Analysis of extinction acquisition to attenuated tones in prenatally stressed and non-stressed offspring following auditory fear conditioning.

Stimulus generalization occurs when stimuli with characteristics similar to a previously conditioned stimulus (CS) become able to evoke a previously conditioned response. Experimental data (Lissek et al., 2005) indicate that patients with post-traumatic stress disorder (PTSD), more often show stimulus generalization following fear conditioning when tested under laboratory conditions. Factors surrounding this observation may contribute to two common features of PTSD: 1) hyper-responsiveness to sensory stimuli reminiscent of those associated with the original trauma, and 2) resistance of PTSD to extinction-based therapies. Adverse early experience is considered a risk factor for the later development of PTSD and in the present experiments we hypothesized that stimulus generalization would occur in an animal model of adverse early experience, the prenatally stressed (PS) rat. Adult PS and control (CON) rats underwent extensive pre-habituation to a conditioning chamber followed by conventional auditory fear conditioning. The next day both groups began an extinction regimen where a series of quieter (attenuated), CSs were administered prior to the full 75 dB training CS. When tested in this manner, PS rats froze at significantly lower tone amplitudes than did CON offspring on the first day of extinction training. This suggests that the PS rats had stimulus-generalized the CS to lower decibel tones. In addition to this finding, we also observed that PS rats froze more often and longer during three ensuing days of extinction training to attenuated tones. Group differences vanished when PS and CON rats were extinguished under conventional conditions. Thus, it appears that the two extinction regimens differed in their aversive cue saliency for the PS vs. CON rats. Follow-up prefrontal cortex transcriptome probing suggests that cholinergic and dopaminergic alterations may be involved.

TLR3 ligation protects human astrocytes against oxidative stress.

Astrocytic Toll-like receptor 3 (TLR3) plays an important role not only in antiviral response but also in regeneration/healing of the CNS. The present study was undertaken to determine whether the neuroprotective effects of TLR3 signaling also include antioxidative protection. TLR3 ligation in human astrocytes induced protracted resistance of the cells to H(2)O(2) toxicity. Similar resistance was induced by conditioned medium from TLR3-ligated astrocytes indicating the involvement of paracrine signaling mechanisms. Out of 13 major antioxidative genes only the gene encoding superoxide dismutase 2 (SOD2) was postligationally upregulated suggesting that SOD2 is the major enzyme responsible for this protection.

Hydrogen peroxide mediates higher order chromatin degradation.

Although a large body of evidence supports a causative link between oxidative stress and neurodegeneration, the mechanisms are still elusive. We have recently demonstrated that hydrogen peroxide (H(2)O(2)), the major mediator of oxidative stress triggers higher order chromatin degradation (HOCD), i.e. excision of chromatin loops at the matrix attachment regions (MARs). The present study was designed to determine the specificity of H(2)O(2) in respect to HOCD induction. Rat glioma C6 cells were exposed to H(2)O(2) and other oxidants, and the fragmentation of genomic DNA was assessed by field inversion gel electrophoresis (FIGE). S1 digestion before FIGE was used to detect single strand fragmentation. The exposure of C6 cells to H(2)O(2) induced a rapid and extensive HOCD. Thus, within 30 min, total chromatin was single strandedly digested into 50 kb fragments. Evident HOCD was elicited by H(2)O(2) at concentrations as low as 5 micro M. HOCD was mostly reversible during 4-8h following the removal of H(2)O(2) from the medium indicating an efficient relegation of the chromatin fragments. No HOCD was induced by H(2)O(2) in isolated nuclei indicating that HOCD-endonuclease is activated indirectly by cytoplasmic signal pathways triggered by H(2)O(2). The exposure of cells to a synthetic peroxide, i.e. tert-butyrylhydroperoxide (tBH) also induced HOCD, but to a lesser extent than H(2)O(2). Contrary to the peroxides, the exposure of cells to equitoxic concentration of hypochlorite and spermine NONOate, a nitric oxide generator, failed to induce rapid HOCD. These results indicate that rapid HOCD is not a result of oxidative stress per se, but is rather triggered by signaling cascades initiated specifically by H(2)O(2). Furthermore, the rapid and extensive HOCD was observed in several rat and human cell lines challenged with H(2)O(2), indicating that the process is not restricted to glial cells, but rather represents a general response of cells to H(2)O(2).

Hydrogen peroxide induces transient dephosphorylation of tau protein in cultured rat oligodendrocytes.

