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Biomedical subjects

A Brusco

Publications and source records attributed to A Brusco.

At least 55 records · Page 3Linked to original sources
PubMed1995

Relationship between glial organization and the establishment of nerve tracts in rat spinal cord.

Embryo and adult rat spinal cord sections immunostained for S-100 protein, vimentin, glial fibrillary acidic protein (GFAP), or phosphorylated 160 kDa neurofilament protein (NF), were used to study glial participation in nerve tissue assembly. Radial glial fibers (RGF) expressed vimentin since E12. In the peripheral ventral zone, RGF formed cephalocaudal plates (CP) within the white matter, ensheathing developing axonal tracts first expressing NF at E13. As axonal tracts increased, CP became denser and extended ventrolaterally. S-100 protein appeared at E17 in the midline and, at E18, it overlapped vimentin expression. Astrocytes expressed GFAP since E20. We conclude that the pattern found in the spinal cord results from interactions between developing axonal tracts and radial glia, and that it could set the structural basis for the organized assembly of the spinal cord found in adults.

Animals↗
PubMed1994

Immunocytochemical localization of tryptamine in the rat brain.

Immunocytochemical localization of tryptamine in rat brain was performed at light and electron microscope level. The immunoreactive cells were present in the mesencephalon, pons and medulla, around the interpendicular nucleus in the reticular substantia nigra and in the giganto cellularis ventralis nucleus. Immunoreactive fibres were present in the dorsal region of the brain stem, and innervated the hippocampus, the ependymal epithelium and the corpus striatum. An ultrastructural study revealed that tryptamine was present only in neuronal perikaryon and processes.

Animals↗
PubMed1994

Structural and immunologic analysis of gene triplications in the Ig heavy chain constant region locus.

The Ig H chain C region is a multigene family often involved in genomic rearrangements leading to deleted and duplicated haplotypes, most probably through unequal crossing over between homologous regions within the locus. The frequency of these haplotypes in Italy is around 2.7% each. Using PFGE analysis in two unrelated Italian families we found an abnormal high m.w. band, inherited in a Mendelian fashion. To assess the extension of the haplotype we performed Southern blot analysis using several specific Ig H chain C probes. In both cases, the haplotype turned out to be triplicated, with three copies of the genes from A1 to E. In one family segregation of a duplication from EP to G4 was also observed. Analysis of polymorphic loci suggests that the two triplications are of independent origin. Serological detection of IgA2 allotypes demonstrated the functional activity of the genes at the 3' end of the triplicated locus, ruling out any major effect of these large genomic rearrangements on Ig class switching. Furthermore, the triplicated haplotype does not seem to give rise to any clinically significant immunological impairment or increase in Ig serum concentrations.

Gene Deletion↗
PubMed1993

Familial clustering of IGHC deletions and duplications: functional and molecular analysis.

The human immunoglobulin heavy chain constant region locus (IGHC) comprises nine genes and two pseudogenes clustered in a 350 kilobase (kb) region on chromosome 14q32. Several IGHC haplotypes with single or multiple gene deletions and duplications have been characterized. The most likely mechanism accounting for these unusual haplotypes is the unequal crossing-over between homologous regions within the locus. Here we report the analysis of an unusual case of familial clustering of deletions/duplications. In the two branches of the BON family, three duplicated and two deleted haplotypes, all probably independent in origin, have been characterized. The structure of the haplotypes, one of which is described here for the first time, supports the hypothesis of homologous unequal crossing-over as the origin of recombinant haplotypes. The analysis of serological markers in a subject carrying one deleted and one duplicated haplotype allowed us the first direct inferences concerning the functions of the duplicated IGHC haplotypes.

Adolescent↗
PubMed1992

Antibodies as molecular probes in neurobiology. Identification of chemically defined neurons and synapses in tissues and tissue cultures.

