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In the brain, extrasynaptically expressed ionotropic, δ subunit-containing γ-aminobutyric acid A-type receptors (δ-GABAARs) 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-GABAARs (δ-KO) has been used to study the roles of δ-GABAARs in brain functions, because a specific antagonist of the δ-GABAARs is still lacking. We have previously observed with these δ-KO mice that activation of δ-GABAARs is needed for morphine-induced conditioning of place preference, and others have suggested that δ-GABAARs 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-GABAARs, 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-GABAARs, our findings on pharmaco-EEG effects of 4-MMC suggest that δ-GABAARs are involved in the secondary indirect regulation of the brain rhythms after 4-MMC.

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.

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 GABAA 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 GABAA 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 GABAA receptors are involved in mediating GABA response.