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G Audesirk

Publications and source records attributed to G Audesirk.

At least 19 recordsLinked to original sources
PubMed2003

Enhancement of dendritic branching in cultured hippocampal neurons by 17beta-estradiol is mediated by nitric oxide.

Both 17beta-estradiol (E2) and nitric oxide (NO) are important in neuronal development, learning and memory, and age-related memory changes. There is growing evidence that a number of estrogen receptor-mediated effects of estradiol utilize nitric oxide as an intermediary. The role of estradiol in hippocampal neuronal differentiation and function has particular implications for learning and memory. Low levels of estradiol (10nM) significantly increase dendritic branching in cultured embryonic rat hippocampal neurons (158% of control). This study investigates the hypothesis that the estrogen-stimulated increase in dendritic branching is mediated by nitric oxide. We found that nitric oxide donors also produce significantly increased dendritic branching S-nitroso-N-acetylpenicillamine (SNAP: 119%; 2,2'-(hydroxynitrosohydrazino)bis-ethanamine (NOC-18): 128% of control). We then determined that the increases in dendritic branching stimulated by estradiol or by a nitric oxide donor were both blocked by an inhibitor of guanylyl cyclase. Dendritic branching was also stimulated by a cell permeable analog of cyclic guanosine monophosphate (dibutyryl-cGMP: 173% of control). Estradiol-stimulated dendritic branching was reversed by the nitric oxide scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl imidazoline-1-oxyl 3-oxide (carboxy-PTIO). This study provides evidence that estradiol influences the development of embryonic hippocampal neurons in culture by increasing the production of nitric oxide or by increasing the sensitivity of the neurons to nitric oxide. Nitric oxide in turn stimulates dendritic branching via activation of guanylyl cyclase.

Animals↗
PubMed2000

Stimulatory and inhibitory effects of inorganic lead on calcineurin.

Calcineurin is a phosphatase with activity dependent on both Ca(2+)/calmodulin binding to the catalytic A subunit and Ca(2+) binding to the regulatory B subunit. We have previously shown that Pb(2+) activates calmodulin with a threshold of about 100 pM free Pb(2+), and that Pb(2+) and Ca(2+) are roughly additive in calmodulin activation (Kern et al., NeuroToxicology 21, 353-364 (2000)). In the present study, we evaluated the effects of Pb(2+), with and without Ca(2+) and calmodulin, on calcineurin activity. In calmodulin-containing, Ca(2+)-free solutions, Pb(2+) activated calcineurin with a threshold of about 100 pM free Pb(2+). Maximum calcineurin activity (comparable to that induced by 10 microM Ca(2+)) was reached at about 200 pM free Pb(2+). Higher Pb(2+) concentrations reduced activity, although some activity remained even at 2000 pM free Pb(2+). Combined with subsaturating Ca(2+) concentrations, as little as 20 pM free Pb(2+) enhanced calcineurin activity, but free Pb(2+) concentrations greater than 200 pM still reduced activity below maximum. Extremely high Ca(2+) concentrations (10 microM) completely reversed the inhibition of activity by 2000 pM free Pb(2+). In the absence of calmodulin, Ca(2+) slightly stimulated calcineurin activity. Pb(2+) did not substitute for Ca(2+) in calmodulin-free activation; in fact, high concentrations of Pb(2+) inhibited Ca(2+)-mediated activation. We tentatively conclude that low concentrations of free Pb(2+) activate calcineurin by activating calmodulin. Higher concentrations reduce calcineurin activity, perhaps by binding to the B subunit.

Calcineurin↗
PubMed2000

Inorganic lead and calcium interact positively in activation of calmodulin.

