Corticosterone and exogenous glucose alter blood glucose levels, neurotoxicity, and vascular toxicity produced by methamphetamine.

Our previous studies have raised the possibility that altered blood glucose levels may influence and/or be predictive of methamphetamine (METH) neurotoxicity. This study evaluated the effects of exogenous glucose and corticosterone (CORT) pretreatment alone or in combination with METH on blood glucose levels and the neural and vascular toxicity produced. METH exposure consisted of four sequential injections of 5, 7.5, 10, and 10 mg/kg (2 h between injections) D-METH. The three groups given METH in combination with saline, glucose (METH+Glucose), or CORT (METH+CORT) had significantly higher glucose levels compared to the corresponding treatment groups without METH except at 3 h after the last injection. At this last time point, the METH and METH+Glucose groups had lower levels than the non-METH groups, while the METH+CORT group did not. CORT alone or glucose alone did not significantly increase blood glucose. Mortality rates for the METH+CORT (40%) and METH+Glucose (44%) groups were substantially higher than the METH (< 10%) group. Additionally, METH+CORT significantly increased neurodegeneration above the other three METH treatment groups (≈ 2.5-fold in the parietal cortex). Thus, maintaining elevated levels of glucose during METH exposure increases lethality and may exacerbate neurodegeneration. Neuroinflammation, specifically microglial activation, was associated with degenerating neurons in the parietal cortex and thalamus after METH exposure. The activated microglia in the parietal cortex were surrounding vasculature in most cases and the extent of microglial activation was exacerbated by CORT pretreatment. Our findings show that acute CORT exposure and elevated blood glucose levels can exacerbate METH-induced vascular damage, neuroinflammation, neurodegeneration and lethality. Cover Image for this issue: doi. 10.1111/jnc.13819.

Vascular-directed responses of microglia produced by methamphetamine exposure: indirect evidence that microglia are involved in vascular repair?

BACKGROUND: Brain microglial activations and damage responses are most commonly associated with neurodegeneration or systemic innate immune system activation. Here, we used histological methods to focus on microglial responses that are directed towards brain vasculature, previously undescribed, after a neurotoxic exposure to methamphetamine. METHODS: Male rats were given doses of methamphetamine that produce pronounced hyperthermia, hypertension, and toxicity. Identification of microglia and microglia-like cells (pericytes and possibly perivascular cells) was done using immunoreactivity to allograft inflammatory factor 1 (Aif1 a.k.a Iba1) and alpha M integrin (Itgam a.k.a. Cd11b) while vasculature endothelium was identified using rat endothelial cell antigen 1 (RECA-1). Regions of neuronal, axonal, and nerve terminal degeneration were determined using Fluoro-Jade C. RESULTS: Dual labeling of vasculature (RECA-1) and microglia (Iba1) showed a strong association of hypertrophied cells surrounding and juxtaposed to vasculature in the septum, medial dorsal hippocampus, piriform cortex, and thalamus. The Iba1 labeling was more pronounced in the cell body while Cd11b more so in the processes of activated microglia. These regions have been previously identified to have vascular leakage after neurotoxic methamphetamine exposure. Dual labeling with Fluoro-Jade C and Iba1 indicated that there was minimal or no evidence of neuronal damage in the septum and hippocampus where many hypertrophied Iba1-labeled cells were found to be associated with vasculature. Although microglial activation around the prominent neurodegeneration was found in the thalamus, there were also many examples of activated microglia associated with vasculature. CONCLUSIONS: The data implicate microglia, and possibly related cell types, in playing a major role in responding to methamphetamine-induced vascular damage, and possibly repair, in the absence of neurodegeneration. Identifying brain regions with hypertrophied/activated microglial-like cells associated with vasculature has the potential for identifying regions of more subtle examples of vascular damage and BBB compromise.

Comparison of the global gene expression of choroid plexus and meninges and associated vasculature under control conditions and after pronounced hyperthermia or amphetamine toxicity.

