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Acute administration of high doses of ibogaine (IBG) to the male rat results in degeneration of Purkinje cells and reactive gliosis in the cerebellar vermis. We examined whether acute and chronic administration of IBG to male and female rats results in gliosis as determined by quantification of the astroglial intermediate filament protein, glial fibrillary acidic protein (GFAP). After acute administration of IBG, rats of both sexes showed dose-related increases in GFAP that were not confined to the cerebellar vermis. After chronic administration of IBG, female, but not male rats, showed large (as much as 200% of control), dose-related increases in GFAP in hippocampus, olfactory bulbs, brain stem and striatum, but not cerebellum. In hippocampus, the cytoskeletal proteins, neurofilament 68 (NF-68) and beta-tubulin were increased in females treated chronically with IBG, findings consistent with a damage-induced sprouting response. Together, the data indicate that IBG damages areas of the brain outside the cerebellum and that the sites damaged are dependent on sex and dosage regimen.

Glial fibrillary acidic protein (GFAP) immunostaining is the most commonly used method to examine the distribution of astrocytes and the hypertrophy of astrocytes in response to neural degeneration or injury. More recently, a variety of biochemical assays for GFAP have been developed. Both qualitative immunocytochemical evaluations of GFAP and quantitative biochemical measurements of GFAP have been used to examine the regional distribution of GFAP within the central nervous system (CNS). The former method has largely been based on aldehyde-fixed tissue, while the latter approach has been based on the use of fresh tissue extracts or homogenates. In the present study, we used ethanol as a fixative to permit both immunocytochemical and biochemical procedure to be carried out on brain tissue from a single animal. Normal adult rats and mice were perfused with a 70% ethanol/saline solution, and each brain was hemisectioned. The concentration of GFAP was measured in regions of 1 hemisection, using an enzyme-linked immunosorbent assay (ELISA), while the other hemisection was used for GFAP immunostaining. Regional differences occurred in the brains of both species, with the highest concentration of GFAP found in the brainstem, and the lowest concentrations found in the striatum and cortex. The specific patterns of GFAP immunoreactivity corresponded to regional concentrations in most brain area of both species. These data show that it is possible to assay GFAP concentrations in tissue prepared for immunocytochemical analysis, providing both qualitative and quantitative information from one set of tissue.

Transgenic mice, expressing the gene for bovine growth hormone (bGH), exhibit increased body size, reduced reproductive capacity, and high basal levels of several hormones including corticosterone. Their shortened life span may be indicative of accelerated aging. As prominent astrogliosis of the CNS accompanies aging in rodents, bGH transgenic mice were examined for astrogliosis, as quantified by an ELISA for the astrocyte-localized protein, glial fibrillary acidic protein (GFAP). Transgenic mice were produced by mating C57BL/6 x C3H F1 hybrid females with male descendants of animals produced by microinjection of fertilized eggs with phosphoenolpyruvate carboxykinase (PEPCK)/bGH-hybrid gene. Transgenic mice (approximately 3.5 and approximately 12 months of age) weighed significantly more than same age or older (approximately 20 month) controls. Most of their internal organs, including the heart, kidneys, adrenals, liver, and spleen, were also heavier. In contrast, the thymus was heavier only in the younger transgenic mice. Serum corticosterone was highest in the older transgenic mice. A small but significant increase in whole brain, cortex, and cerebellar weight, relative to controls and the older transgenic mice, was found in the younger transgenic mice. Control mice exhibited large, significant age-related increases in GFAP. Increases of 35, 70, 68, 89, 79, and 95% for cortex, cerebellum, striatum, hippocampus, midbrain, and brain stem, respectively, were found when comparing the oldest (approximately 20 months) control mice to the youngest (approximately 3.5 months). In contrast, in the olfactory bulbs and the hypothalamus there were no age-related changes in the levels of GFAP in control mice. Transgenic mice (approximately 3.5 months) had significantly elevated GFAP levels relative to the same-age controls in all brain areas examined. In some brain areas, the GFAP levels found in the younger transgenic mice were equivalent to those found in the oldest controls. No differences between controls and transgenics were found in tyrosine hydroxylase protein levels of striatum or hypothalamus. The elevated GFAP levels of transgenic mice may reflect increased neural damage due to accelerated aging processes or damage associated with high circulating levels of bGH or corticosterone. Alternatively, the increased expression of GFAP in the transgenic mice may reflect altered regulation of GFAP rather than an increase signaled by neural damage.

A universal cellular reaction to damage of the CNS is hypertrophy of astrocytes. The hallmark of this response, often termed 'reactive gliosis', is the enhanced expression of the major intermediate filament protein of astrocytes, glial fibrillary acidic protein (GFAP). This latter observation suggests that increased synthesis of GFAP would occur in response to diverse neurotoxic insults. To investigate this possibility, prototype neurotoxicants were administered to experimental animals and the effects of these agents on the tissue content of GFAP was determined by immunoassay. Assays of GFAP were found to reveal dose-, time- and region-dependent patterns of neurotoxicity at toxicant dosages below those that cause light microscopic evidence of cell loss or damage. Moreover, the temporal and regional increments in GFAP correspond to the temporal and regional patterns of argyrophilia, as revealed by the cupric silver degeneration stain of de Olmos. Our findings indicate that assays of GFAP represent a sensitive, simple and quantitative approach for evaluation of nervous system damage. Combining this indirect yet quantitative indicator of neurotoxicity with more traditional neuroanatomical endpoints, should augment the armamentarium of techniques useful for detection and characterization of neurotoxicity.

The sequencing of the human genome in the early days of this millennium was greeted with great fanfare as this accomplishment was expected to revolutionize medicine and result in individualized treatments based on the genetic make-up of the patient. The ultimate promise of personalized medicine would be fulfilled with the identification of disease biomarkers that would be widely available for use in diagnosis and treatment. Progress, however, has been slow in providing disease biomarkers or approved diagnostic tests. This is true for major depressive disorder (MDD), despite its prevalence in the general population and the widespread acceptance of its biological basis. Studies using strategies like genome-wide association and candidate gene analyses have identified a number of possible biomarkers of MDD, including serum levels of neurotrophic factors, inflammatory cytokines and HPA axis hormones, but none have proven sufficiently powerful for clinical use. The lack of biologically based tests available for use in identifying patients with MDD is a significant impediment to personalized and more effective treatment, because it means diagnosis continues to be driven by subjective symptoms. While genetic studies of MDD have not yet led to diagnostic and treatment biomarkers, progress in determining the role of the genome in drug metabolism heralds the first effort in personalized prescribing for the antidepressants. The FDA suggested and approved genotyping tests for common variants of drug metabolism genes, such as the cytochrome p450s. By using these tests a physician can select an appropriate antidepressant for a given patient, as differences in clearance, half-life, and peak blood concentrations are controlled by genetic variability in drug metabolism. Personalization in drug choice can be achieved because these tests: (1) identify responders and non-responders; (2) provide alerts to possible adverse drug events; and (3) help optimize dose. Improved ways of diagnosing and prescribing effective treatments for MDD are needed, as the available methods are inadequate and symptom based. In the foreseeable future, further interrogation of the genome may serve as the basis for development of new personalized medicine strategies for diagnosis and treatment of MDD.

Although organochlorines have been reported more frequently in Parkinson's disease (PD) brains than in controls, the association with brain Lewy pathology is unknown. Honolulu-Asia Aging Study (HAAS) participants, exposed to organochlorines from a variety of sources during midlife, represent a population well suited to determining the relationship of brain organochlorines with Lewy pathology in decedents from the longitudinal HAAS. The study design included the measurement of 21 organochlorine levels in frozen occipital lobe samples from HAAS decedents. Alpha-synuclein immunostaining performed on 225 brains was used to identify Lewy bodies and Lewy neurites. With the potential for spurious associations to appear between Lewy pathology and 17 organochlorine compounds found in at least 1 brain, initial assessments identified heptachlor epoxide isomer b, methoxychlor, and benzene hexachloride b as being most important. The prevalence of Lewy pathology was 75% (6 of 8) among brains with any 2 of the 3 compounds, 48.8% (79 of 162) among those with 1, and 32.7% (18 of 55) for those with neither (P = .007 test for trend). Although findings persisted after removing cases with PD and dementia with Lewy bodies and after adjustment for age at death, body mass index, pack-years of cigarette smoking, and coffee intake (P = .013), the results were insignificant when correcting for multiple testing. Although consistent with earlier accounts of an association between organochlorines and clinical PD, associations with Lewy pathology warrant further study.

