Search

Cholinergic drugs acting at M1/M4 muscarinic receptors hold promise for the treatment of symptoms associated with brain disorders characterized by cognitive impairment, mood disturbances or psychosis, such as Alzheimer’s disease or schizophrenia. However, the brain-wide functional substrates engaged by muscarinic agonists remain poorly understood. Here we used a combination of pharmacological fMRI (phMRI), resting-state fMRI (rsfMRI) and resting-state quantitative EEG (qEEG) to investigate the effects of a behaviorally-active dose of M1/M4 agonist xanomeline on brain functional activity in the rodent brain. We investigated both the effects of xanomeline per se and its modulatory effects on signals elicited by the NMDA-receptor antagonists phencyclidine (PCP) and ketamine. We found that xanomeline induces robust and widespread BOLD signal phMRI amplitude increases and decreased high frequency qEEG spectral activity. rsfMRI mapping in the mouse revealed that xanomeline robustly decreased neocortical and striatal connectivity but induces focal increases in functional connectivity within the nucleus accumbens and basal forebrain. Notably, xanomeline pre-administration robustly attenuated both the cortico-limbic phMRI response and the fronto-hippocampal hyper-connectivity induced by PCP, enhanced PCP-modulated functional connectivity locally within the nucleus accumbens and basal forebrain, and reversed the gamma and high frequency qEEG power increases induced by ketamine. Collectively, these results show that xanomeline robustly induces both cholinergic-like neocortical activation and desynchronization of functional networks in the mammalian brain. These effects could serve as a translatable biomarker for future clinical investigations of muscarinic agents, and bear mechanistic relevance for the putative therapeutic effect of these class of compounds in brain disorders.

One of sleep’s putative functions is mediation of adaptation to waking experiences. Chronic stress is a common waking experience, however, which specific aspect of sleep is most responsive, and how sleep changes relate to behavioral disturbances and molecular correlates remain unknown. We quantified sleep, physical, endocrine and behavioral variables and the brain and blood transcriptome in mice exposed to nine weeks of unpredictable chronic mild stress (UCMS). Comparing 46 phenotypical variables revealed that rapid-eye-movement sleep (REMS), corticosterone regulation and coat state were most responsive to UCMS. REMS theta oscillations were enhanced whereas delta oscillations in non-REMS were unaffected. Transcripts affected by UCMS in the prefrontal cortex, hippocampus, hypothalamus and blood were associated with inflammatory and immune responses. A machine learning approach controlling for unspecific UCMS effects identified transcriptomic predictors for specific phenotypes and their overlap. Transcriptomic predictor sets for the inter-individual variation in REMS continuity and theta activity shared many pathways with corticosterone regulation and in particular pathways implicated in apoptosis, including mitochondrial pathways. Predictor sets for REMS and anhedonia, one of the behavioral changes following UCMS, shared pathways involved in oxidative stress, cell proliferation and apoptosis. RNA predictor sets for non-NREMS parameters showed no overlap with other phenotypes. These novel data identify REMS as a core and early element of the response to chronic stress, and identify apoptotic pathways as a putative mechanism by which REMS mediates adaptation to stressful waking experiences.Significance StatementSleep is responsive to experiences during wakefulness and is altered in stress-related disorders. Whether sleep changes primarily concern rapid-eye-movement sleep (REMS) or non-REM sleep, and how they correlate with stress hormones, behavioral and transcriptomic responses remained unknown. We demonstrate using unpredictable chronic (9-weeks) mild stress that REMS is the most responsive of all the measured sleep characteristics, and correlates with deficiency in corticosterone regulation. An unbiased machine learning, controlling for unspecific effects of stress, revealed that REMS correlated with RNA predictor sets enriched in apoptosis including mitochondrial pathways. Several pathways were shared with predictors of corticosterone and behavioral responses. This unbiased approach point to apoptosis as a molecular mechanism by which REMS mediates adaptation to an ecologically relevant waking experience.

Group II metabotropic glutamate receptors (mGluR2 and mGluR3) are implicated in a number of psychiatric disorders. They also control sleep-wake architecture and may offer novel therapeutic targets. However, the roles of the mGluR2 versus mGluR3 subtypes are not well understood. Here, we have taken advantage of the recently described mutant strain of Han Wistar rats, which do not express mGluR2 receptors, to investigate behavioural, sleep and EEG responses to mGluR2/3 ligands. The mGluR2/3 agonist, LY354740 (10 mg/kg), reversed amphetamine- and phencyclidine-induced locomotion and rearing behaviours in control Wistar but not in mGluR2 lacking Han Wistar rats. In control Wistar but not in Han Wistar rats the mGluR2/3 agonist LY379268 (3 & 10 mg/kg) induced REM sleep suppression with dose-dependent effects on wake and NREM sleep. By contrast, the mGluR2/3 antagonist LY3020371 (3 & 10 mg/kg) had wake-promoting effects in both rat strains, albeit smaller in the mGluR2-lacking Han Wistar rats, indicating both mGluR2 and mGluR3-mediated effects on wakefulness. LY3020371 enhanced wake cortical oscillations in the theta (4–9 Hz) and gamma (30–80 Hz) range in both Wistar and Han Wistar rat strains, whereas LY379268 reduced theta and gamma oscillations in control Wistar rats, with minimal effects in Han Wistar rats. Together these studies illustrate the significant contribution of mGluR2 to the antipsychotic-like, sleep and EEG effects of drugs acting on group II mGluRs. However, we also provide evidence of a role for mGluR3 activity in the control of sleep and wake cortical theta and gamma oscillations.