Immunocytochemical localization of 5-hydroxytryptamine (5-HT) in the nervous system and aggregate tissue cultures was performed employing an antibody to 6-OH-1,2,3,4-tetrahydro-beta-carboline. A number of immunochemical and biochemical tests with the antigen and the antibody and some procedural changes in the methodology applied for immunolocalization revealed the anti-5-HT-like affinity of the antibody, if applied in paraformaldehyde-fixed tissues. Studies in the hypothalamus, striatum, brainstem, spinal cord, and pineal gland show the complexities of the serotoninergic system. Ultrastructural immunocytochemistry with the preembedding technique reveals that 5-HT synapses are of the asymmetric type. The presynaptic element contains clear, round, small vesicles, with some large dense-core vesicles. The contacts are made with the somata and primary, secondary dendrites or with spines of non-5-HT neurons. Presynaptic dendrites are found in the n. raphe dorsalis, contacting non-5-HT dendrites. Double immunocytochemical methods demonstrated contacts of 5-HT fibers on enkephalin containing neurons of the spinal trigeminal nucleus and on somatostatin containing neurons of the medullary reticular formation. In vitro studies of cultured mesencephalic neurons were performed with the method of aggregating cultures. Such development of a miniature organized nerve tissue was followed up to 35 d in culture. Organization of the neuropil and synaptogenesis was studied using standard electron microscopy. The differentiation of neurons and astrocytes was studied using antibodies to 5-HT and GFAP. Serotonin immunoreactivity could be observed in neuronal bodies and processes at light microscope level as early as the fourth day of culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Serotoninergic reinnervation of regenerating tentacular sensory organs in a pulmonate snail, Cryptomphalus aspersa.

Several ontogenetic studies performed in different species suggest a developmental role for 5-HT neurons. The 5-HT system interconnecting the CNS and the tentacular sensory organs in pulmonates is a suitable model for studying the postulated developmental role of 5-HT neurons. In this paper we describe the behavior of the 5-HT fibers during the early stages of blastema reinnervation, primordium formation and differentiation of regenerating tentacular sensory organs in the pulmonate snail Cryptomphalus aspersa. Our results show that the regeneration process allows the development of a normal pattern of 5-HT innervation of the regenerated sensory organs and suggest that 5-HT could be involved in reciprocal developmental interactions with regenerating tentacular tissues.

Animals↗
PubMed1990

Immunocytochemical study of S-100 positive glial cells in the brainstem and spinal cord of the rat embryo.

A light and electronmicroscopic immunocytochemical study of the glial cells in the brainstem and spinal cord of the 18th day rat embryo was performed using an anti-S-100 protein antiserum. Only the radial glia and the free immature glial cells are S-100 immunoreactive. Neurons are devoid of S-100 immunoreactivity. The radial glia form two paramedial plates and a great number of lateral plates, uniformly spaced along the ventral portion of the brainstem from the mesencephalon to the medulla. The S-100 protein was also detected in the perivascular membranes and glial limitans. Embryonic glia adopt a highly organized spatial pattern in the brainstem that could set the structural basis for an organized assembly of the developing nervous tissue. The use of the S-100 protein as a glial marker in the embryonic rat brain proved to be of great value. Antibodies to S-100 protein allow the demonstration of immature glial cells and a highly organized spatial pattern in the brainstem and spinal cord of the rat embryo.

Animals↗
PubMed1988

Immunocytochemical study of the postnatal development of 5-HT-containing neurons and fibers in the cerebroid ganglia of Cryptomphalus aspersa.

In this work we have studied the organization of the serotonergic system in new-hatched snails and the developmental changes that occur postnatally. The serotonergic system of the new-hatched snail is not completely developed. Tracts previously described in the adult were already present at hatching; however immunoreactivity of these tracts was somewhat less intense than that in adults, but, their location was practically the same. At hatch the pro- and postcerebral serotonergic plexi were poorly developed, while the mesocerebral one was nearly absent. The procerebral plexus occupied only the caudal half of the procerebral neuropile. Growth cone profiles were found in all plexi. The paired giant dorsal serotonergic neurons were the only stained neurons constantly found at hatching; they were symmetrically located. The paired medium-sized neurons were not found in all brains examined. Sometimes only one of them was present at hatching; it was located either in the right or in the left ganglion. The small neurons were absent in most cases. They gradually appeared during the first three postnatal months. Following hatching the procerebral plexus had undergone an intense growth. By the third postnatal month it innervated completely the procerebral neuropile. At hatching the area innervated by the procerebral plexus was 4500 micron 2, by the third month this area was about 58,000 micron 2 and in the adult was 80,000 micron 2. The postnatal development of the procerebral serotonergic plexus proceeds through two different modalities of neuritic growth: expansive growth is mediated by centrifugal elongation of terminal branches while the increase in fiber density within the plexus results from lateral sprouting.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

The serotoninergic system in Cryptomphalus aspersa. Immunocytochemical study with an anti-5-HT antiserum.

