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A L Willard

Publications and source records attributed to A L Willard.

15 recordsLinked to original sources
PubMed1999

Upregulation of calcium homeostatic mechanisms in chronically depolarized rat myenteric neurons.

Perturbations of intracellular Ca2+ ion concentration ([Ca2+]i) have important effects on numerous neuronal processes and influence development and survival. Neuronal [Ca2+]i is, in large part, dependent on activity, and changes in activity levels can alter how neurons handle calcium (Ca). To investigate the ability of neuronal Ca homeostatic mechanisms to adapt to the persistent elevation of [Ca2+]i, we used optical and electrophysiological recording techniques to measure [Ca2+]i transients in neurons from the rat myenteric plexus that had been chronically depolarized by growth in culture medium containing elevated (25 mM) KCl. When studied in normal saline, neurons that had previously been chronically depolarized for 3-5 days had briefer action potentials than control neurons, their action potentials produced smaller, more rapidly decaying increases in [Ca2+]i, and voltage-clamp pulses with action potential waveforms evoked smaller Ca currents than in control neurons. Simultaneous voltage-clamp measurements and calcium imaging revealed that increases in the Ca handling capacities of the chronically depolarized neurons permitted them to limit the amplitudes of action potential-evoked [Ca2+]i transients and to restore [Ca2+]i to basal levels more rapidly than control neurons. Release of Ca from endoplasmic reticulum-based Ca stores made smaller contributions to action potential-evoked [Ca2+]i transients in chronically depolarized neurons even though those neurons had larger caffeine-releasable Ca stores. Endoplasmic reticulum-based Ca sequestration mechanisms appeared to contribute to the faster decay of [Ca2+]i transients in chronically depolarized neurons. These results demonstrate that when neurons experience prolonged perturbations of [Ca2+]i, they can adjust multiple components of their Ca homeostatic machinery. Appropriate utilization of this adaptive capability should help neurons resist potentially lethal metabolic and environmental insults.

Action Potentials↗
PubMed1993

Shock trauma!

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Adult↗
PubMed1993

Voltage-dependent sodium and calcium currents of rat myenteric neurons in cell culture.

1. Inward currents of myenteric neurons that had been grown in cell cultures prepared from the small intestines of neonatal or young adult rats were studied with tight seal whole-cell recordings. The kinetic and pharmacological properties of these neurons were analyzed. 2. All neurons had rapidly inactivating, tetrodotoxin (TTX)-sensitive Na+ currents that could be evoked by steps to potentials more positive than -50 mV. Holding potentials more negative than -65 mV were necessary to remove steady-state inactivation. No TTX-insensitive Na+ currents were observed, thus the ability of subsets of myenteric neurons to fire action potentials in TTX must depend upon their density of Ca2+ channels. 3. Ca2+ and Ba2+ currents were studied in neurons perfused internally with CsCl to block K+ currents and bathed with solutions containing TTX and antagonists of K+ channels. Currents were significantly larger when Ba2+ replaced Ca2+ as the charge carrier. Cd2+ and Gd3+ blocked Ca2+ and Ba2+ currents rapidly and reversibly. High-voltage-activated (HVA) Ca2+ and Ba2+ currents were observed in all neurons. Too few neurons possessed detectable low-voltage-activated Ca2+ currents to permit detailed study. 4. HVA Ca2+ and Ba2+ currents evoked from holding potentials more negative than -50 mV could be divided into two kinetically distinguishable components with very different rates of inactivation. A "decaying" component inactivated relatively rapidly with a t1/2 of 25-75 ms. A "sustained" component inactivated quite slowly with a t1/2 of 1-5 s. At more positive holding potentials, only the sustained component was observed. Although the two kinetically distinguishable components had different current-voltage relationships, they had indistinguishable rates of deactivation: a single time constant was sufficient to fit the decay of tail currents. The relative amplitudes of the two components varied considerably among different neurons. 5. Ca2+ and Ba2+ currents could be divided into two pharmacologically distinct components on the basis of sensitivity to omega-conotoxin GVIA (I omega CgTX) and to dihydropyridine antagonists (IDHP). At holding potentials more positive than -70 mV, a combination of omega CgTX and DHPs completely blocked Ca2+ and Ba2+ currents in most neurons. At holding potentials more negative than -50 mV, I omega CgTX and IDHP each contained decaying and sustained components. I omega CgTX activated more slowly than did IDHP. The DHP agonist Bay K8644 increased the amplitude of IDHP and slowed its rate of deactivation. 6. The results suggest that myenteric neurons may have as few as two subtypes of HVA Ca2+ channels; omega CgTX-sensitive ones and DHP-sensitive ones.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Excitatory and neurotoxic actions of platelet-activating factor on rat myenteric neurons in cell culture.

