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Inorganic mercury (Hg2+) in vitro increases spontaneous transmitter release from nerve terminals. The mechanisms of action are not well understood but may involve alterations in intraterminal Ca2+ dynamics. In this study we describe actions of Hg2+ in vitro on isolated mammalian CNS striatal nerve terminals (synaptosomes). Cobalt (2 mM) completely blocked the effect of 2 microM Hg2+ on spontaneous [3H]dopamine release. Cadmium (100 microM) was equipotent to Co2+ in blocking depolarization-dependent [3H]dopamine release, but did not alter the 2 microM Hg2(+)-induced spontaneous [3H]dopamine release. Depolarization-dependent [3H]dopamine release was not altered by 5 microM Hg2+. It appears that the site of action of Hg2+ on spontaneous [3H]dopamine release is not the Ca2+ channel. The effects of Hg2+ on intraterminal ionized Ca2+ [( Ca2+]i) were evaluated using the Ca2(+)-specific fluorescent probe, fura-2. Hg2+ (1-8 microM) had no effect on [Ca2+]i in 1.2 mM Ca2(+)-containing buffers. In nominal Ca2+ media, 4 and 8 microM Hg2+ significantly decreased [Ca2+]i. Following exposure to 4 and 8 microM Hg2+ the quenching of extrasynaptosomal fura-2 by Mn2+ was increased, suggesting that Hg2+ facilitated the leakage of fura-2. This apparent leakage was probably due to a nonspecific increase in membrane permeability since 2 microM Hg2+ produced a Co2(+)-insensitive increase in [3H]deoxyglucose phosphate efflux. Hg2+ did not increase the leakage of either lactate dehydrogenase or soluble protein from synaptosomes. Hg2+ produced a concentration-dependent (1-8 microM) increase in 45Ca2+ efflux from superfused synaptosomes which was insensitive to blockade either by 2 mM Co2+ or by 100 microM Cd2+. These data suggest that the transmitter releasing action of Hg2+ involves interactions with sites that also interact with Co2+ but not with Cd2+. Furthermore, Hg2+ may have direct transmitter releasing actions (i.e., Ca2(+)-mimetic properties), as well as nonspecific actions on plasma membrane permeability which may not necessarily be linked to [3H]dopamine release.

Rats exposed to lead via the maternal milk were tested at maturity on three different visual discrimination tasks. Starting at parturition the dams were given either tap water, 0.20% sodium acetate, 0.02% lead acetate, or 0.20% lead acetate in the drinking water. At weaning, the pups from all the groups were placed on normal chow and tap water. At 20 days of age, the concentration of lead in the blood and brain of the high lead-exposed offspring was approximately 6 times that of controls (11 microgram% vs 66 microgram%). A significant deficit was found in the ability of the high lead-exposed group to acquire a simultaneous visual discrimination task conducted in an operant chamber. No significant differences were observed in the ability of lead-exposed rats to acquire either a successive visual discrimination task or a cued go/no-go discrimination. Thee results suggest that early lead exposure can affect certain behavioral processes and that the effects may persist even after the rat has reached maturity.

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.

Electrophysiological studies employing amphibian neuromuscular preparations have shown that mercuric chloride (HgCl2) in vitro increases both spontaneous and evoked neurotransmitter release. The present study examines the effect of HgCl2 on the release of [3H]dopamine from synaptosomes prepared from mammalian brain tissue. Mercuric chloride (3-10 microM) produces a concentration-dependent increase in spontaneous [3H]dopamine release from "purified" rat striatal synaptosomes, in both the presence and absence of extra-synaptosomal calcium. The effects of HgCl2 on transmitter release from amphibian neuromuscular junction preparations resemble those produced by the Na+, K+-ATPase inhibitor ouabain. Experiments were performed to determine whether the HgCl2 effects on mammalian synaptosomal dopamine release are a consequence of Na+, K+-ATPase inhibition. Na+, K+-ATPase activity in lysed synaptosomal membranes is inhibited by HgCl2 (IC50 = 160 nM). However, mercuric chloride in the presence of 1 mM ouabain still increased [3H]dopamine release. The specific inhibitor of Na+-dependent, high-affinity dopamine transport, RMI81,182 inhibited ouabain-induced [3H]dopamine release whereas it had no effect on HgCl2-induced [3H]dopamine release. These data suggest that augmentation of spontaneous [3H]dopamine release by HgCl2 probably is not mediated by an inhibition of Na+, K+-ATPase and HgCl2 does not act directly on the dopamine transporter.

