I.Arthur Michaelson

Effect of in vitro inorganic lead on dopamine release from superfused rat striatal synaptosomes

The effect of inorganic lead in vitro in several aspects of [3H]dopamine release from superfused rat striatal synaptosomes was examined. Under conditions of spontaneous release, lead (1-30 microM) induced dopamine release in a concentration-dependent manner. The onset of the lead-induced release was delayed by approximately 15-30 sec. The magnitude of dopamine release induced by lead was increased when calcium was removed from the superfusing buffer. Lead-induced release was unaffected in the presence of putative calcium, sodium, and/or potassium channel blockers (nickel, tetrodotoxin, tetraethylammonium, respectively). Depolarization-evoked dopamine release, produced by a 1-sec exposure to 61 mM potassium, was diminished at calcium concentrations below 0.254 mM. The onset of depolarization-evoked release was essentially immediate following exposure of the synaptosomes to high potassium. The combination of lead (3 or 10 microM) with high potassium reduced the magnitude of depolarization-evoked dopamine release. This depression of depolarization-evoked release by lead was greater in the presence of 0.25 mM than 2.54 mM calcium in the superfusing buffer. These findings demonstrate multiple actions of lead on synaptosomal dopamine release. Lead can induce dopamine release by yet unidentified neuronal mechanisms independent of external calcium. Lead can also reduce depolarization-evoked dopamine release by apparent competition with calcium influx at the neuronal membrane calcium channel.

Differential effects of inorganic lead and δ-aminolevulinic acid in vitro on synaptosomal γ-aminobutyric acid release☆☆☆

Several studies have shown that inorganic lead added in vitro does not alter γ-aminobutyric acid (GABA) release from rat brain synaptosomes. The decrease in GABA release observed following chronic neonatal in vivo lead exposure has been proposed to be an indirect effect mediated by the increase in δ-aminolevulinic acid (ALA) accompanying chronic lead exposure. In the present study the effect of both lead and ALA in vitro on several aspects of [3H]GABA release from superfused rat cortical synaptosomes are examined. The present study demonstrates that lead (1–30 μm) added in vitro induces [3H]GABA release from preloaded cortical synaptosomes in a dose-dependent manner. This lead-induced increase in spontaneous [3H]GABA release does not appear to be mediated by inhibition of the membrane Na-K ATPase. ALA also induces a dose-dependent [3H]GABA release, but only at concentrations equal to or greater than 30 μm. Exposure to a combination of 3 μm lead and 100 μm ALA results in an increase in spontaneous [3H]GABA release that is greater than either treatment separately. The depolarization-evoked release of [3H]GABA resulting from a 1-sec exposure to 61 mm potassium chloride is reduced by lead (3 and 10 μm), whereas ALA (30–300 μm) does not alter depolarization-evoked release. These findings indicate that an indirect action of lead (elevated ALA concentrations) need not be proposed to explain the alterations in GABA release observed following chronic lead exposure.

Estimation of daily exposure in neonatal rats receiving lead via dam's milk☆

Abstract Knowledge of the concentration of lead in milk on a specific day of lactation and volume consumed enables estimation of total daily lead exposure. There are limited data available on volume of milk consumed per 24 hr by a neonatal rat. The volume of milk consumed each day can, however, be estimated with knowledge of the caloric requirements necessary to maintain normal daily growth and caloric value of milk during lactation. Average caloric requirements per gram body weight in neonatal rats in approximately 0.40 kcal/g/day. Caloric values of milk range from 2.62 to 1.48 kcal/ml on Day 1 through Day 18 of lactation. Estimated milk consumption increases from 0.65 ml/pup on Day 1 to 15.51 ml on Day 18. Using these values, one can estimate daily lead intake by pups drinking lead-contaminated milk. A 24-hr cross-fostering study in 13-day-old rats indicated that theoretical estimates of lead body burden were 100 ± 5% of the actual lead body burden.

Simultaneous measurement of tyrosine, tryptophan and related monoamines for determination of neurotransmitter turnover in discrete rat brain regions by liquid chromatography with electrochemical detection

Concomitant measurement of monoamine neurotransmitter turnover in discrete rat brain areas with the use of radiolabeled amino acid precursors permits simultaneous evaluation of interacting transmitter systems. [3H]Tyrosine and [3H]tryptophan were administered via indwelling catheters to unrestrained rats. Content and specific activity of norepinephrine, dopamine, 5-hydroxytryptamine, and the metabolites dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid in addition to tyrosine and tryptophan were quantified by liquid chromatography with electrochemical detection and liquid scintillation counting. The method employs a simple extraction procedure without prior cleanup for chromatography. Neurotransmitter turnover rates that incorporated tyrosine- or tryptophan-specific activities were found to be two to four times greater than those that did not include them.

