Biomedical subjects

David M Lovinger

Publications and source records attributed to David M Lovinger.

At least 19 recordsLinked to original sources

PubMed2006

The role of protein synthesis in striatal long-term depression.

Long-term depression (LTD) at the corticostriatal synapse is postsynaptically induced but presynaptically expressed, the depression being a result of retrograde endocannabinoid signaling that activates presynaptic cannabinoid CB1 receptors and reduces the probability of glutamate release. To study the role of protein synthesis in striatal LTD, we used a striatum-only preparation in which the presynaptic cell body is cut off, leaving intact only its axons, whose terminals synapse on medium spiny neurons. LTD (duration >150 min) was induced in this preparation, thus providing evidence that transcription in the presynaptic cell nucleus is not necessary for this form of plasticity. The maintenance of striatal LTD, however, was blocked by bath application of protein translation inhibitors but not by the same inhibitors loaded into the postsynaptic cell. These results suggest that local translation is critical for the expression of striatal LTD, distinguishing this form of mammalian synaptic plasticity from other forms that require postsynaptic protein synthesis. Possible roles of axonal or glial translation in striatal LTD are considered.

Afferent Pathways↗

PubMed2006

Disrupted motor learning and long-term synaptic plasticity in mice lacking NMDAR1 in the striatum.

Much research has implicated the striatum in motor learning, but the underlying mechanisms have not been identified. Although NMDA receptor (NMDAR)-dependent long-term potentiation has been observed in the striatum, its involvement in motor learning remains unclear. To examine the role of striatal NMDAR in motor learning, we created striatum-specific NMDAR1 subunit knockout mice, analyzed the striatal anatomy and neuronal morphology of these mice, evaluated their performance on well established motor tasks, and performed electrophysiological recordings to assay striatal NMDAR function and long-term synaptic plasticity. Our results show that deleting the NMDAR1 subunit of the NMDAR specifically in the striatum, which virtually abolished NMDAR-mediated currents, resulted in only small changes in striatal neuronal morphology but severely impaired motor learning and disrupted dorsal striatal long-term potentiation and ventral striatal long-term depression.

Animals↗

PubMed2006

Ethanol effects on electrophysiological properties of astrocytes in striatal brain slices.

Ethanol (EtOH) is known to alter neuronal physiology, but much less is known about the actions of this drug on glial function. To this end, we examined acute effects of ethanol on resting and voltage-activated membrane currents in striatal astrocytes using rat brain slices. Ten minutes exposure to 50mM EtOH reduced slope conductance by 20%, increased input resistance by 25% and decreased capacitance by 38% but did not affect resting membrane potential. Current generated by a hyperpolarizing pulse was inhibited in a concentration dependent manner in passive astrocytes, while no significant EtOH effect was observed in complex astrocytes or neurons. The EtOH effect was blocked when intracellular KCl was replaced with CsCl, but not during chelation of intracellular calcium with BAPTA. During blockage of gap junction coupling with high intracellular CaCl(2) or extracellular carbenoxolone the EtOH effect persisted but was reduced. Interestingly, EtOH effects were largely irreversible when gap junctions were open, but were fully reversible when gap junctions were closed. Ethanol also reduced the spread to other cells of Lucifer Yellow dye from individual glia filled via the patch pipette. These data suggest that EtOH inhibits a calcium-insensitive potassium channel, most likely a passive potassium channel, but also affects gap junction coupling in a way that is sustained after ethanol withdrawal. Astrocytes play a critical role in brain potassium homeostasis, and therefore EtOH effects on astrocytic function could influence neuronal activity.

1-Octanol↗

PubMed2006

Ethanol inhibits brain-derived neurotrophic factor stimulation of extracellular signal-regulated/mitogen-activated protein kinase in cerebellar granule cells.

