Search

The target of alcohol's effect on the central nervous system has been sought for more than 50 years in the brain's GABA system. The behavioral and emotional effects of alcohol in humans and rodents are very similar to those of barbiturates and benzodiazepines, and GABA A receptors have been shown to be one of the sites of alcohol action. The mechanisms of GABAergic inhibition have been a hotspot of research but have turned out to be complex and controversial. Genetics support the involvement of some GABA A receptor subunits in the development of alcohol dependence and in alcohol use disorders (AUD). Since the effect of alcohol on the GABA A system resembles that of a GABAergic positive modulator, it may be possible to develop GABAergic drug treatments that could substitute for alcohol. The adaptation mechanisms of the GABA system and the plasticity of the brain are a big challenge for drug development: the drugs that act on GABA A receptors developed so far also may cause adaptation and development of additional addiction. Human polymorphisms should be studied further to get insight about how they affect receptor function, expression or other factors to make reasonable predictions/hypotheses about what non-addictive interventions would help in alcohol dependence and AUD., (Copyright © 2024. Published by Elsevier Inc.)

Alcohol is a major cause of pre-mature death and individual suffering worldwide, and the importance of diagnosing and treating AUD cannot be overstated. Given the global burden and the high attributable factor of alcohol in a vast number of diseases, the need for additional interventions and the development of new medicines is considered a priority by the World Health Organization (WHO). As of today, AUD is severely under-treated with a treatment gap nearing 90%, strikingly higher than that for other psychiatric disorders. Patients often seek treatment late in the progress of the disease and even among those who seek treatment only a minority receive medication, mirroring the still-prevailing stigma of the disease, and a lack of access to effective treatments, as well as a reluctance to total abstinence. To increase adherence, treatment goals should focus not only on maintaining abstinence, but also on harm reduction and psychosocial functioning. A personalised approach to AUD treatment, with a holistic view, and tailored therapy has the potential to improve AUD treatment outcomes by targeting the heterogeneity in genetics and pathophysiology, as well as reason for, and reaction to drinking. Also, the psychiatric co-morbidity rates are high in AUD and dual diagnosis can worsen symptoms and influence treatment response and should be considered in the treatment strategies., (Copyright © 2024. Published by Elsevier Inc.)

Expanding knowledge about the cellular composition of subcortical brain regions demonstrates large heterogeneity and differences from the cortical architecture. Previously we described three subtypes of somatostatin-expressing (Sst) neurons in the mouse ventral tegmental area (VTA) and showed their local inhibitory action on the neighboring dopaminergic neurons (Nagaeva et al., 2020). Here, we report that Sst+ neurons especially from the anterolateral part of the mouse VTA also project far outside the VTA and innervate forebrain regions that are mainly involved in the regulation of emotional behavior, including the ventral pallidum, lateral hypothalamus, the medial part of the central amygdala, anterolateral division of the bed nucleus of stria terminalis, and paraventricular thalamic nucleus. Deletion of these VTA Sst neurons in mice affected several behaviors, such as home cage activity, sensitization of locomotor activity to morphine, fear conditioning responses, and reactions to the inescapable stress of forced swimming, often in a sex-dependent manner. Together, these data demonstrate that VTA Sst neurons have selective projection targets distinct from the main targets of VTA dopamine neurons. VTA Sst neurons are involved in the regulation of behaviors primarily associated with the stress response, making them a relevant addition to the efferent VTA pathways and stress-related neuronal network., Competing Interests: Authors declare no competing conflicts of interest., (Copyright © 2023 Nagaeva et al.)

Psychedelics produce fast and persistent antidepressant effects and induce neuroplasticity resembling the effects of clinically approved antidepressants. We recently reported that pharmacologically diverse antidepressants, including fluoxetine and ketamine, act by binding to TrkB, the receptor for BDNF. Here we show that lysergic acid diethylamide (LSD) and psilocin directly bind to TrkB with affinities 1,000-fold higher than those for other antidepressants, and that psychedelics and antidepressants bind to distinct but partially overlapping sites within the transmembrane domain of TrkB dimers. The effects of psychedelics on neurotrophic signaling, plasticity and antidepressant-like behavior in mice depend on TrkB binding and promotion of endogenous BDNF signaling but are independent of serotonin 2A receptor (5-HT 2A ) activation, whereas LSD-induced head twitching is dependent on 5-HT 2A and independent of TrkB binding. Our data confirm TrkB as a common primary target for antidepressants and suggest that high-affinity TrkB positive allosteric modulators lacking 5-HT 2A activity may retain the antidepressant potential of psychedelics without hallucinogenic effects., (© 2023. The Author(s).)

Medications used for the treatment of diseases also affect oral health. We investigated how having/not having periodontitis at baseline in 1985 was associated with purchases of medicines in the long term. The study paradigm is in the oral health-systemic health connections. We hypothesized that periodontitis links to purchases of medicines later in life. The study cohort consisted of 3,276 individuals from the greater Stockholm area, Sweden. Of them, 1,655 were clinically examined at baseline. Patients were followed-up for >35 years, using the national population and patient registers. The burden of systemic diseases and purchases of medicines were statistically analyzed comparing patients with ( n = 285) and without ( n = 1,370) periodontitis. The results showed that patients with periodontitis had purchased more of certain medications than non-periodontitis patients. Periodontitis patients purchased significantly more drugs used in diabetes ( p = 0.035), calcium channel blockers ( p = 0.016), drugs acting on the renin-angiotensin system ( p = 0.024), and nervous system drugs ( p = 0.001). Hence, patients with periodontitis indeed had purchased specific medications statistically significantly more than the periodontally healthy ones. This indicates that periodontitis, over time, might increase the risk for systemic diseases with the subsequent need for medication., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Frankenhaeuser, Söder, Källmén, Korpi and Meurman.)

Background: Psychedelics, like lysergic acid diethylamide (LSD), are again being studied as potential therapies for many neuropsychiatric disorders, including addictions. At the same time, the acute effects of psychedelics on rewarding behaviours have been scarcely studied., Aims: The current study aimed to clarify if LSD decreases binge-like ethanol drinking in mice, and whether the observed acute effects on ethanol consumption are generalizable to a natural reinforcer, sucrose, and if the effects resulted from aversive or reward-attenuating effects caused by LSD., Methods: The effects of acute LSD were examined using 2-bottle choice intermittent ethanol (20%) and sucrose drinking (10%), discrete-trial current-intensity threshold method of intracranial self-stimulation and short-term feeding behaviour assay in C57BL/6 male mice., Results: The results showed that acute 0.1 mg/kg, but not 0.05 mg/kg, dose (i.p.) of LSD reduced 2-h intermittent ethanol drinking transiently without any prolonged effects. No effects were seen in intermittent 2-h sucrose drinking. The tested LSD doses had neither effect on the intracranial self-stimulation current-intensity thresholds, nor did LSD affect the threshold-lowering, or rewarding, effects of simultaneous amphetamine treatment. Furthermore, LSD had small, acute diminishing effects on 2-h food and water intake., Conclusions: Based on these results, LSD decreases binge-like ethanol drinking in mice, but only acutely. This effect is not likely to stem from reward-attenuating effects but could be in part due to reduced consummatory behaviour.

