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The pathogenic mechanisms underlying idiopathic Parkinson's disease (PD) remain enigmatic. Recent findings suggest that inflammatory processes are associated with several neurodegenerative disorders, including PD. Enhanced expression of the proinflammatory cytokine, tumor necrosis factor (TNF)-alpha, has been found in association with glial cells in the substantia nigra of patients with PD. To determine the potential role for TNF-alpha in PD, we examined the effects of the 1-methyl-4-phenyl-1,2,3,4-tetrahydropyridine (MPTP), a dopaminergic neurotoxin that mimics some of the key features associated with PD, using transgenic mice lacking TNF receptors. Administration of MPTP to wild-type (+/+) mice resulted in a time-dependent expression of TNF-alpha in striatum, which preceded the loss of dopaminergic markers and reactive gliosis. In contrast, transgenic mice carrying homozygous mutant alleles for both the TNF receptors (TNFR-DKO), but not the individual receptors, were completely protected against the dopaminergic neurotoxicity of MPTP. The data indicate that the proinflammatory cytokine TNF-alpha is an obligatory component of dopaminergic neurodegeneration. Moreover, because TNF-alpha is synthesized predominantly by microglia and astrocytes, our findings implicate the participation of glial cells in MPTP-induced neurotoxicity. Similar mechanisms may underlie the etiopathogenesis of PD.

Disruptions in homeostasis (ie, stress) place demands on the body that are met by the activation of 2 systems, the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic nervous system (SNS). Stressor-induced activation of the HPA axis and the SNS results in a series of neural and endocrine adaptations known as the "stress response" or "stress cascade." The stress cascade is responsible for allowing the body to make the necessary physiological and metabolic changes required to cope with the demands of a homeostatic challenge. Here we discuss the key elements of the HPA axis and the neuroendocrine response to stress. A challenge to homeostasis (a stressor) initiates the release of corticotropin-releasing hormone (CRH) from the hypothalamus, which in turn results in release of adrenocortiotropin hormone (ACTH) into general circulation. ACTH then acts on the adrenal cortex resulting in release of a species-specific glucocorticoid into blood. Glucocorticoids act in a negative feedback fashion to terminate the release of CRH. The body strives to maintain glucocorticoid levels within certain boundaries and interference at any level of the axis will influence the other components via feedback loops. Over- or underproduction of cortisol can result in the devastating diseases of Cushing's and Addison's, respectively, but less severe dysregulation of the HPA axis can still have adverse health consequences. These include the deposition of visceral fat as well as cardiovascular disease (eg, atherosclerosis). Thus, chronic stress with its physical and psychological ramifications remains a persistent clinical problem for which new pharmacological treatment strategies are aggressively sought. To date, treatments have been based on the existing knowledge concerning the brain areas and neurobiological substrates that subserve the stress response. Thus, the CRH blocker, antalarmin, is being investigated as a treatment for chronic stress because it prevents CRH from having its ultimate effect-a protracted release of glucocorticoids. New therapeutic strategies will depend on the discovery of novel therapeutic targets at the cellular and intracellular level. Advances in molecular biology provide the tools and new opportunities for identifying these therapeutic targets.

The mechanism of 3,4-methylenedioxymethamphetamine (d-MDMA)-induced neurotoxicity may involve formation of toxic radical species. Endogenous defenses against toxic radical species include tissue stores of vitamin E, and thiols. We examined whether vitamin E deficiency could alter d-MDMA-induced neurotoxicity by administration of the drug to animals with diet induced vitamin E deficiency. Brain vitamin E levels in deficient mice were reduced 75% compared to sufficient animals. Animals received d-MDMA 5 or 10 mg/kg or saline (delivered every 2 hx4, s.c.). Diet slightly altered d-MDMA-induced temperature modulation. In brain, MDMA treatment reduced vitamin E, total antioxidant reserve and protein thiols 72 h after the first dose. In liver, MDMA treatment reduced glutathione and total antioxidant reserve at the same time point. The vitamin E-deficient group, treated with the low dose of d-MDMA, exhibited neurotoxic responses, including reduced striatal dopamine (47%) and elevated GFAP protein (3-fold): while the sufficient diet group was not altered. The higher d-MDMA dose caused neurotoxic responses in both diet groups. Liver toxicity was determined by histopathologic examination. d-MDMA caused hepatic necrosis that was more severe in vitamin E deficient than sufficient mice. These data indicate that (1) d-MDMA administration reduces antioxidant measures at a time coincident with d-MDMA-induced neuronal damage and (2) vitamin E deficiency increases susceptibility to d-MDMA-induced neurotoxicity and hepatic necrosis.

Chronic stress and extended periods of elevated circulating glucocorticoids have been reported to exacerbate excitotoxicity-induced hippocampal neuronal injury in rat. Despite continued interest in the effects of protracted exposure to stress or glucocorticoids, there has been little examination of how other types of neurotoxicity may be exacerbated or blocked, by stress. Here we examined the effects of chronic supraphysiologic levels of corticosterone on D-3,4-methylenedioxymethamphetamine (D-MDMA)-induced striatal dopaminergic neurotoxicity in the female C57BL/6J mouse. Corticosterone (5 mg, 15 mg or placebo) pellets were implanted to continuously elevate circulating glucocorticoids and create a model of the ultimate effect of chronic activation of the hypothalamic-pituitary-adrenal axis. After 7 days, a neurotoxic regimen of D-MDMA was administered (20 mg/kg s.c. every 2 hx4); thymus, spleen, striatum and hippocampus were collected 72 h later. Significant involution of thymus and spleen confirmed the bioavailability of the corticosterone at both dosages. D-MDMA increased the striatal levels of the astrocyte-localized protein glial fibrillary acidic protein (GFAP, a marker of gliosis); both dosages of corticosterone exacerbated this increase but only the 15 mg pellet exacerbated the decrease in tyrosine hydroxylase protein. Corticosterone alone or in combination with D-MDMA produced no neural injury in hippocampus, as measured by GFAP. Our work indicates corticosterone was able to increase the vulnerability of the striatum, but not the hippocampus to D-MDMA. An examination of other mouse strains and models of neurotoxic injury would be useful in determining the general validity of the glucocorticoid neuroendangerment hypothesis.