Developmental Neurotoxicity: Evaluation of Testing Procedures with Methylazoxymethanol and Methylmercury. Goldey, E. S., O'Callaghan, J. P., Stanton, M. E., Barone, S., Jr., and Crofton, K. M. (1994). Fundam. Appl. Toxicol. 23, 447-464. Testing procedures for identification of potential developmental neurotoxicants were evaluated using two prototypical developmental neurotoxicants, methylazoxymethanol (MAM) and methylmercury (MeHg). Evaluation of offspring of Long-Evans rats incorporated assessments of developmental toxicity, neurochemistry, histology, and behavior, with most testing being completed near weaning. A number of endpoints in the testing strategy were sensitive to the effects of prenatal exposure to MAM [30 mg/kg on Gestation Day (GD) 15]; (1) MAM caused reduced neonatal body weights but did not effect viability or postnatal survivorship; (2) measurement of total and regional brain weight and histological analysis showed that a number of regions, the cortex and hippocampus in particular, were affected by MAM exposure; (3) an assay for glial fibrillary acidic protein (GFAP) showed that the concentration of this protein was significantly increased in the cortex and hippocampus of treated offspring; (4) a T-maze delayed-alternation procedure indicated that MAM-treated pups were slower in the acquisition phase of the task relative to control pups; (5) motor activity testing revealed hyperactivity in treated offspring that persisted into adulthood; and (6) acoustic startle procedures revealed reduced startle amplitudes in preweanlings. Few endpoints were significantly affected by prenatal MeHg exposure (1, 2, or 4 mg/kg on GD 6-15). High fetal and neonatal mortality and lower neonatal body weights were detected at the highest dose of MeHg. Although minimal effects of MeHg may reflect a relative insensitivity of the test species and/or the test methods, the combined results from both chemicals suggest that some procedures not currently required in the developmental neurotoxicity guideline may be useful in hazard identification, and further evaluation with other chemicals, species, strains, and/or exposure paradigms may be warranted.

Reactive gliosis is a powerful response to brain injury and subsequent neuronal damage in vivo. Neuronal cell cultures are now well established as assays to study this process in vitro. However, equivalent studies of purified glial cell populations have only recently been achieved, following the realization that glial cells produce many of the neuropeptides, transmitters and growth factors that are produced also by neurons. There is now scope for studies in vitro that use mixed, identified populations of glial and neuronal cells to dissect the interactions between the two. Such cultures also lend themselves to assays for potential therapeutic strategies for brain injury that take account of all the different cell types found in the brain.

The neurological effects of organophosphate (OP) pesticides, commonly used on foods and in households, are an important public health concern. Furthermore, subclinical exposure to combinations of organophosphates is implicated in Gulf War illness. Here, we characterized the effects of the broadly used insecticide chlorpyrifos (CPF) on dopamine and glutamatergic neurotransmission effectors in corticostriatal motor/reward circuitry. CPF potentiated protein kinase A (PKA)-dependent phosphorylation of the striatal protein dopamine- and cAMP-regulated phosphoprotein of M(r) 32 kDa (DARPP-32) and the glutamate receptor 1 (GluR1) subunit of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in mouse brain slices. It also increased GluR1 phosphorylation by PKA when administered systemically. This correlated with enhanced glutamate release from cortical projections in rat striatum. Similar effects were induced by the sarin congener, diisopropyl fluorophosphate, alone or in combination with the putative neuroprotectant, pyridostigmine bromide and the pesticide N,N-diethyl-meta-toluamide (DEET). This combination, meant to mimic the neurotoxicant exposure encountered by veterans of the 1991 Persian Gulf War, also induced hyperphosphorylation of the neurofibrillary tangle-associated protein tau. Diisopropyl fluorophosphate and pyrodostigmine bromide, alone or in combination, also increased the aberrant activity of the protein kinase, Cdk5, as indicated by conversion of its activating cofactor p35 to p25. Thus, consistent with recent findings in humans and animals, organophosphate exposure causes dysregulation in the motor/reward circuitry and invokes mechanisms associated with neurological disorders and neurodegeneration.

Our aim was to investigate a model of the morphologic approach proposed in guidelines for developmental neurotoxicity testing (DNT). Hereto, a limited DNT study [EPA Health Effects Test Guidelines OPPTS 870.6300, 1996a. Developmental Neurotoxicity Study "Public Draft", United States Environmental Protection Agency; Prevention, Pesticides and Toxic Substances (7101); EPA 712-C-96-239, June 1996. http://www.epa.gov/opptsfrs/OPPTS_Harmonized/870_Health_Effects_Test_Guidelines/Drafts/870-6300.pdf was carried out with different doses of methylazoxy methanol acetate (MAM), known to affect brain morphology and neuron numbers in the developing brain. After gross examination, the brains of F1-animals were further dissected along neuro-anatomical landmarks to ensure homology between tissues of different individuals. The (relative) weight of the brain (parts) was determined. One brain half (alternating left/right to avoid lateralization) was further used for microscopic slide reading and measurement of brain layer width (linear morphometry); the other was set aside for stereologic investigation in a later phase of the study. In the offspring, a clear reduction in brain size (gross macroscopy) and weight (MAM high- and top-dose groups) was observed on postnatal days (PN) 22 and 62, but this reduction was hard to pinpoint in the microscope as the changes primarily appeared quantitative in nature, rather than qualitative. Linear measurements of brain layer width appeared very sensitive and efficient. This first step of a project is presented and the perspectives of a further stereologic investigation are discussed. © 2005 Published by Elsevier B.V.

The effects of repeated treatment with the phosphodiesterase-4 (PDE4) inhibitors rolipram, piclamilast, and 4-(2-(3-(cyclopentyloxy)-4-methoxyphenyl)-2-phenylethyl)pyridine (CDP840), which differ in their interactions with high- and low-affinity binding conformers of the enzyme, were contrasted to those of acute treatment on cAMP signaling, hippocampal cell proliferation, and immobility in the forced-swim test in rats. Repeated treatment with rolipram (1 and 3 mg/kg), piclamilast (0.3 and 1 mg/kg), or CDP840 (10 and 30 mg/kg) for 16 days increased cAMP and phosphorylation of cAMP response element binding protein (pCREB) in hippocampus and prefrontal cortex. In addition, repeated treatment with the PDE4 inhibitors increased proliferation and survival of newborn cells in the hippocampus and produced antidepressant-like effects on behavior, as evidenced by decreased immobility in the forced-swim test. Acute treatment with rolipram (3 mg/kg), piclamilast (1 mg/kg), or CDP840 (30 mg/kg) induced transient increases in cAMP and pCREB in hippocampus and prefrontal cortex, but the dose and time dependence of these effects did not parallel the behavioral effects. Compared with rolipram and piclamilast, repeated treatment with CDP840 exerted lesser effects on neural and behavioral measures, probably because of its weak interaction with the high-affinity binding conformer of PDE4. This suggests the relative importance of the high-affinity binding conformer in the mediation of the long-term effects of PDE4 inhibition on cAMP/pCREB signaling, hippocampal cell proliferation, and antidepressant-like effects on behavior.

The astrocyte response to central nervous system injury induced by neonatal alcohol exposure was evaluated using radioimmunoassay and immunocytochemistry of glial fibrillary acidic protein (GFAP). Rat pups were exposed to alcohol on postnatal days 4 through 9 via artificial rearing. Alcohol solutions were administered as one of the following treatments: 10.2% (v/v) in two feedings (4.5 g/kg/day), 5.1% (v/v) in four feedings (4.5 g/kg/day), or 2.5% (v/v) in 12 feedings (6.6 g/kg/day), producing mean blood alcohol concentrations (BACs) of approximately 300, 180, and 50 mg/dl, respectively. Littermates were included as gastrostomy controls (GC) and suckle controls (SC). On postnatal day 10, GFAP concentration increased as a function of BAC, and the 10.2% alcohol treatment significantly and dramatically increased GFAP in the cortex (325% of SC). GFAP immunocytochemistry revealed frequent loci of heavily labeled reactive astrocytes surrounding small cortical blood vessels in the 10.2% group. In addition, a generalized increase in GFAP immunoreactivity was present in the deep layers of the cortex in all alcohol groups, marked by astrocytic fibrillary hypertrophy and increased density. Three-dimensional counts in layer V of parietal cortex using confocal microscopy indicated that the density of GFAP-labeled astrocytes of the 10.2% group was twice that of controls. The layer V gliosis was observable even at low BACs, while gliosis around the vasculature occurred only with high BACs. By postnatal day 15, the astroglial effects were no longer evident. These transient astroglial reactions likely constitute an important aspect of cortical pathophysiology resulting from binge alcohol exposure during the brain growth spurt of the third trimester equivalent.