In the brain, extrasynaptically expressed ionotropic, δ subunit-containing γ-aminobutyric acid A-type receptors (δ-GABA A Rs) have been implicated in drug effects at both neuronal and behavioral levels. These alterations are supposed to be caused via drug-induced modulation of receptor ionophores affecting chloride ion-mediated inhibitory tonic currents. Often, a transgenic mouse model genetically lacking the δ-GABA A Rs (δ-KO) has been used to study the roles of δ-GABA A Rs in brain functions, because a specific antagonist of the δ-GABA A Rs is still lacking. We have previously observed with these δ-KO mice that activation of δ-GABA A Rs is needed for morphine-induced conditioning of place preference, and others have suggested that δ-GABA A Rs act as targets selectively for low doses of ethanol. Furthermore, activation of these receptors via drug-mediated agonism induces a robust increase in the slow-wave frequency bands of electroencephalography (EEG). Here, we tested δ-KO mice (compared to littermate wild-type controls) for the pharmaco-EEG responses of a broad spectrum of pharmacologically different drug classes, including alcohol, opioids, stimulants, and psychedelics. Gaboxadol (THIP), a known superagonist of δ-GABA A Rs, was included as the positive control, and as expected, δ-KO mice produced a blunted pharmaco-EEG response to 6 mg/kg THIP. Pharmaco-EEGs showed notable differences between treatments but also differences between δ-KO mice and their wild-type littermates. Interestingly mephedrone (4-MMC, 5 mg/kg), an amphetamine-like stimulant, had reduced effects in the δ-KO mice. The responses to ethanol (1 g/kg), LSD (0.2 mg/kg), and morphine (20 mg/kg) were similar in δ-KO and wild-type mice. Since stimulants are not known to act on δ-GABA A Rs, our findings on pharmaco-EEG effects of 4-MMC suggest that δ-GABA A Rs are involved in the secondary indirect regulation of the brain rhythms after 4-MMC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Grotell, Abdurakhmanova, Elsilä and Korpi.)

Characterization of an electrically active cell, such as a neuron, demands measurement of its electrical properties. Due to differences in gene activation, location, innervation patterns, and functions, the millions of neurons in the mammalian brain are tremendously diverse in their membrane characteristics and abilities to generate action potentials. These features can be measured with a patch-clamp technique in whole-cell current-clamp configuration followed by detailed post-hoc analysis of firing patterns. This analysis can be time-consuming, and different laboratories have their own methods to perform it, either manually or with custom-written scripts. Here, we describe in detail a protocol for firing-pattern registration in neurons of the ventral tegmental area (VTA) as an example and introduce a software for its fast and convenient analysis. With the help of this article, other research groups can easily apply this method and generate unified types of data that are comparable between brain regions and various studies. Graphic abstract: Workflow of the Protocol., Competing Interests: Competing interestsThe authors declare no competing interests., (Copyright © The Authors; exclusive licensee Bio-protocol LLC.)

Mephedrone (4-MMC), despite its illegal status, is still a widely used psychoactive substance. Its effects closely mimic those of the classical stimulant drug methamphetamine (METH). Recent research suggests that unlike METH, 4-MMC is not neurotoxic on its own. However, the neurotoxic effects of 4-MMC may be precipitated under certain circumstances, such as administration at high ambient temperatures. Common use of 4-MMC in conjunction with alcohol raises the question whether this co-consumption could also precipitate neurotoxicity. A total of six groups of adolescent rats were treated twice daily for four consecutive days with vehicle, METH (5 mg/kg) or 4-MMC (30 mg/kg), with or without ethanol (1.5 g/kg). To investigate persistent delayed effects of the administrations at two weeks after the final treatments, manganese-enhanced magnetic resonance imaging brain scans were performed. Following the scans, brains were collected for Golgi staining and spine analysis. 4-MMC alone had only subtle effects on neuronal activity. When administered with ethanol, it produced a widespread pattern of deactivation, similar to what was seen with METH-treated rats. These effects were most profound in brain regions which are known to have high dopamine and serotonin activities including hippocampus, nucleus accumbens and caudate-putamen. In the regions showing the strongest activation changes, no morphological changes were observed in spine analysis. By itself 4-MMC showed few long-term effects. However, when co-administered with ethanol, the apparent functional adaptations were profound and comparable to those of neurotoxic METH., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Grotell, den Hollander, Jalkanen, Törrönen, Ihalainen, de Miguel, Dudek, Kettunen, Hyytiä, Forsberg, Kankuri and Korpi.)

While interest in psychedelic drugs in the fields of psychiatry and neuroscience has re-emerged in recent last decades, the general understanding of the effects of these drugs remains deficient. In particular, there are gaps in knowledge on executive functions and goal-directed behaviors both in humans and in commonly used animal models. The effects of acute doses of psychedelic lysergic acid diethylamide (LSD) on reward-driven decision making were explored using the mouse version of the Iowa Gambling Task. A total of 15 mice were trained to perform in a touch-screen adaptation of the rodent version of the Iowa Gambling Task, after which single acute doses of LSD (0.025, 0.1, 0.2, 0.4 mg/kg), serotonin 2A receptor-selective agonist 25CN-NBOH (1.5 mg/kg), d-amphetamine (2.0 mg/kg), and saline were administered before the trial. 25CN-NBOH and the three lowest doses of LSD showed no statistically significant changes in option selection or in general functioning during the gambling task trials. The highest dose of LSD (0.4 mg/kg) significantly decreased premature responding and increased the omission rate, but had no effect on option selection in comparison with the saline control. Amphetamine significantly decreased the correct responses and premature responding while increasing the omission rate. In conclusion, mice can perform previously learned, reward-driven decision-making tasks while under the acute influence of LSD at a commonly used dose range., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Elsilä, Korhonen, Hyytiä and Korpi.)

The cellular architecture of the ventral tegmental area (VTA), the main hub of the brain reward system, remains only partially characterized. To extend the characterization to inhibitory neurons, we have identified three distinct subtypes of somatostatin (Sst)-expressing neurons in the mouse VTA. These neurons differ in their electrophysiological and morphological properties, anatomical localization, as well as mRNA expression profiles. Importantly, similar to cortical Sst-containing interneurons, most VTA Sst neurons express GABAergic inhibitory markers, but some of them also express glutamatergic excitatory markers and a subpopulation even express dopaminergic markers. Furthermore, only some of the proposed marker genes for cortical Sst neurons were expressed in the VTA Sst neurons. Physiologically, one of the VTA Sst neuron subtypes locally inhibited neighboring dopamine neurons. Overall, our results demonstrate the remarkable complexity and heterogeneity of VTA Sst neurons and suggest that these cells are multifunctional players in the midbrain reward circuitry., Competing Interests: EN, IZ, CB, MF, KN, Ed, LE, AL, JH, GA, EK No competing interests declared, (© 2020, Nagaeva et al.)

Histamine/gamma-aminobutyric acid (GABA) neurons of posterior hypothalamus send wide projections to many brain areas and participate in stabilizing the wake state. Recent research has suggested that GABA released from the histamine/GABA neurons acts on extrasynaptic GABA A receptors and balances the excitatory effect of histamine. In the current study, we show the presence of vesicular GABA transporter mRNA in a majority of quantified hypothalamic histaminergic neurons, which suggest vesicular release of GABA. As histamine/GABA neurons form conventional synapses infrequently, it is possible that GABA released from these neurons diffuses to target areas by volume transmission and acts on extrasynaptic GABA receptors. To investigate this hypothesis, mice lacking extrasynaptic GABA A receptor δ subunit (Gabrd KO) were used. A pharmacological approach was employed to activate histamine/GABA neurons and induce histamine and presumably, GABA, release. Control and Gabrd KO mice were treated with histamine receptor 3 (Hrh3) inverse agonists ciproxifan and pitolisant, which block Hrh3 autoreceptors on histamine/GABA neurons and histamine-dependently promote wakefulness. Low doses of ciproxifan (1 mg/kg) and pitolisant (5 mg/kg) reduced locomotion in Gabrd KO, but not in WT mice. EEG recording showed that Gabrd KO mice were also more sensitive to the wake-promoting effect of ciproxifan (3 mg/kg) than control mice. Low frequency delta waves, associated with NREM sleep, were significantly suppressed in Gabrd KO mice compared with the WT group. Ciproxifan-induced wakefulness was blocked by histamine synthesis inhibitor α-fluoromethylhistidine (αFMH). The findings indicate that both histamine and GABA, released from histamine/GABA neurons, are involved in regulation of brain arousal states and δ-containing subunit GABA A receptors are involved in mediating GABA response., (Copyright © 2020 Abdurakhmanova, Grotell, Kauhanen, Linden, Korpi and Panula.)