Brain-derived neurotrophic factor (BDNF) has emerged as a prominent mediator of neuronal development and synaptic plasticity. BDNF activates multiple signal transduction cascades that regulate cellular function through phosphorylation, transcription, and translation. Ethanol is known to inhibit neurotrophin signaling, but a thorough pharmacological analysis of the effect of ethanol on BDNF signaling in developing neurons has not been performed. These experiments were undertaken to determine the interactions between membrane depolarization, BDNF concentration, and ethanol concentration on extracellular signal-regulated protein kinase (ERK) activation in neurons. We examined cerebellar granule cells grown under physiological (5mM) or elevated (25mM) potassium culture conditions after 3 days in vitro. BDNF-stimulated ERK phosphorylation (pERK) within 10min and supported stimulation from 20 to 60min. Ethanol decreased basal pERK and reduced the magnitude of BDNF stimulation of ERK under both conditions. The NMDA receptor antagonist 2-amino-5-phosphonovalerate did not effect basal pERK or inhibit BDNF stimulation of ERK, suggesting that NMDA receptors do not modulate BDNF stimulation of ERK in short-term cultures. These data characterize the pharmacological effects of ethanol on growth factor signaling and provide the basis of a model for further characterization of the biochemical mechanisms of ERK inhibition by ethanol. Perturbation of BDNF signal transduction by ethanol may underlie some of the cognitive deficits and developmental abnormalities resulting from ethanol exposure.

Animals↗

PubMed2006

Frequency-specific and D2 receptor-mediated inhibition of glutamate release by retrograde endocannabinoid signaling.

The mechanisms underlying modulation of corticostriatal synaptic transmission by D2-like receptors (D2Rs) have been controversial. A recent study suggested that D2Rs inhibit glutamate release at this synapse, but only during high-frequency synaptic activation. Because the release of postsynaptic endocannabinoids (eCBs), which act as retrograde messengers to inhibit presynaptic glutamate release, can be triggered by D2R activation and intense synaptic activation, such a mechanism could mediate dopaminergic modulation of corticostriatal transmission. Here, we show that D2R activation reduces excitatory transmission onto striatal medium spiny neurons at a stimulation frequency of 20 Hz but not at 1 Hz. This form of inhibition requires CB1 receptor activation, as evidenced by the fact that it is blocked by AM251 [N-(piperidin-1-yl)-1-(2,4-dichlorophenyl)-5-(4-chlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide], a CB1 antagonist, and is absent in CB1 knockout mice. It is also blocked by postsynaptic intracellular calcium chelation, by group I metabotropic glutamate receptor antagonism, and by inhibition of postsynaptic phospholipase C. These results demonstrate a previously unrecognized role for retrograde eCB signaling in reversible and frequency-specific inhibition of glutamate release by the activation of striatal D2Rs.

Animals↗

PubMed2006

Dopaminergic control of corticostriatal long-term synaptic depression in medium spiny neurons is mediated by cholinergic interneurons.

Long-term depression (LTD) of the synapse formed between cortical pyramidal neurons and striatal medium spiny neurons is central to many theories of motor plasticity and associative learning. The induction of LTD at this synapse is thought to depend upon D(2) dopamine receptors localized in the postsynaptic membrane. If this were true, LTD should be inducible in neurons from only one of the two projection systems of the striatum. Using transgenic mice in which neurons that contribute to these two systems are labeled, we show that this is not the case. Rather, in both cell types, the D(2) receptor dependence of LTD induction reflects the need to lower M(1) muscarinic receptor activity-a goal accomplished by D(2) receptors on cholinergic interneurons. In addition to reconciling discordant tracts of the striatal literature, these findings point to cholinergic interneurons as key mediators of dopamine-dependent striatal plasticity and learning.

Acetylcholine↗

PubMed2006

Ethanol potentiates GABAergic synaptic transmission in a postsynaptic neuron/synaptic bouton preparation from basolateral amygdala.

