Biomedical subjects

O Bosler

Publications and source records attributed to O Bosler.

At least 37 records · Page 2Linked to original sources

PubMed1995

Efficient immunodetection of various protein antigens in glutaraldehyde-fixed brain tissue.

Optimal ultrastructural preservation of brain tissue for electron microscopy is best achieved with fixatives containing high concentrations of glutaraldehyde, which is generally considered detrimental to the immunogenicity of most protein antigens. We tested seventeen mono- or polyclonal antibodies against peptide or protein antigens, including a majority for which immunoreactivity had previously been reported to be sensitive to glutaraldehyde fixation. Forebrain sections of rats or mice fixed by perfusion with 3.5% glutaraldehyde were processed for pre-embedding immunocytochemistry by the avidin-biotin method. The resulting immunostaining was in most cases at least similar to that obtained in sections fixed with paraformaldehyde. Immunoreactivity against the mouse or human neurofilament protein NF-L was even improved, being similar to that previously reported for unfixed brain tissue. Of all antigens tested, only choline acetyltransferase, phenylethanolamine-N-methyl transferase, and neuropeptide Y were detected with lower sensitivity than after paraformaldehyde fixation, which was attributed to a rather restricted penetration of the primary antibody into glutaraldehyde-fixed tissue sections. These results indicate that glutaraldehyde may be envisaged as a possible fixative for optimal immunocytochemical detection of any tissue antigen at the electron microscopic level, including antigens which, on the basis of results obtained after fixation with paraformaldehyde-glutaraldehyde mixtures, were considered highly sensitive to glutaraldehyde fixation.

Animals↗

PubMed1994

Synaptic connectivity of serotonin graft efferents in the suprachiasmatic and supraoptic nuclei of the hypothalamus.

We have previously reported that a cell suspension from the rostral part of the embryonic raphe grafted to the basal hypothalamus of 5,7-dihydroxytryptamine-denervated rats produced incomplete serotonin (5-HT) re-innervation of the suprachiasmatic nucleus (SCN) as opposed to hyper-innervation of the supraoptic nucleus (SON). We took advantage of this experimental model to investigate whether the graft-derived, 5-HT fibres retained normal ultrastructural features, and, particularly, a normal density of synaptic junctions, irrespective of the extent of target re-innervation. The intrinsic features of immunostained, graft-derived 5-HT axonal varicosities in both the SCN (ventral portion) and the SON were essentially similar to those exhibited by the respective endogenous innervation. Analysis of well-preserved varicosities in uninterrupted series of thin sections allowed us to evaluate directly the proportions of junctional to non-junctional 5-HT varicosities in both regions. Synaptic incidences were also remarkably conserved after grafting (45.5% in the SCN versus 38.5% in the SON; 48% and 38% in normal rats, respectively). Synapses were primarily reestablished on dendritic shafts, which also were identified as the major post-synaptic targets of the normal 5-HT innervations. We noted, however, a tendency toward increased numbers of symmetrical versus asymmetrical synapses in both the SCN and SON of grafted rats. Thus, irrespective of whether hypo- or hyper-innervation patterns developed post-grafting, the transplanted 5-HT neurons essentially retained normal ultrastructural features in their target territories, with a normal incidence of synaptic junctions. The data provide further support to the hypothesis that the innervation territory is the major determinant of the frequency with which ingrowing 5-HT fibres make synaptic junctions.

5,7-Dihydroxytryptamine↗

PubMed1994

Catecholaminergic projections from the solitary tract nucleus to the perifornical hypothalamus.

The source of adrenergic and other catecholaminergic fibers innervating the perifornical lateral hypothalamus was localized in the medulla after combination of Fluoro-Gold retrograde tracing and immunohistochemistry for either tyrosine-hydroxylase or phenylethanolamine-N-methyltransferase. Following perifornical injections, Fluoro-Gold-labeled neurons were observed mainly in regions including the noradrenergic and adrenergic cell groups. In the caudal solitary tract nucleus, two kinds of doubly labeled neurons were found: a) numerous noradrenergic neurons in the A2 group at the level of, or caudal to the area postrema; b) some adrenergic neurons in the C2 group at a level immediately rostral to the area postrema. These catecholaminergic neurons connecting the caudal solitary tract nucleus to the perifornical hypothalamus might convey feeding relevant information such as glycemic level or satiety signals.

