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J A Colombo

Publications and source records attributed to J A Colombo.

At least 37 records · Page 2Linked to original sources

In vitro induction of radial-like cells by leptomeningeal and cortical astroglial conditioned media. Effect of protease inhibitors.

A population of subcultured astroglia from rat fetal cortex was transformed into radial-like cells after exposure to cerebral cortex astroglial conditioned medium in vitro. Such changes were also induced by basal medium modified by fetal leptomeningeal subcultures, but not by postnatal leptomeninges nor by fetal skin fibroblasts. The radializing effects of astroglial conditioned medium were inhibited by previous heat treatment. The addition of protease inhibitors to the basal medium did not cause spontaneous radialization of subcultured cortical astroglia, but increased the length of cell processes and incidence of radial-like forms when added to cortical astroglial conditioned medium. It is concluded that cortical astroglia and leptomeningeal cells share the capability of synthesizing and releasing diffusible molecules into the culture medium which act as morphogenetic inducers in vitro. Based on the present results, it is suggested that such effects would depend on the presence of instructive factor(s) in the conditioned medium which are able to induce rearrangement of the cytoskeleton, rather than on secreted molecules able to modify cell adhesion to the substrate.

Animals↗

Regional differences in in vitro growth of neural cell processes during development.

Primary cell cultures from cerebral cortex, striatum and ventral mesencephalon obtained from rat fetal (embryonic day 17, E17) or postnatal (day 2, PN2) donors were grown either in media conditioned by subcultured astroglia from the same regions, an artificial trophic medium, normal human amniotic fluid, or in normal human cerebrospinal fluid. To estimate the presence of neuronal-like and non-neuronal cells, cell morphology and immunocytochemistry against microtubule-associated proteins and beta-tubulin were taken into consideration. The percentage of emitting neural cells and length of cell processes were determined after 24 hr in culture. Growth of cell processes in neuronal and non-neuronal cells from prenatal striatum was minimal compared with that in cerebral cortex and ventral mesencephalon, regardless of the culture condition. Nerve growth factor, basic fibroblast growth factor or epidermal growth factor did not significantly modify cell growth in E17 cultures, except for epidermal growth factor, which reduced the number of emitting cells in striatal cultures and increased it in cerebral cortex ones. Cultures derived from postnatal striatum showed a significant increase in neurite length when grown in an astroglial conditioned medium as compared to cultures derived from prenatal (E17) striatum. Results suggest significant regional differences in the brain regarding growth of cell processes at age E17, and reversal of striatal ability to grow cell processes by postnatal day 2. Reduced growth of cell processes showed by E17 striatum cultures was rather independent of the culture media. This fact could suggest that such early regional differences would depend on characteristics of sublineages present at this developmental stage, which would modulate the organization of regional neuropils. The restricted growth of cell processes in cultures from E17 striatum, no longer present in postnatal striatum, suggests that inputs to the striatum may modify expression of cell lineages at later stages of development.

Amniotic Fluid↗

Interlaminar astroglial processes in the cerebral cortex of adult monkeys but not of adult rats.

Three adult Cebus apella and two Saimiri sciureus monkeys, as well as four adult rat brains were processed for glial fibrillary acid protein (GFAP) immunocytochemistry. Irregular patches with relatively abundant, long astrocytic processes were found in monkey but not in rat brains, along the cingulate, as well as orbital, medial and dorsolateral cortices of the frontal lobe, and in temporal lobe cortical areas. The observed long astrocytic processes alternated in an apparent irregular fashion with others with a predominantly clear background and scattered GFAP-immunoreactivity (IR) astrocytes or with a predominant GFAP-IR-dense fibrillary meshwork. They usually stemmed from lamina I or II and coursed through deep laminae. Entorhinal cortex in rat brains showed occasional astroglial processes somewhat longer than those commonly observed in other cortical regions, albeit significantly shorter than those observed in Ceboidea monkeys and not truly interlaminar. These observations indicate a clear morphological difference in astroglial organization between nonhuman primates and a rodent brain. They further show that 'interlaminar astrocytes' are distributed nonhomogeneously in the frontal cortex of New World monkeys. No interlaminar processes were observed in cortical areas of rat brains. Even though the physiological role of interlaminar astrocytes in a primate brain remains unknown, their significant departure from the morphology of classical, stellate astrocytes suggest that they may subserve different cortical requirements.

Animals↗

Cortical astroglial conditioned medium induces in vitro radial-like forms.

