Daniel J. Minnema

Calcium efflux and neurotransmitter release from rat hippocampal synaptosomes exposed to lead

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.

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.

Effects of methylmercury on neurotransmitter release from rat brain synaptosomes

Although the effects of methylmercury (MeHg) at the neuromuscular junction have been well characterized, similar studies employing CNS preparations and transmitters have been limited. We found that MeHg (0.5-5.0 microM) produced a concentration-dependent increase in the spontaneous release of [3H]dopamine. gamma-[3H]aminobutyric acid, and [3H]acetylcholine from synaptosomes isolated from rat brain striatum, cortex, and hippocampus, respectively. At these same concentrations MeHg did not attenuate calcium-dependent depolarization-evoked 3H-transmitter release. MeHg did not appear to induce calcium influx into the nerve terminal since the increase in release persists in the absence of extrasynaptosomal calcium. The increase in spontaneous transmitter release induced by MeHg persisted in the presence of low extrasynaptosomal sodium, suggesting that MeHgs effects on release are not mediated by either Na+, K+-ATPase inhibition or selective increases in membrane sodium permeability. MeHg produced only a very small increase in 45Ca efflux from synaptosomes preloaded with 45Ca, whereas these same MeHg concentrations produced large increases in 45Ca efflux from preloaded isolated mitochondria. MeHg did increase the efflux of [3H]deoxyglucose phosphate from synaptosomes. An increase in the efflux of [3H]deoxyglucose phosphate is believed to reflect an increase in neuronal membrane permeability. The quantitative and temporal aspects of the MeHg-induced [3H]-deoxyglucose phosphate efflux were similar to those observed for MeHg-induced neurotransmitter release. These data suggest that the increase in spontaneous transmitter release induced by MeHg is mainly the result of transmitter leakage that occurs subsequent to MeHg-induced increases in synaptosomal membrane permeability. However, these results cannot exclude possible effects of MeHg on intrasynaptosomal calcium homeostasis.

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.

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.

Effects of inorganic mercury on [3H]dopamine release and calcium homeostasis in rat striatal synaptosomes☆

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.

Altered central monoamine response to D-amphetamine in rats chronically exposed to inorganic lead.

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.

Effect of lead exposure on patterns of food intake in weanling rats.

The reduction in growth resulting from lead (PB) exposure in weanling rats is consistent with a lowering of the biological set-point for food intake. In this study the effects of lead on the patterns of food intake were examined. For 10 days (from ages 26 to 36 days), female rats were provided with drinking water containing 250 ppm lead as the acetate (n = 6) or equivalent acetate as sodium acetate (n = 6). A computerized system was used to monitor daily food intake at 5-min intervals over 10 successive 23-h periods (each period consisting of 12 h dark, 11 h light). Control rats consumed approximately 75% to 85% of their food intake during the dark phase. Exposure to lead resulted in decreased body weight, tail length, and cumulative food intake. Decreased food intake associated with lead during the first 6 days of exposure was due to a decrease in the size of each meal during the dark phase, which reflected a decrease in the duration of each meal. These results suggest that lead, at least initially, was affecting food-satiety signals to produce a premature termination of food intake during a meal. After 6 days, the lead-exposed rats appear to have adjusted their meal size and meal duration to approximately control values. However, this compensation appears to have occurred at the expense of the daily (nocturnal) number of meals, which decreased slightly (although not significantly) in lead-exposed animals. Thus, the total daily intake of food in lead-treated animals remained depressed relative to control animals.