At micromolar concentrations, PAF causes intense excitation and elevation of intracellular Ca in a subset of myenteric neurons. When applied for more than about 10 seconds, these concentrations of PAF kill a subset of myenteric neurons. The excitation and elevation of Ca levels are accompanied by increased membrane conductance and enhanced synaptic activity. The effects of brief applications are reversible, but responses to subsequent applications of PAF are substantially reduced. If such responses can be elicited in enteric neurons by the concentrations of PAF that are generated in vivo, they would account for the potent ability of PAF to evoke neurally-mediated secretory responses in GI tissues.

Animals↗
PubMed1992

Long-term regulation of neuronal calcium currents by prolonged changes of membrane potential.

Although rapid-onset, short-term regulation of neuronal Ca currents by neurotransmitters and second messengers is well documented, little is known about conditions that can cause longer-lasting changes in Ca channel function. We report here that persistent depolarization is accompanied by slowly developing long-term reduction of neuronal Ca currents. Rat myenteric neurons grown in cell culture for 1-7 d were studied with the tight-seal whole-cell recording technique. Macroscopic Ca-channel currents had decaying and sustained components at all days studied. When the neurons were grown in medium containing 25 mM KCl, which depolarized them to -40 mV and caused significant elevation of intracellular Ca, the densities of both components of Ca-channel current decreased by 40-80%. Several results suggest that different mechanisms underlie the downregulation of the two components. (1) The density of the decaying component decreased approximately four times faster than did that of the sustained component. (2) When neurons were returned to control medium, which contained 5 mM KCl, the density of the sustained component returned to control levels within 24 hr, while that of the decaying component did not recover significantly. (3) Inhibitors of RNA and protein synthesis reduced or prevented downregulation of the sustained but not of the decaying component. (4) The dihydropyridine antagonist nitrendipine, which prevented the sustained elevation of intracellular Ca in neurons grown in 25 mM KCl, prevented downregulation of the sustained component but had no effect on downregulation of the decaying component. We suggest that these forms of regulation of Ca current density could help neurons adapt to altered levels of electrical activity and may contribute to changes in synaptic strength that occur during periods of increased or decreased electrical activity.

Animals↗
PubMed1990

Substance P mediates synaptic transmission between rat myenteric neurones in cell culture.

1. Whole-cell patch-clamp recordings were made from pairs of neurones in cell cultures of rat myenteric neurones. In some pairs, action potentials evoked in the first neurone evoked a slow excitatory postsynaptic potential (EPSP) in the second neurone. 2. Action potentials at a frequency of at least 5 Hz were required to evoked slow EPSPs. In one group of cells, the slow EPSP followed a series of nicotinic fast EPSPs; in another group, fast EPSPs did not precede the slow EPSP. 3. The slow EPSPs were 2-16 mV in amplitude and were accompanied by decreased resting potassium conductance. 4. Most (17/28) neurones in which action potentials evoked only slow EPSPs in a follower cell contained substance P (SP)-like immunoreactivity; they were not immunoreactive for 5-hydroxytryptamine (0/15) or vasoactive intestinal peptide (0/22). 5. Postsynaptic responses to SP, neurokinin A and a synthetic tachykinin [( pGlu6, Pro9]SP6-11) mimicked the slow EPSPs. The non-tachykinin peptide vasoactive intestinal polypeptide (VIP), which was not found in neurones that evoked only slow EPSPs, also mimicked the slow EPSPs. Responsiveness to SP decreased significantly during slow EPSPs. 6. Desensitization to either SP or VIP reduced or prevented the slow EPSPs and also responses to each other. Two proposed antagonists of SP receptors, [D-Arg1, D-Pro2,D-Trp7,9,Leu11]substance P and [D-Arg1,D-Trp7,9,Leu11]substance P, did not affect the slow EPSPs significantly. 7. Antisera against SP reversibly blocked or reduced slow EPSPs evoked by eight of thirteen presynaptic neurones that evoked slow EPSPs without evoking fast EPSPs. All eight of the presynaptic neurones that evoked anti-SP-sensitive slow EPSPs contained SP-like immunoreactivity. None of the presynaptic neurones that evoked anti-SP-insensitive slow EPSPs contained detectable SP-like immunoreactivity. Normal sera and anti-VIP antisera did not alter the slow EPSPs detectably. 8. It is concluded that subsets of myenteric neurones release an SP-like transmitter to evoke slow EPSPs. These neurones appear to lack a 'classical' neurotransmitter that evokes fast EPSPs.

