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L W Role

Publications and source records attributed to L W Role.

At least 37 records · Page 2Linked to original sources
PubMed1995

Nicotine enhancement of fast excitatory synaptic transmission in CNS by presynaptic receptors.

The behavioral and cognitive effects of nicotine suggest that nicotinic acetylcholine receptors (nAChRs) participate in central nervous system (CNS) function. Although nAChR subunit messenger RNA (mRNA) and nicotine binding sites are common in the brain, there is little evidence for synapses mediated by nAChRs in the CNS. To test whether, CNS nAChRs might modify rather than mediate transmission, the regulation of excitatory synaptic transmission by these receptors was examined. Nanomolar concentrations of nicotine enhanced both glutamatergic and cholinergic synaptic transmission by activation of presynaptic nAChRs that increased presynaptic [Ca2]i. Pharmacological and subunit deletion experiments reveal that these presynaptic nAChRs include the alpha 7 subunit. These findings reveal that CNS nAChRs enhance fast excitatory transmission, providing a likely mechanism for the complex behavioral effects of nicotine.

Animals↗
PubMed1994

Developmental regulation of multiple nicotinic AChR channel subtypes in embryonic chick habenula neurons: contributions of both the alpha 2 and alpha 4 subunit genes.

Habenula neurons from both early and late stage embryonic chickens express multiple subtypes of nicotinic acetylcholine receptor channels (nAChRs). The channel subtypes expressed by habenula neurons are similar in functional properties, but apparently distinct in subunit composition, from their peripheral counterparts in autonomic ganglia. Early in development, nicotine activates four classes of neuronal bungarotoxin (nBGT)-sensitive channels (approx. conductance = 15, 30, 50, 60pS) that are intermingled on the surface of habenula neuronal somata. In neurons removed from older animals, nAChR channel activity has increased 4- to 40-fold and channel subtypes have become spatially segregated from one another. Analysis of the profile of nAChR subunit gene expression by polymerase chain reaction indicates that several of the alpha-type subunit genes, including alpha 2,3,4,5,7, and alpha 8, as well as both beta 2 and beta 4, are expressed. Treatment of the neurons with subunit specific antisense oligonucleotides reveals that the alpha 2 and alpha 4 (but not alpha 3) subunits contribute to the functional profile of native nAChRs expressed by habenula neurons. Consideration of the functional properties and apparent subunit composition of autonomic ganglion nAChRs in the chick suggests that habenula neurons may utilize a very distinct set of subunit combinations to produce an array of nAChR channel subtypes similar in both conductance and pharmacological profile to those expressed by sympathetic neurons.

Acetylcholine↗
PubMed1993

Uptake of antisense oligonucleotides and functional block of acetylcholine receptor subunit gene expression in primary embryonic neurons.

Several recent studies have used antisense oligonucleotides in the nervous system to probe the functional role of particular gene products. Since antisense oligonucleotide-mediated block of gene expression typically involves uptake of the oligonucleotides, we have characterized the mechanism of this uptake into developing neurons from embryonic chickens. Antisense oligonucleotides (15 mers) added to the bathing media are taken up into the embryonic chicken sympathetic neurons maintained in vitro. A portion of the oligonucleotide uptake is temperature dependent and saturates at extracellular oligonucleotide concentrations > or = 20 microM. This temperature sensitive, saturable component is effectively completed by single nucleotides of ATP and AMP and is reminiscent of receptor-mediated endocytosis of oligonucleotides described in non-neuronal cells. The efficiency of the oligonucleotide uptake system is dependent on the developmental stage of the animal but independent of the number of days that the neurons are maintained in vitro. Following the uptake of antisense oligonucleotides directed against ion channel subunit genes expressed by these neurons (nicotinic acetylcholine receptor subunit alpha 3; nAChR alpha 3), biophysical assays reveal that the functional expression of the target gene is largely blocked. Thus the number of wild type nAChR channels expressed is decreased by approximately 80%-90%. Furthermore, following antisense deletion of alpha 3, "mutant" nAChRs with distinct functional characteristics are expressed. In sum, these studies characterize the uptake of antisense oligonucleotide and demonstrate the functional block of specific gene expression in primary developing neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Peptide modulation of ACh receptor desensitization controls neurotransmitter release from chicken sympathetic neurons.