Calmodulin is a ubiquitous calcium-binding protein that mediates many of the intracellular actions of Ca2+ ions. The calcium-binding sites of calmodulin consist of four EF-hand motifs; full activation of calmodulin normally occurs when all four sites are occupied by Ca2+. Inorganic lead (PY2+) has been shown to activate calmodulin at total lead concentrations similar to the concentrations of Ca2+ required for activation (Goldstein and Ar, 1983; Habermann et al., 1983), but the free Pb2+ concentrations required for calmodulin activation have not been determined. In addition, it is possible that activation may occur with different sites occupied by different divalent cations, for example Ca2+ and Pb2+. We investigated the ability of free Pb2+, alone or in combination with Ca2+, to activate calmodulin. In aqueous media, N-phenyl-1-naphthylamine (NPN) and 8-anilino-1-naphthalenesulfonate (ANS) show increased fluorescence when bound to hydrophobic regions of proteins. This increased fluorescence has been used to monitor the conformational change that occurs during calmodulin activation (LaPorte et al., 1980). In the presence of calmodulin, both Ca2+ and Pb2+ stimulated increased fluorescence of NPN and ANS. Threshold and EC50 free metal concentrations were approximately 100 nM and 450-500 nM, respectively, for Ca2+ and 100 pM and 400-550 pM, respectively, for Pb2+. Fluorescence was enhanced by combinations of low concentrations of free Ca2+ and Pb2+; for example, as little as 20 pM free Pb2+ enhanced fluorescence in combination with 200 nM free Ca2+. The activity of the PDE1 isoform of cyclic nucleotide phosphodiesterase is stimulated by Ca2+/calmodulin (Wang et al., 1990). In the presence of calmodulin, we found that Ca2+ and Pb2+ activated calmodulin-stimulated PDE activity, with threshold and EC50 free metal concentrations of approximately 200 nM and 1200 nM, respectively, for Ca2+ and 300 pM and 430 pM, respectively, for Pb2+. PDE activity was stimulated by combinations of Ca2+ and Pb2+. For example, with 100 nM free Ca2+, as little as 50 to 100 pM free Pb2+ further stimulated PDE activity; with 1000 nM free Ca2+, 20 to 50 pM free Pb2+ further stimulated PDE activity. Isobolographic analysis indicated that stimulation of PDE by Ca2+ and Pb2+ was additive. These results show that concentrations of free Pb2+ as low as 100 to 300 pM activate calmodulin and that, in the presence of physiological concentrations of free Ca2+, Pb2+ can activate calmodulin at concentrations below 50 pM. The intracellular free Ca2+ concentration in Ca2+ "hot spots," for example near sites of influx through Ca2+-permeable plasma membrane channels, can reach dozens of pM, with the free Ca2+ concentration decreasing rapidly with distance from the source of the hot spot. Our results suggest that picomolar concentrations of intracellular free Pb2+ should expand both the effective amplitude and volume of Ca2+ hot spots with respect to calmodulin activation, and thus may amplify intracellular Ca2+ signaling in lead-exposed cells.

Calcium↗
PubMed2000

Nanomolar concentrations of inorganic lead increase Ca2+ efflux and decrease intracellular free Ca2+ ion concentrations in cultured rat hippocampal neurons by a calmodulin-dependent mechanism.

Inorganic lead (Pb2+) activates calmodulin, which in turn may stimulate many other cellular processes. The plasma membrane Ca2+ ATPase is a calmodulin-stimulated enzyme that plays the major role in regulating the "resting" intracellular free Ca2+ ion concentration, [Ca2+]i. We hypothesized that exposing neurons to low levels of Pb2+ would cause Pb2+ to enter the cytoplasm, and that intracellular Pb2+, by activating calmodulin, would stimulate plasma membrane Ca2+ ATPase activity, thereby increasing Ca2+ extrusion and reducing [Ca2+]i. We used the ratiometric Ca2+ indicator fura-2 to estimate changes in [Ca2+]i. In vitro calibrations of fura-2 with solutions of defined free Ca2+ and free Pb2+ concentrations showed that, at free Ca2+ concentrations from 10 nM to 1000 nM, adding Pb2+ caused either no significant change in the F340/F380 ratio (free Pb2+ concentrations from 100 fM to 1 pM) or increased the F340/F380 ratio (free Pb2+ concentrations from 5 to 50 pM). Therefore, fura-2 should be suitable for estimating Pb2+-induced decreases in [Ca2+]i, but not increases in [Ca2+]i. We exposed cultured embryonic rat hippocampal neurons to 100 nM Pb2+ for periods from 1 hour to 2 days and measured the F340/F380 ratio; the ratio decreased significantly by 9 to 16% at all time points, indicating that Pb2+ exposure decreased [Ca2+]i. In neurons loaded with 45Ca, Pb2+ exposure increased Ca2+ efflux for at least two hours; by 24 hours, Ca2+ efflux returned to control levels. Influx of 45Ca was not altered by Pb2+ exposure. Low concentrations (250 nM) of the calmodulin inhibitor calmidazolium had no effect on either 45Ca efflux or on the F340/F380 ratio in fura-loaded control neurons, but completely eliminated the increase in 45Ca efflux and decrease in F340/F380 ratio in Pb2+-exposed neurons. Zaldoride, another calmodulin inhibitor, also eliminated the decrease in F340/F380 ratio in Pb2+-exposed neurons. We conclude that Pb2+ exposure decreases [Ca2+]i and increases Ca2+ efflux in cultured hippocampal neurons by a calmodulin-dependent mechanism, probably by stimulating Ca2+ extrusion by the plasma membrane Ca2+ ATPase.

