Neurons In Rat Brain Research Articles

Effects of Microwaves from GSM Mobile Phones on the Blood-brain Barrier and Neurons in Rat Brain

Our group has since 1988 studied the effects of different intensities and modulations of 915MHz RF in a rat model where the exposure takes place in a TEM-cell during various time periods and post exposure recovery times. The power fed into TEM-cells was 0.125, 1.25, 12.5 or 125mW corresponding to whole body SAR (determined experimentally): 0.2, 2, 20 or 200mW/kg. The rats were awake and not restrained during exposure and after the recovery period the animals were anaesthetized and sacrificed by perfusion-fixation with 4% formaldehyde. Paraffin embedded 5 μm. thick brain slices were stained for albumin by applying albumin antibodies (Dakopatts), by which albumin is revealed as brownish discolorations. Dark neurons were revealed by staining for RNA/DNA with cresyl violet. In series of more than 1800 Fisher rats, we have proven that sub thermal power levels from both pulse-modulated and continuous RF fields - including those from real GSM mobile phones - have the potency to significantly open the BBB for the animals' own albumin (but not fibrinogen) to pass out into the brain and to accumulate in the neurons and glial cells surrounding the capillaries. Albumin extravasations are most prominent at the lower SAR values. This dose-response behaviour suggests some kind of energy or electromagnetic field strength windowing effect. A linear dose-response relationship for dark neurons was found at 50 days after exposure, with most prominent occurrence at SAR 200mW/kg. (Less)

Induction of Bcl-2 and Bax was related to hyperphosphorylation of tau and neuronal death induced by okadaic acid in rat brain

Abnormal hyperphosphorylation of the cytoskeletal protein tau is a characteristic feature of neurodegeneration in Alzheimer's disease (AD) brain. Okadaic acid (OA), a protein phosphatase inhibitor, induces neuronal death and hyperphosphorylation of tau. In the present study using a model of microinjection of OA into rat frontal cortex, we aimed to investigate if OA-induced hyperphosphorylation of tau and neuronal death are related to the expression of Bcl-2, an apoptosis inhibitor, or Bax, an apoptosis inducer. Immunohistochemistry and Western blot analysis showed that OA injection dose- and time-dependently induced the expression of Bcl-2 and Bax protein in the surrounding of OA injection areas, which were similar with that of AT8 immunostaining, a marker of hyperphosphorylated tau. However, the ratios of Bcl-2 over Bax had a negative relationship to the expression of AT8. Furthermore, double fluorescent staining showed that AT8-positive neurons mainly costained with terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick-end labeling, a marker of DNA damage, indicating that tau hyperphosphorylation may be associated with DNA damage in the neurons of rat brain. In the areas more adjacent to the OA injection site, most neurons with AT8-positive staining showed vulnerability to OA toxicity and could be triple-stained with Bcl-2 and Bax or double-stained with Bcl-2. However, in the areas further from the OA injection site, neurons with few AT8-positive staining showed resistance to OA toxicity and only stained with Bcl-2, but not Bax. The results suggest that the ratios of Bcl-2 over Bax expression may have an effect on tau hyperphosphorylation and neuronal death following OA injection.

Cytomorphometric changes in rat brain neurons after rapid eye movement sleep deprivation

Rapid eye movement sleep plays a vital role in the survival of animals. Its deprivation causes alterations in brain functions and behaviors including activities of important enzymes, neurotransmitter levels, impairment of neural excitability and memory consolidation. However, there was a lack of knowledge regarding the effects of rapid eye movement sleep deprivation on neuronal morphology that may get affected much earlier than any permanent damage to the neurons. In the present study, some of these issues have been addressed by studying the effects of rapid eye movement sleep deprivation on various morphological parameters viz. neuronal perimeter, area and shape of neurons located in brain areas known to regulate rapid eye movement sleep and as a control in other brain areas which do not regulate rapid eye movement sleep. The results showed that rapid eye movement sleep deprivation differentially affected neurons depending on their physiological correlates of rapid eye movement sleep and neurotransmitter content. The effects could be reversed if the animals were allowed to recover from rapid eye movement sleep loss or by applying alpha1-adrenergic antagonist, prazosin. The findings in rats support reported data and help explaining previous observations.