BACKGROUND: The meninges (arachnoid and pial membranes) and associated vasculature (MAV) and choroid plexus are important in maintaining cerebrospinal fluid (CSF) generation and flow. MAV vasculature was previously observed to be adversely affected by environmentally-induced hyperthermia (EIH) and more so by a neurotoxic amphetamine (AMPH) exposure. Herein, microarray and RT-PCR analysis was used to compare the gene expression profiles between choroid plexus and MAV under control conditions and at 3 hours and 1 day after EIH or AMPH exposure. Since AMPH and EIH are so disruptive to vasculature, genes related to vasculature integrity and function were of interest. RESULTS: Our data shows that, under control conditions, many of the genes with relatively high expression in both the MAV and choroid plexus are also abundant in many epithelial tissues. These genes function in transport of water, ions, and solutes, and likely play a role in CSF regulation. Most genes that help form the blood-brain barrier (BBB) and tight junctions were also highly expressed in MAV but not in choroid plexus. In MAV, exposure to EIH and more so to AMPH decreased the expression of BBB-related genes such as Sox18, Ocln, and Cldn5, but they were much less affected in the choroid plexus. There was a correlation between the genes related to reactive oxidative stress and damage that were significantly altered in the MAV and choroid plexus after either EIH or AMPH. However, AMPH (at 3 hr) significantly affected about 5 times as many genes as EIH in the MAV, while in the choroid plexus EIH affected more genes than AMPH. Several unique genes that are not specifically related to vascular damage increased to a much greater extent after AMPH compared to EIH in the MAV (Lbp, Reg3a, Reg3b, Slc15a1, Sct and Fst) and choroid plexus (Bmp4, Dio2 and Lbp). CONCLUSIONS: Our study indicates that the disruption of choroid plexus function and damage produced by AMPH and EIH is significant, but the changes may not be as pronounced as they are in the MAV, particularly for AMPH. Expression profiles in the MAV and choroid plexus differed to some extent and differences were not restricted to vascular related genes.

Chronic exposure to corticosterone enhances the neuroinflammatory and neurotoxic responses to methamphetamine.

Up-regulation of proinflammatory cytokines and chemokines in brain ("neuroinflammation") accompanies neurological disease and neurotoxicity. Previously, we documented a striatal neuroinflammatory response to acute administration of a neurotoxic dose of methamphetamine (METH), i.e. one associated with evidence of dopaminergic terminal damage and activation of microglia and astroglia. When we used minocycline to suppress METH-induced neuroinflammation, indices of dopaminergic neurotoxicity were not affected, but suppression of neuroinflammation was incomplete. Here, we administered the classic anti-inflammatory glucocorticoid, corticosterone (CORT), in an attempt to completely suppress METH-related neuroinflammation. METH alone caused large increases in striatal proinflammatory cytokine/chemokine mRNA and subsequent astrocytic hypertrophy, microglial activation, and dopaminergic nerve terminal damage. Pre-treatment of mice with acute CORT failed to prevent neuroinflammatory responses to METH. Surprisingly, when mice were pre-treated with chronic CORT in the drinking water, an enhanced striatal neuroinflammatory response to METH was observed, an effect that was accompanied by enhanced METH-induced astrogliosis and dopaminergic neurotoxicity. Chronic CORT pre-treatment also sensitized frontal cortex and hippocampus to mount a neuroinflammatory response to METH. Because the levels of chronic CORT used are associated with high physiological stress, our data suggest that chronic CORT therapy or sustained physiological stress may sensitize the neuroinflammatory and neurotoxicity responses to METH.

Endoplasmic reticulum stress responses differ in meninges and associated vasculature, striatum, and parietal cortex after a neurotoxic amphetamine exposure.

Amphetamine (AMPH) is used to treat attention deficit and hyperactivity disorders, but it can produce neurotoxicity and adverse vascular effects at high doses. The endoplasmic reticulum (ER) stress response (ERSR) entails the unfolded protein response, which helps to avoid or minimize ER dysfunction. ERSR is often associated with toxicities resulting from the accumulation of unfolded or misfolded proteins and has been associated with methamphetamine toxicity in the striatum. The present study evaluates the effect of AMPH on several ERSR elements in meninges and associated vasculature (MAV), parietal cortex, and striatum. Adult, male Sprague-Dawley rats were exposed to saline, environmentally induced hyperthermia (EIH) or four consecutive doses of AMPH that produce hyperthermia. Expression changes (mRNA and protein levels) of key ERSR-related genes in MAV, striatum, and parietal cortex at 3 h or 1 day postdosing were monitored. AMPH increased the expression of some ERSR-related genes in all tissues. Atf4 (activating transcription factor 4, an indicator of Perk pathway activation), Hspa5/Grp78 (Glucose regulated protein 78, master regulator of ERSR), Pdia4 (protein disulfide isomerase, protein-folding enzyme), and Nfkb1 (nuclear factor of kappa b, ERSR sensor) mRNA increased significantly in MAV and parietal cortex 3 h after AMPH. In striatum, Atf4 and Hspa5/Grp78 mRNA significantly increased 3 h after AMPH, but Pdia4 and Nfkb11 did not. Thus, AMPH caused a robust activation of the Perk pathway in all tissues, but significant Ire1 pathway activation occurred only after AMPH treatment in the parietal cortex and striatum. Ddit3/Chop, a downstream effector of the ERSR pathway related to the neurotoxicity, was only increased in striatum and parietal cortex. Conversely, Pdia4, an enzyme protective in the ERSR, was only increased in MAV. The overall ERSR manifestation varied significantly between MAV, striatum, and parietal cortex after a neurotoxic exposure to AMPH.

