Acute Effects of Ethanol on Glutamate Receptors

Transcription

1 Basic & Clinical Pharmacology & Toxicology, 2012, 111, 4 13 Doi: /j x MiniReview Acute Effects of Ethanol on Glutamate Receptors Tommi Möykkynen 1,2 and Esa R. Korpi 1 1 Institute of Biomedicine, Pharmacology, University of Helsinki, Helsinki, Finland, and 2 Department of Biosciences, Division of Biochemistry and Biotechnology, University of Helsinki, Helsinki, Finland (Received 5 January 2012; Accepted 8 March 2012) Abstract: Several studies have revealed that acute ethanol inhibits the function of glutamate receptors. Glutamate receptormediated synaptic plasticity, such as N-methyl-D-aspartate-dependent long-term potentiation, is also inhibited by ethanol. However, the inhibition seems to be restricted to certain brain areas such as the hippocampus, amygdala and striatum. Ethanol inhibition of glutamate receptors generally requires relatively high concentrations and may therefore explain consequences of severe ethanol intoxication such as impairment of motor performance and memory. Effects of ethanol on glutamate system of developing nervous system may have a role in causing foetal alcohol syndrome. Newly found regulatory proteins of a-amino-3-hydroxy-5- methyl-4-isoxazolepropionic acid AMPA receptors seem to affect ethanol inhibition thus opening new lines of research. Ethanol, the active molecule of alcoholic beverages, is without doubt the most widely used drug of abuse in the world along with nicotine. Owing to its legal status and availability, it causes wide addiction/dependency problem with devastating health problems and numerous premature deaths. Ethanol is therefore a widely studied molecule in many research institutes around the world especially devoted for its study. The research has been fruitful in revealing a wide variety of ethanol molecular targets such as many types of ion channels on cell membrane and intracellular signalling pathways. Because of the wide spectrum of actions and mechanisms of ethanol, it has sometimes been difficult to control the experimental conditions to obtain reproducible and reliable results. Some of the variability may stem from the low affinity of ethanol interactions with target molecules and small effect sizes. One can say that this has been an unwanted feature of ethanol, as it has been difficult to pinpoint which of ethanol s actions are involved and important in ethanol intoxication and development of addiction. The first theory of how different alcohols cause their effects was Meyer Overton s lipid theory of anaesthesia coined at the end of 19th century (reviewed in [1]). The theory suggested that ethanol and general anaesthetics produce their action by interaction with lipid membranes, as the actions were related to the lipid solubility of the compounds. However, general anaesthetics could equally well be binding to hydrophobic target sites on proteins in the brain. In the mid-eighties, it was shown that ethanol inhibited the function of soluble nonmembrane-bound firefly luciferase enzyme (reviewed in [1]). Author for correspondence: Tommi Möykkynen, Department of Biosciences, Biochemistry and Biotechnology, PO Box. 56 (Viikinkaari 5), FI-00014, University of Helsinki, Helsinki, Finland (fax , tommi.moykkynen@helsinki.fi). These experiments proved that alcohols and general anaesthetics can induce an action without the primary interference of lipids, and this interaction also interestingly showed a good correlation between potency and lipid solubility in a series of aliphatic alcohols up to a certain carbon chain length. A few years later, it was shown that ethanol alters the function of many neurotransmitter receptor proteins. To date, there are still many discrepancies in published studies of the actions of ethanol as some laboratories fail to repeat the original findings of others (see, for example [2]). This MiniReview focuses on the acute effects of ethanol on the ionotropic glutamate receptors, the most abundant excitatory neurotransmitter system in the mammalian brain. Generally speaking, ethanol inhibits the function of all ionotropic glutamate receptor classes, but experimental conditions seem to produce a lot of variability in the results. A special attention will also be given to immediate consequences of acute ethanol effects on the neurophysiology such as long-term potentiation and depression (LTP and LTD) and neuronal development, which are often dependent on the function and plasticity of glutamate receptors. Ionotropic glutamate receptors are important in brain physiology, for example, by mediating many processes of neuroplasticity needed in cognitive functions and by mediating neurodegeneration in brain ischaemia. They are also important in neuropsychopharmacology by providing targets for drugs such as memantine (low-potency antagonist of N-methyl-D-aspartate (NMDA) receptors, used in Alzheimer s disease), ketamine (a dissociative anaesthetic and putative antidepressant, a more potent NMDA receptor antagonist), and topiramate (an antiepileptic and alcoholism drug, blocking among other targets the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors).