Molecular and cellular mechanisms of neuroprotection by vascular endothelial growth factor

The present view of the neuroprotective functions and mechanisms of action of vascular endothelial growth factor (VEGF) is based on studies of neuronal ischemic/hypoxic models in vivo and in vitro. Endogenous neuronal VEGF increases in the ischemic brain and plays a neuroprotective role in the pathophysiologic processes that follow stroke. Exogenous VEGF, directly administered or overexpressed by gene delivery into rat brains, reduces ischemic brain infarct and decreases hypoxic neuronal death. The main neuroprotective mechanisms of VEGF include: (1) modulation of the phosphatidylinositol 3'-kinase (PI3K)/Akt/nuclear factor-kappaB signaling pathway, inhibition of caspase-3 activity, and reduction of ischemic neuronal apoptosis; (2) inhibition of outward delayed rectifier potassium channel currents and increase of ischemia-induced tyrosine phosphorylation of Kv1.2 potassium channel proteins via activation of the PI3K pathway; and (3) enhancement of proliferation and migration of neural progenitors in the subventricular zone and improvement of striatal neurogenesis and maturation of newborn neurons in adult rat brains after stroke.

Lithium protection from glutamate excitotoxicity: therapeutic implications

Long-term treatment with lithium, the primary drug used to treat bipolar mood disorder, robustly protects against glutamate-induced, NMDA receptor-mediated excitotoxicity in primary cultures of rat brain neurons. The neuroprotective effects of lithium are accompanied by a reduction in NMDA receptor-mediated calcium influx, up-regulation of anti-apoptotic Bcl-2, down-regulation of pro-apoptotic p53 and Bax, and activation of the cell survival signaling pathway. In addition, lithium antagonizes glutamate-induced activation of c-Jun-N-terminal kinase (JNK), p38 kinase and AP-1 binding, and suppresses glutamate-induced loss of phosphorylated CREB due to activation of protein phosphatase 1. Evidence has also been obtained that lithium induces BDNF and activates its receptor TrkB in cortical neurons and this BDNF/TrkB signaling activation is imperative for the neuroprotective effects of this drug. In a rat stroke model, post-insult treatments with therapeutic doses of lithium or valproic acid (VPA) markedly reduce ischemia-induced brain infarction and neurological deficits. These neuroprotective effects are associated with suppression of caspase-3 activation and induction of chaperone proteins. In a rat excitotoxicity model of Huntington's disease (HD) in which an excitotoxin was infused into the striatum, both long- and short-term pretreatment with lithium reduces excitotoxin-induced DNA damage, caspase-3 activation and striatal neuronal loss. This neuroprotection is associated with up-regulation of Bcl-2 in neurons. Lithium also induces cell proliferation near the injury site with a concomitant loss of proliferating cells in the subventricular zone. Some of these proliferating cells display neuronal or astroglial phenotypes. This finding corroborates our in vitro results showing that lithium stimulates the proliferation of neuroblasts in primary cultures of CNS neurons.

Recombinant adeno-associated virus serotype 2 effectively transduces primary rat brain astrocytes and microglia

Recombinant adeno-associated virus-2 (rAAV2) under control of the chicken beta actin promoter/truncated CMV enhancer (CBA) was investigated for its ability to transduce primary cultures of rat brain neurons, microglia and astrocytes. This vector was highly effective in all three cell types in heparin-sensitive manners (astrocytes, microglia and neurons transduced by >98%, 75%, and 95%, respectively). However, astrocytes co-cultured with neurons were not transduced. rAAV2/CBA is an important new method for genetic manipulation of brain cells, though this may be modulated by interactions among cell types.

Branched-chain amino acids and neurotransmitter metabolism: expression of cytosolic branched-chain aminotransferase (BCATc) in the cerebellum and hippocampus.