Animals↗
PubMed1999

Understanding the NIH review process: a brief guide to writing grant proposals in neurotoxicology.

During the past two years, the National Institutes of Health have made significant changes in the review process for investigator-initiated research grant applications in neurotoxicology. First, study sections that formerly dealt with toxicology and alcohol, respectively, have been merged. Neurotoxicology grant applications are now reviewed by ALTX-3, a study section in which the majority of members have expertise in the neuronal, biochemical or behavioral effects of alcohol, but usually not other neurotoxicants. Second, the NIH has instituted new review criteria, in which significance, approach, innovation, investigator expertise, and research environment must all be explicitly addressed by the reviews. In this article, past and present members of the ALTX-3 study section describe the NIH review process, with emphasis on how neurotoxicology applications are handled, and provide guidelines for preparing competitive applications.

Financing, Organized↗
PubMed1998

Low levels of inorganic lead noncompetitively inhibit mu-calpain.

Calpain is a ubiquitous calcium-dependent cysteine protease, whose cytoskeletal protein substrates suggest that it may be important in neuronal differentiation. Lead (Pb2+) is known to substitute for Ca2+ in a variety of intracellular processes, and interferes with the development of hippocampal neurons in vitro. We found that free Pb2+ at 1 nM does not activate calpain in the absence of Ca2+. Pb2+ inhibited the activity of calpain; the degree of calpain inhibition was dependent on an interaction between concentrations of both Ca2+ and Pb2+. In the presence of 1 microM free Ca2+, 10 pM free Pb2+ reduced calpain activity, but in the presence of 100 microM free Ca2+, 1 nM free Pb2+ failed to inhibit calpain. This provides evidence that Pb2+ competes for the Ca2+ binding sites on calpain. In the presence of 40 microM free Ca2+, 1 nM free Pb2+ significantly reduces Vmax without altering Km, suggesting that Pb2+ acts as a noncompetitive inhibitor of calpain. Inhibition of calpain is one mechanism by which Pb2+ may interfere with neuronal development.

Calcium↗
PubMed1997

Modulation of neurite branching by protein phosphorylation in cultured rat hippocampal neurons.

