Newborn Rat Cerebral Cortex Research Articles

Development of l-Dopa-containing diketopiperazines as blood-brain barrier shuttle

In our overall goal to develop anti-Parkinson drugs, we designed novel diketopiperazines (DKP1-6) aiming to both reach the blood-brain barrier and counteract the oxidative stress related to Parkinson's Disease (PD).The anti-Parkinson properties of DKP 1–6 were evaluated using neurotoxin-treated PC12 cells, as in vitro model of PD, while their cytotoxicity and genotoxicity potentials were investigated in newborn rat cerebral cortex (RCC) and primary human whole blood (PHWB) cell cultures. The response against free radicals was evaluated by the total antioxidant capacity (TAC) assay. Comet assay was used to detect DNA damage while the content of 8-hydroxyl-2′-deoxyguanosine (8-OH-dG) was determined as a marker of oxidative DNA damage. PAMPA-BBB and Caco-2 assays were employed to evaluate the capability of DKP1-6 to cross the membranes. Stability studies were conducted in simulated gastric and intestinal fluids and human plasma.Results showed that DKP5-6 attenuate the MPP + -induced cell death on a nanomolar scale, but a remarkable effect was observed for DKP6 on Nrf2 activation that leads to the expression of genes involved in oxidative stress response thus increasing glutathione biosynthesis and ROS buffering. DKP5-6 resulted in no toxicity for RCC neurons and PHWB cells exposed to 10–500 nM concentrations during 24 h as determined by MTT and LDH assays and TAC levels were not altered in both cultured cell types. No significant difference in the induction of DNA damage was observed for DKP5-6. Both DKPs resulted stable in simulated gastric fluids (t1/2 > 22h). In simulated intestinal fluids, DKP5 underwent immediate hydrolysis while DKP6 showed a half-life higher than 3 h. In human plasma, DKP6 resulted quite stable. DKP6 displayed both high BBB and Caco-2 permeability confirming that the DKP scaffold represents a useful tool to improve the crossing of drugs through the biological membranes.

Decrease of protein phosphatase 2A subunit B by glutamate exposure in the cerebral cortex of neonatal rats

Glutamate induces neurotoxicity during brain development, causing nerve damage. Protein phosphatase 2A (PP2A) is a type of serine/threonine phosphatase that regulates various biological functions. Among the PP2A subunit types, subunit B is abundant in brain tissue and plays an essential role in the nervous system. This study investigated changes in PP2A subunit B expression through glutamate exposure in the cerebral cortex of newborn rats. Sprague-Dawley rat pups (7 days after birth) were injected intraperitoneally with vehicle or glutamate (10 mg/kg). After 4 h of drug treatment, the brain tissue was isolated and fixed for morphological study. In addition, the cerebral cortex was collected for RNA and protein works. We observed severe histopathological changes including swollen neuron and atrophied dendrite in the glutamate exposed cerebral cortex. Glutamate exposure leads to a decrease in PP2A subunit B. Reverse-transcription PCR and Western blot analyses confirmed that glutamate induces a decrease of PP2A subunit B in the cerebral cortex of newborn rats. Moreover, immunohistochemical study showed a decrease in PP2A subunit B positive cells. The reduction of PP2A subunit B expression is considered an indicator of neurodegenerative damage. These results suggest that glutamate exposure causes neuronal damage in the cerebral cortex of new born rats through a decrease in PP2A subunit B.