Regions of the basal ganglia and primary olfactory system are most sensitive to neurodegeneration after extended sevoflurane anesthesia in the perinatal rat.

Extended general anesthesia early in life is neurotoxic in multiple species. However, little is known about the temporal progression of neurodegeneration after general anesthesia. It is also unknown if a reduction in natural cell death, or an increase in cell creation, occurs as a form of compensation after perinatal anesthesia exposure. The goal of this study was to evaluate markers of neurodegeneration and cellular division at 2, 24, or 72 h after sevoflurane (Sevo) exposure (6 h) in fully oxygenated postnatal day (PND) 7 rats. Neurodegeneration was observed in areas throughout the forebrain, while the largest changes (fold increase above vehicle) were observed in areas associated with either the primary olfactory learning pathways or the basal ganglia. These regions included the indusium griseum (IG, 25-fold), the posterior dorso medial hippocampal CA1 (17-fold), bed nucleus of the stria terminalis (Bed Nuclei STM, 5-fold), the shell of the nucleus accumbens (Acb, 5-fold), caudate/putamen (CPu, 5-fold), globus pallidus (GP, 9-fold) and associated thalamic (11-fold) and cortical regions (5-fold). Sevo neurodegeneration was minimal or undetectable in the ventral tegmentum, substantia nigra, and most of the hypothalamus and frontal cortex. In most brain regions where neurodegeneration was increased 2 h post Sevo exposure, the levels returned to <4-fold above control levels by 24 h. However, in the IG, CA1, GP, anterior thalamus, medial preoptic nucleus of the hypothalamus (MPO), anterior hypothalamic area (AHP), and the amygdaloid nuclei, neurodegeneration at 24 h was double or more than that at 2 h post exposure. Anesthesia exposure causes either a prolonged period of neurodegeneration in certain brain regions, or a distinct secondary degenerative event occurs after the initial insult. Moreover, regions most sensitive to Sevo neurodegeneration did not necessarily coincide with areas of new cell birth, and new cell birth was not consistently affected by Sevo. The profile of anesthesia related neurotoxicity changes with time, and multiple mechanisms of toxicity may exist in a time-dependent fashion.

Microglial activation and responses to vasculature that result from an acute LPS exposure.

Bacterial cell wall endotoxins, i.e. lipopolysaccharides (LPS), are some of the original compounds shown to evoke the classic signs of systemic inflammation/innate immune response and neuroinflammation. The term neuroinflammation often is used to infer the elaboration of proinflammatory mediators by microglia elicited by neuronal targeted activity. However, it also is possible that the microglia are responding to vasculature through several signaling mechanisms. Microglial activation relative to the vasculature in the hippocampus and parietal cortex was determined after an acute exposure of a single subcutaneous injection of 2 mg/kg LPS. Antibodies to allograft inflammatory factor (Aif1, a.k.a. Iba1) were used to track and quantify morphological changes in microglia. Immunostaining of platelet/endothelial cell adhesion molecule 1 (Pecam1, a.k.a. Cd31) was used to visualize vasculature in the forebrain and glial acidic fibrillary protein (GFAP) to visualize astrocytes. Neuroinflammation and other aspects of neurotoxicity were evaluated histologically at 3 h, 6 h, 12 h, 24 h, 3 d and 14 d following LPS exposure. LPS did not cause neurodegeneration as determined by Fluoro Jade C labeling. Also, there were no signs of mouse IgG leakage from brain vasculature due to LPS. Some changes in microglia size occurred at 6 h, but by 12 h microglial activation had begun with the combined soma and proximal processes size increasing significantly (1.5-fold). At 24 h, almost all the microglia soma and proximal processes in the hippocampus, parietal cortex, and thalamus were closely associated with the vasculature and had increased almost 2.0-fold in size. In many areas where microglia were juxtaposed to vasculature, astrocytic endfeet appeared to be displaced. The microglial activation had subsided slightly by 3 d with microglial size 1.6-fold that of control. We hypothesize that acute LPS activation can result in vascular mediated microglial responses through several mechanisms: 1) binding to Cd14 and Tlr4 receptors on microglia processes residing on vasculature; 2) damaging vasculature and causing the release of cytokines; and 3) possibly astrocytic endfeet damage resulting in cytokine release. These acute responses may serve as an adaptive mechanism to exposure to circulating LPS where the microglia surround the vasculature. This could further prevent the pathogen(s) circulating in blood from entering the brain. However, diverting microglial interactions away from synaptic remodeling and other types of microglial interactions with neurons may have adverse effects on neuronal function.

