Toxic heavy metals potentiate ligand-gated C1-currents in gastropoda neurons.
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Acetylcholine↗
Biomedical subjects
D O Carpenter
Publications and source records attributed to D O Carpenter.
Neurite outgrowth is enhanced by conditioning factor(s) released from central ganglia of Aplysia californica.
Single neurons of Aplysia californica were cultured on control and conditioned medium-treated cover slips (CM, derived from central ganglia of Aplysia) and fed with L-15 medium plus 20% hemolymph. After 5 h 36.9 +/- 3.3% of all neurons plated on CM-treated cover slips sprouted, whereas only 22.9 +/- 2.7% sprouted in the control group. After 24 h the average neurite length in the presence of putative conditioning factor(s) was 621 +/- 42 microns versus 411 +/- 20 microns in control, but the number of primary processes was the same (4.3 +/- 0.5 versus 4.2 +/- 0.9).
Animals↗
Methylmercury induces Ca(2+)-dependent hyperpolarization of mouse thymocytes: a flow cytometric study using fluorescent dyes.
The effect of methylmercury on mouse thymocytes was examined using fluorescent dyes for membrane potential and intracellular Ca2+. Methylmercury at concentrations of 1 microM or higher (up to 30 microM) produced hyperpolarization in a dose-dependent fashion. Charybdotoxin and quinine, but not 4-aminopyridine and tetraethylammonium, greatly suppressed methylmercury-induced hyperpolarization. Removal of external Ca2+ reduced the degree of hyperpolarization. Pretreatment of thymocytes with A23187 under Ca(2+)-free conditions abolished the hyperpolarization induced by methylmercury. Under both normal and Ca(2+)-free conditions methylmercury increased the intracellular concentration of Ca2+. The results suggest that the increase in intracellular Ca2+ is mediated through a Ca2+ release from intracellular stores as well as through influx of external Ca2+. Therefore, it is likely that methylmercury increases the intracellular concentration of Ca2+, resulting in activation of Ca(2+)-dependent K+ conductance of mouse thymocytes.
Animals↗
Delayed increase of cell volume of single pyramidal cells in live rat hippocampal slices upon kainate application.
Cell volume is an important physiologic parameter but is not directly accessible by conventional microscopy in vivo or in slice preparations. After staining of rat hippocampal slices with 1,1'-dioctadecyl-3,3,3',3'-tetramethylinso-carbocyanineperchlor ate (DiI), living single CA1 pyramidal cells were visualized by confocal microscopy while others in the illuminated area were recorded intracellularly. Resting membrane potential and action potentials were not affected by staining with DiI (4 microM) and repeated laser illumination (up to 600 times). A time- and dose-dependent increase of cell volume succeeds membrane depolarization upon bath application of kainate (40 microM and 100 microM) with a delay of several minutes indicating active regulation of cell volume. We conclude that cell swelling as visualized by confocal microscopy is a relatively late event of kainate excitotoxicity.
Animals↗
Aluminum impairs hippocampal long-term potentiation in rats in vitro and in vivo.
