Excitatory Glutamatergic Transmission Research Articles

The Influence of Cold Temperature on Cellular Excitability of Hippocampal Networks

The hippocampus plays an important role in short term memory, learning and spatial navigation. A characteristic feature of the hippocampal region is its expression of different electrical population rhythms and activities during different brain states. Physiological fluctuations in brain temperature affect the activity patterns in hippocampus, but the underlying cellular mechanisms are poorly understood. In this work, we investigated the thermal modulation of hippocampal activity at the cellular network level. Primary cell cultures of mouse E17 hippocampus displayed robust network activation upon light cooling of the extracellular solution from baseline physiological temperatures. The activity generated was dependent on action potential firing and excitatory glutamatergic synaptic transmission. Involvement of thermosensitive channels from the transient receptor potential (TRP) family in network activation by temperature changes was ruled out, whereas pharmacological and immunochemical experiments strongly pointed towards the involvement of temperature-sensitive two-pore-domain potassium channels (K2P), TREK/TRAAK family. In hippocampal slices we could show an increase in evoked and spontaneous synaptic activity produced by mild cooling in the physiological range that was prevented by chloroform, a K2P channel opener. We propose that cold-induced closure of background TREK/TRAAK family channels increases the excitability of some hippocampal neurons, acting as a temperature-sensitive gate of network activation. Our findings in the hippocampus open the possibility that small temperature variations in the brain in vivo, associated with metabolism or blood flow oscillations, act as a switch mechanism of neuronal activity and determination of firing patterns through regulation of thermosensitive background potassium channel activity.

Of apples and oranges: GABA and glutamate transmission in neurones of the nucleus tractus solitarii could not be more different

The nucleus tractus solitarii (NTS) is the principal visceral sensory nucleus in the brain and comprises neurochemically and biophysically distinct neurons located in the dorsomedial medulla oblongata. The NTS conveys information from the gustatory, cardiorespiratory, oesophageal and subdiaphragmatic gastrointestinal visceral systems that is subsequently assimilated with homeostatic signals arriving from an impressive network of connections from other integrative centres of the pons, diencephalon and forebrain. Myelinated A-type and unmyelinated C-type sensory afferent fibres from the trigeminal, facial, glossopharyngeal and vagus nerve form the tractus solitarius (TS) and their terminals overlap along the rostro-caudal extent of the NTS. Regardless of their origin or type, though, afferent fibres entering the brainstem activate NTS neurones via the release of excitatory neurotransmitters, mainly glutamate (Andresen & Kunze, 1994; Baptista et al. 2005). Perhaps the most prominent neurochemical phenotypes encountered in the NTS are GABAergic and glutamatergic, which provide postsynaptic inhibitory and excitatory influences to adjacent motor nuclei such as the dorsal motor nucleus of the vagus, the nucleus ambiguus and the ventrolateral medulla. An impressive amount of work has been conducted in recent times by Andresen's group to characterize the glutamatergic transmission from the tractus solitarius onto NTS neurones. This group has used sophisticated electrophysiological analyses to demonstrate that glutamatergic excitatory transmission is highly effective, has an almost unfailing unitary transmission from primary sensory fibres, and can be associated with forms of synaptic plasticity that are unique to autonomic regulation (Peters et al. 2010). Earlier work by Mifflin (Mifflin, 1996; Mifflin & Felder, 1988) and Smith (Smith et al. 1998), however, showed that TS stimulation can evoke a sequence of excitatory responses that were followed by inhibitory currents, indicating the possible presence of a local GABAergic interneurone. This inhibitory interneurone, however, has not yet been demonstrated with anatomical tools. (Note that the presence of GABAergic neurons within the NTS is not in doubt. What remains to be confirmed, however, is the anatomical proof of their interneurone status, i.e. a neurone that does not project beyond the NTS itself.) The publication by McDougall and Andresen (2012) in this issue of The Journal of Physiology utilises an isolated horizontal brainstem slice in which TS fibres can be stimulated to evoke unitary currents in second order NTS neurones; these currents can then be identified as either mono- or polysynaptic. The authors showed that, in contrast to glutamatergic currents, monosynaptic GABAergic currents are evoked by focal stimulation disproportionately rarely and only following a stimulus that was located in close proximity to the recorded neurone. In addition to the high failure rate, large amplitude variability, low calcium sensitivity and modest frequency-dependent depression, the monosynaptic nature of these GABAergic currents was confirmed by a low jitter (albeit greater than that of glutamatergic synapses) and lack of sensitivity to glutamatergic receptor blockade. Overall, these data suggest rather weak GABAergic transmission whose efficacy must rely on converging activation of inhibitory fibres. These characteristics suggest strongly that the GABA release properties are fundamentally different from the powerful and synaptically reliable glutamatergic inputs impinging onto NTS neurones. When taken together with previous observations reporting that each TS-driven glutamatergic input targets individual NTS neurons selectively, and that the physiological and pharmacological responses of NTS neurones are target-coded (Bailey et al. 2006; Browning et al. 2011; Jin et al. 2004), the overall picture of the NTS strengthens the image of a hyperorganized nucleus with ‘task matching’ capabilities conferring an ability to cope with widely variable demands, both in terms of timing as well as duration and modulation of a response. Furthermore, this type of cellular organization within the NTS should force us, once again, to be extremely cautious in the often simplistic physiological interpretation of observations, often driven by high intensity stimuli, that analyse a single outcome such as cFos expression or calcium oscillations, which provide limited information regarding the excitability of neurones and provide no information at all regarding inhibitory transmission. Several questions remain to be answered before one can elucidate the role of these synaptic connections. For example, what are the inputs that activate these second order GABAergic neurones? What is their origin? Are they anatomically defined interneurones, or do they project beyond the NTS? Are they related to a particular pathway? Do they have a specific physiological role or are they ‘simply’ a low pass filter for excitatory TS-driven inputs?