This immunocytochemical study of 5-HT neurons and fibers in the nervous system of C. aspersa corroborate previous findings and describe new 5-HT neurons and their connections, mainly between the central nervous system and the tentacular sensory organs. We found a number of networks, fascicles, and neurons that show constant and symmetrical location. Three networks were found at the tip of the posterior tentacle: underlying the olfactory epithelium, in the neuropil of the tentacular ganglion (TG), and in the ocular capsule. The TG also contains a ventral 5-HT fascicle. A group of 30-40 serotoninergic fibers run through the tentacular connective from the postcerebrum to the tentacular ganglion. This 5-HT fascicle has a lateral position in the postcerebrum (lateral fascicle of the postcerebrum) and a subcortical location in the procerebrum (subcortical fascicle of the procerebrum). The optic nerve also has a small group of 5-HT fibers. Seven serotoninergic neurons were found in each cerebral ganglion: two giant neurons, one medium-sized, and four small neurons. Three different types of fascicles are in the postcerebrum: fascicles proceeding from the suboesophageal connectives, a lateral fascicle, and a commisural fascicle. Each cerebral ganglion region (pro-, meso- and postcerebrum) has a 5-HT network with a particular pattern of distribution and morphology. The suboesophageal ganglia show the highest concentration of 5-HT neurons (large, medium-sized, and small neurons).

Animals↗
PubMed1986

Further evidence for the specificity of anti-5-HT (serotonin)-like antisera in immunocytochemistry. Existence of cyclic secondary amino groups in the immunogen.

The serotonin antigen (5-HT-BSA formaldehyde conjugate) used for obtaining anti-5-HT antibodies was studied to obtain additional data concerning the nature of its immunogen. Dialysis against 0.1 M acetic acid and then against distilled water proved to be the best way of removing 5-HT condensation products not bound to BSA. The hapten has the configuration of a tetrahydro-beta-carboline (THBC) ring structure that is coupled to protein most probably via the carbon(s) ortho to the phenolic hydroxyl group and the indole nitrogen. The cyclic secondary amine of the THBC remained unsubstituted and was not involved in the bridging to BSA. This functional group was effectively blocked by acetylation and was unreactive to glutaraldehyde. On the other hand, in 5-HT conjugates synthesized using glutaraldehyde as the coupling agent, no cyclization to THBC occurred, and the amino groups were blocked. The chemical reactivity of the secondary amino group of the hapten in the synthesized conjugates was compared to the immunoreactivity of 5-HT conjugates formed in tissues. Immunostaining of formaldehyde-fixed serotoninergic neurons of the raphe of rats was suppressed by acetylation and the use of glutaraldehyde as the primary fixative, but the staining was unaffected when glutaraldehyde was reacted with formaldehyde-fixed 5-HT neurons. It is concluded that the cyclic secondary amine of the THBC structure is not conjugated to protein and forms part of the 5-HT-antibody-binding site in immunogens formed in vitro and in tissues.

Acetylation↗
PubMed1986

A new case for a presynaptic role of dendrites: an immunocytochemical study of the n. raphé dorsalis.

The distribution of serotonin (5-HT) was determined by the application of the preembedding peroxidase-anti-peroxidase (PAP) technique in vibratome and ultrathin sections of the brain stem. The antiserum stained the neuronal groups B1 to B9. Somata, dendrites and axons of multipolar and bipolar neurons were recognized in the usual locations. The most commonly found profiles in the area of the n. raphe dorsalis were dendrites. The search for axon terminals was unsuccessful. The labeled dendrites appear in synaptic contact with unlabeled endings containing round or pleomorphic vesicles, and occasionally some large dense core vesicles. Contacts between two labeled dendrites or processes were not found. Occasionally a dendrodendritic junction between a 5-HT labeled dendrite and an unlabeled dendrite has been found. There are areas of the dendritic membrane free of synaptic junctions and free of glial insulation. Results are discussed in relation with the previously proposed presynaptic role of the dendrites in the neuronal circuitry of the n. raphé dorsalis.

Animals↗
PubMed1984

Basis for the specificity of anti-5-HT-like antisera in immunocytochemistry applied to the central nervous system.