Several recent studies have shown that reducing gamma-aminobutyric acid (GABA)-mediated neurotransmission retards extinction of aversive conditioning. However, relatively little is known about the effect of GABA on extinction of appetitively motivated tasks. We examined the effect of chlordiazepoxide (CDP), a classical benzodiazepine (BZ) and two novel subtype-selective BZs when administered to male C57Bl/6 mice during extinction following training on a discrete-trial fixed-ratio 5 (FR5) food reinforced lever-press procedure. Initially CDP had no effect, but after several extinction sessions CDP significantly facilitated extinction, i.e. slowed responding, compared with vehicle-treated mice. This effect was not due to drug accumulation because mice switched from vehicle treatment to CDP late in extinction showed facilitation immediately. Likewise, this effect could not be attributed to sedation because the dose of CDP used (15 mg/kg i.p.) did not suppress locomotor activity. The two novel subtype-selective BZ partial agonists, L-838417 and TP13, selectively facilitated extinction in similar fashion to CDP. The non-GABAergic anxiolytic buspirone was also tested and found to have similar effects when administered at a non-sedating dose. These studies demonstrate that GABA-mediated processes are important during extinction of an appetitively motivated task, but only after the animals have experienced several extinction sessions.

1. Human GABAA receptors containing different alpha and beta subunits with a gamma 2s subunit were expressed in Xenopus oocytes and the effects of pentobarbitone on these subunit combinations were examined by electrophysiological recording of GABA currents with the two-electrode voltage-clamp method. 2. Pentobarbitone has previously been shown to have three actions on GABAA receptors: a potentiation of GABA responses, a direct activation of GABAA receptors and, at high concentrations, a block of the GABA chloride channel. In this study pentobarbitone activity consisted of the above mentioned three components on all the subunit combinations tested. However, the affinities and efficacies varied with receptor subtype. 3. Potentiation of GABA by pentobarbitone occurred over the same concentration-range for all the subunits with affinities in the range of 20-35 microM. The degree of potentiation obtained, however, varied from 236% of GABA EC20 on alpha 1 beta 2 gamma 2s to 536% on alpha 6 beta 2 gamma 2s. 4. Examination of the direct effect of pentobarbitone revealed that the type of alpha subunit present determines both the degree of affinity and efficacy obtained. Receptors containing an alpha 6 subunit produced maximum direct responses to pentobarbitone larger than that obtainable with maximum GABA (150% to 170% of maximum GABA). The maximum direct pentobarbitone response obtainable with other alpha subunits ranged between 45% of maximum GABA for alpha 5 beta 2 gamma 2s to 82% for alpha 2 beta 2 gamma 2s. The affinity of the direct action of pentobarbitone on alpha 6 beta 2 gamma 2s was 58 microM compared to affinities for the other alpha subunits ranging from 139 microM on alpha 2 beta 2 gamma 2s to 528 microM on alpha 5 beta 2 gamma 2s. 5. The type of beta subunit present did not influence the direct action of pentobarbitone to the same extent as the alpha subunit. There were no significant differences between affinity or efficacy on oocytes expressing alpha 6 and gamma 2s with beta 1, beta 2 or beta 3. Affinities and efficacies on oocytes expressing alpha 1 and gamma 2s with beta 1, beta 2 or beta 3 were significantly different with pentobarbitone having a higher affinity and efficacy on alpha 1 beta 3 gamma 2s followed by alpha 1 beta 2 gamma 2s and then alpha 1 beta 1 gamma 2s. 6. The direct effect of pentobarbitone was blocked by picrotoxin but not by competitive antagonists, such as bicuculline or SR95531, indicating that the direct agonist activity of pentobarbitone was not mediated via the GABA binding site. 7. For the first time the influence of the various alpha and beta subunits on the effects of pentobarbitone were demonstrated. The results indicate that GABAA receptors containing alpha 6 subunits have both a higher affinity and efficacy for direct activation by pentobarbitone, and reveal that pentobarbitone binds to more than one site on the GABAA receptor, and these are dependent on receptor subunit composition.