The effect of lead on oxidative hemolysis and erythrocyte defense mechanisms in the rat

The effect of chronic lead exposure on oxidative hemolysis and on biochemical defense mechanisms against such hemolysis was examined. The interaction between lead (Pb) and phenylhydrazine (PHZ) was studied using four groups of adult male rats treated with lead acetate in a combined oral-ip dosage regimen. After 27 days of exposure the blood lead (PbB) concentrations were (mean ± SD) 2.3 ± 1 (control), 33 ± 4, 67 ± 13, and 104 ± 17 μg/100 ml. On Day 27, PHZ (45 mg/kg sc) was administered to half of the rats in each group, and hemoglobin (Hb) and hematocrit (Hct) determinations were performed on tail blood drawn on Days 28, 29, 34, and 40. The results showed that in the acute hemolytic phase after PHZ both lead alone and PHZ alone reduced Hb and Hct but that the lead-PHZ interaction was not synergistic. A synergistic interaction did occur during the compensatory phase of anemia. The effect of in vivo lead exposure on in vitro hemolysis and biochemical defense mechanisms was studied in a second experiment, the results of which showed that lead caused a dose-dependent increase in oxidative hemolysis in vitro. Superoxide dismutase activity was decreased, whereas pentose shunt activity was increased. The effect of lead on reduced glutathione concentrations and glutathione peroxidase activity was biphasic, being increased at the intermediate dose but returning to baseline at the highest dose. We conclude that the in vivo interaction between Pb concentrations of up to approximately 100 μg/100 ml blood and oxidative hemolytic anemia was due to the ability of lead to inhibit compensatory hematopoiesis after an acute hemolytic episode. The more sensitive in vitro hemolysis test showed that lead caused a dose-dependent increase in oxidative hemolysis, and the biochemical changes observed were consistent with the hypothesis that in vivo lead exposure exerts a moderate pro-oxidant effect on rat erythrocytes.

Determination of gamma-aminobutyric and glutamic acids in rat brain by liquid chromatography with electrochemical detection

The determination of glutamic and gamma-aminobutyric acids in rat brain regions by derivatization with o-phthaldialdehyde-thiol, isocratic separation by liquid chromatography, and quantification by electrochemical detection provides a simple and precise method for assessing changes in glutamatergic and GABAergic neuronal systems. Gamma-aminobutyric acid was eluted in 30-35 minutes followed by a washout step with 90% methanol to remove all amino acid derivatives with longer retention times. Homoserine was used as an internal standard. Significant increases in gamma-aminobutyric acid content in nucleus accumbens and substantia nigra could be detected 20 minutes after injection of 400 mg/kg valproic acid.

Influence of chronic inorganic lead exposure on regional dopamine and 5-hydroxytryptamine turnover in rat brain

The results of previous behavioral studies utilizing chronic exposure to low amounts of inorganic lead (Pb) have suggested alterations in the function of biogenic amine neuronal systems. The following study was performed to provide evidence for the possible bases of these changes in pharmacological responsiveness in exposed animals. Dams were administered 0.2% Pb acetate in drinking water to expose their offspring to Pb via the maternal milk. Males were weaned to the same drinking solution. At 120–140 days a tracer dose of 1.0 mCil-[3H]2,6-tyrosine (3H-TYR) and 0.5 mCil-[3H(G)]tryptophan (3H-TRP) was injected through an indwelling jugular catheter, and norepinephrine (NE), dopamine (DA), 5-hydroxytryptamine (5-HT) and their respective precursors and metabolites were quantified by liquid chromatography with electrochemical detection with column eluate collected for liquid scintillation counting. At this level of exposure (blood lead (PbB) at day 90 in exposed animals=43.1±1.7 μg/dl) no changes were observed in concentration Nf NE or DA mr DA metabolites in any brain region. However, DA turnover was decreased in Pb-exposed animals in nucleus accumbens and frontal cortex. No changes in 5-HT content and turnover were observed in any brain region, but 5-hydroxyindoleacetic acid (5-HIAA) levels were decreased in 6 of the 9 brain regions examined. These findings are consistent with observations of an attenuated behavioral responsiveness to d-amphetamine (AMPH) in exposed animals, and suggest that the changes in DA and 5-HT neurons noted by other workers at higher levels of exposure persist when PbBs are in the range of 40 μg/dl.