Action Potentials↗
PubMed1990

A vasoactive intestinal peptide-like cotransmitter at cholinergic synapses between rat myenteric neurons in cell culture.

Intracellular recording and immunochemical techniques were used to study synaptic transmission between individual pairs of rat myenteric plexus neurons in cell culture. This report describes the synaptic connections made by "dual function" presynaptic neurons that evoked slow postsynaptic depolarizations (slow EPSPs) in the same neurons in which they also evoked fast nicotinic cholinergic EPSPs. The slow EPSPs occurred only when presynaptic neurons were stimulated at frequencies of 5 Hz or higher. During the slow EPSPs, slope input resistance increased. The slow EPSPs were not detectably voltage-dependent, and they reversed sign at the estimated K+ equilibrium potential, suggesting that they resulted from a synaptically mediated decrease in resting K+ conductance. Several lines of evidence suggested that dual-function neurons evoke slow EPSPs by releasing a vasoactive intestinal peptide (VIP)-like cotransmitter. (1) Immunocytochemical staining revealed VIP-like immunoreactivity in all physiologically identified dual-function neurons. (2) Responses to exogenously applied VIP mimicked the slow EPSPs. (3) Superfusion of cultures with anti-VIP antisera blocked the slow EPSPs reversibly, as did application of desensitizing doses of VIP. These findings suggest that during periods of increased activity, subsets of cholinergic myenteric neurons release a VIP-like cotransmitter that enhances postsynaptic excitability. The effects of the cotransmitter may help to compensate for decreases in nicotinic EPSPs that occur during increased presynaptic activity.

Animals↗
PubMed1988

Conditioned medium alters electrophysiological and transmitter-related properties expressed by rat enteric neurons in cell culture.

We have previously shown that rat enteric neurons display many of their in vivo phenotypes when they are dissociated and grown in long-term cell culture. To assess the degree of plasticity of these phenotypes we have examined the effect of medium conditioned by rat heart cells because this treatment strongly affects transmitter properties in rat sympathetic neurons in culture. Growth of enteric neurons for 3-4 weeks in conditioned medium caused several changes that are similar to previously described effects of conditioned medium on other neuronal cell types in culture. When compared to cultures grown in control medium, cultures grown in conditioned medium: (i) contained three times as many large (greater than 25 micron) neurons; (ii) synthesized and stored 3-4 times as much acetylcholine; (iii) contained 4-5 times as many neurons with detectable 5-hydroxytryptamine immunoreactivity; and (iv) contained 10 times as many neurons that fired repetitively during sustained depolarization. Several other changes, which have not been reported in other systems, were also observed. Conditioned medium cultures: (i) contained many fewer neuronal processes with immunohistochemically detectable vasoactive intestinal polypeptide, substance P, somatostatin, and [Met]enkephalin; (ii) contained 70% fewer neuronal cell bodies with vasoactive intestinal polypeptide-like immunoreactivity; and (iii) contained four times as many neurons that had muscarinic responses to acetylcholine. None of the changes in properties described above uniformly affected all enteric neurons, even after 6 weeks of growth in conditioned medium. We conclude that the heterogeneity of enteric neuron phenotypes is established prior to birth and limits the capacity of certain subsets of neurons to respond to exogenous factors in the environment. Nevertheless, the phenotypes of other subsets of neurons displayed considerable plasticity when exposed to conditioned medium.

Acetylcholine↗
PubMed1987

Neuropeptides mark functionally distinguishable cholinergic enteric neurons.

Subpopulations of physiologically identified cholinergic enteric neurons in cell culture contain somatostatin (SOM)- or vasoactive intestinal peptide (VIP)- like immunoreactivity (LIR). These subpopulations differ in their synaptic effects on other neurons: cholinergic neurons that contain SOM-LIR cause fast nicotinic excitatory postsynaptic potentials (EPSPs) that have significantly larger amplitudes than do EPSPs caused by cholinergic neurons that lack SOM-LIR. Cholinergic neurons containing VIP-LIR cause slow non-cholinergic depolarizations in addition to fast nicotinic EPSPs. These findings are the first correlation between neuropeptide content and functional differences in the synaptic effects of subpopulations of cholinergic enteric neurons.