Drugs of abuse induce widespread synaptic adaptations in the mesolimbic dopamine (DA) neurons. Such drug-induced neuroadaptations may constitute an initial cellular mechanism eventually leading to compulsive drug-seeking behavior. To evaluate the impact of GABA B receptors on addiction-related persistent neuroplasticity, we tested the ability of orthosteric agonist baclofen and two positive allosteric modulators (PAMs) of GABA B receptors to suppress neuroadaptations in the ventral tegmental area (VTA) and reward-related behaviors induced by ethanol and cocaine. A novel compound (S)-1-(5-fluoro-2,3-dihydro-1H-inden-2-yl)-4-methyl-6,7,8,9-tetrahydro-[1,2,4]triazolo[4,3-a]quinazolin-5(4H)-one (ORM-27669) was found to be a GABA B PAM of low efficacy as agonist, whereas the reference compound (R,S)-5,7-di-tert-butyl-3-hydroxy-3-trifluoromethyl-3H-benzofuran-2-one (rac-BHFF) had a different allosteric profile being a more potent PAM in the calcium-based assay and an agonist, coupled with potent PAM activity, in the [ 35 S] GTPγS binding assay in rat and human recombinant receptors. Using autoradiography, the high-efficacy rac-BHFF and the low-efficacy ORM-27669 potentiated the effects of baclofen on [ 35 S] GTPγS binding with identical brain regional distribution. Treatment of mice with baclofen, rac-BHFF, or ORM-27669 failed to induce glutamate receptor neuroplasticity in the VTA DA neurons. Pretreatment with rac-BHFF at non-sedative doses effectively reversed both ethanol- and cocaine-induced plasticity and attenuated cocaine i.v. self-administration and ethanol drinking. Pretreatment with ORM-27669 only reversed ethanol-induced neuroplasticity and attenuated ethanol drinking but had no effects on cocaine-induced neuroplasticity or self-administration. These findings encourage further investigation of GABA B receptor PAMs with different efficacies in addiction models to develop novel treatment strategies for drug addiction., (© 2018 Society for the Study of Addiction.)

Extinction and reinstatement of morphine-induced conditioned place preference were studied in glutamate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-receptor GluA1 subunit-deficient mice (global GluA1-KO mice). In line with previous findings, both acquisition and expression of conditioned place preference to morphine (20 mg/kg, subcutaneously) were fully functional in GluA1 KO mice compared with wild-type littermate controls (GluA1-WT), thus enabling the study of extinction. With a 10-session extinction paradigm, the GluA1 KO mice showed complete extinction similar to that of the GluA1-WT mice. Morphine-induced reinstatement (10 mg/kg, subcutaneously) was detected in both mouse lines. GluA1 KO mice moved more during all the phases of the experiment, including the place conditioning trials, extinction sessions, and place preference tests. The results suggest that the GluA1 subunit may be dispensable or prone to compensation at the neural circuitries delineating extinction and reinstatement. The GluA1 KO mice show altered long-term between-session habituation, which extends longer than previously anticipated.

The nonsteroidal anti-inflammatory drug (NSAID) niflumic acid, a fenamate in structure, has many molecular targets, one of them being specific subtypes of the main inhibitory ligand-gated anion channel, the GABA A receptor. Here, we report on the effects of other fenamates and other classes of NSAIDs on brain picrotoxinin-sensitive GABA A receptors, using an autoradiographic assay with [ 35 S]TBPS as a ligand on mouse brain sections. We found that the other fenamates studied (flufenamic acid, meclofenamic acid, mefenamic acid and tolfenamic acid) affected the autoradiographic signal at low micromolar concentrations in a facilitatory-like allosteric fashion, i.e., without having affinity to the [ 35 S]TBPS binding site. Unlike niflumic acid that shows clear preference for inhibiting cerebellar granule cell layer GABA A receptors, the other fenamates showed little brain regional selectivity, indicating that their actions are not receptor-subtype selective. Of the non-fenamate NSAIDs studied at 100 μM concentration, diclofenac induced the greatest inhibition of the binding, which is not surprising as it has close structural similarity with the potent fenamate meclofenamic acid. Using two-electrode voltage-clamp assays on Xenopus oocytes, the effect of niflumic acid was found to be dependent on the β subunit variant and the presence of γ2 subunit in rat recombinant α1β and α1βγ2 GABA A receptors, with the β1 allowing the niflumic acid inhibition and β3 the stimulation of the receptor-mediated currents. In summary, the fenamate NSAIDs constitute an interesting class of compounds that could be used for development of potent GABA A receptor allosteric agonists with other targets to moderate inflammation, pain and associated anxiety/depression., (Copyright © 2019. Published by Elsevier B.V.)

THIP (gaboxadol), a superagonist of the δ subunit-containing extrasynaptic GABA A receptors, produces persistent neuroplasticity in dopamine (DA) neurons of the ventral tegmental area (VTA), similarly to rewarding drugs of abuse. However, unlike them THIP lacks abuse potential and induces conditioned place aversion in mice. The mechanism underlying the aversive effects of THIP remains elusive. Here, we show that mild aversive effects of THIP were detected 2 h after administration likely reflecting an anxiety-like state with increased corticosterone release and with central recruitment of corticotropin-releasing factor corticotropin-releasing factor receptor 1 (CRF 1 ) receptors. A detailed immunohistochemical c-Fos expression mapping for THIP-activated brain areas revealed a correlation between the activation of CRF-expressing neurons in the oval nucleus of the bed nuclei of stria terminalis and THIP-induced aversive effects. In addition, the neuroplasticity of mesolimbic DA system (24 h after administration) and conditioned place aversion by THIP after four daily acute sessions were dependent on extrasynaptic GABA A receptors (abolished in δ-GABA A receptor knockout mice) and activation of the CRF 1 receptors (abolished in wildtype mice by a CRF 1 receptor antagonist). A selective THIP-induced activation of CRF-expressing neurons in the oval part of the bed nucleus of stria terminalis may constitute a novel mechanism for inducing plasticity in a population of VTA DA neurons and aversive behavioral states.

Gene-targeted mice with deficient AMPA receptor GluA1 subunits ( Gria1-/- mice) show robust hyperlocomotion in a novel environment, suggesting them to constitute a model for hyperactivity disorders such as mania, schizophrenia and attention deficit hyperactivity disorder. This behavioral alteration has been associated with increased neuronal activation in the hippocampus, and it can be attenuated by chronic treatment with antimanic drugs, such as lithium, valproic acid, and lamotrigine. Now we found that systemic cannabidiol strongly blunted the hyperactivity and the hippocampal c-Fos expression of the Gria1-/- mice, while not affecting the wild-type littermate controls. Acute bilateral intra-dorsal hippocampal infusion of cannabidiol partially blocked the hyperactivity of the Gria1-/- mice, but had no effect on wild-types. The activation of the inhibitory DREADD receptor hM4Gi in the dorsal hippocampus by clozapine- N -oxide robustly inhibited the hyperactivity of the Gria1-/- mice, but had no effect on the locomotion of wild-type mice. Our results show that enhanced neuronal excitability in the hippocampus is associated with pronounced novelty-induced hyperactivity of GluA1 subunit-deficient mice. When this enhanced response of hippocampal neurons to novel stimuli is specifically reduced in the hippocampus by pharmacological treatment or by chemogenetic inhibition, Gria1-/- mice recover from behavioral hyperactivity, suggesting a hippocampal dysfunction in hyperactive behaviors that can be treated with cannabidiol.