Oxidative stress is a major mediator of neurodegeneration. In this study, we tested the effects of oxidative stress induced by a brief exposure to hydrogen peroxide (H(2)O(2)) on the phosphorylation state of the tau protein in oligodendrocytes (OL). Primary oligodendrocyte cultures prepared from newborn rat brains were exposed to millimolar concentrations of H(2)O(2) for up to 15 min, and then incubated in normal medium for up to 12 h. The treatment caused morphological degeneration of OL characterized by the loss of cellular processes apparent approximately 3 h after H(2)O(2) exposure. The morphological degeneration was preceded by a profound dephosphorylation of tau protein revealed by immunoblot using monoclonal tau-1 antibody that recognizes the dephosphorylated epitope. The dephosphorylated form increased dramatically during H(2)O(2) exposure, peaked after 2 h of post-exposure, and returned to the baseline level within 12 h. Total tau protein levels were not changed in the course of the experiment as judged by immunoblotting with phosphorylation-insensitive tau-5 and 46-1 monoclonal antibodies. Our finding demonstrates that oxidative stress induces a rapid but transient dephosphorylation of tau protein that may underlie morphological degeneration of OL.

Higher order chromatin degradation in glial cells: the role of calcium.

Higher order chromatin degradation (HOCD), i.e. the scission of nuclear chromatin loops at the matrix attachment regions (MARs), is a hallmark of programmed cell death. We have previously demonstrated that hydrogen peroxide (H(2)O(2)) induces rapid HOCD in cultured oligodendrocytes generating two subpopulations of DNA fragments of >or=400 and 50-200 kb. In the present study, we examined the involvement of calcium in this process. HOCD was induced in primary rat oligodendrocytes by exposure to 1 mM H(2)O(2) and assessed by field inversion gel electrophoresis with and without S1 endonuclease digestion, to detect single and double stranded fragmentation, respectively. Chelating intracellular calcium with BAPTA/AM prior to H(2)O(2) exposure inhibited HOCD in a dose-dependent manner. Complete inhibition of HOCD was attained with 50 muM BAPTA/AM. The pretreatment of cells with desferroxamine mesylate, which may lower intracellular calcium levels, also resulted in a profound inhibition of HOCD, but the initial chromatin digestion into >or=400 kb single stranded DNA fragments was unaffected. Neither removing extracellular calcium nor blocking calcium release from intracellular stores with TMB-8 affected HOCD. Moreover, increasing intracellular calcium with A23187 calcium ionophore did not induce HOCD. Subsequent study in nuclei purified from C6 glioma cells revealed that the endonuclease responsible for HOCD is calcium-independent, but is magnesium-dependent. Magnesium-induced HOCD was not affected by the removal of calcium from nuclei with EGTA, but was practically abrogated in nuclei prepared from BAPTA/AM-pretreated cells. These results indicate that although H(2)O(2)-induced HOCD is not directly mediated by an increase of intracellular calcium concentration, normal resting levels of intracellular calcium are required for the maintenance of MAR-associated endonuclease in an active form.

Hydrogen peroxide induces rapid digestion of oligodendrocyte chromatin into high molecular weight fragments.

High molecular weight (HMW) fragmentation of nuclear chromatin was studied in cultured rat oligodendrocytes (OL) exposed to hydrogen peroxide (H2O2). Intact genomic DNA was isolated by agarose embedding, and analyzed by field inversion gel electrophoresis, with and without S1 endonuclease digestion to detect and discriminate between single and double stranded fragmentations, respectively. The exposure of OL to H2O2 resulted in a very rapid degradation of chromosomal DNA into HMW fragments that reflect native chromatin structure. Hence, within 10 min after the addition of 1 mM H2O2, a discrete pool representing approximately 45% of the nuclear chromatin underwent single strand digestion into >400 kb fragments likely at AT-rich matrix attachment regions. Subsequent accumulation of single stand breaks at these regions led to bifilar scission. Ultimately, chromatin within this susceptible pool was cleaved at remaining matrix attachment regions into 50-200 kb fragments. Chromatin digestion could be elicited with H2O2 concentrations as low as 50 microM. After the removal of H2O2, most >400 kb fragments were religated within 2 h; however, digestion into 50-200 kb fragments was irreversible. The DNA digestion was not accompanied by the degradation of nuclear proteins, i.e., lamins A/C and poly (ADP-ribose) polymerase indicating that chromatin fragmentation is unlikely to be mediated by proteolysis. In conclusion, H2O2 at pathologically relevant concentrations induces a very rapid and extensive digestion of OL chromatin into HMW fragments. Because the chromatin fragmentation is only partly reversible, it may be a decisive factor in committing oxidatively stressed OL to degeneration and/or death.

