Lynne A. Fieber

Acetylcholine-evoked currents in cultured neurones dissociated from rat parasympathetic cardiac ganglia.

1. The properties of acetylcholine (ACh)‐activated ion channels of parasympathetic neurones from neonatal rat cardiac ganglia grown in tissue culture were examined using patch clamp recording techniques. Membrane currents evoked by ACh were mimicked by nicotine, attenuated by neuronal bungarotoxin, and unaffected by atropine, suggesting that the ACh‐induced currents are mediated by nicotinic receptor activation. 2. The current‐voltage (I‐V) relationship for whole‐cell ACh‐evoked currents exhibited strong inward rectification and a reversal (zero current) potential of ‐3 mV (NaCl outside, CsCl inside). The rectification was not alleviated by changing the main permeant cation or by removal of divalent cations from the intracellular or extracellular solutions. Unitary ACh‐activated currents exhibited a linear I‐V relationship with slope conductances of 32 pS in cell‐attached membrane patches and 38 pS in excised membrane patches with symmetrical CsCl solutions. 3. Acetylcholine‐induced currents were reversibly inhibited in a dose‐dependent manner by the ganglionic antagonists, mecamylamine (Kd = 37 nM) and hexamethonium (IC50 approximately 1 microM), as well as by the neuromuscular relaxant, d‐tubocurarine (Kd = 3 microM). Inhibition of ACh‐evoked currents by hexamethonium could not be described by a simple blocking model for drug‐receptor interaction. 4. The amplitude of the ionic current through the open channel was dependent on the extracellular Na+ concentration. The direction of the shift in reversal potential upon replacement of NaCl by mannitol indicates that the neuronal nicotinic receptor channel is cation selective and the magnitude suggests a high cation to anion permeability ratio. The cation permeability (PX/PNa) followed the ionic selectivity sequence Cs+ (1.06) greater than Na+ (1.0) greater than Ca2+ (0.93). Anion substitution experiments showed a relative anion permeability, PCl/PNa less than or equal to 0.05. 5. The nicotinic ACh‐activated channels described mediate the responses of postganglionic parasympathetic neurones of the mammalian heart to vagal stimulation.

Brevetoxin derivatives that inhibit toxin activity

BACKGROUND The brevetoxins are marine neurotoxins that interfere with the normal functions of the voltage-gated Na(+) channel. We have identified two brevetoxin derivatives that do not exhibit pharmacological properties typical of the brevetoxins and that function as brevetoxin antagonists. RESULTS PbTx-3 and benzoyl-PbTx-3 elicited Na(+) channel openings during steady-state depolarizations; however, two PbTx-3 derivatives retained their ability to bind to the receptor, but did not elicit Na(+) channel openings. alpha-Naphthoyl-PbTx-3 acted as a PbTx-3 antagonist but did not affect Na(+) channels that were not exposed to PbTx-3. beta-Naphthoyl-PbTx-3 reduced openings of Na(+) channels that were not exposed to PbTx-3. CONCLUSIONS Some modifications to the brevetoxin molecule do not alter either the binding properties or the activity of these toxins. Larger modifications to the K-ring sidechain do not interfere with binding but have profound effects on their pharmacological properties. This implies a critical function for the K-ring sidechain of the native toxin.

Changes in D-aspartate ion currents in the Aplysia nervous system with aging.

D-Aspartate (D-Asp) can substitute for L-glutamate (L-Glu) at excitatory Glu receptors, and occurs as free D-Asp in the mammalian brain. D-Asp electrophysiological responses were studied as a potential correlate of aging in the California sea hare, Aplysia californica. Whole cell voltage- and current clamp measurements were made from primary neuron cultures of the pleural ganglion (PVC) and buccal ganglion S cluster (BSC) in 3 egg cohorts at sexual maturity and senescence. D-Asp activated an inward current at the hyperpolarized voltage of -70 mV, where molluscan NMDA receptors open free of constitutive block by Mg(2+). Half of the cells responded to both D-Asp and L-Glu while the remainder responded only to D-Asp or L-Glu, suggesting that D-Asp activated non-Glu channels in a subpopulation of these cells. The frequency of D-Asp-induced currents and their density were significantly decreased in senescent PVC cells but not in senescent BSC cells. These changes in sensory neurons of the tail predict functional deficits that may contribute to an overall decline in reflexive movement in aged Aplysia.

