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Exposure to the natural pesticide, rotenone, a potent mitochondrial toxin, leads to degeneration in striatal nerve terminals and nigral neurons. Rotenone-induced behavioral, neurochemical and neuropathological changes in rats mimic those observed in Parkinson's disease (PD). Here, protective effects of acetyl-L-carnitine (ALC) in the brain dopaminergic toxicity after a prolonged exposure to rotenone were evaluated using electrophysiological and immunolabeling methods. Adult, male Sprague-Dawley rats were injected i.p. with rotenone alone (1 mg/kg) or rotenone with ALC (either 10 or 100 mg/kg; ALC10 or ALC100, respectively) once daily on days 1, 3, 5, 8, 10, 12, 15, 17, 19, 22, 24, 26, 29, 31, 33 and 37. Control rats received either 100mg/kg ALC or vehicle (30% Solutol HS 15 in 0.9% saline) injections. Animals were weighed on injection days and monitored daily. Motor nerve conduction velocity (MCV) was assessed within two days after treatment using compound muscle action potentials (CMAP) detected from the tail muscle through surface receiver electrodes installed around the distal part of the tail. Rats were perfused immediately after testing with 4% paraformaldehyde and immunohistochemical analysis of dopamine transporter (DAT), tyrosine hydroxylase (TH), glial fibrillary acidic protein (GFAP), and microglial CD11b marker was performed in the caudate-putamen (CPu) and the substantia nigra pars compacta (SNc) in order to estimate dopaminergic neuronal and transporter damage. Additionally, effects of ALC on preventing microglial or astrocytic hypertrophy were also evaluated. In rats exposed to rotenone and rotenone/ACL10, a significant increases in both proximal (S1) and distal (S2) motor latency and a decrease in MCV were detected in tail nerves (p

Various iron-oxide nanoparticles have been in use for a long time as therapeutic and imaging agents and for supplemental delivery in cases of iron-deficiency. While all of these products have a specified size range of ∼40 nm and above, efforts are underway to produce smaller particles, down to ∼1 nm. Here, we show that after a 24-h exposure of SHSY-5Y human neuroblastoma cells to 10 μg/ml of 10 and 30 nm ferric oxide nanoparticles (Fe-NPs), cellular dopamine content was depleted by 68 and 52 %, respectively. Increases in activated tyrosine kinase c-Abl, a molecular switch induced by oxidative stress, and neuronal α-synuclein expression, a protein marker associated with neuronal injury, were also observed (55 and 38 % percent increases, respectively). Inhibition of cell-proliferation, significant reductions in the number of active mitochondria, and a dose-dependent increase in reactive oxygen species (ROS) were observed in neuronal cells. Additionally, using a rat in vitro blood–brain barrier (BBB) model, a dose-dependent increase in ROS accompanied by increased fluorescein efflux demonstrated compromised BBB integrity. To assess translational implications, in vivo Fe-NP-induced neurotoxicity was determined using in vivo MRI and post-mortem neurochemical and neuropathological correlates in adult male rats after exposure to 50 mg/kg of 10 nm Fe-NPs. Significant decrease in T 2 values was observed. Dynamic observations suggested transfer and retention of Fe-NPs from brain vasculature into brain ventricles. A significant decrease in striatal dopamine and its metabolites was also observed, and neuropathological correlates provided additional evidence of significant nerve cell body and dopaminergic terminal damage as well as damage to neuronal vasculature after exposure to 10 nm Fe-NPs. These data demonstrate a neurotoxic potential of very small size iron nanoparticles and suggest that use of these ferric oxide nanoparticles may result in neurotoxicity, thereby limiting their clinical application.

While there are currently no medications approved for methamphetamine (METH) addiction, it has been shown that propentofylline (PPF), an atypical methylxanthine, can suppress the rewarding effects of methamphetamine (METH) in mice. This experiment studied the interactions of PPF with METH in striatal dopaminergic transmission. Herein, the impact of PPF (10-40mM, intrastriatally perfused (80min) on the effect of METH (5mg/kg, i.p.) on striatal dopamine (DA) release was evaluated using brain microdialysis in Sprague-Dawley adult rats. METH was injected at the 60min time point of the 80min PPF perfusion. The extracellular levels of DA and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were determined using high performance liquid chromatography with electrochemical detection (HPLC-ED). PPF induced a concentration-dependent increase in DA release beginning 30min after the onset of PPF perfusion. DA peak levels evoked by 40mM PPF were similar to those induced by 5mg/kg METH i.p. Only the highest concentration of PPF decreased the METH-induced DA peak (circa 70%). The significant decreases in extracellular levels of DOPAC and HVA evoked by METH were partially blocked by 10 and 20mM PPF. Although 40mM of PPF also partially blocked the METH-induced DOPAC decrease, it completely blocked HVA depletion after a transient increase in HVA levels in METH-treated rats. Data indicates for the first time that while PPF increases presynaptic striatal DA dynamics it attenuates METH-induced striatal DA release and metabolism.

Parkinson’s disease (PD) is a common neurodegenerative movement disorder that is characterized pathologically by a progressive loss of midbrain dopaminergic neurons and by protein inclusions, designated Lewy bodies and Lewy neurites. PD is one of the most common neurodegenerative diseases, affecting almost 1% of the population over 60 years old. Although the symptoms and neuropathology of PD have been well characterized, the underlying mechanisms and causes of the disease are still not clear. Genetic mutations can provide important clues to disease mechanism, but most PD cases are sporadic rather than familial; environmental factors have long been suspected to contribute to the disease. Although more than 90% of PD cases occur sporadically and are thought to be due, in part, to oxidative stress and mitochondrial dysfunction, the study of genetic mutations has provided great insight into the molecular mechanisms of PD. Furthermore, rotenone, a widely used pesticide, and paraquat and maneb cause a syndrome in rats and mice that mimics, both behaviorally and neurologically, the symptoms of PD. In the current review, we will discuss various aspects of gene-environment interaction that lead to progressive dopaminergic neurodegenration, mainly focusing on our current finding based on stress-mediated parkin dysfunction.

Acetyl-L-carnitine (ALC) has been shown to be neuroprotective, in a dose and time dependent manner, through a variety of processes, including: membrane stabilization, ionic homeostasis, increased expression of key proteins, decreased expression of iNOS and increased resistance to neurotoxic events. Recently, we successfully demonstrated that pre-treatment with ALC confers effective neuroprotection against MDMA-induced neurotoxicity, preventing mitochondrial oxidative damage and, most importantly, preventing the typical MDMA-induced serotonin loss. However, the mechanisms underlying these actions are still unclear. In the present work, we have exposed PC12 cells to 1 M and 100 M methamphetamine (METH) to evaluate the action of ALC (0.01, 0.05, 0.1, 0.5 and 1 mM) on dopamine (DA) function. Intra and extracellular levels of DA were measured by HPLC-EC, both after a 24 or 72 h incubation period. We show that ALC by itself increases intracellular levels of DA, which may be a direct consequence of the ALC role on tyrosine biosynthesis. We also report that ALC is capable of increasing intracellular DA levels even in the presence of high METH doses and that ALC seems to prevent METH-induced DA release, which may be regulated through ALC direct action on important membrane components.