Animals↗

PubMed1993

Effect of changes in the intrahippocampal vasopressin on memory retrieval and relearning.

Previous results have indicated the involvement of the hippocampus in the behavioral effect of vasopressin, with a better effect when the peptide was injected in the ventral part rather than in the dorsal part of this structure. The purpose of the present study was to determine, in mice, whether the injection of vasopressin or vasopressin antisera into the ventral hippocampus has an effect on retrieval and relearning of a Go-No Go visual discrimination task and, if so, to what extent this involvement of the vasopressin system depends on the integrity of the medial amygdaloid nucleus, the main source of vasopressin innervation in the ventral hippocampus in rats. In the first experiment, we showed that pretest microinjection of Arg8-vasopressin (25 pg per animal) in the ventral hippocampus alleviated forgetting observed after a prolonged interval of 24 days between the acquisition of information and its retrieval. This enhancing effect was characterized by better retrieval and relearning in vasopressin-treated mice than those in control mice. Conversely, an immunoneutralization of endogenous vasopressin in the ventral hippocampus by the microinjection of vasopressin antisera (1/10 dilution) resulted in the drastic impairment of retrieval and relearning. Since the lack of an observable change in a locomotor activity test might explain these results, we postulated that the vasopressin system in the ventral hippocampus is involved in retrieval processes. Moreover, the effects of these treatments in a nonassociative context suggest that the effect of vasopressin could be dependent on the contextual paradigm used. In the second experiment, we localized vasopressin immunoreactive fibers in the CA1-CA2 ventral hippocampal fields and CA4-gyrus dentatus region, and vasopressin perikarya in the medial amygdaloid nucleus. Then, the projection of vasopressin cells from the medial amygdaloid nucleus to the ventral hippocampus was evaluated by studying changes in vasopressin immunoreactive fiber density in the ventral hippocampus after a lesion of the medial amygdaloid nucleus. The results showed the almost complete disappearance of vasopressin fibers in the CA1-CA2 hippocampal fields after the medial amygdaloid lesion. In contrast, vasopressin fibers in the CA4 and gyrus dentatus region remain unchanged. On the basis of our immunohistochemical results, our third experiment tested the repercussions of the change in vasopressin innervation in the ventral hippocampus, due to the medial amygdaloid lesion, on the effects of exogenously administered vasopressin on both retrieval and relearning processes. The medial amygdaloid lesion induced a deleterious effect on retrieval without really affecting the ability to relearn. No observable change in locomotor activity could explain this impairment.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

Convergent serotonin and GABA innervation of VIP neurons in the suprachiasmatic nucleus demonstrated by triple labeling in the rat.

By means of a combination of serotonin (5-HT) uptake radioautography and dual immunocytochemistry, vasoactive intestinal polypeptide (VIP) neurons in the suprachiasmatic nucleus are demonstrated to simultaneously receive both 5-HT and GABA afferents at their somatic and dendritic levels. These data constitute a further step towards the improved characterization of the morphological substrate of the integrative function of these neurons, which are known to play an important role in the delivery of light-mediated rhythmic signals to other parts of the brain.

Animals↗

PubMed1992

Long-term serotonin reinnervation of the suprachiasmatic nucleus after 5,7-dihydroxytryptamine axotomy in the adult rat.

We present here immunohistochemical evidence that serotonin (5-HT) reinnervation in the suprachiasmatic nucleus (SCN) of adult rats, after intraventricular administration of 5,7-dihydroxytryptamine, is more extensive than formerly believed. The first fibers appeared at 4 months and developed until more than 7 months. However, comparison with age-matched controls showed that, even 24 months post-lesion, the density of 5-HT innervation remained abnormally low. At the electron microscopic level, many synaptic contacts were found to have been re-established. The cellular mechanisms possibly responsible for incomplete 5-HT reinnervation in the SCN are discussed.