Therapeutic management of chronic pain has not been widely successful owing to a lack of understanding of factors that initiate and maintain the chronic pain condition. Efforts to delineate the mechanisms underlying pain long have focused on neuronal elements of pain pathways, and both opiate- and non–opiate-based therapeutics are thought largely to target neurons. Abnormal neuronal activity at the level of spinal cord “pain centers” in the dorsal horn leads to hypersensitivity or a hyperalgesic response subsequent to the initial painful stimulus. Only recently has the experimental literature implicated nonneuronal elements in pain because of the realization that glial-derived signaling molecules can contribute to and modulate pain signaling in the spinal cord. Most notably, glial proinflammatory mediators within the dorsal horn of the spinal cord appear to contribute to self-perpetuating pain. Chronic pain is modeled experimentally through a variety of manipulations of sensory nerves including cutting, crushing, resection, and ligation. The cellular and molecular responses in the spinal cord due to these manipulations often reveal activation of 2 types of glia: microglia and astrocytes. The activation states of both microglia and astrocytes are complex and may be driven by underlying chronic neuropathology and/or a chronically “primed” condition that accounts for their contribution to chronic pain. Recent evidence even suggests that opioid tolerance and withdrawal hyperalgesia may be initiated and maintained via actions of microglia and astroglia. Together, these recent findings suggest that glia will serve as novel therapeutic targets for the treatment of chronic pain. To fully exploit glia as novel therapeutic targets will require a greater understanding of glial biology, as well as the identification of agents able to control the glial reactions involved in chronic pain, without interfering with beneficial glial functions.

Repeated exposure to psychostimulant drugs such as d-methamphetamine (d-METH) and cocaine can be associated with extremely long-lived changes in dopamine systems at the behavioral, cellular and molecular level. Sensitization or an enhanced response to drug exposure is one such change. Investigations of these phenomena at the cellular and molecular levels are being conducted in the hope that this will aid in understanding how such adaptations might contribute to drug addition. Repeated exposure to certain amphetamines can also result in damage to dopaminergic pathways. Although some of the same molecular adaptations and mechanisms are suspected to occur or play a role in the neurotoxic sequelae associated with psychostimulant exposure, there has been little attempt to examine the relationship among these phenomena. Here we utilized C57BL/6J female mice to examine whether exposure to a sensitizing regimen of d-METH would impact the degree of neural injury induced by a subsequent exposure to a neurotoxic regimen of the same psychostimulant. Every other day exposure to d-METH (1.0 or 2.0 mg/kg) for 11 days produced a behavioral sensitization, as evidenced by a significant increase in the degree of locomotor activity induced by each subsequent exposure to d-METH. Following a 5-day period of no drug exposure sensitized mice were given a neurotoxic regiment of d-METH (a total of four injections of 10.0 mg/kg, one every 2 hours) and striatal tissue examined 72 hours later. All groups, whether drug-naive or sensitized previously to d-METH, showed exactly the same degree of dopaminergic striatal damage induced by a neurotoxic regimen. This was evidenced by equivalent reductions in dopamine and elevations in GFAP protein, a marker of astrocytic response to injury, GFAP. The inability of a sensitizing regimen to either exacerbate or lessen the neurotoxic actions of the same compound suggests that the molecular and cellular control of these two aspects of psychostimulant exposure may differ.

Objective. This preliminary study assessed possible relationships between plasma and/or cerebrospinal fluid (CSF) concentrations of the pleiotropic cytokine, interleukin (IL)-6, the anti-inflammatory cytokine, IL-10, and levels of pain reported by patients receiving intrathecal (i.t.) opioids. Design. A prospective study quantifying IL-6 and IL-10 concentrations using enzyme-linked immunoassays in samples of plasma and CSF as well as assessment of pain scores in patients receiving intrathecal opioids for management of chronic noncancer pain. Setting. Outpatient pain clinics. Patients. Patients with chronic pain receiving intrathecal morphine or hydromorphone alone or in combination with local anesthetics. Interventions. Two groups of patients were studied. The first group (n = 50) had been receiving long-term i.t. opioids by chronically implanted pump for ∼5 years; paired samples of plasma and CSF were collected at the time of i.t. pump refill. For the second patient group (n = 10), possible temporal changes in the plasma and/or CSF concentrations of IL-6 and IL-10 were investigated for 3 months after initiation of i.t. opioid infusions. Results. For patients receiving long-term i.t. opioid infusions, there were significant inverse correlations ( P ≤ 0.05) between pain intensity and the plasma (but not CSF) IL-10 and IL-6 concentrations. Despite the considerable inter-patient variability in the CSF concentrations of IL-6 in the long-term cohort, the mean CSF IL-6 concentration was approximately fivefold higher in patients receiving long-term i.t. opioids relative to those receiving i.t. opioids for only 3 months. Conclusions. The significant inverse correlations observed between pain intensity and the plasma IL-6 and IL-10 concentrations in patients receiving longterm i.t. opioids for chronic pain management, suggests that these cytokines are worthy of further investigation as possible biomarkers of persistent pain.

Astrogliosis, a cellular reaction with specific structural and functional characteristics, represents a remarkably homotypic response of astrocytes to all kinds of central nervous system (CNS) pathologies. Astrocytes play diverse functions in the brain, both harmful and beneficial. Mounting evidence indicates that astrogliosis is an underlying component of a diverse range of diseases and associated neuropathologies. The mechanisms that lead to astrogliosis are not fully understood, nevertheless, damaged neurons have long been reported to induce astrogliosis and astrogliosis has been used as an index for underlying neuronal damage. As the predominant source of proinflammatory factors in the CNS, microglia are readily activated under certain pathological conditions. An increasing body of evidence suggests that release of cytokines and other soluble products by activated microglia can significantly influence the subsequent development of astrogliosis and scar formation in CNS. It is well known that damaged neurons activate microglia very quickly, therefore, it is possible that activated microglia contribute factors/mediators through which damaged neuron induce astrogliosis. The hypothesis that activated microglia initiate and maintain astrogliosis suggests that suppression of microglial overactivation might effectively attenuate reactive astrogliosis. Development of targeted anti-microglial activation therapies might slow or halt the progression of astrogliosis and, therefore, help achieve a more beneficial environment in various CNS pathologies.

The indirubins long have been used in Chinese medicine for treatment of myelocytic leukemia. Among the many more recently described biological activities of the indirubins, attention has been directed toward the ability of these compounds to inhibit GSK-3 and CDKs, kinases implicated in neurodegenerative conditions. Little information is available on effects of indirubins on chemically-induced neurodegeneration. Here we examined the influence of three indirubins on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- and kainic acid (KA)-induced neurotoxicity in the mouse. The three indirubins examined were 6-bromoindirubin-3'-oxime (6BIO), 5-bromoindirubin-3'-oxime (5BIO) and 5-amino-6-bromoindirubin (5A6BI). The first two derivatives were previously described indirubins with low nanomolar inhibitory activity against GSK-3 and CDKs. The third compound was synthesized by the dimerization of 5-amino-6-bromoisatin with 3-acetoxyindol. The synthesis of the key compound 5-amino-6-bromoisatin was based on the bromination of the ketal of 5-amino-isatin. All indirubins examined decreased various measures associated with dopaminergic neurotransmission in striatum. These effects occurred alone or over and above the decrements seen following administration of the dopaminergic neurotoxicant, MPTP. Striatal serotonin and serotonin turnover were decreased by the indirubins in MPTP-treated mice. None of these striatal effects of the indirubins alone were associated with evidence of astrogliosis, an indicator of underlying neuropathology, nor did they potentiate the astrogliosis accompanying administration of MPTP. In general, the indirubins reduced KA-associated mortality and striatal but not hippocampal astrogliosis due to this toxicant. The data suggest that indirubins affect striatal biogenic amine levels and turnover in intact mice. The data do not indicate a neuroprotective action of indirubins in mice treated with MPTP but that they do suggest that they may be neuroprotective against KA-induced injury of the neostriatum.