The neurotoxicity induced by the mitochondrial inhibitor 3-nitropropionic acid (3-NPA) is associated with a decrease of ATP synthesis and an increase of free radical production which can lead to apoptosis or necrosis. We have used the PC12, neuron-like rat pheochromocytoma cell line, to study further the mechanism of 3-NPA-evoked neurotoxicity and the effects of acetyl-L-carnitine (ALC) which has neuroprotective actions against various types of mitochondrial inhibitors. Cultured PC 12 cells were exposed to a low dose of 3-NPA 50 (microM) in the presence or absence of 5 mM ALC. The dose of 3-NPA was sub toxic and no changes in pro-apoptotic Bax or anti-apoptotic Bcl-2 gene expression were observed. We followed specific genetic markers to look for changes evoked by 3-NPA toxicity and also changes associated with neuroprotection exerted by the ALC treatment, using RT-PCR arrays (delta-delta method). 3-NPA exposure evoked a decrease in expression of the Tp53 gene. This down regulation was prevented by pretreatment of the cells with ALC. The Tp53 gene responds to cellular stresses and the effects seen here are possibly associated with the 3-NPA evoked changes in mitochondrial metabolism. Other genes associated with stress and apoptosis, Parp-1, Bcl-2, and Bax were not affected by 3-NPA or ALC. The decrease of inflammatory response Il-10 gene expression due to 3-NPA was further lowered by presence of ALC. Other inflammation related genes, Il1rn, Nr3c1 and Cxcr4 were not affected. Interestingly, the glutamate transporter slc17a7, carnitine-acylcarnitine translocase Slc25a20 and heat shock proteins genes, Hsp27, Hmox1 (Hsp32, HO1) as well as Hspa 1a (Hsp 70) increased only when both ALC and small dose of 3-NPA were present. The alterations in gene expression detected in this study suggest role of several intracellular pathways in the neurotoxicity of 3-NPA and the neuroprotection against 3-NPA-induced neurotoxicity by ALC.

Oxidative stress and secondary excitotoxicity, due to cellular energy deficit, are major factors playing roles in 3-nitropropionic acid (3-NPA) induced mitochondrial dysfunction. Acute or chronic exposure to 3-NPA also leads to neuronal degeneration in different brain regions. The present study quantitatively assessed peripheral neuropathy induced by chronic exposure to 3-NPA in rats. The neuroprotective abilities of two antioxidants, acetyl-l-carnitine and resveratrol, were investigated as well. Rats were exposed for up to four weeks to 3-NPA alone or 3-NPA combined with acetyl-l-carnitine or resveratrol, administered peripherally. The experimental outcome was evaluated by neurophysiological, histological, and morphometric analyses. Rats exposed to 3-NPA developed hind limb paresis. Furthermore, a significant decrease in motor nerve conduction velocity (MCV) was detected in tail nerves and axonal degeneration in sciatic nerves (p

Encephalopathy is evidenced as an altered mental state with various neurological symptoms, such as memory and cognitive problems. The type of a substance-evoked encephalopathy will depend on the drug, substance, or combination being abused. The categories into which we could place the various abused substances could be tentatively divided into stimulants, amphetamines, hallucinogens, narcotics, inhalants, anesthetics, anabolic steroids, and antipsychotics/antidepressants. Other factors that may underlie encephalopathy, such as infectious agents, environmental, and other factors have also to be taken into account. Drugs of abuse can be highly toxic to the CNS following acute, but more so in chronic exposure, and can produce significant damage to other organs, such as the heart, lungs, liver, and kidneys. The damage to these organs may be at least partially reversible when drug abuse is stopped but CNS damage from repeated or prolonged abuse is often irreversible. The major pathways for the organ and CNS toxicity could be related to ischemic events as well as increased cell damage due to metabolic or mitochondrial dysfunction resulting in increased excitotoxicity, reduced energy production, and lowered antioxidant potential. These susceptibilities could be strengthened by the use of antioxidants to combat free radicals (e.g., vitamin E, lipoic acid); trying to improve energy generation by using mitochondriotropic/metabolic compounds (e.g., thiamine, coenzyme Q10, carnitine, riboflavin); by reducing excitotoxicity (e.g., glutamate antagonists) and other possible strategies, such as robust gene response, need to be investigated further. The drug-abuse-evoked encephalopathy still needs to be studied further to enable better preventative and protective strategies.

Methamphetamine (METH) abuse and addiction present a major problem in the United States and globally. Oxidative stress associated with exposure to METH mediates to the large extent METH-evoked neurotoxicity. While there are currently no medications approved for treating METH addiction, its pharmacology provides opportunities for potential pharmacotherapeutic adjuncts to behavioral therapy in the treatment of METH addiction. Opioid receptor agonists can modulate the activity of dopamine neurons and could, therefore, modify the pharmacodynamic effects of METH in the dopaminergic system. Efficacy of the adjunctive medication with buprenorphine has been demonstrated in the treatment of cocaine addiction extending beyond opiate addiction. We investigated the interactions of morphine (10 mg/kg) and buprenorphine (0.01 and 10 mg/kg) with METH (2 mg/kg) affecting striatal dopaminergic transmission. The extracellular concentration of dopamine (DA) and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were determined using brain microdialysis coupled with high performance liquid chromatography with electrochemical detection (HPLC-ED) in the caudate nucleus of adult, awake, male Sprague-Dawley rats. Compared to METH alone, extracellular DA release was prolonged for 140 min without changes in DA peak-effect by combined treatment with morphine/METH. Morphine did not change DOPAC efflux evoked by METH. On the other hand, both buprenorphine doses attenuated the METH-induced DA peak-effect. However, whereas high buprenorphine dose extended DA outflow for 190 min, the low-dose abbreviated DA release. High buprenorphine dose also shortened METH-induced decrease in DOPAC efflux. Data confirm that opiates modulate dopaminergic neurotransmission evoked by METH. Alteration of dopaminergic response to METH challenge under buprenorphine may suggest effectiveness of buprenorphine treatment in METH addiction.

A novel gold phosphate complex called Black-Gold II with improved myelin staining properties has been developed. It differs from its predecessor, Black-Gold, in that it is highly water soluble at room temperature. This unique physical property confers a number of advantages for the high resolution staining of myelinated fibers. Specifically, it 1) allows for easier solution preparation, eliminating the need for extended heating or sonicating; 2) produces a more uniform and consistent tracer concentration, resulting in more consistent staining and 3) can be used at a 50% higher concentration, resulting in faster and more intense staining without the need for subsequent treatment with gold chloride intensifiers. To characterize the stain, both normal rat brains as well as those exposed to the neurotoxins kainic acid or methamphetamine were examined. The study also incorporates the first application of such stains to examine peripheral nerves of control and acrylamide-exposed rats.

Excitotoxicity and disrupted energy metabolism are major events leading to nerve cell death in neurodegenerative disorders. These cooperative pathways share one common aspect: triggering of oxidative stress by free radical formation. In this work, we evaluated the effects of the antioxidant and energy precursor, levocarnitine (L-CAR), on the oxidative damage and the behavioral, morphological, and neurochemical alterations produced in nerve tissue by the excitotoxin and free radical precursor, quinolinic acid (2,3-pyrindin dicarboxylic acid; QUIN), and the mitochondrial toxin, 3-nitropropionic acid (3-NP). Oxidative damage was assessed by the estimation of reactive oxygen species formation, lipid peroxidation, and mitochondrial dysfunction in synaptosomal fractions. Behavioral, morphological, and neurochemical alterations were evaluated as markers of neurotoxicity in animals systemically administered with L-CAR, chronically injected with 3-NP and/or intrastriatally infused with QUIN. At micromolar concentrations, L-CAR reduced the three markers of oxidative stress stimulated by both toxins alone or in combination. L-CAR also prevented the rotation behavior evoked by QUIN and the hypokinetic pattern induced by 3-NP in rats. Morphological alterations produced by both toxins (increased striatal glial fibrillary acidic protein-immunoreactivity for QUIN and enhanced neuronal damage in different brain regions for 3-NP) were reduced by L-CAR. In addition, L-CAR prevented the synergistic action of 3-NP and QUIN to increase motor asymmetry and depleted striatal GABA levels. Our results suggest that the protective properties of L-CAR in the neurotoxic models tested are mostly mediated by its characteristics as an antioxidant agent.