Doubly transgenic mice expressing both a mutated amyloid precursor protein and a mutated presenilin-1 protein accumulate A(beta) deposits as they age. The early A(beta) deposits were found to be primarily composed of fibrillar A(beta) and resembled compact amyloid plaques. As the mice aged, nonfibrillar A(beta) deposits increased in number and spread to regions not typically associated with amyloid plaques in Alzheimer's disease. The fibrillar, amyloid-containing deposits remained restricted to cortical and hippocampal structures and did not increase substantially beyond the 12-month time point. Even at early time points, the fibrillar deposits were associated with dystrophic neurites and activated astrocytes expressing elevated levels of glial fibrillary acidic protein. Microglia similarly demonstrated increased staining for complement receptor-3 in the vicinity of A(beta) deposits at early time points. However, when MHC-II staining was used to assess the degree of microglial activation, full activation was not detected until mice were 12 months or older. Overall, the regional pattern of A(beta) staining resembles that found in Alzheimer disease; however, a progression from diffuse A(beta) to more compact amyloid deposits is not observed in the mouse model. It is noted that the activation of microglia at 12 months is coincident with the apparent stabilization of fibrillar A(beta) deposits, raising the possibility that activated microglia might clear fibrillar A(beta) deposits at a rate similar to their rate of formation, thereby establishing a relatively steady-state level of amyloid-containing deposits.

Toxicant-induced injury of the adult or developing central nervous system results in hypertrophy of astrocytes at the site of injury. The hallmark of this response is enhanced expression of the major intermediate filament protein of astrocytes, glial fibrillary acid protein (GFAP). A protocol is provided for assaying GFAP levels in detergent homogenates of brain tissue using a sandwich ELISA.

Astrogliosis, a cellular response characterized by astrocytic hypertrophy and accumulation of GFAP, is a hallmark of all types of central nervous system (CNS) injuries. Potential signaling mechanisms driving the conversion of astrocytes into "reactive" phenotypes differ with respect to the injury models employed and can be complicated by factors such as disruption of the blood-brain barrier (BBB). As denervation tools, neurotoxicants have the advantage of selective targeting of brain regions and cell types, often with sparing of the BBB. Previously, we found that neuroinflammation and activation of the JAK2-STAT3 pathway in astrocytes precedes up regulation of GFAP in the MPTP mouse model of dopaminergic neurotoxicity. Here we show that multiple mechanistically distinct mouse models of neurotoxicity (MPTP, AMP, METH, MDA, MDMA, KA, TMT) engender the same neuroinflammatory and STAT3 activation responses in specific regions of the brain targeted by each neurotoxicant. The STAT3 effects seen for TMT in the mouse could be generalized to the rat, demonstrating cross-species validity for STAT3 activation. Pharmacological antagonists of the neurotoxic effects blocked neuroinflammatory responses, pSTAT3tyr705 and GFAP induction, indicating that damage to neuronal targets instigated astrogliosis. Selective deletion of STAT3 from astrocytes in STAT3 conditional knockout mice markedly attenuated MPTP-induced astrogliosis. Monitoring STAT3 translocation in GFAP-positive cells indicated that effects of MPTP, METH and KA on pSTAT3tyr705 were localized to astrocytes. These findings strongly implicate the STAT3 pathway in astrocytes as a broadly triggered signaling pathway for astrogliosis. We also observed, however, that the acute neuroinflammatory response to the known inflammogen, LPS, can activate STAT3 in CNS tissue without inducing classical signs of astrogliosis. Thus, acute phase neuroinflammatory responses and neurotoxicity-induced astrogliosis both signal through STAT3 but appear to do so through different modules, perhaps localized to different cell types.

Body temperature profiles observed during methamphetamine (METH) exposure are known to affect dopamine and tyrosine hydroxylase (TH) levels in the striatum of mice; hyperthermia potentiates depletion while hypothermia is protective against depletions. In the current study, the doses of METH were sufficiently great that significant dopamine and TH depletions occurred even though hypothermia occurred. Four doses, administered at 2 h intervals, of 15 mg/kg (4×15 mg/kg) d -METH significantly decreased TH and dopamine levels to 50% of control in mice becoming hypothermic during dosing in a 13°C environment. Phenobarbital or dizocilpine during METH exposure blocked the depletions while diazepam did not. Phenobarbital and dizocilpine did not block depletions by altering the hypothermic profiles from that observed during METH only exposure. Here we show that phenobarbital and dizocilpine can block measures of METH neurotoxicity by non-thermoregulatory mechanisms.