The dopamine D 3 receptor (D 3 R), in the nucleus accumbens (NAc), plays an important role in alcohol reward mechanisms. The major neuronal type within the NAc is the GABAergic medium spiny neuron (MSN), whose activity is regulated by dopaminergic inputs. We previously reported that genetic deletion or pharmacological blockade of D 3 R increases GABA A α6 subunit in the ventral striatum. Here we tested the hypothesis that D 3 R-dependent changes in GABA A α6 subunit in the NAc affect voluntary alcohol intake, by influencing the inhibitory transmission of MSNs. We performed in vivo and ex vivo experiments in D 3 R knockout (D 3 R -/- ) mice and wild type littermates (D 3 R +/+ ). Ro 15-4513, a high affinity α6-GABA A ligand was used to study α6 activity. At baseline, NAc α6 expression was negligible in D 3 R +/+ , whereas it was robust in D 3 R -/- ; other relevant GABA A subunits were not changed. In situ hybridization and qPCR confirmed α6 subunit mRNA expression especially in the NAc. In the drinking-in-the-dark paradigm, systemic administration of Ro 15-4513 inhibited alcohol intake in D 3 R +/+ , but increased it in D 3 R -/- ; this was confirmed by intra-NAc administration of Ro 15-4513 and furosemide, a selective α6-GABA A antagonist. Whole-cell patch-clamp showed peak amplitudes of miniature inhibitory postsynaptic currents in NAc medium spiny neurons higher in D 3 R -/- compared to D 3 R +/+ ; Ro 15-4513 reduced the peak amplitude in the NAc of D 3 R -/- , but not in D 3 R +/+ . We conclude that D 3 R-dependent enhanced expression of α6 GABA A subunit inhibits voluntary alcohol intake by increasing GABA inhibition in the NAc., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Extrasynaptic δ subunit-containing γ-aminobutyric acid type A receptors (δ-GABA A Rs) are emerging as targets for a number of neuropsychopharmacological drugs, including the direct GABA site agonist gaboxadol and neuroactive steroids. Among other regions, these δ-GABA A Rs are functionally expressed in the ventral tegmental area (VTA), the cell body region of mesocorticolimbic dopamine (DA) system important for motivated behaviours, and in the target region, the nucleus accumbens. Gaboxadol and neurosteroids induce VTA DA neuron plasticity ex vivo, by inhibiting the VTA GABA neurons, and aversive place conditioning, which are absent in the δ-GABA A R knockout mice (δ-KO). It is not known whether δ-GABA A Rs are important for the effects of other drugs, such as opioids (that also inhibit GABA neurons) and stimulants (that primarily elevate monoamine levels). Here, we used δ-KO mice and conditioned place preference (CPP) test to study the rewarding effects of morphine (20 mg/kg), methamphetamine (1 mg/kg) and mephedrone (5 mg/kg). Morphine-induced nociception was also assessed using tail-flick and hot-plate tests. We found that the δ-KO mice failed to express morphine-induced CPP, but that they were more sensitive to morphine-induced analgesia in the tail-flick test. In contrast, stimulant-induced CPP in the δ-KO mice was similar to that in the wild-type controls. Thus, the conditioned rewarding effect by opioids, but not that of stimulants, was impaired in the absence of δ-GABA A Rs. Further studies are warranted to assess the potential of δ-GABA A R antagonists as possible targets for reducing morphine reward and potentiating morphine analgesia., (© 2018 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

The gabapentinoid pregabalin is a rapid-acting anxiolytic and analgesic, possibly suitable in supervised opioid detoxification. However, clinicians have been cautious in using it because of its unknown addictive risk and rising number of mortalities after pregabalin self-medication in opioid abusers. Here, we studied interactions of pregabalin and morphine on reward functions of the dopamine system in mice and the efficacy of pregabalin on withdrawal in opioid addicts. After the treatment of mice with pregabalin and morphine, we used electrophysiology to study neuroplasticity in midbrain slices, self-administration and conditioned place preference tests to investigate the rewarding potential of pregabalin and naloxone-precipitated morphine withdrawal to evaluate opioid withdrawal symptoms. Further, we ran a pilot single-blind, randomized, controlled trial (34 heroin addicts) to evaluate the efficacy and safety of pregabalin in the treatment of opioid withdrawal syndrome. Pregabalin alone did not induce glutamate receptor neuroplasticity of dopamine neurons in the ventral tegmental area, but pre-treatment with pregabalin suppressed morphine-induced neuroplasticity, hyperlocomotion and morphine self-administration. Pregabalin administration after chronic morphine exposure failed to induce any rewarding effects. Instead, pregabalin suppressed withdrawal symptoms in both morphine-treated mice and opioid addicts and was well tolerated. Intriguingly, pregabalin administration after a low dose of morphine strongly facilitated ventral tegmental area neuroplasticity and led to increased conditioned place preference. Pregabalin appears to have the efficacy to counteract both reinforcing and withdrawal effects of opioids, but it also has a potentiating effect when given to mice with existing opioid levels., (© 2017 Society for the Study of Addiction.)

Opioid antagonist treatments reduce alcohol drinking in rodent models and in alcohol-dependent patients, with variable efficacy across different studies. These treatments may suffer from the development of tolerance and opioid receptor supersensitivity, as suggested by preclinical models showing activation of these processes during and after subchronic high-dose administration of the short-acting opioid antagonist naloxone. In the present study, we compared equipotent low and moderate daily doses of naltrexone and nalmefene, two opioid antagonists in the clinical practice for treatment of alcoholism. The antagonists were given here subcutaneously for 7 days either as daily injections or continuous osmotic minipump-driven infusions to alcohol-preferring AA rats having trained to drink 10% alcohol in a limited access protocol. One day after stopping the antagonist treatment, [ 35 S]GTPγS autoradiography on brain cryostat sections was carried out to examine the coupling of receptors to G protein activation. The results prove the efficacy of repeated injections over infused opioid antagonists in reducing alcohol drinking. Tolerance to the reducing effect on alcohol drinking and to the enhancement of G protein coupling to μ-opioid receptors in various brain regions were consistently detected only after infused antagonists. Supersensitivity of κ-opioid receptors was seen in the ventral and dorsal striatal regions especially by infused nalmefene. Nalmefene showed no clear agonistic activity in rat brain sections or at human recombinant κ-opioid receptors. The findings support the as-needed dosing practice, rather than the standard continual dosing, in the treatment of alcoholism with opioid receptor antagonists., (© 2016 Society for the Study of Addiction.)

Abnormal liver function has dramatic effects on brain functions. Hyperammonemia interferes profoundly with brain metabolism, astrocyte volume regulation, and in particular mitochondrial functions. Gene expression in the brain and excitatory and inhibitory neurotransmission circuits are also affected. Experiments with a number of pertinent animal models have revealed several potential mechanisms which could underlie the pathological phenomena occurring in hepatic encephalopathy.

Endogenous neurosteroids and neuroactive steroids have potent and widespread actions on the brain via inhibitory GABA A receptors. In recombinant receptors and genetic mouse models their actions depend on the α, β, and δ subunits of the receptor, especially on those that form extrasynaptic GABA A receptors responsible for non-synaptic (tonic) inhibition, but they also act on synaptically enriched γ2 subunit-containing receptors and even on αβ binary receptors. Here we tested whether behavioral sensitivity to the neuroactive steroid agonist 5β-pregnan-3α-ol-20-one is altered in genetically engineered mouse models that have deficient GABA A receptor-mediated synaptic inhibition in selected neuronal populations. Mouse lines with the GABA A receptor γ2 subunit gene selectively deleted either in parvalbumin-containing cells (including cerebellar Purkinje cells), cerebellar granule cells, or just in cerebellar Purkinje cells were trained on the accelerated rotating rod and then tested for motor impairment after cumulative intraperitoneal dosing of 5β-pregnan-3α-ol-20-one. Motor-impairing effects of 5β-pregnan-3α-ol-20-one were strongly increased in all three mouse models in which γ2 subunit-dependent synaptic GABA A responses in cerebellar neurons were genetically abolished. Furthermore, rescue of postsynaptic GABA A receptors in Purkinje cells normalized the effect of the steroid. Anxiolytic/explorative effects of the steroid in elevated plus maze and light:dark exploration tests in mice with Purkinje cell γ2 subunit inactivation were similar to those in control mice. The results suggest that, when the deletion of γ2 subunit has removed synaptic GABA A receptors from the specific cerebellar neuronal populations, the effects of neuroactive steroids solely on extrasynaptic αβ or αβδ receptors lead to enhanced changes in the cerebellum-generated behavior.