The mRNA expression and histological integrity in rat forebrain motor and sensory regions are minimally affected by acrylamide exposure through drinking water.

A study was undertaken to determine whether alterations in the gene expression or overt histological signs of neurotoxicity in selected regions of the forebrain might occur from acrylamide exposure via drinking water. Gene expression at the mRNA level was evaluated by cDNA array and/or RT-PCR analysis in the striatum, substantia nigra and parietal cortex of rat after a 2-week acrylamide exposure. The highest dose tested (maximally tolerated) of approximately 44 mg/kg/day resulted in a significant decreased body weight, sluggishness, and locomotor activity reduction. These physiological effects were not accompanied by prominent changes in gene expression in the forebrain. All the expression changes seen in the 1200 genes that were evaluated in the three brain regions were < or =1.5-fold, and most not significant. Very few, if any, statistically significant changes were seen in mRNA levels of the more than 50 genes directly related to the cholinergic, noradrenergic, GABAergic or glutamatergic neurotransmitter systems in the striatum, substantia nigra or parietal cortex. All the expression changes observed in genes related to dopaminergic function were less than 1.5-fold and not statistically significant and the 5HT1b receptor was the only serotonin-related gene affected. Therefore, gene expression changes were few and modest in basal ganglia and sensory cortex at a time when the behavioral manifestations of acrylamide toxicity had become prominent. No histological evidence of axonal, dendritic or neuronal cell body damage was found in the forebrain due to the acrylamide exposure. As well, microglial activation was not present. These findings are consistent with the absence of expression changes in genes related to changes in neuroinflammation or neurotoxicity. Over all, these data suggest that oral ingestion of acrylamide in drinking water or food, even at maximally tolerable levels, induced neither marked changes in gene expression nor neurotoxicity in the motor and somatosensory areas of the central nervous system.

Amphetamine and environmentally induced hyperthermia differentially alter the expression of genes regulating vascular tone and angiogenesis in the meninges and associated vasculature.

An amphetamine (AMPH) regimen that does not produce a prominent blood-brain barrier breakdown was shown to significantly alter the expression of genes regulating vascular tone, immune function, and angiogenesis in vasculature associated with arachnoid and pia membranes of the forebrain. Adult-male Sprague-Dawley rats were given either saline injections during environmentally-induced hyperthermia (EIH) or four doses of AMPH with 2 h between each dose (5, 7.5, 10, and 10 mg/kg d-AMPH, s.c.) that produced hyperthermia. Rats were sacrificed either 3 h or 1 day after dosing, and total RNA and protein was isolated from the meninges, arachnoid and pia membranes, and associated vasculature (MAV) that surround the forebrain. Vip, eNos, Drd1a, and Edn1 (genes regulating vascular tone) were increased by either EIH or AMPH to varying degrees in MAV, indicating that EIH and AMPH produce differential responses to enhance vasodilatation. AMPH, and EIH to a lesser extent, elicited a significant inflammatory response at 3 h as indicated by an increased MAV expression of cytokines Il1b, Il6, Ccl-2, Cxcl1, and Cxcl2. Also, genes related to heat shock/stress and disruption of vascular homeostasis such as Icam1 and Hsp72 were also observed. The increased expression of Ctgf and Timp1 and the decreased expression of Akt1, Anpep, and Mmp2 and Tek (genes involved in stimulating angiogenesis) from AMPH exposure suggest that angiogenesis was arrested or disrupted in MAV to a greater extent by AMPH compared to EIH. Alterations in vascular-related gene expression in the parietal cortex and striatum after AMPH were less in magnitude than in MAV, indicating less of a disruption of vascular homeostasis in these two regions. Changes in the levels of insulin-like growth factor binding proteins Igfbp1, 2, and 5 in MAV, compared to those in striatum and parietal cortex, imply an interaction between these regions to regulate the levels of insulin-like growth factor after AMPH damage. Thus, the vasculature and meninges surrounding the surface of the forebrain may be an important region in which AMPHs can disrupt vascular homeostasis.