Decrease of 14\u20133-3 proteins by glutamate exposure in the cerebral cortex of newborn rats

Glutamate is a representative excitatory neurotransmitter. However, excessive glutamate exposure causes neuronal cell damage by generating neuronal excitotoxicity. Excitotoxicity in neonates caused by glutamate treatment induces neurological deficits in adults. The 14–3-3 family proteins are conserved proteins that are expressed ubiquitously in a variety of tissues. These proteins contribute to cellular processes, including signal transduction, protein synthesis, and cell cycle control. We proposed that glutamate induces neuronal cell damage by regulating 14–3-3 protein expression in newborn animals. In this study, we investigated the histopathological changes and 14–3-3 proteins expressions as a result of glutamate exposure in the neonatal cerebral cortex. Rat pups at post-natal day 7 were intraperitoneally administrated with vehicle or glutamate (10 mg/kg). Animals were sacrificed 4 h after treatment, and brain tissues were fixed for histological study. Cerebral cortices were isolated and frozen for proteomic study. We observed serious histopathological damages including shrunken dendrites and atypical neurons in glutamate-treated cerebral cortices. In addition, we identified that 14–3-3 family proteins decreased in glutamate-exposed cerebral cortices using a proteomic approach. Moreover, Western blot analysis provided results that glutamate treatment in neonates decreased 14–3-3 family proteins expressions, including the β/α, ζ/δ, γ, ε, τ, and η isoforms. 14–3-3 proteins are involved in signal transduction, metabolism, and anti-apoptotic functions. Thus, our findings suggest that glutamate induces neonatal neuronal cell damage by modulating 14–3-3 protein expression.

2,4 Dinitrophenol Attenuates Mitochondrial Dysfunction and Improves Neurobehavioral Outcomes Postanoxia in Neonatal Rats.

Following anoxia, a rapid and marked mitochondrial-linked cell death occurs in the cerebral cortex of newborn rats which leads to insult advancement within a couple of days and causes lifelong neurobehavioral abnormalities. The present study investigated the role of 2,4 dinitrophenol (2,4 DNP) in three doses, i.e.,1, 2.5, and 5mg/kg on anoxia-induced time-dependent mitochondrial dysfunction and associated neurobehavioral outcome using a well-established global model of anoxia. Briefly, rat pups of 30-h age (P2) were subjected to two episodes of anoxia (10min each) at 24h of the time interval in an enclosed chamber supplied with 100% N2 and immersed in a water bath (35-37°C) to avoid hypothermia. Results demonstrated that the uncoupler 2,4 DNP, in the dose 2.5 and 5mg/kg injected i.p. within 5min after second anoxic episode significantly (P < 0.05) preserved mitochondrial function on day 7 preferentially by maintaining mitochondrial membrane potential (MMP) and inhibiting mitochondrial permeability transition (MPT) pore. Further, 2,4 DNP preserved mitochondrial function by improving different states of mitochondrial respiration (s2, s3, s4, s5), respiratory control ratio (RCR), antioxidant enzyme system like superoxide dismutase (SOD) and catalase (CAT), and mitochondrial complex enzymes (I, II, IV, V) after anoxia. Furthermore, a marked decrease in the levels of expression of cytochrome C (cyt C) and pro-apoptotic (Bcl-2 family) and apoptotic (caspase-9/3) proteins was observed on day 7 indicating that the treatment with 2,4 DNP prevented mitochondrial dysfunction and further insult progression (day 1 to day 7). Moreover, 2,4 DNP decreased the apoptotic cell death on day 7 and overall improved the neurobehavioral outcomes like reflex latency and hanging latency which suggests its role in treating neonatal anoxia.

Intracerebral Glycine Administration Impairs Energy and Redox Homeostasis and Induces Glial Reactivity in Cerebral Cortex of Newborn Rats.

Accumulation of glycine (GLY) is the biochemical hallmark of glycine encephalopathy (GE), an aminoacidopathy characterized by severe neurological dysfunction that may lead to early death. In the present study, we evaluated the effect of a single intracerebroventricular administration of GLY on bioenergetics, redox homeostasis, and histopathology in brain of neonatal rats. Our results demonstrated that GLY decreased the activities of the respiratory chain complex IV and creatine kinase, induced reactive species generation, and diminished glutathione (GSH) levels 1, 5, and 10days after GLY injection in cerebral cortex of 1-day-old rats. GLY also increased malondialdehyde (MDA) levels 5days after GLY infusion in this brain region. Furthermore, GLY differentially modulated the activities of superoxide dismutase, catalase, and glutathione peroxidase depending on the period tested after GLY administration. In contrast, bioenergetics and redox parameters were not altered in brain of 5-day-old rats. Regarding the histopathological analysis, GLY increased S100β staining in cerebral cortex and striatum, and GFAP in corpus callosum of 1-day-old rats 5days after injection. Finally, we verified that melatonin prevented the decrease of complex IV and CK activities and GSH concentrations, and the increase of MDA levels and S100β staining caused by GLY. Based on our findings, it may be presumed that impairment of redox and energy homeostasis and glial reactivity induced by GLY may contribute to the neurological dysfunction observed in GE.

