Biomedical subjects

D M Lovinger

Publications and source records attributed to D M Lovinger.

At least 55 records · Page 3Linked to original sources

PubMed1995

Heterologous expression of metabotropic glutamate receptors in adult rat sympathetic neurons: subtype-specific coupling to ion channels.

A novel heterologous expression system was used to examine the coupling of metabotropic glutamate receptors (mGluRs) to neuronal voltage-gated ion channels. Cytoplasmic injection of mGluR2 cRNA into adult rat sympathetic neurons resulted in the expression of receptors that negatively coupled to N-type Ca2+ channels through a pertussis toxin-sensitive pathway. Injection of mGluR1 alpha cRNA resulted in the expression of receptors that inhibited M-type K+ channels via a pertussis toxin-insensitive pathway. Coupling was restricted to specific transduction elements and effectors, since mGluR2 did not inhibit M channels and mGluR1 alpha had minimal effects on Ca2+ channels. These findings demonstrate that heterologously expressed, and thus unambiguously identified, mGluR subtypes modulate specific neuronal ion channels through discrete signal transduction pathways.

Animals↗

PubMed1995

Metabotropic glutamate receptor-mediated presynaptic depression at corticostriatal synapses involves mGLuR2 or 3.

1. The pharmacology of the metabotropic glutamate receptor (mGluR) that mediates synaptic depression at corticostriatal synapses was investigated with the use of field potential and whole cell patch-clamp recording from striatal slices and whole cell recordings from isolated striatal neurons. 2. The mGluR2,3-selective agonists (R,S)-4-carboxy-3-hydroxyphenylglycine (CHPG), (2S, 1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl) glycine (DCG-IV), and (2S, 3S, 4S)-alpha-(carboxycyclopropyl) glycine (L-CCG-I) inhibited the synaptically driven population spike (PS) evoked by afferent stimulation during field potential recording in striatal slices. These agonists also inhibited excitatory postsynaptic potentials (EPSPs) evoked by afferent stimulation during whole cell recordings. The metabotropic receptor antagonist R,S-alpha-methyl-4-carboxyphenylglycine (MCPG) blocked the synaptic depressant actions of DCG-IV and trans-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD). 3. The mGluR4,6,7-selective agonist L-serine-O-phosphate (L-SOP) did not alter corticostriatal synaptic transmission, but both this agonist and the mGluR4,6,7 agonist D,L-2-amino-4-phosphonobutyric acid (AP4) reduced the amplitude of the population EPSP and PS evoked in the dentate gyrus (DG) by stimulation of the lateral perforant path (LPP). These data are consistent with earlier observations that AP4 does not inhibit corticostriatal transmission, but produces presynaptic depression at LPP-DG synapses. 4. Application of mGluR agonists that inhibited transmission did not alter the input resistance or excitability of striatal neurons and did not inhibit responses evoked by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Developmental decrease in ethanol inhibition of N-methyl-D-aspartate receptors in rat neocortical neurons: relation to the actions of ifenprodil.

Inhibition of N-methyl-D-aspartate (NMDA) receptor function by ethanol (EtOH) and interactions between EtOH and the noncompetitive NMDA receptor antagonist ifenprodil were examined in neocortical neurons from rat and human embryonic kidney (HEK) 293 cells expressing recombinant NMDA receptors. Ethanol inhibited receptor function at concentrations in the 10 to 100 mM range in cortical neurons. EtOH inhibition of NMDA receptor function decreased as a function of time in culture over a 4-wk period. No difference in EtOH inhibition of AMPA/kainate receptor function was observed in neurons from 2- to 4-wk-old cultures. The time course of decreased EtOH inhibition paralleled a developmental decrease in inhibition by the noncompetitive NMDA receptor antagonist ifenprodil in these cortical neurons. Inhibition by EtOH was decreased in magnitude in the presence of 10 microM ifenprodil in 2- to 3-wk-old cortical neurons, but not in 1-wk-old neurons. Ifenprodil inhibited the function of recombinant NMDA receptors expressed in HEK 293 cells. Consistent with earlier reports, ifenprodil selectively inhibited responses mediated by recombinant receptors containing the NMDAR2B subunit at concentrations up to 10 microM. EtOH also inhibited the function of recombinant NMDA receptors expressed in HEK 293 cells. The potency of EtOH for inhibiting responses differed slightly among receptors containing different R2 subunits, especially at low EtOH concentrations. Finally, ifenprodil did not alter the effect of EtOH on recombinant R2A or 2B-containing NMDA receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Cellular and behavioral neurobiology of alcohol: receptor-mediated neuronal processes.