2 MiniReview ACUTE EFFECTS OF ETHANOL ON GLUTAMATE RECEPTORS 5 Ionotropic Glutamate Receptors Ionotropic glutamate receptors include AMPA, NMDA and kainate types of glutamate receptors, which were named after the synthetic agonists, which best activate the currents of the receptor in question (reviewed in [3]). Their activation is coupled to Na +,K + and Ca 2+ conductances as opposed to metabotropic glutamate receptors that couple to G-protein activation [3]. The function of ionotropic glutamate receptors is to quickly excite the neuron during fast synaptic transmission, whereas metabotropic glutamate receptors have several effects, including reducing transmission, often presynaptically. NMDA receptors (NMDARs), and to a smaller extent also GluA2- lacking AMPA receptors (AMPARs), play a role also in modulation of the strength of neurotransmission as they can serve channels for the entry of calcium, an important second messenger. This MiniReview will concentrate only on the effect of ethanol on ionotropic glutamate receptors (iglurs, later often called glutamate receptors ). Glutamate receptors are widely expressed through the brain in most of neurons, and they mediate most of the fast excitatory neurotransmission. The structure of glutamate receptor and list of subunits according the latest nomenclature [4] are provided in fig. 1. AMPA and kainate receptors form functional tetrameric receptors as combinations of any subunits within their class, whereas in NMDARs the GluN1 is a mandatory subunit. In addition to different subunit assembly possibilities, alternative splicing and RNA editing increase the structural variation of glutamate receptors [3]. a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors mediate fast depolarization of neurons, whereas NMDARs play a special role in synaptic transmission. Mg 2+ block of the ion channel within the NMDAR is relieved by depolarization, and thus, current through these channels will normally occur mainly in depolarized synapses/dendrites. Another important feature of NMDA receptors is the Ca 2+ permeability. These properties make NMDARs coincidence detectors converting a strong synaptic transmission to calcium influx which then alters synaptic strength via downstream effects on protein kinase cascades and increase in synaptic AMPAR-mediated function. Studies carried out during the last decade suggest that all native AMPARs are in companion with auxiliary proteins when they are expressed at the neuronal cell surface. To date, three auxiliary protein families [transmembrane AMPA receptor-regulating proteins (TARPs), cornichon proteins (CNIH-1/2), transmembrane protein CKAMP44, (reviewed in [5])] have been discovered (fig. 1). They seem to be involved in (i) promotion of the surface expression and synaptic targeting of AMPARs, (ii) modulation of channel kinetics such as desensitization and deactivation and (iii) modulation of agonist/antagonist pharmacology. Interestingly, at least the TARPs also affect ethanol sensitivity of AMPARs [6], which is covered in more detail later in this MiniReview. The significance of kainate receptors in the function of neural connections is not as well understood as that of AMPA and NMDA receptors. Highly selective agonists and antagonists have only become available in recent years, which has Fig. 1. Subunits and the structure of glutamate receptors. In the box, main ionotropic glutamate receptor subunits according to the latest nomenclature [4] and currently known auxiliary proteins are listed. Schematic drawing of tetrameric glutamate receptor and a drawing showing the main domains in amino acid sequence: N-terminal domain (NTD); ligand-binding domain (SI S2); transmembrane domains (TM1 3); carboxyl-tail (C). Membrane loop (P) forms the channel pore as depicted in the drawing.

3 6 TOMMI MÖYKKYNEN AND ESA R. KORPI MiniReview made it possible to investigate kainate receptors reliably in native neural preparations. In the CA3 area of the hippocampus, kainate receptors are located mainly in presynaptic cell membranes and function as autoreceptors regulating glutamate release and also mediate synaptic plasticity. In the CA1 area of hippocampus, the function of presynaptic kainate receptors seem to be the tuning of the developing synaptic connections to respond on high-frequency burst activity, which typically occurs in the hippocampus of the neonatal rat [7]. Kainate receptors in some brain areas are also inhibited by ethanol, which is suggested to affect the formation of neural circuits during brain development and the anxiolytic effects of ethanol [8 11]. Acute Effect of Ethanol on Glutamate Receptors All glutamate receptors are subject to inhibition by ethanol, if not in all, but at least in some experimental conditions. NMDARs are generally regarded as the most sensitive glutamate receptors to ethanol as they are inhibited by clinically relevant concentrations of ethanol (20 mm, ~1& BAC) in brain slice experiments where the receptors and synaptic transmission are intact. However, in some experimental conditions, AMPA and kainate receptors (non-nmdars) can be as sensitive as NMDARs