1. The distribution and release of substance P (SP) in embryonic chicken lumbar sympathetic ganglia was examined with the use of immunohistochemistry and radioimmunoassay, respectively. SP immunoreactivity was detected in nerve fibers surrounding individual sympathetic neurons and was released by ganglionic depolarization. 2. Effects of SP on nicotinic acetylcholine receptor (AChR) function was assayed in embryonic sympathetic neurons in vitro by whole-cell patch clamp. SP (0.1-20 microM) accelerated the rate of decay (desensitization) of ACh-induced currents. The AChR desensitization time course is biphasic and described by the sum of two exponential functions dependent on agonist concentration (time constant of the faster component, tau f = 1-2 s, and the slower time constant, tau s = 10-25 s). SP selectively decreased tau s and the contribution of the slow component to the overall rate of current decay. The effects of SP on desensitization were concentration dependent and reversible. SP slowed recovery from desensitization by 2.5-fold. 3. SP shifted the dose-response curve for ACh-induced desensitization, reducing the concentration of ACh required to produce half-maximal desensitization by approximately twofold. 4. Preapplication of SP was equivalent to SP applied together with ACh in accelerating AChR desensitization. SP did not alter the time course of currents elicited by nondesensitizing concentrations of ACh, carbamylcholine (CARB), or dimethylphenylpiperazinium (DMPP). These data suggest that AChR activation is neither necessary nor sufficient for the peptide to modulate receptor function. A kinetic model of the effects of SP on specific steps in AChR desensitization is presented. 5. SP enhanced the rate of decay of synaptic currents in sympathetic neurons innervated in vitro, decreasing the synaptic current duration by up to 80%. 6. Effects of SP on neurotransmitter release from sympathetic neurons were evaluated by measuring the release of [3H]-norepinephrine (NE). ACh and CARB stimulated NE release in a concentration- and calcium-dependent manner. SP alone had no effect on NE secretion, but the peptide inhibited NE release induced by ACh or CARB by 40-50%. 7. Although agonists specific for either nicotinic or muscarinic receptors stimulated release of NE, SP selectively inhibited the nicotinic component of transmitter secretion. Thus SP suppressed NE release induced by DMPP by up to 80% but had no effect on muscarine or depolarization-induced NE secretion. 8. Parallel studies of the modulatory effects of SP on whole-cell currents and NE secretion revealed that SP inhibition of transmitter release from sympathetic neurons is directly proportional to the extent of potentiation of AChR desensitization.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Protein kinase C blocks somatostatin-induced modulation of calcium current in chick sympathetic neurons.

1. Somatostatin produces a voltage-dependent inhibition of N-type Ca2+ current in chick sympathetic neurons. Pretreatment of chick sympathetic ganglion neurons with protein kinase C (PKC) activators has no effect on calcium current (ICa) but reduces the inhibition of ICa by somatostatin. 2. The effects of the alkaloid PKC activator (-)-indolactam V were indistinguishable from those of 4 beta-phorbol-12-myristate-13-acetate (4 beta-PMA). The inactive isomers (+)-indolactam V and 4 alpha-PMA did not alter the modulation of ICa by somatostatin. 3. Modulation of ICa by somatostatin desensitizes, with a time for half desensitization of approximately 3 min. PKC activation mimics the normal desensitization process in that responses to 30 nM somatostatin are inhibited to a greater extent than are responses to 1 microM somatostatin. 4. PKC appears to act at the level of the somatostatin receptor or receptor-G protein interaction because PKC activation does not alter Ca2+ current inhibition in response to a nonhydrolyzable analog of GTP, GTP-gamma-S, which directly activates G proteins. 5. The specific PKC inhibitor calphostin C largely reverses the effects of phorbol esters, but does not slow the normal rate of desensitization of somatostatin responses. This indicates that PKC is not involved in the homologous desensitization of the somatostatin receptor. 6. Neither substance P, which activates PKC in these cells, nor arachidonic acid, another PKC activator, altered the action of somatostatin on ICa.

Animals↗
PubMed1993

Enhanced ACh sensitivity is accompanied by changes in ACh receptor channel properties and segregation of ACh receptor subtypes on sympathetic neurons during innervation in vivo.