Ethanol can be viewed as a receptor modulator, selectively altering neurochemical processes in discrete regions of the CNS. The actions of ethanol at two inotropic receptor systems, gamma-aminobutyric acid (GABA) and N-methyl-D-aspartate (NMDA) glutamate receptors, are highlighted in this review. In addition, evidence is presented and discussed concerning the possible effects of ethanol that arise from interactions between these two receptor systems, the interactions of ethanol with phosphorylation states of receptor proteins and with metabotropic receptor systems.

Ethanol↗

PubMed1994

Alcohols potentiate ion current mediated by recombinant 5-HT3RA receptors expressed in a mammalian cell line.

The effect of acute exposure to alcohols on ion current mediated by recombinant 5-HT3RA receptors transiently expressed in human embryonic kidney 293 cells was investigated. Cells transfected with 5-HT3RA cDNA expressed receptors with pharmacological and functional properties similar to those of native 5-HT3 receptors. Potentiation of receptor-mediated cation current was observed in the presence of ethanol (10-100 mM), butanol (0.1-20 mM), isopentanol (0.01-25 mM) and trichloroethanol (0.5-25 mM). Potentiation increased in a concentration-dependent manner until saturation was achieved for all alcohols tested. The maximal efficacies of potentiation differed among the alcohols with isopentanol > butanol = trichloroethanol > ethanol. Potentiation by butanol and isopentanol appeared to show acute tolerance such that the percent increase in current amplitude was largest upon the first of a series of alcohol applications and decreased during subsequent applications. The effect of ethanol was variable with potentiation occurring in 74% of cells examined, but not in the remaining cells. These observations indicate that the potentiating action of alcohols is similar in recombinant receptors to that previously observed in neuroblastoma cells and neurons expressing native receptors. These findings indicate that this recombinant system is suitable for studying the molecular basis of alcohol actions on the 5-HT3 receptor.

Alcohols↗

PubMed1994

Involvement of N- and non-N-type calcium channels in synaptic transmission at corticostriatal synapses.

Calcium channels participate in the events linking axon terminal depolarization to neurotransmitter secretion. We wished to evaluate the role of N-type and non-N-type calcium channels in glutamatergic transmission at corticostriatal synapses, since this is a well defined excitatory synapse. In addition, these synapses are subject to a variety of forms of presynaptic modulation, some of which may involve alterations in calcium channel function. Application of the selective N-type channel blocker omega-conotoxin GVIA produced an irreversible depression of excitatory synaptic transmission in rat neostriatal slices shown by a decrease of approximately 50% in the amplitude of the synaptically driven population spike during field potential recording and a similar decrease in the amplitude of excitatory postsynaptic potentials during whole-cell recording. The component of transmission which was resistant to omega-conotoxin GVIA was blocked by omega-conotoxin MVIIC. omega-Agatoxin IVA had little effect on transmission. Activation of a presynaptic metabotropic glutamate receptor depressed transmission to a similar extent before and after omega-conotoxin GVIA treatment. Likewise, protein kinase C-activating phorbol esters potentiated transmission to the same extent before and after omega-conotoxin GVIA treatment. N-type calcium channels appear to be crucial for a component of excitation-secretion coupling at corticostriatal synapses. A component of transmission involves non-N-, non-L-type high-voltage-activated calcium channels. The effects of presynaptic metabotropic receptors and protein kinase C activation cannot be accounted for solely by alterations in the N-type channel function.

Animals↗

PubMed1994

The brain Kv1.1 potassium channel: in vitro and in vivo studies on subunit assembly and posttranslational processing.