to ethanol inhibition. Non-NMDARs are especially sensitive to ethanol inhibition when they are studied in heterologous expression systems and in isolated cells, but there are also some cases where they are also potently inhibited in more intact conditions of native receptors. Ethanol Inhibition of NMDA Receptors Vast number of studies have shown that the NMDARs are particularly important sites of ethanol action. Acute ethanol potently inhibits NMDARs and prolonged ethanol exposure leads to compensatory up-regulation of NMDAR-mediated functions. It is therefore understandable that NMDAR is widely regarded as the most important glutamate receptor in a variety of ethanol-associated behavioural phenotypes such as tolerance, dependence, withdrawal, craving and relapse [12]. Potent inhibition of NMDAR function in brain slices by physiologically relevant concentration of ethanol starting from 25 mm (~1.1& BAC) was first shown in hippocampal preparations [13]. Early experiments established that ethanol inhibited NMDARs non-competitively and in a reversible manner, as the inhibition was easily washable and reproducible. Inhibition seems to be dependent on subunit composition of NMDARs as studies generally indicate that GluN1/N2A and GluN1/N2B are slightly more sensitive to ethanol than GluN1 combined with either GluN2C or 2D. For instance, a study of rat GluN1/N2A, GluN1/N2B and GluN1/N2C subunit combinations expressed in Xenopus laevis oocytes showed that GluN1/N2C channels were slightly less sensitive to ethanol inhibition than the other channels in Ca 2+ -deficient, Ba 2+ -containing medium [14]. In native neurons, the most sensitive receptors seem to be GluN2B-containing ifenprodil-sensitive NMDARs [15]. There are studies reporting that elevating extracellular Mg 2+ from close to zero magnesium to more physiological concentrations increases ethanol sensitivity of NMDARs [16]. In HEK 293 cell expression system, ethanol inhibition of GluN1/N2D receptors was found to be less sensitive to magnesium than those of GluN1/N2A and N2B receptors [17]. In the same study, it was also reported that when NR3 subunit was expressed with GluN1/N2B subunits, Mg 2+ effect on ethanol sensitivity was retained but it was absent in GluN1/N2A/N3 receptors. These results suggest that the extent of ethanol inhibition of native NMDA receptors has been underestimated, because the usual way to measure NMDARs function in brain slices has been in the presence of low extracellular Mg 2+ to enhance the ion currents when the cells have been voltage-clamped close to resting membrane potential. There are evidences that intracellular calcium plays a role in ethanol inhibition of NMDARs. The target site of intracellular calcium was located at the C-terminal tail of GluN1 subunit that contains the C0 domain that binds calmodulin- and actinin-binding protein a-actinin-2 [18,19]. Calmodulin binding to the C0 domain mediates the calcium-dependent reduction in NMDAR function [19]. Calmodulin and a-actinin-2 seem to compete for NMDA receptor interactions, calcium concentration being the factor that determines the outcome of this competition. It is suggested that in the presence of elevated calcium, the inactivation of NMDA receptor occurs after C0 dissociates from a-actinin-2 by two distinct but converging calcium-dependent processes: competitive displacement of a-actinin-2 by calmodulin and the reduction in the affinity of a-actinin-2 for C0 after calcium binds to it. In keeping, ethanol inhibition is stronger if calcium- and calmodulindependent inactivation of NMDA receptors are allowed to function properly, suggesting that ethanol promotes calmodulin-dependent inactivation of NMDA receptors [18]. The mechanism of ethanol inhibition of NMDARs is poorly understood. In single-channel recordings, ethanol decreases burst frequency and burst duration and intraburst open channel lifetime but it does not alter closed-time distribution [20]. Because of the low potency of ethanol to cause an effect on target molecules, in other words, effective ethanol concentrations are needed in mm-range; there are no high-affinity binding sites for ethanol in target molecules to perform binding studies. It has therefore been difficult to obtain direct evidence that ethanol binds to NMDAR but some indirect evidence exists. Alcohol potency to inhibit NMDARs increases as the carbon chain length grows reaching a maximum at six to eight carbon atoms and then decreases (fig. 2A) [21]. This is called the cut-off phenomenon. Lengthening of the carbon chain increases the lipid solubility of alcohols. The cut-off phenomenon is interpreted as indicating that alcohol molecules accommodate into a physical cavity in NMDARs, which is reachable from a lipophilic milieu such as via the lipid-rich cell membrane. This lipophilic milieu favours entry and affinity/efficacy of longer-chain alcohols on the receptor. The cut-off carbon length is presumably reached when the alcohol molecule becomes too large to fit in the binding cavity formed by the tertiary structure of the receptor. There is a caveat, however, to date we have no direct physical evidence of alcohol