Zingiberene attenuates hydrogen peroxide-induced toxicity in neuronal cells.

In this experimental design, we explored the neuroprotective potential of zingiberene (ZGB), a monocyclic sesquiterpene, in hydrogen peroxide (H2O2)-induced toxicity in newborn rat cerebral cortex cell cultures for the first time. The rats were exposed to H2O2 for 6 h to determine the oxidative stress levels. To evaluate cell viability, both 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays were carried out. Total antioxidant capacity (TAC) and total oxidative stress (TOS) parameters were used to evaluate oxidative changes. Besides determining 8-hydroxy-2-deoxyguanosine (8-OH-dG) levels in vitro, single-cell gel electrophoresis was also performed to measure the resistance of neuronal DNA to H2O2- exposed rats. Our results showed that survival and TAC levels of the cells decreased, while TOS, 8-OH-dG levels and the mean values of the total scores of cells showing DNA damage increased in the H2O2 alone-treated cultures. But pretreatment of ZGB suppressed the cytotoxicity, genotoxicity and oxidative stress that were increased by H2O2. Based on these observations, it is suggested that the sesquiterpene ZGB can be used as a novel and natural potential therapeutic in counteracting oxidative damages in the field of neurodegenerative disorders.

Protective effects of cyclosativene on H2O2-induced injury in cultured rat primary cerebral cortex cells

Sesquiterpenes have attracted much interest with respect to their protective effect against oxidative damage that may be the cause of many diseases including several neurodegenerative disorders and cancer. Our previous unpublished work suggested that cyclosativene (CSV), a tetracyclic sesquiterpene, has antioxidant and anticarcinogenic features. However, little is known about the effects of CSV on oxidative stress induced neurotoxicity. We used hydrogen peroxide (H2O2) exposure for 6h to model oxidative stress. Therefore, this experimental design allowed us to explore the neuroprotective potential of CSV in H2O2-induced toxicity in new-born rat cerebral cortex cell cultures for the first time. For this aim, MTT and lactate dehydrogenase release assays were carried out to evaluate cytotoxicity. Total antioxidant capacity (TAC) and total oxidative stress (TOS) parameters were used to evaluate oxidative changes. In addition to determining of 8-hydroxy-2-deoxyguanosine (8-OH-dG) levels, the single cell gel electrophoresis (or Comet assay) was also performed for measuring the resistance of neuronal DNA to H2O2-induced challenge. Our results showed that survival and TAC levels of the cells decreased, while TOS, 8-OH-dG levels and the mean values of the total scores of cells showing DNA damage (Comet assay) increased in the H2O2 alone treated cultures. But pre-treatment of CSV suppressed the cytotoxicity, genotoxicity and oxidative stress which were increased by H2O2. On the basis of these observations, it is suggested that CSV as a natural product with an antioxidant capacity in mitigating oxidative injuries in the field of neurodegenerative disorders.

Neuroprotective effects of farnesene against hydrogen peroxide-induced neurotoxicity in vitro.