Action Potentials↗
PubMed1985

Neurons dissociated from rat myenteric plexus retain differentiated properties when grown in cell culture. I. Morphological properties and immunocytochemical localization of transmitter candidates.

We have developed procedures for dissociating neurons from the myenteric plexus of the small intestine of newborn rats and for growing those neurons in cell cultures for up to 3 months. Neurons in these cultures retain many of the differentiated properties of myenteric neurons in vivo. This is the first of a series of 3 papers describing those properties. In this paper, we describe the morphology of cultured neurons that we have observed with light and electron microscopy; we also describe the patterns of straining observed when immunocytochemical techniques were used to localize neurotransmitter candidates in the cultured neurons. Intracellular injections of a fluorescent dye, Lucifer yellow, revealed that many of the cultured neurons had morphologies similar to those of myenteric neurons in vivo. When thin sections of cultures were viewed in an electron microscope, many neurons were observed to have numerous small (40-60 nm), clear synaptic vesicles and/or large (80-150 nm), opaque-cored (p-type) vesicles. Synaptic profiles were most often observed on neuronal somata. Neurons containing immunoreactive serotonin, substance P, somatostatin, enkephalin, bombesin and gastrin/cholecystokinin were observed in about the same proportions as they occur in the intact myenteric plexus. Neurons containing immunoreactive vasoactive intestinal polypeptide were found in higher numbers than reported in vivo. Neurons containing immunoreactive neurotensin, secretin and glutamate decarboxylase were not observed. An antiserum directed against choline acetyltransferase stained 40-50% of the neurons. We conclude that myenteric neurons continue to express much of their normal differentiated properties even when they are removed from the gut, dissociated into a suspension of single cells and grown in culture. Such cultures will be useful for correlating the morphological, biophysical, pharmacological and synaptic properties of individual myenteric neurons and for testing the ability of altered environmental conditions to change those properties.

Animals↗
PubMed1985

Neurons dissociated from rat myenteric plexus retain differentiated properties when grown in cell culture. II. Electrophysiological properties and responses to neurotransmitter candidates.

We have used intracellular recordings to study the electrophysiological and pharmacological properties of neurons that have been grown in cell cultures after having been dissociated from the myenteric plexus of the small intestine of newborn rats. Studies of action potential mechanisms revealed that all of the neurons could generate Na+-dependent action potentials in the presence of Ca2+-channel blockers and that about 70% could generate Ca2+-dependent action potentials when Na+ channels were blocked with tetrodotoxin. No neurons generated long afterhyperpolarizations after single action potentials but about 50% of neurons did so following trains of action potentials. Over 95% of the neurons tested accommodated rapidly to sustained depolarization. The effects of several enteric neurotransmitter candidates were studied by superfusing or pressure-ejecting test solutions while recording neuronal responses. All of the cultured neurons tested had nicotinic responses to acetylcholine. Subsets of neurons responded to muscarinic cholinergic agonists (slow depolarization and increased excitability), serotonin (fast depolarization or slow depolarization and increased excitability), gamma-aminobutyrate (fast depolarization), substance P (slow depolarization, biphasic fast and slow depolarization or increased excitability without a change in membrane potential), vasoactive intestinal peptide (slow depolarization and increased excitability), or [Met]enkephalin (slow hyperpolarization and/or decreased action potential duration). We conclude that myenteric neurons grown in cell culture retain many of the physiological and pharmacological properties that they have in situ. Such cultures will permit detailed biophysical and pharmacological studies of the mechanisms of action of enteric neurotransmitter candidates.

Acetylcholine↗
PubMed1985

Neurons dissociated from rat myenteric plexus retain differentiated properties when grown in cell culture. III. Synaptic interactions and modulatory effects of neurotransmitter candidates.