4 MiniReview ACUTE EFFECTS OF ETHANOL ON GLUTAMATE RECEPTORS 7 binding at these sites, at least in the NMDAR, and thus, these ideas are conjectural at this point. The actual binding sites of ethanol within the NMDARs have been searched by studying point mutations altering the ethanol sensitivity. The strongest effects have been with mutations in various transmembrane regions (TMs). Point mutations Met813Ala and Leu819Ala in TM4 of GluN1 subunit enhance the ethanol inhibition [22]. When previously identified ethanol sensitivity-reducing Phe639Ala (TM3) point mutation is combined with either Leu819Ala (TM4) or Gly822Trp (TM4) point mutations, the ethanol inhibition returns to the level of wild-type receptor (fig. 2B) [22]. These data can be interpreted as evidence that the TM3 and TM4 domains form a binding cavity for ethanol and increasing the volume of this cavity by substituting the phenylalanine with smaller amino acids at position 639 increases ethanol inhibition of NMDA receptors. The size of the binding site can be compensated by the expression of larger amino acid residues such as tryptophan at the TM4 domain which then restores the ethanol inhibition to the control level. A simple antiparallel alignment of the amino acids forming TM3 and TM4 domains in the GluN1 subunit suggests that these residues may reside close enough to each other to make this sort of interaction possible. It is good to keep in mind that because of the lack of direct physical evidence for alcohol binding in the NMDAR, the abovementioned reasoning is hypothetical. Ethanol administration has been shown to cause acute tolerance that is mediated by enhancement of NMDAR function. One candidate protein mediating this effect is fyn kinase, which phosphorylates GluN2B subunit, which enhances the NMDAR function [23]. Fyn kinase is linked to scaffold proteins located in close proximity of NMDARs in a brain areaspecific manner, which may explain variations in ethanol actions between brain areas [24]. time (e.g. seconds) both in native neurons and in expression systems [27,28]. In these experiments, ethanol inhibits AM- PARs in a non-competitive manner, and the ethanol inhibition is reproducible and reversible as currents return back to control level after ethanol application [28]. Ethanol inhibits various AMPARs usually with equal efficacy, but some differences in potency (IC 50 -values) have been reported, which could be tracked to subunit composition and type of agonist used. Heteromeric AMPARs have a higher IC 50 than homomeric; homomeric GluA1 and GluA4 receptors had IC 50 values of 119 and 133 mm, respectively; co-expressed GluA1/A4 heteromers had an IC 50 value of 165 mm [27]. In cultured cortical neurons, the IC 50 value seemed to depend on the agonist used as kainate-evoked AMPAR currents gave an IC 50 value for ethanol of approximately 400 mm, whereas AMPAevoked currents were inhibited by ethanol with an IC 50 -value of 162 mm [27]. Straight-chain alcohols had a cut-off phenomenon in ethanol inhibition of homomeric GluA1 and GluA3 AMPA receptors expressed in Xenopus laevis oocytes, suggesting that ethanol interacts with a specific binding site or sites in the molecular structure of the receptors [29]. In the afore-mentioned study, n-alcohols inhibited kainate-activated ion currents with increasing potency up to the carbon length of seven, whereas longer-chain alcohols did not markedly inhibit the currents. As with NMDA receptors, the exact mechanism through which ethanol acts on AMPARs has not been clarified. Our studies have produced experimental evidence that ethanol inhibits AMPARs by interacting with the desensitization process of the receptor. As mentioned previously, synaptic AM- PARs are not sensitive to ethanol in brain slice studies, whereas in experiments performed with isolated neurons or cultured cells, non-synaptic currents are sensitive to ethanol. The most obvious difference between these two types of Ethanol Inhibition of AMPA Receptors a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors AMPARs are not generally thought to be as sensitive to ethanol as NMDARs. They are not much inhibited in intact synapses, when the currents are activated by synaptically released glutamate evoked by electrical stimulation of afferent axons [13]. However, ethanol has been found to inhibit synaptic AMPAR currents in the central amygdala [25] and in hippocampus of neonatal rats [10,26]. Interestingly, in the CA3 area of hippocampus of 3 6 days old rats, AMPAR currents were sensitive to ethanol and NMDAR currents were not, whereas the opposite was true in day-old rats, suggesting that ethanol inhibition of glutamate receptors is dependent on the developmental stage [10]. AMPARs in the developing hippocampus may be especially sensitive to ethanol as it was recently reported that a moderate ethanol concentration of 40 mm inhibits significantly AMPARs in 7 9-day-old rats [26], an effect which apparently is absent in older, dayold rats [9]. On the other hand, AMPARs seem to be consistently as sensitive to ethanol as NMDARs in experiments where an agonist is applied