Oxidative stress is highly damaging to cellular macromolecules and is also considered a main cause of the loss and impairment of neurons in several neurodegenerative disorders. Recent reports indicate that farnesene (FNS), an acyclic sesquiterpene, has antioxidant properties. However, little is known about the effects of FNS on oxidative stress-induced neurotoxicity. We used hydrogen peroxide (H2O2) exposure for 6h to model oxidative stress. Therefore, this experimental design allowed us to explore the neuroprotective potential of different FNS isomers (α-FNS and β-FNS) and their mixture (Mix-FNS) in H2O2-induced toxicity in newborn rat cerebral cortex cell cultures for the first time. For this aim, both MTT and lactate dehydrogenase assays were carried out to evaluate cell viability. Total antioxidant capacity (TAC) and total oxidative stress (TOS) parameters were used to assess oxidative alterations. In addition to determining of 8-hydroxy-2-deoxyguanosine (8-OH-dG) levels in vitro, the comet assay was also performed for measuring the resistance of neuronal DNA to H2O2-induced challenge. Our results showed that survival and TAC levels of the cells decreased, while TOS, 8-OH-dG levels and the mean values of the total scores of cells showing DNA damage (comet assay) increased in the group treated with H2O2 alone. But pretreatment of FNS suppressed the cytotoxicity, genotoxicity and oxidative stress, which were increased by H2O2 in clear type of isomers and applied concentration-dependent manners. The order of antioxidant effectiveness for modulating H2O2-induced oxidative stress-based neurotoxicity and genotoxicity is as β-FNS>Mix-FNS>α-FNS.

Immunohistochemistry of synaptic proteins for synaptic exocytosis

The synaptic proteins synaptobrevin/VAMP, SNAP-25, Syntaxin1, NSF and α-SNAP were revealed by means of immunocytochemistry. Materials from the cerebral cortex of adult, newborn and postnatal rats (P6 and P11) were used. Immunostaining for synaptobrevin/VAMP was mainly around the synaptic vesicles, whereas the immunolabeling for SNAP-25 and syntaxin1 was revealed in most cases on the cytoplasmic surface of the presynaptic membrane and to a lesser extend - on synaptic vesicles. Immunostaning for NSF and α-SNAP was found out not only on the axoplasm of axonal endings and varicosities, but also in perikarya and dendrites. In the cerebral cortex of newborn rats, a small number of immunopositive presynaptic parts could be observed. The number of these immunolabeled structures increases evidently with increasing age of rats. Scripta Scientifica Medica 2013; 45(1): 29-34.

Hypoxia–Ischemia Alters Nucleotide and Nucleoside Catabolism and Na+,K+-ATPase Activity in the Cerebral Cortex of Newborn Rats

It is well known that the levels of adenosine in the brain increase dramatically during cerebral hypoxic-ischemic (HI) insults. Its levels are tightly regulated by physiological and pathophysiological changes that occur during the injury acute phase. The aim of the present study was to examine the effects of the neonatal HI event on cytosolic and ecto-enzymes of purinergic system--NTPDase, 5'-nucleotidase (5'-NT) and adenosine deaminase (ADA)--in cerebral cortex of rats immediately post insult. Furthermore, the Na(+)/K(+)-ATPase activity, adenosine kinase (ADK) expression and thiobarbituric acid reactive species (TBARS) levels were assessed. Immediately after the HI event the cytosolic NTPDase and 5'-NT activities were increased in the cerebral cortex. In synaptosomes there was an increase in the ecto-ADA activity while the Na(+)/K(+) ATPase activity presented a decrease. The difference between ATP, ADP, AMP and adenosine degradation in synaptosomal and cytosolic fractions could indicate that NTPDase, 5'-NT and ADA were differently affected after insult. Interestingly, no alterations in the ADK expression were observed. Furthermore, the Na(+)/K(+)-ATPase activity was correlated negatively with the cytosolic NTPDase activity and TBARS content. The increased hydrolysis of nucleotides ATP, ADP and AMP in the cytosol could contribute to increased adenosine levels, which could be related to a possible innate neuroprotective mechanism aiming at potentiating the ambient levels of adenosine. Together, these results may help the understanding of the mechanism by which adenosine is produced following neonatal HI injury, therefore highlighting putative therapeutical targets to minimize ischemic injury and enhance recovery.