The increased use of cathinone-type designer drugs, known as legal highs, has led to concerns about their potential neurotoxicity due to their similarity to methamphetamine (METH). Therefore, closer investigations of their toxic effects are needed. We investigated the effects of the cathinones 4-methylmethcathinone (4-MMC) and 3,4-methylenedioxymethcathinone (MDMC) and the amphetamine METH on cytotoxicity and mitochondrial respiration in SH-SY5Y neuroblastoma cells. We also investigated the contribution of reactive species, dopamine, Bcl-2 and tumor necrosis factor α (TNFα) on toxicity. Finally, we investigated the effect of cathinone breakdown products using ultra-high performance liquid chromatography/high-resolution time-of-flight mass spectrometry and studied their involvement in toxicity. We observed dose-dependent increases in cytotoxicity and decreases in mitochondrial respiration following treatment with all cathinones and amphetamines. Glutathione depletion increases amphetamine, but not cathinone toxicity. Bcl-2 and TNFα pathways are involved in toxicity but dopamine levels are not. We also show that cathinones, but not amphetamines, spontaneously produce reactive species and cytotoxic methylbenzamide breakdown products when in aqueous solution. These results provide an important first insight into the mechanisms of cathinone cytotoxicity and pave the way for further studies on cathinone toxicity in vivo.

Adaptation of the nervous system to different chemical and physiologic conditions is important for the homeostasis of brain processes and for learning and remembering appropriate responses to challenges. Although processes such as tolerance and dependence to various drugs of abuse have been known for a long time, it was recently discovered that even a single pharmacologically relevant dose of various drugs of abuse induces neuroplasticity in selected neuronal populations, such as the dopamine neurons of the ventral tegmental area, which persist long after the drug has been excreted. Prolonged (self-) administration of drugs induces gene expression, neurochemical, neurophysiological, and structural changes in many brain cell populations. These region-specific changes correlate with addiction, drug intake, and conditioned drugs effects, such as cue- or stress-induced reinstatement of drug seeking. In rodents, adolescent drug exposure often causes significantly more behavioral changes later in adulthood than a corresponding exposure in adults. Clinically the most impairing and devastating effects on the brain are produced by alcohol during fetal development. In adult recreational drug users or in medicated patients, it has been difficult to find persistent functional or behavioral changes, suggesting that heavy exposure to drugs of abuse is needed for neurotoxicity and for persistent emotional and cognitive alterations. This review describes recent advances in this important area of research, which harbors the aim of translating this knowledge to better treatments for addictions and related neuropsychiatric illnesses., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

A GABA-site agonist gaboxadol (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) at 3 mg/kg induces strong anxiolytic response in a transgenic Thy1α6 mouse line ectopically expressing the GABA(A) receptor α6 subunit gene under the Thy-1.2 promoter. Now, we compared brain activation patterns between Thy1α6 and wild-type mice to identify brain structures potentially mediating this anxiolytic response. Acutely efficient anxiolytics such as benzodiazepines typically depress most brain regions while activating specifically neurons within the central extended amygdala. Gaboxadol treatment (3 mg/kg, i.p., 2 h) induced a significant increase in c-Fos expression selectively in many Thy1α6 brain regions including the limbic cortex, anterior olfactory nucleus, septal area and central and basolateral nuclei of amygdala. It failed to activate the lateral part of mediodorsal thalamic nucleus (MDL) in the Thy1α6 mice that was activated in the wild-type mice. Detailed mapping of the α6 subunit mRNA by in situ hybridization revealed expression in the middle layers of the isocortex, olfactory areas, hippocampal formation and basolateral nucleus of amygdala (BLA) in the Thy1α6 forebrain. The ligand autoradiographies (t-butylbicyclophosphoro[(35)S]thionate ([(35)S]TBPS) and [(3)H]Ro 15-4513) revealed high levels of pharmacologically active extrasynaptic α6β and α6βγ2 GABA(A) receptors in these same areas. However, c-Fos induction by gaboxadol treatment in Thy1α6 brain was not restricted to areas highly expressing the α6-containing GABA(A) receptors suggesting that indirect pathways lead to the paradoxically widespread activation. Interestingly, the activation pattern by gaboxadol at the dose that is anxiolytic in Thy1α6 mice resembled closely that observed after various fear- and stress-provoking challenges. However, our results are consistent with a recent observation that optogenetic activation of specific neuronal pathways in the extended amygdala mediates anxiolytic responses. Our results suggest that the widespread neuronal inhibition as typically associated with benzodiazepines is not the exclusive mechanism of anxiolysis., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

The GABAergic system is the main inhibitory neurotransmitter system in the central nervous system (CNS) of vertebrates. Signalling of the transmitter γ-aminobutyric acid (GABA) via GABA type A receptor channels or G-protein-coupled type B receptors is implicated in multiple CNS functions. Recent findings have implicated the GABAergic system in immune cell functions, inflammatory conditions and diseases in peripheral tissues. Interestingly, the specific effects may vary between immune cell types, with stage of activation and be altered by infectious agents. GABA/GABA-A receptor-mediated immunomodulatory functions have been unveiled in immune cells, being present in T lymphocytes and regulating the migration of Toxoplasma-infected dendritic cells. The GABAergic system may also play a role in the regulation of brain resident immune cells, the microglial cells. Activation of microglia appears to regulate the function of GABAergic neurotransmission in neighbouring neurones through changes induced by secretion of brain-derived neurotrophic factor. The neurotransmitter-driven immunomodulation is a new but rapidly growing field of science. Herein, we review the present knowledge of the GABA signalling in immune cells of the periphery and the CNS and raise questions for future research., (© 2015 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

Malfunction of glutamate transmission is implicated in several neuropsychiatric disorders. Gria1-/- mouse line with knocked-out GluA1 subunits of ionotropic AMPA glutamate receptor displays several behavioural features of schizoaffective disorder. Typically, these mice show hyperactivity provoked by environmental novelty, which is attenuated after 4-week treatment with the standard mood-stabilisers lithium and valproate and the mood-stabilising anticonvulsants topiramate and lamotrigine (Maksimovic, M., Vekovischeva, O.Y., Aitta-Aho, T., Korpi, E.R., 2014. Chronic treatment with mood-stabilizers attenuates abnormal hyperlocomotion of GluA1-subunit deficient mice. PloS One. 9, e100188). Here, we complement our study by treating these mice chronically with perampanel, a novel non-competitive antagonist of AMPA receptors, for 4 weeks at the dose of 60 mg/kg diet, and found reduced locomotor hyperactivity in the Gria1-/- animals, while not affecting the wild-type littermates. To study the cellular mechanism by which chronic treatments with glutamate-modulating mood-stabilizing drugs alleviate this hyperactivity, we used the immediate early gene c-Fos protein expression as a marker of neuronal activity in the brain. Chronic lithium, valproate and topiramate blunted the c-Fos expression especially in the dorsal hippocampus of the Gria1-/- mice, with all of them reducing the number of c-Fos-positive cells in the CA3 region and valproate and topiramate also in the dentate gyrus (DG). Lamotrigine and perampanel treatments had the same effect in the all CA1, CA3 and DG subfields of the dorsal hippocampus of Gria1-/- mice. The results suggest that abnormal (hippocampal) glutamatergic transmission underlies the hyperactive phenotype of the Gria1-/- mice in a novel environment, and based on the efficacies of the present chronic drug treatments, this mouse model may serve as a predictive tool for studying novel mood-stabilisers., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: In recent years there has been a large increase in the use of substituted cathinones such as mephedrone (4-methylmethcathinone, 4-MMC), a psychostimulant drug that shows a strong resemblance to methamphetamine (METH). Unlike METH, which can produce clear long-term effects, the effects of 4-MMC have so far remained elusive. We employ manganese-enhanced magnetic resonance imaging (MEMRI), a highly sensitive method for detecting changes in neuronal activation, to investigate the effects of METH and 4-MMC on the brain., Methods: In Wistar rats we performed a MEMRI scan two weeks after binge treatments (twice daily for 4 consecutive days) of METH (5 mg/kg) or 4-MMC (30 mg/kg). Furthermore, locomotor activity measurements and novel object recognition tests were performed., Results: METH produced a widespread pattern of decreased bilateral activity in several regions, including the nucleus accumbens, caudate putamen, globus pallidus, thalamus, and hippocampus, as well as several other cortical and subcortical areas. Conversely, 4-MMC produced increased bilateral activity, anatomically limited to the hypothalamus and hippocampus. Drug treatments did not affect the development of locomotor sensitization or novel object recognition performance., Conclusions: The pattern of decreased brain activity seen after METH corresponds closely to regions known to be affected by this drug and confirms the validity of MEMRI for detecting neuroadaptation two weeks after amphetamine binge treatment. 4-MMC, unlike METH, produced increased activity in a limited number of different brain regions. This highlights an important difference in the long-term effects of these drugs on neural function and shows precisely the anatomical localization of 4-MMC-induced neuroadaptation., (© The Author 2015. Published by Oxford University Press on behalf of CINP.)