High levels of stress or stress hormones have been reported to exacerbate a variety of human disorders of the cardiovascular, gastrointestinal, immune, reproductive, and nervous systems. In rats, high glucocorticoid levels have been reported to cause neuronal death and injury as well as enhance susceptibility to neurotoxic agents and attenuate repair mechanisms; however, the impact of high dosages of CORT in mice has not been fully evaluated. We investigated the ability of supraphysiological levels of CORT to cause hippocampal neuronal death, and to modulate the neurotoxicity of kainic acid (KA) in male C57BL/6J mice. Timed-release CORT pellets (10, 35, 100 mg/21 d) were implanted subcutaneously in the back of mice, and the sustained release of glucocorticoid caused involution of the thymus and decreased the weight of the spleen. Kainic acid caused stage 1 seizures that were unaffected by CORT; however, steroid treatment decreased KA-associated mortality. Little neuronal damage was detected by the cupric-silver neurodegeneration stain. Neurotoxicity caused by an intraperitoneal injection of 25mg/kg KA was attenuated by seven days of CORT pre-treatment. The KA-induced increase in cupric-silver staining, reactive gliosis, microglial activation, and blood-brain barrier disruption was attenuated indicating neuroprotection. Our data indicate supraphysiological levels of CORT do not cause neuronal death or injury in hippocampus of C57BL/6J mice and provide neuroprotection against KA-induced neural damage.

Diverse neurotoxic insults result in proliferation and hypertrophy of astrocytes, a subtype of central nervous system glia. The hallmark of this response, often termed “reactive 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. To investigate this possibility we have administered prototype neurotoxicants to experimental animals and then assessed the effects of these agents on the tissue content of GFAP, as determined by radioimmunoassay. We found that assays of GFAP reveal dose-, time-, and region-dependent patterns of neurotoxicity at toxicant dosages below those that cause light microscopic evidence of cell loss or damage. No false positives have been seen following exposure to a variety of pharmacological agents. By using regional assessments of GFAP in a first-tier evaluation, it should be possible to localize areas of damage. A second-tier evaluation, using assays of proteins or transmitters associated with cells in the affected region, may reveal the cellular targets of neurotoxicity. This two-tiered approach should serve as a foundation for guiding studies aimed at determining mechanisms of neurotoxicity.

A enzyme-linked immunosorbent assay has been developed for tyrosine hydroxylase (TH). The method uses a polyclonal antibody to trap TH, a monoclonal antibody to bind the immobilized TH, a biotinylated, anti-mouse immunoglobulin to bind the monoclonal antibody, and streptavidin covalently coupled to horseradish peroxidase (SA-HRP). The antigen-antibody complex is detected colorometrically following incubation with an HRP substrate. The method detects less than 1 ng (16 fmol) of TH and can be performed in 3 h. The high specificity of the assay is attributed to the use of both polyclonal and monoclonal antibodies, each of which are specific for TH. Data acquisition and reduction is rapid and linked directly to a common desktop computer. Levels of TH protein average 1 ng/microgram protein in striatum and, following treatment with the neurotoxicant MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), are decreased to a similar extent as is catalytic activity. In contrast, MPTP did not alter TH homospecific activity. The monoamine oxidase B inhibitor deprenyl blocked both the decrease in activity and the decrease in immunoreactive protein caused by MPTP.

Diethyldithiocarbamic acid (DDC) potentiates in vivo neurotoxicity of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) and in vitro neurotoxicity of 1-methyl-4-phenylpyridinium (MPP+). Male C57B1/6 mice were given two or five injections of MPTP (30 rag/kg i.p.) preceded 0.5 h by DDC (400 mg/kg i.p.). The mice were tested for catalepsy, akinesia, or motor activity during and after the period of dosing. Striatal and hippocampal tissues were obtained at 2 and 7 days following the last injection and evaluated for dopamine and norepinephrine levels, respectively. These same tissues were also analyzed for the levels of glial fibrillary acidic protein (GFAP), an astrocyte-localized protein known to increase in response to neural injury. Pretreatment with DDC potentiated the effect of MPTP in striatum and resulted in substantially greater dopamine depletion, as well as a more pronounced elevation in GFAP. In hippocampus, the levels of norepinephrine and GFAP were not different from controls in mice receiving only MPTP, but pretreatment with DDC resulted in a sustained depletion of norepinephrine and an elevation of GFAP, suggesting that damage was extended to this brain area by the combined treatment. Mice receiving MPTP preceded by DDC also demonstrated a more profound, but reversible, catalepsy and akinesia compared to those receiving MPTP alone. Systemically administered MPP+ decreased heart norepinephrine, but did not alter the striatal levels of dopamine or GFAP, and pretreatment with DDC did not alter these effects, but did increase lethality. DDC is known to increase brain levels of MPP+ after MPTP, but our data indicate that this is not due to a movement of peripherally generated MPP+ into CNS. In cultured bovine adrenal medullary cells, MPP+ (300 μM) slightly decreased catecholamine levels, but had no effect on tyrosine hydroxylase activity or cellular protein. However, the incubation of these cells with both MPP+ and DDC (1.5 or 3.0 mM) caused large decreases in all indicators of cell viability. The enhancement of MPP+ neurotoxicity by DDC in an in vitro system, where distributional factors are limited, raises die possibility that mechanisms in addition to altered kinetics may account for DDC-induced potentiation of MPTP neurotoxicity in vivo.

Kindling is a process in which episodic electrical stimulation permanently increases seizure susceptibility. One mechanism to account for a change in seizure susceptibility is some alteration in signal transduction, possibly at the level of second messenger systems. In this study, male Long-Evans rats were kindled in the amygdala, and Ca 2+ /calmodulin (Ca 2+ /CaM)-dependent protein phosphorylation was assessed at the site of the primary kindled focus using one- and two-dimensional gel electrophoresis. In vitro phosphorylation of membrane and cytosol fractions in the presence or absence of Ca 2+ /CaM did not differentiate kindled from nonkindled amygdaloid tissue. These results suggest that changes in Ca 2+ /CaM-dependent phosphorylation are not related to the mechanism(s) underlying the establishment of an amygdaloid kindled focus.

3,4-Methylenedioxymethamphetamine (MDMA), a substituted amphetamine analogue which stimulates serotonin release in the CNS, has been shown to induce near lethal elevations in core temperature in the rat. To characterize the effects of MDMA on temperature regulation, we measured metabolic rate (MR), evaporative water loss (EWL), motor activity (MA), and colonic temperature (Tc) in male, Long-Evans rats at 60 min following 30 mg/kg (SC) MDMA or saline at ambient temperatures (Ta) of 10, 20, and 30°C. MDMA caused an elevation in MR at Ta's of 20 and 30°C but had no effect at 10°C. At a Ta of 30°C, MR of the MDMA group was double that of the saline group. EWL was elevated by MDMA, an effect which was potentiated with increasing Ta. MDMA also elicited an increase in MA at all three Ta's. MDMA led to a 3.2°C increase in Tc at 30°C, no change in Tc at 20°C, and a 2.0°C decrease in Tc at 10°C. A second study found that treatment with 20 mg/kg MDMA failed to elicit an increase in blood flow to the tail in spite of a hyperthermic core temperature of 41.4°C. Preliminary studies using radiotelemetry methodology suggested that MDMA lethality is preceded by precipitous elevations in heart rate and core temperature. The data suggest that, at relatively warm Ta's, MDMA-induced stimulation of serotonergic pathway causes an elevation in MR and peripheral vasoconstriction, thus producing life-threatening elevations in Tc. The increase in EWL following MDMA partially attenuates the hyperthermia at warm Ta's, but leads to hypothermia in the rat maintained at a cold Ta of 10°C.

The aim of this randomized double-blind, placebo-controlled, parallel-group study was to evaluate the efficacy, safety, and tolerability of pregabalin in combination with oxycodone or placebo, in patients with either postherpetic neuralgia (PHN) or painful diabetic neuropathy (PDN). After a 7-day washout period, 62 patients were randomized to receive either oxycodone mixture 10 mg/day or placebo mixture for 1 week. Patients were then started on open-label pregabalin (75, 150, 300 and 600 mg/day) according to a forced titration dosing regimen, while continuing the same dosage of oxycodone or placebo for 4 weeks. The primary efficacy measure was a decrease in the pain-intensity score of at least 2cm and a pain score4cm measured using a 10-cm visual analogue scale (VAS) following pregabalin dosage escalation and treatment for 4 weeks. Secondary efficacy measures included sleep interference and the Neuropathic Pain Scale. There were similar levels of overall efficacy between pregabalin/oxycodone and pregabalin/placebo groups in relieving PHN and PDN related pain.Peripheral neuropathic pain presents commonly in clinical practice, and 2 of its most prevalent types are PHN and PDN. Currently available treatments provide some degree of pain relief in approximately 40-60% of patients, leaving the remainder with unremitting pain. Although this study supports the effectiveness of pregabalin in the treatment of PHN or PDN, it also shows that the addition of a low dose of oxycodone at 10mg/day does not enhance the pain-relieving effects of pregabalin.