Effect of Xenon on Excitatory and Inhibitory Transmission in Rat Spinal Ventral Horn Neurons

The minimum alveolar concentration is determined in the spinal cord rather than in the brain. Xenon inhibits glutamatergic excitatory synaptic transmission in the dorsal horn neurons. However, its actions in the ventral horn neurons have not been investigated. The effects of 50 or 75% xenon on excitatory and inhibitory synaptic transmission were examined in the spinal lamina IX neurons of neonatal rats by using a whole cell patch clamp technique. Fifty percent xenon inhibited the α-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid-induced currents (amplitudes = 72 ± 9% and integrated area = 73 ± 13% of the control values), and α-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid receptor-mediated electrically evoked excitatory postsynaptic currents (amplitudes = 69 ± 13% of the control values). Seventy-five percent xenon similarly inhibited α-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid-induced currents. However, xenon had no effect on the N-methyl-D-aspartate-induced currents or N-methyl-D-aspartate receptor-mediated electrically evoked excitatory postsynaptic currents. Xenon decreased the amplitude, but not the frequency, of miniature excitatory postsynaptic currents. There were no discernible effects on the currents induced by γ-aminobutyric acid or glycine or on miniature inhibitory postsynaptic currents. Xenon inhibits α-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid receptor-mediated glutamatergic excitatory transmission in the spinal lamina IX neurons via a postsynaptic mechanism. In contrast, there are no substantial effects on N-methyl-D-aspartate receptor-mediated or inhibitory synaptic transmission. The suppressive effects on excitatory synaptic transmission in the ventral horn neurons partly account for the mechanism behind xenon's ability to produce immobility in response to noxious stimuli and to determine the minimum alveolar concentration.

Presynaptic enhancement by eugenol of spontaneous excitatory transmission in rat spinal substantia gelatinosa neurons is mediated by transient receptor potential A1 channels

Eugenol, which is contained in several plants including clove, has been widely used as an analgesic and anti-inflammatory drug in the dental clinic. Eugenol also has anesthetic effects and produces sedation and the reduction of convulsion threshold. These benefits have been partly attributed to the effects of eugenol on neural tissues, such as inhibition of voltage-gated ion channels. As expected from the fact that eugenol is a vanilloid compound, this drug activates transient receptor potential (TRP) V1 channels in the peripheral nervous system (PNS). Although eugenol affects synaptic transmission in the central nervous system (CNS), this has not yet been fully examined. We investigated how eugenol affects spontaneous glutamatergic excitatory transmission in substantia gelatinosa (SG; lamina II of Rexed) neurons of adult rat spinal cord slices by use of the blind whole-cell patch-clamp technique. Bath-applied eugenol reversibly enhanced spontaneous excitatory transmission and produced an outward current at −70 mV in SG neurons. The former action was due to a large increase in the frequency of spontaneous excitatory postsynaptic current (sEPSC) with a small increase in the amplitude. These actions of eugenol were seen by its repeated application and resistant to a voltage-gated Na+ channel blocker tetrodotoxin. The effect of eugenol on sEPSC frequency was concentration-dependent with an EC50 value of 3.8 mM and unaffected by a TRPV1 antagonist capsazepine, whereas inhibited by a nonspecific TRP antagonist ruthenium red and a TRPA1 antagonist HC-030031. On the other hand, the eugenol-induced outward current was not affected by these TRP antagonists. It is concluded that eugenol activates TRPA1 channels in the SG, leading to an increase in the spontaneous release of l-glutamate to SG neurons, and that eugenol also produces a membrane hyperpolarization that is not mediated by TRP channels. Eugenol is suggested to activate different types of TRP channel between the PNS and CNS.

