Acute inflammation alters adult hippocampal neurogenesis in a multiple sclerosis mouse model.

Neural precursor cells (NPCs) located in the subgranular zone (SGZ) of the dentate gyrus (DG) give rise to thousands of new cells every day, mainly hippocampal neurons, which are integrated into existing neuronal circuits. Aging and chronic degenerative disorders have been shown to impair hippocampal neurogenesis, but the consequence of inflammation is somewhat controversial. The present study demonstrates that the inflammatory environment prevailing in the brain of experimental autoimmune encephalomyelitis (EAE) mice enhances the proliferation of NPCs in SGZ of the dorsal DG and alters the proportion between radial glial cells and newborn neuroblasts. The injection protocol of the cell cycle marker bromodeoxyuridine and the immunohistochemical techniques that were employed revealed that the proliferation of NPCs is increased approximately twofold in the SGZ of the dorsal DG of EAE mice, at the acute phase of the disease. However, although EAE animals exhibited significant higher percentage of newborn radial-glia-like NPCs, the mean percentage of newborn neuroblasts rather was decreased, indicating that the robust NPCs proliferation is not followed by a proportional production of newborn neurons. Significant positive correlations were detected between the number of proliferating cells in the SGZ and the clinical score or degree of brain inflammation of diseased animals. Finally, enhanced neuroproliferation in the acute phase of EAE was not found to trigger compensatory apoptotic mechanisms. The possible causes of altered neurogenesis observed in this study emphasize the need to understand more precisely the mechanisms regulating adult neurogenesis under both normal and pathological conditions.

The milk-ejection reflex in the sheep: an anatomical study on the afferent pathway.

The present study is an attempt to reveal the spinal and supraspinal organization of the ascending branch of the milk-ejection reflex in the ewe by means of a tract-tracing technique. For this purpose, injections of horseradish peroxidase (HRP) were performed into the lateral cervical nucleus (LCN) and into the hypothalamic paraventricular nucleus (PVN). Peroxidase injections into the LCN revealed retrogradely labelled neurons in the medial part of laminae I-III of the ipsilateral L3 and L4 spinal segments, while injections of HRP into the PVN revealed retrogradely labelled cells in the contralateral LCN and the medial cuneate nucleus. Taking into account the results obtained, it is concluded that the transmission of the afferent input from the nipples to the PVN is accomplished by at least two pathways: one employing a single relay station located in the medial cuneate nucleus, and another possessing two relay stations located in the medial part of laminae I-III of the dorsal horn of L3 and L4 spinal segments and in the LCN.

Juvenile laryngeal paralysis in three Siberian husky x Alaskan malamute puppies.

Three three-month-old Siberian husky x Alaskan malamute crossbreds had suffered episodic inspiratory dyspnoea and stridor for four to eight weeks and their endurance had decreased. In two of them bilateral, and in the other unilateral, laryngeal paralysis was diagnosed by laryngoscopy. In the nucleus ambiguus of the dogs there was a depletion of motor neurons, neuronal degeneration and mild gliosis, but there were no lesions in the root and peripheral segments of the recurrent laryngeal nerves.

Vascular network of the rat hippocampus is not homogeneous along the septotemporal axis.

Qualitative and quantitative image analysis of hippocampal vascular bed, after transcardial perfusion of India ink, reveals significant differences among hippocampal subfields and along the septotemporal axis of the rat hippocampus. Ventral hippocampus exhibits significantly higher levels of vascularization compared to dorsal hippocampus, which, however, is characterized by significantly higher capillary density. These results may explain the selective ischemia vulnerability of hippocampus along its septotemporal axis.

Analysis of transient expression of estrogen receptor-alpha in newborn rat primary auditory cortex.