The carnitine palmitoyl transferase (CPT) system is a multiprotein complex with catalytic activity localized within a core represented by CPT1 and CPT2 in the outer and inner membrane of the mitochondria, respectively. Two proteins, the acyl-CoA synthase and a translocase also form part of this system. This system is crucial for the mitochondrial beta-oxidation of long-chain fatty acids. CPT1 has two well-known isoforms, CPT1a and CPT1b. CPT1a is the hepatic isoform and CPT1b is typically muscular; both are normally utilized by the organism for metabolic processes throughout the body. There is a strong evidence for their involvement in various disease states, e.g., metabolic syndrome, cardiovascular diseases, and in diabetes mellitus type 2. Recently, a new, third isoform of CPT was described, CPT1c. This is a neuronal isoform and is prevalently localized in brain regions such as hypothalamus, amygdala, and hippocampus. These brain regions play an important role in control of food intake and neuropsychiatric and neurological diseases. CPT activity has been implicated in several neurological and social diseases mainly related to the alteration of insulin equilibrium in the brain. These pathologies include Parkinson's disease, Alzheimer's disease, and schizophrenia. Evolution of both Parkinson's disease and Alzheimer's disease is in some way linked to brain insulin and related metabolic dysfunctions with putative links also with the diabetes type 2. Studies show that in the CNS, CPT1c affects ceramide levels, endocannabionoids, and oxidative processes and may play an important role in various brain functions such as learning.

This study tested the hypothesis that the expression of uncoupling proteins (UCPs) and dopamine (DA) system genes is responsive to 3-nitropropionic acid (3-NPA) neurotoxic effects and to the neuroprotective effects of the mitochondrial enhancer, L-carnitine (LC), in the rat striatum. Inactivation of mitochondrial succinate dehydrogenase (SDH) by 3-NPA results in hypoxic brain damage. Hypoxic conditions induce uncoupling protein-2 (UCP-2). An increase in UCP-2 expression may lead to a decrease in production of reactive oxygen species (ROS) associated with energy depletion. However, this adaptive response can also lead to a reduction of ATP that may further contribute to energy deficit and mitochondrial dysfunction. Here, male adult Sprague-Dawley rats (n=5/group) were injected either with saline or 3-NPA at 30 mg/kg, s.c. alone or 30 min after pre-treatment with LC (100mg/kg, i.p.). Rectal temperature was monitored before treatment and 4h following 3-NPA administration. Animals were sacrificed 4h post-treatment. Total RNA was isolated from the striatum and transcripts of UCP-2, UCP-4 and UCP-5 genes, as well as genes related to dopamine metabolism, such as DA D(1) and D(2) receptors, tyrosine hydroxylase (TH), monoamine oxidase-B (MAO-B), and vesicular monoamine transporter-2 (VMAT-2), were measured using real-time reverse transcription polymerase chain reaction (RT-PCR). While core temperature decreased significantly in 3-NPA-treated rats, LC significantly inhibited the hypothermic effect of 3-NPA (p

Nutritional deficiency in combination with drug abuse may increase risk of developing the metabolic syndrome by augmenting cell damage, excitotoxicity, reducing energy production, and lowering the antioxidant potential of the cells. We have reviewed here the following points: effects of drugs of abuse on nutrition and brain metabolism; effects of nutrition on actions of the drugs of abuse; drug abuse and probability of developing metabolic syndrome; role of genetic vulnerability in nutrition/drug abuse and brain damage; and the role of neuroprotective supplements in drug abuse. Nutrition education is an essential component of substance abuse treatment programs and can enhance substance abuse treatment outcomes. The strategies available, in particular the nutritional approach to protect the drug abusers from the metabolic syndrome and other diseases are discussed.

In humans or animals, symptoms of mitochondrial energy dysfunction may be produced by mutations or inborn errors of the necessary enzymes, as well as by enzyme inhibitors or uncouplers of the oxidative phosphorylation process. 3-Nitropropionic acid (3-NPA) is a toxin that is sometimes produced on moldy crops (sugarcane, peanuts, etc.) in amounts sufficient to cause severe neuromuscular disorders when consumed by humans. In vitro, 3-NPA irreversibly inactivates SDH, a Complex II respiratory enzyme important for mitochondrial energy production. We have been studying biomarkers of 3-NPA exposure in the expectation that such markers may be useful in the screening process to identify neuroprotective agents against neurotoxicity produced by mitochondrial energy dysfunction. Animals were sacrificed at various times after 3-NPA exposure for histochemical visualization of SDH activity and measurement of immediate postmortem rectal temperature. 3-NPA-treated rats experienced progressive hypothermia that reached a loss of 3 degrees C or more in core body temperature by three hours after dosing. The optical density of the SDH stain in brain was reduced, following a similar time course, most prominently in the cerebellum and least sharply in the thalamus. Some rats were given injections of L-carnitine (an enhancer of fatty acid transport) either alone, or as a pretreatment prior to a dose of 3-NPA. Although L-carnitine deficiency by itself can produce mitochondrial dysfunction, pretreatment with L-carnitine was of limited efficacy at overcoming the effects of 3-NPA on either body temperature or quantitative SDH histochemistry. Body temperature and SDH histochemistry may be useful biomarkers for evaluating the efficacy of neuroprotective agents against lower doses of 3-NPA, against other pharmacological models of mitochondrial dysfunction, or even against genetic mitochondrial diseases.

L-Carnitine (L-C) is a naturally occurring quaternary ammonium compound endogenous in all mammalian species and is a vital cofactor for the mitochondrial oxidation of fatty acids. Fatty acids are utilized as an energy substrate in all tissues, and although glucose is the main energetic substrate in adult brain, fatty acids have also been shown to be utilized by brain as an energy substrate. L-C also participates in the control of the mitochondrial acyl-CoA/CoA ratio, peroxisomal oxidation of fatty acids, and the production of ketone bodies. Due to their intrinsic interaction with the bioenergetic processes, they play an important role in diseases associated with metabolic compromise, especially mitochondrial-related disorders. A deficiency of carnitine is known to have major deleterious effects on the CNS. Several syndromes of secondary carnitine deficiency have been described that may result from defects in intermediary metabolism and alterations principally involving mitochondrial oxidative pathways. Mitochondrial superoxide formation resulting from disturbed electron transfer within the respiratory chain may affect the activities of respiratory chain complexes I, II, III, IV, and V and underlie some CNS pathologies. This mitochondrial dysfunction may be ameliorated by L-C and its esters. In addition to its metabolic role, L-C and its esters such as acetyl-L-carnitine (ALC) poses unique neuroprotective, neuromodulatory, and neurotrophic properties which may play an important role in counteracting various disease processes. Neural dysfunction and metabolic imbalances underlie many diseases, and the inclusion of metabolic modifiers may provide an alternative and early intervention approach, which may limit further developmental damage, cognitive loss, and improve long-term therapeutic outcomes. The neurophysiological and neuroprotective actions of L-C and ALC on cellular processes in the central and peripheral nervous system show such effects. Indeed, many studies have shown improvement in processes, such as memory and learning, and are discussed in this review.

The damage to the central nervous system that is observed after administration of either methamphetamine (METH) or 1-methyl-4-phenylpy-ridinium (MPP + ), the neurotoxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), is known to be linked to dopamine (DA). The underlying neurotoxicity mechanism for both METH and MPP + seem to involve free radical formation and impaired mitochondrial function. The MPP + is thought to selectively kill nigrostriatal dopaminergic neurons by inhibiting mitochondrial complex I, with cell death being attributed to oxidative stress damage to these vulnerable DA neurons. In the present study, MPP + was shown to significantly inhibit the response to MTT by cultured PC12 cells. This inhibitory action of MPP + could be partially reversed by the co-incubation of the cells with the acetylated form of carnitine, acetyl-L-carnitine (ALC). Since at least part of the toxic action of MPP + is related to mitochondrial inhibition, the partial reversal of the inhibition of MTT response by ALC could involve a partial restoration of mitochondrial function. The role carnitine derivatives, such as ALC, play in attenuating MPP + and METH-evoked toxicity is still under investigation to elucidate the contribution of mitochondrial dysfunction in mechanisms of neurotoxicity.