Neuroprotective effect of ace inhibitors in glutamate-induced neurotoxicity: rat neuron culture study

Glutamate is known to be neurotoxic at concentrations of 10-6M and 10-7M. Angiotensin converting enzyme (ACE) inhibitors can be assumed to be neuroprotective as they open the mitochondrial adenosine triphosphate-sensitive potassium channels by inhibiting the degradation of bradykinin. In this study, we investigated whether the ACE inhibitors captopril, ramipril and perindopril have protective effects in glutamate-induced neurotoxicity in newborn rat cerebral cortex cell cultures. Viability tests were performed among ACE inhibitors by constituting groups of control and 10-7M and 10-6M glutamate doses in newborn rat cortex cultures. While the mean viable cell number was 0.47±0.06 in the control group, it was 0.37±0.03 in the group exposed to 10-7M glutamate (p < 0.05) and 0.37±0.01 in the group exposed to 10-6M glutamate (p < 0.05). Captopril was used at a dose of 10 μM, perindopril was used at a dose of 1 μM, and ramipril was used at a dose of 30 μM against 10-7M and 10-6M glutamate. Ramipril and perindopril reversed the toxicity against 10-6M glutamate (p < 0.05). The neuroprotective properties of captopril, perindopril and ramipril were not found to be statistically significant against 10-7M glutamate at the doses mentioned above. Data obtained from this study indicate that ramipril and perindopril can prevent 10-6M glutamate-induced neurotoxicity.

Primary Cultures From Cerebral Cortex and Hippocampus Enriched in Glutamatergic and GABAergic Neurons

The aim was to define a primary culture system enriched in neurons using a defined culture medium, and characterize the model system as to cellular morphology and neuronal phenotypes. We found that these primary neuron enriched cultures from either newborn rat cerebral cortex or hippocampus contain small GABAergic and large glutamatergic neurons as well as astrocytes and microglia. Astrocytes in these cultures are morphologically differentiated with long, slender processes and interact with soluble factors responsible for induction and expression of the glutamate transporter GLT-1. The cultures achieve the highest expression of the vesicular glutamate transporter 1 (VGLUT1) and GLT-1 after 20 days in vitro. Conditioned media from these neuron enriched cultures also induced GLT-1 expression in primary astrocytic cultures, which were free from neurons. The amount of glutamatergic neurons guides the morphological maturation of astrocytes and GLT-1 expression both in the neuron enriched cultures and in the conditioned media supplemented astrocytic cultures. Interestingly, these cultures were not influenced or activated by the inflammatory stimulus lipopolysaccharide. This suggests that soluble factors from neurons protect microglia and astrocytes to become inflammatory reactive. In conclusion we have developed a well characterized culture model system enriched in neurons, taken from newborn rats and cultured in defined media. The neurons express different neuronal phenotypes. Such a model system is valuable when studying interactions between neurons and glial cells.

μ-Opioid agonists inhibit the enhanced intracellular Ca 2+ responses in inflammatory activated astrocytes co-cultured with brain endothelial cells

In order to imitate the in vivo situation with constituents from the blood–brain barrier, astrocytes from newborn rat cerebral cortex were co-cultured with adult rat brain microvascular endothelial cells. These astrocytes exhibited a morphologically differentiated appearance with long processes. 5-HT, synthetic μ-, δ- or κ-opioid agonists, and the endogenous opioids endomorphin-1, β-endorphin, and dynorphin induced higher Ca 2+ amplitudes and/or more Ca 2+ transients in these cells than in astrocytes in monoculture, as a sign of more developed signal transduction systems. Furthermore, stimulation of the co-cultured astrocytes with 5-HT generated a pronounced increase in intracellular Ca 2+ release in the presence of the inflammatory or pain mediating activators substance P, calcitonin gene-related peptide (CGRP), lipopolysaccharide (LPS), or leptin. These Ca 2+ responses were restored by opioids so that the δ- and κ-opioid receptor agonists reduced the number of Ca 2+ transients elicited after incubation in substance P+CGRP or leptin, while the μ- and δ-opioid receptor agonists attenuated the Ca 2+ amplitudes elicited in the presence of LPS or leptin. In LPS treated co-cultured astrocytes the μ-opioid receptor antagonist naloxone attenuated not only the endomorphin-1, but also the 5-HT evoked Ca 2+ transients. These results suggest that opioids, especially μ-opioid agonists, play a role in the control of neuroinflammatory activity in astrocytes and that naloxone, in addition to its interaction with μ-opioid receptors, also may act through some binding site on astrocytes, other than the classical opioid receptor.