Chronic consumption of alcohol by humans has been shown to lead to impairment of executive and cognitive functions. Here, we have studied the mRNA expression of ion channel receptors for glutamate and GABA in the dorsal striatum of post-mortem brains from alcoholics (n = 29) and normal controls (n = 29), with the focus on the caudate nucleus that is associated with the frontal cortex executive functions and automatic thinking and on the putamen area that is linked to motor cortices and automatic movements. The results obtained by qPCR assay revealed significant changes in the expression of specific excitatory ionotropic glutamate and inhibitory GABA-A receptor subunit genes in the caudate but not the putamen. Thus, in the caudate we found reduced levels of mRNAs encoding the GluN2A glutamate receptor and the δ, ε, and ρ2 GABA-A receptor subunits, and increased levels of the mRNAs encoding GluD1, GluD2, and GABA-A γ1 subunits in the alcoholics as compared to controls. Interestingly in the controls, 11 glutamate and 5 GABA-A receptor genes were more prominently expressed in the caudate than the putamen (fold-increase varied from 1.24 to 2.91). Differences in gene expression patterns between the striatal regions may underlie differences in associated behavioral outputs. Our results suggest an altered balance between caudate-mediated voluntarily controlled and automatic behaviors in alcoholics, including diminished executive control on goal-directed alcohol-seeking behavior.

GABAA receptors are the main fast inhibitory neurotransmitter receptors in the mammalian brain, and targets for many clinically important drugs widely used in the treatment of anxiety disorders, insomnia and in anesthesia. Nonetheless, there are significant risks associated with the long-term use of these drugs particularly related to development of tolerance and addiction. Addictive mechanisms of GABAA receptor drugs are poorly known, but recent findings suggest that those drugs may induce aberrant neuroadaptations in the brain reward circuitry. Recently, benzodiazepines, acting on synaptic GABAA receptors, and modulators of extrasynaptic GABAA receptors (THIP and neurosteroids) have been found to induce plasticity in the ventral tegmental area (VTA) dopamine neurons and their main target projections. Furthermore, depending whether synaptic or extrasynaptic GABAA receptor populations are activated, the behavioral outcome of repeated administration seems to correlate with rewarding or aversive behavioral responses, respectively. The VTA dopamine neurons project to forebrain centers such as the nucleus accumbens and medial prefrontal cortex, and receive afferent projections from these brain regions and especially from the extended amygdala and lateral habenula, forming the major part of the reward and aversion circuitry. Both synaptic and extrasynaptic GABAA drugs inhibit the VTA GABAergic interneurons, thus activating the VTA DA neurons by disinhibition and this way inducing glutamatergic synaptic plasticity. However, the GABAA drugs failed to alter synaptic spine numbers as studied from Golgi-Cox-stained VTA dendrites. Since the GABAergic drugs are known to depress the brain metabolism and gene expression, their likely way of inducing neuroplasticity in mature neurons is by disinhibiting the principal neurons, which remains to be rigorously tested for a number of clinically important anxiolytics, sedatives and anesthetics in different parts of the circuitry.

The central amygdala (CeA) has a role for mediating fear and anxiety responses. It is also involved in emotional imbalance caused by alcohol abuse and dependence and in regulating relapse to alcohol abuse. Growing evidences suggest that excitatory glutamatergic and inhibitory γ-aminobutyric acid-ergic (GABAergic) transmissions in the CeA are affected by chronic alcohol exposure. Human post-mortem CeA samples from male alcoholics (n = 9) and matched controls (n = 9) were assayed for the expression level of ionotropic glutamate and GABA-A receptors subunit mRNAs using quantitative real-time reverse transcription-PCR (RT-qPCR). Our data revealed that out of the 16 ionotropic glutamate receptor subunits, mRNAs encoding two AMPA [2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid] receptor subunits GluA1 and GluA4; one kainate receptor subunit GluK2; one NMDA (N-methyl-D-aspartate) receptor subunit GluN2D and one delta receptor subunit GluD2 were significantly decreased in the CeA of alcoholics. In contrast, of the 19 GABA-A receptor subunits, only the mRNA encoding the α2 subunit was significantly down-regulated in the CeA of the alcoholics as compared with control subjects. Our findings imply that the down-regulation of specific ionotropic glutamate and GABA-A receptor subunits in the CeA of alcoholics may represent one of the molecular substrates underlying the new balance between excitatory and inhibitory neurotransmission in alcohol dependence.

The β-keto amphetamine (cathinone, β-KA) designer drugs such as mephedrone (4-methylmethcathinone, 4-MMC) show a large degree of structural similarity to amphetamines like methamphetamine (METH). However, little is currently known about whether these substances also share the potential neurotoxic properties of their non-keto amphetamine counterparts, or what mechanisms could be involved. Here, we evaluate the cytotoxicity of β-KAs in SH-SY5Y cells using lactate dehydrogenase (LDH) assays, assess the redox potential of a range of β-KAs and non-keto amphetamines using the sensitive redox indicator 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1), and explore the effect of 4-MMC on the formation of protein adducts using ultra-high performance liquid chromatography/high-resolution time-of-flight mass spectrometry (UHPLC-HR-TOFMS) and on the mitochondrial respiratory chain using high-resolution respirometry. We show that treatment with β-KAs increases LDH release. Further, we demonstrate that even under physiological pH, β-KAs are effective and selective-as compared with their non-keto analogues-reductants in the presence of electron acceptors. Increased pH (range 7.6-8.0) greatly enhanced the reactivity up to sixfold. We found no evidence of protein adduct formation, suggesting the reactivity is due to direct electron transfer by the β-KAs. Finally, we show that 4-MMC and METH produce dissimilar effects on the respiratory chain. Our results indicate that β-KAs such as 4-MMC possess cytotoxic properties in vitro. Furthermore, in the presence of an electron-accepting redox partner, the ketone moiety of β-KAs is vital for pH-dependent redox reactivity. Further work is needed to establish the importance of β-KA redox properties and its potential toxicological importance in vivo., (© The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Abnormal excitatory glutamate neurotransmission and plasticity have been implicated in schizophrenia and affective disorders. Gria1-/- mice lacking GluA1 subunit (encoded by Gria1 gene) of AMPA-type glutamate receptor show robust novelty-induced hyperactivity, social deficits and heightened approach features, suggesting that they could be used to test for anti-manic activity of drugs. Here, we tested the efficacy of chronic treatment with established anti-manic drugs on behavioural properties of the Gria1-/- mice. The mice received standard mood stabilizers (lithium and valproate) and novel ones (topiramate and lamotrigine, used more as anticonvulsants) as supplements in rodent chow for at least 4 weeks. All drugs attenuated novelty-induced locomotor hyperactivity of the Gria1-/- mice, especially by promoting the habituation, while none of them attenuated 2-mg/kg amphetamine-induced hyperactivity as compared to control diet. Treatment with lithium and valproate reversed the elevated exploratory activity of Gria1-/- mice. Valproate treatment also reduced struggling behaviour in tail suspension test and restored reciprocally-initiated social contacts of Gria1-/- mice to the level shown by the wild-type Gria1+/+ mice. Gria1-/- mice consumed slightly more sucrose during intermittent sucrose exposure than the wild-types, but ran similar distances on running wheels. These behaviours were not consistently affected by lithium and valproate in the Gria1-/- mice. The efficacy of various anti-manic drug treatments on novelty-induced hyperactivity suggests that the Gria1-/- mouse line can be utilized in screening for new therapeutics.