Domoic acid, a potent excitotoxic analogue of glutamate and kainate, may cause seizures, amnesia, and sometimes death in humans consuming contaminated shellfish. Continuous behavioral observations and recordings of the electrocorticogram (ECoG, via bipolar, epidural electrodes) were obtained from nonanesthetized rats for 2 h after intraperitoneal injection with either saline, 2.2, or 4.4 mg/kg of domoic acid. Rats were then sacrificed for c- fos immunohistochemistry. Fast Fourier transformation (FFT) of the ECoG data to obtain the voltage as a function of frequency indicated that the lower frequency bands (theta, 4.75–6.75 Hz and delta, 1.25–4.50 Hz) were the first to respond, with a significant elevation by 30 min after the high dose of domoic acid. The lower dose of domoic acid also caused a significant elevation of ECoG voltage, but not until later in the session. Sixty minutes after dosing, the behavioral biomarkers of “ear scratching” and “rearing, praying” (RP) seizures became significantly elevated in the high-dose rats. The low-dose rats showed no significant alterations in behavior at any time during the session. In postmortem brains obtained immediately after the sessions, c- fos was activated in the anterior olfactory nucleus by both the low and high doses of domoic acid. However, only the high dose increased c- fos immunoreactivity in the hippocampus, affecting both the granule and pyramidal neurons. These data indicate that electroencephalographic and c- fos responses can be obtained at a dose of domoic acid that fails to activate the behavioral response most commonly used as a bioassay for this marine toxin: ear scratching with the ipsilateral foot.

The neuroprotective action of l-carnitine (LC) in the rat model of 3-nitropropionic acid (3-NPA)-induced mitochondrial dysfunction was examined. 3-NPA is known to produce decreases in neuronal ATP levels via inhibition of the succinate dehydrogenase (SDH) at complex II of the mitochondrial electron transport chain. SDH is involved in reactions of the Krebs cycle and oxidative phosphorylation, and its inhibition leads to both necrosis and apoptosis. LC enhances mitochondrial metabolism and, together with its acetylated form, acetyl-l-carnitine (ALC), via the LC-ALC-mediated transfer of acetyl groups, plays an important modulatory role in neurotransmitter signal transduction pathways and gene expression in neuronal cells. In the study described here, adult male Sprague-Dawley rats were injected with 3-NPA alone or treated with LC prior to 3-NPA administration. Pretreatment with LC totally prevented the 3-NPA-induced decrease in brain temperature measured using temperature probes implanted intracranially. It appears that the protective effects of LC against 3-NPA-induced neurotoxicity are achieved via compensatory enhancement of several pathways of mitochondrial energy metabolism. The results of this and previous studies conducted by our division in the 3-NPA model of mitochondrial dysfunction demonstrate that 3-NPA may be employed in vivo to evaluate enhancers of mitochondrial function that might exert neuroprotective effects.

Some of the damage to the CNS that is observed following amphetamine and methamphetamine (METH) administration is known to be linked to increased formation of free radicals. This increase could be, in part, related to mitochondrial dysfunction and/or cause damage to the mitochondria, thereby leading to a failure of cellular energy metabolism and an increase in secondary excitotoxicity. The actual neuronal damage that occurs with METH-induced toxicity seems to affect dopaminergic cells in particular. METH-induced toxicity is related to an increase in the generation of both reactive oxygen (hydroxyl, superoxide, peroxide) and nitrogen (nitric oxide) species. Peroxynitrite (ONOO(-)), which is a reaction product of either superoxide or nitric oxide, is the most damaging radical. It can be reduced by antioxidants such as selenium, melatonin, and the selective nNOS inhibitor, 7-nitroindazole. METH-induced toxicity has been previously shown to increase production of the peroxynitrite stress marker, 3-nitrotyrosine (3-NT), in vitro, in cultured PC12 cells, and also in vivo, in the striatum of adult male mice. Pre- and post-treatment of mice with l-carnitine (LC) significantly attenuated the production of 3-NT in the striatum after METH exposure. LC is a mitochondriotropic compound in that it carries long-chain fatty acyl groups into mitochondria for beta-oxidation. It was shown also to play a protective role against various mitochondrial toxins, such as 3-nitropropionic acid. The protective effects of LC against METH-induced toxicity could be related to its prevention of possible metabolic compromise produced by METH and the resulting energy deficits. In particular, LC may be maintaining the mitochondrial permeability transition (MPT) and modulating the activation of the mitochondrial permeability transition pores (mPTP), especially the cyclosporin-dependent mPTP. The possible neuroprotective mechanism of LC against METH-toxicity and the role of the mitochondrial respiratory chain and the generation of free radicals and their subsequent action on the MPT and mPTP are also being examined using an in vitro model of NGF-differentiated pheochromocytoma cells (PC12). In preliminary experiments, the pretreatment of PC12 cells with LC (5 mM), added 10 min before METH (500 micro M), indicated that LC enhances METH-induced DA depletion. The role of LC in attenuating METH-evoked toxicity is still under investigation and promises to reveal information regarding the underlying mechanisms and role of mitochondria in the triggering of cell death.

3-Nitropropionic acid (3-NPA) is an inhibitor of the mitochondrial enzyme succinate dehydrogenase (SDH, a part of complex II) that links the tricarboxylic acid (TCA) cycle to the respiratory electron transport chain. 3-NPA inactivates SDH by covalently and irreversibly binding to its active site. We previously examined the effects of 3-NPA on the histochemical activity of SDH in vivo, by using the reduction of a yellow tetrazolium dye (nitro blue tetrazolium) to a blue formazan as an indicator. In studies of cultured cells, the related dye methylthiazoletetrazolium (MTT) has commonly been used as an indicator of the presence and number of viable cells; that is cells that are capable of producing energy via the TCA cycle. Here we observed that doses of 3-NPA as low as 10(-8) M inhibited formazan production in an in vitro model system using CHO cells. This effect was antagonized by l-carnitine, which greatly increased the production of formazan, indicating a considerable improvement in energy production by the cultured cells. CHO cells appear to be a convenient model for the evaluation of therapeutic compounds that may modulate cellular bioenergetics.

L-carnitine (LC) plays an important regulatory role in the mitochondrial transport of long-chain free fatty acids (FFA). 3-Nitropropionic acid (3-NPA) is known to induce cellular energy deficit and oxidative stress related neurotoxicity via an irreversible inhibition of the mitochondrial enzyme succinate dehydrogenase (SDH). Protective effects of L-carnitine on the neurotoxicity induced by 3-NPA have been shown in vitro. Here, the activities of SDH as well as the activity of the antioxidant enzymes, catalase (CAT), and superoxide dismutase (SOD) were measured in order to evaluate the protective action of LC against 3-NPA-induced neurotoxicity. Male, CD Sprague-Dawley rats, 3-month old, were injected with either 50 or 100 mg/kg of LC, i.p., 30-60 min prior to 3-NPA (30 mg/kg, s.c.) or with 3-NPA alone. Enzyme activities were assayed in caudate nucleus (CN), frontal cortex (FC), and hippocampus (HIP) post sacrifice. Increased activities of CAT and SOD were observed after treatment with 3-NPA alone. Pretreatment with low or high doses of LC was associated with attenuation of these increases equivalent to, or below, the control levels. In rats treated with 3-NPA alone, SDH activity was inhibited by 62% (CN), 50% (FC), and 65% (HIP) of controls. Pretreatment with LC prior to 3-NPA attenuated decreases of SDH activity in a dose-dependent manner. However, compared with control, the activity of SDH remained significantly lower in brain regions of treated rats despite the attenuation of inhibition by LC pretreatment (P