Negative regulation of caspase-3 expression in the neonatal cerebral cortex by α2A-adrenoceptors

Antisense oligonucleotide to alpha2A-adrenoceptors increased the levels of mRNA (reverse polymerase chain reaction) and protein for a key executioner protease of apoptosis caspase-3 (immunoblotting assay) in the cerebral cortex of newborn rats. The relationship between the observed effect and low expression of alpha2-adrenoceptors was confirmed by the possibility of correcting this phenomenon by clonidine (stimulator of alpha2-adrenoceptors).

Host/parasite relationship in the in vitro infection of rat gliocytes by Neospora caninum: Evaluation of cell respiration

Neospora caninum is a protozoon that causes abortion in cattle and neuromuscular lesions in dogs, with the formation of cysts mainly in the central nervous system. Since N. caninum is an intracellular parasite with tropism for the cells of nervous system, this study evaluated the respiratory metabolism of glial cells infected by this* parasite. Glial cultures obtained from the cerebral cortex of newborn rats were kept in DMEM enriched with 10% fetal bovine serum, 1mM pyruvic acid and 2 mM of l-glutamine. They were infected at a ratio of approximately 1:1 (cell/parasite). Oxygen consumption was evaluated by polarography in the non infected and N. caninum infected groups, 24 and 72 h following infection. Glial cell respiration after 24 and 72 h was 307.2 ± 34.7 and 308.9 ± 64.1 μL of oxygen per μg of total protein per minute, and 566.2 ± 54.6 and 579 ± 117.5 μL O 2/μg of total protein/minute in the control and infected groups, respectively. These results show that N. caninum does not interfere with glial respiration in vitro.

Specificity of glutamate receptors in P2 synaptosomal fraction from rat brain cortex

Specificity of glutamate receptors in the P2 synaptosomal fraction from the cerebral cortex of newborn rats was studied by measuring (45)Ca(2+) uptake by synaptosomes in the presence of agonists of ionotropic and metabotropic glutamate receptors. It was shown that P2 synaptosomal fraction from rat cortex contains NMDA receptors, kainate receptors, and group 1 metabotropic receptors.

Culture of neural stem cells from cerebral cortex of newborn rats and its effect in cell replace in siblings

目的 从新生鼠大脑皮层分离培养神经干细胞(neural stem cells,NSC),并将这些NSC植入局灶性缺血损伤鼠的大脑皮层,观察其植入同胞鼠后的细胞替代作用.方法应用含表皮生长因子(EGF)和碱性成纤维细胞生长因子(bFGF或FGF-2)的无血清DMEM/F12(1:1)培养液获得新生鼠NSC球,酶免疫技术检测NSC标志nestin和诱导分化实验证明所获得的细胞球是NSC.在细胞球的体外培养中掺入BrdU,标记NSC,用于移植实验.鼠出生后第4天,开颅去脑膜阻断脑皮层血液供应建立新生鼠脑皮层局灶性缺血动物模型,术后第4天(出生后第8天)将标记好的NSC直接注射到缺血损伤灶的边缘区.实验鼠分为损伤移植组、损伤对照组和假手术对照组.于移植后第4、7、14、30天杀鼠取脑,进行BrdU标记的酶免疫组化染色,检查植入细胞的存活和迁移情况.结果新生鼠大脑皮层在体外培养一周,可以获得漂浮生长的、表达nestin的、具有分化为神经元和星形胶质细胞潜能的NSC球.移植实验发现:植入细胞有向大脑皮层缺血损伤灶周边迁移的特点,促进损伤灶组织结构的修复,损伤获得了很好的细胞修复作用.镜下可见灶区有满视野BrdU标记的阳性细胞,颗粒层集聚密度最大,未见有远部位的细胞迁移.假手术对照鼠脑内细胞逐渐减少,至移植后第14天只能检测到零星散在阳性细胞,明显较损伤移植鼠少.结论新生鼠大脑皮层内存在NSC.缺血性损伤可刺激移植的NSC增殖与迁移.移植的NSC对于大脑皮层局灶性缺血损伤灶的完整修复发挥了重要作用。