to the receptors by relatively long A Fig. 2. Evidence suggesting that ethanol inhibits N-methyl-D-aspartate receptors (NMDAR) function by direct interaction with the protein. (A) Cut-off phenomenon in the inhibition of NMDAR currents by alcohols of varying carbon chain length. The potency to inhibit ion currents grows with the length of alcohol up to a certain cut-off point (6 8 carbons) as shown in the bar chart. Nonanol and decanol do not inhibit NMDAR current, therefore their IC 50 values are not shown. The cut-off in potency is interpreted as the existence of a binding site in the tertiary structure of NMDARs. Note the logarithmic y-axis. Bar chart was generated from the IC 50 values reported in [21]. (B) One possible site of ethanol (E) interaction is located in GluN1 subunit of NMDAR formed by TM3 and TM4 based on point mutation experiments [22]. Numbers inside TM domains indicate which amino acids form the possible binding site. B

5 8 TOMMI MÖYKKYNEN AND ESA R. KORPI MiniReview experiments is that in the former, the electrical stimulation evokes the ion currents by endogenous transmitter release, and in the latter, the exogenous agonist application is used to activate the ion currents. This means that in brain slice experiments, the ion current resembles the endogenous synaptic transmission during which the receptors do not have time to desensitize before the agonist is cleaned off from the synaptic cleft. Agonist application, on the other hand, lasts generally seconds, during which AMPARs desensitize strongly, over 90%. However, if the agonist is applied very shortly, for example, for 1 ms, and very rapid piezo-driven solution application to excised membrane patches is used, then AMPAR currents resembling the synaptic transmission can be produced. These AMPAR currents are insensitive to ethanol in cultured cortical neurons [30]. Additional evidence that ethanol inhibition depends on the AMPAR desensitization comes from the observation that the steady-state current is significantly more inhibited than the peak current in isolated hippocampal neurons and in HEK cells (fig. 3A) [6,30]. For instance, 100 mm ethanol inhibited 100 lm AMPA-evoked peak currents by 15% and steady-state current by 35%. In addition, blocking the desensitization with cyclothiazide significantly decreased the ethanol inhibition in acutely isolated hippocampal neurons. Ethanol inhibition was also decreased when kainate that does not cause full desensitization of AMPARs was used as agonist. In addition, a point-mutated AMPAR (GluA1-flip Leu497Tyr), which has markedly decreased desensitization as compared to wild-type receptor, exhibited decreased ethanol inhibition when expressed in HEK cells. Accumulating evidence suggests that the native AMPARs are expressed in brain together with auxiliary proteins that participate in their trafficking to the cell membrane and modulate their function. As mentioned previously, to date three families of auxiliary proteins have been found, which are transmembrane AMPA receptor-regulating proteins (TARPs), CKAM44 and CHIN (reviewed in [5]). Of these, the TARPs have been studied most extensively. TARPs slow the rate of AMPAR desensitization, which tempted us to investigate the possibility that TARPs may alter the ethanol sensitivity of the AMPARs as we had previously shown that ethanol affects more the desensitized receptors [30]. As in isolated hippocampal neurons [30], we observed that ethanol inhibited more the steadystate than peak current in GluA4 alone and in GluA4 with the TARPs stargazin (c2) and c4 expressed in HEK cells [6]. In addition, we observed that ethanol increased the rate of desensitization measured as the time constant of 10 mm glutamateevoked whole cell peak current decay of homomeric GluA4 AMPARs. Co-expression of c2 and c4 TARPs further increased this effect on the desensitization (fig. 3B,C). Our two studies [6,30] suggest that the mechanism of ethanol inhibition of AMPAR is the promotion of receptor desensitization. In native brain, desensitization is affected by TARPs, which show a distinct regional developmental expression pattern in brain [31]. However, it is currently uncertain A control current level pa 10 mm Glu mm EtOH mm EtOH 25 pa 25 ms B GluA4 i +EtOH 200 GluA4 i + γ2 + EtOH 200 GluA4 i + γ4 + EtOH 200 Glu 10 mm 10 ms Glu 10 mm 10 ms Glu 10 mm C Normalized effect on τ-value GluA4 i + γ 2 ## * # [EtOH] (mm) Normalized effect on τ-value ## * GluA4 i +γ4 ### [EtOH] (mm) Fig. 3. The effect of ethanol on desensitization of homomeric GluA4 receptors and TARPs expressed in HEK cells. (A) Representative traces showing the ethanol inhibition of homomeric GluA4 receptors. Horizontal bar indicates the agonist application. Note that the peak current is inhibited less than the steady-state currents (enlarged inset). (B) Co-expression of c2 and c4 TARPs on the desensitization of GluA4 a-amino- 3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor currents. The currents are scaled to the same size in amplitude to better illustrate the difference in current decay, in other words, desensitization (figure-modified form [6]).