Motor learning and neuro-adaptations to drugs of abuse rely upon neuronal signaling in the striatum. Cyclin-dependent kinase 5 (Cdk5) regulates striatal dopamine neurotransmission and behavioral responses to cocaine. Although the role for Cdk5 in neurodegeneration in the cortex and hippocampus and in hippocampal-dependent learning has been demonstrated, its dysregulation in the striatum has not been examined. Here we show that strong activation of striatal NMDA receptors produced p25, the truncated form of the Cdk5 co-activator p35. Furthermore, inducible overexpression of p25 in the striatum prevented locomotor sensitization to cocaine and attenuated motor coordination and learning. This corresponded with reduced dendritic spine density, increased neuro-inflammation, altered dopamine signaling, and shifted Cdk5 specificity with regard to physiological and aberrant substrates, but no apparent loss of striatal neurons. Thus, dysregulation of Cdk5 dramatically affects striatal-dependent brain function and may be relevant to non-neurodegenerative disorders involving dopamine neurotransmission.

Phosphodiesterase (PDE) is a critical regulator of cAMP/protein kinase A (PKA) signaling in cells. Multiple PDEs with different substrate specificities and subcellular localization are expressed in neurons. Dopamine plays a central role in the regulation of motor and cognitive functions. The effect of dopamine is largely mediated through the cAMP/PKA signaling cascade, and therefore controlled by PDE activity. We usedin vitroandin vivobiochemical techniques to dissect the roles of PDE4 and PDE10A in dopaminergic neurotransmission in mouse striatum by monitoring the ability of selective PDE inhibitors to regulate phosphorylation of presynaptic [e.g., tyrosine hydroxylase (TH)] and postsynaptic [e.g., dopamine- and cAMP-regulated phosphoprotein of Mr32 kDa (DARPP-32)] PKA substrates. The PDE4 inhibitor, rolipram, induced a large increase in TH Ser40 phosphorylation at dopaminergic terminals that was associated with a commensurate increase in dopamine synthesis and turnover in striatumin vivo. Rolipram induced a small increase in DARPP-32 Thr34 phosphorylation preferentially in striatopallidal neurons by activating adenosine A2Areceptor signaling in striatum. In contrast, the PDE10A inhibitor, papaverine, had no effect on TH phosphorylation or dopamine turnover, but instead robustly increased DARPP-32 Thr34 and GluR1 Ser845 phosphorylation in striatal neurons. Inhibition of PDE10A by papaverine activated cAMP/PKA signaling in both striatonigral and striatopallidal neurons, resulting in potentiation of dopamine D1receptor signaling and inhibition of dopamine D2receptor signaling. These biochemical results are supported by immunohistochemical data demonstrating differential localization of PDE10A and PDE4 in striatum. These data underscore the importance of individual brain-enriched cyclic-nucleotide PDE isoforms as therapeutic targets for neuropsychiatric and neurodegenerative disorders affecting dopamine neurotransmission.

Acute administration of the dopaminergic neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to the C57BL/6 mouse caused a rapid decrease in the amount of striatal tyrosine hydroxylase (TH), a marker of the nigrostriatal dopaminergic pathway, followed by a large increase in the astrocyte protein, glial fibrillary acidic protein (GFAP). The astrocyte (GFAP) response declined to baseline three weeks after administration of MPTP. Administration of a second dosage of MPTP at this time evoked a second GFAP response. The magnitude of the second response, however, was decreased in comparison to the response seen after only a single exposure to MPTP. Increasing the initial dosage of MPTP resulted in greater reductions of the second GFAP response. These data indicate that MPTP-induced damage or loss of striatal dopaminergic neurons reduces the signal available for initiating astrogliosis and thereby reduces the astrocyte response to a second exposure to MPTP.

We used the dopaminergic neurotoxicant, 1-methyl-1,2,3,6-tetrahydropyridine (MPTP), as a tool to characterize the origins of astroglial response to injury. Radioimmunoassay of the astrocyte protein, glial fibrillary acidic protein (GFAP), was used to quantify the astrocyte reaction to MPTP. Assays of neuron-localized proteins and of dopamine were used to assess neuronal damage caused by MPTP. A single administration of MPTP (12.5 mg/kg, s.c.) to the C57BL/6J mouse resulted in more than a 3-fold increase in striatal GFAP within 48 h, followed by a decline to baseline at 3 weeks. A decrease in the amount of striatal tyrosine hydroxylase (TH), a marker of dopaminergic neurons, preceded the rise in GFAP. The concentration of striatal DARPP-32, a phosphoprotein enriched in neurons receiving dopaminergic input, was not affected by MPTP. Protecting the dopaminergic neurons from the neurotoxic metabolite of MPTP, 1-methyl-4-phenylpyridinium (MPP+), either by blocking its formation or by preventing its uptake into dopaminergic neurons, completely blocked the increase in GFAP. MPTP did not appear to disrupt the blood-brain barrier, therefore, blood-borne elements probably did not mediate the increase in GFAP. In addition, immunoblot data indicated that brain-derived interleukin 1, an astrocyte growth factor, also did not play a role in MPTP-induced gliosis. Together, these findings suggest that diffusible factors derived from damaged dopaminergic neurons initiate the astrocyte response to MPTP and that large increases in GFAP can be induced without the participation of serum-derived growth factors. Because the transient nature of GFAP reaction to MPTP coincides with the rapid elimination of the active toxicant, MPP+, suggests that maintenance of reactive gliosis, in general, may require the continued presence of the damage-inducing insult.

AMP-activated protein kinase is activated when the catalytic α subunit is phosphorylated on Thr172 and therefore, phosphorylation of the α subunit is used as a measure of activation. However, measurement of α-AMP-activated protein kinase phosphorylation in vivo can be technically challenging. To determine the most accurate method for measuring α-AMP-activated protein kinase phosphorylation in the mouse brain, we compared different methods of sacrifice and tissue preparation. We found that freeze/thawing samples after homogenization on ice dramatically increased α-AMP-activated protein kinase phosphorylation in mice sacrificed by cervical dislocation. Sacrifice of mice by focused microwave irradiation, which rapidly heats the brain and causes enzymatic inactivation, prevented the freeze/thaw-induced increase in α-AMP-activated protein kinase phosphorylation and similar levels of phosphorylation were observed compared to mice sacrificed with cervical dislocation without freeze/thawing of samples. Sonication of samples in hot 1% sodium dodecyl sulfate blocked the freeze/thaw-induced increase in α-AMP-activated protein kinase phosphorylation, but phosphorylation was higher in mice sacrificed by cervical dislocation compared to mice sacrificed by focused microwave irradiation. These results demonstrate that α-AMP-activated protein kinase phosphorylation is dependent on method of sacrifice and tissue preparation and that α-AMP-activated protein kinase phosphorylation can increase in a manner that does not reflect biological alterations.