In the primary temporal cortex (Te1) of newborn rats, we detected transient expression of alpha-type estrogen receptor (ER alpha). Since they have a pyramidal-like shape, they are considered neurons. By immunohistochemistry we found that they were absolutely devoid of glial fibrillary acidic protein but some of them contained calretinin, a calcium binding protein. It is already known that neurons in layer V of the Te1 extend their projections to the contralateral side of the Te1, the ipsilateral inferior colliculus, or the ipsilateral medial geniculate nucleus. Thus, we applied a retrograde track tracer into those regions of newborn rats and examined the possible colocalization of ER alpha signals and the tracer in the same cells. So far no clear colocalization of both signals has been detected in cells in the Te1. Thus, the cells expressing ER alpha transiently are not projecting to the assumed regions, at least at the newborn age examined in the present experiment. The possibility exists that (1) they are not projection neurons but local interneurons, (2) even though they are projection neurons, they did not have any synaptic contacts with their target region(s), (3) they may die after they are attached to the target neurons. Further analyses are needed to clarify the biological roles of ER alpha expressed transiently in these neurons. On the other hand, no ER beta cells were detected in the same region of the brain under the same condition. Thus, this finding was limited to the ER alpha.

Vasoactive intestinal polypeptide in neuroglia? Immunoelectron microscopic localization in astrocytes of the rat mesencephalon.

The dorsal region of the rat interpeduncular nucleus (IPN) was found highly immunoreactive for vasoactive intestinal polypeptide (VIP). This area appeared as a cap-like structure at the midcaudal level of the nucleus. Unlike other brain areas, however, VIP immunoreactivity within the "cap" appeared vaguely punctuate with no light microscopically identifiable cell structures. Ultrastructurally, a dense meshwork of VIP-immunopositive bouton-laden axons was revealed. Labeled neuronal perikarya were not encountered. Some lightly immunoreactive dendrites were observed. In addition, immunopositive glial profiles were frequently seen. Perikarya and numerous fine processes, occasionally perivascular, identified as astroglia by established ultrastructural criteria, exhibited VIP immunoreactivity. Constant feature of the peptide immunolocalization was the predilection for the intermediate filament bundles of astrocytic perikarya and processes. This was usually accompanied by a thick coating of the inner aspect of glial plasmalemma and, in perikarya, by highly reactive vesicular profiles. Glial immunopositive elements were never seen beyond the boundaries of the diffuse "cap." In view of the repertoire of metabolic, neurotrophic, and neuroprotective mechanisms in which VIP neurons are involved in conjunction with astroglia, the presence of VIP-immunoreactive astrocytes in a circumscribed area, confirms the heterogeneity of astrocyte populations.

Serotoninergic afferents preferentially innervate distinct subclasses of peptidergic interneurons in the rat visual cortex.

Although it is well documented that the non-pyramidal neurons of the cerebral cortex are under the influence of the vast serotoninergic input, the ultrastructural substrate for such functional interactions appears largely obscure. We sought to address this issue by dual immunoelectron microscopy, combining antibodies against serotonin (5-HT) and three neurochemical markers for peptidergic interneurons, namely somatostatin (SRIF), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP). The gold-substituted silver-peroxidase method was employed to intensify and differentiate the end-product of the peptide-immunoreaction from the non-intensified 5-HT fibers. Mainly the SRIF but also the NPY neurons were encountered among the postsynaptic targets of the 5-HT boutons. Recipients of synapses were perikarya and proximal dendrites of SRIF and NPY cells but also distal dendrites of the SRIF neurons. Neither synaptic relationships nor close appositions were ever identified between 5-HT boutons and VIP-immunoreactive elements. This remarkable synaptic preference/avoidance of 5-HT afferents for specific peptidergic subpopulations reveals a 'wired' component of cortical serotonin neurotransmission, which should be carefully interpreted within the frame of the available literature for extrasynaptic serotonin release.

Ovarian cycle-related changes of glial fibrillary acidic protein (GFAP) immunoreactivity in the rat interpeduncular nucleus.

The interpeduncular nucleus (IPN) of female rats was studied across the estrous cycle to observe whether the expression of the astroglial marker, glial fibrillary acidic protein (GFAP) reacts to hormonal changes in an area not belonging to the 'endocrine brain'. A marked reduction of immunoreactive GFAP was observed in estrus as compared to the immunoreactivities in met- and proestrus. This finding is consistent with earlier observations in the endocrine hypothalamus, but also proves that gonadal steroids influence astroglia in brain regions not involved in neuroendocrine regulation. Since cyclic fluctuations of synaptic numbers in the female have been described only for the endocrine hypothalamus, decrease of immunoreactive GFAP in the IPN during estrus may reflect a down-regulation of GFAP synthesis.