Delayed oligodendrocyte degeneration induced by brief exposure to hydrogen peroxide.

An in vitro model system of cultured oligodendrocytes was used to determine the susceptibility of these cells to oxidative stress induced by 15 min exposure to millimolar concentrations of hydrogen peroxide (H2O2). Following the exposure, the cells were incubated in normal growth medium, and analyzed at different time points. Although no cell loss was observed during the exposure period, there was a progressive depletion of adherent cells during the postexposure period as seen from either the number of recoverable nuclei, or from total RNA content of the cultures. Both the rate and the extent of cell deletion was directly dependent on H2O2 concentration. Cell death was preceded by structural alterations in the nuclear envelope resulting in "fragile" nuclei which disintegrated during isolation. Northern blot analysis showed that the expression of myelin-specific genes was rapidly downregulated in H2O2-treated cells. On the other hand, the expression of antiapoptotic gene, bcl-2 featured massive but transient upregulation. Oligodendrocyte degeneration also featured genomic DNA degradation into high molecular weight fragments, which are likely to represent cleaved chromosomal loops. The results demonstrate vulnerability of oligodendrocytes to oxidative stress that induces rapid degeneration and ultimately leads to delayed cell death. This feature is highly relevant to oligodendrocyte damage and depletion following ischemic, traumatic, or inflammatory insults to the central nervous system (CNS).

Differentiation dependent activation of the myelin genes in purified oligodendrocytes is highly resistant to hypoglycemia.

We have previously demonstrated that the developmental upregulation of myelin-specific genes in mixed glial cultures is strongly attenuated by hypoglycemia. The present study was designed to evaluate the effect of hypoglycemia on differentiation-dependent upregulation of myelin genes in purified oligodendrocyte cultures. The expression of major myelin protein genes, i.e., proteolipid protein (PLP), basic protein (BP) and myelin associated glycoprotein (MAG) were monitored by Northern blot analysis. In control cultures maintained at 6 mg/ml of glucose, the expression of all the genes upregulated rapidly, and plateaued at approximately day 4. A similar pattern of differentiation-dependent upregulation was observed for the gene encoding a lipogenic enzyme, i.e., malic enzyme (ME). In contrast to mixed glial cultures, however, this developmental gene upregulation was not significantly affected by severe hypoglycemia (approximately 0.02 mg/ml). The results indicate that the effect of glucose deprivation on oligodendrocyte genes observed in mixed glial cultures is mediated by other cells. The upregulation of the genes in differentiating oligodendrocytes was accompanied by the production of myelin-related membrane that was isolated by density gradient fractionation. In contrast to the effect on gene expression, this anabolic activity was highly dependent on glucose, as seen from a profound suppression by severe hypoglycemia.

Antisense oligodeoxynucleotides targeted to MAG mRNA profoundly alter BP and PLP mRNA expression in differentiating oligodendrocytes: a caution.

The applicability of antisense technology to suppress the expression of myelin associated glycoprotein (MAG) in cultured oligodendrocytes was evaluated. Differentiating oligodendrocyte precursor cells obtained by the shake-off method were exposed to nine unmodified antisense oligodeoxynucleotides (ODNs) targeted to the first seven exons of MAG mRNA. After four days, steady-state levels of MAG, proteolipid protein (PLP) and basic protein (BP) mRNAs were determined by Northern blot analysis. Only ODN annealing to 599-618 nt of the MAG mRNA (the junction of exon 5 and 6) resulted in a significant, 75% decrease in the MAG mRNA level. Unexpectedly, six other anti-MAG ODNs which had no significant effect on the MAG message, greatly increased the level of BP mRNA. The highest upregulation of approximately 12 fold was observed with ODN annealing to 139-168 nt (junction of exon 3 and 4). On the other hand, the 997-1016 ODN decreased the levels of BP and PLP messages by 70-80%. The 599-618 ODN also decreased the PLP mRNA by 85%. The results demonstrate that antisense ODNs targeted to one gene may profoundly alter the expression of other genes, and hence, complicate functional analysis of the targeted protein.

Selenium is required for normal upregulation of myelin genes in differentiating oligodendrocytes.