Our objective was to construct a physiologically-based pharmacokinetic (PBPK) model describing the kinetic behavior of 2,4-dichlorophenoxyacetic acid (2,4-D) on rats after long-term exposures to low doses. Our study demonstrated the model's ability to simulate uptake of 2,4-D in discrete areas of the rat brain. The model was derived from the generic PBPK model that was first developed for high-dose, single exposures of 2,4-D to rats or rabbits (Kim, C.S., Gargas, M.L., Andersen, M.E., 1994. Pharmacokinetic modeling of 2,4-dichlorophenoxyacetic acid (2,4-D) in rats and rabbits brain following single dose administration. Toxicol. Lett. 74, 189–201; Kim, C.S., Slikker, W., Jr., Binienda, Z., Gargas, M.L., Andersen, M.E., 1995. Development of a physiologically based pharmacokinetic (PBPK) model for 2,4-dichlorophenoxyacetic acid (2,4-D) dosimetry in discrete areas of the brain following a single intraperitoneal or intravenous dose. Neurotox. Teratol. 17, 111–120.), to which a subcutaneous (hypodermal) compartment was incorporated for low-dose, long-term infusion. It consisted of two body compartments, along with compartments for venous and arterial blood, cerebrospinal fluid, brain plasma and six brain regions. Uptake of the toxin was membrane-limited by the blood-brain barrier with clearance from the brain provided by cerebrospinal fluid ‘sink’ mechanisms. This model predicted profiles of 2,4-D levels in brain and blood over a 28-day period that compared well with concentrations measured in vivo with rats that had been given 2,4-D (1 or 10 mg/kg per day) with [ 14 C]-2,4-D subcutaneously (s.c.) for 7, 14, or 28 days, respectively. This PBPK model should be an effective tool for evaluating the target tissue doses that may produce the neurotoxicity of organic acid toxicants after low-dose, long-term exposures to contaminated foods or the environment.

The psychoactive indole alkaloid, ibogaine (IBO), has been investigated for over a decade concerning its reported anti-addictive properties for opioids as well as psychomotor stimulants. The mechanism for the anti-addictive action of IBO is still unclear. IBO interactions with opioid, NMDA, nicotinic, adrenergic, and serotonergic receptor sites have been suggested. The involvement of the dopaminergic system in IBO action is well documented. Increased or decreased levels of dopamine (DA) in specific brain regions following IBO pretreatment have been seen concomitantly with increased or decreased motor activity after subsequent amphetamine or cocaine administration. In this report, in vivo electrophysiological measures were monitored in awake adult male rats in order to investigate alterations of the electrocorticogram (ECoG) resulting from interactions between IBO and cocaine (COC). Rats were implanted bilaterally with bipolar ECoG electrodes. They were either injected with saline, COC alone (20 mg/kg, i.p.) or IBO (50 mg/kg, i.p.) and COC 1 hr later. The concentrations of DA, 5-HT, and their metabolites DOPAC, HVA, and 5-HIAA were assessed in the caudate nucleus in separate groups of saline-, COC-, and IBO/COC-treated rats. An alpha1 power increase was observed within 10 min after COC injection, which lasted for less than 20 min. A desynchronization over alpha2 and both beta power bands was observed throughout the recording. In IBO/COC-treated rats, a significant increase in delta, theta, and alpha1 power occurred within 20 min after COC injection (p

Background The overall goal of human immunodeficiency virus (HIV) therapy during pregnancy is to maintain maternal health and reduce the probability of vertical transmission during gestation and delivery, while keeping toxicity risks low. Azidothymidine (AZT) is currently recommended for pregnant women infected with HIV; however, many pregnant women are unable to tolerate AZT because of toxicity. In the present study, the placental transfer and fetal accumulation of the anti-HIV compound 2',3'-didehydro-3'-deoxythymidine (d4T) and its active (triphosphorylated) and inactive (thymine and beta-aminoisobutyric acid) metabolites were examined at steady state in late-term rhesus macaques. Methods On the day of the hysterotomy, the mother was administered an intravenous loading dose of d4T, followed by a 3-hr steady-state intravenous infusion that also included [(3)H]d4T as a tracer. After 3 hr of infusion, the fetus was delivered by cesarean section under halothane/N(2)O anesthesia. Plasma, amniotic fluid, and tissues were analyzed for d4T and its inactive metabolites by HPLC; tissue samples were analyzed for d4T and active (phosphorylated) metabolites by strong anion-exchange HPLC. Results Maternal steady-state plasma concentrations of d4T were 1-2 microg/ml, with a fetal-to-maternal plasma ratio of 0.85 +/- 0.09. The fetal tissue distribution of radioactivity was highest in the kidney and lowest in the brain. D4T, thymine, and beta-aminoisobutyric acid were detected in all fetal tissues examined. Conclusions Our data indicate that d4T readily crosses the placenta and is present in the fetus as parent compound or its inactive metabolites after maternal infusion. Although fetal plasma concentrations of d4T were similar to clinical d4T concentrations, no phosphorylated metabolites were detected. Teratology 62:93-99, 2000. Published 2000 Wiley-Liss, Inc.

3-nitropropionic acid (3-NPA) is a toxin sometimes produced on moldy crops (sugarcane, peanuts, etc.) in amounts sufficient to cause severe neurological disorders when consumed by humans. In vitro, 3-NPA irreversibly inactivates SDH, a Complex II respiratory enzyme required for mitochondrial energy production. A single dose of 3-NPA (30 mg/kg S.C.) was given to singly-caged adult male Sprague-Dawley rats. Rectal temperature was measured after dosing as a potential biomarker of exposure to 3-NPA, and animals were sacrificed at various times after 3-NPA exposure for histochemical visualization of SDH activity. 3-NPA-treated rats experienced a progressive hypothermia, which reached a loss of 3°C or more in core body temperature by 3 hours after dosing. The optical density of the SDH stain in brain was reduced according to a similar time-course, most prominently in the cerebellum and least sharply in the thalamus. The caudate nucleus had the greatest density of SDH staining that we measured in brain; it also has been reported to be the region most consistently lesioned by 3-NPA. However, within other areas of brain such as subdivisions of the hippocampus, neither endogenous SDH activity nor its sensitivity to inhibition by 3-NPA could predict the susceptibility to neurodegenerative changes. Although SDH activity remained significantly reduced in most areas of brain (except thalamus) for up to 5 days after dosing, core temperatures had returned to control values by 5 days suggesting that animals can utilize an alternate method of heat production to withstand insult by 3-NPA.

Ibogaine (IBO) is a psychoactive indole alkaloid that has antiaddictive properties. However, treatment with IBO may lead to neurotoxicity, since IBO and its metabolites interact persistently with many neurotransmitter systems. Here, we recorded cortical electroencephalogram (EEG) signals from rats anesthetized with isoflurane. The heart rate (HR) was monitored via electrocardiogram (EKG) electrodes. After the baseline EEG was recorded, rats received one intraperitoneal (i.p.) dose of 50 mg/kg IBO. EEG signals were recorded for 2 hr. Rats were then sacrificed and brains dissected into frontal cortex (FC), caudate nucleus (CN), hippocampus (HIP), and brain stem (BS). The level of dopamine (DA), serotonin (5-HT), and their metabolites were determined by high-performance liquid chromatography with electrochemical detection (HPLC-ECD). Compared with baseline, a decrease in HR immediately after IBO injection and a decrease in delta, theta, alpha and beta power spectra frequency bands (1-4, 4-8, 8-13, 13-32 Hz) during the first 30 min after IBO administration was observed. EEG recovered within the next 15 min. In CN, the level of DA decreased and DA turnover rate increased significantly. The levels of 5-HT increased in FC. The pattern of EKG AND EEG response to IBO may be due to multiple receptor interactions of IBO.