Arsenic toxicity in the human nerve cell line SK-N-SH in the presence of chromium and copper

As, Cr, and Cu represent one potential combination of multiple metals/metalloids exposures since these three elements are simultaneously leached from chromated copper arsenate (CCA)-treated wood, a common product used for building construction, at levels that can be potentially harmful. This study investigated the neurotoxicity of As associated with CCA-treated wood when accompanied by Cr and Cu. The toxicity was evaluated on the basis of a cytotoxicity model using human neuroblastoma cell line SK-N-SH. The cells were cultured with CCA-treated wood leachates or with solutions containing arsenate [As(V)], divalent copper [Cu(II)], trivalent chromium [Cr(III)] alone or in different combinations of the three elements. The toxicity was evaluated using variations in cell replication compared to controls after 96 h exposure. Among the three elements present in wood leachates, As played the primary role in the observed toxic effects, which were exerted through multiple pathways, including the generation of oxidative stress. DOM affected the absorption of metals/metalloids into the test cells, which however did not obviously appear to impact toxicity. As toxicity was enhanced by Cu(II) and inhibited by Cr(III) at concentrations below U.S. EPAs allowable maximum contaminant levels in drinking waters. Thus assessing As toxicity in real environments is not sufficient if based solely on the result from As.

Behavioral aging is associated with reduced sensory neuron excitability in Aplysia californica

Invertebrate models have advantages for understanding the basis of behavioral aging due to their simple nervous systems and short lifespans. The potential usefulness of Aplysia californica in aging research is apparent from its long history of neurobiological research, but it has been underexploited in this model use. Aging of simple reflexes at both single sensory neuron and neural circuit levels was studied to connect behavioral aging to neurophysiological aging. The tail withdrawal reflex (TWR), righting reflex, and biting response were measured throughout sexual maturity in 3 cohorts of hatchery-reared animals of known age. Reflex times increased and reflex amplitudes decreased significantly during aging. Aging in sensory neurons of animals with deficits in measures of the TWR and biting response resulted in significantly reduced excitability in old animals compared to their younger siblings. The threshold for firing increased while the number of action potentials in response to depolarizing current injection decreased during aging in sensory neurons, but not in tail motoneurons. Glutamate receptor-activated responses in sensory neurons also decreased with aging. In old tail motoneurons, the amplitude of evoked EPSPs following tail shock decreased, presumably due to reduced sensory neuron excitability during aging. The results were used to develop stages of aging relevant to both hatchery-reared and wild-caught Aplysia. Aplysia is a viable aging model in which the contributions of differential aging of components of neural circuits may be assessed.

Physiological evidence that D-aspartate activates a current distinct from ionotropic glutamate receptor currents in Aplysia californica neurons

D-Aspartate (D-Asp) activates an excitatory current in neurons of Aplysia californica. Although D-Asp is presumed to activate a subset of L-glutamate (L-Glu) channels, the identities of putative d-Asp receptors and channels are unclear. Whole cell voltage- and current-clamp studies using primary cultures of Aplysia buccal S cluster (BSC) neurons were executed to characterize D-Asp-activated ion channels. Both D-Asp and L-Glu evoked currents with similar current-voltage relationships, amplitudes, and relatively slow time courses of activation and inactivation when agonists were pressure applied. D-Asp-induced currents, however, were faster and desensitized longer, requiring 40 s to return to full amplitude. Of cells exposed to both agonists, 25% had D-Asp- but not L-Glu-induced currents, suggesting a receptor for D-Asp that was independent of l-Glu receptors. D-Asp channels were permeable to Na(+) and K(+), but not Ca²⁺, and were vulnerable to voltage-dependent Mg²⁺ block similarly to vertebrate NMDA receptor (NMDAR) channels. d-Asp may activate both NMDARs and non-l-Glu receptors in the nervous system of Aplysia.

The development of excitatory capability in Aplysia californica bag cells observed in cohorts

The bag cells of Aplysia release egg laying hormone in sexually mature animals. Bag cells cannot sustain the long-lasting excitatory afterdischarge (AD) required for hormone release prior to sexual maturity (T.A. Nick, L.K. Kaczmarek, T.J. Carew, Ionic currents underlying developmental regulation of repetitive firing in Aplysia bag cell neurons, J. Neurosci. 1996;16:7583-7598; L.A. Fieber, Characterization of Na(+) and Ca(2+) currents in bag cells of sexually immature Aplysia californica, J. Exp. Biol. 1998;201:745-754). To investigate the development of bag cell excitability, whole-cell voltage-clamp experiments were executed in dissociated bag cells from four cohorts (batches) of hatchery-reared A. californica maintained at 13-15 degrees C. K(+) current densities, representing the sum of at least four different outward K(+) currents (Nick et al., 1996), declined significantly as a function of age, beginning at least 2-3 months before sexual maturity. The K(+) current decreases coincided with the first appearance of Na(+) and Ca(2+) currents in bag cells, which occurred at ages 6-7 months. Whole cell K(+) currents were not decreased significantly by a cAMP analog earlier than 1 month prior to the onset of reproductive activity. The frequency of observing Na(+) currents in whole cell recordings was low for developmental times earlier than sexual maturity. In one winter batch, both control and PMA-treated Na(+) currents increased significantly with age, and PMA-treated current densities were significantly greater than controls, but the other two batches studied had significant differences in Na(+) current frequency only at sexual maturity. Ca(2+) currents were reliably measured in more cells than were Na(+) currents. The Ca(2+) current frequency increased significantly with maturity in one winter batch. Ca(2+) currents were significantly increased by phorbol ester treatment beginning 6-8 weeks before reproductive activity in the two winter batches. These observations support the hypothesis that bag cell excitability is not fully developed until shortly before sexual maturity.