Although aluminum (Al) contributes to a variety of cognitive dysfunctions and mental diseases, the underlying mechanisms of Al interactions with the nervous system are still unknown. We have studied the action of Al on synaptic transmission and long-term potentiation (LTP) by performing electrophysiological recordings both in vivo, using freely moving animals, and in vitro, using hippocampal slices. In vivo recordings of the population spikes (PSs) of dentate gyrus granule cells in response to medial perforant path stimulation were performed on both acutely and chronically (Al each day for 5 days) intraventricularly injected animals. Acute Al-infusion (calculated brain concentrations of 0.27, 0.68, and 2.7 micrograms/ml) had no influence on baseline values. Al at 0.27 microgram/ml did not alter the induction and maintenance of LTP, but 0.68 and especially 2.7 micrograms/ml Al lead to a reduction in LTP, and the potentiation declined to baseline within 2 h. In chronic animals their neuronal responsiveness was reduced and in 30% of the rats the PS was completely lost. High-frequency tetanization failed to induce LTP. In slices, field potentials were evoked stimulating Schaffer collaterals and recording pyramidal cells of the CA1 region. Bath application of 0.68 microgram/ml Al increased the baseline amplitude of the PS slightly, whereas 2.7 micrograms/ml decreased the amplitude and concentrations > 5.4 micrograms/ml blocked the PS completely. Induction of LTP in the presence of 0.68 microgram/ml Al led to a smaller increase of the PS amplitude compared to controls, but the duration of LTP was not affected. In the presence of 2.7 micrograms/ml Al LTP was further reduced and declined to baseline levels within 60 min. Given that LTP is a form of synaptic plasticity underlying some forms of learning, our data suggest that both preparations are suitable models for investigating actions of Al-induced neurotoxicity.
Aluminum↗
Opioid peptides in the nervous system of Aplysia: a combined biochemical, immunocytochemical, and electrophysiological study.
1. We have used biochemical, immunocytochemical, and electrophysiological techniques to evaluate the role of opioid peptides in the central nervous system of the marine mollusc, Aplysia californica. 2. Binding studies using 3H-D-Ala2, met-enkephalinamide (3H-DAMA) showed a single class of high-affinity binding sites with a Kd of 1.3 nM and a binding density of 45 pmol/g. 3. HPLC extracts of ganglia revealed multiple peaks with immunoreactivity for either leu (LEU-IR)- or met-enkephalin (MET-IR), but the amounts were not uniformly distributed in all ganglia. 4. LEU-IR and MET-IR neurons were demonstrated immunocytochemically in all ganglia, but MET-IR neurons were more frequent and were concentrated in pedal and pleural ganglia. While absorption control studies abolished MET-IR, LEU-IR was only partially abolished in the neuropil. 5. In electrophysiological studies, both depolarizing and hyperpolarizing responses were found to D-Ala2-leu-enkephalin (DALEU) and D-Ala2-met enkephalin (DAMET) on some and different neurons. 6. HPLC fractions from regions with retention times corresponding to authentic leu- or met-enkephalin showed physiologic responses similar to those of DALEU and DAMET, respectively. 7. These studies suggest that a variety of endogeneous opioid peptides play physiologically important roles in the nervous system of Aplysia, including but not necessarily limited to leu- and met-enkephalin.
Analgesics, Opioid↗
Failure of neuronal ion exchange, not potentiated excitation, causes excitotoxicity after inhibition of oxidative phosphorylation.
Neuronal cell death during impaired energy metabolism is often attributed to increased activity at glutamate receptors, but this increase has not been directly demonstrated. We recorded responses to glutamate and N-methyl-D-aspartate in hippocampal slice CA1 neurons and glia while inhibiting mitochondrial complex II with 3-nitropropionic acid. As the period of inhibition increased, neuronal depolarization following bath application of glutamate (5 mM) or N-methyl-D-aspartate (50 microM) increased dramatically. However, depolarization upon iontophoresis of glutamate and N-methyl-D-aspartate decreased with time. A transient hyperpolarization, reflecting electrogenic sodium pump activity, was present immediately after responses to iontophoretic glutamate agonists. In the presence of the inhibitor, this hyperpolarization decreased and eventually disappeared. Even the repolarization seen upon washing of the iontophoretic or bath application of glutamate or N-methyl-D-aspartate was incomplete. Glial depolarization upon bath application of glutamate increased during inhibition, while glial depolarization upon application of N-methyl-D-aspartate decreased. Application of the N-methyl-D-aspartate antagonists aminophosphonovaleric acid (100 microM) or MK-801 (20 microM) resulted in a delay of further depolarization when applied early during the terminal decay of membrane potential following metabolic inhibition. We conclude that during impaired oxidative phosphorylation the failure of repolarizing mechanisms, not potentiated neuronal depolarization by excitants, is the primary cause of the terminal depolarization. Large glial depolarization increases the demand for neuronal ion exchange which cannot be met in situations of reduced energy metabolism. Our results provide further evidence that acute and chronic blockade of energy metabolism have different effects.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Communicating with the public on issues of science and public health.