Ethanol induces GAD67 and VGAT in slice cultures of newborn rat cerebral cortex

Exposure of the developing brain to ethanol disposes to the fetal alcohol syndrome, causing changes in the neurotransmitter systems of glutamate and gamma-amino butyric acid (GABA). However, expression of genes involved in GABA synthesis, vesicular and transmembrane transport has not been investigated so far. We exposed organotypic slice cultures of newborn rat cerebral cortex to ethanol (100 mM) for 4 days and observed a significant induction of the enzyme producing GABA (GAD67, +21%) and the vesicular transporter VGAT (+112%), whereas the expression of the transmembrane transporters did not differ from control conditions. In summary, ethanol exposure of the developing cortex induces GABAergic genes, which might facilitate the excitatory functions of GABA during development.

Differential regulation of activity-dependent neuroprotective protein in rat astrocytes by VIP and PACAP

Activity-dependent neuroprotective protein (ADNP) was shown to be a vasoactive intestinal peptide (VIP) responsive gene in astrocytes derived from the cerebral cortex of newborn rats. The present study was set out to identify VIP receptors that are associated with increases in ADNP expression in developing astrocytes. Using VIP analogues specific for the VPAC1 and the VPAC2 receptors, it was discovered that VIP induced changes in ADNP expression in astrocytes via the VPAC2 receptor. The constitutive synthesis of ADNP and VPAC2 was shown to be age-dependent and increased as the astrocyte culture developed. Pituitary adenylate cyclase-activating polypeptide (PACAP) also induced changes in ADNP expression. The apparent changes induced by VIP and PACAP on ADNP expression were developmentally dependent, and while stimulating expression in young astrocytes, an inhibition was demonstrated in older cultures. In conclusion, VIP, PACAP and the VPAC2 receptor may all contribute to the regulation of ADNP gene expression in the developing astrocyte.

Oligodendrocyte progenitor migration in response to injury of glial monolayers requires the polysialic neural cell-adhesion molecule.

Injury to the nervous system results in reactive astrogliosis that is a critical determinant of neuronal regeneration. To analyze glial responses to mechanical injury and the role of the polysialic neural cell adhesion molecule (PSA-NCAM) in this process, we established primary glia cultures from newborn rat cerebral cortex. Scratching a confluent monolayer of primary glial cells resulted in two major events: rapid migration of oligodendrocyte progenitor-like (O-2A) cells into the wounded area and development of polarized morphology of type 1 astrocytes at the wound edge. Migrating O-2A progenitors had a bipolar morphology and exhibited A2B5 and O4 immunolabeling. Once these cells were established inside the wounded area, they lost A2B5 immunoreactivity and differentiated into glial fibrillary acidic protein-positive astrocytes. Migrating O-2A cells expressed PSA-NCAM, but type 1 astrocytes at the wound edge did not. Treatment of wounded cultures with Endo-N, which specifically removes PSA from the surface of cells, resulted in a significant decrease in O-2A cell migration into the wounded area and completely blocked the wound closure. Video time-lapse analysis showed that, in the presence of Endo-N, O-2A cells remained motile and migrated short distances but did not move away from the monolayer. These results demonstrate that O-2A progenitors contribute to reactive astrogliosis in culture and that PSA-NCAM is involved in this process by regulating cell migration.