THE SUBCELLULAR DISTRIBUTION OF ACETYLCHOLINE, CHOLINE ACETYLTRANSFERASE AND ACETYL CHOLINESTERASE IN NERVE TISSUE

It is well established that acetylcholine (ACh) is the chemical transmitter at peripheral synapses, including the neuromuscular junction. Its role as a central transmitter has been confirmed by the classical criterion of increased release during nerve stimulation (Mitchell, 1963). Only within the last few years have biochemical studies, correlated with electron microscopy, worked out the subcellular localization of acetylcholine and the enzymes involved in its synthesis (choline acetyltransferase, CHAc) and in its destruction (acetyl cholinesterase, ChE). Nature of Bound Acetylcholine. Acetylcholine of nervous tissue is not freely diffusible but is largely bound to particulate material of the cell. Early work suggested that it was held in some kind of subcellular structure such as mitochondria (Bodian, 1942; Brodkin & Elliot, 1953; Braganca & Quastel, 1952). Even earlier work (Stedman & Stedman, 1959; Mann et al., 1938, 1939a, b) , on the activating effects of organic solvents on the synthesis of ACh by brain suspension, indicated that the rate of synthesis might be limited by membrane permeability. The implication was that the enzyme was occluded as well. Feldberg (1945) viewed the nature of bound ACh as “synthesized into its linkage.” In other words, within the cell the ester has a close spatial relationship with the enzymes involved in its synthesis. Brodie and Bain (1952) showed that brain tissue homogenized in 0.25 M sucrose could be fractionated into nuclear, mitochondrial, microsomal, and supernatant fractions, the last representing the soluble cytoplasmic components of the cell. With similar techniques of tissue disruption and differential centrifugation, Hebb and Smallman (1956) studied the intracellular distribution of choline acetyltransferase, the enzyme that catalyzes the transfer of the acetyl moiety from acetyl CoA to choline. They found that choline acetyltransferase activity is associated with the mitochondrial fraction as defined by Brodie and Bain ( 1952) ; this fraction also possesses staining reactions characteristic of mitochondria. In the light of earlier evidence, it was logical to suggest that the mitochondria were also the storage sites of the sequestered acetylcholine found in nerve tissue. A mitochondrial localization of choline acetyltransferase and acetylcholine was of physiological interest, since Jordan (1955) observed many mitochondria at synapses of the central nervous system. The Synapse. The general features of elements of mitochondrial dimensions or larger are visible by light microscopy, but resolution of sub-mitochondrial particles requires electron microscopy. By light microscopy, synaptic boutons or end feet are observed as solid bulbs or reticulated expansions of axonal stalks. By electron microscopy, the synaptic bouton appears as a bulb-shaped or bag-like expansion of the axon. The first clear electron micrographs of the synaptic region showed that the nerve ending has a number of characteristic structural com-

A superfusion apparatus for the examination of neurotransmitter release from synaptosomes

A superfusion apparatus for examining the release of neurotransmitter from synaptosomes is described. The apparatus provides for accurate temperature control, rapid switching and continuous gassing of superfusing buffers, short (i.e. 1-s) pulses of high-potassium-containing buffer for examining depolarized release, rapid collection of superfusing eluate, and low tissue chamber dead volume. Depolarization (high-potassium) evoked release and to some degree spontaneous release of [3H]dopamine from rat striatal synaptosomes are dependent on buffer calcium concentration. The putative calcium channel blocker nickel reduces both spontaneous and high-potassium-evoked [3H]dopamine release.

Drug induced activity in lead-exposed mice.

Three interrelated studies were conducted to examine the locomotor activity of lead-exposed mice. The effects of lead were examined as a function of the dose and duration of exposure. Exposure during the first three weeks occurred via the maternal milk supply. Exposure following weaning was achieved via the water supply. Mice received challenges with various pharmaceutical agents, including d-amphetamine, methylphenidate, apomorphine and phenobarbital. The spontaneous activity prior to injection and the drug-induced activity were monitored. Lead-exposed mice usually displayed spontaneous activity which was indistinguishable from that of the control animals. In only one set of observations did lead exposure result in a modest increase in spontaneous activity. The drug-induced activity varied in a complex manner as a function of the magnitude and duration of the lead exposure. Depressed body weight, which was concurrent with high lead exposure (0.5% Pb(Ac)2) was also a significant parameter affecting both the spontaneous and drug-induced activity.