Cytokines and brain excitability

Cytokines are molecules secreted by peripheral immune cells, microglia, astrocytes and neurons in the central nervous system. Peripheral or central inflammation is characterized by an upregulation of cytokines and their receptors in the brain. Emerging evidence indicates that pro-inflammatory cytokines modulate brain excitability. Findings from both the clinical literature and from in vivo and in vitro laboratory studies suggest that cytokines can increase seizure susceptibility and may be involved in epileptogenesis. Cellular mechanisms that underlie these effects include upregulation of excitatory glutamatergic transmission and downregulation of inhibitory GABAergic transmission.

Purinergic modulation of the excitatory synaptic input onto rat striatal neurons

There is no in situ evidence hitherto for a modulation by ATP of the glutamatergic excitatory transmission onto medium spiny neurons (MSNs) in the rat striatum. In order to resolve this question, we used the patch-clamp technique in brain slice preparations to record excitatory postsynaptic currents (EPSCs) evoked by intrastriatal electrical stimulation and applied N-methyl-d-aspartate (NMDA) or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) to activate transmembrane currents of MSNs. In the absence of external Mg2+, ATP caused a higher maximum inhibition of the EPSCs than adenosine. Only P1 (A1), but not P2 receptor antagonists interfered with the effects of both ATP and adenosine. Moreover, A1 receptor antagonists were less potent in blocking the inhibition by ATP than that by adenosine. Eventually, adenosine deaminase (ADA) almost abolished the adenosine-induced inhibition, but only moderately decreased the ATP-induced inhibition. Antagonists of A1 receptors (but not of P2 receptors) counteracted the depression by ATP of the current responses to exogenous NMDA, without altering those to AMPA. It is suggested that ATP indirectly, via its degradation product adenosine, stimulates presynaptic inhibitory A1 receptors situated at glutamatergic nerve terminals of striatal afferents; these nerve terminals are devoid of P2 receptors. However, ATP, in contrast to adenosine, also activates postsynaptic A1 receptors at the MSN neurons themselves. The resulting negative interaction with NMDA receptors requires localized extracellular catabolism of ATP by ectonucleotidases.

GABAA receptors facilitate spontaneous glutamate release in rat periaqueductal gray neurons

The functional role of presynaptic γ-aminobutyric acid (GABA)(A) receptors in excitatory glutamatergic transmission was examined in rat periaqueductal gray neurons recorded using a conventional whole-cell patch clamp technique. Muscimol, a GABA(A) receptor agonist, significantly increased the frequency of spontaneous excitatory postsynaptic currents without affecting their amplitude, and this effect was completely blocked by the selective GABA(A) receptor antagonist. The muscimol-induced facilitation of spontaneous excitatory postsynaptic current frequency disappeared either in the presence of tetrodotoxin or Cd. The results suggest that the activation of presynaptic GABA(A) receptors directly depolarizes glutamatergic terminals resulting in the facilitation of spontaneous glutamate release, and that presynaptic GABA(A) receptors play an important role in the regulation of various physiological functions mediated by the periaqueductal gray.

Endogenous brain-derived neurotrophic factor in the nucleus tractus solitarius tonically regulates synaptic and autonomic function.

