The Dopamine D1 Receptor Positive Allosteric Modulator Mevidalen (LY3154207) Enhances Wakefulness in the Humanized D1 Mouse and in Sleep-Deprived Healthy Male Volunteers.

Dopamine (DA) plays a key role in several central functions including cognition, motor activity, and wakefulness. Although efforts to develop dopamine receptor 1 (D1) agonists have been challenging, a positive allosteric modulator represents an attractive approach with potential better drug-like properties. Our previous study demonstrated an acceptable safety and tolerability profile of the dopamine receptor 1 positive allosteric modulator (D1PAM) mevidalen (LY3154207) in single and multiple ascending dose studies in healthy volunteers (Wilbraham et al., 2021). Herein, we describe the effects of mevidalen on sleep and wakefulness in humanized dopamine receptor 1 (hD1) mice and in sleep-deprived healthy male volunteers. Mevidalen enhanced wakefulness (latency to fall asleep) in the hD1 mouse in a dose dependent [3-100 mg/kg, orally (PO)] fashion when measured during the light (zeitgeber time 5) and predominantly inactive phase. Mevidalen promoted wakefulness in mice after prior sleep deprivation and delayed sleep onset by 5.5- and 15.2-fold compared with vehicle-treated animals, after the 20 and 60 mg/kg PO doses, respectively, when compared with vehicle-treated animals. In humans, mevidalen demonstrated a dose-dependent increase in latency to sleep onset as measured by the multiple sleep latency test and all doses (15, 30, and 75 mg) separated from placebo at the first 2-hour postdose time point with a circadian effect at the 6-hour postdose time point. Sleep wakefulness should be considered a translational biomarker for the dopamine receptor 1 positive allosteric modulator mechanism. SIGNIFICANCE STATEMENT: This is the first translational study describing the effects of a selective dopamine receptor 1 positive allosteric modulator (D1PAM) on sleep and wakefulness in the human dopamine receptor 1 mouse and in sleep-deprived healthy male volunteers. In both species, drug exposure correlated with sleep latency, supporting the use of sleep-wake activity as a translational central biomarker for D1PAM. Wake-promoting effects of D1PAMs may offer therapeutic opportunities in several conditions, including sleep disorders and excessive daytime sleepiness related to neurodegenerative disorders.

REM sleep's unique associations with corticosterone regulation, apoptotic pathways, and behavior in chronic stress in mice.

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, as well as the brain and blood transcriptome in mice exposed to 9 weeks of unpredictable chronic mild stress (UCMS). Comparing 46 phenotypic 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 predictor sets for REMS parameters that were enriched in 193 pathways, including some involved in stem cells, immune response, and apoptosis and survival. Only three pathways were enriched in predictor sets for non-REMS. Transcriptomic predictor sets for variation in REMS continuity and theta activity shared many pathways with corticosterone regulation, in particular pathways implicated in apoptosis and survival, including mitochondrial apoptotic machinery. Predictor sets for REMS and anhedonia shared pathways involved in oxidative stress, cell proliferation, and apoptosis. These data identify REMS as a core and early element of the response to chronic stress, and identify apoptosis and survival pathways as a putative mechanism by which REMS may mediate the response to stressful waking experiences.

Effects of Aging on Cortical Neural Dynamics and Local Sleep Homeostasis in Mice.

Healthy aging is associated with marked effects on sleep, including its daily amount and architecture, as well as the specific EEG oscillations. Neither the neurophysiological underpinnings nor the biological significance of these changes are understood, and crucially the question remains whether aging is associated with reduced sleep need or a diminished capacity to generate sufficient sleep. Here we tested the hypothesis that aging may affect local cortical networks, disrupting the capacity to generate and sustain sleep oscillations, and with it the local homeostatic response to sleep loss. We performed chronic recordings of cortical neural activity and local field potentials from the motor cortex in young and older male C57BL/6J mice, during spontaneous waking and sleep, as well as during sleep after sleep deprivation. In older animals, we observed an increase in the incidence of non-rapid eye movement sleep local field potential slow waves and their associated neuronal silent (OFF) periods, whereas the overall pattern of state-dependent cortical neuronal firing was generally similar between ages. Furthermore, we observed that the response to sleep deprivation at the level of local cortical network activity was not affected by aging. Our data thus suggest that the local cortical neural dynamics and local sleep homeostatic mechanisms, at least in the motor cortex, are not impaired during healthy senescence in mice. This indicates that powerful protective or compensatory mechanisms may exist to maintain neuronal function stable across the life span, counteracting global changes in sleep amount and architecture.SIGNIFICANCE STATEMENT The biological significance of age-dependent changes in sleep is unknown but may reflect either a diminished sleep need or a reduced capacity to generate deep sleep stages. As aging has been linked to profound disruptions in cortical sleep oscillations and because sleep need is reflected in specific patterns of cortical activity, we performed chronic electrophysiological recordings of cortical neural activity during waking, sleep, and after sleep deprivation from young and older mice. We found that all main hallmarks of cortical activity during spontaneous sleep and recovery sleep after sleep deprivation were largely intact in older mice, suggesting that the well-described age-related changes in global sleep are unlikely to arise from a disruption of local network dynamics within the neocortex.