The purpose of this study was to characterize the selenium requirement for the normal differentiation of oligodendrocyte lineage cells. In primary mixed glial cultures prepared from newborn rat brains, the overall growth of cultures, as seen from the total RNA yield, was not significantly affected by selenium. However, 30 nM selenium was required for the normal upregulation the proteolipid protein, basic protein, and myelin-associated glycoprotein gene expression assessed by Northern blot analysis. Selenium deprivation during initial, rapid phase of the gene upregulation irreversibly suppressed the genes, indicating the existence of a critical period in oligodendrocyte differentiation. In purified oligodendrocyte cultures prepared by mechanical dislodging of progenitor (O-2A) cells from mixed glial cultures, total cell number and total RNA yield were virtually unaffected by selenium deprivation; however, the developmental upregulation of the myelin genes was profoundly attenuated. Immunocytochemical analysis confirmed the suppressive effect of selenium deficiency on the differentiation of oligodendrocyte lineage cells, as seen from a significant decrease in the population of GalC+ and O4+ cells. Because the number of GC+ cells was more reduced than the number of O4+ cells, the results indicate that selenium deficiency may specifically inhibit the progression from immature to mature oligodendrocytes.

Structural characterization of myelin-associated glycoprotein gene core promoter.

Myelin-associated glycoprotein (MAG) is emerging as an important molecule involved in the plasticity and regeneration of the central nervous system. In this study, the structure of MAG gene promoter was characterized in cultured rat oligodendrocyte lineage cells. Heterogeneous transcription initiation with five major and eight minor start sites scattered within 72 bp was shown by primer extension analysis. This TATA-less core promoter contains no prominent initiator (Inr) elements associated with the transcription initiation sites, and hence, appears to utilize novel positioning mechanisms. Genomic footprinting analysis revealed several putative protein-binding regions overlapping the initiation sites and containing a multitude of CG-rich sequences. However, no conspicuous alterations in the protein-binding pattern were evident between O2A progenitors in which the gene is inactive, and mature oligodendrocytes with fully upregulated gene. The core promoter DNA features a differentiation-dependent demethylation as shown by genomic sequencing analysis. Three of eight cytosines are totally demethylated in oligodendrocyte chromosomes, indicating that these unmodified bases may be critical for full activation of the promoter. The core promoter is located within an internucleosomal linker, and the upstream regulatory region appears to be organized into an array of nucleosomes with hypersensitive linkers.

Chromatin structure and transcriptional activity of MAG gene.

Myelin associated glycoprotein (MAG) is an essential component of the periaxonal architecture of the myelin sheath. Because of its potent neurite growth repressive activity, MAG is also likely to play an important role in axonal guidance during the CNS development, and to be responsible for abortive neuronal regeneration in adult CNS. The MAG gene chromatin from approximately -1.6 to +0.6 kb features MNase hypersensitivity that may delineate the gene control region. The proximal upstream region of the gene is organized into an array of five nucleosomes with hypersensitive linkers. The core promoter is located within the first upstream linker that becomes highly hypersensitive in the course of oligodendrocyte differentiation. The adjacent upstream region contains positive and negative enhancers that are likely to streamline oligodendrocyte specific expression of the gene. The TATA-less core promoter contains novel, as yet uncharacterized initiator elements that direct the assembly of transcriptional complexes. The promoter appears to be controlled by both, the addition of activating trans-factors and removal of inhibitory trans-factors as progenitor cells differentiate into mature oligodendrocyte. The developmental activation of the gene is also concomitant with profound DNA demethylation that may provide auxiliary regulatory mechanisms. Hence, the upregulation of the MAG gene in differentiating oligodendrocytes entails chromatin remodeling as well as changes in the assortment of nuclear trans-factors.

Generation of high efficiency ssDNA hybridization probes by linear polymerase chain reaction (LPCR).

The polymerase chain reaction (PCR) methodology can be employed to produce DNA hybridization probes. The major advantages of this paradigm over other techniques include superior specific activity of the probes, the versatility of sequence selection, the ability to produce short probes, and the simplicity of the procedure. We have further improved the efficiency of PCR probes by generating single stranded (ssDNA) probes that do not reanneal with themselves in solution, and hence, their availability for the interaction with the complementary sequences of the target is profoundly increased. Protocols for 32P-dCTP labeled and digoxigenin-dUTP labeled probes have been elaborated to maximize the incorporation rate of the label as well as to provide for the production of full-length probes. The ssDNA probes may be particularly suitable for nucleic acid detection in tissues by in situ hybridization.

Glioma-stimulated chemoattraction of endothelial cells and fibroblasts in vitro: a model for the study of glioma-induced angiogenesis.