Noradrenergic innervation of peptidergic interneurons in the rat visual cortex.

Previous studies have shown that cortical interneurons, presumably GABAergic, are among the targets of the noradrenaline (NA)-containing cortical afferents and that NA interacts with neuropeptides at various cellular levels. The present study attempts to characterize further the cortical targets of the NA afferents by examining, at the light and the electron microscopic level, the anatomical relationships of the NA fibers with three subpopulations of cortical interneurons, those containing somatostatin (SRIF), neuropeptide Y (NPY) or vasoactive intestinal polypeptide (VIP). For this purpose, a double preembedding immunoprocedure with antibodies against NA and SRIF, NPY or VIP was combined with the gold-substituted silver peroxidase method. Light microscopic examination showed that NA fibers contact perikarya and proximal dendrites of the SRIF, NPY and VIP neurons. However, NA fibers, while found to form pericellular arrays around NPY neurons and, to a lesser extent, around SRIF neurons, were seen to target VIP cortical cells with single terminal varicosities. Electron microscopy revealed that all peptidergic populations examined represent synaptic targets for the NA fibers. The NAergic synapses, localized onto the cell body and proximal dendrites of the peptidergic neurons, were always of the symmetrical variety. Results of the present study provide the morphological basis for the explanation of the functional interaction between the NA cortical afferent system and the intrinsic cortical elements.

Expression of the prion protein in the rat forebrain--an immunohistochemical study.

The cellular prion protein (PrP(C)) is crucial for the development of transmissible spongiform encephalopathies (TSEs), where the pathogenic scrapie isoform (PrP(Sc)) of the same protein, is considered to be the principal or sole infectious agent. Here, we report findings on PrP(C) expression in the rat forebrain, using immunohistochemical techniques on free floating sections of 60 microm thickness. Along with neurons and astrocytes in the gray matter, PrP(c) was detected for the first time in glial cells of the white matter and in cells of circumventricular organs. PrP(C) positive cellular processes were also found to be closely associated with intraparenchymal blood vessels, often in the form of end feet. Interestingly, PrP(C) expression was observed in areas where PrP(Sc) deposition in late stages of infection has been earlier reported in the rat and other species.

Apoptosis in the visual system of normal and dark-reared rats.

Apoptotic cell death, known to play a role in the developing brain, was immunocytochemically mapped in cortical and subcortical visual centers of Wistar rats developing under normal lighting conditions or in complete darkness. Apoptosis was relatively pronounced and exhibited the same developmental pattern in all visual centers examined. Visual deprivation was found to have negligible effects on apoptosis in subcortical areas, or to amplify it temporarily in the cerebral cortex.

Mast cells in the sheep, hedgehog and rat forebrain.

The study was designed to reveal the distribution of various mast cell types in the forebrain of the adult sheep, hedgehog and rat. Based on their histochemical and immunocytochemical characteristics, mast cells were categorised as (1) connective tissue-type mast cells, staining metachromatically purple with the toluidine blue method, or pale red with the Alcian blue/safranin method, (2) mucosal-type or immature mast cells staining blue with the Alcian blue/safranin method and (3) serotonin immunopositive mast cells. All 3 types of brain mast cells in all species studied were located in both white and grey matter, often associated with intraparenchymal blood vessels. Their distribution pattern exhibited interspecies differences, while their number varied considerably not only between species but also between individuals of each species. A distributional left-right asymmetry, with more cells present on the left side, was observed in all species studied but it was most prominent in the sheep brain. In the sheep, mast cells were abundantly distributed in forebrain areas, while in the hedgehog and the rat forebrain, mast cells were less widely distributed and were relatively or substantially fewer in number respectively. A limited number of brain mast cells, in all 3 species, but primarily in the rat, were found to react both immunocytochemically to 5-HT antibody and histochemically with Alcian blue/safranin staining.