The Carnitine Palmitoyl Transferase (CPT) system comprises several metabolically important enzymes of the carnitine/choline acyl transferase family. CPTs are transferases that allow the energy-neutral replacement of coenzyme A (CoA) by L-carnitine, and vice versa, when bound to acyl chains as (thio) esters. Together with acylCoA synthases and a carnitine/acyl carnitine translocase, the mitochondrial beta-oxidation of long-chain fatty acids is facilitated by distinct CPT proteins. CPT deficiencies manifest as disorders of mitochondrial fatty acid oxidation. Recently a new CPT isoform, CPT1c has been described which is found in entusiasmoc reticulum (ER) of neurons. Unlike CPT1a and CPT1b it has lower palmitoyl transferase activity. It is localised mainly in hypothalamus, amygdala and hippocampus, i.e., brain regions with roles in the control of food intake. It plays a role in energy homeostasis but its exact role in physiology of neurons is still not clear. Studies suggest a biosynthetic rather than catabolic role in long chain acyl carnitine production. The role of CPT1c may extend beyond simply the interchange of CoA and L-carnitine to acyl groups. Studies show that in the CNS the CPT1c affects ceramide levels, endocannabionoids and oxidative processes, and may play an important role in various brain functions such as learning. It is a player in insulin resistance that may occur as a result of oxidative damage and in altered redox status diseases such as metabolic cognitive syndrome, type 2 diabetes, neurodegenerative diseases and cancer. Unhealthy lifestyles, especially high sugar and fat diet in combination with other genetic and environmental risk factors, probably compromise metabolism at various levels. At the cellular level there would be increased ER stress as well as mitochondrial dysfunction leading to impaired oxidative phosphorylation and increased reliance on glucose (Warburg effect). Targeting CPT1c may provide insight on treatment of many metabolically-related diseases as well as pervasive developmental disorders.

To explore the effects of gestational cocaine exposure in a nonhuman primate model, pregnant rhesus monkeys were treated from about 1 month of gestation until term with either 0 (N = 3), 0.3 (N = 3), 1.0 (N = 3), or escalating doses up to 8.5 (N = 3) mg/kg (IM), three times per day, 5 consecutive days per week. Despite these differences in cocaine exposure, the experimental groups did not differ significantly with respect to maternal outcome, as measured by body weight gain during pregnancy and length of pregnancy. A clear dose-response relationship was observed between the cumulative dose of cocaine administered during gestation and the levels of both cocaine and its major metabolite, benzoylecgonine, in samples of infant hair taken at birth. However, the experimental groups did not differ significantly with respect to infant outcome, as measured at birth by body weight, overall length, crown-to-rump length, rump-to-heel length, biparietal diameter, and crown circumference. Furthermore, the experimental groups did not differ significantly with respect to the integrity of a variety of infant reflexes tested at birth. It was concluded that, in a rhesus monkey model, chronic cocaine exposure during pregnancy had no significant effect on maternal and infant outcomes as assessed in this investigation.

Diet in human health is no longer simple nutrition, but in light of recent research, especially nutrigenomics, it is linked via evolution and genetics to cell health status capable of modulating apoptosis, detoxification, and appropriate gene response. Nutritional deficiency and disease especially lack of vitamins and minerals is well known, but more recently, epidemiological studies suggest a role of fruits and vegetables, as well as essential fatty acids and even red wine (French paradox), in protection against disease. In the early 1990s, various research groups started considering the use of antioxidants (e.g., melatonin, resveratrol, green tea, lipoic acid) and metabolic compounds (e.g., nicotinamide, acetyl-l-carnitine, creatine, coenzyme Q10) as possible candidates in neuroprotection. They were of course considered on par with snake oil salesman (women) at the time. The positive actions of nutritional supplements, minerals, and plant extracts in disease prevention are now mainstream and commercial health claims being made are subject to regulation in most countries. Apart from efficacy and finding, the right dosages, the safety, and especially the level of purification and lack of contamination are all issues that are important as their use becomes widespread. From the mechanistic point of view, most of the time these substances replenish the body’s deficiency and restore normal function. However, they also exert actions that are not sensu stricto nutritive and could be considered pharmacological especially that, at times, higher intake than recommended (RDA) is needed to see these effects. Free radicals and neuroinflammation processes underlie many neurodegenerative conditions, even Parkinson’s disease and Alzheimer’s disease. Curcumin, carotenoids, acetyl-l-carnitine, coenzyme Q10, vitamin D, and polyphenols and other nutraceuticals have the potential to target multiple pathways in these conditions. In summary, augmenting neuroprotective pathways using diet and finding new natural substances that can be more efficacious, i.e., induction of health-promoting genes and reduction of the expression of disease-promoting genes, could be incorporated into neuroprotective strategies of the future.

Experimental evidence suggests that oxidative and nitrative mechanisms account for much of the dopaminergic neuronal injury in Parkinson’s disease (PD). The ubiquitously expressed non-receptor tyrosine kinase c-Abl is activated by oxidative stress and thus, may play a role in redox-mediated neurodegeneration. Recently, we reported that c-Abl is activated in PD and that a c-Abl inhibitor mitigated neuronal damage in a PD animal model, suggesting a novel neuroprotective therapeutic approach. In the studies presented here, we evaluated the efficacy of a potent and clinically relevant second-generation irreversible Abl kinase inhibitor, INNO-406, as a therapeutic agent for PD. Our studies reveal that INNO-406 is capable of preventing the progression of dopaminergic neuronal damage in a toxin-induced C57 mouse model of PD. Using bovine brain microvessel endothelium as an in vitro blood-brain barrier (BBB) model, we detected rapid and significant transfer of INNO-406. Additionally, pharmacokinetic analyses demonstrated significant nanomolar concentrations of INNO-406 in brain in the presence or absence of MPTP administration, however, INNO-406 did not alter the brain levels of MPP+ in MPTP-treated mice. Finally, we showed that 10 mg/kg of INNO-406 given to C57 mice for one week before MPTP treatment (4×20 mg/kg i.p., every 2 h) and then for one week after MPTP treatment decreased the loss of dopamine in the striatum by 45% and the loss of TH+ neurons in substantia nigra pars compacts by 40%. This treatment regimen also abrogated activation of c-Abl, tyrosine phosphorylation of the Abl substrate and E3-ubiquitin ligase parkin, and accumulation of the toxic parkin substrate AIMP2. We propose that compounds of the INNO-406 class of Abl inhibitors will be useful new neuroprotective drugs for the treatment of PD-like pathology in preclinical systems that should be easily translated to the clinic.

The effects of acute perinatal ischemia-hypoxia on fetal liver and brain energy metabolism, fetal brain total free fatty acid concentration and subsequent offspring behavior were investigated in rats. Ischemia-hypoxia was induced at term either by ligation of the uterine blood vessels or submersion of the entire uterine horn in warmed saline. Fetuses of the adjacent horn served as within-dam controls for all assessments and fetuses of dams which had not undergone the surgical stress served as independent controls for enzyme assays. Ischemia-hypoxia was associated with reduced activity of fatty acid synthase in the liver and brain. Total free fatty acid concentration significantly increased in the fetal hypoxic brain. Pups not used for enzyme analyses were cross-fostered for behavioral assessments. Relative to the enzymatic alterations, there were few behavioral alterations associated with ischemia-hypoxia. At postnatal day 30, rats made hypoxic by ligation of the uterine blood vessels had decreased caudate nucleus and brain stem weights relative to within-dam controls. At postnatal day 85, rats made hypoxic by submersion of the uterine horn had decreased olfactory bulb weight. The results of this study indicate an initial acute response to a brief period of ischemia-hypoxia at term pregnancy in the fetal rat brain and liver.

The combined effects of amphetamine (AMPH) and 3-nitropropionic acid (3-NPA) were investigated to determine how the energy depletion proposed to be produced by AMPH interacts with an inhibitor of mitochondrial respiration to produce striatal neurotoxicity. Neither two doses (2 h apart) of 3.75 mg/kg AMPH alone nor a single dose of 30 mg/kg 3-NPA i.p. produced neurotoxicity in the striatum or lowered striatal dopamine content in rat. Administration of 40 mg/kg of 3-NPA alone almost invariably produced either lethality or did not produce neurotoxicity in the striatum of surviving animals. However, 30 mg/kg of 3-NPA administered along with 2 doses of 3.75 mg/kg AMPH to 47 animals produced striatal damage in the 31 survivors with 15 of the surviving rats showing muscle rigidity/catatonia for several days after dosing, along with decreased food consumption. Thirteen of these 15 rats showed degeneration of axons and cell bodies in the medial caudate-putamen with minimal damage to the globus pallidus. However, two rats exhibited hindlimb paralysis and signs of axonal and neuronal soma degeneration in the thalamus and cerebellar nuclei as well as striatum. Sixteen of the rats given both AMPH and 3-NPA exhibited only torpidity and loss of muscle tone 1-3 h after dosing. Such rats showed no signs of neuronal cell degeneration in the striatum, but did show significant dopamine depletions (60% of control) and reductions in tyrosine hydroxylase immunoreactivity at 14 days postexposure. The mitochondrial dysfunction produced by 3-NPA combined with activation of neuronal pathways by AMPH may have predisposed terminals, axons and cell bodies in striatum to degeneration.

