found in the ventral part of the lateral ventricles and in the dorsal part of the third ventricle. A large number of PHA-L-immunoreactive fibers were observed in the hypendymal layer of the lateral part of the SCO. Terminal fibers near the ependymal cells were also observed. In all cases, the PHA-L injections labeled innervating fibers both within the ependyma and in the SCO, whereas injections into the median raphe nucleus or in other raphe nuclei (i.e., the raphe pallidus and the raphe pontis) labeled fibers neither in the SCO nor in the ependyma. This study shows that a specific group of predominantly serotoninergic neurons innervates both the ependyma and the SCO and is probably involved in cerebrospinal fluid regulation.

Immunohistochemical localization of the somatostatin receptor subtype 2 (sst2) in the central nervous system of the golden hamster (Mesocricetus auratus).

The many actions of somatostatin in the central nervous system are mediated through specific membrane receptors of which five have been cloned. In this study, we have investigated the distribution of one of these receptors, the sst2 subtype, in the brain and spinal cord of the golden hamster (Mesocricetus auratus). Immunohistochemistry was carried out by using polyclonal antibodies raised against the C-terminal part of the human sst2 receptor. sst2 immunoreactivity was found in the forebrain, brainstem, cerebellum, and spinal cord. In the forebrain, strong immunoreactivity was observed in the deep layers of the neocortex as well as in the endopiriform cortex, claustrum, and basolateral amygdaloid nucleus. Immunoreactivity was also found in the CA1 area of the hippocampus and in the subiculum. In the diencephalon, staining was observed in the periventricular area, the dorsomedial and arcuate nuclei of the hypothalamus, and the medial habenular nucleus. Other areas such as the thalamus, striatum, and globus pallidus were almost devoid of staining. In the brainstem, strong immunoreactivity was observed in the locus coeruleus and the parabrachial nucleus. In addition, immunostaining was observed in the cortex of the cerebellum. In the spinal cord, intense immunoreactivity was seen in lamina I and II of the dorsal horn. Finally, immunoreactive cells were widely distributed in the anterior pituitary. The localization of the sst2 receptor in many brain regions suggests that this receptor subtype is involved in different neuromodulatory actions of somatostatin such as somatosensory, motor, memory, and neuroendocrine functions.

Origin of projections from the midbrain raphe nuclei to the hypothalamic paraventricular nucleus in the rat: a combined retrograde and anterograde tracing study.

A number of neuronal functions governed by the hypothalamic paraventricular nucleus are influenced by serotonin, and it is generally believed that the moderate density of serotonin-immunoreactive fibres and terminals within the paraventricular nucleus originates from the midbrain dorsal and median raphe nuclei. To further evaluate the intricate anatomy of projections from brain stem raphe nuclei of the rat, a combination of retrograde and anterograde tracing experiments were conducted to determine the medullary raphe nuclei projection to the paraventricular nucleus. Rhodamine-labelled latex microspheres, Cholera toxin subunit B and FluoroGold we used as retrograde tracers. Intracerebroventricular injections into the third ventricle of all retrograde tracers labelled a distinct population of neurons in the dorsal raphe situated in the subependymal stratum adjacent to the cerebral aqueduct indicating that these cells take up the tracer from the cerebrospinal fluid. Very few retrogradely labelled neurons were seen in the median raphe after i.c.v. administration of the tracers. Retrograde tracers delivered into the medial part of the paraventricular nucleus labelled no further cells in the midbrain dorsal and median raphe nuclei, whereas a substantial number of retrogradely labelled cells emerged in the pontine raphe magnus. However, when the retrograde tracers were delivered into the lateral part of the paraventricular nucleus, avoiding leakage of the tracer into the ventricle, very few labelled neurons were seen in the dorsal and median raphe, whereas the prominent labelling of raphe magnus neurons persisted. The anatomical organization of nerve fibres terminating in the area of the paraventricular nucleus originating from midbrain raphe nuclei was studied in a series of anterograde tracing experiments using the plant lectin Phaseolus vulgaris leucoagglutinin. Injections delivered into the dorsal raphe or median raphe labelled but a few fibres in the paraventricular nucleus proper. A high number of fine calibered nerve fibres overlying the ependyma adjacent to the paraventricular nucleus was, however, seen after the injections into the subependymal rostral part of the dorsal raphe. Injections delivered into the raphe magnus gave rise to a dense plexus of terminating fibres in the parvicellular parts of the paraventricular nucleus and moderately innervated the posterior magnocellular part of the paraventricular nucleus as well as the magnocellular supraoptic nucleus. Concomitant visualization of serotonin-immunoreactive neurons and retrograde FluoroGold-tracing from the paraventricular nucleus revealed that none of the serotonergic neurons of the raphe magnus projects to this nucleus, while a few of the neurons putatively projecting to the paraventricular nucleus from the median raphe are serotonergic. The current observations suggest that the raphe magnus constitute by far the largest raphe input to the paraventricular nucleus and strongly questions the earlier held view that most raphe fibres innervating the paraventricular nucleus are derived from the midbrain dorsal and median raphe. However, the source of serotonergic innervation of the paraventricular nucleus remains elusive.