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) is an autosomal recessively inherited neurodegenerative disease, manifesting with myoclonus, seizures and ataxia, caused by mutations in the cystatin B (CSTB) gene. With the aim of understanding the molecular basis of pathogenetic events in EPM1 we characterized gene expression changes in the cerebella of pre-symptomatic postnatal day 7 (P7) and symptomatic P30 cystatin B -deficient (Cstb(-/-) ) mice, a model for the disease, and in cultured Cstb(-/-) cerebellar granule cells using a pathway-based approach. Differentially expressed genes in P7 cerebella were connected to synaptic function and plasticity, and in cultured cerebellar granule cells, to cell cycle, cytoskeleton, and intracellular transport. In particular, the gene expression data pinpointed alterations in GABAergic pathway. Electrophysiological recordings from Cstb(-/-) cerebellar Purkinje cells revealed a shift of the balance towards decreased inhibition, yet the amount of inhibitory interneurons was not declined in young animals. Instead, we found diminished number of GABAergic terminals and reduced ligand binding to GABAA receptors in Cstb(-/-) cerebellum. These results suggest that alterations in GABAergic signaling could result in reduced inhibition in Cstb(-/-) cerebellum leading to the hyperexcitable phenotype of Cstb(-/-) mice. At P30, the microarray data revealed a marked upregulation of immune and defense response genes, compatible with the previously reported early glial activation that precedes neuronal degeneration. This further implies the role of early-onset neuroinflammation in the pathogenesis of EPM1.

The main fast-acting inhibitory receptors in the mammalian brain are γ-aminobutyric acid type-A (GABAA) receptors for which neurosteroids, a subclass of steroids synthesized de novo in the brain, constitute a group of endogenous ligands with the most potent positive modulatory actions known. Neurosteroids can act on all subtypes of GABAA receptors, with a preference for δ-subunit-containing receptors that mediate extrasynaptic tonic inhibition. Pathological conditions characterized by emotional and motivational disturbances are often associated with perturbation in the levels of endogenous neurosteroids. We studied the effects of ganaxolone (GAN)-a synthetic analog of endogenous allopregnanolone that lacks activity on nuclear steroid receptors-on the mesolimbic dopamine (DA) system involved in emotions and motivation. A single dose of GAN in young mice induced a dose-dependent, long-lasting neuroplasticity of glutamate synapses of DA neurons ex vivo in the ventral tegmental area (VTA). Increased α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/N-methyl-D-aspartate ratio and rectification of AMPA receptor responses even at 6 days after GAN administration suggested persistent synaptic targeting of GluA2-lacking AMPA receptors. This glutamate neuroplasticity was not observed in GABAA receptor δ-subunit-knockout (δ-KO) mice. GAN (500 nM) applied locally to VTA selectively increased tonic inhibition of GABA interneurons and triggered potentiation of DA neurons within 4 h in vitro. Place-conditioning experiments in adult wild-type C57BL/6J and δ-KO mice revealed aversive properties of repeated GAN administration that were dependent on the δ-subunits. Prolonged neuroadaptation to neurosteroids in the VTA might contribute to both the physiology and pathophysiology underlying processes and changes in motivation, mood, cognition, and drug addiction.

Glutamate is the main excitatory transmitter in the human brain. Drugs that affect the glutamatergic signaling will alter neuronal excitability. Ethanol inhibits glutamate receptors. We examined the expression level of glutamate receptor subunit mRNAs in human post-mortem samples from alcoholics and compared the results to brain samples from control subjects. RNA from hippocampal dentate gyrus (HP-DG), orbitofrontal cortex (OFC), and dorso-lateral prefrontal cortex (DL-PFC) samples from 21 controls and 19 individuals with chronic alcohol dependence were included in the study. Total RNA was assayed using quantitative RT-PCR. Out of the 16 glutamate receptor subunits, mRNAs encoding two AMPA [2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid] receptor subunits GluA2 and GluA3; three kainate receptor subunits GluK2, GluK3 and GluK5 and five NMDA (N-methyl-D-aspartate) receptor subunits GluN1, GluN2A, GluN2C, GluN2D, and GluN3A were significantly increased in the HP-DG region in alcoholics. In the OFC, mRNA encoding the NMDA receptor subunit GluN3A was increased, whereas in the DL-PFC, no differences in mRNA levels were observed. Our laboratory has previously shown that the expression of genes encoding inhibitory GABA-A receptors is altered in the HP-DG and OFC of alcoholics (Jin et al., 2011). Whether the changes in one neurotransmitter system drives changes in the other or if they change independently is currently not known. The results demonstrate that excessive long-term alcohol consumption is associated with altered expression of genes encoding glutamate receptors in a brain region-specific manner. It is an intriguing possibility that genetic predisposition to alcoholism may contribute to these gene expression changes.

Dysfunctional glutamatergic neurotransmission has been implicated in schizophrenia and mood disorders. As a putative model for these disorders, a mouse line lacking the GluA1 subunit (GluA1-KO) of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor displays a robust novelty-induced hyperlocomotion associated with excessive neuronal activation in the hippocampus. Agonists of metabotropic glutamate 2/3 receptors (mGluR2/3) inhibit glutamate release in various brain regions and they have been shown to inhibit neuronal activation in the hippocampus. Here, we tested a hypothesis that novelty-induced hyperlocomotion in the GluA1-KO mice is mediated via excessive hippocampal neuronal activation by analyzing whether an mGluR2/3 agonist inhibits this phenotypic feature. GluA1-KO mice and littermate wildtype (WT) controls were administered with (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY354740) (15 mg/kg, i.p.) 30 min before a 2-h exposure to novel arenas after which c-Fos immunopositive cells were analyzed in the hippocampus. LY354740 (15 mg/kg) decreased hyperactivity in male GluA1-KO mice, with only a minimal effect in WT controls. This was observed in two cohorts of animals, one naïve to handling and injections, another pre-handled and accustomed to injections. LY354740 (15 mg/kg) also reduced the excessive c-Fos expression in the dorsal hippocampal CA1 pyramidal cell layer in maleGluA1-KO mice, while not affecting c-Fos levels in WT mice. In female mice, no significant effect for LY354740 (15 mg/kg) on hyperactive behavior or hippocampal c-Fos was observed in either genotype or treatment cohort. A higher dose of LY354740 (30 mg/kg) alleviated hyperlocomotion of GluA1-KO males, but not that of GluA1-KO females. In conclusion, the excessive behavioral hyperactivity of GluA1-KO mice can be partly prevented by reducing neuronal excitability in the hippocampus with the mGluR2/3 agonist suggesting that the hippocampal reactivity is strongly involved in the behavioral phenotype of GluA1-KO mice., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