The control of branching of axons and dendrites is poorly understood. It has been hypothesized that branching may be produced by changes in the cytoskeleton [F.J. Diez-Guerra, J. Avila, MAP2 phosphorylation parallels dendrite arborization in hippocampal neurones in culture, NeuroReport 4 (1993) 412-419; P. Friedrich, A. Aszodi, MAP2: a sensitive cross-linker and adjustable spacer in dendritic architecture, FEBS Lett. 295 (1991) 5-9]. The assembly and stability of microtubules, which are prominent cytoskeletal elements in both axons and dendrites, are regulated by microtubule-associated proteins, including tau (predominantly found in axons) and MAP2 (predominantly found in dendrites). The phosphorylation state of tau and MAP2 modulates their interactions with microtubules. In their low-phosphorylation states, tau and MAP2 bind to microtubules and increase microtubule assembly and/or stability. Increased phosphorylation decreases these effects. Diez-Guerra and Avila [F.J. Diez-Guerra, J. Avila, MAP2 phosphorylation parallels dendrite arborization in hippocampal neurones in culture, NeuroReport 4 (1993) 412-419] found that protein phosphorylation correlates with neurite branching in cultured rat hippocampal neurons, and hypothesized that increased protein phosphorylation stimulates neurite branching. To test this hypothesis, we cultured rat hippocampal neurons in the presence of specific modulators of serine-threonine protein kinases and phosphatases. Inhibitors of several protein kinases, which would be expected to decrease protein phosphorylation, reduced branching. KT5720, an inhibitor of cyclic AMP-dependent protein kinase, and KN62, an inhibitor of Ca(2+)-calmodulin-dependent protein kinases, inhibited branching of both axons and dendrites. Calphostin C and chelerythrine, inhibitors of protein kinase C, inhibited branching of axons but not dendrites. Treatments that would be expected to increase protein phosphorylation, including inhibitors of protein phosphatases (okadaic acid, cyclosporin A and FK506) and stimulators of PKA (SP-cAMPS) or PKC (phorbol 12-myristate 13-acetate), increased dendrite branching. Only FK506 and phorbol 12-myristate 13-acetate stimulated axon branching. A subset of these agents was tested to confirm their effects on protein phosphorylation in this preparation. Okadaic acid, FK506 and SP-cAMPS all increased protein phosphorylation; KT5720 and KN62 decreased protein phosphorylation. On Western blots, the position of MAP2c extracted from cultures exposed to okadaic acid was slightly shifted toward higher molecular weight, suggesting greater phosphorylation, while the position of MAP2c from cultures exposed to KT5720 and KN62 was slightly shifted toward lower molecular weight, suggesting less phosphorylation. We conclude that protein phosphorylation modulates both dendrite branching and axon branching, but with differences in sensitivity to phosphorylation and/or dephosphorylation by specific kinases and phosphatases.

Animals↗
PubMed1995

Inorganic lead may inhibit neurite development in cultured rat hippocampal neurons through hyperphosphorylation.

Inorganic lead inhibits neurite initiation in cultured rat hippocampal neurons at concentrations as low as 100 nM. Conflicting reports suggest that Pb2+ may stimulate or inhibit protein kinase C, adenylyl cyclase, phosphodiesterase, and calmodulin, or increase intracellular free Ca2+ concentrations. Therefore, Pb2+ may alter the activities of Ca2+/calmodulin-dependent protein kinase (CaM kinase) or protein kinases C or A. We cultured rat hippocampal neurons in 100 nM PbCI2 alone or in combination with kinase or calmodulin inhibitors. Inhibiting protein kinase C with calphostin C exacerbated the inhibition of neurite initiation caused by PbCI2, but inhibiting protein kinase A with KT5720, CaM kinase with KN62, or calmodulin with calmidazolium completely reversed the effects of PbCI2. These results indicate that Pb2+ may inhibit neurite initiation by inappropriately stimulating protein phosphorylation by CaM kinase or cyclic AMP-dependent protein kinase (PKA), possibly by stimulating calmodulin. This hypothesis is supported by findings that other treatments that should increase protein phosphorylation (okadaic acid, a protein phosphatase inhibitor, and Sp-cAMPS, a PKA activator) also reduced neurite initiation. Whole-cell intracellular free Ca2+ ion concentrations were not significantly altered by 100 nM PbCI2 at 4, 12, 24, or 48 hr. Therefore, the hypothesized stimulatory effects of Pb2+ exposure on calmodulin, CaM kinase, or PKA are probably not caused by increases in whole-cell intracellular free Ca2+, but may be attributable either to intracellular Pb2+ or to localized increases in [Ca2+]in that are not reflected in whole-cell measurements.

Animals↗
PubMed1993

Effects of selective inhibition of protein kinase C, cyclic AMP-dependent protein kinase, and Ca(2+)-calmodulin-dependent protein kinase on neurite development in cultured rat hippocampal neurons.