Enhanced expression of glial fibrillary acidic protein (GFAP) is known to be associated with toxicant-induced gliosis, a homotypic response of the central nervous system to neural injury. A variety of neurochemical and neurophysiological effects have been observed in experimental animals exposed to white spirit, but a linkage of such effects to neural damage has not been established. Here we evaluated the regional levels of GFAP to assess potential sites of CNS damage in the rat, following exposure to dearomatized and aromatic white spirit. Samples from rats exposed to dearomatized white spirit were assayed for GFAP levels in the United States and Denmark. The results were remarkably similar between countries. Small region-dependent increases and decreases in GFAP were observed with the cerebellum showing the most consistent effects (increases). In contrast, samples from rats exposed to aromatic white spirit showed large (as much as 150% of control) increases in regional levels of GFAP; again, the cerebellum showed the most consistent effects. The data are indicative of an aromatic white-spirit-induced astrogliosis in several regions of the rat CNS and suggest that chronic exposure to this solvent may be associated with underlying neural damage.

Reactive gliosis is the most prominent response to diverse forms of central nervous system (CNS) injury. The signaling events that mediate this characteristic response to neural injury are under intense investigation. Several studies have demonstrated the activation of phosphoproteins within the mitogen-activated protein kinase (MAPK) and Janus kinase (JAK) pathways following neural insult. These signaling pathways may be involved or responsible for the glial response following injury, by virtue of their ability to phosphorylate and dynamically regulate the activity of various transcription factors. This study sought to delineate, in vivo, the relative contribution of MAPK- and JAK-signaling components to reactive gliosis as measured by induction of glial-fibrillary acidic protein (GFAP), following chemical-induced neural damage. At time points (6, 24, and 48 h) following methamphetamine (METH, 10 mg/kg x 4, s.c.) administration, female C57BL/6J mice were sacrificed by focused microwave irradiation, a technique that preserves steady-state phosphorylation. Striatal (target) and nontarget (hippocampus) homogenates were assayed for METH-induced changes in markers of dopamine (DA) neuron integrity as well as differences in the levels of activated phosphoproteins. GFAP upregulation occurred as early as 6 h, reaching a threefold induction 48 h following METH exposure. Neurotoxicant-induced reductions in striatal levels of DA and tyrosine hydroxylase (TH) paralleled the temporal profile of GFAP induction. Blots of striatal homogenates, probed with phosphorylation-state specific antibodies, demonstrated significant changes in activated forms of extracellular-regulated kinase 1/2 (ERK 1/2), c-jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), MAPK/ERK kinase (MEK1/2), 70-kDa ribosomal S6 kinase (p70 S6), cAMP responsive element binding protein (CREB), and signal transducer and activator of transcription 3 (STAT3). MAPK-related phosphoproteins exhibited an activation profile that peaked at 6 h, remained significantly increased at 24, and fell to baseline levels 48 h following neurotoxicant treatment. The ribosomal S6 kinase was enhanced over 60% for all time points examined. Immunoreactivity profiles for the transcription factors CREB and STAT3 indicated maximal increases in phosphorylation occurring at 24 h, and measuring greater than 2- or 17-fold, respectively. Specific signaling events were found to occur with a time course suggestive of their involvement in the gliotic response. The toxicant-induced activation of these growth-associated signaling cascades suggests that these pathways could be obligatory for the triggering and/or persistence of reactive gliosis and may therefore serve as potential targets for modulation of glial response to neural damage.

DARPP-32, a dopamine- and adenosine 3′,5′-monophosphate (cAMP)–regulated phosphoprotein (32 kilodaltons in size), is an obligate intermediate in progesterone (P)–facilitated sexual receptivity in female rats and mice. The facilitative effect of P on sexual receptivity in female rats was blocked by antisense oligonucleotides to DARPP-32. Homozygous mice carrying a null mutation for the DARPP-32 gene exhibited minimal levels of P-facilitated sexual receptivity when compared to their wild-type littermates. P significantly increased hypothalamic cAMP levels and cAMP-dependent protein kinase activity. These increases were not inhibited by a D 1 subclass dopamine receptor antagonist. P also enhanced phosphorylation of DARPP-32 on threonine 34 in the hypothalamus of mice. DARPP-32 activation is thus an obligatory step in progestin receptor regulation of sexual receptivity in rats and mice.

The distribution and levels of glial fibrillary acidic protein (GFAP) were determined in the adult rat hypothalamus following axotomy of serotonin (5-HT) neurons. Seven days after unilateral intrahypothalamic injection of the 5-HT neurotoxin, 5,7-dihydroxytryptamine, there was a marked increase in the number of GFAP-labelled astrocytes in the ipsilateral hypothalamus of 5,7-DHT-treated as compared to sham-treated rats. In addition, levels of GFAP were significantly increased 7 days after 5,7-DHT injection.

Estimation of the concentration of a specific protein in a biological sample often is obtained by analysis of immunoblots. We used this technique to estimate the concentration of three proteins present in homogenates of brain: glial fibrillary acidic protein (GFAP), myelin basic protein (MBP), and synapsin I. Homogenates prepared from rat brains known to contain more than 6-fold increases in GFAP, based on a GFAP enzyme-linked immunosorbent assay (ELISA), showed only small relative increases in this protein when the same samples were subjected to immunoblot analysis with polyclonal or monoclonal anti-GFAP; quantification was based on PhosphorImager analysis of [(125)I] protein A bound to the antibodies. Estimates of GFAP in the GFAP-enriched samples approached the expected 6-fold increase when the total protein load per gel lane was reduced from 30 to 1 microgram. Pure GFAP run as standard was not affected by 10-fold increases in protein load, but spiking brain homogenates with pure GFAP "quenched" the values obtained for the standard run alone. Examination of the quenching potential of pure brain tubulin, a protein that nearly comigrates with GFAP on SDS gels, showed that it may be one component of brain homogenates that contributes to masking of immunodetection of GFAP. The effect of total brain homogenate proteins on the signal obtained for a specific protein was not limited to GFAP; similar effects were observed for MBP and synapsin I. The data indicate that estimates of the concentration of a specific protein, whether as a function of its relative amount in a given protein mixture or its relative amount in one mixture compared to another, are influenced by other homogenate proteins present in the mixture.

