Gary B. Freeman

Selective alteration of mouse brain neurotransmitter release with age

The release of acetylcholine (ACh), glutamate (GLU) and dopamine (DA) from various brain regions was investigated in young (3 month) and old (30 month) Balb/c mice. Aging increased the basal release of GLU (77%) and DA (29%) in striatum and GLU in hippocampus (94%); the concentrations of these neurotransmitters in the media after K+ stimulation were unaltered by aging. Although the basal release of ACh was not altered by age, K+-stimulated ACh release was reduced in striatum. The age-related increases in basal GLU and DA release may be important in the pathophysiology of cell death during aging.

Cytosolic-free calcium and neurotransmitter release with decreased availability of glucose or oxygen

Exposing brain slices to reduced oxygen tensions or impairing their ability to utilize oxygen with KCN decreases acetylcholine (ACh) but increases dopamine (DA) and glutamate in the medium at the end of a release incubation. To determine if these changes are due to alterations in the presynaptic terminals, release from isolated nerve endings (i.e. synaptosomes) was determined during histotoxic hypoxia (KCN). KCN reduced potassium-stimulated synaptosomal ACh release and increased dopamine and glutamate release. Since several lines of evidence suggest that altered calcium homeostasis underlies these changes in release, the effects of reducing medium calcium concentrations from 2.3 to 0.1-mM were determined. In low calcium medium, KCN still increased dopamine and glutamate release, but had no effect on ACh release. Hypoxia increased cytosolic-free calcium in both the normal and low calcium medium, although the elevation was less in the low calcium medium. Thus, the effects of histotoxic hypoxia on cytosolic free calcium concentration paralleled those on glutamate and dopamine release. Reducing the glucose concentration of the medium also increased cytosolic-free calcium. The data are consistent with the hypothesis that hypoxia and hypoglycemia increase cytosolic-free calcium, which stimulates the release of dopamine and glutamate, whose excessive release may lead to subsequent cellular damage postsynaptically.

Differential alteration of dopamine, acetylcholine, and glutamate release during anoxia and/or 3,4-diaminopyridine treatment

The potassium-stimulated release of acetylcholine (ACh), glutamate (GLU) and dopamine (DA) from mouse striatal slices was studied during anoxia and/or 3,4-diaminopyridine (DAP) treatment. Anoxia, in the presence of calcium, increased DA and GLU release, but depressed ACh release. Omission of calcium from an anoxic incubation further stimulated GLU and DA release and impaired ACh release. Under normoxic conditions, DAP (100 μM) increased the release of all three neurotransmitters; the sensitivity of the slices to DAP changed with the presence or absence of an acetylcholinesterase inhibitor in the preincubation media. During an anoxic incubation, DAP did not ameliorate the anoxic-induced, K+-stimulated impairment of ACh release, but significantly reduced the K+-stimulated release of GLU and DA. These results are consistent with the hypothesis that hypoxia induces a presynaptic deficit that may underlie postsynaptic ischemic-induced changes. Amelioration of these presynaptic alterations in neurotransmitter release may be an effective approach to preventing hypoxic-induced damage.

Monoamine Neurotransmitter Metabolism and Locomotor Activity During Chemical Hypoxia

Abstract: The effects of hypoxia on metabolism of 5‐hydroxytryptamine (5‐HT or serotonin) and 3,4‐dihydroxy‐phenylethylamine (DA or dopamine) were compared with those on open‐field activity in male CD‐1 mice. Chemical hypoxia was induced with NaNO2. Hypoxia did not alter striatal concentrations of DA, 5HT, Trp, Tyr, 5‐hydroxyindoleacetic acid, or homovanillic acid. However, NaNO2 (75 mg/kg) reduced the rates of conversion of [3H]Tyr to [3H]DA (−41%) and [3H]Trp to [3H]5‐HT (39%). Hypoxia also reduced dihydroxy‐phenylacetic acid (DOPAC) levels (−27%) and DOPAC/DA ratios (−20%). Open‐field behavior, as measured in an automated activity monitor, decreased in a dose‐dependent fashion with 75–150 mg/kg of NaNO2 (−35 to −90%). Comparison with previous studies suggests that the syntheses of dopamine, serotonin, and the amino acids are equally vulnerable to hypoxic insults but may be less sensitive than the synthesis of acetylcholine.

Locomotor activity as a predictor of times and dosages for studies of nicotine's neurochemical actions

Nicotines action on the central nervous system is complex and likely involves an interaction of neurotransmitters. To determine the time after administration of nicotine and dosage for neurochemical studies, locomotor activity of CD-1 mice was determined at 5 min intervals between 0-60 min. A low nicotine dosage (0.05 mg/kg) did not alter activity 5-15 min after drug injection, but increased activity 28% at 15-25 min post-injection. A high dosage (0.8 mg/kg) reduced total distance 62% and rearing 87% at 5-15 min; at 15-25 minutes total distance declined 56% and rearing 69%; all measures returned to control values after 30 minutes; rearing then increased at 40 min after nicotine. Pretreatment (15 min before nicotine) with mecamylamine (1.0 mg/kg), but not hexamethonium (1.0 mg/kg), prevented the depressant effect of nicotine. Dopamine (DA) and its metabolites as well as acetylcholine (ACh) synthesis were measured at the point of nicotines maximal depressant action. Striatal levels of dihydroxyphenylacetic acid (DOPAC) were increased and ACh utilization was reduced in striatum (-25%) and cortex (-24%) 10 min after nicotine (0.8 mg/kg). Mecamylamine, while preventing the depressant effect of nicotine on locomotor activity, did not alter its effects on DA metabolism. These results demonstrate that the behavioral outcome of acute nicotine treatment is time and dose-dependent. Nicotines depressant action appears not to be due to altered DA but may be related to changes in carbohydrate and acetylcholine metabolism.