The results of several studies, employing various tissue preparations, have demonstrated that in vitro Pb exposure has similar effects on the release of several different transmitter substances. Pb has been observed to attenuate depolarization-evoked release and increase spontaneous (depolarization-independent) release. The current study confirms that Pb in vitro increases the spontaneous release of [3H]acetylcholine (ACh) from superfused synaptosomes prepared from rat hippocampus. Additionally, hippocampal synaptosomes, preloaded with 45Ca, were superfused under conditions similar to those used in the [3H]ACh-release studies. Exposure to 1-30 microM Pb produced a concentration-dependent increase in the efflux of 45Ca that was quantitatively and temporally related to the Pb-induced release of [3H]ACh from the hippocampal synaptosomes. Depolarization-evoked [3H]ACh release with high potassium did not produce a corresponding increase in 45Ca efflux. It is concluded that the Pb-induced increase in spontaneous transmitter release is apparently due to either an increase in intraneuronal ionized calcium or the stimulation by Pb of Ca-activated molecules mediating transmitter release.

Locomotor activity and brain catecholamine concentrations following d-amphetamine challenge were evaluated in lead-exposed rats. Sucklings were exposed to lead via its transfer from maternal drinking solutions (0.02% Pb (Ac)2, 109 ppm Pb) to the maternal milk supply. At 21 days of age half of each exposed litter was weaned to the maternal regimen, the remainder to distilled water. Spontaneous locomotor activity and lead in blood were qualified at 21, 30 and 90 days of age. At 90 days of age rats were also challenged with d-amphetamine sulfate (1.0 mg/kg, SC) and their drug-induced activity recorded for a 2 hours period. One week later the same animals received a similar dose of d-amphetamine and were sacrificed at 0, 0.5, 1, 3, 6, 12, 24 and 48 hours post injection for analysis of whole brain dopamine (DA) and norepinephrine (NE). Catecholamine concentrations did not differ between lead-exposed and control rats in the absence of a d-amphetamine challenge. However, over the 48 hr period following drug challenge, lead-exposed rats had lower whole brain steady state levels of NE than control animals. Similar changes were evident for rats exposed to lead continuously and for those exposed to lead only during the 21 day period from birth to weaning. The spontaneous locomotor activity of lead-exposed and control rats was comparable at all ages tested. However, drug-induced activity was greater in lead-exposed animals than controls. This facilitated drug response was statistically greater than that of controls for continuously lead-exposed rats. Those exposed only during the neonatal period showed similar trends.

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.

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.

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.

Rats exposed to lead via maternal milk were tested at various stages of development on a number of behavioral tasks. Beginning at paturition, the dams were given either tap water, 0.02%, or 0.10% lead acetate in the drinking water. Pups from all three groups were weaned to normal chow and tap water at 21 days of age. The mean lead concentration of the dam's blood and of neonatal (20 days of age) brain and blood were all below 50 microgram/100 ml. No significant differences were found between the high lead-exposed group and controls in general as measured by wheel running over a 21 day period beginning at 30 days of age. However, there was a significant difference in wheel running behavior during the first three hr of testing. Both lead-exposed groups were found to display significantly less aggressive behavior as measured by the shock-elicited aggression test. Low level lead exposure had no discernable effect on the acquisition and subsequent reversal of a successive brightness discrimination task. Lead exposure under these conditions appears to affect some aspects of emotional behavior, while having little effect on general activity or cognitive function.

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.

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 mCi L-[3H]2,6-tyrosine (3H-TYR) and 0.5 mCi L-[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 micrograms/dl) no changes were observed in concentration of NE or DA or 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 micrograms/dl.

Neonatal rats were given aqueous lead acetate intragastrically from d 2--20 of life at doses of 0, 25, 75, and 225 mg Pb/kg.d. Blood Pb concentrations on d 21 were (mean +/- SE) 27 +/- 4 (control), 150 +/- 26, 263 +/- 63, and 518 +/- 97 microgram/100 ml, respectively. Growth was significantly depressed only in animals given the highest dose of Pb (225 mg/kg.d). Hematocrits were significantly decreased by d21 at all doses of Pb. Malondialdehyde (MDA) formation in 750 x g (10 min) brain supernatants induced spontaneously by aerobic incubation at 37 degrees C was not altered by Pb on d 7 and 14, but a slight decrease was observed on d 21. The extent of MDA formation induced by enzymatically generated superoxide anion was not altered by Pb toxicity during the first 21 d of life. Addition of Pb to 750 x g (10 min) brain supernatants in vitro significantly decreased MDA formation at Pb concentrations of 10(-5) M and higher. These results show that the central nervous system toxicity of Pb in neonatal rats is not associated with accelerated in vitro lipid peroxidation of brain tissue.