Ultrastructural evidence for combined action of noradrenaline and vasoactive intestinal polypeptide upon neurons, astrocytes, and blood vessels of the rat cerebral cortex.

The intracortical organization of the noradrenaline (NA) and vasoactive intestinal polypeptide (VIP) systems provides ample opportunity for functional convergence, and accumulated evidence indicates that NA and VIP share certain cellular actions upon both neuronal and nonneuronal cortical elements. In the present study, a double immunolabeling method was combined with a silver-gold intensification procedure to examine the ultrastructural relationships of the NA coeruleocortical afferents and the intrinsic VIP neurons with three main constituents of the cortex: neurons, astrocytes, and blood vessels. Electron microscopy of singly or doubly labeled material indicated that NA and VIP boutons are engaged in a variety of anatomical relationships with both neuronal and nonneuronal elements. Dendritic shafts and perikarya of nonpyramidal neurons, some of which are VIP positive, receive combined NA and VIP synapses. A significant number of cortical microvessels are in intimate contact with NA or VIP profiles. NA axons often form perivascular loops, and VIP dendritic shafts of large diameter are frequently observed to bend around the vessel circumference. Serial section examination demonstrates that some NA boutons are directly apposed to the capillary wall at sites of glial end-feet discontinuities, whereas VIP boutons contact astrocytic sleeves of capillaries but never cross the perivascular astroglial barrier. Some VIP dendrites containing coated vesicles make intimate contact with the capillary basal lamina. Astrocytic perikarya, mainly in the supragranular layers, are also directly apposed to NA and/or VIP elements. These complex anatomical relationships provide a structural basis for the known interactions between NA and VIP in the control of cortical metabolism and function.

The noradrenergic innervation of identified hypothalamic magnocellular somata and its contribution to lactation-induced synaptic plasticity.

Despite several studies showing that the rat supraoptic (SON) and paraventricular (PVN) nuclei are innervated by noradrenergic afferents, the respective contribution of these inputs to the oxytocinergic and vasopressinergic neuronal populations remains to be clearly defined. In the present study, we used the unbiased disector method to estimate the numerical density of noradrenergic varicosities on identified oxytocinergic and vasopressinergic somata in the rat SON and PVN. The analysis was carried out on semithin (1 micron) plastic sections cut from vibratome slices (50 microns) of the SON and PVN which had been double-labelled for noradrenaline (NA) and oxytocin- or vasopressin-related neurophysin. These preparations displayed many noradrenergic varicosities which electron microscopy showed to represent, in the main, synaptic boutons. Our quantitative analysis revealed that noradrenergic varicosities contacted oxytocinergic and vasopressinergic somata to a similar extent in male and female rats, under basal conditions of hormone secretion. The incidence of these axo-somatic contacts was similar in the SON and PVN. In contrast, in lactating rats, in which oxytocin secretion is enhanced, there was a significant increase in the density of noradrenergic varicosities apposed to oxytocinergic somata, in both nuclei. Our observations indicate that, in male and female rats under normal conditions, noradrenergic afferents innervate each type of neurosecretory somata, in both magnocellular nuclei, in a similar fashion. They reveal, moreover, that noradrenergic afferents participate in lactation-induced structural plasticity of synapses impinging on oxytocinergic somata.

Neuronal expression of Fos-like protein along the afferent pathway of the milk-ejection reflex in the sheep.

This study was designed to reveal the relay stations of the afferent branch of the milk-ejection reflex in the sheep, by examining the effect of nipple stimulation on the expression of Fos protein along the spino-hypothalamic axis. Immunocytochemical detection of Fos protein after manual nipple stimulation in nonlactating ewes revealed immunolabeled neurons located exclusively ipsilaterally in the 3rd and 4th lumbar spinal ganglia, the medial part of laminae I-III of the 3rd and 4th lumbar spinal segments, the lateral cervical nucleus, the dorsal column nuclei, and bilaterally in the hypothalamic paraventricular nucleus. These findings selectively demonstrate for the first time those cell groups mediating the neuroendocrine effects of nipple stimulation.