In the brain, catabolism of the branched-chain amino acids (BCAAs) provides nitrogen for the synthesis of glutamate and glutamine. Glutamate is formed through transfer of an amino group from BCAA to alpha-ketoglutarate in reaction catalyzed by branched-chain aminotransferases (BCAT). There are two isozymes of BCAT: cytosolic BCATc, which is found in the nervous system, ovary, and placenta, and mitochondrial BCATm, which is found in all organs except rat liver. In cell culture systems, BCATc is found only in neurons and developing oligodendrocytes, whereas BCATm is the isoform in astroglia. In this study, we used immunohistochemistry to examine the distribution of BCATc in the rat brain, focusing on the well-known neural architecture of the cerebellum and hippocampus. We show that BCATc is expressed only in neurons in the adult rat brain. In glutamatergic neurons such as granule cells of the cerebellar cortex and of the dentate gyrus, BCATc is localized to axons and nerve terminals. In contrast, in GABAergic neurons such as cerebellar Purkinje cells and hippocampal pyramidal basket cells, BCATc is concentrated in cell bodies. A common function for BCATc in these neurotransmitter systems may be to modulate amounts of glutamate available either for release as neurotransmitter or for use as precursor for synthesis of GABA. Particularly striking in our findings is the strong expression of BCATc in the mossy fiber pathway of the hippocampal formation. This result is discussed in light of the effectiveness of the anticonvulsant drug gabapentin, which is a specific inhibitor of BCATc.

Dopamine D1 and D2 Receptor Co-activation Generates a Novel Phospholipase C-mediated Calcium Signal

Although dopamine D1 and D2 receptors belong to distinct subfamilies of dopamine receptors, several lines of evidence indicate that they are functionally linked. However, a mechanism for this linkage has not been elucidated. In this study, we demonstrate that agonist stimulation of co-expressed D1 and D2 receptors resulted in an increase of intracellular calcium levels via a signaling pathway not activated by either receptor alone or when only one of the co-expressed receptors was activated by a selective agonist. Calcium signaling by D1-D2 receptor co-activation was abolished following treatment with a phospholipase C inhibitor but not with pertussis toxin or inhibitors of protein kinase A or protein kinase C, indicating coupling to the G(q) pathway. We also show, by co-immunoprecipitation from rat brain and from cells co-expressing the receptors, that D1 and D2 receptors are part of the same heteromeric protein complex and, by immunohistochemistry, that these receptors are co-expressed and co-localized within neurons of human and rat brain. This demonstration that D1 and D2 receptors have a novel cellular function when co-activated in the same cell represents a significant step toward elucidating the mechanism of the functional link observed between these two receptors in brain.

Nicotinic receptor inhibition by Tetraponera ant alkaloids.

1. Tetraponerines are a group of alkaloids occurring in the venoms of ants belonging to the genus Tetraponera. Eight compounds had been isolated and their structures elucidated, but their mechanisms of action had not yet been reported. We have studied the actions of several of these tetraponerines on vertebrate neuromuscular, ganglionic, and brain nicotinic acetylcholine receptors (nAChRs) using a variety of techniques including muscle contracture, cultured cell functional assays, neuronal patch clamping, and radioligand binding methods. 2. Potency for inhibition of the frog muscle carbachol-elicited contracture increased as the carbon 9 side chain alkyl group was increased in length to 10-12 carbons, then decreased when the chain was 18-carbons long. Potency differences between T-7 and T-8, which differ only in the stereochemistry of the carbon pentyl side chain were rather small. Quaternization of either N atom in a T-8 analog bearing a 10-carbon length alkyl substituent did not greatly affect potency for inhibition of the muscle response; thus the ionized form is an active form of this tetraponerine. 3. T-7 inhibited the nicotine-stimulated efflux of 86Rb from cultured PC12 cells, which primarily express alpha3-beta4 ganglionic type nicotinic receptors. T-8 blockade of BTX-sensitive and insensitive neuronal nAChRs, as studied by patchclamp recordings from cultured rat brain neurons, was also consistent with a noncompetitive type of inhibition. 4. T-7 displaced binding of the nAChR ion channel binding ligand thienylcyclophenidyl (TCP), an analog of PCP, to Torpedo neuromuscular type receptors. The affinity of the TCP binding site for T-7 did not depend upon the desensitization state of the receptor. 5. We conclude that the tetraponerines act at a site on nAChRs different from the ACh binding site which is probably located within the ion channel.

