Thomas N. Ferraro

Free and conjugated amino acids in human CSF: Influence of age and sex

An extended baseline characterization of amino acids (AAs) and related amino compounds in CSF is reported. Thirty-one amino compounds were measured in deproteinized CSF before and after acid hydrolysis using a triple-column HPLC/fluorometric analyzer. CSF specimens were collected under strictly controlled conditions from neurologically normal myelogram patients and carefully pooled with regard to subject age and sex. Consideration was given to factors which may produce artifactual alterations in AA levels during CSF collection, storage and handling. Conjugated AAs were determined as the difference between levels of free AAs (measured in CSF prior to hydrolysis) and total AAs (measured in hydrolyzed CSF) and are taken as an index of total CSF peptide AAs. Results documented conjugated forms of all non-acid-labile CSF AAs except citrulline and ethanolamine. In general, ratios of conjugated to free AAs were relatively low, however for the neurotransmitter AAs aspartate, glutamate, glycine and GABA as well as for beta-alanine hydrolysis produced marked increases indicating that these compounds are present predominantly in bound form in CSF. Results also revealed the significant influence of both age and sex on levels of a number of CSF free and conjugated AAs. Compared to younger individuals (those less than 40 years of age), older individuals exhibited significantly higher levels of free aspartate, glycine, alpha-aminobutyric acid, valine, isoleucine, leucine, phenylalanine and 3-methylhistidine as well as significantly lower levels of free phosphoethanolamine, serine, GABA, homocarnosine, conjugated GABA and conjugated beta-alanine. Additionally, significantly higher levels of free tyrosine, ethanolamine, arginine and conjugated aspartate were documented in males compared to females.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential susceptibility to seizures induced by systemic kainic acid treatment in mature DBA/2J and C57BL/6J mice

Summary: Mature DBA/2J (D2) and C57BL/6J (B6) mice aged P.10 weeks were studied to determine susceptibility to behavioral seizures induced by kainic acid (KA) and the possible influence exerted by differences in metabolism and blood‐brain barrier (BBB) transport. Mice were observed for 4 h after subcutaneous (s.c.) KA injection. Behavioral seizure parameters included latency to first seizure (clonus), latency to tonic/clonic seizure, and latency to status epilepticus (SE). At a KA dose of 25 mg/kg, 80% of D2 mice exhibited tonicklonic seizures, whereas all B6 mice remained seizure‐free. At 30 mg/kg, tonic/ clonic seizures were observed in 100% of D2 mice and 25% of B6 mice. Of D2 mice exhibiting at least one clonic seizure in response to KA at a dose of 25 mg/kg, 50% entered SE and eventually died. Administration of [3H]KA (6.6 × 106dpm) at doses of 25 mg/kg (convulsive) or 11.1 μ/g (nonconvulsive) to mice of both strains resulted in similar levels of radioactivity in cortex, hippocampus, and cerebellum 30 and 60 min after injection. Bioconversion of [3H]KA to a radiolabeled brain metabolite in vivo could not be documented in mice from either strain. Results confirm previously reported differences between D2 and B6 mice in their relative susceptibility to seizures induced by systemic KA administration and suggest that these differences are not related to strain‐specific variation in metabolism or BBB transport of KA. Further studies of these two strains of mice may be useful for investigating genetic influences upon seizure susceptibility.

Human mu opioid receptor gene polymorphisms and vulnerability to substance abuse

Two polymorphisms of the human mu opioid receptor gene are described. A non‐coding region polymorphism (G to T) occurs at nucleotide 175 preceding the initiation of translation. A coding polymorphism in exon 1 (C to T) at nucleotide 229 changes an alanine residue to a valine residue. Frequencies of these polymorphisms were examined in groups of cocaine and/or opioid dependent individuals and matched controls. There were no significant differences between groups, although a trend (p= 0.05) towards a higher frequency of the 229 valine allele was observed in the substance abuse group, suggesting a need for large, well‐controlled studies of this polymorphism in severe substance abusers.

Rat strain and age differences in kainic acid induced seizures

This study reports comparative dose-response data for kainic acid (KA) induced seizures in juvenile (35-40 days old) and adult (70-90 days old) Wistar-Furth (WF), Fisher 344 (F344), Sprague-Dawley (SD) and Long-Evans Hooded (LEH) rats. Juvenile male WF (n = 51), F344 (n = 55), SD (n = 60), LEH (n = 50) and adult male WF (n = 48), F344 (n = 52), SD (n = 52), LEH (n = 53) rats were given KA 6, 8, 10, 12 or 14 mg/kg, sc. As previously demonstrated adult WF and F344 rats showed the greatest sensitivity and most reliable convulsant responses to kainic acid; SD and LEH rats were less sensitive and showed more variable convulsant responses. Regardless of strain, all juvenile rats exhibited greater sensitivity and less variable convulsant response to KA compared to adults. This was most evident in juvenile SD and LEH rats. Results suggest that while seizure sensitivity to KA decreases with age, genetic factors may regulate the expression of this resistance.

Effect of 1-methyl-4-phemyl-1,2,3,6-tetrahydropyridine (MPTP) on levels of glutathione in the extrapyramidal system of the mouse

Treatment of mice with the proximate neurotoxin MPTP depletes striatal dopamine levels. Depletion of striatal dopamine and metabolites in MPTP-treated mice is accompanied by depletion of glutathione (GSH) in the substantia nigra (SN). Striatal GSH and nigral amino acid levels were not significantly affected by MPTP. Results suggest that GSH depletion in SN may represent an index of regional vulnerability to metabolic oxidative stress and also of selective susceptibility to the toxic effects of MPTP.

Systemic acetyl-L-carnitine elevates nigral levels of glutathione and GABA.