Developing accessible biomarkers of neurotoxic effects which are readily applicable to human populations poses a challenge for neurotoxicology. In the past, the neurotoxic organometal trimethyltin (TMT) has been used as a denervation tool to validate the enhanced expression of GFAP as a biomarker of astrogliosis and neurotoxicity resulting from chemical exposures. In the present study, TMT was used to assess the detection of serum autoantibodies as biomarkers of neurotoxicity. Previous studies in both human and animals have demonstrated the presence of serum autoantibodies to neurotypic [e.g., neurofilament triplet (NF)] and gliotypic proteins [myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP)] as a peripheral marker of neurodegeneration that may be applicable to humans and experimental studies. Male Long-Evans rats (45 days of age) were administered either TMT (8 mg/kg; s) or an equal volume of sterile 0.9% saline. At 1, 2, and 3 weeks post-administration, serum was collected, and rats were sacrificed for the collection of brains. Serum autoantibodies (both IgM and IgG isotypes) to NF68, NF160, NF200, MBP, and GFAP were assayed using an ELISA. Saline only rats did not have detectable levels of autoantibodies. Only sera from TMT-exposed rats had detectable titers of autoantibodies to NFs with IgG predominating starting week 2. Anti-NF68 titers were highest compared to NF160, or NF200. Autoantibodies to MBP and GFAP also were detected; however, there was no significant increase in their titers until week 3. Hippocampal GFAP, detected at these time points, was significantly (p

Astroglial activation constitutes a dominant response to all types of injuries of the CNS. Despite the ubiquitous nature of this cellular reaction to neural injury, a little is known concerning the signaling mechanisms that initiate it. Recently, we demonstrated that astrocytic hypertrophy and enhanced expression of glial fibrillary acidic protein resulting from toxicant-induced neurodegeneration are linked to activation of the janus kinase (JAK)-signal transducer and activator of transcription-3 (STAT3) pathway. These observations implicate ligands at the gp130 receptor as potential upstream effectors of astrogliosis. Here we used the brain slice preparation to examine potential activators of the JAK-STAT3 pathway. Following incubation of freshly cut striatal slices in phosphate-free oxygenated buffer for up to 75 min, we found that slicing the striatum itself was a sufficient stimulus to initiate a rapid activation of the JAK-STAT3 pathway as assessed with immunoblots of pSTAT3((tyr705)) using phospho-state specific antibodies. The mRNA for the gp130 cytokines, leukemia inhibitory factor, interleukin-6 and oncostatin M or the beta-chemokine, monocyte chemoattractive protein (CCl2) also were up-regulated in the slice. Moreover, we could enhance the activation of STAT3((tyr705)) by adding exogenous cytokines to the slice and we could inhibit phosphorylation of STAT3((tyr705)) by addition of tyrosine kinase inhibitors (Lav A and AG490) or neutralizing antibodies directed against leukemia inhibitory factor or oncostatin M. These data suggest that STAT3 activation is an early event in slice-induced glial activation and establishes the brain slice preparation method as a reliable model to examine the signaling mechanisms that underlie glial activation.

Compulsive drug abuse has been conceptualized as a behavioral state where behavioral stimuli override normal decision making. Clinical studies of methamphetamine users have detailed decision making changes and imaging studies have found altered metabolism and activation in the parietal cortex. To examine the molecular effects of amphetamine (AMPH) on the parietal cortex, gene expression responses to amphetamine challenge (7.5 mg/kg) were examined in the parietal cortex of rats pretreated for nine days with either saline, non-neurotoxic amphetamine, or neurotoxic AMPH dosing regimens. The neurotoxic AMPH exposure [three doses of 7.5 mg/kg/day AMPH (6 h between doses), for nine days] produced histological signs of neurotoxicity in the parietal cortex while a non-neurotoxic dosing regimen (2.0 mg/kg/day x 3) did not. Neurotoxic AMPH pretreatment resulted in significantly diminished AMPH challenge-induced mRNA increases of activity-regulated cytoskeletal protein (ARC), nerve growth-factor inducible protein A (NGFI-A), and nerve growth-factor inducible protein B (NGFI-B) in the parietal cortex while neither saline pretreatment nor non-neurotoxic AMPH pretreatment did. This effect was specific to these genes as tissue plasminogen activator (t-PA), neuropeptide Y (NPY) and c-jun expression in response to AMPH challenge was unaltered or enhanced by amphetamine pretreatments. In the striatum, there were no differences between saline, neurotoxic AMPH, and non-neurotoxic AMPH pretreatments on ARC, NGFI-A or NGFI-B expression elicited by the AMPH challenge. These data indicate that the responsiveness of synaptic plasticity-related genes is sensitive to disruption specifically in the parietal cortex by threshold neurotoxic AMPH exposures.

Being awake, alert, and able to function in our 24-7 world is a challenge in the face of the fatigue and sleepiness engendered by long work hours, unusual work schedules, sickness, and other factors. Development of effective treatments to combat fatigue and sleepiness requires an understanding of the neurobiology of wakefulness. In this brief review, we examine the neuroanatomical, neurochemical, and molecular basis of the wakeful state to provide a framework for understanding current and future pharmacologic approaches to modification of wakefulness. The spontaneously awake state can be defined as a natural state of vigilance or arousal differing from natural sleep in both behavior and neural activity. These differences have long intrigued researchers and largely have been characterized in the brain areas and neurochemical systems affecting the sleep and wake states. Many of the strategies for promoting the awake condition involve manipulation or modulation of specific neurochemical systems with the ultimate goal of enhancing wakefulness, diminishing sleepiness, or both. Wakefulness is an important cortical function that depends on the coordinated effort of multiple brain areas including the thalamus, hypothalamus, and basal forebrain to integrate and relay information from the brainstem to the cortex. Norepinephrine and serotonin-long considered arousal-enhancing transmitters as well as glutamate, acetylcholine, histamine, and the neuromodulators hypocretin-orexins and adenosine, are known to affect the signal transduction in these brain areas and initiate, promote, or enhance wakefulness. Use of molecular tools to evaluate the awake, asleep, and sleep-deprived state has revealed novel insights concerning the gene expression events associated with wakefulness. Understanding wakefulness at this level undoubtedly will contribute to the development of pharmacologic approaches to promote or enhance the wakeful state. We caution, however, that sleep may have a necessary, restorative function for the brain; therefore, prolonging wakefulness for long periods through artificial means could have unexpected and perhaps detrimental consequences on brain health.

Capillary liquid chromatography (CLC) coupled off-line with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and TOF/TOF-MS were explored for identification and quantification of neuropeptides in microwave-fixed rat brain tissue. Sample was separated by gradient elution on 50-mum-inner diameter reversed-phase columns at 180 nL/min. Effluent was mixed with matrix solution and transferred to a MALDI target plate by pulsed electric field deposition, yielding sample spots with 200-300-mum diameter. Mass detection limits as low as 2 amol, corresponding to 1 pM concentration, were achieved for neuropeptides. CLC-MALDI-TOF-MS analysis of microwave-fixed rat striatum tissue yielded detection of over 400 distinctive peaks. CLC-MALDI-TOF/TOF-MS allowed identification of 10 peptides including 3 novel peptides. Quantification was evaluated using substance P as analyte and 15N3-labeled substance P as an internal standard. Quantification of substance P revealed approximately 6.8-fold higher levels than previously reported in the rat striatum. This increase is attributed to use of microwave fixation, which prevented degradation of the peptide, aggressive extraction procedures, and accounting for oxidation of substance P in the analysis. These results demonstrate that CLC-MALDI-TOF-MS is a versatile tool for neuropeptide analysis in brain tissue by allowing for detection, identification, and quantification.

Enhanced expression of tumor necrosis factor (TNF) -alpha, is associated with the neuropathological effects underlying disease-, trauma- and chemically induced neurodegeneration. Previously, we have shown that deficiency of TNF receptors protects against MPTP-induced striatal dopaminergic neurotoxicity, findings suggestive of a role for TNF-alpha in neurodegeneration. Here, we demonstrate that deficiency of TNF receptors suppresses microglial activation and alters the susceptibility of brain regions to MPTP. MPTP-induced expression of microglia-derived factors, TNF-alpha, MCP-1, and IL-1alpha, preceded the degeneration of striatal dopaminergic nerve terminals and astrogliosis, as assessed by loss of striatal dopamine and TH, and an increase in striatal GFAP. Pharmacological neuroprotection with the dopamine reuptake inhibitor, nomifensine, abolished striatal dopaminergic neurotoxicity and associated microglial activation. Similarly, in mice lacking TNF receptors, microglial activation was suppressed, findings consistent with a role for TNF-alpha in striatal MPTP neurotoxicity. In the hippocampus, however, TNF receptor-deficient mice showed exacerbated neuronal damage after MPTP, as evidenced by Fluoro Jade-B staining (to identify degenerating neurons) and decreased microtubule-associated protein-2 (MAP-2) immunoreactivity. These effects were not accompanied by microglial activation, but were associated with increased oxidative stress (nitrosylation of tyrosine residues). These findings suggest that TNF-alpha exerts a neurotrophic/neuroprotective effect in hippocampus. The marked differences we observed in the regional density, distribution and/or activity of microglia and microglia-derived factors may influence the region-specific role for this cell type. Taken together, our results are indicative of a region-specific and dual role for TNF-alpha in the brain: a promoter of neurodegeneration in striatum and a protector against neurodegeneration in hippocampus.