5,7-Dihydroxytryptamine↗

PubMed1992

Co-localization of tyrosine hydroxylase, GABA and neuropeptide Y within axon terminals innervating the intermediate lobe of the frog Rana ridibunda.

Possible co-existence of gamma-aminobutyric acid (GABA), catecholamines, and neuropeptide Y (NPY) in the same nerve terminals of the frog intermediate lobe was investigated by immunocytochemistry at the electron microscopic level. Co-localization of GABA and tyrosine hydroxylase (TH) was studied by using a double immunogold labeling procedure. Co-localization of glutamate decarboxylase (GAD) and NPY was studied by combining, respectively, the peroxidase-antiperoxidase method and a radioimmunocytochemical labeling procedure. Catecholamines and GABA were systematically co-localized in nerve endings of the pars intermedia. Most of the NPY-immunoreactive fibers also contained GAD-like immunoreactivity. However, a few NPY-positive nerve terminals were not immunoreactive for GAD. These data provide evidence for co-existence of a regulatory peptide (NPY) and several neurotransmitters (i.e., GABA and catecholamines) within the same axon terminals in the intermediate lobe. Since GABA, dopamine, and NPY have all been shown to inhibit the activity of frog melanotrope cells, the present findings suggest that these neuroendocrine factors may interact either at the pre-synaptic or post-synaptic level.

Animals↗

PubMed1992

Association of neurotensin receptors with VIP-containing neurons and serotonin-containing axons in the suprachiasmatic nucleus of the rat.

The aim of the present study was to identify cellular elements bearing high-affinity neurotensin (NT) binding sites in the suprachiasmatic nucleus (SCN) of the rat hypothalamus. Because the distribution of these binding sites had previously been reported to conform to that of both vasoactive intestinal peptide (VIP)-containing nerve cell bodies and serotonin (5-HT)-containing axons, the following experimental approaches were used: (1) the overlap between autoradiographically labeled NT binding sites and immunocytochemically labeled VIP neurons was examined in adjacent 5-microns-thick sections taken across the entire rostrocaudal extent of the SCN; and (2) the density of NT binding sites was examined by quantitative autoradiography following cytotoxic lesioning of 5-HT afferents. Double-labeling studies demonstrated precise overlap between 125I-NT binding and VIP immunostaining throughout the SCN. Moreover, at high magnification intensely VIP-immunoreactive neurons were found in direct register with 125I-NT-labeled cells visualized in adjacent sections. Densitometric autoradiographic studies demonstrated a significant reduction in specific 125I-NT binding within the SCN following intracerebroventricular injection of the neurotoxin, 5,7-dihydroxytryptamine. Taken together, these results indicate that within the SCN, NT receptors are present both presynaptically on serotonin axons and postsynaptically on the perikarya and dendrites of VIP-containing neurons.

5,7-Dihydroxytryptamine↗

PubMed1991

Dual immunocytochemistry using 125I-labeled protein A: a new electron microscopic technique applied to the investigation of chemical connectivity and axonal transmitter co-localization in the brain.

We have developed a double labeling immunocytochemical method utilizing peroxidase conjugated Fab fragments and 125I-labeled protein A to localize two neuronal markers on the same light or electron microscopic section with primary antibodies raised in the same animal species. The technique is applicable to the study of chemical connectivity in the brain, as illustrated by data obtained in the hypothalamus using rabbit polyclonal antisera against tyrosine hydroxylase (TH), phenylethanolamine-N-methyltransferase (PNMT), neuropeptide Y (NPY), and vasoactive intestinal peptide (VIP). Moreover, due to a high level of sensitivity and resolution, the technique offers considerable advantages over many previously developed dual labeling immunocytochemical methods for the demonstration of transmitter axonal co-localizations. Utilizing the peroxidase Fab/[125I]protein A method, we present here the first direct evidence that PNMT is present in many endings also containing NPY in the thalamic and hypothalamic paraventricular nuclei and in the arcuate nucleus. The method also may be combined as required with other labeling methods for localizing more than two neurochemical markers on one and the same electron microscopic section.