Female mice of the AKR/J (AK) strain were fed a control diet (Purina Rodent Laboratory Chow) or a lipotrope-supplemented diet (Purina Rodent Chow plus 2% D,L-methionine and 1% choline chloride) beginning at 1 day after weaning. Food consumption and weight gain were found to be the same in both groups of animals. Mice of this inbred strain spontaneously develop thymic lymphoma, with close to 100% mortality expected by 12-13 months of age. Two separate experiments were carried out with 50 mice per group in one, and 40 mice per group in the other. The slopes of the survival curves for the animals in the control group and supplemented group of mice diverged after the animals reached 6.5 months of age. In both experiments, 20% of the mice receiving supplemented diet were still alive at 1 year, while 3% in one experiment and 8% in the other experiment survived in the control groups. Each experiment was terminated when the animals reached 13 months of age. At that time the survival rate of the controls was 2 and 4%, and survival in the groups of mice receiving supplemented diet was 14 and 18%. Necropsy revealed that the animals in both groups had advanced malignant lymphoma. Our results demonstrate that intake of a chow diet that is supplemented with moderate quantities of methionine and choline results in enhanced survival of spontaneously leukemic AK mice, in comparison with animals of this strain fed the same diet without supplements of choline and methionine.

Testosterone reversed the age-related increase in glial fibrillary acidic protein (GFAP) in the male rat cerebellum, a brain region not generally associated with gonadal steroid hormone sensitivity. This supports the hypothesis that a decrease in circulating testosterone contributes to age-related increase in GFAP. These data also suggest that reductions in circulating gonadal steroids during aging could render the brain more susceptible to neurodegeneration and that hormone replacement therapy might have value in neurodegenerative disease intervention.

Enhanced expression of glial fibrillary acidic protein (GFAP) has been shown to be associated with gliosis, a generic response of the CNS to neural injury. The effects of aluminum (Al) on regional GFAP concentrations were evaluated to determine potential sites of Al-induced neural injury. Rabbits received 20 Al (100 mumol/kg) or sodium lactate injections over 1 month. Frontal cortical GFAP increased (approximately twofold above control) in Al-loaded rabbits; whereas hippocampal and cerebellar GFAP concentrations were not affected. Frontal cortical synaptophysin, neurofilament 68, and myelin basic protein concentrations were then examined in an attempt to determine cell-specific targets of Al neurotoxicity. These proteins were not affected by Al. The ability of chelators to influence brain Al concentrations and the Al effect on GFAP were assessed. Desferrioxamine (DFO) and six 3-hydroxypyridin-4-ones (CPs) were given 12 times, over 1 month, to Al-loaded rabbits. CP24 significantly reduced brain Al. CP93, CP52, and CP24 significantly reduced frontal cortical GFAP. The data suggest an Al-induced gliosis consequent to subtle damage in the frontal cortex and a protective role of some chelators against this CNS injury.

Parenterally administered domoic acid, a structural analog of the excitatory amino acids glutamic acid and kainic acid, has specific effects on brain histology in rats, as measured usung different anatomic markers. Domoic acid-induced convulsions affects limbic structures such as hippocampus and entorhinal cortex, and different anatomic markers can detect these neurotoxic effects to varying degrees. Here we report effects of domoic acid administration on quantitative indicators of brain metabolism and gliosis. Domoic acid, 2.25 mg/kg i.p., caused stereotyped behavior and convulsions in approximately 60% of rats which received it. Six to eight days after domoic acid or vehicle administration, the animals were processed to measure regional brain incorporation of the long-chain fatty acids [1- 14 C]arachidonic acid ([ 14 C]AA) and [9,10- 3 H]palmitic acid ([ 3 H]PA), or regional cerebral glucose utilization ( rCMR glc ) using 2-[1- 14 C]deoxy- d -glucose, by quantitative autoradiography. Others rats were processed to measure brain glial fibrillary acidic protein (GFAP) by enzyme-linked immunosorbent assay. Domoic acid increased GFAP in the anterior portion of cerebral cortex, the caudate putamen and thalamus compared with vehicle. However, in rats that convulsed after domoic acid GFAP was significantly increased throughout the cerebral cortex, as well as in the hippocampus, septum, caudate putamen, and thalamus. Domoic acid, in the absence of convulsions, decreased relative [ 14 C]AA incorporation in the claustrum and pyramidal cell layer of the hippocampus compared with vehicle-injected controls. In the presence of convulsions, relative [ 14 C]AA incorporation was decreased in hippocampus regions CA1 and CA2. Uptake of [ 3 H]PA into brain was unaffected. Relative rCMR glc decreased in entorhinal cortex following domoic acid administration with or without convulsions. These results suggest that acute domoic acid exposure affects discrete brain circuits by inducing convulsions, and that domoic acid-induced convulsions cause chronic effects on brain function that are reflected in altered fatty acid metabolism and gliosis. © 1977 Elsevier Science B.V. All rights reserved.