RNA interference suggests a primary role for monoacylglycerol lipase in the degradation of the endocannabinoid 2-arachidonoylglycerol.

The endogenous cannabinoid 2-arachidonoylglycerol (2-AG) is produced by neurons and other cells in a stimulus-dependent manner and undergoes rapid biological inactivation through transport into cells and catalytic hydrolysis. The enzymatic pathways responsible for 2-AG degradation are only partially understood. We have shown previously that overexpression of monoacylglycerol lipase (MGL), a cytosolic serine hydrolase that cleaves 1- and 2-monoacylglycerols to fatty acid and glycerol, reduces stimulus-dependent 2-AG accumulation in primary cultures of rat brain neurons. We report here that RNA interference-mediated silencing of MGL expression greatly enhances 2-AG accumulation in HeLa cells. After stimulation with the calcium ionophore ionomycin, 2-AG levels in MGL-silenced cells were comparable with those found in cells in which 2-AG degradation had been blocked using methyl arachidonyl fluorophosphonate, a nonselective inhibitor of 2-AG hydrolysis. The results indicate that MGL plays an important role in the degradation of endogenous 2-AG in intact HeLa cells. Furthermore, immunodepletion experiments show that MGL accounts for at least 50% of the total 2-AG-hydrolyzing activity in soluble fractions of rat brain, suggesting that this enzyme also contributes to 2-AG deactivation in the central nervous system.

Cellular localization of TRPC3 channel in rat brain: preferential distribution to oligodendrocytes

In the present work we describe the cellular localization of TRPC3 in non-excitable cells as compared to the neurons in normal rat brain. We performed a double labeling study for TRPC3 and one of the following cell-specific markers: mouse anti-glial fibrillary acidic protein (GFAP) for astrocytes; mouse anti-RIP for oligodendrocytes, or mouse anti-OX42 for microglia, or mouse anti-NeUN for neuronal nuclei or mouse anti-tyrosine hydroxylase (TH) for detection of dopaminergic neurons of the substantia nigra. Our double label immunofluorescence study showed that that TRPC3 is mainly localized in oligodendrocytes. These result were confirmed by the electron microscopy study, which showed TRPC3 immunoreactivity in oligodendrocytes. Consistent with the evidence that calcium homeostasis is important to oligodendrocytes for development, myelination, and demyelination [Microsc. Res. Tech. 52 (2001) 672], we can speculate that the distribution of TRPC3 in oligodendrocytes plays a role in myelination and or demyelination processes.

Survival of trauma-injured neurons in rat brain by treatment with proline-rich peptide (PRP-1): an immunohistochemical study.

The objective of this immunohistochemical research was to reveal the distribution of a proline-rich peptide-1 (PRP-1) in various brain structures of intact and trauma-injured rats and to identify the mechanisms of promotion of neuronal recovery processes following PRP-1 treatment. PRP-1, produced by bovine hypothalamic magnocellular cells and consisting of 15 amino acid residues, is a fragment of neurophysin vasopressin associated glycoprotein isolated from bovine neurohypophysis neurosecretory granules. PRP-1-immunoreactivity (PRP-1-IR) was detected in the brain of intact rats in the neurons of paraventricular (PVN) and supraoptic (SON) nuclei in the hypothalamus, in almost all cell groups in the medulla oblongata, in Purkinje and some cerebellar nuclei cells, and in nerve fibers. At 3 weeks after hemisection of the spinal cord (SC) an asymmetry of PRP-1 localization in the PVN and SON was observed: no PRP-1-IR was exhibited at the affected sides of both nuclei. Daily intramuscular administration of PRP-1 for 3 weeks significantly increased the number of PRP-1-immunoreactive (PRP-1-Ir) varicose nerve fibers, and cells in PVN and SON and in cell groups of the limbic system and brain stem. Tanycytes in the median eminence and covering ependyma also demonstrated strong PRP-1-IR. PRP-1 treatment also activated neuropeptide Y-IR (NPY-IR) in nerve fibers and immunophilin fragment-IR (IphF-IR) in lymphocytes and nerve cells. A strong increase of PRP-1-IR was observed in the PVN and SON of SC-injured rats following the treatment with another PRP (PRP-3). Preliminary physiological data demonstrate that PRP-3 is more "aggressive" in the recovery processes than PRP-1. Based on the findings regarding PRP action on neurons survival, axons regeneration, and the number of IphF-Ir lymphocytes and NPY-Ir nerve fibers, PRP is suggested to act as a neuroprotector, functioning as a putative neurotransmitter and immunomodulator.