Identification of whole blood mRNA and microRNA biomarkers of tissue damage and immune function resulting from amphetamine exposure or heat stroke in adult male rats.

This work extends the understanding of how toxic exposures to amphetamine (AMPH) adversely affect the immune system and lead to tissue damage. Importantly, it determines which effects of AMPH are and are not due to pronounced hyperthermia. Whole blood messenger RNA (mRNA) and whole blood and serum microRNA (miRNA) transcripts were identified in adult male Sprague-Dawley rats after exposure to toxic AMPH under normothermic conditions, AMPH when it produces pronounced hyperthermia, or environmentally-induced hyperthermia (EIH). mRNA transcripts with large increases in fold-change in treated relative to control rats and very low expression in the control group were a rich source of organ-specific transcripts in blood. When severe hyperthermia was produced by either EIH or AMPH, significant increases in circulating organ-specific transcripts for liver (Alb, Fbg, F2), pancreas (Spink1), bronchi/lungs (F3, Cyp4b1), bone marrow (Np4, RatNP-3b), and kidney (Cesl1, Slc22a8) were observed. Liver damage was suggested also by increased miR-122 levels in the serum. Increases in muscle/heart-enriched transcripts were produced by AMPH even in the absence of hyperthermia. Expression increases in immune-related transcripts, particularly Cd14 and Vcan, indicate that AMPH can activate the innate immune system in the absence of hyperthermia. Most transcripts specific for T-cells decreased 50-70% after AMPH exposure or EIH, with the noted exception of Ccr5 and Chst12. This is probably due to T-cells leaving the circulation and down-regulation of these genes. Transcript changes specific for B-cells or B-lymphoblasts in the AMPH and EIH groups ranged widely from decreasing ≈ 40% (Cd19, Cd180) to increasing 30 to 100% (Tk1, Ahsa1) to increasing ≥500% (Stip1, Ackr3). The marked increases in Ccr2, Ccr5, Pld1, and Ackr3 produced by either AMPH or EIH observed in vivo provide further insight into the initial immune system alterations that result from methamphetamine and AMPH abuse and could modify risk for HIV and other viral infections.

Microglial activation and vascular responses that are associated with early thalamic neurodegeneration resulting from thiamine deficiency.

Thiamine/vitamin B1 deficiency can lead to behavioral changes and neurotoxicity in humans. This may due in part to vascular damage, neuroinflammation and neuronal degeneration in the diencephalon, which is seen in animal models of pyrithiamine-enhanced thiamine deficiency. However, the time course of the progression of these changes in the animal models has been poorly characterized. Therefore, in this study, the progression of: 1) activated microglial association with vasculature; 2) neurodegeneration; and 3) any vascular leakage in the forebrain during the progress of thiamine deficiency were determined. A thiamine deficient diet along with 0.25 mg/kg/d of pyrithiamine was used as the mouse model. Vasculature was identified with Cd31 and microglia with Cd11b and Iba1 immunoreactivity. Neurodegeneration was determined by FJc labeling. The first sign of activated microglia within the thalamic nuclei were detected after 8 d of thiamine deficiency, and by 9 d activated microglia associated primarily with vasculature were clearly present but only in thalamus. At the 8 d time point neurodegeneration was not present in thalamus. However at 9 d, the first signs of neurodegeneration (FJc + neurons) were seen in most animals. Over 80% of the microglia were activated at 10 d but almost exclusively in the thalamus and the number of degenerating neurons was less than 10% of the activated microglia. At 10 d, there were sporadic minor changes in IgG presence in thalamus indicating minor vascular leakage. Dizocilpine (0.2-0.4 mg/kg) or phenobarbital (10-20 mg/kg) was administered to groups of mice from day 8 through day 10 to block neurodegeneration but neither did. In summary, activated microglia start to surround vasculature 1-2 d prior to the start of neurodegeneration. This response may be a means of controlling or repairing vascular damage and leakage. Glutamate excitotoxicity and vascular leakage likely only play a minor role in the early neurodegeneration resulting from thiamine deficiency. However, failure of dysfunctional vasculature endothelium to supply sufficient nutrients to neurons could be contributing to the neurodegeneration.

The time course of blood brain barrier leakage and its implications on the progression of methamphetamine-induced seizures.