Substantial evidence suggests that stress can alter the general toxicological properties of the substituted amphetamines (AMPs) as well as their psychostimulant properties. Research concerning the interactions between stress and the neurotoxicity associated with the AMPs is, however, limited. Our previous work demonstrated that a variety of AMPs, including d-METH, d-MDA, d-MDMA but not d-FEN are able to damage dopaminergic elements of the striatum as shown by decreases in dopamine and tyrosine hydroxylase. The neurotoxic capabilities of these AMPs appear linked to their hyperpyrexic actions as diverse manipulations able to block AMP-induced hyperthermia are also neuroprotective. Surprising, since stress usually potentiates the actions of the AMPs, it is our finding that restraint, a commonly used stressor, is protective against the injurious actions of all neurotoxic AMPs evaluated to date. In the mouse restraint acts to elevate blood levels of corticosterone (CORT) by activating the hypothalamic-pituitary-adrenal (HPA) axis as well as inducing a profound hypothermia. The role CORT may play in the neuroprotective actions of restraint, if any, is unknown. Here, data is presented showing the impact of several HPA axis manipulations, including restraint, supplementation with CORT in the drinking water and removal of CORT by adrenalectomy (ADX) on the striatal dopaminergic neurotoxicity of d-AMP. As strain is known to be a powerful determinant of the actions of stress an essential element of these experiments was the evaluation of both an inbred, C57BL/6J and outbred, CD-1, mouse strain. Exposure to d-AMP caused hyperthermia and substantial striatal dopaminergic neurotoxicity in both strains suggesting that an elevation in body temperature is as important a component of the neurotoxicity of d-AMP, as it is of the other neurotoxic AMPs. Restraint was equally effective in both strains and completely blocked the hyperthermia and striatal neurotoxicity induced by d-AMP. CORT supplementation, evaluated in only the C57BL/6J mouse at dosages not capable of involuting either the thymus or the spleen, did not alter d-AMP-induced neurotoxicity. Although the immune system organs of the two strains responded differentially to the removal of CORT, ADX provided equivalent partial protection against the loss of dopaminergic elements in striatum for both strains. Adrenal status clearly affects d-AMP neurotoxicity but the interaction is complex. Future work should examine the roles of the cortical and medullary components of the adrenal gland in the neuroprotective actions of ADX. A precise assessment of the role of circulating CORT In the neurotoxicity of the AMPs will require additional work in which a wider range of CORT dosages, including those capable of involuting thymus and spleen, are evaluated.

We have investigated the response of astrocytes and microglia to trimethyl tin intoxication in the septum, hippocampus, olfactory bulb, and pyriform cortex of the rat. Microglia were studied qualitatively using lectin histochemistry, and astrocytes were examined both qualitatively with immunohistochemistry, and quantitatively using an immunoassay for glial fibrillary acidic protein. Our results show that activated microglia first appeared 2 days after trimethyl tin intoxication in the lateral septum and hippocampus. Four days after trimethyl tin intoxication, the same regions revealed a most intense microglial reaction characterized by microglial hypertrophy and the formation of phagocytic clusters. By day 7, microglial activation in the septum and hippocampus had lessened, suggesting that the cells were reverting to the resting phenotype. The microglial response in the pyriform cortex and olfactory bulb, while being later in onset than in the septum and hippocampus, showed a similar progression of microglial changes reaching maximal intensity 7 days after trimethyl tin intoxication. Significant increases in the expression of glial fibrillary acidic protein were observed in all regions examined and typically occurred after microglial activation was already underway. We conclude that microglial and astroglial reactions which occur in response to trimethyl tin-induced neuronal necrosis are separated in time, with microglial activation preceding astrogliosis. In addition, our study stresses the importance of microglia as an endogenous source of CNS macrophages, and illustrates the merit of histochemical analysis with microglial markers for the early delineation of neurotoxicant-induced brain damage.

Publisher Summary Diverse types of injury to the central nervous system result in hypertrophy of astrocytes, called reactive gliosis. The hallmark of this gliosis is the enhanced expression of the major intermediate filament protein of astrocytes, glial fibrillary acidic protein (GFAP). These morphological observations suggest that GFAP may be a useful biochemical indicator of neurotoxicity. This chapter focuses on studies that investigate this possibility by administering prototype neurotoxicants to experimental animals and then assessing the effects of these agents on the tissue content of GFAP by immunoassay. The assays of GFAP reveal dose-, time-, and region-dependent patterns of neural injury, often at toxicant dosages below those that result in light microscopic evidence of cell loss or damage. In rodent studies, measurement of GFAP has proven useful for distinguishing neurotoxic amphetamines, such as methamphetamine and methylenedioxymethamphetamine (MDMA), from non-neurotoxic non-amphetaminic anorectic agents, such as dexfenfluramine. The major conclusions that emerged from studies show that assays of GFAP provide a sensitive means for assessing neurotoxicity; increase in GFAP are specific and can differentiate long-term neurochemical changesfrom neurotoxic effects. In summary, assays of GFAP represent one approach for neurotoxicity assessment that can be used to differentiate pharmacological from neurotoxicological responses.