Activated microglia are implicated in the pathogenesis of disease-, trauma- and toxicant-induced damage to the CNS, and strategies to modulate microglial activation are gaining impetus. A novel action of the tetracycline derivative minocycline is the ability to inhibit inflammation and free radical formation, factors that influence microglial activation. Minocycline is therefore being tested as a neuroprotective agent to alleviate CNS damage, although findings so far have yielded mixed results. Here, we showed that administration of a single low dose of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or methamphetamine (METH), a paradigm that causes selective degeneration of striatal dopaminergic nerve terminals without affecting the cell body in substantia nigra, increased the expression of mRNAs encoding microglia-associated factors F4/80, interleukin (IL)-1alpha, IL-6, monocyte chemoattractant protein-1 (MCP-1, CCL2) and tumor necrosis factor (TNF)-alpha. Minocycline treatment attenuated MPTP- or METH-mediated microglial activation, but failed to afford neuroprotection. Lack of neuroprotection was shown to be due to the inability of minocycline to abolish the induction of TNF-alpha and its receptors, thereby failing to modulate TNF signaling. Thus, TNF-alpha appeared to be an obligatory component of dopaminergic neurotoxicity. To address this possibility, we examined the effects of MPTP or METH in mice lacking genes encoding IL-6, CCL2 or TNF receptor (TNFR)1/2. Deficiency of either IL-6 or CCL2 did not alter MPTP neurotoxicity. However, deficiency of both TNFRs protected against the dopaminergic neurotoxicity of MPTP. Taken together, our findings suggest that attenuation of microglial activation is insufficient to modulate neurotoxicity as transient activation of microglia may suffice to initiate neurodegeneration. These findings support the hypothesis that TNF-alpha may play a role in the selective vulnerability of the nigrostriatal pathway associated with dopaminergic neurotoxicity and perhaps Parkinson's disease.

Serious questions have been raised regarding a possible causal association between neurological effects in welders and the presence of manganese in welding fume. An experimental model is needed that could examine the potential neurotoxic effect of manganese after pulmonary exposure to welding fume. The National Institute for Occupational Safety and Health (NIOSH) has recently finished construction of a completely automated, computer controlled welding fume generation and inhalation exposure system for laboratory animals. The system is comprised of a programmable six-axis robotic welding arm and a water-cooled arc welding torch. A flexible trunk has been attached to the robotic arm of the welder and is used to collect and transport fume from the vicinity of the arc to the animal exposure chamber. Preliminary fume characterization studies have indicated that particle morphology, size, and chemical composition were comparable to welding fume generated in the workplace. Animal inhalation studies are currently underway. With the development of this novel system, an animal model has been established using controlled welding exposures to investigate the possible mechanisms by which welding fume may affect the central nervous system.

We evaluated regional neuropathological changes in adult and aged male mice treated systemically with kainic acid (KA) in a strain reported to be resistant to excitotoxic neuronal damage, C57BL/6. KA was administered in a single intraperitoneal injection. Adult animals were dosed with 35 mg/kg KA, while aged animals received a dose of 20 mg/kg in order to prevent excessive mortality. At time-points ranging from 12 h to 7 days post-treatment, animals were sacrificed and prepared for histological evaluation utilizing the cupric-silver neurodegeneration stain, immunohistochemistry for GFAP and IgG, and lectin staining. In animals of both ages, KA produced argyrophilia in neurons throughout cortex, hippocampus, thalamus, and amygdala. Semi-quantitative analysis of neuropathology revealed a similar magnitude of damage in animals of both ages, even though aged animals received less toxicant. Additional animals were evaluated for KA-induced reactive gliosis, assayed by an ELISA for GFAP, which revealed a 2-fold elevation in protein levels in adult mice, and a 2.5-fold elevation in aged animals. Histochemical evaluation of GFAP and lectin staining revealed activation of astrocytes and microglia in regions with corresponding argyrophilia. IgG immunostaining revealed a KA-induced breach of the blood-brain barrier in animals of both ages. Our data indicate widespread neurotoxicity following kainic acid treatment in C57BL/6J mice, and reveal increased sensitivity to this excitotoxicant in aged animals.

A variety of '-omic' technologies are being increasingly applied in preclinical safety assessments. Such approaches, however, have not been implemented in neurotoxicity safety evaluations. Current regulatory guidelines for assessing neurotoxicity emphasise reliance on traditional histopathological stains and behavioural testing batteries. Although these methods may be sufficient to detect some neurotoxic effects, they lack both the sensitivity and specificity required for broad-scale neurotoxicity screening. The glial reaction to nervous system damage, often termed gliosis, represents a hallmark of all types of nervous system injury. As such, the development and implementation of gliosis biomarkers represents a broadly applicable approach for neurotoxicity safety assessment. Using a panel of known neurotoxic agents, the authors have shown that the astroglial protein, glial fibrillary acidic protein (GFAP), can serve as one such biomarker of neurotoxicity. Qualitative and quantitative analysis of GFAP has shown this biomarker to be a sensitive and specific indicator of the neurotoxic condition. The implementation of GFAP and related glial biomarkers in neurotoxicity screens may serve as the basis for further development of molecular signatures predictive of adverse effects on the nervous system.

Functional loss often occurs in many body systems (e.g., endocrine, cognitive, motor) with the passage of years, but there is great individual variation in the degree of compromise shown. The current focus on brain aging will continue because demographic trends indicate that the average lifespan will show a continued increase. There is increasing emphasis on understanding how aging contributes to a decline in brain functions, cognition being a prime example. This is due in part to the fact that dementias and other losses in brain function that sometimes accompany aging cause an obvious decline in the quality of life and these deficits are of more concern as the number of elderly increase. Stress also is a ubiquitous aspect of life and there is now a greater interest in understanding the role of stress and the stress response in brain aging. The key role of the hippocampus and its related brain structures in cognition, as well as in the feedback control of the response to stress, have made this brain area a logical focus of investigation for those interested in the impact of stress on brain aging. Here, we describe how the hippocampus changes with age and we examine the idea that age-related changes in the secretion patterns of the hypothalamic-pituitary adrenal (HPA) axis can contribute to aging of this structure. We also examine the proposal that stress, perhaps due to compromised HPA axis function, can contribute to hippocampal aging through exposure to excessive levels of glucocorticoids. The aging hippocampus does not appear to suffer a generalized loss of cells or synapses, although atrophy of the structure may occur in humans. Thus, age-related cognitive impairments are likely related to other neurobiological alterations that could include changes in the signaling, information encoding, plasticity, electrophysiological or neurochemical properties of neurons or glia. Although excessive levels of glucocorticoids are able to interfere with cognition, as well as hippocampal neuronal integrity, and aging is sometimes accompanied by an increase in these steroids because of inadequate feedback control of the HPA axis, none of these are a foregone consequence of aging. The general preservation of cells and the plastic potential of the hippocampus provide a focus for the development of pharmacological, nutritive or lifestyle strategies to combat age-related declines in the hippocampus as well as other brain areas.

To evaluate the ability of a tiered quantitative morphological approach to reveal developmental neurotoxicity, morphometric parameters were measured in the offspring of rats treated with methylazoxymethanol (MAM) during days 13-15 of pregnancy. Treatment was aimed at inhibiting the proliferation phase of hippocampal neurons while leaving cerebellar neurons unaffected. 2D and 3D assessment of brain morphology combined with straightforward measurement of brain size, weight and volume, and the usefulness of estimation of total neuron numbers were studied. Each tier indicated major effects of MAM, from macroscopic effects in the cerebrum (first tier) to a considerable loss of neurons in the hippocampal CA1 pyramidal layer (third tier). The cerebellum and the number of cerebellar granular neurons were not changed. Along with each step of the proposed tiered approach (brain size → linear morphometry → stereology), the discriminative strength of the endpoints, and thus the probability to pinpoint the extent and location of developmental brain lesions increased. © 2005 Elsevier Inc. All rights reserved.