Investigations of the mechanisms involved in the neurotoxicity resulting from chronic inorganic lead (Pb) exposure have centered on CNS biogenic amine function on the basis of behavioral and neurochemical findings. The following study examined the time course of the response of dopamine (DA) and 5-hydroxytryptamine (5-HT) neurons to d-amphetamine (AMPH) in rats chronically exposed to Pb from birth in order to further examine neurochemical mechanisms implicated by previous work. Offspring were exposed to 0.2% Pb acetate via the lactating dam and then weaned to the same drinking solution. At 120-140 days animals were injected with 1.0 mg/kg s.c. of the drug or with saline and sacrificed after various intervals. DA content in nucleus accumbens and corpus striatum in Pb-exposed animals was significantly higher than corresponding levels in controls at 20 minutes post-drug and remained significantly higher than baseline values at 80 minutes after the drug when DA concentrations in controls had returned to normal. These data suggest enhanced AMPH-induced DA synthesis in exposed rats. 5-Hydroxyindoleacetic acid (5-HIAA) content was significantly increased in three brain regions in exposed rats given AMPH compared to values in saline-injected exposed animals, indicating a compensation in these areas for the decreases in 5-HIAA values produced by Pb exposure alone. The results of this study reinforce the hypothesis that DA and 5-HT neurons are sensitive to relatively low levels of Pb exposure.

The primary objective of this research was to test the hypothesis that low level lead (Pb) exposure during early life leads to disruption in blood-brain barrier (BBB) function in the young rat. Newborn rats received lead via milk from lactating dams that were drinking water containing 0.1% lead acetate Pb(Ac)2. Pups were weaned to, and maintained on, 0.1% Pb(Ac)2-containing solution up to 70 days of age. Growth was no different from that of coetaneous controls. Experimental animals displayed elevated blood lead (15 microgram/dl) within 2 days from the onset of exposure, and it increased to 35-40 microgram/dl between 13 and 22 days of age. Following weaning to the higher lead source, blood lead values continued to increase (55 microgram/dl) but, then, appeared to decline after 55 days of age. Control animals consistently possessed blood lead values of less than 5 microgram/dl. The brain capillary (BBB) transport of the neurotransmitter precursors, choline and tyrosine, was studied at 55 and 70 days of age using intracarotid injections of a bolus containing 14C-labeled substrate and 3HOH as a diffusable reference (Brain Uptake Index). There was no difference in the transport of either choline of tyrosine in lead-intoxicated rats compared to controls. Suspected adverse psychoneurological effects of low level inorganic lead probably relate to the parenchymal cells of the CNS and not to the brain capillary endothelial cells.

Inorganic lead produces cerebral dysfunction and clinically definable encephalopathies in man. To date there have been few studies on the biochemical changes in brain following exposure to inorganic lead. Studies correlating toxicity with behavioral and brain neurochemical changes following lead exposure have been hindered because adult laboratory animals are resistant to the central nervous system effects of lead poisoning. Such studies have been impeded by lack of suitable experimental models until Pentschew and Garro showed that brain lesions develop in neonatal rats when a pregnant rat newly delivered of her litter is placed on a 4% lead carbonate containing diet. Lead passes into the developing sucklings via maternal milk. Lead-poisoned new-borns have pronounced retardation of growth and during the fourth week of ilfe develop the severe signs of lead encephalopathy, namely, extensive histological lesions of the cerebellum, brain edema, and paraplegia. There is an approximate 85-fold increase in the lead concentration of both the cerebellum and cerebral cortex relative to controls, but edema and gross vascular changes are confined to the cerebellum. Ingested lead had little effect on RNA, DNA, and protein concentrations of developing rat cerebellum and cerebral cortex. However, there was a reduction of between 10 and 20% in the DNA content of the cerebellum around 3 weeks of age in the lead-exposed sucklings. This suggests a failure of cell multiplication in this part of the brain.A critical evaluation of this experimental approach indicated that under similar dietary conditions experimental lactating rats eat 30% less food than controls resulting in: (a) sustained loss in body weight of nursing mothers and that (b) offsprings who develop paraplegia and cerebellar damage do so after gaining access to lead containing diet. We have studied mothers' food consumption and body weight changes and blood, milk, and brain lead content; and newborns' body and brain weight changes, blood and brain lead content, and brain serotonin (5HT), norepinephrine (NE), dopamine (DA), and gamma-aminobutyric acid (GABA). We have found that a lactating mother rat eating 5% lead acetate (2.73% Pb) produced milk containing 25 ppm lead. When the mothers' diet is changed at day 16 from 5% PbAc to one containing 25 ppm Pb, and neonates allowed free access to the solid diet, the sucklings still have retarded body growth but do not develop paraplegia or grossly apparent vascular damage of the cerebellum. However, during the fourth week these animals exhibit a less severe form of "encephalopathy" consisting of hyperactivity, tremors, and stereotype behavior. Pair-fed controls coetaneous to experimental groups do not display such activities. There was no change in brain 5HT, GABA, or NE, but a 15-20% decrease in brain DA. Change in DA relative to other monoamines suggests a relationship between CNS dysfunction due to lead and DA metabolism in the brain.The experimental design as discribed provides a model of CNS dysfunction due to lead exposure without debilitating histopathologies. It is possible that our findings on increased motor activity and changes in brain dopamine may correspond to early responses to lead exposure before recognized overt signs of toxicity.