Amino acid and reduced glutathione (GSH) levels in substantia nigra (SN) as well as striatal monoamine levels were measured in acetyl-L-carnitine (ALCar) treated and control Swiss-Webster mice. ALCar, L carnitine, or saline were administered i.p. to mice for 5 days and mice were decapitated 24 hours following the last injection. Substantia nigra and striata were isolated within 2.5 and 3 min., respectively, and frozen immediately on dry ice. A significant dose-dependent increase of nigral GABA was observed following ALCar treatment; GABA levels were also increased by administration of carnitine. Nigral GSH levels were also increased. Striatal levels of dopamine and metabolites were not significantly affected by ALCar or carnitine. These results, suggest that ALCar may be useful in treating symptoms of neuronal dysfunction related to accumulation of metabolic waste.

In vivo modulation of excitatory amino acid receptors: microdialysis studies on N-methyl-d-aspartate-evoked striatal dopamine release and effects of antagonists

Striatal dopamine (DA) release was measured following intrastriatal (i.s.) administration of N-methyl-D-aspartate (NMDA) to unanesthetized, freely-moving rats. One hour after insertion of a removable microdialysis probe and perfusion with normal Ringers solution, a modified Ringers solution containing 100 mM potassium (high-K+ Ringers) was used to standardize the preparation. DA release following i.s. administration of NMDA (12.5 mM in normal Ringers) was dose-dependent. When NMDA (12.5 mM) was administered in high-K+ Ringers, DA release was greatly potentiated. Administration of the competitive NMDA receptor antagonist aminophosphonovalerate (APV) in normal Ringers prior to treatment with NMDA in high-K+ Ringers resulted in a significant reduction of DA release compared to control animals. In contrast, administration of APV priot to treatment with NMDA in normal Ringers resulted in a significantly increased release of DA compared to controls. Administration of the non-competitive NMDA antagonist, dextromethorphan (DXT) prior to treatment with NMDA in normal Ringers or NMDA in high-K+ Ringers caused significant reductions of DA release compared to controls. Intrastriatal DXT also caused dose-dependent inhibition of high-K+ Ringers-induced DA release. Similarly, administration of the non-specific calcium channel blocker, cadmium, prior to treatment with NMDA resulted in a significant decrease when compared to control values. Results of this study indicate that dose-dependent NMDA-induced striatal DA release is greatly potentiated by potassium suggesting that under physiological conditions in vivo, striatal NMDA receptors are mostly inactivated.(ABSTRACT TRUNCATED AT 250 WORDS)

Continuous monitoring of brain ethanol levels by intracerebral microdialysis

A method is described which allows simultaneous collection of blood and perfusate of discrete brain regions from an individual animal over several hours. This procedure involves catheterization of a peripheral blood vessel (jugular vein) and the insertion of a microdialysis probe into a specified brain area (lateral hypothalamus) for sampling of blood and brain perfusate, respectively. Using this procedure, levels of ethanol in blood and brain perfusates were determined by scintillation counting following administration of [14C]-ethanol (20 mu Ci) to adult male rats at a dose of 0.8 or 2.4 g/kg. Ethanol levels in brain and blood as well as the time-course of disappearance were dependent on the dose administered. Peak blood levels were observed in the first sample taken (i.e., at 10 min), whereas a slight delay was noted in the time to peak level in brain. At subsequent time points, a good correlation was observed between blood and brain perfusate radioactivity levels although perfusate levels were slightly lower. It is concluded that this approach will prove useful for investigating the molecular and cellular mechanisms of action of ethanol by enabling the direct correlation of blood and brain ethanol levels with various behavioral, electrophysiological and/or biochemical measures.

Triple-column ion-exchange physiological amino acid analysis with fluorescent detection: Baseline characterization of human cerebrospinal fluid

A highly resolving triple-column amino acid analyzer with fluorometric detection is described. The reliability of this technique was evaluated and it was used in a baseline investigation of amino acids and related compounds in human cerebrospinal fluid (CSF). The procedure employs three distinct ion-exchange columns to elute the acidic, neutral, and basic amino acids, respectively. Each column is run isocratically with lithium citrate buffers designed to provide overlapping elution profiles. Studies using CSF collected under strictly controlled conditions documented nanomolar concentrations of aspartate, gamma-aminobutyric acid (GABA), beta-alanine, 1-methylhistidine, and 3-methylhistidine, as well as low levels of glutamate, methyllysine, and ammonia. In addition, other common amino acids were also quantified. Chromatograms of CSF from all three systems (acidic, neutral, and basic) exhibited numerous uncharacterized compounds emphasizing the resolution and sensitivity of the analytical procedure. In vitro stability studies revealed that levels of aspartate, glutamate, GABA, homocarnosine, and ammonia are subject to significant change when CSF is maintained at room temperature for various periods of time up to 24 h. It is concluded that the valid and accurate measurement of CSF amino compounds, especially the neurotransmitter amino acids, requires a highly specific and sensitive assay procedure as well as strict control of CSF manipulation in vitro.

Cerebral metabolism of parkinsonian primates 21 days after MPTP

This study evaluates the changes in the local cerebral metabolic rate for glucose (LCMRg) in primates exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The LCMRg was evaluated 21 days following the last dose of MPTP. At this time, all MPTP-injected animals demonstrated parkinsonism and striatal dopamine was reduced to less than 3% of control values. The structures whose LCMRg was most affected were the motor cortex, the intermediate zone of the putamen, the external segment of the globus pallidus, the medial part of the ventrolateral nucleus of the thalamus (VLm), visual cortex, locus ceruleus, and the dorsolateral segment of the substantia nigra pars compacta. The structure whose increase in LCMRg correlated most closely to the clinical severity of parkinsonism was the external segment of the globus pallidus.