The presence of 3'-5' exonuclease activity in rat brain neurons and its role in template-driven extension of 3'-mismatched primers by DNA-polymerase beta in aging neurons.

A three-step reaction strategy has been developed to examine the mechanism of extension of a mismatched primer in an oligoduplex substrate by rat neuronal extracts and DNA polymerase beta. The results revealed that in the case of duplexes with a mismatch at 3'-end of primer, significant extension by DNA polymerase beta has taken place only after the removal of the mismatched base, thus indicating the presence of a proof reading 3'-5' exonuclease activity in neuronal extracts of all ages. A closer examination of the neuronal exonuclease activity revealed that bases are excised from the 3' end in a sequential and nonspecific manner, although initial excision of a mismatched base was slightly faster. Further, the excision efficiency is seen to decrease with the age of the animal but apparently does not go below a critical level so as to become a rate-limiting factor for the DNA-repair activity.

The Hippocampal Cholinergic Neurostimulating Peptide, the N-terminal Fragment of the Secreted Phosphatidylethanolamine-binding Protein, Possesses a New Biological Activity on Cardiac Physiology

Phosphatidylethanolamine-binding protein (PEBP), alternatively named Raf-1 kinase inhibitor protein, is the precursor of the hippocampal cholinergic neurostimulating peptide (HCNP) corresponding to its natural N-terminal fragment, previously described to be released by hippocampal neurons. PEBP is a soluble cytoplasmic protein, also associated with plasma and reticulum membranes of numerous cell types. In the present report, using biochemistry and cell biology techniques, we report for the first time the presence of PEBP in bovine chromaffin cell, a well described secretion model. We have examined its presence at the subcellular level and characterized this protein on both secretory granule membranes and intragranular matrix. In addition, its presence in bovine chromaffin cell and platelet exocytotic medium, as well as in serum, was reported showing that it is secreted. Like many other proteins that lack signal sequence, PEBP may be secreted through non-classic signal secretory mechanisms, which could be due to interactions with granule membrane lipids and lipid rafts. By two-dimensional liquid chromatography-tandem mass spectrometry, HCNP was detected among the intragranular matrix components. The observation that PEBP and HCNP were secreted with catecholamines into the circulation prompted us to investigate endocrine effects of this peptide on cardiovascular system. By using as bioassay an isolated and perfused frog (Rana esculenta) heart preparation, we show here that HCNP acts on the cardiac mechanical performance exerting a negative inotropism and counteracting the adrenergic stimulation of isoproterenol. All together, these data suggest that PEBP and HCNP might be considered as new endocrine factors involved in cardiac physiology.

Neuroprotective and Neurotrophic Actions of the Mood Stabilizer Lithium: Can It Be Used to Treat Neurodegenerative Diseases?