Induction of angiogenesis is essential for the continued growth of solid tumors, and one critical component of tumor-induced angiogenesis involves the stimulation of microvascular cells to migrate into the growing mass. We have developed a convenient model system utilizing dual co-culture chambers to study cellular chemotaxis induced by glioma cells in vitro. In this system, rat C6 glioma cells induced migration of fibroblasts and brain capillary endothelial cells. The migratory response was directly related to the number of C6 cells serving as stimulus in the lower chamber. Similar migratory responses were induced by C6 cell conditioned medium in a concentration dependent fashion. Medium conditioned by cultured human anaplastic astrocytoma cells was also found to contain potent chemotactic factor(s). This system may ultimately be employed in the identification of particular glioma cell population(s) and secreted factor(s) responsible for the chemoattraction of microvascular cells.

Differentiation-specific demethylation of myelin associated glycoprotein gene in cultured oligodendrocytes.

The methylation status of a 4.4-kb 5' end of the myelin-associated glycoprotein (MAG) gene was assessed in cells with different levels of transcriptional activity of the gene, i.e., liver, brain, O-2A oligodendrocyte precursors cells, mature oligodendrocytes, and glioma C6 cells. Purified DNA was digested with methylation-sensitive restriction enzymes, and the cuts were mapped by the indirect end-labeling technique. The restriction sites within the 4.4-kb fragment revealed a highly heterogenous methylation pattern among cells and tissues, and liver DNA was the most heavily methylated. Most of the restriction sites were partly demethylated in the nervous system cells. Notably, two adjacent Hha1 sites at +94 and +96 were fully methylated in liver, but partially demethylated in the brain, OL, and O2A. Two Hpa2 site located at -1836 and at -39 were progressively demethylated in oligodendrocyte lineage cells, indicating specific hypomethylation associated with the oligodendrocytic differentiation. Most of the restriction sites were weakly methylated in the DNA from neoplastic C6 cells, although the Hha1 sites were fully methylated. No clear-cut correlation between the extent of CpG dinucleotide methylation and the chromatin conformation was found. For example, out of four heavily methylated sites only two comapped with MNase hypersensitive sites. Also, the -1836 Hpa2 site whose demethylation is concomitant with oligodendrocytic differentiation seems to be localized within precisely positioned nucleosomal arrays of the MAG gene chromatin. The results indicate that the MAG gene undergoes progressive demethylation concomitant with the oligodendrocyte differentiation/maturation. However, certain CpG dinucleotides remain heavily methylated even in the fully active gene in mature oligodendrocytes, indicating that they may be essential in maintaining proper chromatin structure.

Transcriptional regulation of myelin associated glycoprotein gene expression by cyclic AMP.

The treatment of rat glioma C6 cells with 10 microM isoproterenol (Ipt) for 4 days upregulated the expression of the myelin-associated glycoprotein (MAG) gene by approximately 55-fold over the control value. The constant presence of Ipt in the medium was required for the maximal upregulation, as time-restricted exposures to the drug produced only partial, or no upregulation of the gene. No difference in the MAG mRNA stability could be detected in Ipt-treated vs untreated cells indicating that the drug upregulates the MAG gene at transcriptional level. Serum (FCS) strongly attenuated the response of the MAG gene to Ipt. The stimulatory effect of Ipt was profoundly reduced by spermine and H-89, indicating that protein kinase A-dependent protein phosphorylation is involved in the MAG gene activation. Within 30 min after Ipt administration, the c-fos gene was upregulated by 10-fold, and thereafter, its message level decreased and stabilized at approximately 3-fold over control. In contrast, the c-jun gene was downregulated to approximately 20% of control within 30 min after Ipt administration. Subsequently, its message level rose and fell once again within 12 h to approximately half of control, and returned to control level within 72 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucocorticoid-induced upregulation of proteolipid protein and myelin-associated glycoprotein genes in C6 cells.

The effect of dexamethasone on the expression of proteolipid protein (PLP) and myelin-associated glycoprotein (MAG) genes was investigated in rat C6 glioma cells. The steady state level of the respective mRNAs was quantitated by Northern blot analysis. The treatment of cells with dexamethasone transiently upregulated the expression of both genes with peak mRNA levels of approximately 10-fold over control levels occurring at day 3 for the PLP gene and at day 5 for the MAG gene. The effect was directly related to the drug concentration in the range from 10(-9) to 10(-5) M. Combined exposure of the cells to dexamethasone and retinoic acid featured an additive effect on PLP gene expression, whereas MAG gene expression was depressed below detectability level. The dissimilarity in the response of the genes to dexamethasone and retinoic acid supports the contention that the genes are controlled by different mechanisms. Furthermore, the results indicate that the effects of dexamethasone and retinoic acid on the myelin genes are mediated by different regulatory pathways.