Amphetamines (AMPs) can cause long-term depletions in striatal dopamine (DA) and serotonin (5-HT), and these decrements are often accepted as prima facie evidence of AMP-induced damage to the dopaminergic and serotonergic projections to striatum. Rarely are indices linked to neural damage used to evaluate the neurotoxicity of the AMPs. Here, we determined the potential neurotoxic effects of two substituted AMPs, d-methylenedioxymethamphetamine (d-MDMA) and d-fenfluramine (d-FEN) in group-housed female C57BL6/J mice. Astrogliosis, assessed by quantification of glial fibrillary acidic protein (GFAP), was the main indicator of d-MDMA-induced neural damage. Assays of tyrosine hydroxylase (TH), DA, and 5-HT were used to determine effects on DA and 5-HT systems. Since AMPs are noted for both their stimulatory and hyperthermia-inducing properties, activity, as well as core temperature, was monitored in several experiments. To extend the generality of our findings, these same end points were examined in singly housed female C57bL6/J mice and in group-housed male C57BL6/J or female B6C3F1 mice after treatment with d-MDMA. Mice received either d-MDMA (20 mg/kg) (singly housed mice received dosages of 20, 30, or 40 mg/kg) or d-FEN (25 mg/kg) every 2 h for a total of four sc injections. d-MDMA caused hyperthermia, whereas d-FEN induced hypothermia. d-MDMA cause a large (300%) increase in striatal GFAP that resolved by 3 wk and a 50-75% decrease in TH and DA that was still apparent at 3 wk, d-FEN did not affect any parameters in striatum. d-MDMA is a striatal dopaminergic neurotoxicant in both male and female C57BL6/mice, as evidenced by astrogliosis and depletions of DA in this area in both sexes. The greater lethality to males suggests they may be more sensitive, at least to the general toxicity of d-MDMA, that females. d-MDMA (20 mg/kg) induced the same degree of damage whether mice were housed singly or in groups. Higher dosages in singly housed mice induced greater lethality, but not greater neurotoxicity. d-MDMA was also effective in inducing striatal damage in mice of the B6C3F1 strain. Significant increases in activity were induced by d-MDMA, and these increases were not blocked by pretreatment with MK-801, despite the profound lowering of body temperature induced by this combination. A lowering of body temperature, whether by a 15 degree C ambient temperature (approx 2 degree drop), pretreatment with MK-801 (1.0 mg/kg prior to the first and third d-MDMA injections; approx 5-6 degrees C drop) or restraint (approx 5-6 degrees C drop) was effective in blocking the neurotoxicity of d-MDMA in both C57BL6/J and B6C3F1. The stimulatory effects of d-MDMA appeared to have little impact on the neurotoxicity induced by d-MDMA or the protection conferred by MK-801. These data suggest that in the mouse, the neurotoxic effects of d-MDMA, and most likly other AMPs, are linked to an effect on body temperature.

An altered form of an esterase has been implicated in the development of neurotoxicity after exposure to organophosphates. Mice deficient in this enzyme should be less susceptible to toxicity, but the opposite turns out to be the case.

Mechanical injury to the brain results in enhanced immunostaining for glial fibrillary acidic protein (GFAP) that is markedly inhibited by difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase. In the current study, systemic exposure of mice to the dopaminergic neurotoxicant, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), also increased GFAP but, unlike mechanical injury, this increase was not prevented by DFMO pretreatment. These results indicate that de novo polyamine biosynthesis is not obligatory for the MPTP-induced increase in GFAP. MPTP administration, unlike mechanical injury, does not disrupt the blood-brain barrier; thus, a role for polyamine biosynthesis in the astrocyte response to injury may be restricted to insults involving a compromised blood-brain barrier.

Administration of pargyline or gamma-butyrolactone enhanced immunostaining of tyrosine hydroxylase immunoreactive axons in the striatum. The qualitative characteristics of the enhancement were compound dependent and the enhancement was not associated with an increase in the amount of tyrosine hydroxylase within the striatum. These results demonstrate the pharmacological enhancement of immunostaining for neurotransmitter synthetic enzymes in the absence of increased synthesis.

Detailed protocols are presented for assaying glial fibrillary acidic protein (GFAP), an astrocyte localized protein which serves as a quantitative marker of toxicant-induced injury to the central nervous system. Two different solid-phase assay procedures are described: 1) a nitrocellulose based slot-immunobinding assay and 2) a novel microtiter plate based sandwich ELISA. The performance of both assays was assessed by measuring the content of GFAP in homogenates of specific regions of the rat brain and in homogenates of brain regions damaged by the prototype neurotoxicants, trimethyltin (TMT) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Both procedures gave similar results that were consistent with previously published observations. By comparing the simplicity, cost effectiveness, safety and speed of the two methods, it appears likely that the sandwich ELISA has several advantages over slot-immunobinding assays.

The role of aging in the expression of the astrocyte protein, glial fibrillary acidic protein (GFAP), was examined. In both mice and rats the concentration of GFAP increased throughout the brain as a function of aging. The largest increase (2-fold) was observed in striatum for both species. The neuron-specific proteins, synapsin I and neurofilament-200 (Mr 200 kilodaltons), were not altered by aging in any region of the mouse or rat brain. Brains of aged rats, but not mice, showed a decrease in beta-tubulin. The data suggest that astrocytic hypertrophy observed with aging involves an accumulation of glial filaments.

The brain is a dynamic and changing organ in which synapses, dendrites and the neurochemicals of synaptic neurotransmission are continually being renewed and remodeled during the entire lifespan of an individual. Gene activity, controlled by environmental signals and mediated by circulating hormones, is fundamental to this plasticity (1). Our understanding of these relationships has arisen in part from studies that have identified and characterized the receptor sites for adrenal, gonadal and thyroid hormones in the brain (2). Together with ongoing advances in many aspects of neuroscience, and in our understanding of how steroid and thyroid hormone receptors regulate gene expression (3,4), this information has stimulated a new field of investigation into how the brain changes in response to circulating hormones.

The effects of a single oral dose of 750 mg/kg tri-o-cresyl phosphate (TOCP) on the endogenous phosphorylation of specific brain proteins were assessed in male adult chickens following the development of delayed neurotoxicity. Phosphorylation of crude synaptosomal (P2) membrane and synaptosomal cytosolic proteins was assayed in vitro by using [gamma-32P]ATP as phosphate donor. Following resolution of brain proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis, specific protein phosphorylation was detected by autoradiography and quantified by microdensitometry. TOCP administration enhanced the phosphorylation of both cytosolic (Mr 65,000 and 55,000) and membrane (20,000) proteins by as much as 146% and 200%, respectively.