The mood stabilizing drug lithium has emerged as a robust neuroprotective agent in preventing apoptosis of neurons. Long-term treatment with lithium effectively protects primary cultures of rat brain neurons from glutamate-induced, NMDA receptor-mediated excitotoxicity. This neuroprotection is accompanied by an inhibition of NMDA-receptor-mediated calcium influx, upregulation of anti-apoptotic Bcl-2, downregulation of pro-apoptotic p53 and Bax, and activation of cell survival factors. Lithium treatment antagonizes glutamate-induced activation of c-Jun-N-terminal kinase (JNK), p38 kinase, and AP-1 binding, which has a major role in cytotoxicity, and suppresses glutamate-induced loss of phosphorylated cAMP responsive element binding protein (CREB). Lithium also induces the expression of brain-derived neurotrophic factor (BDNF) and subsequent activation TrkB, the receptor for BDNF, in cortical neurons. The activation of BDNF/TrkB signaling is essential for the neuroprotective effects of this drug. In addition, lithium stimulates the proliferation of neuroblasts in primary cultures of CNS neurons. Lithium also shows neuroprotective effects in rodent models of diseases. In a rat model of stroke, post-insult treatment with lithium or valproate, another mood stabilizer, at therapeutic doses markedly reduces brain infarction and neurological deficits. This neuroprotection is associated with suppression of caspase-3 activation and induction of chaperone proteins such as heat shock protein 70. In a rat model of Huntington's disease (HD) in which an excitotoxin is unilaterally infused into the striatum, both long- and short-term pretreatment with lithium reduces DNA damage, caspase-3 activation, and loss of striatal neurons. This neuroprotection is associated with upregulation of Bcl-2. Lithium also induces cell proliferation near the injury site with a concomitant loss of proliferating cells in the subventricular zone. Some of these proliferating cells display neuronal or astroglial phenotypes. These results corroborate our findings obtained in primary neuronal cultures. The neuroprotective and neurotrophic actions of lithium have profound clinical implications. In addition to its present use in bipolar patients, lithium could be used to treat acute brain injuries such as stroke and chronic progressive neurodegenerative diseases.

Effects of amyloid-beta on cholinergic and acetylcholinesterase-positive cells in cultured basal forebrain neurons of embryonic rat brain

The neurotoxic effects of amyloid-β 1–42 and amyloid-β 25–35 (Aβ) on cholinergic and acetylcholinesterase-positive neurons were investigated in primary cultures derived from embryonic 18-day-old rat basal forebrain. After various time intervals, the cultures were treated with 1, 5, 10 or 20 μM Aβ for different time periods. The cholinergic neurons and their axon terminals were revealed by vesicular acetylcholine transporter immunohistochemistry and the cholinoceptive cells by acetylcholinesterase histochemical staining. To assess the toxic effects of these Aβ peptides on the cholinergic neurons, image analysis was applied for quantitative determination of the numbers of axon varicosities/terminals and cells. The results demonstrate that, following treatment with 1 or 5 μM Aβ for 5, 10, 30, 60 or 120 min, no changes in vesicular acetylcholine transporter immunohistochemical staining were observed. However, after treatment for 30 min with 10 or 20 μM Aβ, the number of stained axon varicosities was reduced, and treatment for 2 h they had disappeared. In contrast, vesicular acetylcholine transporter-positivity could be seen in some of the neuronal perikarya even after 3 days after treatment. The acetylcholinesterase staining was homogeneously distributed in the control neurons. After Aβ treatment, the histochemical reaction end-product was detected in some of the neuronal perikarya or in the dendritic processes near to the soma. It is concluded that the neurotoxic effects of Aβ appear more rapidly in the cholinergic axon terminals than in the cholinergic and acetylcholinesterase-positive neuronal perikarya.

Single-walled Carbon Nanotubes Are a New Class of Ion Channel Blockers

Here we identify a novel class of biological membrane ion channel blockers called single-walled carbon nanotubes (SWNTs). SWNTs with diameter distributions peaked at approximately 0.9 and 1.3 nm, C60 fullerenes, multi wall nanotubes (MWNTs), and hyperfullerenes (nano-"onions") were synthesized by several techniques and applied to diverse channel types heterologously expressed in mammalian cells. External as-fabricated and purified SWNTs blocked K+ channel subunits in a dose-dependent manner. Blockage was dependent on the shape and dimensions of the nanoparticles used and did not require any electrochemical interaction. SWNTs were more effective than the spherical fullerenes and, for both, diameter was the determining factor. These findings postulate new uses for SWNTs in biological applications and provide unexpected insights into the current view of mechanisms governing the interaction of ion channels with blocking molecules.

