Joanne M. Miller

Plasma norepinephrine, epinephrine and corticosterone stress responses to restraint in individual male and female rats, and their correlations

Norepinephrine (NE), epinephrine (E), and corticosterone (C) levels were determined in the same male and female rats before and during a 30-min period of restraint. Individual values were compared within and among NE, E and C. Individual rats responded differently to immobilization and females usually showed higher stress values. NE and E levels rose smoothly and simultaneously in all animals and peaked early. The early (5 min) NE and E levels, but not the baseline values (0 min), predicted the total extent of the NE and E stress responses in both sexes. The C response was relatively independent of either E or NE. The male C response, but not the female C response, was strongly predicted by the C 5-min level, and significantly predicted by the C 15-min level. Additionally, the male C response, unlike the E, NE or female C response, was biphasic, dropping significantly below baseline at 5 min before rising to peak levels at the end of immobilization (30 min).

Rat plasma levels of amino acids and related compounds during stress

Forty-one amino acids and related compounds were measured (using an HPLC physiological amino acid analysis procedure fully validated for plasma studies) in rat plasma obtained through an indwelling jugular catheter before, during and following a 30 min period of immobilization. Taurine, phosphoethanolamine, aspartic acid, glutamic acid, alanine, cystine, tyrosine, beta-alanine and ethanolamine were increased during the period of stress; whereas, valine, tryptophan and arginine were decreased. Most of these alterations were restored toward normal during the 30 min of rest following the stress period. However, cystine, ethanolamine and beta-alanine remained significantly elevated, and valine, tryptophan and arginine remained significantly reduced. Serine, isoleucine, leucine and glutamine were not significantly altered during the stress period, but became significantly reduced during the 30 min following the stress period. While the patterns of amino acid alterations were generally consistent from animal to animal, the magnitude of the responses were variable with some rats demonstrating much larger responses than others. These results may implicate amino acids as important markers for stress related pathologies. The individual differences noticed may explain why some individuals show more stress effects than others.

Effects of psychoactive drugs on plasma catecholamines during stress in rats

In unstressed rats, morphine, pentobarbital, diazepam and alprazolam were without effect on plasma norepinephrine (NE) and epinephrine (E) levels. Amphetamine increased the levels of both catecholamines. In stressed rats, morphine did not affect the stress-induced increases in NE and E. Amphetamine enhanced the catecholamine stress-response further. Diazepam reduced stress-induced increases of NE levels. Pentobarbital and alprazolam attenuated stress-induced increases of both NE and E levels; this effect was particularly marked with alprazolam.

Comparison of amino acid levels in rat blood obtained by catheterization and decapitation

A novel, sensitive and highly resolving amino acid analysis procedure was developed and used to compare two methods of obtaining blood from experimental animals. The procedure, utilizing a meter long microbore HPLC column containing spherical cation-exchange resin and fluorescence detection following postcolumn reaction with o-phthaldialdehyde, was shown to reliably measure forty-one primary amine components in rat plasma. Comparison of values from blood obtained by decapitation and by catheterization documented the significant artifactual influence of the decapitation procedure on approximately half of the measured constituents.

Effects of acute ethanol administration on rat plasma amino acids and related compounds

Using high performance liquid chromatography (HPLC) with fluorometric detection, thirty-three amino acids (AA) and related compounds were measured in plasma obtained from catheterized rats over a 3-hr period following a 2 g/kg, i.p., injection of ethanol. The concentrations of twenty-three of these compounds had decreased significantly 15 min after the injection, and twenty-three remained depressed for the 3-hr period. Marked reductions were noted for alanine and arginine. Glutamic acid, 1-methylhistidine and 3-methylhistidine were unaffected by ethanol. During these studies individual differences were observed in that some rats showed marked biochemical changes, whereas other rats showed only minimal responses. These observations indicate that ethanol administration may have a significant and long-lasting impact on plasma amino acid biochemistry.

Brain amino acid concentrations in rats killed by decapitation and microwave irradiation

The effect of death by decapitation or focused beam microwave irradiation (FBMI) on rat brain amino acid concentrations was investigated. Twenty-nine amino acids and related compounds were measured by ion-exchange chromatography in the cerebral cortex, hippocampus, striatum and substantia nigra of male Sprague-Dawley rats killed by decapitation (n = 5) or by FBMI (n = 5). Alanine, GABA, ethanolamine and NH3 concentrations were significantly lower in all 4 brain regions of the FBMI group animals. Valine, leucine, tyrosine and phenylalanine levels were also lower in the hippocampus, striatum and substantia nigra of the FBMI group. The FBMI group showed less aspartate in the hippocampus and substantia nigra as well as less glycine in the cortex, hippocampus and striatum. In the FBMI group, the only amino acids exhibiting significantly higher levels were GSH in the striatum and substantia nigra and glutamate in the substantia nigra. These results show a significant impact of method of killing on the determination of baseline concentrations of brain amino acids.