Myelin gene activation: a glucose sensitive critical period in development.

Glucose deprivation was employed to model caloric undernutrition in newborn rat mixed glial cultures. Six day-old cultures were placed in serum-free media containing glucose concentrations from 0.55 mg/ml to 10 mg/ml. The expression of the myelin PLP, BP, and MAG genes was determined by Northern blot analysis. The activation of the myelin genes began at approximately 6 days in vitro (DIV), and a period of rapid upregulation followed through 14 DIV. The gene activity was directly related to the glucose concentration. The increase in glucose concentration from 0.55 to 1.5 mg/ml (which spans the physiological range) resulted in 2-3 fold increases in expression of the myelin genes, whereas further increases in glucose (2-10 mg/ml) produced only slight additional elevation in the gene activity. We used high glucose (5-6 mg/ml) as control, or low glucose (0.55 mg/ml) as deprived, to delineate possible critical periods of oligodendrocytic differentiation. Cultures were deprived for 4-day intervals, staggered from 6 to 22 DIV. Deprivation from 6 to 10 DIV produced an 80-90% suppression of the myelin gene upregulation at 22 DIV; deprivation from 10 to 14 DIV produced 60-70% suppression; whereas deprivation from 14 to 18 DIV was fully recoverable by 22 DIV. These results show that mixed glial cultures model the developmental pattern of myelin gene expression, as well as their vulnerability. Furthermore, the period of rapid upregulation of the myelin genes appears to be a critical period in development, during which glucose deprivation irreversibly alters oligodendrocyte differentiation.

Abnormal upregulation of myelin genes underlies the critical period of myelination in undernourished developing rat brain.

Since myelin gene expression is suppressed during active myelination of the undernourished brain, this study was designed to determine the effects of undernourishment on the upregulation of myelin genes and the relationship between upregulation and the 'critical period' associated with permanent hypomyelination of the brain. Long-Evans rat dams were given either ad libitum or restricted access to rat chow to produce two populations of developing offsprings. The food deprivation schedule was designed to produce a degree of growth retardation comparable to our earlier studies of hypomyelination in undernourished brain. The expression of myelin genes, at various developmental ages, was determined in the forebrains from undernourished and normal, well fed controls by Northern analysis. In well nourished forebrain, proteolipid protein (PLP), myelin associated glycoprotein (MAG), and basic protein (BP) messages began to increase polynomially after day 8 post partum, leading to a rapid accumulation of message during the following several days. In undernourished forebrain, PLP, MAG, and BP messages did not show any increase until day 10, and then increased at a diminished rate as compared to well nourished forebrain. Additionally, the two PLP messages (1.6 kb and 3.2 kb) showed different vulnerabilities to protein-calorie undernourishment, which explains the abnormal ratio of the 3.2 and 1.6 kb forms we previously found in undernourished brain. This study shows a pattern of temporal specificity when the myelin PLP, MAG, and BP genes are synchronously upregulated in the normal forebrain to a high rate of transcription between day 7 to 9, which is several days before the onset of rapid myelination of the brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Cocaine cytotoxicity in serum-free environment: C6 glioma cell culture.

Rat glioma C6 cells were employed to determine the vulnerability of the CNS-derived cells to cocaine. The cells were cultured either in the presence of serum or in serum-free (defined) medium to model glial development in the normal brain. In serum-containing medium, cocaine in amounts up to 100 micrograms/ml neither retarded cell proliferation nor altered cell morphology. In the absence of serum, the culture growth was profoundly retarded and cell death was observed with amounts as small as 0.1 micrograms/ml. Even brief (24 hrs) exposure to low cocaine concentrations in serum-free medium irreversibly decreased the cell number. However, an initial 24 hr exposure to 0.1-2.5 micrograms/ml cocaine prolonged survival of cells subsequently exposed to a lethal concentration (100 micrograms/ml). Benzoylecgonine in amounts up to 100 micrograms/ml had no effect on cell proliferation, with or without serum. These data show that cocaine in the absence of serum is highly cytotoxic, which indicates a possibility that the blood-brain-barrier-protected CNS cells may be particularly vulnerable to the drug when it enters the nervous system.