A basic PBPK model for the dosimetry of organic acids in discrete areas of the brain was constructed using 2,4-D as a model compound. The PBPK model describes distribution of 2,4-D throughout the body and within discrete areas of the brain. The brain compartments in the model were the hypothalamus, caudate nucleus, hippocampus, forebrain, brainstem, and cerebellum. The remainder of the model consisted of two-body compartments and venous and arterial blood compartments. In the model, chemical uptake by the brain was membrane-limited by the blood-brain barrier (BBB) with saturable clearance from the cerebrospinal fluid (CSF) into the venous blood by the choroid plexus. The body has both a central and a deep compartment with saturable clearance from the central compartment. The model was used to examine the brain and CSF concentrations of 2,4-D as a function of plasma 2,4-D, as well as plasma time-course behavior using experimental data from rabbits given 2,4-D 40 or 100 mg/kg, IP or Na-2,4-D 40 mg/kg, i.v. administration. Model parameters were adjusted to fit the observed 2,4-D concentrations in blood and brain regions over a 2-h time frame. PBPK models could be a useful tool for evaluating the safety of this class of organic acids.

Rotenone, a widely used pesticide, causes a syndrome in rats that replicates, both pathologically and behaviorally, the symptoms of Parkinson's disease (PD). In the present study, we sought to determine if a chronic exposure to rotenone, resulting in dopaminergic loss, could also lead to peripheral neuronal damage related to motor dysfunction. Adult male Sprague-Dawley rats (n=14) were treated with rotenone (1 or 2mg/kg, s.c., once daily) on days 1, 3, 6, 8, 10, 13, 15, 17, 21, 22, and 27 to minimize mortality. Control rats received vehicle (DMSO) injections. Animals were weighed on the days of injection and monitored daily. A mortality of 21% was observed in rotenone treated rats. The motor nerve conduction velocity (MCV) was assessed using action potentials detected from the tail muscle through surface receiver electrodes installed around the distal portion of the tail. Rats exposed to rotenone often developed hind limb paresis with a significant decrease in MCV as detected in tail nerves (p

Metabolic stressors such as hyperthermia, seizures and ischemic hypoxia result in the induction of C-fos and heat-shock proteins (HSP) in affected brain cells of the adult rodent, especially within the hippocampal region, which normally has high metabolic demands. Here we ligated the uterine vessels of gestational day (GD) 21 rat pups to produce ischemic hypoxia. We confirmed that HSP-72 protein, as previously reported, was activated in the perinatal rat pup, especially in the hippocampal CA3 region. However, the capability of hippocampal cells to produce C-fos protein following drug-induced seizures has been reported to develop only after postnatal day 13. Here, ischemic hypoxia caused CA1 hippocampal cells to produce immunohistochemically detectable C-fos protein in GD-21 rats. These results seem to contradict the previous reports of no C-fos induction in rats this young by demonstrating a functional C-fos translational mechanism by GD-21. However, seizure vs ischemic hypoxia-induced C-fos expression may involve several different pre-translational pathways. A delayed development of a receptor, second messenger, or genomic element for regulating C-fos transcription remain as possible explanations for the late maturity of responsivity to seizures.

The Effect of Dideoxycytidine on Lymphocyte Subpopulations in Nonhuman Primates. Taylor, L. D., Binienda, Z., Schmued, L., and Slikker, W., Jr. (1994). Fundam. Appl. Toxicol. 23, 434-438. In the present study, 2′,3′dideoxycytidine (ddC), which has antiretroviral activity, was given chronically to uninfected nonhuman primates to determine whether it produces adverse immunological or hematological effects. Nine healthy adult male rhesus monkeys were divided into three groups and given the following doses of ddC in a gelatin vehicle; group A, 0.06, 6.0, 3.0, and 1.5 mg/kg; group B, 0.6 mg/kg; group C, 0 mg/kg. Blood samples were collected for hematologic analysis and flow cytometric analyses of lymphocyte subpopulations. Chronic ddC exposure did not cause significant changes in the number of red blood cells, monocytes, or reticulocytes. The number of white blood cells and neutrophils increased and these changes were observed only in group A animals at the 1.5 mg/kg dose. The most significant alterations observed were decreases in the number of T helper cells (CD4) and B cells (CD20). CD4 + and CD20 + lymphocytes exhibited dose-related shifts that were reversible over time and after drug withdrawal. The results indicate that ddC has few hematologic effects but it does have profound but transient effects on the number of cells in lymphocyte subpopulations in normal primates.

In order to clarify the mechanism of action of cerulenin analog, C75, known to suppress feeding behavior, food intake was measured in adult CD-1 male mice n=5 per group, treated i.p. with 10 and 20mg/kg of C75. Animals in both treatment groups had significantly lower 24h food consumption rate relative to the control group injected with vehicle. Striatal monoamine neurotransmitters and striatal as well as liver long chain free fatty acids concentrations were subsequently evaluated in another group treated i.p. with 20mg/kg C75. Acute exposure to C75 at 20mg/kg led to approximately 50% increase in the striatal dopamine levels and a decrease in dopamine turnover for up to 24h following the injection. The concentration of serotonin remained unchanged. Concentration of saturated fatty acids in the liver and striatum did not change, while striatal unsaturated myristoleic acid (cis-9-tetradecenoic acid) levels were significantly higher as early as 2h post-injection and remained elevated at 24h post-injection. These preliminary data suggest a central regulatory role of unsaturated fatty acids under dopaminergic control in the C75-induced anorexia. Pharmacological alterations in fatty acid metabolism may prove beneficial in the treatment of obesity.

Advances in computer technology have allowed quantification of the electroencephalogram (EEG) and expansion of quantitative EEG (qEEG) analysis in neurophysiology, as well as clinical neurology, with great success. Among the variety of techniques in this field, frequency (spectral) analysis using Fast Fourier Transforms (FFT) provides a sensitive tool for time-course studies of different compounds acting on particular neurotransmitter systems. Studies presented here include Electrocorticogram (ECoG) analysis following exposure to a glutamic acid analogue - domoic acid (DOM), psychoactive indole alkaloid - ibogaine, as well as cocaine and gamma-hydroxybutyrate (GHB). The ECoG was recorded in conscious rats via a tether and swivel system. The EEG signal frequency analysis revealed an association between slow-wave EEG activity delta and theta and the type of behavioral seizures following DOM administration. Analyses of power spectra obtained in rats exposed to cocaine alone or after pretreatment with ibogaine indicated the contribution of the serotonergic system in ibogaine mediated response to cocaine (increased power in alpha(1) band). Ibogaine also lowered the threshold for cocaine-induced electrographic seizures (increased power in the low-frequency bands, delta and theta). Daily intraperitoneal administration of cocaine for two weeks was associated with a reduction in slow-wave ECoG activity 24 hrs following the last injection when compared with controls. Similar decreased cortical activity in low-frequency bands observed in chronic cocaine users has been associated with reduced metabolic activity in the frontal cortex. The FFT analyses of power spectra relative to baseline indicated a significant energy increase over all except beta(2) frequency bands following exposure to 400 and 800 mg/kg GHB. The EEG alterations detected in rats following exposure to GHB resemble absence seizures observed in human petit mal epilepsy. Spectral analysis of the EEG signals combined with behavioral observations may prove to be a useful approach in studying chronic exposure to drugs of abuse and treatment of drug dependence.