Pharmacological evidence that D-aspartate activates a current distinct from ionotropic glutamate receptor currents in Aplysia californica

D‐Aspartate (D‐Asp) activates a nonspecific cation current of unknown identity independent of L‐glutamate (L‐Glu) in neurons of Aplysia californica. Whole‐cell voltage clamp studies were conducted using primary cultures of Aplysia buccal S cluster (BSC) neurons to characterize these receptor channels pharmacologically. The N‐methyl‐D‐aspartate receptor (NMDAR) coagonist glycine potentiated D‐Asp currents only at −30 mV, while D‐serine did not potentiate D‐Asp currents at any amplitude. Portions of D‐Asp currents were blocked by the L‐Glu antagonists kynurenate, DL‐2‐amino‐5‐phosphonopentanoic acid (APV), (2S,3R)‐1‐(phenanthren‐2‐carbonyl)piperazine‐2,3‐dicarboxylic acid (PPDA), and 1,3‐dihydro‐5‐[3‐[4‐(phenylmethyl)‐1–2H‐benzimidazol‐2‐one (TCS46b), suggesting that L‐Glu channels, particularly NMDAR‐like channels, may partially contribute to D‐Asp whole‐cell currents. In contrast, L‐Glu currents were unaffected by APV, and showed greater block by kynurenate, suggesting that D‐Asp and L‐Glu act, in part, at different sites. The excitatory amino acid transport blocker DL‐threo‐b‐Benzyloxyaspartic acid (TBOA) blocked a fraction of D‐Asp currents, suggesting that currents associated with these transporters also contribute. Non‐NMDA L‐GluR antagonists that preferentially block alpha‐amino‐3‐hydroxyl‐5‐methyl‐4‐isoxazole‐propionic acid (AMPA)/kainate receptors significantly increased D‐Asp currents, suggesting a possible allosteric potentiating effect of these antagonists on D‐Asp receptors. L‐Glu‐induced currents were significantly reduced in the presence of bath‐applied D‐Asp, whereas bath‐applied L‐Glu had no effect on D‐Asp‐induced currents. The mixed effects of these agents on D‐Asp‐induced currents in Aplysia illustrate that the underlying channels are not uniformly characteristic of any known agonist associated channel type.

Transport and interaction of arsenic, chromium, and copper associated with CCA-treated wood in columns of sand and sand amended with peat

Laboratory column leaching experiments were conducted to investigate the transport and interaction of As, Cr, and Cu associated with CCA-treated wood in sand with and without peat amendment. Results showed that leaching behavior of As, Cr, and Cu in these substrates were totally different. Substrate characteristics and microorganism activity posed distinct effects on the transport and transformation of these three elements. Arsenic was rapidly leached out from the columns with or without the amendment of peat, while Cr remained in all columns during the entire experimental period (215d). Copper was leached out only in the substrate column without peat. The presence of microorganism clearly facilitated the transport of As, while it did not show obvious effects on the transport of Cr and Cu. Interactions among these three elements were observed during the processes of adsorption and transport. The adsorption of Cu on soil was enhanced with the adsorption of As, likely caused by a more negatively charged soil surface because of As adsorption. The adsorption of Cr on soil increased the adsorption of As due to the additional As binding sites induced by Cr adsorption. These results suggest that As concentrations in the soil affected by CCA-treated wood could largely exceed predictions based on soil adsorption capacity for As. The evaluation of the impact on human health associated with CCA-treated wood should take consideration of the distinct transport characteristics of three elements and their interactions in soils.

Aging in sensory and motor neurons results in learning failure in Aplysia californica

The physiological and molecular mechanisms of age-related memory loss are complicated by the complexity of vertebrate nervous systems. This study takes advantage of a simple neural model to investigate nervous system aging, focusing on changes in learning and memory in the form of behavioral sensitization in vivo and synaptic facilitation in vitro. The effect of aging on the tail withdrawal reflex (TWR) was studied in Aplysia californica at maturity and late in the annual lifecycle. We found that short-term sensitization in TWR was absent in aged Aplysia. This implied that the neuronal machinery governing nonassociative learning was compromised during aging. Synaptic plasticity in the form of short-term facilitation between tail sensory and motor neurons decreased during aging whether the sensitizing stimulus was tail shock or the heterosynaptic modulator serotonin (5-HT). Together, these results suggest that the cellular mechanisms governing behavioral sensitization are compromised during aging, thereby nearly eliminating sensitization in aged Aplysia.