Interactions between ethanol and synaptic transmission mediated by gamma -amino-N-butyric acid (GABA) have been suggested to contribute to alcohol intoxication. Ethanol effects on postsynaptic GABAA receptors have been the major focus of this line of research. There is increasing evidence that ethanol potentiation of GABAergic transmission involves increased GABA release from presynaptic terminals. In the present study, a mechanically isolated neuron/bouton preparation from the basolateral amygdala was used to examine the effects of ethanol on spontaneous GABAergic synaptic currents elicited by GABA release from the presynaptic terminals. We found that ethanol application produced a rapid increase in the frequency of spontaneous GABAergic synaptic currents. An acute tolerance to ethanol was also observed, and this tolerance involved GABAB receptor activation. The ethanol-induced potentiation did not involve alterations in the function of postsynaptic GABAA receptors and was independent of presynaptic action potential firing. These findings indicate that ethanol potentiates GABA release, most likely via a direct action on presynaptic boutons.

Action Potentials↗

PubMed2006

Temperature: an important experimental variable in studying PKC modulation of ligand-gated ion channels.

Amphibian oocyte and mammalian heterologous expression systems are often used to investigate the function of recombinant ion channels using electrophysiological techniques. Although both systems have yielded important information, the results obtained in these systems are sometimes conflicting. Oocytes and mammalian cells differ in their physiological temperature requirements. While room temperature is within the physiological temperature range for oocytes, this temperature is far below that required by mammalian cells. Since electrophysiological studies are often performed in both oocytes and mammalian cells at room temperature, we sought to determine if recording temperature could be a factor in some disparate results obtained in these cell types. For these studies, we examined phorbol ester modulation of GABA(A) and glycine receptors. Consistent with the literature, at room temperature, PMA (phorbol 12-myristate 13-acetate) produced a large reproducible decrease in the peak amplitude of GABA and glycine-gated currents in Xenopus oocytes. In contrast, PMA was ineffective in modulating these heterologously expressed receptors at room temperature in human embryonic kidney (HEK) 293 cells. However, when electrophysiological experiments were performed at 35 degrees C in HEK 293 cells, PMA decreased the function of these receptors. Our results indicate that the temperature at which electrophysiological studies are conducted is an important experimental variable. To determine the extent to which electrophysiological recordings are performed at physiological temperatures in HEK 293 cells, a PubMed search was conducted using the search terms "patch clamp" and "HEK" for the years 2003-2004. This search revealed that only 15% of the patch clamp studies were reported to have been conducted in the temperature range of 32-37 degrees C. The results of our study indicate that temperature is an important experimental variable that requires rational consideration in the design of electrophysiological experiments.

Analysis of Variance↗

PubMed2006

Mixing proteomics and alcohol.

Techniques for high-throughput measurement of protein expression and posttranslational modification are just beginning to be applied to alcohol research. Studies using this proteomic approach to examine tissue from alcoholic humans and alcohol-exposed nonhuman primates have appeared in the last year. In the present issue of Alcohol, Bell et al. present the first such analysis of brain protein expression in relation to alcohol drinking in rodents. This study found changes in several interesting classes of proteins, including molecules involved in vesicular neurotransmitter release, cellular metabolism, and cell structure. These new and exciting findings are discussed in relation to the proteomics studies in primates, and the future of proteomics in the alcohol research field.

Alcohol Drinking↗

PubMed2006

Mutations of L293 in transmembrane two of the mouse 5-hydroxytryptamine3A receptor alter gating and alcohol modulatory actions.

1 The goal of this study was to determine whether mutations of L293 at the 15' position of TM2 in the 5-HT(3A) receptor alter macroscopic current kinetics, and if these changes could account for alterations in alcohol modulation. Receptor function was assessed in Xenopus oocytes under voltage-clamp and in HEK293 cells with whole-cell patch-clamp recording and rapid drug application. 2 Examination of responses of L293C and L293S receptors to agonist alone revealed enhanced activation, deactivation, and desensitization rates relative to the wild-type receptor. The L293G mutation produced marked slowing of deactivation and desensitization rates. Increased potency of 5-HT and increased efficacy of the partial agonist, DA, was also observed in these mutant receptors. 3 Ethanol and trichloroethanol (TCEt) enhancement of receptor function was reduced or eliminated in receptors containing L293 mutations to C, G, or S. The L293I mutant receptor retained ethanol and TCEt sensitivity. Ethanol and TCEt enhanced activation rate in the wild-type, but not the L293G and L293S receptors. No relationship was observed between any physicochemical property of the substituted amino acids and the change in alcohol potentiation of function. 4 The changes in receptor-channel properties in the mutant receptors support the idea that the L293 residue has important roles in channel gating. Our findings indicate that loss of allosteric modulation by alcohols is not related in any simple way to changes in channel kinetic properties brought about by L293 mutants. We did not observe any evidence that L293 is part of an alcohol binding site.