Cerebral cortex and striatal cell dissociates obtained from rat fetuses (E 17) were subcultured and enriched in astroglial cells before being grown in regional (cerebral cortex, striatum) astroglial conditioned media (CM) or defined basal medium. Incidence of radial-like astroglia (vimentin+ or glial fibrillary acid protein, GFAP+) and length of processes in cortical cell subcultures showed a greater increase when exposed to cerebral cortex CM than to striatal CM or basal medium. Stellate (GFAP+) forms prevailed in subcultures grown in basal medium while striatal cells exposed to CM of either origin remained undifferentiated. Additionally, cultures were treated with various concentrations of cAMP (0.25 and 0.5 mM) and calcitonin gene related peptide (CGRP) (0.1, 0.5, and 1.0 microM). Under these conditions CM-exposed cultures (with predominant "radial-like" forms) did not increase stellate glial numbers, while fetal calf serum (FCS)-exposed cultures (morphologically undifferentiated) underwent significant degrees of stellate transformation. When CM-exposed cultures were shifted to FCS supplemented basal medium for 24-48 hr and then to basal medium alone prior to treatment, cAMP and CGRP were effective in transforming flat astroglia into stellate morphology. Results are indicative of the existence of astroglial diffusible factors affecting the in vitro expression of astroglial morphotypes from the cerebral cortex. Previous exposure to CM interferes with cytoskeletal astrocytic changes induced by cAMP and CGRP. It is speculated that astroglial factors could act in vivo to maintain the expression of radial-like cells during early developmental stages of the cerebral cortex, but it would not be effective in E 17 striatum.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Long, interlaminar astroglial cell processes in the cortex of adult monkeys.

At variance with current descriptions stressing the stellate geometry of cortical astrocytes in the brain of adult mammals, GFAP-immunoreactive astrocytes from prefrontal and rostral cingulate cortices in two adult New World monkey species, Cebus apella and Saimiri sciureus, were found to have long cellular processes traversing several cortical lamina. These unreported features of cortical astroglial cells in adult nonhuman primates pose new issues for the understanding of iso- and allocortical organization and processing in higher mammals.

Animals↗

Long-term MPTP-treated monkeys are resistant to GM1 systemic therapy.

GM1 has been reported to promote sprouting of dopaminergic mesencephalic neurons when administered at the time of MPTP treatment. Owing to its potential clinical significance, we evaluated behavioral effects of GM1 treatment in three Cebus apella monkeys with a persistent hemiparkinsonian syndrome after 20-22 mo of an intracarotid infusion of MPTP. MPTP monkeys compared with normal ones presented difficulty in solving motor cognitive tests and reversal of circling activity after apomorphine treatment. Monkeys were treated during 3 wk with daily saline, followed by 4 wk with GM1 (20 mg/kg, im). Neither during saline nor GM1 treatment, nor 30 d afterwards, did the animals improve their performances nor did the apomorphine tests reveal significant changes in circling behavior. These results are discussed in terms of their possible implications for Parkinson disease treatment.

Animals↗

"In vitro" effect of GM1 ganglioside on adrenal chromaffin cells.

Exposure of "in vitro" grown immature and adult adrenal chromaffin cells to concentrations of 10(-3) or 10(-5) M but not 10(-7) M GM1 ganglioside, resulted in significant increase in cell diameter, coupled with reduction of adhesion to substrate within 48 hrs of exposure. None of the GM1 concentrations, with or without serum supplementation, did significantly increase neuritogenesis in chromaffin cells. Immature chromaffin cells underwent neuritogenesis when grown in co-cultures with actively growing astroglia from striatum or cerebral cortex, an effect that was potentiated by NGF administration and blocked by anti-NGF. In neither of the former conditions did 10(-6) M GM1 prove to increase the number of neurite emitting cells nor their mean neuritic length further. It is speculated that GM1 does not perform the neuritogenic role described for central neurons in chromaffin cells, nor does it potentiate NGF effect on neuritogenesis observed in other peripheral neurons.

Animals↗

Leptomeningeal and skin fibroblasts: two different cell types?

Leptomeningeal and skin fetal (E16-17) fibroblasts were subcultured in vitro either in DMEM/F12 basal medium (with or without 10% FCS) or in astroglial conditioned medium (ACM). Both populations were characteristically composed of flat, undifferentiated, fibronectin(+), GFAP(-)cells where cultured in fetal serum supplemented basal media. When exposed to ACM leptomeningeal cells developed a population of thin, elongated, fibronectin(+) cells with radial type long processes while skin fibroblasts did not show significant changes in their characteristic morphotype. Exposure to db cAMP in basal medium resulted within 3 hr in their transformation to an astrocytic-like morphotype characterized by a condensed soma and multiple, short processes. Twenty-four hours later skin fibroblasts had returned to their flat appearance while leptomeningeal ones showed elongated, radial-like forms. Results indicate the possible existence of different receptors (to ACM factors) and/or cytoskeletal properties, and suggest that ACM-reactive fibroblasts of leptomeningeal origin represent a different cell type from those of skin origin. The hypothetical role of leptomeningeal cells during brain development is considered.