The evolution of the serotonergic nervous system.

The pattern of development of the serotonergic nervous system is described from the larvae of ctenophores, platyhelminths, nemerteans, entoprocts, ectoprocts (bryozoans), molluscs, polychaetes, brachiopods, phoronids, echinoderms, enteropneusts and lampreys. The larval brain (apical ganglion) of spiralian protostomes (except nermerteans) generally has three serotonergic neurons and the lateral pair always innervates the ciliary band of the prototroch. In contrast, brachiopods, phoronids, echinoderms and enteropneusts have numerous serotonergic neurons in the apical ganglion from which the ciliary band is innervated. This pattern of development is much like the pattern seen in lamprey embryos and larvae, which leads the author to conclude that the serotonergic raphe system found in vertebrates originated in the larval brain of deuterostome invertebrates. Further, the neural tube of chordates appears to be derived, at least in part, from the ciliary band of deuterostome invertebrate larvae. The evidence shows no sign of a shift in the dorsal ventral orientation within the line leading to the chordates.

Vasoactive intestinal peptide and substance P-like immunoreactivities in the enteric nervous system of the pig correlate with the severity of pathological changes induced by Schistosoma japonicum.

Limited studies have shown that in intestinal schistosomosis, the enteric nervous tissue becomes inflamed, disrupted and destroyed by granulomas and peptides, amines and neurofilaments contents are altered. Therefore, immunoreactivities of vasoactive intestinal peptide and substance P were correlated to pathological lesions in the large intestine from pigs infected with Schistosoma japonicum. Ganglia situated within or near granulomas showed ganglionitis, and necrosis of neurons as well as infiltration by eosinophils, mast cells, lymphocytes, plasma cells, neutrophils and macrophages. The inner submucous and mucous plexuses were the most damaged. In all categories of inflamed areas, the vasoactive intestinal peptide-like immunoreactive was reduced in all plexuses whereas, that of substance P was increased both in the enteric nerve plexuses and enterochromaffin cells in lightly, moderately and severely inflamed tissues. However, both peptides were highly diminished or absent in very severe lesions and areas surrounding schistosome eggs and mature worms laying eggs in the submucosal veins. The alterations of the levels of vasoactive intestinal peptide and substance P were correlated with severity of inflammation. Our observations show alterations of vasoactive intestinal peptide and substance P contents in the local microenvironment in the vasoactive intestinal peptide- and substance P-mediated reflex pathways which regulate intestinal motility, epithelial transport and modulate immunity. These changes could cause alterations in bowel motility, electrolyte and fluid secretion, vascular and immune functions during S. japonicum infections in the pig. This may, therefore, partly play a role in the pathobiology of migration and egress of schistosome eggs as well as influence trapping of eggs in granulomas, and account for diarrhoea, loss of body weight and failure to thrive, which are recorded in schistosomosis.

Demonstration of a neuronal projection from the entopeduncular nucleus to the substantia nigra of the rat.

The two neuronal tracers, Phaseolus vulgaris leucoagglutinin (PHA-L) and Cholera toxin subunit B (CHB) were used in order to study a possible neuronal projection from the entopeduncular nucleus to the ventral mesencephalon in the rat. Both tracers were identified in brain sections by means of immunohistochemistry. After injection of PHA-L in the entopeduncular nucleus PHA-L-positive nerve fibers observed in the mesencephalon were moderate in number and mostly restricted to the mediodorsal part of the substantia nigra, pars reticulata. In addition, a low number of PHA-L-immunoreactive nerve fibers was found in the substantia nigra, pars compacta, the rostral part of the ventral tegmental area, and in the deep mesencephalic nucleus. In agreement with these observations, several labeled neurons were observed in the ipsilateral entopeduncular nucleus after injections of CHB in the substantia nigra. These results indicate the presence of a direct neuronal projection between the two major output channels of the basal ganglia.

Neuronal nitric oxide synthase activity is increased during granulomatous inflammation in the colon and caecum of pigs infected with Schistosoma japonicum.

Neuronal nitric oxide is a non-adrenergic non-cholinergic neurotransmitter in the enteric nervous system and plays a role in a variety of enteropathies including Crohn's and Chagas' diseases, ulcerative colitis, diabetes, atrophy and hypertrophy. The content of neuronal nitric oxide synthase (nNOS) in the colon and the caecum from pigs infected with Schistosoma japonicum was studied using immunohistochemical and histochemical staining for nNOS and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase), respectively. In the infected pigs, lightly, moderately and less severely inflamed tissues showed increased nNOS and NADPH-diaphorase activities in nerve cell bodies and nerve fibres in the enteric plexuses compared to control pigs. There was a significant increase in the nerve cell body density of nNOS immunoreactive nerve cell bodies in the inner submucous plexus, outer submucous plexus and in the myenteric plexus. More intensely stained nerve cell bodies and varicosities were observed in tissue from prenatally infected and prenatally infected, postnatally re-infected pigs compared to postnatally infected pigs. However, the latter showed the highest numerical density of nNOS immunoreactive nerve cell bodies. Marked increases were seen in the inner submucous plexus followed by myenteric plexus, inner circular muscle, outer submucous plexus and mucous plexus. However, in very severe inflamed tissues, the number and staining intensity of nerve cell bodies and nerve fibre varicosities were reduced in plexuses located in the lesions with the inner submucous and mucous plexuses being the most affected. There was no staining in the nervous tissue within the eosinophilic cell abscesses and productive granulomas. The apparent alterations in the activities of enzymes responsible for the generation of nitric oxide (NO) show possible alterations in the NO mediated non-adrenergic non-cholinergic reflexes in the enteric nervous tissue. These alterations might contribute to impaired intestinal motility and absorption, and other pathophysiological conditions seen during S. japonicum infections.