Differential effects of ethanol on plasma catecholamine levels in rats

Acute ethanol administration (1-4 g/kg, i.p.) had no effect on plasma catecholamine levels in nonstressed animals except at the highest dose where levels of both catecholamines increased. In animals stressed for 30 min, the higher doses had a biphasic effect on plasma catecholamines; at earlier times during stress a reduction in stress-induced increases in both catecholamines was seen, whereas later during stress or after release from stress an increase was noted. Semi-chronic ethanol administration (0.5 and 2 g/kg/day, i.p.) had no significant effect on plasma catecholamine levels in nonstressed rats. In stressed rats, ethanol reduced stress-induced catecholamine increases but these reductions were less than those seen after acute administration. Although ethanol reduced the gross behavioral stress response, no correlation between gross behavioral and biochemical responses was detected. These data show that ethanol can indeed reduce the behavioral and biochemical stress responses in rats but that effects seen depend on the state (nonstressed vs stressed) of the animal, the dose of ethanol (low vs high) used, the length of ethanol administration (acute vs semi-chronic), and the time of measurement of the catecholamine level after ethanol administration.

Chronic lithium treatment and status epilepticus induced by lithium and pilocarpine cause selective changes of amino acid concentrations in rat brain regions

We measured the effects of four weeks of dietary lithium treatment and of status epilepticus induced by administration of pilocarpine to lithium-treated rats on the concentrations of amino acids in four regions of rat brain: cerebral cortex, hippocampus, striatum, and substantia nigra. To ensure accurate quantitation of the amino acids, animals were sacrificed by focussed beam microwave irradiation and amino acids were measured using a fully validated triple-column ion-exchanged amino acid analyzer with post-column o-phthalaldehyde derivatization and fluorometric detection. The concentrations of four amino acids, threonine, methionine, lysine and tyrosine, were increased significantly in two to four brain regions by chronic lithium treatment. Their concentrations remained elevated, or were further increased, during status epilepticus. The concentrations of eight amino acids and ammonia were not altered by lithium treatment but increased in concentration during status epilepticus in some brain regions. Glycine, serine, arginine and citrulline were decreased by chronic lithium treatment. Status epilepticus increased the concentrations of these four amino acids above that found in the lithium-treated samples in some of the brain regions that were examined. Six amino acids and glutathione were generally unaltered by both treatments. These results are related to the effects of lithium treatment and are compared with changes reported by others following treatment with a variety of convulsive stimuli.

Biochemical and behavioral changes in rats during and after chronic d-amphetamine exposure

Two groups of rats were implanted with ALZET minipumps to deliver vehicle or a theoretical amount of 1 mg/kg per h of d-amphetamine (A) for 12 days. After 3 days of A-exposure, motor movements and stereotypic behavior were markedly increased. Subsequent testing during A-exposure showed that motor movements and stereotypic behavior remained significantly increased but declined. After removal of the pumps, these effects disappeared and no differences at rest, during stress or A challenge, were apparent in either group. Animals sacrificed after 3 days of drug exposure, showed a drastic decrease in cardiac, but not adrenal, catecholamine levels. In the brain, norepinephrine (NE) levels were markedly decreased in the frontal cortex, hypothalamus, caudate, pons-medulla and cerebellum. Epinephrine (E) levels were unaffected and dopamine (DA) levels were decreased in most areas without reaching statistical significance. Plasma corticosterone levels were similar in both groups. Animals in both groups sacrificed about 25 days after pump removal were biochemically similar. Under our conditions, A-exposure produced marked behavioral and biochemical changes but there was no evidence of residual abnormalities after cessation of drug treatment.

Detection of Several Novel γ-Aminobutyric Acid-Containing Compounds in Human CSF

Abstract: γ‐Aminobutyric acid (GABA) concentrations in human CSF are known to increase significantly after hydrolysis; however, the source of this increase has been unknown. Using either ion‐exchange or reverse‐phase chromatography coupled with on‐line alkaline hydrolysis, we have shown 2‐pyrrolidinone, the lactam of GABA, to be present in insufficient quantity to account for this increase. Subsequent experiments involving fraction collection of column eluents followed by acid hydrolysis and rechromatography demonstrated the presence of several previously undetected GABA‐containing compounds.