6 MiniReview ACUTE EFFECTS OF ETHANOL ON GLUTAMATE RECEPTORS 9 which proteins in intact brain make up the complexes of AMPAR subunits and regulatory proteins. A recent study suggests that hippocampal AMPARs are not combined only with TARPs but with both TARPs and cornichons [32]. Native AMPARs may therefore form highly varying complexes with auxiliary regulatory proteins, and some of these combinations may be sensitive to ethanol in intact neurotransmission. Expression of ethanol-sensitive AMPARs complexes may be restricted only to certain brain areas and developmental stages, which may explain why ethanol-sensitive AMPARs have been found only in some brain areas and in mature nervous system [10,25,26]. One would also expect to find ethanol-sensitive AMPARs in synapses where desensitization probably plays a role in synaptic transmission as AMPAR currents are reported to decay at the rate of desensitization rather than deactivation [33]. To date, according to the best of our knowledge, there are no studies that have investigated the ethanol sensitivity of the afore-mentioned synapses. Ethanol Inhibition of Kainate Receptors Kainate receptors have not been as extensively studied as the other two iglur classes. Kainate receptors in synaptic transmission seem to be sensitive to ethanol in pyramidal neurons in the CA3 region of the hippocampus even more than NMDA and AMPA receptors [8]. Ethanol-sensitive kainate receptors have also been found in the basolateral amygdala, where a relatively low concentration of ethanol, 20 mm, inhibited kainate receptors by 26%, making them four times more sensitive to ethanol than NMDA receptors in that brain area [11]. In the same study, basolateral amygdala kainate receptors were shown to play a role in anxiety-related behaviour in rats. Ethanol inhibition of kainate receptors may thus explain the ethanol-induced anxiolysis, perhaps offering an explanation for the relief of anxiety after just a few drinks in human beings. In the CA1 region of the hippocampus, kainate receptors are located in presynaptic terminals of interneurons, where their activation increases GABA release. These kainate receptors seem to be sensitive to low ethanol concentrations such as 5 10 mm, which has been reported to lead to decreased interneuron firing, thus decreasing the GABA-mediated inhibitory input to the CA1 pyramidal neurons [9]. This is suggested to contribute to the stimulating effect related to consumption of small amounts of alcohol. The mechanism of ethanol inhibition of kainate receptors, such as the possible interaction with desensitization, is not currently known. Effect of Ethanol on Glutamatergic Short-Term Plasticity Synaptic transmission is modulated by short-term plasticity that involves the alteration of the probability of synaptic vesicle release. The main modulator of the vesicle release is calcium that can increase neurotransmitter release via several related mechanisms. For example, during repetitive synaptic activity, residual calcium can either increase or decrease the neurotransmitter release caused by the subsequent presynaptic depolarization. A common way to measure the short-term plasticity experimentally has been paired-pulse ratio measurement, in which two stimuli with varying intervals are used to evoke synaptic currents. If the second current has higher amplitude than the first, paired-pulse facilitation has occurred giving a pairedpulse ratio higher than 1. If the second pulse is smaller in amplitude, there is a paired-pulse depression giving a pairedpulse ratio <1. Paired-pulse facilitation usually indicates low intrinsic release probability or that the treatment (e.g. drug) decreases the release probability. Paired-pulse depression, on the other hand, indicates high intrinsic release probability or that the treatment increases neurotransmitter release probability. When paired-pulse ratio measurement has been employed in the studies of ethanol effects, varying results have been acquired which seem to depend on the brain region and the developmental age of the preparation. To begin with, there are numerous studies showing that ethanol does not alter glutamate release from presynaptic terminals. However, there are studies reporting that ethanol increases paired-pulse ratio suggesting that it inhibits glutamate release in some neural preparations such as neonatal rat hippocampal CA3 synapses [10] and adult central amygdala synapses [25]. The mechanism of the inhibition of glutamate release is suggested to be the inhibition of presynaptic calcium entry [10,25]. Acute ethanol can also increase the release of glutamate as has been reported to take place in the CA1 area of the hippocampus from 3 to 4-day-old rats. In that preparation, ethanol increases the production of pregnanolone-like neuroactive steroids that then elevate the expression of presynaptic calcium-permeable NMDA receptors enhancing the glutamate release [34]. Effect of Ethanol on Long-Term Plasticity Long-term plasticity differs from short-term plasticity in mechanisms and in the length of the time it persists. Longterm plasticity, either depression or potentiation of synaptic transmission, lasts for hours or days, possibly years in a living animal. The mechanisms underlying synaptic plasticity can be presynaptic or post-synaptic and can involve several different induction mechanisms (reviewed in [35]). Some forms of long-term plasticity involve the glutamate receptors in their induction and expression, and, therefore, it is not surprising that they are affected by acute ethanol. This MiniReview concentrates mainly on NMDA receptor-dependent LTP and LTD (summarized in fig. 4). In the hippocampus, LTP requires robust synaptic glutamate release, post-synaptic depolarization and activation of NMDA receptors. Function of calcium-permeable NMDARs is a source of calcium needed for activation of protein kinases [calcium-/calmodulin-dependent protein kinase 2 (CaMKII) and protein kinase C (PKC)]. The activation of kinases then starts biochemical cascades that lead to an increase in the trafficking of additional AMPARs to synapses. NMDA receptor-dependent LTD, on the other hand, follows after relatively long and moderate elevation of intracellular Ca 2+ leading to preferential activation of protein phosphatases, which leads to dephosphorylation of proteins in synapses and removal of AMPA receptors from the synapses (reviewed in [35]).