Brain-derived neurotrophic factor (BDNF) and its receptor, TrkB, are highly expressed in the nucleus tractus solitarius (nTS), the principal target of cardiovascular primary afferent input to the brainstem. However, little is known about the role of BDNF signaling in nTS in cardiovascular homeostasis. We examined whether BDNF in nTS modulates cardiovascular function in vivo and regulates synaptic and/or neuronal activity in isolated brainstem slices. Microinjection of BDNF into the rat medial nTS (mnTS), a region critical for baroreflex control of sympathetic outflow, produced dose-dependent increases in mean arterial pressure (MAP), heart rate (HR), and lumbar sympathetic nerve activity (LSNA) that were blocked by the tyrosine kinase inhibitor K252a. In contrast, immunoneutralization of endogenous BDNF (anti-BDNF), or microinjection of K252a alone, decreased MAP, HR, and LSNA. The effects of anti-BDNF were abolished by blockade of ionotropic glutamate receptors, indicating a role for glutamate signaling in the response to BDNF. In vitro, BDNF reduced the amplitude of miniature EPSCs as well as solitary tract (TS) evoked EPSC amplitude and action potential discharge (APD) in second-order nTS neurons. BDNF effects on EPSCs were independent of GABAergic signaling and abolished by AMPA receptor blockade. In contrast, K252a increased spontaneous EPSC frequency and TS evoked EPSC amplitude. BDNF also attenuated APD evoked by injection of depolarizing current into second-order neurons, indicating reduced intrinsic neuronal excitability. Our data demonstrate that BDNF signaling in mnTS plays a tonic role in regulating cardiovascular function, likely via modulation of primary afferent glutamatergic excitatory transmission and neural activity.

Glutamatergic spontaneous excitatory transmission enhancement produced by eugenol is due to TRPA1 channel activation in the rat spinal dorsal horn

Glutamatergic spontaneous excitatory transmission enhancement produced by eugenol is due to TRPA1 channel activation in the rat spinal dorsal horn

Cortical cultures coupled to Micro-Electrode Arrays: A novel approach to perform in vitro excitotoxicity testing

In vitro neuronal cultures exhibit spontaneous electrophysiological activity that can be modulated by chemical stimulation and can be monitored over time by using Micro-Electrode Arrays (MEAs), devices composed by a glass substrate and metal electrodes. Dissociated networks respond to transmitters, their blockers and many other pharmacological substances, including neurotoxic compounds. In this paper we present results related to the effects, both acute (i.e. 1hour after the treatment) and chronic (3days after the treatment), of increasing glutamatergic transmission induced by the application of rising concentrations of glutamate and its agonists (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid — AMPA, N-methyl-D-aspartate — NMDA and AMPA together with cyclothiazide — CTZ). Increase of available glutamate was obtained in two ways: 1) by direct application of exogenous glutamate and 2) by inhibiting the clearance of the endogenously released glutamate through DL-threo-β-benzyloxyaspartate (TBOA). Our findings show that fine modulations (i.e. low concentrations of drug) of the excitatory synaptic transmission are reflected in the electrophysiological activation of the network, while intervention leading to excessive direct stimulation of glutamatergic pathways (i.e. medium and high concentrations of drug) results in the abolishment of the electrophysiological activity and eventually cell death. The results obtained by means of the MEA recordings have been compared to the analysis of cell viability to confirm the excitotoxic effect of the applied drug. In conclusion, our study demonstrates that MEA-coupled cortical networks are very sensitive to pharmacological manipulation of the excitatory ionotropic glutamatergic transmission and might provide sensitive endpoints to detect acute and chronic neurotoxic effects of chemicals and drugs for predictive toxicity testing.

Zaltoprofen Inhibits Bradykinin-Mediated Enhancement of Glutamate Receptor Activity in Substantia Gelatinosa Neurons

Zaltoprofen, a propionic acid derivative of nonsteroidal anti-inflammatory drugs, has been proposed to inhibit the nociception mediated by bradykinin. Here, I attempted to clarify the molecular mechanisms underlying the blocking effect of zaltoprofen on bradykinin-mediated enhancement of excitatory glutamatergic transmission in the superficial dorsal horn of the spinal cord. The effects of zaltoprofen on the response to exogenous administration of α-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid (AMPA) currents were examined in lamina II neurons of adult rat spinal cord slices using the whole-cell patch-clamp technique. AMPA currents were significantly enhanced by preapplication of bradykinin (10 μM). However, zaltoprofen (1, 10 μM) and a nonselective cyclooxygenase (COX)-1 and COX-2 inhibitor, ibuprofen, blocked the bradykinin-mediated enhancing effect. The inhibitory effect of ibuprofen, but not zaltoprofen, was removed by adding prostaglandin E(2). Furthermore, the inhibitory effect of zaltoprofen was removed in the presence of protein kinase C (PKC) activator, whereas the effect of zaltoprofen was still present in the presence of phospholipase C activator. These findings suggest that the antinociceptive effect of zaltoprofen may block the augmenting effect of bradykinin on AMPA currents through inhibition of protein kinase C activation, without affecting COX in the superficial dorsal horn.