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Communication↗
The role of glutamate reuptake in regulation of glutamate responses in Aplysia neurons.
Glutamate elicits several different responses on neurons of isolated ganglia of Aplysia, the most common of which is a hyperpolarization due to conductance increases to either chloride or potassium. We have investigated the actions of aspartate and cysteate on the responses to glutamate. Neither aspartate nor cysteate is potent in activation of glutamate receptors. However both aspartate and cysteate cause a dramatic increase in the response to glutamate when ionophoretically applied before the glutamate application. This potentiating effect of aspartate and cysteate is a result of competition with glutamate for the glutamate transport system, since the potentiation is blocked by cooling and by perfusion with sodium-free sea water. Blockade of glutamate re-uptake by perfusion of sodium-free sea water also causes a significant increase in the response to ionophoretically applied glutamate, which in some neurons may be very large. These results demonstrate that the glutamate reuptake system has an important role in regulation of the responses to glutamate which is similar to that of acetylcholinesterase in regulation of responses to acetylcholine. These observations may be of particular importance in mammalian systems where excess glutamate is associated with neuronal excitotoxicity and cell death.
Animals↗
HgCl2 potentiates GABA activated currents in Lymnaea stagnalis L. neurones.
Using the concentration clamp technique the effects of inorganic mercury (HgCl2) on gamma-aminobutyric acid (GABA) activated Cl- currents were studied on Lymnaea neurons. Hg2+ at concentrations of 0.01-1 microM caused a significant increase in the GABA induced chloride permeability of the cell membrane. With simultaneous application of GABA and 0.1 microM Hg2+ the amplitudes of the GABA activated inward Cl- currents were increased to a mean of 168%. The threshold concentration for potentiation was 0.01 microM Hg2+, while the GABA induced Cl- current was suppressed at concentrations higher than 100 microM. HgCl2 was also effective in decreasing the time-to-peak value and the time of inactivation of the GABA evoked Cl- current. In the presence of 0.1 microM mercury chloride the peak reached the maximum 1.6 times faster than without mercury. The effect was independent of the membrane potential, and mercury did not change the reversal potential of the GABA activated Cl- current. Application of mercury alone at concentrations effective to enhance GABA evoked Cl- permeability caused no or only a very small sustained inward current. However, higher mercury concentrations or repeated mercury application greatly increased the membrane permeability, and this effect could not be reversed by a 15 min wash. The results suggest, that low mercury concentrations act on GABA-ergic synapses at a postsynaptic site, and the potentiation of the ligand gated Cl- current may be an important factor in the mechanism of Hg2+ neurotoxicity.
Animals↗
Hg2+ increases the open probability of carbachol-activated Cl- channels in Aplysia neurons.
Carbachol and acetylcholine (ACh)-activated whole-cell chloride currents were recorded in identified neurons of Aplysia californica. Application of 0.1-10 microM HgCl2 potentiated the chloride response and reduced the rate of desensitization. To investigate the underlying mechanism we recorded single chloride channels in a cell-attached patch configuration. Recordings were performed successively on the same neuron with 1 microM carbachol and with 1 microM carbachol + 1 microM HgCl2, respectively. The slope conductance did not change significantly, but there was a manyfold increase in the open probability, Po. This may underlie the Hg(2+)-induced enhancement of carbachol- and ACh-evoked chloride currents in Aplysia neurons and may serve as a model for the neurotoxic effect of Hg2+ on the mammalian ACh receptor.
Animals↗
Direct excitatory opiate effects mediated by non-synaptic actions on rat medial vestibular neurons.