Most antidepressants suppress rapid eye movement (REM) sleep, which is thought to be important to brain function, yet the resulting REM sleep restriction is well tolerated. This study investigated the impact of antidepressants with different mechanisms of action, such as selective serotonin reuptake inhibitors (SSRIs) and tricyclic antidepressants (TCA), on the regulation of REM sleep in rats. REM sleep was first demonstrated to be homeostatically regulated using 5, 8 and 10 h of REM-sleep specific restriction through EEG-triggered arousals, with an average of 91 ± 10% of lost REM sleep recovered following a 26–29 -hour recovery period. Acute treatment with the antidepressants paroxetine, citalopram and imipramine inhibited REM sleep by 84 ± 8, 84 ± 8 and 69 ± 9% respectively relative to vehicle control. The pharmacologically-induced REM sleep deficits by paroxetine and citalopram were not fully recovered, whereas, after imipramine the REM sleep deficit was fully compensated. Given the marked difference between REM sleep recovery following the administration of paroxetine, citalopram, imipramine and REM sleep restriction, the homeostatic response was further examined by pairing REM sleep specific restriction with the three antidepressants. Surprisingly, the physiologically-induced REM sleep deficits incurred prior to suppression of REM sleep by all antidepressants was consistently recovered. The data indicate that REM sleep homeostasis remains operative following subsequent treatment with antidepressants and is unaffected by additional pharmacological inhibition of REM sleep.

SummaryRhythmic neural network activity patterns are defining features of sleep, but interdependencies between limbic and cortical oscillations at different frequencies and their functional roles have not been fully resolved. This is particularly important given evidence linking abnormal sleep architecture and memory consolidation in psychiatric diseases. Using EEG, local field potential (LFP), and unit recordings in rats, we show that anteroposterior propagation of neocortical slow-waves coordinates timing of hippocampal ripples and prefrontal cortical spindles during NREM sleep. This coordination is selectively disrupted in a rat neurodevelopmental model of schizophrenia: fragmented NREM sleep and impaired slow-wave propagation in the model culminate in deficient ripple-spindle coordination and disrupted spike timing, potentially as a consequence of interneuronal abnormalities reflected by reduced parvalbumin expression. These data further define the interrelationships among slow-wave, spindle, and ripple events, indicating that sleep disturbances may be associated with state-dependent decoupling of hippocampal and cortical circuits in psychiatric diseases.