Calcium stimulated the phosphorylation of several specific synaptosomal cytosolic proteins. The effects of calcium were both concentration and time dependent and were most apparent for proteins with molecular weights of 50,000, 55,000, and 60,000. Exogenous calcium (1.0-100 microM) enhanced the net incorporation of phosphate into protein by as much as 23-fold. In the absence of added calcium, the calcium chelator [ethylenebis(oxyethylenenitriolo)]tetraacetic acid did not lower the phosphorylation of any protein below control levels. The antipsychotic, fluphenazine (1.0-100 microM), caused a concentration-dependent decrease in calcium-stimulated protein phosphorylation. When the heat-stable calcium-binding protein, calmodulin, was removed from synaptosomal cytosol by affinity chromatography on fluphenazine-Sepharose, calcium-stimulated protein phosphorylation was abolished. Responsiveness to calcium could be restored by the addition of calmodulin to the phosphorylation assay. These results indicate that calcium-dependent protein kinases are of major importance in regulating the phosphorylation of specific cytosolic proteins in neuronal tissue. Furthermore, it would appear that one of the three substrates under investigation is specific to synaptosomal cytosol whereas the other two are present in both the cytosol and membrane fractions.

Trimethyltin is a neurotoxicant which produces a distinct pattern of neuronal cell death following peripheral administration of a single dose (8 mg/kg, i.p.) in rats. The cupric-silver degeneration stain was used to produce an atlas documenting the distribution and time course of trimethyltin-induced neuronal damage in adult, male Long-Evans rats. Animals were examined at survival times of 1, 2, 3, 4, 5, 7, 10 and 18 days after intoxication. The earliest degeneration was observed at day 1 in the intermediate and ventral divisions of the lateral septal nucleus, followed by development of degeneration on days 2-4 in neuron populations including the septohippocampal nucleus, septohypothalamic nucleus, anterior olfactory nucleus, bed nucleus of the stria terminalis, endopiriform nucleus, parafascicular nucleus, superior colliculus, interstitial nucleus of the posterior commissure, inferior colliculus, pontine nuclei, raphe nuclei, pars caudalis of the spinal trigeminal nucleus, the caudal aspect of nucleus tractus solitarius, dorsal vagal motor nucleus, granule cells in the dentate gyrus, pyramidal cells in CA fields of the hippocampus, and of neurons in the subiculum, pyriform cortex, entorhinal cortex and neocortex (mainly layer Vb and VI). This was followed by degenerative changes on days 5-7 in other structures, including the amygdaloid nuclei, the ventral posterolateral and ventral posteromedial thalamic nuclei and the periaqueductal gray. The distribution of terminal degeneration from these neurons indicate that specific pools of cells are affected in each structure, and the time course suggests somatofugal degeneration. The trimethyltin damage was also assessed with immunocytochemical visualization of a neuronotypic protein, protein-O-carboxyl methyltransferase and a radioimmunoassay for glial fibrillary acidic protein. Protein-O-carboxyl methyltransferase immunoreactivity was altered in neuronal populations damaged by trimethyltin, but did not appear to be either as sensitive or selective an assay of neuronal damage as the silver stain, especially at short survival times. Glial fibrillary acidic proteins were dramatically elevated 21 days after trimethyltin intoxication, particularly in areas of extensive damage. These studies revealed advantages and problems encountered in the use of each technique in assessing neurotoxic effects, forming a basis for discussion of the relative merits of using a battery of specific molecular probes for neurotoxicity evaluations.

Rationale Schizophrenia remains among the most prevalent neuropsychiatric disorders, and current treatment options are accompanied by unwanted side effects. New treatments that better address core features of the disease with minimal side effects are needed. Objectives As a new therapeutic approach, 1-(4-fluoro-phenyl)-4-((6bR, 10aS)-3-methyl-2,3,6b,9,10,10a-hexahydro-1H,7H-pyrido[3′,4′:4,5]pyrrolo[1,2,3-de]quinoxalin-8-yl)-butan-1-one (ITI-007) is currently in human clinical trials for the treatment of schizophrenia. Here, we characterize the preclinical functional activity of ITI-007. Results ITI-007 is a potent 5-HT2A receptor ligand (Ki = 0.5 nM) with strong affinity for dopamine (DA) D2 receptors (Ki = 32 nM) and the serotonin transporter (SERT) (Ki = 62 nM) but negligible binding to receptors (e.g., H1 histaminergic, 5-HT2C, and muscarinic) associated with cognitive and metabolic side effects of antipsychotic drugs. In vivo it is a 5-HT2A antagonist, blocking (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI)-induced headtwitch in mice with an inhibitory dose 50 (ID50) = 0.09 mg/kg, per oral (p.o.), and has dual properties at D2 receptors, acting as a postsynaptic D2 receptor antagonist to block D-amphetamine hydrochloride (D-AMPH) hyperlocomotion (ID50 = 0.95 mg/kg, p.o.), yet acting as a partial agonist at presynaptic striatal D2 receptors in assays measuring striatal DA neurotransmission. Further, in microdialysis studies, this compound significantly and preferentially enhances mesocortical DA release. At doses relevant for antipsychotic activity in rodents, ITI-007 has no demonstrable cataleptogenic activity. ITI-007 indirectly modulates glutamatergic neurotransmission by increasing phosphorylation of GluN2B-type N-methyl-d-aspartate (NMDA) receptors and preferentially increases phosphorylation of glycogen synthase kinase 3β (GSK-3β) in mesolimbic/mesocortical dopamine systems. Conclusion The combination of in vitro and in vivo activities of this compound support its development for the treatment of schizophrenia and other psychiatric and neurologic disorders.