The initial goals of these experiments were to determine: 1) if blood-brain barrier (BBB) breakdown was a cause or an effect of METH-induced seizures; 2) all the brain regions where BBB is disrupted as seizures progress; and 3) the correlations between body temperature and vascular leakage and neurodegeneration. A fourth objective was added after initial experimentation to determine if sub-strain differences existed in adult male C57 B6 J (Jackson laboratories, JAX) versus C57 B6N (Charles River, CR) mice involving their susceptibility to BBB breakdown and seizure severity. With the 1st "maximal" intensity myoclonic-tonic seizure (MCT) varying degrees of IgG infiltration across the BBB (≤1 mm2) were prominent in olfactory system (OS) associated regions and in thalamus, hypothalamus and neocortex. IgG infiltration areas in the OS-associated regions of the bed nucleus of the stria terminalis, septum and more medial amygdala nuclei, and the hypothalamus were increased significantly by the time continuous behavioral seizures (CBS) developed. Mice receiving METH that had body temperatures of ≥40 °C had IgG infiltration along with MCT or CBS but peak body temperatures above 40 °C did not significantly increase IgG infiltration. Neurodegeneration seen at ≥6 h was restricted to the OS in both JAX and CR mice and was most prominent in the posteromedial cortical amygdaloid nucleus. Neurodegeneration in the anterior septum (tenia tecta) was seen only in the JAX mice. We hypothesize that METH-induced hypertension and hyperthermia lead to BBB breakdown and other vascular dysfunctions in the OS brain regions resulting in OS hyperexcitation. Excitation of the OS neural network then leads to the development of seizures. These seizures in turn exacerbate the energy depletions and the reactive oxygen stress produced by hyperthermia further damaging the BBB and vascular function. These events form a recurrent cycle that results in ever increasing seizure activity and neurotoxicity.

Multi-class computational evolution: development, benchmark evaluation and application to RNA-Seq biomarker discovery.

BACKGROUND: A computational evolution system (CES) is a knowledge discovery engine that can identify subtle, synergistic relationships in large datasets. Pareto optimization allows CESs to balance accuracy with model complexity when evolving classifiers. Using Pareto optimization, a CES is able to identify a very small number of features while maintaining high classification accuracy. A CES can be designed for various types of data, and the user can exploit expert knowledge about the classification problem in order to improve discrimination between classes. These characteristics give CES an advantage over other classification and feature selection algorithms, particularly when the goal is to identify a small number of highly relevant, non-redundant biomarkers. Previously, CESs have been developed only for binary class datasets. In this study, we developed a multi-class CES. RESULTS: The multi-class CES was compared to three common feature selection and classification algorithms: support vector machine (SVM), random k-nearest neighbor (RKNN), and random forest (RF). The algorithms were evaluated on three distinct multi-class RNA sequencing datasets. The comparison criteria were run-time, classification accuracy, number of selected features, and stability of selected feature set (as measured by the Tanimoto distance). The performance of each algorithm was data-dependent. CES performed best on the dataset with the smallest sample size, indicating that CES has a unique advantage since the accuracy of most classification methods suffer when sample size is small. CONCLUSION: The multi-class extension of CES increases the appeal of its application to complex, multi-class datasets in order to identify important biomarkers and features.

Effect of arylformamidase (kynurenine formamidase) gene inactivation in mice on enzymatic activity, kynurenine pathway metabolites and phenotype.

The gene coding for arylformamidase (Afmid, also known as kynurenine formamidase) was inactivated in mice through the removal of a shared bidirectional promoter region regulating expression of the Afmid and thymidine kinase (Tk) genes. Afmid/Tk -deficient mice are known to develop sclerosis of glomeruli and to have an abnormal immune system. Afmid-catalyzed hydrolysis of N-formyl-kynurenine is a key step in tryptophan metabolism and biosynthesis of kynurenine-derived products including kynurenic acid, quinolinic acid, nicotinamide, NAD, and NADP. A disruption of these pathways is implicated in neurotoxicity and immunotoxicity. In wild-type (WT) mice, Afmid-specific activity (as measured by formyl-kynurenine hydrolysis) was 2-fold higher in the liver than in the kidney. Formyl-kynurenine hydrolysis was reduced by approximately 50% in mice heterozygous (HZ) for Afmid/Tk and almost completely eliminated in Afmid/Tk knockout (KO) mice. However, there was 13% residual formyl-kynurenine hydrolysis in the kidney of KO mice, suggesting the existence of a formamidase other than Afmid. Liver and kidney levels of nicotinamide plus NAD/NADP remained the same in WT, HZ and KO mice. Plasma concentrations of formyl-kynurenine, kynurenine, and kynurenic acid were elevated in KO mice (but not HZ mice) relative to WT mice, further suggesting that there must be enzymes other than Afmid (possibly in the kidney) capable of metabolizing formyl-kynurenine into kynurenine. Gradual kidney deterioration and subsequent failure in KO mice is consistent with high levels of tissue-specific Afmid expression in the kidney of WT but not KO mice. On this basis, the most significant function of the kynurenine pathway and Afmid in mice may be in eliminating toxic metabolites and to a lesser extent in providing intermediates for other processes.