We have used intracellular recordings to study synaptic interactions between myenteric neurons grown in dissociated cell culture. Intracellular stimulation of individual myenteric neurons caused several types of synaptic effects in nearby neurons: fast excitatory synaptic potentials mediated by nicotinic acetylcholine receptors; slow, non-cholinergic synaptic potentials; dual transmission having both fast cholinergic and slow non-cholinergic components and inhibition of spontaneously occurring fast nicotinic synaptic potentials. Fast nicotinic synaptic potentials were elicited by about 40% of neurons tested and often occurred spontaneously. The fast synaptic potentials were similar to those that have been studied in other autonomic neurons with respect to their estimated reversal potential and their sensitivity to cholinergic antagonists. The amplitudes of the fast synaptic potentials declined if evoked at frequencies greater than 0.5 Hz. Potentiation of the fast synaptic potentials was observed following high-frequency stimulation of presynaptic neurons. Several transmitter candidates modulated fast cholinergic transmission. Substance P and vasoactive intestinal peptide promoted nicotinic transmission by causing increased amplitudes of evoked and spontaneous fast synaptic potentials and an increased frequency of spontaneous synaptic potentials. gamma-Aminobutyrate and [Met]enkephalin both caused decreased amplitudes and frequency of nicotinic synaptic potentials. Serotonin depressed synaptic potentials in some neurons while enhancing them or having no effect in others. Slow, non-cholinergic, synaptic potentials were elicited by about 10% of neurons tested. These synaptic effects lasted 15-300s, caused depolarizations of 3-15 mv and were accompanied by increased neuronal input resistance. The transmitter(s) causing these slow synaptic potentials has not yet been identified.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Insulin promotes electrical coupling between cultured sympathetic neurons.

Placing neurons in tissue culture is one way to study how environmental factors affect their differentiation. Replacement of serum-supplementation of the culture medium with defined ingredients extends the experimenter's control of the culture environment; however it also introduces additional potential influences. In this report, we confirm the observation of Higgins and Burton (Higgins, D., and H. Burton (1982) Neuroscience 7:2241-2253) of increased frequency of electrical coupling in serum-free compared to serum-supplemented cultures of rat sympathetic neurons. In addition, experiments were performed to determine whether this effect results from the removal of serum or from the addition of the defined medium components to the culture environment. The results of testing individual ingredients of the defined medium recipe adapted for use on sympathetic neurons (Bottenstein, J.E., and G. H. Sato (1979) Proc. Natl. Acad. Sci. U. S. A. 76:514-517) show that insulin is capable of inducing electrical coupling in serum-free cultures. Thus, the formation of electrical synapses by sympathetic neurons can be hormonally regulated.

Animals↗
PubMed1980

Electrical excitability of outgrowing neurites of embryonic neurones in cultures of dissociated neural plate of Xenopus laevis.

1. I have studied the electrical excitability of outgrowing processes of individual neurones in cultures made from dissociated neural plates of embryos of Xenopus laevis prior to the time of neurite outgrowth in vivo. 2. The electrical excitability of neurites was tested by stimulating them extracellularly and recording responses with an intracellular electrode in their cell bodies; neurites were excitable at all times examined. 3. The ionic basis of the excitability of neurites was tested by recording from cells while changing the composition of the salines perfusing the cultures. 4. In cultures less than 10 hr old, all neurites tested made responses which depended on Ca2+. The action potentials of the cell bodies were also Ca2+-dependent at these times. 5. Between 10 and 12 hr in culture, a time at which the cell bodies still made Ca2+-dependent action potentials, neurites acquired the ability to make Na+-dependent responses. At these times, two-thirds of neurites tested retained the ability to produce divalent cation-dependent action potentials when perfused with solutions of isotonic Ba2+. 6. After 12 hr in culture, no neurites were observed to make Ca2+-or Ba2+-dependent responses; only Na+-dependent responses were observed. Cells continued to initiate and elongate new neurites until about 24 hr in culture. Thus neurites sent out at different times in culture differed in their development of excitability. 7. Cell bodies making exclusively Ca2+-dependent action potentials could be found until about 15 hr in culture, after which time a Na+-dependent component appeared. Cell bodies could then be observed to make action potentials which depended on both Ca2+ and Na+ until about 3 days in culture. After 3 days, most cell bodies made predominately Na+-dependent action potentials. Unlike the neurites, cell bodies retained the ability to make action potentials in isotonic Ba2+ for as long as the cultures were maintained (up to 5 days). 8. The possibility that changes in the ionic basis of action potentials reflected the death of one population of cells and the simultaneous appearance of another population with different properties was eliminated by observing the fate of single cells while changes in the physiological properties were occurring. Such observations showed that the majority of cells in each culture were surviving throughout the period of study. 9. Thus the membranes of the neurites and cell bodies of neurones in these cultures appeared to undergo independently timed changes in the ionic basis of their action potentials.

Action Potentials↗