Drug abuse is associated with significant health risk. Whether drug abusers are at a higher risk of suffering the metabolic syndrome is not widely known. The metabolic syndrome is a cluster of metabolic abnormalities, including hyperinsulinemia, hypertension, dyslipidemia, and abdominal obesity, and is probably triggered by initial imbalances at the cellular level in various critical metabolic pathways. These initially small metabolic imbalances are believed to cascade with time and lead to larger problems. Some indications that drug abuse may increase the risk of the metabolic syndrome include the following: Drug-abusing patients have higher rates of diabetes complications. Substance abuse is a significant contributing factor for treatment noncompliance in diabetes. Nutrition education can enhance substance abuse treatment outcomes. Each type of drug/substance abuse has a unique profile of toxicity. For example, the amphetamines generally affect the cardiovascular and neurological systems, worsening the risk factors for the metabolic syndrome. Methamphetamine (meth) abusers suffer cognitive deficits and abnormal metabolic activity, which affect nutritional status. This condition is further worsened by a drastic reduction in oral health in meth abusers, resulting in improper chewing and, therefore, digestion. Nutritional deficiency in combination with drug abuse would increase the risk of developing the metabolic syndrome by increasing cell damage, augmenting excitotoxicity, reducing energy production, and lowering the antioxidant potential of the cells. Another potential risk factor in the development of the metabolic syndrome is genetic vulnerability, especially in combination with drug abuse and nutritional deficiencies. The strategies available to treat this problem include pharmacological agents as well as dietary antioxidants. Such measures may be useful in reducing drug abuse-related toxicity that may lead to the metabolic syndrome.

Adult, male Sprague-Dawley rats were injected with 3-ni-tropropionic acid (3-NPA) at 30 mg/kg or methamphetamine (METH) at 20 mg/kg alone or following pretreatment with L-cartnitine (LC) at 100 mg/kg. Rectal temperature was measured before and 4 h following treatment. Animals were sacrificed at 4 h posttreatment. Monoamine neurotransmitters, dopamine (DA) and serotonin (5-HT), and their metabolites were analyzed in the striatum using high-performance liquid chromatography method coupled with electrochemical detection (HPLC/ED). Transcripts of several genes related to DA metabolism were quantified using real time reverse transciption polymerase chain reaction (RT-PCR). Core temperature decreased significantly after 3-NPA acid and increased in METH-treated rats (P < 0.05). Temperature change at 4 h exhibited a significant LC effect for 3-NPA, preventing hypothermia (P < 0.05) and no effect for METH. Concentration of DA and 5-HT, and their metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA), increased significantly in 3-NPA and decreased in METH-treated rats. An increase in DOPAC/DA turnover and serotonin observed after 3-NPA was abolished in LC-/3-NPA-treated rats. In both 3-NPA- and METH-treated rats, LC prevented an increase in DA receptor D(1) gene expression. It appears that carnitine effect preventing hypothermia after 3-NPA treatments may be related not only to its mitochondriotropic actions but also to inhibitory effect on the DA and 5-HT systems activated after the exposure to 3-NPA. The same effect observed at the transcriptional level, at least for the DA receptor D(1), may account for protection against METH toxicity.

Acute exposure to a neurotoxin, 3-nitropropionic acid (3-NPA), in rats results in an increase in total free fatty acid (FFA) concentration in selective brain regions. We investigated the effect of 3-NPA administration on the cerebral concentrations of FFA used as a marker of oxidative stress. Rats (n = 3/group) were dosed subcutaneously (s.c.) either with a vehicle (phosphate buffer) or 3-NPA in phosphate buffer at 30 mg/kg body weight. Animals were sacrificed after 1, 2, 3, and 6 h of injection. Brains were then dissected into frontal cortex (FC), caudate nucleus (CN), and hippocampus (HIP). The concentration of total FFA increased from 130 to 300% within 1-2 h after 3-NPA injection in all brain regions when compared with the baseline level obtained from the control rats and taken as 100%. In CN, FFA returned to the baseline level within 3 h of treatment. However, in FC and HIP the concentration of FFA remained significantly elevated above the baseline until 6 h. The released FFA provide a substrate for free radicals formation. The results of this study suggest a role of oxidative stress in the mechanism of 3-NPA toxicity.

A number of strategies using the nutritional approach are emerging for the protection of the brain from damage caused by metabolic toxins, age, or disease. Neural dysfunction and metabolic imbalances underlie many diseases, and the inclusion of metabolic modifiers may provide an alternative and early intervention approach that may prevent further damage. Various models have been developed to study the impact of metabolism on brain function. These have also proven useful in expanding our understanding of neurodegeneration processes. For example, the metabolic compromise induced by inhibitors such as 3-nitropropionic acid (3-NPA), rotenone, and 1-methyl-4-phenylpyridinium (MPP+) can cause neurodegeneration in animal models and these models are thought to simulate the processes that may lead to diseases such as Huntington's and Parkinson's diseases. These inhibitors of metabolism are thought to selectively kill neurons by inhibiting various mitochondrial enzymes. However, the eventual cell death is attributed to oxidative stress damage of selectively vulnerable cells, especially highly differentiated neurons. Various studies indicate that the neurotoxicity resulting from these types of metabolic compromise is related to mitochondrial dysfunction and may be ameliorated by metabolic modifiers such as L-carnitine (L-C), creatine, and coenzyme Q10, as well as by antioxidants such as lipoic acid, vitamin E, and resveratrol. Mitochondrial function and cellular metabolism are also affected by the dietary intake of essential polyunsaturated fatty acids (PUFAs), which may regulate membrane composition and influence cellular processes, especially the inflammatory pathways. Cellular metabolic function may also be ameliorated by caloric restriction diets. L-C is a naturally occurring quaternary ammonium compound that is a vital cofactor for the mitochondrial entry and oxidation of fatty acids. Any factors affecting L-C levels may also affect ATP levels. This endogenous compound, L-C, together with its acetyl ester, acetyl-L-carnitine (ALC), also participates in the control of the mitochondrial acyl-CoA/CoA ratio, peroxisomal oxidation of fatty acids, and production of ketone bodies. A deficiency of carnitine is known to have major deleterious effects on the CNS. We have examined L-C and its acetylated derivative, ALC, as potential neuroprotective compounds using various known metabolic inhibitors, as well as against drugs of abuse such as methamphetamine.

3-Nitropropionic acid (3-NPA) is a model mitochondrial inhibitor that causes selective neurodegeneration in brain. 3-NPA-induced neurodegeneration occurs via a secondary neurotoxicity, caused initially by ATP depletion and redox changes in the cell. It is known that the hippocampal degeneration caused by mitochondrial dysfunction affects learning and memory, cognitive functions commonly disturbed in neurodegenerative diseases. The 3-NPA- treated animal model can be used to study molecular mechanisms underlying selective degeneration in the brain. In this study, a microarray approach was utilized to define changes in the expression of 530 genes in the rat hippocampus after acute exposure to 3-NPA at 30 mg/kg, sc. The microarray data were collected at 30 min, 2 h, and 4 h post-3-NPA. Statistical modeling using an ANOVA mixed model applied to Van der Waerden scores of rank-transformed intensity data was used to assign statistical significance to 44 transcripts. These transcripts represent genes associated with energy metabolism, calcium homeostasis, the cytoskeleton, neurotransmitter metabolism, and other cellular functions. Changes in the transcripts of genes encoding 2 transporters [blood-brain specific anion transporter (Slco1c1) and sodium-dependent inorganic phosphate cotransporter (Slc17a7)] were confirmed by real-time RT-PCR. In conclusion, this study identified 2 new potential targets for enhancement of neuroprotection or inhibition of neurodegeneration associated with ATP depletion in the hippocampus.