3,3'-Diaminobenzidine↗

PubMed1990

Serotonin reinnervation of the rat organum vasculosum laminae terminalis (OVLT) after 5,7-dihydroxytryptamine deafferentation.

The time course and pattern of serotonin (5-HT) reinnervation in the rat organum vasculosum laminae terminalis (OVLT) following intracerebroventricular administration of 5,7-dihydroxytryptamine were examined by means of [3H]5-HT uptake radioautography. 5-HT axonal varicosities reappeared in the OVLT within 3 months post-lesion. Six months later, they were found to have preferentially reinvested the neurohemal contact area (juxtavascular zone) of the organ. Regenerated terminals further increased in number. At the end of a 16-month survival time, they displayed normal-looking distributional patterns, similar to those of age-matched control animals, and formed new synaptic junctions in the juxtaventricular zone. The cellular mechanisms possibly responsible for 5-HT recovery in the OVLT are discussed.

5,7-Dihydroxytryptamine↗

PubMed1990

Adrenergic innervation of noradrenergic locus coeruleus neurons. A dual labeling immunocytochemical study in the rat.

By means of dual immunocytochemistry, synaptic associations between adrenergic terminals and noradrenergic neurons were directly demonstrated in the rat locus ceruleus (LC). It could be estimated that every adrenergic afferent contacts at least one noradrenergic dendrite in the nucleus. An adrenergic innervation of non-noradrenergic targets was also evidenced. These data add to our knowledge on the synaptic circuitry by which activation of the adrenergic input could affect central mechanisms known to be influenced by LC neurons.

Adrenergic Fibers↗

PubMed1990

GABA neurons in the rat suprachiasmatic nucleus: involvement in chemospecific synaptic circuitry and evidence for GAD-peptide colocalization.

Dual labelling methods were employed for the electron microscopic detection of glutamate decarboxylase (GAD) immunoreactivity, together with vasoactive intestinal peptide (VIP) or neuropeptide Y (NPY) immunoreactivity in the suprachiasmatic nucleus (SCN) of colchicine pretreated and untreated rats. These methods involved the combined use of diaminobenzidine and benzidine dihydrochloride as distinct chromogens to visualize peroxidase-anti-peroxidase (PAP) immunostaining, and a combination of the PAP procedure with a radioimmunocytochemical method employing 125I-labelled secondary antisera. We were thereby able to demonstrate that gamma-aminobutyric acid (GABA) terminals provide an important afferent synaptic input to VIP neurons. Some of these VIP-immunoreactive neurons also exhibited GAD immunoreactivity. Examples of direct appositions between GABA and NPY terminals, and of a convergence of the two types of terminals on to the same postsynaptic targets, were frequently encountered. NPY/GAD colocalization within a few axonal varicosities was also demonstrated. These data provide additional information concerning chemospecific neuronal interactions that could be of functional importance in the regulation of circadian rhythmicity at the level of the SCN.

Animals↗

PubMed1989

Adrenergic neurons in the nucleus tractus solitarius receive GABAergic synapses. Demonstration by dual immunocytochemistry in the rat.

By means of a dual immunocytochemical labeling for phenylethanolamine-N-methyltransferase and glutamate decarboxylase, synaptic associations between adrenaline-synthesizing neurons and GABAergic terminals are demonstrated in the medial nucleus tractus solitarius of the rat. These relationships could constitute an anatomical substrate for the presumed role of GABA in modulation of baroreceptor reflexes at this level.

Adrenergic Fibers↗

PubMed1989

Ultrastructural correlates of functional relationships between nigral dopaminergic or cortical afferent fibers and neuropeptide Y-containing neurons in the rat striatum.

This study examines the ultrastructural relationships established by the nigrostriatal dopaminergic and the corticostriatal afferent fibers with neuropeptide Y (NPY)-containing neurons in the rat striatum. By means of dual immunolabeling procedures using peroxidase conjugated F(ab) fragments and 125I-labeled protein A, direct appositions and morphologically defined synaptic contacts of the symmetrical type were visualized between tyrosine hydroxylase-labeled nerve terminals and NPY-labeled neurons. After deafferentation of the striatum from its cortical input direct appositions and asymmetrical synaptic contacts were evidenced between characteristic degenerative boutons and NPY-positive neurons in the striatum. These results suggest that striatal NPY interneurons undergo direct influence from both nigrostriatal dopaminergic and corticostriatal neuronal systems.