Several studies have shown that inorganic lead added in vitro does not alter gamma-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 delta-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 microM) 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 AT-Pase. ALA also induces a dose-dependent [3H]GABA release, but only at concentrations equal to or greater than 30 microM. Exposure to a combination of 3 microM lead and 100 microM 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 microM), whereas ALA (30-300 microM) 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.

Daily oral administration of lead to newborn rats has no adverse effect on their body growth. Lead-treated rats were more active than age-matched controls. Endogenous levels of brain dopamine were unchanged, whereas norepinephrine was increased, suggesting a possible relationship between lead exposure during earliest developmental periods, increased motor activity, and brain norepinephrine, and not brain dopamine as previously postulated.

An automated technique for the study of visual discrimination learning in mice has been developed. The technique utilizes a nose-poke as the operant response. The nose-poke response requires no shaping, has a relatively high operant level and can be used to measure preacquisition exploratory behavior. CD-1 mice acquired a simultaneous brightness discrimination readily but a successive brightness discrimination proved more difficult. A 20 sec intertrial interval was optimal for acquisition of the simultaneous discrimination. Reversal learning was slow. This procedure should prove useful in the study of the effects of pharmacologic and toxic agents on learning and performance in both weanlings and adult mice.

Neonatal rats were given aqueous lead acetate intragastrically from d 2-20 of life at doses of 0, 10, 50, and 225 mg Pb/kg.d. Blood Pb concentrations on d 21 were (mean +/- SE) 23 +/- 3 (control), 63 +/- 19, 246 +/- 55, and 994 +/- 223 microgram/100 ml, and brain Pb concentrations were 14 +/- 2, 60 +/- 5, 114 +/- 15, and 275 +/- 26 microgram/100 g, respectively. Growth was significantly depressed only in rats given the highest dose of Pb (225 mg/kg.d). Solvent-extractable lipofuscin pigment concentration of brain tissue progressively decreased over the 21-d duration of the experiment but was not significantly altered at any dose of Pb. Brain glutathione peroxidase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase activities were stimulated on d 20 at the maximal dose of Pb, but the activities of brain superoxide dismutases and catalase were not altered by Pb exposure. Locomotor activity was significantly increased in the male animals on d 20, but only at the highest dose of Pb. These results indicate that Pb toxicity in neonatal rats is not associated with accelerated in vivo lipid peroxidation in the brain, but that certain oxidant defense mechanisms in the brain are stimulated by Pb.

The distribution of dopamine (3,4-dihydroxyphenylethylamine) between the subcellular fractions from homogenates of dog caudate nucleus was compared with the distributions of acetylcholine, 5-hydroxytryptamine and lactate dehydrogenase activity. The distributions of noradrenaline and 5-hydroxytryptamine between the subcellular fractions from homogenates of dog hypothalamus were also determined. Most of the dopamine, in contrast to acetylcholine, occurred in the soluble supernatant fraction; the remainder was associated with the fractions rich in pinched-off nerve ending particles, but localization in any one fraction was not as sharp as that of acetylcholine. Evidence is presented that suggests that the dopamine occurs in a free or easily released form throughout cell cytoplasm.

Newborn rats that suckled mothers eating a diet containing 4 percent lead carbonate display hyperactivity, aggressiveness, and excessive stereotyped behavior starting at 4 weeks of age. There is an eightfold increase in the concentration of lead in brain, no change in norepinephrine, but a 20 percent decrease in dopamine relative to coetaneous controls. This suggests a relationship between central nervous system dysfunction due to lead and dopamine metabolism in brain.