Opiates increase firing of rat medial vestibular nucleus neurons. We have attempted to determine the mechanism of these excitatory opiate actions by extracellular recording of neuronal activity with ionophoretic application of opiate agonists and bath application of antagonists. The spontaneous activity of approximately 30% of medial vestibular neurons, scattered throughout the nucleus, was increased by ionophoretic application of either morphine or [D-Ala2]leucine enkephalin, implicating the presence of both mu and delta opiate receptors. The responses to both were blocked by the opiate receptor antagonist, naloxone. In only a few neurons opiates decreased firing. Most previous reports of direct opiate excitation have proven to be due to disinhibition. This is not the case here, as indicated by three observations: 1) the excitatory opiate response was sustained when gamma-aminobutyric acid (GABA) receptors were blocked by bicuculline; 2) perfusion of a solution containing 0.1 mM Ca2+ and 6.3 mM Mg2+ blocks synaptic transmission, but does not block the excitatory responses to both opiates and 3) the opiate-induced depolarization and action potential generation was evident in neurons whose spontaneous firing was almost totally depressed by adenosine. These results indicate that the excitation is neither due to disinhibition nor to a presynaptic opiate action. We conclude that medial vestibular neurons have postsynaptic opiate receptors that mediate direct neuronal excitation.
Action Potentials↗
Transient ischemia causes a reduction of Mg2+ blockade of NMDA receptors.
After transient ischemia, synaptic activation of neurons in CA1 results in an abnormally long depolarization. This is due to the appearance of an NMDA component which results from a decrease in the normal voltage-dependent blockade by Mg2+. The reduction of NMDA channel blockade is likely the cause of the hyperexcitability which follows transient ischemia and the subsequent delayed neuronal cell death, resulting from entry of calcium through hyperactive NMDA channels.
2-Amino-5-phosphonovalerate↗
Functional and morphological changes induced by transient in vivo ischemia.
Brief transient ischemia causes a delayed neuronal death of pyramidal neurons in the CA1 area of hippocampus after a period of hyperexcitability. We have previously shown that the hyperexcitability is due to an increase in an N-methyl-D-aspartate (NMDA) component of the response. In the present study, we recorded intracellularly from pyramidal neurons in CA1 and find that there is little change in membrane potential or input resistance at this point in time. The dramatic increase in the NMDA component of the synaptic response is a result of a significant reduction in the ability of Mg2+ to induce a normal voltage-dependent blockade of the response. In spite of the relatively normal membrane properties, there is at this time a significant reduction in the amplitude of the population excitatory potential and a near total loss of long-term potentiation. In contrast, post-tetanic potentiation is unchanged in magnitude and character. These observations suggest more severe damage to the neuron than indicated by the membrane potential and resistance. When single neurons were injected with horseradish peroxidase and visualized after the electrophysiological recording, we found extensive beading of the dendrites in both the apical and basal regions, presumably reflecting a disproportionate damage to the dendritic areas, which are the primary sites of the excitatory amino acid synapses onto the neuron. These observations are consistent with the hypothesis that transient ischemia causes a fundamental change in the NMDA-activated ion channel such that Mg2+ is no longer able to block the response, resulting in increased entry of calcium into synaptic regions, which causes dendritic damage that progresses to neuronal cell death.
Animals↗
The public health significance of metal neurotoxicity.
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Aluminum↗
Effect of HgCl2 on acetylcholine, carbachol, and glutamate currents of Aplysia neurons.