Modelling maintenance of wakefulness in rats: comparing potential non-invasive sleep-restriction methods and their effects on sleep and attentional performance.

While several methods have been used to restrict the sleep of experimental animals, it is often unclear whether these different forms of sleep restriction have comparable effects on sleep-wake architecture or functional capacity. The present study compared four models of sleep restriction, using enforced wakefulness by rotation of cylindrical home cages over 11 h in male Wistar rats. These included an electroencephalographic-driven 'Biofeedback' method and three non-invasive methods where rotation was triggered according to a 'Constant', 'Decreasing' or random protocol based upon the 'Weibull' distribution fit to an archival Biofeedback dataset. Sleep-wake architecture was determined using polysomnography, and functional capacity was assessed immediately post-restriction with a simple response latency task, as a potential homologue of the human psychomotor vigilance task. All sleep restriction protocols resulted in sleep loss, behavioural task disengagement and rebound sleep, although no model was as effective as real-time electroencephalographic-Biofeedback. Decreasing and Weibull protocols produced greater recovery sleep than the Constant protocol, mirrored by comparably poorer simple response latency task performance. Increases in urinary corticosterone levels following Constant and Decreasing protocols suggested that stress levels may differ between protocols. Overall, these results provide insight into the value of choosing a specific sleep restriction protocol, not only from the perspective of animal welfare and the use of less invasive procedures, but also translational validity. A more considered choice of the physiological and functional effects of sleep-restriction protocols in rodents may improve correspondence with specific types of excessive daytime sleepiness in humans.

Novel bicyclo[3.1.0]hexane analogs as antagonists of metabotropic glutamate 2/3 receptors for the treatment of depression.

Negative modulators of metabotropic glutamate 2 & 3 receptors demonstrate antidepressant-like activity in animal models and hold promise as novel therapeutic agents for the treatment of major depressive disorder. Herein we describe our efforts to prepare and optimize a series of conformationally constrained 3,4-disubstituted bicyclo[3.1.0]hexane glutamic acid analogs as orthosteric (glutamate site) mGlu2/3 receptor antagonists. This work led to the discovery of a highly potent and efficacious tool compound 18 (hmGlu2 IC50 46±14.2nM, hmGlu3 IC50=46.1±36.2nM). Compound 18 showed activity in the mouse forced swim test with a minimal effective dose (MED) of 1mg/kg ip. While in rat EEG studies it exhibited wake promoting effects at 3 and 10mg/kg ip without any significant effects on locomotor activity. Compound 18 thus represents a novel tool molecule for studying the impact of blocking mGlu2/3 receptors both in vitro and in vivo.

N-(4-((2-(trifluoromethyl)-3-hydroxy-4-(isobutyryl)phenoxy)methyl)benzyl)-1-methyl-1H-imidazole-4-carboxamide (THIIC), a novel metabotropic glutamate 2 potentiator with potential anxiolytic/antidepressant properties: in vivo profiling suggests a link between behavioral and central nervous system neurochemical changes.

The normalization of excessive glutamatergic neurotransmission through the activation of metabotropic glutamate 2 (mGlu2) receptors may have therapeutic potential in a variety of psychiatric disorders, including anxiety/depression and schizophrenia. Here, we characterize the pharmacological properties of N-(4-((2-(trifluoromethyl)-3-hydroxy-4-(isobutyryl)phenoxy)methyl)benzyl)-1-methyl-1H-imidazole-4-carboxamide (THIIC), a structurally novel, potent, and selective allosteric potentiator of human and rat mGlu2 receptors (EC(50) = 23 and 13 nM, respectively). THIIC produced anxiolytic-like efficacy in the rat stress-induced hyperthermia assay and the mouse stress-induced elevation of cerebellar cGMP and marble-burying assays. THIIC also produced robust activity in three assays that detect antidepressant-like activity, including the mouse forced-swim test, the rat differential reinforcement of low rate 72-s assay, and the rat dominant-submissive test, with a maximal response similar to that of imipramine. Effects of THIIC in the forced-swim test and marble burying were deleted in mGlu2 receptor null mice. Analysis of sleep electroencephalogram (EEG) showed that THIIC had a sleep-promoting profile with increased non-rapid eye movement (REM) and decreased REM sleep. THIIC also decreased the dark phase increase in extracellular histamine in the medial prefrontal cortex and decreased levels of the histamine metabolite tele-methylhistamine (t-MeHA) in rat cerebrospinal fluid. Collectively, these results indicate that the novel mGlu2-positive allosteric modulator THIIC has robust activity in models used to predict anxiolytic/antidepressant efficacy, substantiating, at least with this molecule, differentiation in the biological impact of mGlu2 potentiation versus mGlu2/3 orthosteric agonism. In addition, we provide evidence that sleep EEG and CSF t-MeHA might function as viable biomarker approaches to facilitate the translational development of THIIC and other mGlu2 potentiators.

Aberrant waste disposal in neurodegeneration: why improved sleep could be the solution.