Fluoro-Ruby labeling prior to an amphetamine neurotoxic insult shows a definitive massive loss of dopaminergic terminals and axons in the caudate-putamen.

Fluoro-Ruby (FR) was injected into the substantia nigra (SNc) to label dopaminergic axons and terminals in the caudate putamen (CPu) of rats 7 days prior to a neurotoxic d-amphetamine (AMPH) exposure. Three days after AMPH exposure, a massive loss in the TH immunoreactive (TH(+)) axons and terminals was seen in the CPu. The FR-labeled (FR(+)) axons and terminals in the CPu were greatly diminished with those remaining being enlarged or swollen after AMPH. Fluoro-Jade C (FJ-C) labeling was used to verify AMPH-induced axonal and terminal degeneration. This study demonstrates that fluorescent anterograde tract tracers can be used to show the subsequent axonal and terminal degeneration after systemic exposures to toxins and provides direct evidence that CPu axons and terminals from SNc dopaminergic neurons can be destroyed after neurotoxic exposure to AMPH.

An Iterative Leave-One-Out Approach to Outlier Detection in RNA-Seq Data.

The discrete data structure and large sequencing depth of RNA sequencing (RNA-seq) experiments can often generate outlier read counts in one or more RNA samples within a homogeneous group. Thus, how to identify and manage outlier observations in RNA-seq data is an emerging topic of interest. One of the main objectives in these research efforts is to develop statistical methodology that effectively balances the impact of outlier observations and achieves maximal power for statistical testing. To reach that goal, strengthening the accuracy of outlier detection is an important precursor. Current outlier detection algorithms for RNA-seq data are executed within a testing framework and may be sensitive to sparse data and heavy-tailed distributions. Therefore, we propose a univariate algorithm that utilizes a probabilistic approach to measure the deviation between an observation and the distribution generating the remaining data and implement it within in an iterative leave-one-out design strategy. Analyses of real and simulated RNA-seq data show that the proposed methodology has higher outlier detection rates for both non-normalized and normalized negative binomial distributed data.

Evaluating the Stability of RNA-Seq Transcriptome Profiles and Drug-Induced Immune-Related Expression Changes in Whole Blood.

Methods were developed to evaluate the stability of rat whole blood expression obtained from RNA sequencing (RNA-seq) and assess changes in whole blood transcriptome profiles in experiments replicated over time. Expression was measured in globin-depleted RNA extracted from the whole blood of Sprague-Dawley rats, given either saline (control) or neurotoxic doses of amphetamine (AMPH). The experiment was repeated four times (paired control and AMPH groups) over a 2-year span. The transcriptome of the control and AMPH-treated groups was evaluated on: 1) transcript levels for ribosomal protein subunits; 2) relative expression of immune-related genes; 3) stability of the control transcriptome over 2 years; and 4) stability of the effects of AMPH on immune-related genes over 2 years. All, except one, of the 70 genes that encode the 80s ribosome had levels that ranked in the top 5% of all mean expression levels. Deviations in sequencing performance led to significant changes in the ribosomal transcripts. The overall expression profile of immune-related genes and genes specific to monocytes, T-cells or B-cells were well represented and consistent within treatment groups. There were no differences between the levels of ribosomal transcripts in time-matched control and AMPH groups but significant differences in the expression of immune-related genes between control and AMPH groups. AMPH significantly increased expression of some genes related to monocytes but down-regulated those specific to T-cells. These changes were partially due to changes in the two types of leukocytes present in blood, which indicate an activation of the innate immune system by AMPH. Thus, the stability of RNA-seq whole blood transcriptome can be verified by assessing ribosomal protein subunits and immune-related gene expression. Such stability enables the pooling of samples from replicate experiments to carry out differential expression analysis with acceptable power.

Classification of cDNA array genes that have a highly significant discriminative power due to their unique distribution in four brain regions.