NMDA receptor activation enhances inhibitory GABAergic transmission onto hippocampal pyramidal neurons via presynaptic and postsynaptic mechanisms

N-methyl-D-aspartate (NMDA) receptors (NMDARs) are implicated in synaptic plasticity and modulation of glutamatergic excitatory transmission. Effect of NMDAR activation on inhibitory GABAergic transmission remains largely unknown. Here, we report that a brief application of NMDA could induce two distinct actions in CA1 pyramidal neurons in mouse hippocampal slices: 1) an inward current attributed to activation of postsynaptic NMDARs; and 2) fast phasic synaptic currents, namely spontaneous inhibitory postsynaptic currents (sIPSCs), mediated by GABA(A) receptors in pyramidal neurons. The mean amplitude of sIPSCs was also increased by NMDA. This profound increase in the sIPSC frequency and amplitude was markedly suppressed by the sodium channel blocker TTX, whereas the frequency and mean amplitude of miniature IPSCs were not significantly affected by NMDA, suggesting that NMDA elicits repetitive firing in GABAergic interneurons, thereby leading to GABA release from multiple synaptic sites of single GABAergic axons. We found that the NMDAR open-channel blocker MK-801 injected into recorded pyramidal neurons suppressed the NMDA-induced increase of sIPSCs, which raises the possibility that the firing of interneurons may not be the sole factor and certain retrograde messengers may also be involved in the NMDA-mediated enhancement of GABAergic transmission. Our results from pharmacological tests suggest that the nitric oxide signaling pathway is mobilized by NMDAR activation in CA1 pyramidal neurons, which in turn retrogradely facilitates GABA release from the presynaptic terminals. Thus NMDARs at glutamatergic synapses on both CA1 pyramidal neurons and interneurons appear to exert feedback and feedforward inhibition for determining the spike timing of the hippocampal microcircuit.

Chronic nicotine treatment differentially modifies acute nicotine and alcohol actions on GABA(A) and glutamate receptors in hippocampal brain slices.

Tobacco and alcohol are often co-abused producing interactive effects in the brain. Although nicotine enhances memory while ethanol impairs it, variable cognitive changes have been reported from concomitant use. This study was designed to determine how nicotine and alcohol interact at synaptic sites to modulate neuronal processes. Acute effects of nicotine, ethanol, and both drugs on synaptic excitatory glutamatergic and inhibitory GABAergic transmission were measured using whole-cell recording in hippocampal CA1 pyramidal neurons from brain slices of mice on control or nicotine-containing diets. Acute nicotine (50 nM) enhanced both GABAergic and glutamatergic synaptic transmission; potentiated GABA(A) receptor currents via activation of α7* and α4β2* nAChRs, and increased N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor currents through α7* receptors. While ethanol (80 mM) also increased GABA(A) currents, it inhibited NMDA currents. Although ethanol had no effect on AMPA currents, it blocked nicotine-induced increases in NMDA and AMPA currents. Following chronic nicotine treatment, acute nicotine or ethanol did not affect NMDA currents, while the effects of GABAergic responses were not altered. Acute ethanol ingestion selectively attenuated nicotine enhancement of excitatory glutamatergic NMDA and AMPA receptor function, suggesting an overall reduction in excitatory output from the hippocampus. It also indicated that ethanol could decrease the beneficial effects of nicotine on memory performance. In addition, chronic nicotine treatment produced tolerance to the effects of nicotine and cross-tolerance to the effects of ethanol on glutamatergic activity, leading to a potential increase in the use of these drugs.

Opioid Actions in Primary-Afferent Fibers—Involvement in Analgesia and Anesthesia

Opioids inhibit glutamatergic excitatory transmission from the periphery by activating G-protein coupled opioid receptors in the central terminals of primary-afferent neurons in the spinal substantia gelatinosa, resulting in antinociception. Opioid receptor activation in the peripheral terminals of primary-afferent neurons inhibits the production of action potentials in response to nociceptive stimuli given to the periphery, leading to antinociception. Opioids also exhibit a local anesthetic effect without opioid receptor activation in peripheral nerve fibers. This review article will focus on analgesia and anesthesia produced by the actions of opioids on primary-afferent fibers.

