Tissue printing of astroglial interlaminar processes from human and non-human primate cerebral cortex.

Astroglial interlaminar processes are unique features of the cerebral cortex of adult primates, including man. The functional role of these processes in the primate cerebral cortex is largely unknown. The development and standardization of procedures that could maximize the utilization of primate brain samples is required for the experimental analysis of the individual and collective dynamic properties of interlaminar glial processes. With this aim and in order to assess the relative stability of these glial processes in ex vivo conditions, "tissue printing" procedures were applied. "Tissue printing" allows for the acute transfer of cellular elements from fresh tissue onto an artificial substrate. Human, monkey (Cebus apella), and rat brain samples were subjected to "tissue printing" procedures followed by cell culture and immunohistochemistry. For the purpose of comparing the efficiency of this procedure on the transfer of other long glial processes, "tissue prints" of radial glial processes from neonatal rats and of Bergmann glia from cerebellar samples of adult rats were included. Nitrocellulose (with and without added fibronectin or laminin) produced the best attachment results. Interlaminar processes were not modified following 24-h incubation in a cell culture medium, with the addition of agents known to modify astroglial morphotypes in vitro (cyclic adenosine monophosphate, 40 mM K(+), or fetal calf serum). It is concluded that glia with interlaminar processes can be detached from fresh tissue using "tissue printing" procedures, can be maintained for at least 24 h in standard culture conditions, and showed a stable morphological phenotype.

Heterochronous maturation of regional brain astroglia: neuronal modulation of striatal glial cells differentiation ex vivo.

Subcultured astroglial cells from striatum, cerebral cortex and ventral mesencephalon obtained from primary cultures of fetal (E14, E17 and E21) or postnatal (days 5-6) rats showed different regional, age-dependent morphological response (stellation) to cyclic AMP. While most of the cerebral cortex and ventral mesencephalic astroglial cell population was responsive at all ages tested, striatal cells at E14 and E17 were not. At age E21 striatal astroglia showed a significant shift toward a mature-like type of response to cyclic AMP. Postnatal striatal astroglia responded to cyclic AMP as the cortical and ventral mesencephalic astroglia did, with generalized stellation. Prenatal striatal astroglia was characterized immunocytochemically as A2B5+, fibronectin+, vimentin+, S-100+ and GFAP-. Failure of early prenatal (E14, E17) striatal astroglia to differentiate in response to cyclic AMP, was overcome by previous (5-7 days) co-culture with primary cell dissociates from postnatal-, but not from prenatal donors, from all brain regions tested including a non-target region for striatal cells, such as septum. This effect was duplicated when striatal astroglia was co-cultured with cell populations enriched in neurons through Percoll gradients. Only cell-to-cell contact co-cultures were able to induce a change in the studied response. Dead neuron-enriched populations obtained following various types of physical treatments were also able to change significantly striatal cell response toward cyclic AMP. Enriched astroglial populations from postnatal donors did not change striatal astroglial response toward cyclic AMP, except for ventral mesencephalic astroglia which induced a comparatively reduced but significant increase in striatal cell responsiveness. It is concluded that astroglial maturation and potential for phenotype expression during brain development proceeds with regional heterochrony. Also, that maturation of prenatal striatal astroglia responsiveness toward cyclic AMP is inducible by non-diffusible factors, probably of neuronal origin, expressed in live or dead primary cultures from various, homotopic and heterotopic, postnatal brain regions. It is further suggested that striatal afferents and/or mature local striatal neurons express membrane associated molecules that regulate responsiveness for phenotype expression of striatal glial cells, thus reinforcing the concept of a highly interactive, continuous neuron-glial developmental process that takes place during brain organization.

Cerebrospinal fluid from L-dopa-treated Parkinson's disease patients is dystrophic for various neural cell types ex vivo: effects of astroglia.

Cerebrospinal fluid from L-dopa-treated Parkinson's disease patients and subjects without neurodegenerative diseases (controls) was explored in its trophic properties as culture medium on a variety of cells from neural origin. Primary cultures of regional brain dissociates from rat and Cebus apella monkey fetuses, immature rat adrenal chromaffin cells, phaeochromocytoma (PC12), and neuroblastoma (NB69) cell lines as well as subcultured fetal rat astroglia were used as target cells for 24- to 48-h culture periods. Most cerebrospinal fluid samples from L-dopa-treated patients had a general dystrophic effect. This phenomenon was more apparent on striatum and ventral mesencephalon than on cerebral cortex cell dissociates. The deleterious effect of these samples was abolished by previous exposure to fetal astroglial cells. Neuroblastoma cells showed no differential response when exposed to samples from control and L-dopa-treated patients. Phaeochromocytoma cells did not grow processes under any of the samples assayed in the time interval explored, but neither showed evidence of dystrophy. The relevance of these findings to the transplantation of different cell types as one of the possible therapies for Parkinson's disease is discussed. The suggestion is made that CSF testing prior to transplantation may aid in anticipating its possible outcome. Cotransplantation of neuronal cells with subcultured astroglia may foster survival and growth of the former cells.

Cold stress related alteration of RNA biosynthesis in brain cortex of mother-deprived newborn rats.

We studied the influence of maternal deprivation on the RNA biosynthesis in the brain cortex of 10 day-old rats. Mother-deprived pups, placed at 25 degrees C showed a reduction in body temperature of 6 +/- 1 degree C. After mother retrieval, RNA biosynthesis decreased 27% and 34% in total brain cortex and in isolated neurons, respectively. This fall is proportional to the body temperature reduction and can be avoided placing the pups at 37 degrees C immediately after the separation. Rethermostatization of offsprings, after one hour at 25 degrees C, showed an overshoot of RNA biosynthesis (145%) with further stabilization of synthesis rates to normal levels after 100 min. This classical physiological mechanism was further studied in vitro. Comparing in vivo and in vitro experiments, it is concluded that overshooting can not be observed in vitro if temperature reduction was not previously performed in vivo. Thus, this phenomenon seems to respond to humoral factors in order to be triggered. Afterwards, in vitro overshooting following cold stress in vivo, demonstrates that the depressed tissue by itself has the capability to turn back to normal RNA levels in the same way as observed in vivo.

Ex vivo astroglial-induced radial glia express in vivo markers.

Ex vivo induction of radial-like glia has been previously reported to occur following exposure of cerebral cortex subcultures from fetal origin to cerebral cortex astroglial-conditioned medium. The present report further confirms similarities between in vivo and ex vivo radial glia, using additional criteria: adhesion of primary cell dissociates to glial processes, with presumptive cell migration along them, punctuate labelling for laminin, and immunolabelling with Rat-401 antisera.

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.

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.