A variety of experimental evidence suggests that calmodulin and protein kinases, especially protein kinase C, may participate in regulating neurite development in cultured neurons, particularly neurite initiation. However, the results are somewhat contradictory. Further, the roles of calmodulin and protein kinases on many aspects of neurite development, such as branching or elongation of axons vs dendrites, have not been extensively studied. Cultured embryonic rat hippocampal pyramidal neurons develop readily identifiable axons and dendrites. We used this culture system and the new generation of highly specific protein kinase inhibitors to investigate the roles of protein kinases and calmodulin in neurite development. Neurons were cultured for 2 days in the continuous presence of calphostin C (a specific inhibitor of protein kinase C), KT5720 (inhibitor of cyclic AMP-dependent protein kinase), KN62 (inhibitor of Ca(2+)-calmodulin-dependent protein kinase II), or calmidazolium (inhibitor of calmodulin), each at concentrations from approximately 1 to 10 times the concentration reported in the literature to inhibit each kinase by 50%. The effects of phorbol 12-myristate 13-acetate (an activator of protein kinase C) and 4 alpha-phorbol 12,13-didecanoate (an inactive phorbol ester) were also tested. At concentrations that had no effect on neuronal viability, calphostin C reduced neurite initiation and axon branching without significantly affecting the number of dendrites per neuron, dendrite branching, dendrite length, or axon length. Phorbol 12-myristate 13-acetate increased axon branching and the number of dendrites per cell, compared to the inactive 4 alpha-phorbol 12,13-didecanoate. KT5720 inhibited only axon branching. KN62 reduced axon length, the number of dendrites per neuron, and both axon and dendrite branching. At low concentrations, calmidazolium had no effect on any aspect of neurite development, but at high concentrations, calmidazolium inhibited every parameter that was measured (including viability). These results suggest that these three protein kinases selectively modulate different aspects of neurite development. The university of effects caused by calmodulin inhibition make it impossible to determine if there are specific targets of calmodulin action involved in neurite development. Finally, our data indicate that some superficially similar characteristics of neuronal differentiation, such as neurite initiation and branching, may be controlled by quite different molecular mechanisms.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

Effects of inorganic lead on voltage-sensitive calcium channels in N1E-115 neuroblastoma cells.

N1E-115 mouse neuroblastoma cells have been reported to possess two types of voltage-sensitive calcium channels: Low voltage activated, rapidly inactivating T-type (type I) and high voltage activated, slowly inactivating L-type (type II). We studied the effects of acute in vitro exposure to inorganic lead on these calcium channels, using the whole-cell variant of patch clamping. Using salines with a high lead-buffering capacity, we found that both T-type and L-type channels are reversibly inhibited in a dose-dependent manner at free Pb2+ concentrations ranging from 20 nM to 14 microM. L-type channels are somewhat more sensitive to Pb2+ than T-type channels are (L-type: IC50 approx. 0.7 microM; T-type: IC50 approx. 1.3 microM). Both channels show small but significant inhibition (approx. 10%) at 20 nM free Pb2+. Pb2+ affects neither activation nor inactivation of T-type channels, but enhances inactivation of L-type channels at holding potentials around -60 to -40 mV. A peculiar phenomenon was observed in cells exposed to 2.3 microM free Pb2+. T-type channels were inhibited in all 20 cells studied. In 15 cells, L-type channels were also inhibited, but in the remaining 5 cells, current flow through L-type channels was enhanced by Pb2+ exposure.

Animals↗
PubMed1991

Effects of inorganic lead on the differentiation and growth of cultured hippocampal and neuroblastoma cells.