Introduction/aims: The use of cathinone-derivative designer drugs methylone and mephedrone has increased rapidly in recent years. Our aim was to investigate the possible long-term effects of these drugs on a range of behavioral tests in mice. Further, we investigated the long-term effects of these drugs on brain neurochemistry in both rats and mice., Methods: We treated animals with a binge-like regimen of methylone or mephedrone (30 mg/kg, twice daily for 4 days) and, starting 2 weeks later, we performed behavioral tests of memory, anxiety and depression and measured brain levels of dopamine (DA), serotonin (5-HT), their metabolites and norepinephrine (NE). 5-HT and DA transporter (5-HTT and DAT) levels were also measured in rats by [(3)H]paroxetine and [(3)H]mazindol binding., Results: Mephedrone reduced working memory performance in the T-maze spontaneous alternation task but did not affect neurotransmitter levels aside from a 22% decrease in striatal homovanillic acid (HVA) levels in mice. Methylone had little effect on behavior or neurotransmitter levels in mice but produced a widespread depletion of 5-HT and 5-HTT levels in rats., Conclusions: Both methylone and mephedrone appeared to have a long-term effect on either behavioral or biochemical gauges of neurotoxicity in rodents., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Several studies have revealed that acute ethanol inhibits the function of glutamate receptors. Glutamate receptor-mediated 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 α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid AMPA receptors seem to affect ethanol inhibition thus opening new lines of research., (© 2012 The Authors Basic & Clinical Pharmacology & Toxicology © 2012 Nordic Pharmacological Society.)

Dopamine neurons of the ventral tegmental area (VTA) are involved at early phases of drug addiction. Even the first in vivo dose of various abused drugs induces glutamate receptor plasticity at the excitatory synapses of these neurons. Benzodiazepines that suppress the inhibitory GABAergic interneurons in the VTA via facilitation of synaptic GABA(A) receptors have induced neuroplasticity in dopamine neurons due to this disinhibitory mechanism. Here, we have tested a non-benzodiazepine direct GABA site agonist 4,5,6,7-tetrahydroisoxazolol[4,5-c]pyridine-3-ol (THIP) (also known as gaboxadol) that acts preferentially via high-affinity extrasynaptic GABA(A) receptors. A single sedative dose of THIP (6 mg/kg) to mice induced glutamate receptor plasticity for at least 6 d after administration. Increased AMPA/NMDA receptor current ratio and increased frequency, amplitude, and rectification of AMPA receptor responses suggested persistent targeting of GluA2-lacking AMPA receptors in excitatory synapses of VTA dopamine neurons ex vivo after THIP administration. This effect was abolished in GABA(A) receptor δ(-/-) mice, which have a loss of extrasynaptic GABA(A) receptors. In behavioral experiments, we found neither acute reinforcement in intravenous self-administration sessions with THIP at relevant doses using a yoked control paradigm in mice nor in baboons using a standard paradigm for assessing drug abuse liability; nor was any place preference found after conditioning sessions with various doses of THIP but rather a persistent aversion in 6 mg/kg THIP-conditioned mice. In summary, we found that activation of extrasynaptic δ-subunit-containing GABA(A) receptors leads to glutamate receptor plasticity of VTA dopamine neurons, but is not rewarding, and, instead, induces aversion.

The classical benzodiazepine diazepam (DZ) induces anxiolysis at low doses and sedation and hypnosis at higher doses. Different brain areas and neuronal populations most likely mediate these different behavioral effects. We used c-Fos immunohistochemistry as an indirect way to study neuronal activation or inhibition induced by DZ at anxiolytic and sedative doses (0.5 and 5mg/kg, respectively) in various brain areas involved in anxiety, arousal, sedation and addiction in C57BL/6J mice. We also focused on the two neuronal populations, orexinergic and dopaminergic neuronal populations, with the help of double-immunohistochemistry using c-Fos and orexin-A antibodies and c-Fos and tyrosine hydroxylase antibodies. We found that different brain areas of unhabituated mice reacted differently to the mild stress induced by vehicle injection. Also the response to anxiolytic or sedative doses of DZ differed between the areas, suggesting that distinct brain areas mediate the behavioral effects of low and high DZ doses. Our findings propose a role for inhibition of orexin neurons in the anxiolytic and sleep-promoting effects of DZ. In addition, the activation of central amygdala neurons by DZ treatment was associated with anxiolytic and sedative effects. On the other hand, the ventral hippocampus, basolateral amygdala, ventral tegmental area and prefrontal cortex were sensitive even to the mild injection stress, but not to the anxiolytic dose of DZ., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Alcohol dependence is a common chronic relapsing disorder. The development of alcohol dependence has been associated with changes in brain GABA(A) channel-mediated neurotransmission and plasticity. We have examined mRNA expression of the GABA(A) channel subunit genes in three brain regions in individuals with or without alcohol dependence using quantitative real-time PCR assay. The levels of selective GABA(A) channel subunit mRNAs were altered in specific brain regions in alcoholic subjects. Significant increase in the α1, α4, α5, β1, and γ1 subunit mRNAs in the hippocampal dentate gyrus region, and decrease in the β2 and δ subunit mRNAs in the orbitofrontal cortex were identified whereas no changes in the dorsolateral prefrontal cortex were detected. The data increase our understanding of the role of GABA(A) channels in the development of alcohol dependence.

Agonist responses and channel kinetics of native α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors are modulated by transmembrane accessory proteins. Stargazin, the prototypical accessory protein, decreases desensitization and increases agonist potency at AMPA receptors. Furthermore, in the presence of stargazin, the steady-state responses of AMPA receptors show a gradual decline at higher glutamate concentrations. This "autoinactivation" has been assigned to physical dissociation of the stargazin-AMPA receptor complex and suggested to serve as a protective mechanism against overactivation. Here, we analyzed autoinactivation of GluA1-A4 AMPA receptors (all flip isoform) expressed in the presence of stargazin. Homomeric GluA1, GluA3, and GluA4 channels showed pronounced autoinactivation indicated by the bell-shaped steady-state dose response curves for glutamate. In contrast, homomeric GluA2i channels did not show significant autoinactivation. The resistance of GluA2 to autoinactivation showed striking dependence on the splice form as GluA2-flop receptors displayed clear autoinactivation. Interestingly, the resistance of GluA2-flip containing receptors to autoinactivation was transferred onto heteromeric receptors in a dominant fashion. To examine the relationship of autoinactivation to physical separation of stargazin from the AMPA receptor, we analyzed a GluA4-stargazin fusion protein. Notably, the covalently linked complex and separately expressed proteins expressed a similar level of autoinactivation. We conclude that autoinactivation is a subunit and splice form dependent property of AMPA receptor-stargazin complexes, which involves structural rearrangements within the complex rather than any physical dissociation.

In state-dependency, information retrieval is most efficient when the animal is in the same state as it was during the information acquisition. State-dependency has been implicated in a variety of learning and memory processes, but its mechanisms remain to be resolved. Here, mice deficient in AMPA-type glutamate receptor GluA1 subunits were first conditioned to morphine (10 or 20 mg/kg s.c. during eight sessions over four days) using an unbiased procedure, followed by testing for conditioned place preference at morphine states that were the same as or different from the one the mice were conditioned to. In GluA1 wildtype littermate mice the same-state morphine dose produced the greatest expression of place preference, while in the knockout mice no place preference was then detected. Both wildtype and knockout mice expressed moderate morphine-induced place preference when not at the morphine state (saline treatment at the test); in this case, place preference was weaker than that in the same-state test in wildtype mice. No correlation between place preference scores and locomotor activity during testing was found. Additionally, as compared to the controls, the knockout mice showed unchanged sensitization to morphine, morphine drug discrimination and brain regional μ-opioid receptor signal transduction at the G-protein level. However, the knockout mice failed to show increased AMPA/NMDA receptor current ratios in the ventral tegmental area dopamine neurons of midbrain slices after a single injection of morphine (10 mg/kg, s.c., sliced prepared 24 h afterwards), in contrast to the wildtype mice. The results indicate impaired drug-induced state-dependency in GluA1 knockout mice, correlating with impaired opioid-induced glutamate receptor neuroplasticity.