While combined cloning, mutagenesis, and electrophysiological techniques have provided great insight into K+ channel structure/function relationships, little is known about K+ channel biosynthesis. To examine K+ channel biosynthesis, immune purifications were conducted on Triton X-100 extracts of 35S-met-labeled channels from in vitro translations and transfected mouse L-cells. When Kv1.1 and Kv1.4 were cotranslated in vitro, isoform-specific antisera copurified both proteins even at early time points, suggesting rapid subunit assembly. The non-Shaker Kv2.1 channel did not assemble with Kv1.1 or Kv1.4. Mouse L-cells transfected with Kv1.1 cDNA yielded 1000-4000 functional surface channels, and immune purification from Kv1.1 cells with Kv1.1 antisera produced a 57-59 kDa doublet on SDS-PAGE but not in sham-transfected cells. Immune purification of surface channels isolated both the 57 and 59 kDa proteins, suggesting cell surface channels are represented by two species. Pulse-chase metabolic labeling studies were consistent with a precursor-product relationship with the 57 kDa species giving rise to the 59 kDa protein within several minutes of synthesis. At longer chase times, the 57 kDa species reappeared, indicating both an early precursor and a mature protein ran with identical electrophoretic mobility. Mutation of the extracellular glycosylation site (N207) yielded two proteins at steady state, a 55 kDa core peptide and a 57 kDa species. Lack of glycosylation at N207 had little effect on channel synthesis, turnover, or function. Together these results suggest (1) heteromeric assembly of Shaker-like channels is cotranslational, and (2) N207 glycosylation of Kv1.1 occurs but is not required for subunit assembly, transport, or function.

Amino Acid Sequence↗

PubMed1993

High ethanol sensitivity of recombinant AMPA-type glutamate receptors expressed in mammalian cells.

The effect of ethanol (EtOH) on ion current mediated by recombinant alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate (KA) receptors was examined in transfected human embryonic kidney 293 cells using whole-cell recording. Inhibition of KA-activated current was observed in the presence of intoxicating EtOH concentrations. The potency with which EtOH inhibited current was similar for receptors formed by different subunits or subunit combinations. EtOH also inhibited KA-activated current in cultured neurons from fetal rat cortex. However, the potency of EtOH inhibition in cortical neurons was lower than that observed in 293 cells expressing recombinant receptors. The properties of receptors in cultured neurons, other than EtOH sensitivity, were similar to those displayed by recombinant AMPA/KA receptors. These observations indicate that some forms of non-NMDA ionotropic glutamate receptors have relatively high EtOH sensitivity. These receptors appear to differ in some respect from AMPA/kainate receptors expressed endogenously in cortical neurons.

Ethanol↗

PubMed1993

Protein kinase C modulates glutamate receptor inhibition of Ca2+ channels and synaptic transmission.

Fast synaptic transmission in the central nervous system can be modulated by neurotransmitters and second-messenger pathways. For example, transmission at glutamatergic synapses can be depressed by the metabotropic glutamate receptor, providing autoreceptor-mediated negative feedback. Metabotropic glutamate receptor inhibition of Ca2+ channels may contribute to this pathway. In contrast, stimulation of protein kinase C can enhance excitatory synaptic transmission, whereas both depression and enhancement of Ca2+ current have been reported. Here we show that in hippocampal CA3 and cortical pyramidal neurons, activation of protein kinase C enhances current through N-type Ca2+ channels and, in addition, dramatically reduces G protein-dependent inhibition of these same channels by the metabotropic glutamate receptor. In parallel experiments on fast excitatory transmission at corticostriatal synapses, kinase C activators were similarly found to reduce the inhibitory effect produced by stimulation of the metabotropic glutamate receptor. The results show that second-to-second control of Ca2+ channels by the metabotropic glutamate receptor can itself be modulated on a slower timescale by protein kinase C. These mechanisms may be used in the control of fast excitatory synaptic transmission.

Animals↗

PubMed1993

Excitotoxicity and alcohol-related brain damage.