Novel statistical methods were used to distinguish functionally distinct brain regions using their cDNA array gene expression profiles, and it was found that one of four specific factors is often associated with the most regionally discriminative genes. The gene expression profiles for the substantia nigra (SN), striatum (STR), parietal cortex (PC), and posterolateral cortical amygdaloid nucleus (PLCo) brain regions were determined from each brain region. An F-test identified 339 genes of the 1185 array genes as having a P < or = 0.01 and applied a gene ranking and selection method based on Soft Independent Modeling of Class Analogy (SIMCA) to obtain 59 of the most discriminative genes. Their discriminative power was validated in three steps. The most convincing step showed their ability to correctly predict the brain regional classifications for 18 "test" gene expression sets obtained from the four regions. A two-way Hierarchical Cluster Analysis organized the 59 genes in six clusters according to their expression differences in the brain regions. Expression patterns in the SN and STR regions greatly differed from each other and the PC and PLCo. The closer similarity in the gene expression patterns of the PC and PLCo was probably due to their functional similarity. The important factors in determining differences in the regional gene expression profiles in six clusters were (1) regional myelin/oligodendrocyte levels, (2) resident neuron types, (3) neurotransmitter innervation profiles, and (4) Ca++-dependent signaling and second messenger systems.

A statistical approach in using cDNA array analysis to determine modest changes in gene expression in several brain regions after neurotoxic insult.

Modest changes in gene expression of three-fold or less might be expected after mild to moderate neurotoxic exposure to classes of compounds, such as the substituted amphetamines, or at time points that are weeks after more severe neurotoxic exposures. When many genes appear to change expression by less than two-fold, it is crucial to run several pairs of arrays and use statistical analysis to determine which genes are really changing. This limits the number of genes that have to undergo the time consuming task of performing RT-PCR to validate change in expression levels. We describe here methods for statistically determining which genes are being expressed above background levels. These methods are used to compare expression differences among the striatum, parietal cortex, posterior lateral amygdaloid nucleus, and substantia nigra brain regions, all of which differ significantly in their gene expression profiles. In these comparisons, it was possible to distinguish differences among hundreds of genes with manageable estimated false discovery rates. The effect of amphetamine treatment on gene expression in posterior lateral amygdaloid nucleus was also evaluated. The expression data indicate that many genes have changed, but in this case it is more difficult to separate affected genes from false positives. The optimum list has 50 genes, of which 32% are expected to be false positives.

Parvalbumin neuron circuits and microglia in three dopamine-poor cortical regions remain sensitive to amphetamine exposure in the absence of hyperthermia, seizure and stroke.

The dopamine-releasing and depleting substance amphetamine (AMPH) can make cortical neurons susceptible to damage, and the prevention of hyperthermia, seizures and stroke is thought to block these effects. Here we report a 2-day AMPH treatment paradigm which affected only interneurons in three cortical regions with average or below-average dopamine input. AMPH (six escalating doses/day ranging from 5 to 30 mg/kg for 2 days) was given at 17-18 degrees C ambient temperature (T) to adult male rats. During the 2-day AMPH treatment, peak body T stayed below 38.9 degrees C in 40% of the AMPH treated rats. In 60% of the rats, deliberate cooling suppressed (<39.5 degrees C) or minimized (<40.0 degrees C) hyperthermia. Escalation of stereotypes to seizure-like behaviors was rare and post-mortem morphological signs of stroke were absent. Neurons labeled with the anionic, neurodegeneration-marker dye Fluoro-Jade (F-J) were seen 1 day after dosing, peaked 3 days later, but were barely detectable 14 days after dosing. Only nonpyramidal neurons in layer IV of the somatosensory barrel cortex and in layer II of the piriform cortex and posterolateral cortical amygdaloid nucleus were labeled with Fluoro-Jade. Isolectin B-labeled activated microglia were only detected in their neighborhood. F-J labeled neurons were extremely rare in cortical regions rich in dopamine (e.g. cingulate cortex), and were absent in cortical regions with no dopamine (e.g. visual cortex). Parvalbumin was seen in some Fluoro-Jade-labeled neurons and parvalbumin immunostaining in local axon plexuses intensified. This AMPH paradigm affected fewer cortical regions, and caused smaller reduction in striatal tyrosine hydroxylase (TH) immunoreactivity than previous 1-day AMPH regimens generating seizures or severe (above 40 degrees C) hyperthermia. Correlation between peak or mean body T and the extent of neurodegeneration or microgliosis was below statistical significance. Astrogliosis (elevated levels of the astroglia-marker, glial fibrillary acidic protein (GFAP)) was detected in many brain regions. In the striatum and midbrain, F-J labeled neurons and activated microglia were absent, but astrogliosis, decreased TH immunolabel, and swollen TH fibers were detected. In sum, after this AMPH treatment, cortical pyramidal neurons were spared, but astrogliosis was brain-wide and some interneurons and microglia in three cortical regions with average or below-average dopamine input remained sensitive to AMPH exposure.