Animals↗

Trophic influences of human and rat amniotic fluid on neural tube-derived rat fetal cells.

Normal human (week 17-20) and rat (E16-17) amniotic fluids were used as culture media for primary cultures of rat fetal (E 16) cortical, mesencephalic and striatal cell dissociates, or astroglial subcultures from the same brain regions. Phase-bright and dark cells were identified under phase contrast microscopy and their cell processes were measured utilizing semi-automated procedures. Subcultured astroglia were immuno-reacted against glial fibrillary acidic protein and fibronectin. Rat and human amniotic fluid allowed survival and growth of neuronal and non-neuronal cells. Human amniotic fluid samples were trophic in variable degrees. Cerebral cortex subcultured astroglia usually expressed a radial-like morphotype. Although charcoal-adsorbed human amniotic fluid was trophic for primary cultures, its ability to sustain neuritic growth depended on its degree of trophism before treatment. Growth of cell processes in neuronal- and glial-like cells in primary cultures was inhibited to different degrees by the addition of antisera towards nerve or epidermal growth factors. It is concluded that amniotic fluid constitutes a trophic medium for astroglia and neurons. Both, nerve and epidermal growth factors appear to be necessary for growth of cell processes in neuronal and glial primary cultures in amniotic fluid. Trophic effect of amniotic fluid on subcultured astroglia did not seem to be diminished by nerve growth factor antiserum. The role of amniotic fluid during the early phases of brain organogenesis is discussed.

Amniotic Fluid↗

In vitro culture and labeling of neural cell aggregates followed by transplantation.

Septal, cortical, or whole brain fetal (E15-17) cells were dissociated and cultured in serum-supplemented Dulbecco minimum essential medium under rotating culture conditions. Preincubation and exposure to cytosine arabinoside was utilized to produce "neuron-rich" cultures. Fluorescent latex microbeads were added to cultures at seeding time or early after aggregate formation. All cell types were found to incorporate the fluorescent beads, although apparently not to the same extent. Two- to five-day-old aggregates tended to attach and grow neurites after their transfer to poly-l-lysine- or Matrigel-coated dishes under stationary conditions. Early aggregates transplanted to the hippocampus of adult rats developed into identifiable grafts, with fluorescent-labeled cells. We conclude that "young" neural cell aggregates maintain their ability to undergo two basic phenomena for cellular interaction, i.e., attachment and neuritic growth. Floating aggregates may provide a convenient cellular condition whenever culturing of neural cells is to be used before grafting them into a host animal.

Animals↗

Neural projections of the preoptic-suprachiasmatic region to the median eminence in the rat: a quantitative autoradiographic study.

The possibility of a preferential distribution of projections from the preoptic-suprachiasmatic region (POA-Sch) to the median eminence in the male rat was studied using autoradiographic and computer procedures. A cocktail of [3H]leucine-[3H]proline was injected into the POA-Sch. Animals were killed after either 1, 4, or 24 h of incorporation time (IT). The number of silver grains per 25-micron square was counted throughout the median eminence. Squares with silver grain densities at or above the 75 percentile of the population for any given IT group were labeled as "concentrating areas." The distribution of these concentrating areas in median eminence tissue was analyzed utilizing univariate procedures. Visual inspection of the distribution of silver grain densities in a rostrocaudal fashion indicated the likelihood of uneven distribution; however, these differences were not statistically significant. In the dorsoventral plane, results indicated that the highest silver grain density was at 50 to 75 micron from the floor of the 3rd ventricle. Such distribution was apparent both ipsi- and contralateral to the injection site, but was greater on the ipsilateral side.

Animals↗

Experimental dissociation of hypothalamic functions: an analysis of neural structural components for prolactin release control in the male rat.