Allosteric Regulation↗

PubMed2006

Effects of ethanol on adenosine 5'-triphosphate-gated purinergic and 5-hydroxytryptamine receptors.

This report of the proceedings of a symposium presented at the 2005 annual meeting of the Research Society on Alcoholism highlights the actions of ethanol on purinergic (P2XRs) and 5-hydroxytryptamine3 (5-HT3Rs) receptors. Both P2XRs and 5-HT3Rs, are modulated by pharmacologically relevant concentrations of ethanol, with inhibition or stimulation of P2XR subtypes and stimulation of 5-HT3Rs, respectively. With regard to ethanol-modulatory actions, these 2 distinctly different receptor classes have been studied to a much lesser extent than other LGICs. The organizers and chairs were Daryl L. Davies and Tina K. Machu. John J. Woodward discusses the molecular pharmacology and physiology of P2XRs and 5-HT3Rs and sets the stage for a detailed investigation into the ethanol sensitivity of these channels by the invited speakers. Daryl L. Davies discusses the results from recent electrophysiological studies conducted in his and Dr. Woodward's laboratories, highlighting the actions of ethanol on P2XR subtypes. Jiang-Hong Ye discusses results from recent studies using loose-patch and whole-cell recordings on purinergic receptors expressed on neurons from the ventral tegmental area (VTA) in rats. Tina K. Machu discusses electrophysiological studies conducted in her and Dr. David Lovinger's laboratories on nonpore lining residues of the second transmembrane domain (TM2) of the 5-HT3A receptor. Li Zhang presents data demonstrating that F-actin cytoskeletons play a critical role in 5-HT3 receptor clustering in hippocampal neurons. Collectively, the presentations provided strong evidence that P2X and 5-HT3 receptors are important targets for ethanol action.

Animals↗

PubMed2005

Activation of NR2A-containing NMDA receptors is not obligatory for NMDA receptor-dependent long-term potentiation.

Activation of NMDA receptors (NMDARs) within the CNS represents a major signal for persistent alterations in glutamatergic signaling, such as long-term potentiation (LTP) and long-term depression. NMDARs are composed of a combination of NR1 and NR2 subunits, with distinct NR2 subunits imparting distinct characteristics on the receptor. One particular NR2 subunit, NR2A (NRepsilon1), has been proposed to play an integral role in LTP induction in the hippocampus and cortex. Here, we report studies investigating the role of NR2A in LTP induction in the dorsolateral bed nucleus of the stria terminalis (dlBNST). The putative NR2A-specific inhibitor NVP-AAM077 (AAM077) has been used previously to demonstrate the dependence of cortical and hippocampal LTP on NMDARs containing NR2A subunits. We report here the same sensitivity of LTP to pretreatment with AAM077 (0.4 microm) in the dlBNST. However, inconsistent with the conclusion that LTP in the dlBNST is NR2A dependent, we see intact LTP in the dlBNST of NR2A knock-out mice. Because we also see blockade of this dlBNST LTP in NR2A knock-out mice after pretreatment with AAM077, we conclude that the antagonist is targeting non-NR2A subunit-containing receptors. Using a variety of cultured cell types, we find that AAM077 (0.4 microm) can attenuate transmission of NR2B subunit-containing NMDARs when preapplied rather than coapplied with an agonist. Therefore, we conclude that NR2A is not obligatory for the induction of LTP in the dlBNST. Furthermore, our data demonstrate that care must be exercised in the interpretation of data generated with AAM077 when the compound is applied before an agonist.

Animals↗

PubMed2005

Role of aspartate 298 in mouse 5-HT3A receptor gating and modulation by extracellular Ca2+.