Dopamine, Acetylcholine, and Glutamate Interactions in Aging Behavioral and Neurochemical Correlates

Aging, hypoxia, and thiamin deficiency diminish motor performance. Similar alterations of ACh, DA, and glutamate metabolism accompany hypoxia, thiamin deficiency, and aging. Both aging and hypoxia reduce ACh release and stimulate DA and glutamate release. Presynaptic enhancement of DA and glutamate release may be important in the production of cell damage that may contribute, in part, to age-related deficits in motor as well as cognitive function. The decline in ACh release may be important in the production of the cognitive deficits. An understanding of the interactions of neurotransmitters in hypoxia and thiamin deficiency aids our understanding of normal aging and increases the possibility of developing better treatments for the multiple neurotransmitter deficiencies that accompany many metabolic, age-related, and chronic degenerative disorders.

Effect of age on behavioral and enzymatic changes during thiamin deficiency

Open field behavior and whole brain enzymatic activities were determined during thiamin deficiency in two strains of young, as well as in aged mice. In young CD-1 mice, thiamin deficiency reduced total distance traveled and vertical movements after 7 days and the decline was more than 50% by day 9. The behavioral deficit was highly correlated to decreases in 2-oxoglutarate dehydrogenase activity (KGDH). The open field behavior of Balb/c mice was about 40% less than in CD-1 mice and responded in a qualitatively different manner to thiamin deficiency. The activity of the Balb/c mice increased and then decreased with thiamin deficiency. The activity of 3 month old mice peaked on day 6 (126% of initial score), whereas 10 and 30 month mice showed a much greater increase (about 175% of initial scores), but on day 7. Although the activity of the thiamin dependent enzyme transketolase (TK) was affected similarly at all ages, the activity of KGDH in the aged brain was more sensitive to thiamin deficiency than in the young; KGDH activity declined 41%, 57% or 74% at 3, 10, or 30 months, respectively. Thus, the current mouse model is an attractive one to study the interaction of thiamin deficiency with aging.

Selective damage in striatum and hippocampus with in vitro anoxia

An in vitro model of anoxia-induced brain damage was utilized to help elucidate the biochemical basis of cell damage due to reduced oxygen availability. Previous studies suggest that anoxia-induced damage may vary presynaptically, post-synaptically or in the cell body. Thus, the consequences of an anoxic treatment incubation were examined with hippocampal slices, which contain cholinergic nerve terminals but not cell bodies, and with slices from whole striatum or its subregions, which contain both cholinergic cell bodies and nerve terminals. Slices were preincubated with either oxygen or nitrogen (treatment incubation) and the persistent effects of this treatment on [14C]acetylcholine and14CO2 production from [U-14C]glucose were assessed in a subsequent incubation under optimal conditions (test incubation). An anoxic treatment incubation reduced the subsequent test incubation production of CO2 about 40% in the hippocampus and striatum, The anoxic treatment incubation diminished ACh production by 46% in the striatum, but only minimally affected that in the hippocampus. Anoxic treatment incubations of synaptosomes did not alter test-incubation ACh synthesis or CO2 production. Omission of calcium from the anoxic treatment incubation increased striatal ACh synthesis by 88% and CO2 production in both regions. These results suggest that anoxia produces persistent changes in postsynaptic processes or cell bodies (in this model cholinergic ones) that differ from those in nerve terminals and that calcium is important in the production of these deficits.

Neurotransmitters and Calcium During Hypoxia

An interaction of calcium homeostasis and neutrotransmitter metabolism may underlie deficits induced by hypoxia and ischemia. Both in vitro and in vivo results suggest that altered calcium homeostasis has an integral role in hypoxic/ischemic induced cell damage. Cause-effect relations to cell damage in vivo are difficult to determine because so many variables change simultaneously. In addition, the changes in calcium homeostasis differ between presynaptic and postsynaptic processes and these are also difficult to distinguish in vivo. Thus, the studies on calcium that are described here emphasize in vitro approaches. Similarly, numerous studies suggest that altered neurotransmitter metabolism accompanies hypoxia/ischemia. The precise role of neurotransmitters in the production of hypoxic/ischemic induced tissue damage is not well documented. Much of the research on the role of neurotransmitters in ischemic cell damage has emphasized their postsynaptic action. The present studies concentrate on the presynaptic changes that lead to increased extracellular concentrations of these neurotransmitters during hypoxia/ischemia. The interaction of calcium homeostasis with neurotransmitter metabolism appears important to hypoxic-induced alterations.

Automated method to estimate catecholamine and indoleamine content and turnover rates

A double-label isotopic method for estimation of the rate of formation of serotonin (5-HT) and dopamine (DA) in mouse striatum, hippocampus and cortex was standardized. Mice received an intravenous pulse injection of [3H]tryptophan (TRP) and [3H]tyrosine (TYR) at 2.5, 5, 10 or 20 min before sacrifice by microwave irradiation. Compounds of interest were separated by automated high-performance liquid chromatography and their contents were determined by electrochemical detection. Programmed collection of the TYR, DA, 5-HT and TRP peaks allowed determination of their radioactivity by liquid scintillation. Conversion of [3H]TYR to [3H]DA was nearly ten times greater in striatum than cortex, whereas the formation of [3H]5-HT from [3H]TRP was similar in striatum, cortex and hippocampus.