A herpesvirus vector can transduce axotomized brain neurons

If gene therapy is to be used to promote axon regeneration after spinal cord injury, a suitable vector for transgene delivery must be obtained. Replication-defective herpes simplex virus (HSV) vectors are promising candidates. We have examined whether they can express a LacZ transgene in injured neurons of adult rat brain. We transected the medial forebrain bundle, injected replication-defective HSV/ LacZ vectors close to the lesion site, and looked for transgene expression at 2–14 days after the lesion. The vectors carried the LacZ transgene controlled either by the cytomegalovirus immediate-early promoter (vector CS5) or the HSV latency-associated promoter (vector CS1). CS5 transfected many cells near the lesion at 2 days, but did not give persistent expression at 5 days. CS1, in contrast, labeled many neurons in midbrain regions remote from the injection site at 5 days, and much of this expression remained at 12–14 days. The neurons of most interest were in the substantia nigra pars compacta and parabrachial nuclei, which were axotomized by the lesion. Vector-driven β-galactosidase expression was detected in neurons in both regions. These were confirmed as axotomized by double immunofluorescence for c-Jun. By 12–14 days, many substantia nigra neurons had disappeared but some transduced neurons remained; there was no net loss of transduced neurons from the parabrachial nuclei. These results show that an HSV vector is capable of transducing axotomized cells in the central nervous system and producing transgene expression in them for at least 2 weeks after injection.

KCC2 mediates NH4+ uptake in cultured rat brain neurons.

Elevated levels of NH4+ in the brain impair neuronal function. We studied the effects of NH4+ on postsynaptic inhibition of cultured rat brain neurons using whole cell recording under nominally HCO3- -free conditions. Application of NH4+ shifted the reversal potentials for spontaneous inhibitory postsynaptic currents and currents elicited by dendritic GABA applications in a positive direction because [Cl-]i increased. The positive shift of the reversal potentials of GABA-induced Cl- currents was equal on equimolar elevation of [NH4+]o or [K+]o, respectively. The NH4+-induced increase in [Cl-]i was reversed by an inhibitor of cation-anion cotransport, furosemide (0.1 mM), but not by bumetanide (0.01 mM) or by replacement of [Na+]o by Li+. We conclude that neuron-specific K-Cl cotransporter (KCC2) transports NH4+ similar to K+. Despite this fact, the small increase of [NH4+]o during metabolic encephalopathies will barely elevate [Cl-]i. However, an impairment of neuronal function may result because KCC2 provides a pathway to accumulate NH4+, and thereby, a continuous acid load to neurons.

Differential effects of natural polyphenols on neuronal survival in primary cultured central neurons against glutamate- and glucose deprivation-induced neuronal death

Neuronal injury in the central nervous system following ischemic insult is believed to result from glutamate toxicity and glucose deprivation. In this study, polyphenols isolated from Scutellaria baicalensis Georgi, including baicalin, baicalein, and wogonin, were investigated for their neuroprotective effects against glutamate/NMDA (Glu/NMDA) stimulation and glucose deprivation in primary cultured rat brain neurons. Cell death was accessed by lactate dehydrogenase (LDH) release assay for necrosis, and mitochondrial activity was accessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction activity assay. It was found that both baicalin and baicalein decreased LDH release of the cultured neurons after 24 h treatment, whereas wogonin profoundly increased LDH release after 2 h treatment and resulted in neuronal death after 24 h. Glu/NMDA treatment profoundly increased LDH release and moderately decreased MTT reduction activity in an NMDA receptor-dependent manner. Both baicalin and baicalein significantly reduced Glu/NMDA-increased LDH release, in which baicalein is much more potent than baicalin. Glu/NMDA-increased intracellular calcium was also significantly attenuated by baicalin and baicalein. Baicalin and baicalein did not affect glutamate receptor binding activity, but baicalein did moderately decrease Glu/NMDA-induced nitric oxide (NO) production. In the glucose deprivation (GD) study, baicalein but not baicalin showed significant protective effects on the GD-increased LDH release, without affecting the GD-induced NO production, in cultured rat brain neurons. These results suggest that baicalein is the most effective compound among three polyphenols tested in preventing neurotoxicity induced by both glutamate and GD, whereas baicalin was only effective in preventing glutamate toxicity. Wogonin might have a neurotoxic effect on the brain.