The TM2-TM3 extracellular loop is critical for activation of the Cys-loop family of ligand-gated ion channels. The contribution of aspartate 298 (D298), an amino acid that links the transmembrane domain 2 (TM2) to the TM2-TM3 loop, in mouse 5-hydroxytryptamine(3A) (5-HT(3A)) receptor function was probed with site-directed mutagenesis in the present study. This negatively charged residue was replaced with an alanine to neutralize the charge, with a glutamate to conserve the charge, or with an arginine to reverse the charge. Human embryonic kidney 293 (HEK 293) cells transfected with the wild-type and mutant receptors were studied by combining whole-cell patch-clamp recording with fast agonist application. The D-->A or D-->R mutations resulted in a receptor with reduced 5-HT potency, and accelerated kinetics of desensitization and deactivation. In addition, the efficacy of partial agonists was reduced by the D-->A mutation. The D-->E mutation produced a receptor with properties similar to those of the wild-type receptor. In addition, the potential role of this residue in modulation of the receptor by extracellular calcium ([Ca(2)(+)](o)) was investigated. Increasing [Ca(2)(+)](o) inhibited 5-HT-activated currents and altered receptor kinetics in a similar manner in the wild-type and D298E receptors, and this alteration was eliminated by the D-->A and D-->R mutations. Our data suggest that the charge at D298 participates in transitions between functional states of the 5-HT(3A) receptor, and provide evidence that the charge of the side-chain at residue D298 contributes to channel gating kinetics and is crucial for Ca(2)(+) modulation.

Amino Acid Sequence↗

PubMed2005

Retrograde endocannabinoid signaling in a postsynaptic neuron/synaptic bouton preparation from basolateral amygdala.

Retrograde synaptic signaling by endogenous cannabinoids (endocannabinoids) is a recently discovered form of neuromodulation in the brain. In the basolateral amygdala (BLA), endocannabinoid signaling has been implicated in learning and memory, specifically in extinction of aversive memories. To examine retrograde endocannabinoid signaling in this brain region, BLA neurons were freshly isolated using an enzyme-free procedure. These isolated neurons retain attached functional excitatory and inhibitory synaptic boutons. Spontaneous GABAergic IPSCs (sIPSCs) were isolated from these freshly isolated neurons and a 4 s step of depolarization from -60 to 0 mV produced suppression of sIPSC frequency and amplitude. A similar depolarization-induced suppression of inhibition (DSI) was observed in neurons in BLA slices. DSI in the single-cell preparation was abolished by the CB1 receptor antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide, and DSI duration was shortened in the presence of 2-methyl-6-(phenylethynyl) pyridine, an mGluR5 (metabotropic glutamate receptor 5) antagonist. The initial decrease in sIPSCs induced by the DSI procedure was greatly attenuated in recordings with 20 mm BAPTA containing postsynaptic internal solution, but a delayed-onset decrease was observed under this recording condition. A CB1 agonist decreased sIPSC frequency and amplitude, whereas CB1 antagonists increased these responses. The antagonist-induced increase was abolished in 20 mm BAPTA-filled cells. These data provide solid evidence for retrograde endocannabinoid signaling in the BLA and also indicate that this retrograde signaling requires only a postsynaptic neuron and attached synaptic boutons.

Amygdala↗

PubMed2005

CaMKIIalpha enhances the desensitization of NR2B-containing NMDA receptors by an autophosphorylation-dependent mechanism.

Long-term potentiation or depression of synaptic function often requires Ca2+ influx via NMDA-type glutamate receptors (NMDARs) and changes in the autophosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) at Thr286. Autophosphorylated CaMKII binds directly to NMDAR subunits, co-localizes with NMDARs in the postsynaptic density, and phosphorylates NR2B subunits at Ser1303. Here, we demonstrate that CaMKIIalpha enhances the extent and/or rate of desensitization of NMDA-induced macroscopic currents in HEK293 cells co-expressing NR2B with either the NR1(011) or NR1(101) splice variants, without significantly changing other current parameters. In contrast, the extent of desensitization of NMDARs containing NR2A in place of NR2B is significantly decreased by co-expression of CaMKIIalpha. Kinases harboring K42R (inactive kinase) or T286A (autophosphorylation-deficient) mutations are defective in enhancing the desensitization of NR1/NR2B channels. In addition, the CaMKII-dependent enhancement of NR1/NR2B channel desensitization is abrogated by intracellular loading with BAPTA. These data suggest a novel mechanism for Ca2+-dependent negative-feedback regulation of NR2B-containing NMDARs in a CaMKII activity- and autophosphorylation-dependent manner that may modulate NMDAR-mediated synaptic plasticity.