Hyperexcitability following chronic alcohol exposure appears to result in enhanced activation of glutamatergic synapses in the brain. This enhanced glutamatergic transmission probably results from a combination of increased NMDA receptor activation, decreased GABAA receptor activation and increased function of voltage-activated calcium channels. Prolonged or repetitive bouts of enhanced excitatory transmission during withdrawal may destroy central neurons via "excitotoxic" mechanisms. Increased NMDA receptor activation might initiate toxicity by increasing intracellular calcium. Summation of these effects with increased intracellular calcium from voltage-activated channels might promote disinhibition and enhance cellular damage. Recent studies suggest that NMDA receptor-initiated excitotoxicity may result from thiamine deficiency. Alterations in neurotransmitter levels and receptor function during alcohol-related thiamine deficiency may contribute to this neuropathology. Thus, excitotoxic damage due to neural compensation for sustained alcohol levels and nutritional deficits may underlie aspects of alcohol-related brain damage.

Alcoholism↗

PubMed1993

Properties of a presynaptic metabotropic glutamate receptor in rat neostriatal slices.

1. The effect of the metabotropic glutamate receptor agonist trans-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD) on glutamatergic transmission at corticostriate synapses was examined using slices of neostriatum. Field potential recordings were performed in slices from adult animals, and the effects of t-ACPD on the synaptically driven population spike were examined. Tight-seal whole-cell recordings were made in slices from 2 to 4-wk-old rats, and effects of t-ACPD on the amplitude of excitatory postsynaptic potentials (EPSPs) and postsynaptic neuronal membrane properties were examined. In addition, the effects of putative metabotropic receptor agonists and antagonists and 4-aminopyridine were examined. The ability of these compounds to mimic t-ACPD or block its actions were determined. 2. Application of t-ACPD (5-100 microM) depressed the maximal amplitude of the synaptically driven population spike during field potential recording. This compound likewise depressed the amplitude of EPSPs observed with whole-cell recording. The 1S,3R isomer of t-ACPD was effective in depressing transmission, whereas the 1R,3S isomer was without effect at 50 microM. The cis isomer of ACPD (c-ACPD) also depressed transmission at concentrations from 25 to 100 microM. 3. Depression of population spike or EPSP amplitude by t-ACPD was not altered in the presence of the putative metabotropic receptor antagonist L-aminophosphonopropionic acid (AP3, 1 mM). In addition, the depressant action of t-ACPD on the population spike was not mimicked by aminophosphonobutyric acid, which has been shown to produce synaptic depression at other excitatory synapses.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Aminopyridine↗

PubMed1993

Short- and long-term synaptic depression in rat neostriatum.

1. We have examined plasticity at glutamatergic synapses on neurons in slices of neostriatum, a forebrain area involved in movement and cognitive function. 2. High-frequency stimulation of afferent inputs to neostriatal neurons induced depression of glutamatergic synaptic transmission. Depression could be induced using either prolonged trains or short repetitive bursts of high-frequency stimulation. Depression developed within seconds after such stimulation. Responses recovered to baseline levels within 10 min in most slices but persisted for up to 60 min in others. 3. Postsynaptic passive electrical properties and the ability to elicit action potentials by postsynaptic depolarization were not altered during depression. 4. The magnitude and time course of depression was similar whether postsynaptic responses were mediated by alpha amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) or N-methyl-D-aspartate (NMDA) type glutamate receptors. Depression was not altered by antagonism of AMPA or NMDA receptors or potentiation of AMPA receptor function with aniracetam. 5. Depression was blocked by treatments that increase transmitter release including increased extracellular Ca2+, application of 4-aminopyridine, or application of phorbol ester. 6. Our findings indicate that glutamatergic synapses in neostriatum are capable of expressing a form of synaptic depression that may involve decreased glutamate release.

Afferent Pathways↗

PubMed1993

Ethanol action on excitatory amino acid activated ion channels.