In the present study, we examined whether calcium/calmodulin-dependent protein kinase II (CaMKII) is affected by chronic developmental Pb2+ exposure. The effects of Pb2+ exposure on rat hippocampal CaMKII were assessed by measuring CaMKII activity, phosphorylation of CaMKII at threonine-286, and CaMKII alpha and beta protein levels. In the hippocampus of Pb2+-exposed 50-day-old rats known to exhibit deficits in hippocampal long-term potentiation (LTP) and spatial learning, there was a marked reduction (41%) in the apparent maximal velocity (Vmax) of CaMKII and a significant increase (22%) in apparent affinity of the enzyme. These Pb2+-induced changes in CaMKII activity could not be explained by changes in enzyme phosphorylation at threonine-286 or sensitivity to calmodulin. In vitro incubation of hippocampal homogenates from control rats, but not from Pb2+-exposed rats, with Pb2+ prior to assay recapitulated the increase in the affinity of the enzyme observed with in vivo exposure to Pb2+. Western blots of cytosolic and membrane fractions from hippocampus showed a significant decrease in the levels of CaMKII-beta but not alpha protein in the cytosolic fraction of Pb2+-exposed rats. These findings indicate effects of developmental Pb2+ exposure on CaMKII, a component of calcium signaling associated with synaptic plasticity, learning, and memory.

Sensitive indices of neural injury were used to evaluate the time course of kainic acid (KA)-induced hippocampal damage in adult C57BL/6J mice (4 months), a strain previously reported to be resistant to kainate-induced neurotoxicity. Mice were injected systemically with saline or kainate, scored for seizure severity (Racine scale), and allowed to survive 12 h, one, three, or seven days following which they were evaluated for neuropathological changes using histological or biochemical endpoints. Most kainate-treated mice exhibited limited seizure activity (stage 1); however, cupric-silver and Fluoro-Jade B stains revealed significant damage by 12 h post-treatment. Immunohistochemistry and immunoassay of glial fibrillary acidic protein and lectin staining revealed a strong treatment-induced reactive gliosis and microglial activation. Immunostaining for immunoglobulin G revealed a kainate-induced breach in the blood-brain barrier. Nissl and hematoxylin stains provided little information regarding neuronal damage, but revealed the identity of non-resident cells which infiltrated the pyramidal layer. Our data suggest sensitive indicators of neural injury evaluated over a time course, both proximal and distal to treatment, are necessary to reveal the full extent of neuropathological changes which may be underestimated by traditional histological stains. The battery of neuropathological indices reported here reveals the C57BL/6J mouse is sensitive to excitotoxic neural damage caused by kainic acid, in the absence of tonic-clonic seizures.

Reactive gliosis is a hallmark of disease-, trauma-, and chemical-induced damage to the central nervous system. The signaling pathways associated with this response to neural injury remain to be elucidated, but recent evidence implicates the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway. Here, we used the known dopaminergic neurotoxicant, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), to selectively damage striatal dopaminergic nerve terminals and elicit a glial response. We then analyzed changes in gene expression and protein phosphorylation, in vivo, to identify ligands and mediators of the JAK-STAT pathway that accompany glial activation. Administration of MPTP caused rapid tyrosine (Tyr-705) phosphorylation and nuclear translocation of STAT3 in striatal astrocytes, prior to the induction of glial fibrillary acidic protein mRNA and protein. Pharmacological protection of dopaminergic nerve terminals with nomifensine abolished MPTP-mediated phosphorylation and translocation of STAT3 and prevented induction of astrogliosis. Among the Janus kinase family of tyrosine kinases, only JAK2 was associated with the phosphorylation of STAT3 after MPTP and, inhibition of JAK2 by AG490, in vivo, attenuated both the phosphorylation of STAT3 and induction of GFAP. The p44/42 mitogen-activated protein kinase (MAPK; ERK1/2) also was activated by MPTP, but was not associated with activation of STAT3, because serine (Ser-727) was not phosphorylated. The mRNA for ligands of the gp130-JAK/STAT3 signaling pathway, interleukin-6, leukemia inhibitory factor, and oncostatin M were elevated prior to activation of STAT3 and induction of astrogliosis; neuroprotection with nomifensine blocked these effects of MPTP. Taken together, our results suggest that the gp130-mediated activation of JAK2/STAT3 signaling pathway may play a key role in the induction of astrogliosis.

Aging is often simply defined as the decline in various body systems and functions (eg, endocrine, cognitive, motor, etc) that occur with the passage of time, although the degree of deterioration can vary greatly across individuals. Increases in average life span have brought a greater focus on brain aging. There is an emphasis on understanding how aging contributes to a decline in brain functions (eg, cognition) because such a decline adversely affects the quality of life. The hippocampus is a key brain structure for cognition and the feedback control of the stress response. Herein we describe how the hippocampus changes with age and we examine the idea that age-related changes in the secretory patterns of the hypothalamic-pituitary adrenal (HPA) axis can contribute to hippocampal aging. We also examine the proposal that cumulative stress, perhaps due to compromised HPA axis function, can contribute to hippocampal aging by subjecting it to exposure to excessive levels of glucocorticoids. The aging hippocampus does not appear to suffer a generalized loss of cells or synapses, although atrophy of the structure may occur in humans. Thus, age-related cognitive impairments are likely related to other neurobiological alterations that could include changes in the signaling, information encoding, and plastic, electrophysiological, or neurochemical properties of neurons or glia. Dysfunction of the HPA axis sometimes occurs with aging, and while excessive glucocorticoids can disrupt cognition as well as hippocampal neuronal integrity, these are not an inevitable consequence of aging. The general preservation of cells and the plastic potential of the hippocampus provide a focus for the development of pharmacological, nutritional, or life-style strategies to combat age-related declines.

Strain differences represent an overlooked variable that may play an important role in neurotoxic outcomes that can impact regulatory decision making. Here, we examined the strain-dependent effects of trimethyltin (TMT), a compound used as a positive control for behavioral and neurochemical assessments of neurotoxicity. Adult male Long-Evans (LE) and Fischer 344 (F344) rats (n=12 each) were trained to respond under a multiple, fixed-interval 3-min fixed-ratio 10-response (multi FI 3-min FR10) schedule of milk reinforcement. Acquisition was characterized by time-dependent changes in several behavioral endpoints in both strains, although rate of acquisition of the fixed-interval pattern of responding was slower in F344 rats. Steady-state (baseline) performance was characterized by slower overall rates of responding in F344 rats. There was little evidence of strain differences in many of the other baseline performance measures. Rats of each strain were then divided into two equal groups that received either 1 ml/kg saline or 8.0 mg/kg iv TMT approximately 18 h before the next test session. TMT produced transient changes in the performance of LE and F344 rats that lasted for several sessions. For many behavioral measures, F344 rats were more affected by TMT than were LE rats. TMT-induced reactive gliosis, as assessed by assaying glial fibrillary acidic protein (GFAP), was also greater in F344 rats than in LE rats. These results suggest F344 rats may be more susceptible to TMT-induced neurotoxicity than are LE rats.

Gene expression profiles in several brain regions of adult male rats were evaluated following a d -amphetamine (AMPH) exposure paradigm previously established to produce AMPH neurotoxicity. Escalating doses of AMPH (5–30 mg/kg) were given over the course of 16 h per day in an 18 °C environment for 2 days. This paradigm produces neurotoxicity but eliminates or minimizes the hyperthermia and seizure activity that might influence gene expression in a manner unrelated to the neurotoxic effects of AMPH. The expression of 1185 genes was monitored in the striatum, parietal cortex, piriform cortex and posteriolateral cortical amygdaloid nucleus (PLCo) using cDNA array technology, and potentially significant changes were verified by RT–PCR. Gene expression was determined at time points after AMPH when neurodegeneration was beginning to appear (16 h) or maximal (64 h). Expression was also determined 14 days after AMPH to find long-term changes in gene expression that might be biomarkers of a neurotoxic event. In the parietal cortex there was a two-fold increase in neuropeptide Y precursor protein mRNA whereas nerve growth factor-induced receptor protein I-A and I-B mRNA decreased 50% at 16 h after the end of AMPH exposure. Although these changes in expression were not observed in the PLCo, insulin-like growth factor binding protein 1 mRNA was increased two-fold in the PLCo at 16 and 64 h after AMPH. Changes in gene expression in the cortical regions were all between 1.2- and 1.5-fold 14 days after AMPH but some of these changes, such as annexin V increases, may be relevant to neurotoxicity. Gene expression was not affected by more than 1.5-fold at the time points in the striatum, although 65% dopamine depletions occurred, but the plasma membrane-associated dopamine transporter and dopamine D2 receptor were decreased about 40% in the substantia nigra at 64 h and 14 days post-AMPH. Thus, the 2-day AMPH treatment produced a few changes in gene expression in the two-fold range at time points 16 h or more after exposure but the majority of expression changes were less than 1.5-fold of control. Nonetheless, some of these lesser fold-changes appeared to be relevant to the neurotoxic process.