Although presynaptic input can influence the number and distribution of ACh receptors (AChRs) on muscle, the role of cellular interactions in the development of transmitter sensitivity in neurons is less clear. To determine whether presynaptic input modifies neuronal AChR channel function and distribution, we must first ascertain the profile of changes in receptor properties relative to the timing of synapse formation. We have examined the temporal aspects of synaptogenesis in the lumbar sympathetic ganglia of the embryonic chick in anatomical experiments with anterograde 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate labeling of presynaptic inputs and cytochrome oxidase histochemistry. Biophysical studies of sympathetic neurons, within hours of removal from animals at different stages relative to synapse formation, show that both the properties and distribution of AChR channels are modified concurrent with a significant increase in presynaptic input to the neurons. The most striking change in AChR channel distribution is revealed by patching multiple sites on the surface of individual neurons. Following innervation in vivo, many neurons express only one of the four AChR channel subtypes and the AChRs are clustered in discrete, high-activity patches. Furthermore, when neurons at this stage express more than one AChR channel subtype, the different classes are often spatially segregated from one another on the cell surface. This contrasts with patches from neurons removed earlier on, which have lower overall activity, often comprised of multiple channel subtypes. Comparison of the AChR properties of acutely dispersed neurons to those of neurons maintained in vitro indicates that most features of AChR channels are conserved despite their removal from presynaptic and other in vivo influences. These findings are consistent with inductive interactions between pre- and postsynaptic neurons playing an important regulatory role in transmitter receptor expression.

Acetylcholine↗
PubMed1992

Activation of phosphoinositide turnover and protein kinase C by neurotransmitters that modulate calcium channels in embryonic chick sensory neurons.

Gamma aminobutyric acid (GABA) and norepinephrine modulate the excitability of primary chick sensory neurons by decreasing the voltage dependent Ca current. Although previous electrophysiological studies indicate that neurotransmitter modulation of the Ca current in these neurons involves protein kinase C, the biochemical aspects of this mechanism have not been examined directly. We find that both norepinephrine (via a unique alpha receptor subtype) and GABA (via GABAb receptors) linked to pertussis toxin sensitive pathways, stimulate the metabolism of membrane phosphatidylinositol phospholipids in primary chick sensory neurons. In addition, norepinephrine causes the rapid translocation of C kinase activity from cytosolic to membrane associated distribution, consistent with its rapid activation in response to applied neurotransmitter. The pharmacology, pertussis toxin sensitivity and time course of the biochemical changes due to neurotransmitter treatment parallel the effects of these transmitters on calcium current modulation. These biochemical studies confirm the hypothesis that activation of protein kinase C is critically involved in calcium channel modulation in embryonic chick sensory neurons.

Animals↗
PubMed1992

Diversity in primary structure and function of neuronal nicotinic acetylcholine receptor channels.

Neuronal nicotinic acetylcholine receptors are oligomeric protein complexes whose component subunits are each encoded by a family of homologous genes. The current challenge is to determine the functional contributions of the related subunits to the receptor-linked ion channels they compose and to uncover the physiological impact of the distinct channel classes expressed in vivo. In the past year, new approaches to the analysis of these receptors have yielded important insights into their stoichiometry, pharmacology and functional properties.

Animals↗
PubMed1991

Functional contribution of neuronal AChR subunits revealed by antisense oligonucleotides.

Although multiple related genes encoding nicotinic acetylcholine receptor (AChR) subunits have been identified, how each of these subunits contributes to AChRs in neurons is not known. Sympathetic neurons express four classes of AChR channels and six AChR subunit genes (alpha 3, alpha 4, alpha 5, alpha 7, beta 2, and beta 4). The contribution of individual subunits to AChR channel subtypes in these neurons was examined by selective deletion with antisense oligonucleotides. An alpha 3 antisense oligonucleotide decreased the number and altered the properties of the normally expressed ACh-activated channels. The remaining AChR channels have distinct biophysical and pharmacological properties that indicate an important functional contribution of the alpha 7 subunit.

Animals↗
PubMed1991

Developmental changes in transmitter sensitivity and synaptic transmission in embryonic chicken sympathetic neurons innervated in vitro.

Dispersed neurons from embryonic chicken sympathetic ganglia were innervated in vitro by explants of spinal cord containing the autonomic preganglionic nucleus or somatic motor nucleus. The maturation of postsynaptic acetylcholine (ACh) sensitivity and synaptic activity was evaluated from ACh and synaptically evoked currents in voltage-clamped neurons at several stages of innervation. All innervated cells are more sensitive to ACh than uninnervated neurons regardless of the source of cholinergic input. Similarly, medium conditioned by either dorsal or ventral explants mimics innervation by enhancing neuronal ACh sensitivity. This increase is due to changes in the rate of appearance of ACh receptors on the cell surface. There are also several changes in the nature of synaptic transmission with development in vitro, including an increased frequency of synaptic events and the appearance of larger amplitude synaptic currents. In addition, the mean amplitude of the unit synaptic current mode increases, as predicted from the observed changes in postsynaptic sensitivity. Although spontaneous synaptic current amplitude histograms with multimodal distributions are seen at all stages of development, histograms from early synapses are typically unimodal. Changes in the synaptic currents and ACh sensitivity between 1 and 4 days of innervation were paralleled by an increase in the number of synaptic events that evoked suprathreshold activity in the postsynaptic neurons. The early pre- and postsynaptic differentiation described here for interneuronal synapses formed in vitro may be responsible for increased efficacy of synaptic transmission during development in vivo.