7 10 TOMMI MÖYKKYNEN AND ESA R. KORPI MiniReview HFS LFS LTP induction AMPAR NMDAR LTD induction P Phosphate group HFS P 845 Mg 2+ ser Mg 2+ + Second Ca Second messengers messengers (CaMKII) (phosphatases) EtOH - Ca 2+ LFS Mg ser P Mg2+ + Second + + messengers (CaMKII) Ca Second Ca messengers 2+ (phosphatases) Fig. 4. Schematic illustration showing general mechanisms and ethanol inhibition of N-methyl-D-aspartate receptors (NMDAR)-dependent long-term potentiation (LTP) (right) and long-term depression (LTD) (left). Upper part of the picture presents the normal situation and the lower part presents the situation in the presence of ethanol. In the absence of ethanol, high-frequency stimulation (HFS) activates strong glutamate release which leads to a strong activation of NMDARs and to high concentration of intracellular calcium. High intracellular calcium activates calcium-/calmodulindependent protein kinase II (CaMKII), which leads after a few steps to an increased trafficking of additional a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors to the synapses. Low-frequency stimulation (LFS) causes LTD via moderate activation of AMPA and NMDA receptors, leading to subsequent moderate elevation of intracellular calcium and activation of calcium-/calmodulin-dependent phosphatases. One of the most important dephosphorylations performed by phosphatases is at serine residue 845 of GluA1 subunit leading to decreased function of the receptor. Ethanol (EtOH) inhibits NMDA and AMPA receptors thus opposing the elevation of intracellular calcium and the activation of second messenger systems required for full expression of NMDAR-dependent synaptic plasticity. The strength of arrows indicates the magnitude of cellular events. There are many studies reporting ethanol inhibition of LTP in the CA1 area of the hippocampus (for example [26]). The concentration of ethanol required for a significant inhibition of LTP induction has often been relatively high, around 50 mm and above, but concentrations as low as 5 mm have been reported to be inhibitory [36]. In the dorsomedial striatum, NMDAR-dependent LTP is decreased by a low concentration of ethanol of 2 mm, with 10 mm completely blocking it [37]. In addition, in these synapses, 50 mm ethanol reversed the direction of synaptic plasticity and induced LTD. This is especially interesting regarding the development of ethanol addiction, because depression of the dorsomedial striatal pathway may increase the significance of the parallel dorsolateral striatal pathway in a striatal output pathway, thus increasing habitual learning associated with ethanol consumption [38]. The reason for varying sensitivity of ethanol to inhibit hippocampal LTP is probably a consequence of different experimental conditions (e.g. slice preparation protocol, recording temperature, LTP induction paradigm, and developmental stage of experimental animal). For instance, young animals might be more sensitive to ethanol as it has been reported that hippocampal LTP was virtually abolished by 60 mm ethanol in slices from 15 to 25-day-old rats, but not in those from 70 to 100-day-old ones [39]. The way ethanol is applied to brain slices also seems to matter: When ethanol is applied on a slice in a stepwise fashion, which more closely resembles the way ethanol is consumed in real life, neural circuits undergo adaptation leading to a tolerance to the inhibitory effect of ethanol on LTP [40]. The induction of this type of ethanol-tolerant LTP was not dependent on NMDARs, L-type calcium channels or metabotropic glutamate receptors. However, LTP still required extracellular calcium, as a decrease in extracellular Ca 2+ concentration and blocking of the intracellular calcium stores prevented the induction of LTP. However, NMDARs seem to be involved also in the process that leads to the formation of ethanol-tolerant LTP as it was blocked if NMDARs were blocked during the stepwise increase in ethanol. This suggests that NMDARs participate in formation of ethanol-tolerant LTP, for example, making the intracellular calcium stores more susceptible to releasing calcium. The mechanism by which ethanol blocks LTP in the CA1 region of the hippocampus seems also to be dependent on the

8 MiniReview ACUTE EFFECTS OF ETHANOL ON GLUTAMATE RECEPTORS 11 age of the animal. In the developing hippocampus, inhibition of LTP probably involves the inhibition of NMDARs because the dose dependency of ethanol s effect on NMDAR-mediated fepsps (excitatory post-synaptic field potentials) matches to that of its effect on LTP [36]. In older animals ( dayold rats), the inhibition of NMDARs by ethanol was not sufficient to inhibit LTP as such, but additional indirect inhibition possibly through potentiation of GABA A receptors activity was needed [41]. Ethanol also inhibits both AMPA and NMDA receptors in the CA1 area of hippocampus of 7 9- day-old rats [26]. AMPARs can therefore contribute to inhibition of LTP because their activation is needed to remove the Mg 2+ block from NMDARs. The effects of ethanol on synaptic plasticity in the hippocampal formation has long been used as a model to study the effects of prenatal ethanol exposure serving as a valuable model for the development of foetal alcohol syndrome [10,26]. Hippocampal LTP is also important in learning and memory processes, and their inhibition by ethanol most likely contributes to the decrease in cognitive performance and memory impairment related to heavy ethanol intoxication [42]. Long-term potentiation-like synaptic plasticity in the ventral tegmantal area (VTA) dopamine neurons has become an important early step in the mechanisms of action of ethanol and many other drugs of abuse. A single IP injection of ethanol has been shown to elevate