1. Using conventional two-microelectrode voltage-clamp techniques we studied the effects of inorganic mercury (HgCl2) on acetylcholine-, carbachol-, and glutamate-activated currents on Aplysia neurons. Hg2+ was applied with microperfusion. 2. Acetylcholine and carbachol activated an inward, sodium-dependent current in the anterior neurons of the pleural ganglion. The medial neurons gave a biphasic current to acetylcholine and carbachol, which was outward at resting membrane potential. The faster component was Cl- dependent and reversed at about -60 mV, while the slower component was K+ dependent and reversed at greater than -80 mV. 3. Hg2+ (0.1-10 microM) caused a dramatic increase in the acetylcholine- and carbachol-induced inward current in anterior neurons and the fast Cl- current in medial neurons. With only a 1-min preapplication of Hg2+, the acetylcholine- or carbachol-activated sodium or chloride currents were increased to 300% and the effect was only partly reversible. The threshold concentration was 0.1 microM Hg2+. 4. Contrary to the effects on sodium and chloride currents, concentrations of 0.1-10 microM Hg2+ caused a complete and irreversible blockade of K(+)-dependent acetylcholine and carbachol currents. The block of the potassium current was relatively fast and increased with time. The concentration of HgCl2 that gave a half-maximal blockade of the carbachol-activated potassium current was 0.89 microM. The chloride-dependent current elicited by glutamate on medial neurons was increased by HgCl2 as well. 5. These results suggest that actions at agonist-activated channels must be considered as contributing to mercury neurotoxicity. It is possible that the toxic actions of Hg2+ on synaptic transmission at both pre- and postsynaptic sites are important factors in the mechanism of Hg2+ toxicity.
Acetylcholine↗
Mercuric(II) chloride modulates single-channel properties of carbachol-activated Cl- channels in cultured neurons of Aplysia californica.
1. The effect of mercuric(II) chloride on kinetic parameters of carbachol-activated single chloride channels were studied in cultured neurons of the marine mollusk, Aplysia californica. 2. Single neurons of Aplysia were cultured in L-15 medium containing 1 mM beta-D-xyloside, which improved the success rate for gigaseal formation by 46%. Carbachol-activated single chloride channels were recorded in the cell-attached patch clamp configuration. Recordings with control solution (1 microM carbachol) and with test solution (1 microM carbachol + 1 microM HgCl2) were performed successively on the same neuron. 3. In both the control and the test solution the open and closed time distributions were fitted with a double-exponential function. However, kinetic analysis revealed that Hg2+ caused a significant reduction of the mean closed time (10.37 +/- 1.08 vs. 3.32 +/- 0.02 msec) and of the second time constant tau 2 of the closed time distribution (2.09 +/- 0.05 vs. 0.66 +/- 0.5 msec). The reduction of tau 2, i.e., fewer events in the longer closed state under the action of Hg2+, may be the physical cause for the reduction of the mean closed time and thus underlies the increased open probability p0 (0.13 +/- 0.01 vs. 0.29 +/- 0.01 msec) of carbachol-activated chloride channels. 4. Inorganic Hg2+ affects the acetylcholine receptor at lower concentrations than previously reported.
Animals↗
Long-term potentiation in the piriform cortex is blocked by lead.
1. Long-term potentiation (LTP) is a prolonged increase in synaptic efficacy that is triggered by a brief tetanic stimulation at certain central synapses. LTP is one of the best available model systems available to the neurophysiologist of neuronal plasticity such as that underlying learning and memory. 2. We have studied the susceptibility of LTP to blockade by lead as a test of the hypothesis that the negative effect of lead on intelligence in children may result from interference with this process. LTP was studied in slices of rat piriform cortex. At this site, as in many other central synapses, LTP requires activation of postsynaptic N-methyl-D-aspartate (NMDA) receptors, and we investigated whether lead actions, if any, were mediated via effects on NMDA-activation ion channels or, alternatively, at voltage-activated calcium channels. 3. We find that lead blocks LTP at low micromolar concentrations. However, concentrations of lead that totally block LTP had no apparent effect on either NMDA-activated responses or presynaptic calcium channels, as monitored by transmitter release from presynaptic terminals. 4. While the mechanism of lead blockade of LTP remains to be determined, these observations are consistent with the hypothesis that the cognitive effects of lead neurotoxicity may result from effects on LTP.
2-Amino-5-phosphonovalerate↗