Calcium↗

PubMed2005

Laboratory models of alcoholism: treatment target identification and insight into mechanisms.

Laboratory models, including animal tissues and live animals, have proven useful for discovery of molecular targets of alcohol action as well as for characterization of genetic and environmental factors that influence alcohol's neural actions. Here we consider strengths and weaknesses of laboratory models used in alcohol research and analyze the limitations of using animals to model a complex human disease. We describe targets for the neural actions of alcohol, and we review studies in which animal models were used to examine excessive alcohol drinking and to discover genes that may contribute to risk for alcoholism. Despite some limitations of the laboratory models used in alcohol research, these experimental approaches are likely to contribute to the development of new therapies for alcohol abuse and alcoholism.

Alcoholism↗

PubMed2004

Induction of striatal long-term synaptic depression by moderate frequency activation of cortical afferents in rat.

The striatum regulates motor output, and it is thought that changes in the synaptic efficacy of inputs to the striatum contribute to motor learning and habit formation. Previously, several laboratories have observed that brief high frequency stimulation (HFS) of cortical afferents innervating the dorsolateral striatum induces a long-term decrease in synaptic efficacy called long-term depression (LTD). We recently showed that HFS-induced striatal LTD requires retrograde signalling involving postsynaptic release of endocannabinoids and activation of presynaptic CB1 cannabinoid receptors. In the present study we have employed whole-cell recording in brain slices to examine a new form of LTD at corticostriatal synapses that can be induced by a 10 Hz, 5 min train. The decrease in synaptic efficacy is associated with a decrease in presynaptic release probability, as demonstrated by a decrease in frequency but not amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) and an increase in the paired pulse ratio (PPR). This form of LTD is blocked by antagonists for CB1 and D2 dopamine receptors and impaired by blockers of L-type calcium channels. However, 10 Hz-induced LTD does not depend on postsynaptic depolarization, unlike HFS-induced LTD. Furthermore, this new form of LTD is not prevented by treatments known to block HFS-induced LTD, including antagonism of metabotropic glutamate receptors (mGluRs), chelation of postsynaptic calcium, or intracellular application of an anandamide membrane transport inhibitor (VDM11). From these findings it is not clear that the endocannabinoid responsible for this form of LTD acts in a retrograde fashion, and the cellular source of endocannabinoid necessary for 10 Hz-induced LTD is as yet unknown. Our results demonstrate that a prolonged moderate frequency train induces cannabinoid-dependent LTD, further supporting the idea that endocannabinoids play a prominent role in the regulation of long-lasting changes in striatal output.

Animals↗

PubMed2004

Disruption of endocannabinoid release and striatal long-term depression by postsynaptic blockade of endocannabinoid membrane transport.

Activation of the CB1 cannabinoid receptor inhibits neurotransmission at numerous synapses in the brain. Indeed, CB1 is essential for certain types of both short- and long-term synaptic depression. It was demonstrated recently that CB1 is critical for activity-dependent long-term depression (LTD) at glutamatergic corticostriatal synapses in acute brain slice preparations. Here, we show that CB1 activation is necessary, but not solely sufficient, for induction of LTD and that the requisite signaling by endocannabinoids (eCBs) occurs during a time window limited to the first few minutes after high-frequency stimulation delivery. In addition, we have applied intracellularly anandamide membrane transporter inhibitors to provide novel evidence that postsynaptic transport mechanisms are responsible for the release of eCBs from striatal medium spiny neurons. These findings shed new light on the mechanisms by which transient eCB formation participates in the induction of long-lasting changes in synaptic efficacy that could contribute to brain information storage.

Animals↗