Catecholaminergic and serotoninergic fibres innervate the ventricular system of the hedgehog CNS.

Immunocytochemistry with antisera against serotonin (5-HT), dopamine (DA) and noradrenaline (NA) was used to detect monoaminergic (MA) fibres in the ventricular system of the hedgehog Erinaceus europaeus. Light microscopic examination of immunocytochemically stained sections revealed that the ventricular system of the hedgehog is unique among mammals in that the choroid plexuses receive CA axons and that the supraependyma and subependyma of the cerebral ventricles and the spinal central canal are innervated both by serotoninergic and catecholaminergic (CA) fibres. Supraependymal 5-HT axons were generally more abundant and created at places a large number of interconnected basket-like structures, whereas CA fibres were usually directed towards the ventricular lumen. In the lateral ventricles, CA fibres were more numerous in the ependyma lining grey matter, whereas a higher 5-HT innervation density was observed in the area between the corpus callosum and the caudate nucleus or the septum. In the 3rd ventricle, the ependyma of its dorsal part exhibited a higher 5-HT and NA innervation density, whereas DA fibres were preferentially distributed in the ventral half of the basal region. The ependyma lining the cerebral aqueduct displayed a higher MA innervation density in its ventral part. The ependymal wall of the 4th ventricle exhibited an extremely dense 5-HT innervation, mainly in the floor of the ventricle, relatively fewer NA fibres and only sparse DA ones. Few NA and relatively more 5-HT fibres were detected in the ependyma of the central canal. Finally, the circumventricular organs were unevenly innervated by the 3 types of MA fibres. The extensive monoaminergic innervation of the hedgehog ventricular system described here probably reflects a transitory evolutionary stage in the phylogeny of the MA systems with presently unknown functional implications.

Ultrastructural relationships between noradrenergic nerve fibers and non-neuronal elements in the rat cerebral cortex.

Pharmacological and biochemical data suggest that noradrenaline (NA)-containing fibers not only regulate the activity of cortical neurons but also influence the functional state of non-neuronal elements. In the present study, immunocytochemistry with an antiserum against NA, followed by silver-gold intensification of the immunoreaction end-product, was employed to examine the ultrastructural relationships between the NA fiber system and the intraparenchymal blood vessels, oligodendrocytes, and astrocytes in the rat visual cortex. Electron microscopy revealed a large number of fine varicose NA fibers to be in intimate contact with cortical capillaries. Examination of single thin sections showed that NA boutons were usually separated from the capillary wall by a fine astroglial sleeve. However, serial section analysis revealed that the continuity of the astrocytic end feet was interrupted at sites, resulting in direct apposition of the perivascular NA fibers to the capillary basal lamina. Noradrenergic fibers were found to contact both types of macroglial cells. Single or clustered oligodendrocytes in intimate contact with NA fibers were observed throughout the cortical depth. Individual contacts could be followed in more than six successive thin sections, and oligodendrocyte plasma membrane frequently exhibited a light thickening at the sites of the NA fiber apposition. NA fiber-astroglial relationships were largely encountered in supragranular layers. In these layers, astrocytic cell bodies were characteristically outlined by fine varicose NA fibers. However, no plasma membrane differentiations were observed at the sites of intimate NA fiber apposition. The present ultrastructural findings provide the anatomical substrate for the control exerted by the NA fiber system over cortical microvasculature and macroglia.

Interruption of visual deprivation during development restores the normal density of peptidergic neurons in the rat visual cortex.

We have previously shown that dark rearing moderates the normal decline in the number of somatostatin (SRIF) and neuropeptide (NPY) neurons in the developing rat visual cortex. Thus, cortical areas of visually deprived animals at postnatal day (P) 60 contain consistently more SRIF and NPY neurons than do the same areas of rats reared in normal lighting conditions. In present study animals were reared in darkness from birth to P14, P21, P30 or P60 and then placed in ambient light conditions up to the day of sacrifice (P60 or P90). Counting of immunocytochemically identified SRIF and NPY neurons in all cortical visual areas of these and of undeprived animals, showed that interruption of visual deprivation during both the early or the late stages of postnatal development restores the normal density of the above peptidergic neurons.