Acetylcholine↗
PubMed1990

Development of synaptic transmission at autonomic synapses in vitro revealed by cytochrome oxidase histochemistry.

We have studied the development of synaptic transmission by innervating sympathetic neurons in vitro and monitoring synaptic activity with both physiological recording and cytochrome oxidase histochemistry. The onset of synaptic transmission was reflected in increased cytochrome oxidase reaction product within individual neurons. Within 24 hours of co-culture, relatively low frequency suprathreshold potentials were recorded in approximately 20% of the innervated neurons. At this stage the cytochrome oxidase activity of innervated neurons, as assessed by optical density of the histochemical reaction product, was increased twofold compared with uninnervated neurons. Over the next 2-4 days of innervation, changes in the pattern and extent of synaptic activity and superthreshold events were accompanied by a net fourfold increase in cytochrome oxidase activity levels compared with noninnervated neurons. The increase in density of cytochrome oxidase reaction product observed after innervation was reversed completely by blockade of synaptic transmission. Differences in the efficacy of synaptic input provided to the sympathetic neurons by appropriate versus inappropriate presynaptic sources was determined by co-culturing sympathetic neurons with explants that contained either preganglionic neurons or somatic motor neurons. Although sympathetic neurons innervated by motor neuron explants had increased levels of cytochrome oxidase activity compared with noninnervated controls, the density of cytochrome oxidase reaction product was even greater in sympathetic neurons innervated by preganglionic explants. We conclude that both the onset of innervation of sympathetic neurons as well as the subsequent maturation of synaptic function is directly reflected in graded increases in cytochrome oxidase reaction product.

Adrenergic Fibers↗
PubMed1990

Substance P modulates single-channel properties of neuronal nicotinic acetylcholine receptors.

Substance P (SP) is present in avian sympathetic ganglia and accelerates the decay rate of acetylcholine (ACh)-evoked macroscopic currents in sympathetic neurons. We demonstrate here that SP modulates ACh-elicited single channels in a manner consistent with an enhancement of ACh receptor (AChR) desensitization. Furthermore, since AChR channel function was monitored in cell-attached patches with SP applied to the extra-patch membrane, the peptide must act via a second messenger mechanism. SP specifically decreases the net ACh-activated single-channel current across the patch membrane by decreasing both channel opening frequency and mean open time kinetics. These experiments demonstrate that a peptide can modulate neuronal AChR function by a second messenger mechanism.

Acetylcholine↗
PubMed1989

Functional properties and developmental regulation of nicotinic acetylcholine receptors on embryonic chicken sympathetic neurons.

Measurement of acetylcholine (ACh)-induced currents indicates that the sensitivity of embryonic sympathetic neurons increases following innervation in vivo and in vitro. We have used single-channel recording to assess the contribution of changes in ACh receptor properties to this increase. Early in development (before synaptogenesis), we detect three classes of ACh-activated channels that differ in their conductance and kinetics. Molecular studies indicating a variety of neuronal receptor subunit clones suggest a similar diversity. Later in development (after innervation), changes in functional properties include increases in conductance and apparent mean open time, the addition of a new conductance class, as well as apparent clustering and segregation of channel types. These changes in channel function are compatible with the developmental increase in ACh sensitivity.

Acetylcholine↗
PubMed1988

Neural regulation of acetylcholine sensitivity in embryonic sympathetic neurons.

The development of transmitter sensitivity is an important component of synaptic differentiation. Despite a wealth of information about the appearance of acetylcholine (AcCho) sensitivity at the neuromuscular junction, the onset and regulation of this critical aspect of synaptogenesis has not previously been examined for synapse formation between neurons. To determine whether there is a role of presynaptic input in the induction of AcCho sensitivity at interneuronal synapses, AcCho-induced currents were measured in embryonic sympathetic neurons before and after synapse formation in vitro. The total AcCho sensitivity of postsynaptic neurons was increased nearly 10-fold after innervation. The effects of innervation are mimicked by medium conditioned by preganglionic neurons, suggesting that presynaptic neurons regulate postsynaptic AcCho sensitivity by release of a soluble factor. These observations provide evidence that presynaptic input regulates neuronal sensitivity to an identified synaptic transmitter.