AMPA/NMDA ratio in VTA dopamine neuron. The increase in AMPA/NMDA ratio is thought to be caused by NMDA-dependent LTP as the NMDA receptor blocker dizocilpine inhibits this plasticity. The drugs of abuse affect by many different mechanisms but the end result seems to be the NMDA receptor activation in VTA dopamine neurons and subsequent NMDA receptormediated calcium signalling and the trafficking of additional AMPA receptors to the synapses (reviewed in [43]). The elevation of AMPA receptor function in the VTA by ethanol putatively leads to an elevation of dopamine release in the nucleus accumbens [44], one of the target areas of VTA dopamine neurons. Elevated accumbal dopamine may be one of the first steps of the development of addiction by increasing the hedonic value related to the drug use. Later, when the ethanol or drug exposure is repeated, addiction-related behaviour comes under the control of habit-forming neural circuits such as those involving the dorsal striatum. As noted previously, these circuits also seem to be very sensitive to ethanol, putatively contributing to habit formation related to excessive ethanol consumption [37]. Interestingly, it has also been reported that ethanol itself can cause an elevation of NMDAR function in dorsomedial striatum via phosphorylation of GluN2B by fyn kinase [24]. This can play a role in the formation of alcohol addiction as elevated NMDAR function in the dorsomedial striatum might lower the action potential firing threshold and subsequently increase the ethanol seeking and intake. Ethanol has been reported to inhibit LTD in some brain areas, such as in the hippocampus and nucleus accumbens [45,46], where the inhibition of LTD seems to be a result of the inhibition of NMDARs. In the hippocampus, lowfrequency stimulation produces LTD, which was blocked by mm ethanol, probably due to inhibition of GluN2B containing NMDARs as the effects of ethanol and ifenprodil were similar and overlapping [45]. In the nucleus accubens, ethanol concentrations from 20 to 60 mm inhibited LTD in 1 2-month-old mice [46]. Accumbal LTD was also mediated by GluN2B subunit-containing NMDA receptors and inhibition of these receptors by ethanol most likely underlies the inhibition of LTD. Effect of Ethanol on Glutamate Receptors in Living Animals In living animals, acute ethanol causes many behavioural effects such as decreased anxiety, impaired motor performance and increased sedation. In addition, ethanol impairs the learning (reviewed in [47]). It has been difficult to study which neurotransmitter system underlies ethanol s effect on particular behaviours, as ethanol has so many molecular targets in the nervous system. However, some indirect conclusions can be made. Ethanol-induced impairment of hippocampus-related learning and memory may be related to inhibition of NMDAdependent LTP in the hippocampus. This can be indirectly concluded from the fact that both ethanol and NMDA receptor blocker dizocilpine (MK-801) have been shown to inhibit spatial learning tasks (reviewed in [47]). Experiments performed on living animals have not revealed much about which NMDA and AMPA receptor subunits are important in behavioural effects of ethanol despite the availability of knockout mice and subunit selective drugs. In one study, the role of NMDA receptor subunits in the effects of acute ethanol on ataxia, hypothermia and sedation was investigated using GluN2A knockout mice and ifenprodil, a GluN2B-selective antagonist [48]. AMPA receptor GluA1 subunit knockouts were also included in the study. No major roles for any of the studied glutamate receptor subunits in the ethanol effects emerged. A minor finding was that GluN2A knockout mice had reduced sedation hypnosis when treated with a combination of MK-801 and ethanol. Another study found that mice that had genetically engineered NMDA receptor GluN2A subunits lacking the C-terminal tail had shortened time to loss of righting reflex and an increased sleep time when ethanol was administered the second time, a week after the first administration [49]. The same study found also alterations in ethanol inhibition of NMDA receptor activity in C-terminal truncated mice in field potential electrophysiology in the CA1 area: GluN2A-mediated field potentials were inhibited more while GluN2B-mediated field potentials were inhibited less than in wild-type controls. AMPA receptor subunit GluA3 has been linked to reinstatement of ethanol drinking behaviour, as GluA3 knockout mice display decreased cue-induced reinstatement response and alcohol deprivation effect as compared to wild-type controls [50]. Clearly, more detailed studies on the roles of glutamate receptors on ethanol behaviours are needed, especially in relation to auxiliary proteins and altered subunit trafficking in response to phosphorylation/dephosphorylation processes.

9 12 TOMMI MÖYKKYNEN AND ESA R. KORPI MiniReview Concluding Remarks N-methyl-D-aspartate receptors are presently the most important target molecules of ethanol among glutamate receptors, but we should not ignore the other classes, kainate and AMPA receptors in ethanol actions, as they have been shown to be sensitive to ethanol as well. Their inhibition may be confined to only certain brain areas and developmental stages, but they clearly have significance in brain function. Kainate receptors may contribute to the anxiolytic effects of ethanol, and the ethanol inhibition of AMPA receptors, along with other glutamate receptors, may contribute to foetal alcohol spectrum disorders. In the near future, this line of research will likely concentrate on the effects of ethanol on brain pathways that are involved in the generation of addiction. 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