Animals↗

PubMed1989

Are adrenergic neurons subject to a serotoninergic influence in the nucleus tractus solitarii? A morphological and biochemical study in the rat.

The possible relationships between adrenaline-synthesizing neurons and serotoninergic afferent fibers in the nucleus tractus solitarii of the rat were investigated both morphologically and biochemically. Adrenergic elements (cell bodies, dendrites and nerve endings) were detected simultaneously with serotoninergic axonal varicosities in the same electron-microscopic sections by means of combined phenylethanolamine-N-methyltransferase immunocytochemistry and [3H]serotonin-uptake radioautography. Among some 500 serotoninergic varicosities scanned in the areas of significant overlap between the 2 types of labeling, only 3 were directly apposed to an adrenergic process, identified as a dendrite in each case. No synaptic membrane differentiations were seen at these occasional sites of contact. Destruction of the serotonin input by 5,7-dihydroxytryptamine had no significant effect on the tyrosine hydroxylase dopamine-beta-hydroxylase and phenylethanolamine-N-methyltransferase enzymatic activities in the C2 adrenergic region, but induced 22% and 38% increases of tyrosine hydroxylase and dopamine-beta-hydroxylase activities, respectively, in the neighboring A2 noradrenergic area. Taken together, these results suggest that serotoninergic and adrenergic neurons do not significantly interact in the nucleus tractus solitarii; this implies that the possible catecholaminergic relays for the action of serotonin in autonomic regulation at this level could consist of noradrenergic neurons rather than of their adrenergic counterparts.

Animals↗

PubMed1989

Ultrastructural relationships of serotonin and GABA terminals in the rat suprachiasmatic nucleus. Evidence for a close interconnection between the two afferent systems.

Serotonin (5-HT) and gamma-aminobutyric acid (GABA) nerve endings were identified in the rat suprachiasmatic nucleus (SCN) by combined [3H]5-HT uptake radioautography and glutamate decarboxylase (GAD) immunocytochemistry at the electron microscope level. In areas of good overlap between radioautographic and immunocytochemical labellings, there were no axonal varicosities exhibiting both labellings, indicating that 5-HT and GABA are not co-localized in the SCN. The systematic survey in these areas of all profiles that had accumulated [3H]5-HT and of all GAD-immunoreactive varicosities allowed the analysis of 247 of the former and 896, i.e. an almost four-fold greater number, of the latter. This seems concordant with the view that GABA endings would be the most numerous of all classes of nerve terminals so far identified in the SCN. More than 22% of the [3H]5-HT labelled profiles showed the membrane specialization typically associated with synaptic junctions. Thereby, it was possible to evaluate that about 45% of the 5-HT terminals actually form a synapse in the SCN. Some 37% of the GAD-positive varicose profiles which could be formally interpreted also showed well differentiated synaptic contacts, suggesting that the GABAergic innervation of the SCN could be entirely junctional. Whereas 5-HT terminals usually innervated only one dendritic or somatic element, a convergence of several GABAergic terminals onto the same postsynaptic target also receiving a 5-HT input was frequently observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Host afferents into intrastriatal transplants of fetal ventral mesencephalon.