Lead exposure has devastating effects on the developing nervous system, and has been implicated in variety of behavioral and cognitive deficits as well as neural morphological abnormalities. Since lead impacts many calcium-dependent processes, one likely mechanism of lead toxicity is its disruption of calcium dependent processes, among which is neuronal differentiation. We investigated the effects of inorganic lead on survival and several parameters of differentiation of cultured neurons. Three different cell types were used: Rat hippocampal neurons (a primary CNS cell type), B50 rat neuroblastoma cells (a transformed CNS-derived cell line), and N1E-115 mouse neuroblastoma cells (a transformed peripherally-derived cell line). Lead concentrations ranged from low nM to 1 mM. Lead effects differed considerably among the three cell types, with B50 cells least affected. Lead effects were generally multimodal, with fewest effects observed at intermediate concentrations. Lead inhibited neurite initiation in hippocampal neurons, but stimulated initiation in N1E-115 cells. In those cells that differentiated, lead increased dendrite numbers in hippocampal neurons and neurite numbers in N1E-115 cells. Lead exposure increased both the length and the degree of branching of axons in hippocampal neurons and the length of neurites in N1E-115 cells. We hypothesize that lead impacts multiple regulatory processes that influence neuron survival and differentiation, and that its effects show differing dose-dependencies. The differing responses of the different cell types to lead suggests that differentiation may be regulated in different ways by the three types of cells. Alternatively, or additionally, the cell types may differ in their ability to compensate for, sequester, or expel lead.

Animals↗
PubMed1990

L-type calcium channels may regulate neurite initiation in cultured chick embryo brain neurons and N1E-115 neuroblastoma cells.

The intracellular free Ca2+ concentration, [Ca2+]i, plays an important role in regulating neurite growth in cultured neurons. Insofar as [Ca2+]i is partly a function of Ca2+ influx through voltage-sensitive calcium channels (VSCC), Ca2+ entry through VSCC should influence neurite growth. Vertebrate neurons may possess several types of VSCC. The most frequently described VSCC types are usually designated L, T and N. In most preparations, these VSCC types respond differently to certain pharmacological agents, including Cd2+, Ni2+, the dihydropyridines nifedipine and BAY K8644, and the aminoglycoside antibiotics. We used these agents to study the role of Ca2+ influx in regulating neurite initiation and length in cultures of chick embryo brain neurons and N1E-115 mouse neuroblastoma cells. In chick neurons, nifedipine and Cd2+ (less than 50 microM), which have been reported to inhibit L-type channels, reduced neurite initiation, but not mean neurite length. Ni2+ (less than 100 microM), reported to inhibit T-type channels, had no effect on either initiation or length. Low concentrations of most aminoglycosides (less than 300 microM), reported to inhibit N-type channels, had no effect on neurite initiation, but high concentrations of streptomycin (great than 300 microM), reported to inhibit both L- and N-type channels, reduced neurite initiation. BAY K8644, which enhances current flow through L-type channels, had no effect except at high concentration (50 microM), which inhibited initiation. N1E-115 neuroblastoma cells have been reported to contain L-type and T-type channels, but thus far no channel similar to the N-type has been described. In cultured N1E-115 cells, nifedipine (5 microM), Cd2+ (5 microM), and streptomycin (200 microM) reduced neurite initiation, while nickel (50 microM) and neomycin (100 microM) did not affect initiation. None of these agents altered neurite length. In N1E-115 cells, whole-cell voltage clamp recordings showed that nifedipine and Cd2+ inhibited L-type channels but not T-type channels, while Ni2+ inhibited T-type channels but not L-type channels. Streptomycin slightly inhibited L-type channels but enhanced current flow through T-type channels. Neomycin slightly inhibited both channel types. These data indicated that neurite initiation in these two cell types may be modulated by Ca2+ influx through L-type channels, but not T- or N-type channels. Neurite length was not significantly influenced by any of the agents tested, suggesting that Ca2+ influx through VSCC may not affect neurite elongation.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1989

Organic and inorganic lead inhibit neurite growth in vertebrate and invertebrate neurons in culture.

Neurons from brains of chick embryos and pond snails (Lymnaea stagnalis) were cultured for 3 to 4 d in the presence of no toxins, inorganic lead (PbCl2), or organic lead (triethyl lead chloride). In chick neurons, inorganic lead reduced the percentage of cells that grew neurites (IC50 = 270 microM total lead, approximately 70 nM free Pb2+) but did not reduce the number of neurites per cell or the mean neurite length. Triethyl lead reduced the percentage of cells that grew neurites (IC50 = 0.24 microM) and the mean neurite length (extrapolated IC50 = 3.6 microM) but did not reduce the number of neurites per cell. In Lymnaea neurons, inorganic lead reduced the percentage of cells that grew neurites (IC50 = 13 microM total lead; approximately 10 nM free Pb2+). Triethyl lead reduced the percentage of cells that grew neurites (IC50 = 0.4 microM) and exerted significant toxicity at 0.2 microM. The two forms of lead affected neurite growth in qualitatively different ways, which suggests that their mechanisms of action are different.