The effects of ethanol on excitatory amino acid activated ion channels were investigated using patch-clamp recording methods. Intoxicating concentrations of ethanol (5-50 mM) inhibited ion current activated by the glutamate receptor agonist N-methyl-D-aspartate (NMDA) in a concentration-dependent manner (IC50 = 30 mM). The intoxicating potency of different alcohols was correlated with their potency for inhibiting NMDA-activated current, suggesting that alcohol-induced inhibition of NMDA channel function may contribute to the neural and cognitive impairments associated with intoxication. Analysis of mechanism suggests that ethanol inhibits NMDA-activated current by altering gating of the channel, rather than by affecting channel conductance, ion permeance or regulatory sites on the channel. Anesthetic concentrations of ethanol (> 50 mM) produced a concentration-dependent inhibition of kainate- and quisqualate-activated currents (IC50 = 220 mM), suggesting that this inhibition may contribute to the general anesthetic effects of ethanol. This hypothesis is supported by the observations that the general anesthetic agents, trichloroethanol (the active metabolite of chloral hydrate), pentobarbital and halothane, all inhibit kainate- and quisqualate-activated currents.

Animals↗

PubMed1993

Trichloroethanol potentiates synaptic transmission mediated by gamma-aminobutyric acidA receptors in hippocampal neurons.

We have examined the actions of trichloroethanol (TCEt), the active metabolite of the general anesthetic chloral hydrate, on responses mediated by gamma-aminobutyric acid (GABA)A receptors in response to application of exogenous GABA and activation of endogenous GABAergic transmission, by using hippocampal neurons in cell culture and in brain slices. In the presence of TCEt, Cl- current activated by exogenous GABA was both enhanced in amplitude and prolonged, leading to a net increase in total charge passing through GABAA receptor channels. Prolongation of GABA-activated current increased in magnitude in a concentration-dependent manner from 0.2 to 10 mM TCEt. Inhibitory postsynaptic currents produced at synapses between pairs of cultured GABAergic neurons or by activation of interneurons in hippocampal slices were also prolonged by TCEt, at concentrations from 0.5 to 10 mM. Application of TCEt at concentrations of 1 mM and above produced a small amplitude current which was directed outwardly at -40 mV in neurons in which methylsulfate or gluconate was the major intracellular anion and directed inwardly in neurons filled with Cl-. Our observations indicate that TCEt potentiates GABAergic transmission; presumably by potentiating the function of GABAA receptors in a manner similar to barbiturate or steroid anesthetics. This action is likely to contribute to the general anesthetic effect of TCEt which occurs after chloral hydrate administration.

Action Potentials↗

PubMed1993

Modulation of gating of cloned rat and human K+ channels by micromolar Zn2+.

The actions of zinc ions on three species of K+ channels were studied using mouse fibroblasts stably transfected with a plasmid containing both the appropriate K+ channel gene and a steroid-inducible promotor. The channels studied were rKv1.1 and hKv1.5, delayed rectifiers cloned from rat and human tissue, respectively, and hKv1.4, an inactivating human K+ channel. Zn2+ shifted the activation curves for all three K+ currents in the depolarizing direction and also shifted the steady state inactivation curve for hKv1.4 in the depolarizing direction. The effect of Zn2+ was concentration dependent between 2 and 1000 microM. As a consequence of the modulation of gating, the activation kinetics of the K+ currents were slowed by Zn2+, an effect likely to delay repolarization of the neuronal action potential. The action of Zn2+ on these diverse K+ channels suggests the existence of a common Zn2+ binding domain, the occupation of which influences the voltage sensor. The resulting modulation of gating of hKv1.4 by Zn2+ may well be of physiological significance, in view of the localization of this channel in mossy fiber nerve terminals in the hippocampus, where Zn2+ is found in abundance.

Animals↗

PubMed1993

Trichloroethanol potentiation of 5-hydroxytryptamine3 receptor-mediated ion current in nodose ganglion neurons from the adult rat.