Vapor condensates of baseline gasoline (BGVC), or gasoline-blended with methyl tertiary butyl ether (G/MTBE), ethyl t-butyl ether (G/ETBE), t-amyl methyl ether (G/TAME), diisopropyl ether (G/DIPE), ethanol (G/EtOH), or t-butyl alcohol (G/TBA) were evaluated for reproductive toxicity in rats at target concentrations of 2000, 10,000, or 20,000mg/m(3), 6h/day, 7days/week. BGVC and G/MTBE were assessed over two generations, the others for one generation. BGVC and G/MTBE F1 offspring were evaluated for neuropathology and changes in regional brain glial fibrillary acidic protein content. No neurotoxicity was observed. Male kidney weight was increased consistent with light hydrocarbon nephropathy. In adult rats, decreased body weight gain and increased liver weight were seen. Spleen weight decreased in adults and pups exposed to G/TBA. No pathological changes to reproductive organs occurred in any study. Decreased food consumption was seen in G/TAME lactating females. Transient decreases in G/TAME offspring weights were observed during lactation. Except for a minor increase in time to mating in G/TBA which did not affect other reproductive parameters, there were no adverse reproductive findings. The NOAEL for reproductive and offspring parameters was 20,000mg/m(3) for all vapor condensates except for lower offspring NOAELs of 10,000mg/m(3) for G/TBA and 2000mg/m(3) for G/TAME.

Recent advances in neuroimmunology and protein purification methodology have led to the identification of nervous-system specific proteins. Their intimate relationship to the cellular and functional heterogeneity of the nervous system, makes these proteins ideal biochemical markers for the detection and characterization of neurotoxicity.

Numerous severe pathologic changes occur following damage to the central nervous system (CNS) of mammals. Formation of a neuroglial scar following damage is seen throughout the neuraxis and is attributed to both hyperplastic (1) and hypertrophic responses (2) of non-neuronal cells near the lesion. One conceptual approach has been to consider the glial scar as a mechanical impediment for the regrowth of axons across this barrier (3, 4). Thus, numerous attempts have been made to reduce the extent of the glial scar via surgical (5) and pharmacologic means (6). However, there have been few attempts to establish a rational pharmacologic approach to the problem of inhibition of glial scars. Clearly, attempts at pharmacotherapy must follow several guidelines which can established based on the metabolic differences between neurons and glial cells. The therapeutic modalities currently used in the treatment of head trauma focus primarily on reduction of brain edema (7) and maintenance of adequate cardiovascular perfusion to the nervous system (8). Beyond this initial stage of therapeutic intervention, there are no currently acceptable pharmacologic approaches which can either prevent the induction of or inhibit neuroglial scar formation. The goal of this manuscript is to outline possible therapeutic modalities which may prove useful in the prevention of neuroglial proliferation following trauma. We will also explore several experimental models of in vivo glial cell proliferation which may prove useful in evaluating experimental therapeutic approaches.

Dithiocarbamates exhibit potent metal-binding properties which have been exploited in a variety of applications, one of which is chelation therapy for heavy metal toxicity. Such therapy, however, promotes the accumulation of metals in the brain, a side effect which may result in neurotoxicity. To examine this possibility we used morphological and biochemical indices to assess the effects of diethyldithiocarbamate (DDC) on cadmium-induced neurotoxicity in the newborn rat. Co-administration of DDC prevented the neurotoxic effects of cadmium while causing a persistent increase in the distribution of cadmium to brain.

The time course of tin distribution in homogenates and subcellular fractions of rat brain was determined following the acute administration of trimethyl tin (TMT) and triethyl tin (TET) to the rat. Exposure to TMT resulted in lower concentrations but greater persistence of tin in subcellular fractions compared to exposure to TET. A delayed accumulation of tin in the mitochondrial fraction was observed following the administration of TMT but not TET. Analysis of total protein and mitochondrial markers did not reveal differences between the compositions of mitochondrial fractions prepared from control and TMT-treated subjects.

Tryptophan hydroxylase [tryptophan 5-monooxygenase, L-tryptophan,tetrahydropterin:oxygen oxidoreductase (5-hydroxylating), EC 1.14.16.4] is activated by phosphorylating conditions (ATP-Mg2+) in a calcium-dependent, cyclic AMP-independent manner. Addition to the phosphorylation reaction of certain antipsychotic drugs that bind to calmodulin, the heat-stable calcium-binding protein, prevents the activation of tryptophan hydroxylase by ATP-Mg2+ in a concentration-dependent fashion. External addition of purified calmodulin protects the enzyme from the drug-induced effects. Calmodulin-free tryptophan hydroxylase prepared by affinity chromatography on fluphenazine-Sepharose is not activated by ATP-Mg2+ whereas addition of calmodulin to calmodulin-free enzyme restores the responsiveness of the hydroxylase to ATP-MG2+ only in the presence of Ca2+. These results indicate that the activation of tryptophan hydroxylase by phosphorylating conditions is dependent on both calcium and calmodulin.

The role of glucocorticoids in the in vivo regulation of glial fibrillary acidic protein (GFAP) was examined. Corticosterone administration to adult rats resulted in decreased levels of GFAP throughout the brain whereas adrenalectomy caused levels of GFAP to increase. Corticosterone administration to adrenalectomized rats lowered GFAP levels to values below those of sham controls. Thus, the expression of GFAP throughout the brain appears to be physiologically regulated by adrenal glucocorticoids.