Acetylcholine↗
PubMed1987

Activators of protein kinase C enhance acetylcholine receptor desensitization in sympathetic ganglion neurons.

Recent studies suggest that phosphorylation may regulate the rate of desensitization of nicotinic acetylcholine (AcCho) receptors (AcChoR) in vertebrate muscle and Torpedo. It is not known if phosphorylation is involved in regulation of the neuronal AcChoR, however. In this study we examine the possibility that protein kinase C might regulate nicotinic AcChoR function in neurons. Several activators of protein kinase C (1-oleoyl-2-acetylglycerol, phorbol 12,13-diacetate, and phorbol 12,13-dibutyrate) were tested for their ability to modulate AcChoR function in embryonic chicken sympathetic ganglion neurons. Neurons were voltage-clamped at the resting potential, and the response to AcCho was tested before and after treatment with activators of protein kinase C. We find that all of these agents enhance the rate of decay of AcCho-induced current without affecting peak current amplitude or cellular input resistance. The drugs were ineffective if applied concurrently with AcCho: significant effects could be detected after 60 sec of pretreatment. A phorbol that does not increase protein kinase C activity (4 beta-phorbol) was ineffective in enhancing the decay of AcCho-induced current. Thus, the effects of these agents on AcChoR function are likely to be mediated by their interaction with C kinase, rather than by direct interaction with the AcChoR channel. Our data suggest that kinase C may regulate agonist-induced desensitization of the neuronal AcChoR channel.

Animals↗
PubMed1987

Changes in the number of chick ciliary ganglion neuron processes with time in cell culture.

The purpose of this study was to describe the shape of chick ciliary ganglion neurons dissociated from embryonic day 8 or 9 ganglia and maintained in vitro. Most of the neurons were multipolar during the first three days after plating, with an average of 6.0 processes extending directly from the cell body. The neurons became unipolar with time. The remaining primary process accounted for greater than 90% of the total neuritic arbor. This striking change in morphology was not due to the selective loss of multipolar cells, or to an obvious decline in the health of apparently intact cells. The retraction of processes was neither prevented nor promoted by the presence of embryonic muscle cells. Process pruning occurred to the same extent and over the same time course whether the cells were plated on a monolayer of embryonic myotubes or on a layer of lysed fibroblasts. Process retraction is not an inevitable consequence of our culture conditions. Motoneurons dissociated from embryonic spinal cords remained multipolar over the same period of time. We conclude that ciliary ganglion neurons breed true in dissociated cell culture in that the multipolar-unipolar transition reflects their normal, in vivo, developmental program.

Animals↗
PubMed1987

The distribution of acetylcholine receptor clusters and sites of transmitter release along chick ciliary ganglion neurite-myotube contacts in culture.

Acetylcholine receptors accumulate along the length of cholinergic neuron-skeletal muscle contacts in vitro. The main purpose of this study was to describe, in a quantitative way, the distribution of acetylcholine receptor clusters induced by ciliary ganglion neurons over a period of time extending from hours to weeks after contacts are established. Neurites were filled with Lucifer Yellow and receptor clusters were identified with rhodamine-bungarotoxin. A cluster located within 5 micron of a nerve process or 10 micron of the base of a growth cone was considered to be a neurite-associated receptor patch (NARP). The first synaptic potentials were evoked 20 min after growth cone-myotube contact, and, after 24 h of co-culture, greater than 60% of the nerve-muscle pairs tested were functionally connected. NARPs appear rapidly; the first clusters were detected approximately 6 h after the neurons were plated. They were composed of several small subclusters or speckles of rhodamine-bungarotoxin fluorescence. The initial accumulation of receptors may occur at the advancing tips of nerve processes because NARPs were found at greater than 80% of the growth cone-muscle contacts examined between 12 and 24 h of co-culture. Over the 3-wk period examined, the mean incidence of NARPs ranged between 1.0 and 2.6 per 100 micron of neurite-myotube contact, with the peak observed on the second day of co-culture. During the first 3 d in culture, when the neurons were multipolar, nearly all of the primary processes induced one or more clusters. With time, as the neurons become unipolar (Role and Fischbach, 1987) NARPs persisted along the remaining dominant process. Measurements made during the third day of co-culture suggest that NARPs disappear along shorter neurites before they retract. Synaptic currents were detected by focal extracellular recording at 55% of the NARPs. The fact that spontaneous or evoked responses were not recorded at 45% suggests that contacts with clusters exhibit two functional states. Two types of presynaptic specialization at identified NARPs observed by scanning electron microscopy appear to be correlated with the functional state.

Animals↗