Host afferents into fetal ventral mesencephalic tissue grafted to the neostriatum of adult rats have been studied by using anterograde transport of Phaseolus vulgaris leucoagglutinin (PHA-L) and immunocytochemistry for serotonin (5-HT), Substance P (SP), and dopamine-adenosine 3':5'-monophosphate-regulated-phosphoprotein-32 (DARPP-32). Numerous fibers of cortical origin were detected in the transplants following multiple (11-15) iontophoretic injections of PHA-L into the frontal and anterior cingulate cortex. The labeled fibers occurred with an apparently random distribution throughout the graft tissue. Their overall density was lower than that of the surrounding striatum but similar to that found in the host nigra-ventral tegmental area. The majority of the PHA-L-labeled fibers in the grafts were thin and tortuous with varicosities or lateral clubs with terminal boutons. Dual labeling showed frequent close appositions between PHA-L-labeled terminals and dopamine-immunoreactive cell bodies. In parallel electron microscopy, synaptic contacts were observed between PHA-L-labeled terminals and unlabeled neuronal profiles in the graft. Other labeled fibers in the grafts were thick and smooth, corresponding probably to labeled myelinated axons observed in the electron microscope. These thick fibers were often seen to give off collaterals of the thin type. The virtual absence of such thick fibers in the normal striatal neuropil suggests that at least some of the cortical afferents to the grafts may have sprouted from axons normally projecting to diencephalic or brain stem regions. Serotonin fibers occurred in patches or as scattered single fibers in both deep and superficial portions of the nigral transplants. In the electron microscope some of these terminals were seen to establish synaptic contacts with nonimmunoreactive elements in the graft. These fibers were present also when the graft tissue had been pretreated with 5,7-dihydroxytryptamine at the time of transplantation. This treatment eliminated all 5-HT-containing neurons from the grafts without any noticeable adverse effect on the survival of the dopaminergic neurons. The serotonin fibers in the grafts were thus most likely of host origin. SP-positive fibers formed a dense plexus inside the grafts. Since many SP-positive cell bodies were visualized inside the transplant after colchicine pretreatment, it is unclear, however, whether any of these fibers were of host origin. Intrastriatal injections of PHA-L or DARPP-32 immunocytochemistry indicated that the deep portions of the nigral grafts were entirely devoid of host striatal afferents.(ABSTRACT TRUNCATED AT 400 WORDS)

Afferent Pathways↗

PubMed1988

Monoamine innervation of the organum vasculosum laminae terminalis (OVLT): a high resolution radioautographic study in the rat.

The monoamine innervation of the organum vasculosum laminae terminalis (OVLT) was examined in the adult rat by light and electron microscope radioautography after intraventricular administration of tritiated serotonin [( 3H]5-HT) or dopamine [( 3H]DA). Radioautographic and biochemical controls after 5,7-dihydroxytryptamine or 6-hydroxydopamine lesioning established the respective serotonin (5-HT) and catecholamine (CA) identities of the axonal varicosities labeled under the conditions of the present experiments. For descriptive purposes, the OVLT was subdivided in three parts: two parenchymal zones, one juxtaventricular, the other juxtavascular, and the vascular core. Almost 10% of all axonal varicosities in the OVLT were found to be labeled with [3H]5-HT. This 5-HT innervation was most prominent in the rostrocaudal and ventrodorsal portions of the juxtaventricular zone and the dorsal aspect of the juxtavascular zone; there was none in the vascular core. [3H]DA-labeled varicosities were much less abundant and yet more numerous than earlier histofluorescent and immunohistochemical studies would have predicted. They predominated in the juxtavascular zone, where a majority presumably had a dopamine (DA) rather than a noradrenaline identity. Some were also found in the vascular core, where they most likely corresponded to peripheral autonomic noradrenaline endings. In the juxtaventricular zone of the OVLT, a significant proportion of the [3H]5-HT-labeled varicosity profiles could be observed to form axodendritic synapses, but in the juxtavascular zone no 5-HT or any [3H]DA-labeled ones were ever seen in synaptic junction. In the juxtavascular zone, the 5-HT and the presumed DA endings established close relationships with neurosecretory axons, and with astrocytic or tanycytic processes on which they occasionally formed "synaptoid contacts." A few endings of either type were also seen to about directly on the outer basement membrane of the perivascular space. It therefore appears probable that in OVLT monoamines influence neural and nonneural elements. At a proximal level of regulation (juxtaventricular zone), 5-HT could act both synaptically and nonsynaptically as an interneuronal transmitter or modulator. In contrast, distally (juxtavascular zone), both DA and 5-HT could be released as neurohormones in addition to modulating neurosecretion. 5-HT and DA varicosities in the OVLT could also behave as sensors for circulating factors that do not cross the blood-brain barrier.

Animals↗