Lesions of the enteric nervous system and the possible role of mast cells in the pathogenic mechanisms of migration of schistosome eggs in the small intestine of cattle during Schistosoma bovis infection.

The enteric nervous system in the small intestine of cattle during Schistosoma bovis infection was studied by histological stains and immunohistochemical methods. Lesions due to migration of schistosoma eggs were located mainly in the mucous and the submucous layer overlaying the submucous vascular arcades. Granulomas destroyed ganglia, neurons, nerves fibre strands and nerve fibres. Ganglia situated within or near granulomas were infiltrated by mast cells, eosinophils, lymphocytes, globule leukocytes, neutrophils and macrophages. Mast cells were in close contact with degenerating neuronal perikarya. Whereas vasoactive intestinal peptide-like immunoreactivity in the nerves and neurons in the ganglia within and around granulomas was increased, the neurofilament-like immunoreactivity was reduced. Compared to the myenteric and external submucous plexuses, the internal submucous and mucous plexuses were the most damaged. These changes imply reduced functional capacity in the nervous tissue which might cause reduced motility, malabsorption and partly account for the loss of body weight and condition and failure to thrive which occur in schistosomosis. Biotinylated affinity purified swine anti-rabbit and mouse anti-rabbit immunoglobulins reacted nonspecifically with a subset of mast cells. The reaction revealed many mast cells in early forming granulomas and around schistosome egg tracts and infiltration of mast cells into the ganglia of intestinal nerve plexuses. The observation shows a localized, Type I hypersensitivity reaction suggesting for the release of mast cell-derived chemical mediators in the intestinal reaction to trap or evict S. bovis eggs and to cause diarrhoea.

Transcriptional activity in in vivo developed early cleavage stage bovine embryos.

Bovine embryos developed in vivo from the first to the fourth post-fertilization cell cycles were processed for ultrastructural autoradiography after incubation with 3H-uridine for 10 h. We wished to detect and localize transcriptional activity. During the first (1-cell stage) and second (2-cell stage) cell cycles we observed electron-dense fibrillar spheres (nucleolus precursor bodies) and fibrillo-granular complexes in the nuclei. During these cell cycles, autoradiographic labeling was observed in heterochromatic areas and at the periphery of the fibrillo-granular complexes. During the third cell cycle (4-cell stage) the electron dense fibrillar spheres exhibited vacuolization. Autoradiographic labeling was found in heterochromatic areas and in the vacuoles of the fibrillar spheres. During the fourth cell cycle (8-cell stage), the electron dense fibrillar spheres exhibited both a large eccentric vacuole and peripheral smaller vacuoles. Autoradiographic labeling was found in heterochromatic areas throughout the nucleus and over the substance of the vacuolated fibrillar spheres, especially where chromatin penetrated into them and where presumptive fibrillar centers were formed. In conclusion, a low level of transcription can be detected in in vivo developed bovine embryos as early as the one-cell stage. Moreover, nuclear entities that probably prepare for nucleolus formation during the fourth cell cycle, display a progressive autoradiographic labeling that signals a possible initiation of transcription of the ribosomal RNA genes during the third cell cycle.

[Human serum albumin as the protein source in culture of human oocytes, spermatozoa and pre-embryos]. / Humant serum-albumin som proteinkilde ved dyrkning af humane oocytter, spermatozoer og praeembryoer.

In the treatment of infertility employing in vitro fertilisation and embryo transfer (IVF-EF), oocytes, spermatozoa and pre-embryos are cultured for 48 hours outside the woman's body before they are introduced into the uterus. In addition to the necessary salts, the media in which this culture takes place, also consists of a source of protein. In order to eliminate the variability of patient sera, a prospective, randomized investigation was performed to elucidate whether a well-defined source of protein such as human serum albumin (hSA-hSA 200 mg/ml, Statens Seruminstitute) can replace patient serum as source of protein in the culture of oocytes, spermatozoa and pre-embryos in IVF-ET treatment. The pregnancy rate per transplantation was increased from 30% in the serum group (21 pregnant out of 69 transplantations) to 39% in the albumin group (26 pregnant out of 66 transplantations) but the difference is not significant. On the other hand, the quality of the pre-embryos as assessed by morphological criteria became significantly better and the implantation rate per transplanted pre-embryo was found to be significantly increased in the albumin group. On the basis of this investigation, hSA is recommended as the source of protein, rather than the patient's own serum in the culture of oocytes, spermatozoa and pre-embryos in IVF-ET treatment.