GABAA receptors are ligand gated ion channels that are targeted by drugs with multiple therapeutic applications. Most notably, such drugs include the benzodiazepines, the hypnotic Z-drugs (zolpidem, zopiclone, and zaleplon), and the barbiturates. The benzodiazepines rapidly achieved pre-eminence as minor tranquilizers when they were introduced in the 1960s but thereafter lost favour, in a story that exemplifies how a “good drug can go bad” [1]. Nevertheless, benzodiazepines remain a mainstay of psychopharmacology but the decline in their public image is encapsulated by the colourful comment of Gorman [2] who stated that “Prescribing benzodiazepines is like watching pornography. If you ask a person at random if he watches pornography, he will vehemently deny it, but someone must be because it is a billion dollar a year business. Similarly, if you ask a physician if he prescribes Valium or Xanax or Ativan, he or she will say of course not…. Yet, like pornography, benzodiazepine prescriptions generate billions of dollars of revenue around the world, so somebody must be prescribing them”. Recently, there has been a resurgence of interest in GABAA receptor pharmacology which has formed the basis for the development of novel modulators of discrete populations of this receptor family [3]. Over the last 25 years or so considerable advances in the molecular biology of GABAA receptors have identified multiple subtypes of GABAA receptor. Hence, GABAA receptors are heteropentameric assemblies of proteins derived from a family comprising 16 members (a1–6, β1–3, γ1–3, δ, e, θ, π). Despite the enormous number of theoretical pentameric combinations, thankfully only around 20 configurations have been described in native receptors [4]. A variety of approaches have been used to develop a greater understanding of the roles of these different subtypes in the physiology and pathophysiology of the CNS. These strategies include the generation of transgenic mouse models that have been used to delineate the functions of different subtypes, and the identification of subtype-selective pharmacological compounds that have formed the basis of hypotheses that subtype-selectivity can lead to therapeutic selectivity (e.g., anxioselective drugs without sedation). A combination of these strategies has resulted in emerging scientific evidence for novel therapeutic applications for subtype-selective drugs (e.g., in neuropathic pain, autism, schizophrenia, Alzheimer's disease, and stroke), and consistent with these various hypotheses clinical data on subtype-selective drugs in human trials indicate a novel pharmacology relative to known drugs targeting GABAA receptors. In this special issue, the articles focus on various aspects of the molecular genetics as well as preclinical and clinical pharmacology of GABAA receptors. S. Nickoll's et al. in his paper focus on the novel therapeutic opportunity afforded by drugs that positively modulate GABAA-α2/α3 receptors selectively as novel analgesics without sedation, cognition impairment, or abuse. Testing such compounds in animal models of inflammatory and neuropathic pain, complemented by in vivo electrophysiology, they highlight the need for sufficient efficacy at relevant subtypes of GABAA receptor in addition to selectivity. C. Vinkers and B. Olivier explore mechanisms underlying benzodiazepine tolerance and conclude that no unifying underlying mechanisms can be discerned but emphasize that since this is an important therapeutic issue with respects to currently marketed GABAergic drugs, it is an important area to investigate with respects to emerging subtype selective compounds. GABAA-α5 and GABAA-δ receptors are located extrasynaptically, mediate tonic inhibitory neurotransmission in the CNS, and have different biophysical, physiological, and pharmacological properties relative to GABAA-α1/α2/α3 receptors which are predominantly synaptically located. J. Braudeau et al. focus on selective negative modulators of GABAA-α5 receptors as a unique cognition enhancing strategy in Down's syndrome (DS), by demonstrating that the selective compound α5IA induces gene expression (c-fos and Arc) and rescues impaired gene expression in the Ts65Dn mouse model of DS. The compound α5IA has already shown evidence of cognition enhancement in man. Currently, Roche pharmaceuticals is assessing a compound (RG1662) in Down's syndrome, and by extension the relevance of this line of research in potentially reducing cognitive burden in Alzheimer's disease is clear. A. Clarkson reviews the complex role of GABAA receptors in cerebral ischaemia/stroke, suggesting that in an animal model of stroke, treatment with the α5-selective negative allosteric modulator L-655708 can enhance functional recovery when given after a delay but not, interestingly, when given at the time of stroke. The assumption is that the attenuation of extrasynaptic GABAergic function can be beneficial by counteracting an increase in tonic inhibition that results from increased GABA concentrations in the peri-infarct region. Consequently, therapeutic strategies based upon a disinhibition of GABAA-α5 or GABAA-δ receptors may aid functional recovery in vivo. A. C. Errington et al. suggest negative modulators of GABAA-δ receptors as a novel treatment option for absence epilepsy given that enhanced GABAergic function has been demonstrated in animal models of absence epilepsy, and that compounds enhancing GABAA function can engender absence seizures. Certainly this suggestion is contrary to the classical approach of treating seizures by increasing inhibitory neurotransmission, but the authors elegantly demonstrate the relevance of key thalamocortical circuits in generating EEG signatures common to animals and man and the high expression and relevance of a α4β2δ receptor population in this circuit. M. W. Hulin et al. focus on literature implicating GABAA receptors and neurosteroids in mediating actions of alcohol. They describe the neurosteroid dehydroepiandrosterone (DHEA) which decreases alcohol intake in rats and broadly behaves as a negative modulator of GABAA receptors, although genomic effects of this molecule complicate matters. Nonetheless, the authors emphasize the potential role of GABAA-δ receptors in mediating the effects of DHEA in reducing alcohol intake; albeit this is a controversial area. X. Chen et al. use a comparative pharmacology approach in human volunteers to demonstrate that subtype-selective GABAA drugs engender pharmacodynamic effects that only partly overlap with those of benzodiazepines. For example, the GABAA-α2/α3 selective drug TPA023 induces impairment of saccadic peak velocity but does not induce body sway or impair attention in volunteers. The novel pharmacological profile of subtype selective compounds in man mirrors preclinical data using transgenic mouse models and subtype-selective tools, indicating that molecular neuroscience can be translated to the clinic and guide drug development. The paper by B. H. Bentzen and M. Grunnet gives some insight into the drug development of new chemical entities, specifically focusing on cardiovascular (CV) safety. Clearly many benzodiazepines were registered for clinical use in a regulatory environment considerably different from today's. Therefore, in developing novel subtype-selective modulators, assumptions cannot be made in terms of safety and toxicology. These authors use anaesthetized animals and isolated hearts to emphasize that the CV effects of benzodiazepines are highly dependent on the conscious state of the animal, with implications for CV assessment of future novel GABAergic molecules. Given the wide-spread therapeutic success of GABAergic drugs across a variety of indications and an emerging understanding of the pharmacology of subtype-selective drugs preclinically and clinically, GABAA receptors are clearly a highly druggable target class for which preclinical to clinical transition/translation strategies exist—desirable qualities in today's highly complex drug development and regulatory environment. The articles in this special issue give a snap-shot of the diverse range of topics being explored regarding the therapeutic utility of selectively targeting GABAA receptors, providing a ground base of understanding for potentially important drugs of the future. Naheed R. Mirza John Atack Keith Wafford