Trichloroethanol (TCEt) potentiated ion current mediated by 5-hydroxytryptamine (5-HT)3 receptors in isolated adult rat nodose ganglion neurons. The magnitude of potentiation of peak current amplitude increased with increasing TCEt concentrations from 0.5 to 5 mM. The rate of decay of current also increased as a function of TCEt concentration. Steady-state current was unaffected by TCEt at concentrations up to 10 mM. A high concentration of TCEt (25 mM) potentiated peak current but inhibited steady-state current. Potentiation appeared to involve an increase in agonist potency as the magnitude of potentiation of peak current decreased with increasing agonist concentration. Agonist application before TCEt treatment decreased the magnitude of potentiation elicited by 5 mM TCEt. These observations indicate that the potentiating action of TCEt arises from an increase in the efficacy with which 5-HT activates current. TCEt appears to facilitate transitions from closed to open states more readily than transitions from desensitized to open states. These observations are consistent with the hypothesis that 5-HT3 receptor function may contribute to the behavioral pharmacology of alcohols and related sedative/hypnotic agents.

Animals↗

PubMed1992

The actions of 3-aminopropanephosphinic acid at GABAB receptors in rat hippocampus.

The actions of 3-aminopropanephosphinic acid (APPA) were examined using whole-cell patch-clamp recording in rat hippocampal slice. In recordings from neurons in subfield CA1 of slices from young (2-4 weeks) and adult (greater than 2 month) rats, APPA (0.5-50 microM) produced membrane hyperpolarization and outward current under voltage-clamp. APPA also inhibited excitatory postsynaptic potentials with an IC50 of 2.3 microM, and reduced inhibitory postsynaptic potentials at concentrations from 0.1 to 1 microM. The hyperpolarizing and synaptic depressant effects of APPA were reduced by 2-OH-saclofen an antagonist at the B-type receptor for the neurotransmitter gamma-aminobutyric acid (GABA). In this preparation APPA exhibited potencies similar to those previously reported for the GABAB receptor agonist baclofen. APPA was much less effective in inhibiting synaptic transmission measured using field potential recordings. The observations made with whole-cell patch-clamp recording indicate that in hippocampus APPA acts as a potent agonist at presynaptic GABAB receptors associated with both excitatory and inhibitory synapses, and also activates postsynaptic GABAB receptors.

Action Potentials↗

PubMed1992

GABA- and glutamate-gated ion channels as molecular sites of alcohol and anesthetic action.

The evidence presented above indicates that GABA- and glutamate-activated ion channels are molecular sites of alcohol and anesthetic action. In view of the important role that these channels play in CNS excitability, it seems likely that the actions of alcohol and anesthetics on these channels contribute significantly to the behavioral effects of these agents. Although the behavioral effects of alcohol and anesthetics may well result from a combination of actions on different ion channels and other molecular sites in the CNS, it is of interest to consider whether the actions of these agents on particular types of ion channels may contribute to particular behavioral effects. In this regard, it should be noted that benzodiazepines potentiate GABAA responses, but do not produce intoxication or general anesthesia in their clinical dose range. Benzodiazepines are widely used clinically, primarily for their anxiolytic actions (26), suggesting that the potentiation of GABAA responses by ethanol and barbiturates may contribute to the anxiolytic effects of these agents. Since kainate and quisqualate channels mediate fast excitatory transmission in the CNS, inhibition of kainate and quisqualate receptor-activated responses would be expected to result in general CNS depression. This suggests that inhibition of kainate and quisqualate receptor-mediated responses may contribute to the general anesthetic effects of ethanol, trichloroethanol and barbiturates. NMDA channels are thought to mediate complex excitatory neural phenomena and cognitive function. In view of this, the observation that ethanol inhibits NMDA receptor-mediated responses over the concentration range that produces intoxication and the correlation between the potency of different alcohols for inhibiting NMDA-activated current and their potency for producing intoxication suggest that ethanol-induced inhibition of NMDA receptor-mediated responses may contribute to the intoxicating effects of ethanol. Although these speculations are no doubt oversimplifications, the recognition that GABA- and glutamate-gated ion channels are molecular sites of alcohol and anesthetic action provides a basis for investigating the molecular mechanisms involved in the action of these agents and the behavioral significance of those actions.

Alcohols↗