Recently (Pecci Saavedra et al. 1982; Brusco et al. 1982, 1983) we have showed that the actual specificity of the rabbit anti-5-HT antibodies, is for the beta-carboline derivatives of 5-HT as a result of cyclization of the lateral chain. We explained this as resulting from the use of formaldehyde which acted both as a fixative in the preparation of the tissues, and as the coupling agent in the preparation of the immunogen. Following this line we have fixed several brain stem specimens with 0.5% p-benzoquinone; 3% glutaraldehyde; 4% paraformaldehyde plus 0.25% glutaraldehyde and compare the results with tissues fixed in 4% paraformaldehyde. Glutaraldehyde and p-benzoquinone do not produce cyclization of 5-HT but immobilize monoamines in situ. As expected, the antibodies applied according to the PAP technique did not stain the neuronal bodies of the raphe system, known to contain 5-HT when 3-4% glutaraldehyde or 0.5% p-benzoquinone were used. Good staining was obtained with 4% paraformaldehyde alone or with 4% paraformaldehyde plus 0.25% glutaraldehyde. A quantitative assay of the spot test of Larsson (1981) was devised for measuring in vitro the inhibitory effects of 5-HT, of the 5-HT-BSA complex and of the cyclic derivative, 6-OH-1,2,3,4-tetrahydro-beta-carboline. The results confirmed that the avidity of the antiserum is much greater for the cyclic derivatives contained in the 5-HT-BSA complex and for 6-OH-1,2,3,4-tetrahydro-beta-carboline than for 5-HT. It is concluded that the formation of a new ring by the lateral chain of 5-HT is responsible of the in-vitro and in the tissue immunoreactivity of the anti-5-HT-antibodies.

Animals↗
PubMed1983

A quantitative study of visual cortex synapses during the postnatal development of dark-reared rats.

The method of Aghajanian and Bloom (1967) was applied to the visual cortex of normal and neonatally visually deprived rats. The rats were kept with their mothers in total darkness since birth, in a ventilated and temperature-controlled animal quarter. Controls were rats from the same stock, maintained in a regular 12-h-light/12-h-dark rhythm. The animals were killed at 15, 23, 40, and 65 days of age, and the visual cortices fixed in 4% glutaraldehyde and processed for electron micrography with ethanol-phosphotungstic acid. A total of 6249 synaptic profiles were counted and their numerical density (DS) determined in both conditions. Features of the presynaptic grid were used for classifying the synaptic profiles in: type A [with one presynaptic dense projection (PsDP)]; type B (with two or three PsDPS); and type C (with four or more PsDPS). In the visually deprived rats the DS increases with a rate similar to controls, but the values for each age are slightly lower (P less than 0.01). Type B synapses predominate in the visually deprived group while types A and C are scarcer. The differences found between types were maximal at 65 days of age and the results were highly significant (P less than 0.001). It is concluded that major effects of dark-rearing are manifested when the individual features of the presynaptic grid are considered. It seems that a selected population of synapses is affected by the alteration of the normal epigenetic influence of early visual experience.

Animals↗
PubMed1983

Serotonin-like immunoreactivity and anti-5-hydroxytryptamine (5-HT) antibodies: ultrastructural application in the central nervous system.

Serotonin immunoreactivity of rat neurons was studied using antibodies to 5-hydroxytryptamine (5-HT) raised in rabbits and the peroxidase-antiperoxidase (PAP) technique. The brains were fixed by perfusion with 4% paraformaldehyde + 0.25% glutaraldehyde. Vibratome sections were incubated in 1:1,000 to 25,000 antiserum dilutions. Specificity of 5-HT immunoreactivity was investigated following immunochemical and immunocytochemical criteria. Ultrastructural analysis of the raphe neurons allowed intracellular localization of 5-HT immunoreactivity in somata, dendrites, and synaptic terminals. Granular reaction product deposits were found attached to microtubules, endoplasmic reticulum membranes, synaptic vesicles, and outer mitochondrial membranes. In all cases the reaction product was confined within the limits of the plasma membrane. It is proposed that binding of 5-HT molecules to tissue proteins via a Mannich reaction and formation of intramolecular condensation products (fluorophores) result from aldehyde fixation. The immunogen (5-HT-bovine serum albumin (BSA) complex) exhibits spectrofluorometric emission and excitation maxima similar to those previously described for the 5-HT fluorophore studied in protein films and in fixed tissue. It is concluded that our antibody detects 5-HT-like immunoreactivity preserved in the tissue with the configuration of the fluorophore (i.e., 3,4-dihydrocarboline). Close agreement between the results and previous studies of 5-HT neurons with other techniques (formaldehyde-induced fluorescence, biochemical assays) support the potential value of this antibody.

Animals↗