Noradrenergic and dopaminergic systems in the central nervous system of the hedgehog (Erinaceus europaeus).

The distribution of the noradrenaline (NA)- and dopamine (DA)-containing neuronal structures in the central nervous system of the hedgehog (Erinaceus europaeus), a phylogenetically old mammalian species, was immunocytochemically studied employing antibodies directed against the catecholamines (CA) themselves. Groups of DA cell bodies observed in this study were similar to those present in other species but the distributional map of the NA-containing cell bodies exhibited some peculiarities. Prominent among them were the absence of the A3 group and the paucity of CA cells in the A2 group. DA neurons in the hypothalamus, apart from the densely populated paraventricular and arcuate nuclei, were fewer and less widely distributed than in other species. In the hedgehog mesencepha- Ion, in contrast to what has been described in other species, the major DA cell group was present in the ventral tegmental area. CA immunoreactive fibers were widely distributed in the CNS of the hedgehog. However, similarly to what has been observed in other species, terminal fields of DA neurons were much more restricted when compared to those of the NA neurons. The neocortical DA projection system of the hedgehog appeared less developed but organized similar to that of the rat, and even less developed than that of the primates. The lack of profound regional and laminar variations in the density of cortical NA fibers in the hedgehog enhances the suggestion that the elaboration and differentiation of the NA cortical system parallels the phylogenetic development of the cortex. In the brainstem, interspecies differences in the distribution of the CA fibers were found to concern primarily some hypothalamic areas (medial preoptic area, suprachiasmatic nucleus, arcuate nucleus). Such differences in the thalamus concerned the NA innervation and they were notably present in the visual thalamic nuclei (dorsal lateral geniculate nucleus, lateral posterior thalamic nucleus). In the spinal cord, which was found to receive fewer CA afferents than those found in other species, the density of the DA fibers was much lower than that of the NA axons. In addition to the CNS areas that have been described in other species to receive catecholaminergic innervation, the present study showed that both types of catecholaminergic fibers are distributed in the choroid plexus and along the ventricular wall of the brain ventricles and the central canal of the hedgehog.

Atlas of the serotonin-containing cell bodies and fibers in the central nervous system of the hedgehog.

Immunocytochemistry with an antiserum raised against serotonin was used to provide detailed description of the serotonin (5-HT)-containing cell bodies and fibers in the CNS of the hedgehog (Erinaceus europeaus). The distribution of the 5-HT-positive cells and the pattern of 5-HT innervation resembled those described in other mammalian species but several differences were also noticed. The nine B groups of Dahlstrom and Fuxe (1964) were observed, except for the B 4 group which was not detected in the hedgehog. Neuron-like immunoreactive cells were also observed in brain regions located outside the brainstem reticular formation. Among the latter the olfactory tubercle and the thalamus have not been described to contain 5-HT cell bodies in other mammals. The number and the "lateralization" of 5-HT-positive neurons in areas outside the midline raphe were more limited than those described in the rodents and much more limited than in the carnivores and the primates. 5-HT-positive axons were unevenly distributed throughout the hedgehog's CNS. From all neocortical areas, which displayed the highest innervation density in the layer I, somatosensory and visual areas were the most heavily and lightly innervated ones respectively. Other regions of the forebrain exhibiting dense serotoninergic innervation were: olfactory cortex; olfactory tubercle; subiculum; lateral septal nucleus; stratum; medial and basal amygdaloid nuclei. Dense serotoninergic innervation were also found to receive the following brainstem structures: lateral hypothalamic area; dorsal part of the suprachiasmatic nucleus; arcuate nucleus; median eminence; habenular nuclei; centromedian thalamic nucleus; ventral tegmental area; medial part of the substantia nigra; interpeduncular nucleus; locus coeruleus; cuneiform nucleus; motor nucleus of the trigeminal nerve; facial nucleus; parabrachial nucleus; prepositus hypoglossi nucleus; medial subnucleus of the nucleus of the solitary tract; hypoglossal nucleus; dorsal motor nucleus of the vagus. In the spinal cord, laminae IX and X received the heaviest innervation.