A review of the research work done in male rats regarding the possible anatomical substrate underlying the control of prolactin release is presented. Also, a basis for the experimental dissociation of prolactin and LH release responses from a single brain locus is offered. It is proposed that the preoptic suprachiasmatic region (POA-Sch) encompasses neural elements involved in the facilitation and inhibition of prolactin release: the dorsal aspect of this region seems to encompass predominantly inhibitory neural elements for prolactin release, while more ventrally placed elements appear to be mostly facilitatory; inhibitory components of this hypothetical dual system for the control of pituitary prolactin release are significantly represented by incoming fibers coursing at some point as pericommissural, while facilitatory ones appear to be taking origin within the ventral half of the POA-Sch; and although direct stimulation of the ventral half of the POA-Sch often results in the combined release of prolactin and LH, it is possible to dissociate such responses by modifying the temporal structure of the applied electrical stimulus.

Action Potentials↗

Dissociation of prolactin and LH release responses after stimulation within the preoptic-suprachiasmatic region in male rats.

The relative efficiency of unilateral electrical stimuli applied within the preoptic-suprachiasmatic region (POA-Sch) to induce the release of prolactin and/or LH was studied in male rats under pentobarbital anesthesia. It was observed that unilateral stimulation with 10 Hz, 1.0 msec width pulses applied in the ventral half of the POA-Sch resulted primarily in the release of prolactin rather than LH. Fifty Hz stimuli applied at this same locus resulted also in LH release. It is concluded that the probability to dissociate the induced release of prolactin and LH from the anterior pituitary in male rats after stimulation from a given locus within the ventral POA-Sch is significantly affected by the characteristics of the applied stimulus.

Animals↗

Prolactin and LH release induced by kainic acid administration within the preoptic-suprachiasmatic region in behaving male rats.

Adult male rats were chronically implanted with a multiple metal cannula assembly to allow for the administration of kainic acid within the POA-SCH region. Kainic acid was applied in its solid form at two dose levels: 2 micrograms and 4 micrograms. A chronic heart cannula implanted through the external jugular vein allowed for sequential blood sampling. Animals were subjected to two different regimes, with 2-3 days difference, one in which blood sampling took place without kainic acid administration, and a second one in which the drug was administered after a first, control sample was obtained. Administration of kainic acid at both dose levels induced motor hyperactivity that lasted for more than 1 h in the group that received the higher dose. Prolactin concentration in plasma rose by 30 min and declined thereafter in the "low" dose group, or remained high for at least 90 min in the "high" dose group. LH levels increased significantly only after administration of the "high" dose. These results are indicative of the presence of facilitatory elements for the release of prolactin (and LH) which originate within the POA-SCH region, probably involving glutamatergic receptors. Stimulation of these elements, rather than passing fibers, are probably responsible for the observed hormonal results.

Animals↗

Cathodal versus anodal stimulation within the preoptic-suprachiasmatic region in male rats: effects on the release of prolactin and luteinizing hormone.

The relative efficiency for the release of luteinizing hormone (LH) and prolactin after anodal and cathodal stimulation applied within the preoptic-suprachiasmatic region (POA-SCH) was studied in adult male rats under pentobarbital anesthesia. Single monopolar pulses 0.5 ms duration, 100 or 200 microA intensity, were applied within the dorsal [vertical (V) 7.3] or ventral (V 7.8 to 7.9) aspects of the POA-SCH at a mean rate of 10 Hz in 30-s trains every other 30 s for a period of 30 min. Blood samples were obtained at 30-min intervals starting immediately before onset of stimulation. Although there were no obvious effect on plasma LH titers, prolactin concentrations tended to be higher when cathodal stimulation was applied at V 7.9 and when anodal pulses were applied at V 7.3. Stimulation with paired twin pulses (1.0 ms duration, 20.0 ms intrapair interval) resulted in clearer hormonal effects, namely, a significant increase in plasma prolactin concentration at 30 min only when cathodal stimuli were applied to the ventral aspect of the POA-SCH. Plasma LH concentrations were higher also at 30 min in the same experimental group, although the increase was not statistically significant. These results indicate the effectiveness of cathodal stimuli to induce hormonal release when applied within the ventral half of the POA-SCH in male rats. Present results suggest the existence of predominantly facilitatory neural elements for the release of prolactin (and LH) in this brain region.

Animals↗

Immediate prolactin release after pericommissural deafferentation in behaving male rats.

Adult male rats received a chronic midline implant of Halász-type knife placed at the level of the anterior commissure. A chronically implanted heart cannula allowed for sequential blood sampling in unrestrained conditions. Anterior knife rotation of 180 degrees induced a significant increase of plasma prolactin concentration in 30 min. Plasma LH levels remained within control levels. It is suggested that neural projections dorsally entering the preoptic-suprachiasmatic region are significantly involved in the control of prolactin release in male rats.

Afferent Pathways↗