A novel non‐CB1/TRPV1 endocannabinoid‐mediated mechanism depresses excitatory synapses on hippocampal CA1 interneurons

Endocannabinoids (eCBs) mediate various forms of synaptic plasticity at excitatory and inhibitory synapses in the brain. The eCB anandamide binds to several receptors including the transient receptor potential vanilloid 1 (TRPV1) and cannabinoid receptor 1 (CB1). We recently identified that TRPV1 is required for long-term depression at excitatory synapses on CA1 hippocampal stratum radiatum interneurons. Here we performed whole-cell patch clamp recordings from CA1 stratum radiatum interneurons in rat brain slices to investigate the effect of the eCB anandamide on excitatory synapses as well as the involvement of Group I metabotropic glutamate receptors (mGluRs), which have been reported to produce eCBs endogenously. Application of the nonhydrolysable anandamide analog R-methanandamide depressed excitatory transmission to CA1 stratum radiatum interneurons by ∼50%. The Group I mGluR agonist DHPG also depressed excitatory glutamatergic transmission onto interneurons to a similar degree, and this depression was blocked by the mGluR5 antagonist MPEP (10 μM) but not by the mGluR1 antagonist CPCCOEt (50 μM). Interestingly, however, neither DHPG-mediated nor R-methanandamide-mediated depression was blocked by the TRPV1 antagonist capsazepine (10 μM), the CB1 antagonist AM-251 (2 μM) or a combination of both, suggesting the presence of a novel eCB receptor or anandamide target at excitatory hippocampal synapses. DHPG also occluded R-methanandamide depression, suggesting the possibility that the two drugs elicit synaptic depression via a shared signaling mechanism. Collectively, this study illustrates a novel CB1/TRPV1-independent eCB pathway present in the hippocampus that mediates depression at excitatory synapses on CA1 stratum radiatum interneurons.

VGLUT2-Dependent Glutamate Release from Nociceptors Is Required to Sense Pain and Suppress Itch

Itch can be suppressed by painful stimuli, but the underlying neural basis is unknown. We generated conditional null mice in which vesicular glutamate transporter type 2 (VGLUT2)-dependent synaptic glutamate release from mainly Nav1.8-expressing nociceptors was abolished. These mice showed deficits in pain behaviors, including mechanical pain, heat pain, capsaicin-evoked pain, inflammatory pain, and neuropathic pain. The pain deficits were accompanied by greatly enhanced itching, as suggested by (1) sensitization of both histamine-dependent and histamine-independent itch pathways and (2) development of spontaneous scratching and skin lesions. Strikingly, intradermal capsaicin injection promotes itch responses in these mutant mice,as opposed to pain responses in control littermates. Consequently, coinjection of capsaicin was no longer able to mask itch evoked by pruritogenic compounds. Our studies suggest that synaptic glutamate release from a group of peripheral nociceptors is required to sense pain and suppress itch. Elimination of VGLUT2 in these nociceptors creates a mouse model of chronic neurogenic itch.

Correlated changes in NMDA receptor phosphorylation, functional activity, and sedation by chronic ethanol consumption

Alcohol abuse leads to tolerance, dependence, and memory impairments that involve excitatory glutamatergic NMDA synaptic transmission. The NMDA receptor (NMDAR) is known to undergo activity-dependent adaptive functional changes. Since we observed that acute ethanol inhibition of the NMDAR was regulated by protein tyrosine phosphorylation, we investigated the role of protein tyrosine kinases and phosphatases on the NMDAR functions by chronic ethanol treatment. We carried out whole-cell recording, western blotting, and behavioral righting reflex measurements to assess the impact of chronic ethanol treatment on NMDAR function. Our results indicated that these receptors became resistant to the acute ethanol inhibition following chronic ethanol consumption. This resistance occurred without an increase in the NMDAR subunit expression but was associated with decreases in the levels of phospho-Y-1472 NR2B, increases in the levels of STEP33, increases in phospho-p38 mitogen-activated protein kinase (pp38 MAPK), and acquisition of tolerance to the sedative effects of ethanol. These data suggested that altered protein tyrosine phosphorylation of the NMDAR subunits significantly contributes to functional changes of this receptor by chronic ethanol ingestion. Therefore, preservation of the integrity of tyrosine phosphorylation mechanisms of the NMDAR may be important in controlling the progression of alcohol tolerance and dependence.