Animals↗
PubMed1989

Effects of in vitro lead exposure on voltage-sensitive calcium channels differ among cell types in central neurons of Lymnaea stagnalis.

The effects of acute in vitro lead exposure on slowly inactivating voltage-sensitive calcium channels in central neurons of the freshwater pond snail Lymnaea stagnalis were studied under voltage clamp. Three physiologically distinct cell types were used: two subsets of the B cell cluster (Bpos and Bneg) and the pedal giant neuron (RPeD1). In Bpos neurons, 5 nM free Pb2+ irreversibly inhibited current flow through calcium channels by 38 +/- 10%. In Bneg neurons, 5 nM free Pb2+ slightly inhibited inward currents (12 +/- 6%) and may have shifted their voltage dependence to more depolarized voltages. The inhibition and voltage shift were irreversible. In RPeD1 neurons, Pb2+ caused a small, statistically insignificant inhibition of inward current (5 nM free Pb2+; 18 +/- 19%; 30 nM free Pb2+: 31 +/- 23%). The effects of Pb2+ were fully reversible. These data indicate that (1) voltage-sensitive calcium channels in Lymnaea neurons are inhibited by nanomolar concentrations of free Pb2+; (2) there are multiple types of calcium channels in Lymnaea neurons; and (3) the effects of in vitro lead exposure differ qualitatively among channel types.

Animals↗
PubMed1987

Effects of in vitro and in vivo lead exposure on voltage-dependent calcium channels in central neurons of Lymnaea stagnalis.

Currents through calcium channels of members of an identified cluster of neurons (B cells) in the pond snail Lymnaea stagnalis were studied under voltage clamp. The normal physiological saline was modified to maximize the visibility of voltage-dependent calcium currents and minimize contamination by other currents. Barium was used as the charge carrier for the calcium channels. Depolarizing voltage steps induce an inward current, the magnitude of which varies with the barium concentration. In brains taken from animals not exposed in vivo to lead, in vitro addition of lead acetate to the recording medium (0.25 to 14 microM) inhibits the barium current by 59 +/- 14% (mean +/- s.d.), in a manner that is independent of the lead concentration. The magnitude of the residual current still varies with the barium concentration. The voltage dependence of the current appears to be unaffected by lead. In contrast to some other calcium-channel blockers, such as cobalt, the inhibition of barium currents by in vitro lead exposure is irreversible, at least in short-term experiments. Contrary to expectations based on these in vitro results, barium currents in B cells of animals exposed to 5 microM lead for 6 to 12 weeks in vivo were approximately twice as large as barium currents in B cells from unexposed controls, when both were recorded in lead-free saline. It is possible that chronic in vivo lead exposure causes an increase in the number of calcium channels in these neurons.

Animals↗
PubMed1985

Amine-containing neurons in the brain of Lymnaea stagnalis: distribution and effects of precursors.

Glyoxylic acid-induced fluorescence in whole-brain preparations of the central nervous system of the freshwater pond snail, Lymnaea stagnalis, was used to map the distribution of serotonin-and dopamine-containing neurons. Serotonin and dopamine were easily distinguishable by differences in color of fluorescence. Serotonin-containing neurons were consistently found in the cerebral, pedal, right parietal and visceral ganglia. Dopamine-containing neurons were found in the pedal, and buccal ganglia. Prior incubation of brains in 5-hydroxytryptophan (5-HTP), the immediate precursor to serotonin, produced serotonin-like fluoresence in neurons which do not normally fluoresce. These neurons thus probably possess specific uptake mechanisms for 5-HTP. Since 5-HTP itself fluoresces yellow, the glyoxylic acid technique cannot determine if these neurons contain the enzyme aromatic amino acid decarboxylase, which converts 5-HTP to serotonin, or merely fluoresce because of the 5-HTP taken into the cells.

5-Hydroxytryptophan↗