Opiate-Induced Suppression of Rat Hypoglossal Motoneuron Activity and Its Reversal by Ampakine Therapy

BackgroundHypoglossal (XII) motoneurons innervate tongue muscles and are vital for maintaining upper-airway patency during inspiration. Depression of XII nerve activity by opioid analgesics is a significant clinical problem, but underlying mechanisms are poorly understood. Currently there are no suitable pharmacological approaches to counter opiate-induced suppression of XII nerve activity while maintaining analgesia. Ampakines accentuate α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor responses. The AMPA family of glutamate receptors mediate excitatory transmission to XII motoneurons. Therefore the objectives were to determine whether the depressant actions of μ-opioid receptor activation on inspiratory activity includes a direct inhibitory action at the inspiratory premotoneuron to XII motoneuron synapse, and to identify underlying mechanism(s). We then examined whether ampakines counteract opioid-induced depression of XII motoneuron activity.Methodology/Principal FindingsA medullary slice preparation from neonatal rat that produces inspiratory-related output in vitro was used. Measurements of inspiratory burst amplitude and frequency were made from XII nerve roots. Whole-cell patch recordings from XII motoneurons were used to measure membrane currents and synaptic events. Application of the μ-opioid receptor agonist, DAMGO, to the XII nucleus depressed the output of inspiratory XII motoneurons via presynaptic inhibition of excitatory glutamatergic transmission. Ampakines (CX614 and CX717) alleviated DAMGO-induced depression of XII MN activity through postsynaptic actions on XII motoneurons.Conclusions/SignificanceThe inspiratory-depressant actions of opioid analgesics include presynaptic inhibition of XII motoneuron output. Ampakines counteract μ-opioid receptor-mediated depression of XII motoneuron inspiratory activity. These results suggest that ampakines may be beneficial in countering opiate-induced suppression of XII motoneuron activity and resultant impairment of airway patency.

Anandamide potentiation of miniature spontaneous excitatory synaptic transmission is mediated via IP3 pathway

Although arachidonoyl ethanolamide (AEA or anandamide) is the first identified endocannabinoid, its roles in synaptic signaling and neuronal survival are still controversial. Here we report that AEA induced a dose-dependent elevation of the frequency of miniature excitatory postsynaptic currents (mEPSCs) in mouse hippocampal neurons in culture. This potentiation was not blocked by SR141716 or AM251, selective cannabinoid receptor antagonists, indicating that the AEA elevation of mEPSCs is not mediated via the CB1 receptor. Similarly, capsazepine and iodoresiniferatoxin, selective vanilloid receptor antagonists, and ryanodine also failed to inhibit the effect of AEA on mEPSCs. However, 2-APB and Xestospongin C, IP3 inhibitors, significantly attenuated AEA-induced increase in hippocampal excitatory synaptic transmission. Application of 3-deoxy-3-fluoro-d-myo-inositol 1,4,5-trisphosphate enhanced the frequency of mEPSCs and occluded the effect of AEA on mEPSCs. Our results suggest that AEA-produced stimulatory effect on excitatory glutamatergic synaptic transmission is likely mediated via an IP3 pathway.

Reduced expression of glutamate receptors and phosphorylation of CREB are responsible for in vivoΔ9‐THC exposure‐impaired hippocampal synaptic plasticity

Chronic use of marijuana impairs synaptic plasticity and cognitive function. However, the molecular mechanisms by which marijuana alters long-term synaptic plasticity are largely unknown. Here, we show that repeated in vivo exposures to Delta9-THC for 7 consecutive days significantly impaired hippocampal long-term potentiation (LTP) of excitatory glutamatergic synaptic transmission. The Delta9-THC exposure-induced decrease in LTP was prevented by pharmacological inhibition or deletion of the cannabinoid 1 receptor (CB1R). To determine the molecular mechanisms underlying Delta9-THC-altered LTP, we targeted expression and function of the glutamate receptors (GluR) and phosphorylation status of cAMP-response element-binding protein (CREB). Chronic in vivo exposure to Delta9-THC produced CB1R-dependent decreases in expression of hippocampal GluR1, NR2A, and NR2B, the ratio of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/NMDA receptor-gated currents, and phosphorylation of CREB. Our results suggest that reduced expression and function of the GluR subunits and phosphorylation of CREB may underlie the impaired long-term synaptic plasticity induced by repeated in vivo exposure to Delta9-THC.