Sleep takes up a large percentage of our lives and the full functions of this state are still not understood. However, over the last 10 years a new and important function has emerged as a mediator of brain clearance. Removal of toxic metabolites and proteins from the brain parenchyma generated during waking activity and high levels of synaptic processing is critical to normal brain function and only enabled during deep sleep. Understanding of this process is revealing how impaired sleep contributes an important and likely causative role in the accumulation and aggregation of aberrant proteins such as ß-amyloid and phosphorylated tau, as well as inflammation and neuronal damage. We are also beginning to understand how brain slow-wave activity interacts with vascular function allowing the flow of CSF and interstitial fluid to drain into the body's lymphatic system. New methodology is enabling visualization of this process in both animals and humans and is revealing how these processes break down during ageing and disease. With this understanding we can begin to envisage novel therapeutic approaches to the treatment of neurodegeneration, and how reversing sleep impairment in the correct manner may provide a way to slow these processes and improve brain function.

In vitro and in vivo properties of 3-tert-butyl-7-(5-methylisoxazol-3-yl)-2-(1-methyl-1H-1,2,4-triazol-5-ylmethoxy)-pyrazolo[1,5-d]-[1,2,4]triazine (MRK-016), a GABAA receptor alpha5 subtype-selective inverse agonist.

3-tert-Butyl-7-(5-methylisoxazol-3-yl)-2-(1-methyl-1H-1,2,4-triazol-5-ylmethoxy)-pyrazolo[1,5-d][1,2,4]triazine (MRK-016) is a pyrazolotriazine with an affinity of between 0.8 and 1.5 nM for the benzodiazepine binding site of native rat brain and recombinant human alpha1-, alpha2-, alpha3-, and alpha5-containing GABA(A) receptors. It has inverse agonist efficacy selective for the alpha5 subtype, and this alpha5 inverse agonism is greater than that of the prototypic alpha5-selective compound 3-(5-methylisoxazol-3-yl)-6-[(1-methyl-1,2,3-triazol-4-hdyl)methyloxy]-1,2,4-triazolo[3,4-a]phthalazine (alpha5IA). Consistent with its greater alpha5 inverse agonism, MRK-016 increased long-term potentiation in mouse hippocampal slices to a greater extent than alpha5IA. MRK-016 gave good receptor occupancy after oral dosing in rats, with the dose required to produce 50% occupancy being 0.39 mg/kg and a corresponding rat plasma EC(50) value of 15 ng/ml that was similar to the rhesus monkey plasma EC(50) value of 21 ng/ml obtained using [(11)C]flumazenil positron emission tomography. In normal rats, MRK-016 enhanced cognitive performance in the delayed matching-to-position version of the Morris water maze but was not anxiogenic, and in mice it was not proconvulsant and did not produce kindling. MRK-016 had a short half-life in rat, dog, and rhesus monkey (0.3-0.5 h) but had a much lower rate of turnover in human compared with rat, dog, or rhesus monkey hepatocytes. Accordingly, in human, MRK-016 had a longer half-life than in preclinical species ( approximately 3.5 h). Although it was well tolerated in young males, with a maximal tolerated single dose of 5 mg corresponding to an estimated occupancy in the region of 75%, MRK-016 was poorly tolerated in elderly subjects, even at a dose of 0.5 mg, which, along with its variable human pharmacokinetics, precluded its further development.

Pharmacological Modulation of Sleep Homeostasis in Rat: Novel Effects of an mGluR2/3 Antagonist.

Increasing vigilance without incurring the negative consequences of extended wakefulness such as daytime sleepiness and cognitive impairment is a major challenge in treating many sleep disorders. The present work compares two closely related mGluR2/3 antagonists LY3020371 and LY341495 with two well-known wake-promoting compounds caffeine and d-amphetamine. Sleep homeostasis properties were explored in male Wistar rats by manipulating levels of wakefulness via (1) physiological sleep restriction (SR), (2) pharmacological action, or (3) a combination of these. A two-phase nonlinear mixed-effects model combining a quadratic and exponential function at an empirically estimated join point allowed the quantification of wake-promoting properties and any subsequent sleep rebound. A simple response latency task (SRLT) following SR assessed functional capacity of sleep-restricted animals treated with our test compounds. Caffeine and d-amphetamine increased wakefulness with a subsequent full recovery of non-rapid eye movement (NREM) and rapid eye movement (REM) sleep and were unable to fully reverse SR-induced impairments in SRLT. In contrast, LY3020371 increased wakefulness with no subsequent elevation of NREM sleep, delta power, delta energy, or sleep bout length and count, yet REM sleep recovered above baseline levels. Prior sleep pressure obtained using an SR protocol had no impact on the wake-promoting effect of LY3020371 and NREM sleep rebound remained blocked. Furthermore, LY341495 increased functional capacity across SRLT measures following SR. These results establish the critical role of glutamate in sleep homeostasis and support the existence of independent mechanisms for NREM and REM sleep homeostasis.

The expression of GABAA beta subunit isoforms in synaptic and extrasynaptic receptor populations of mouse dentate gyrus granule cells.

The subunit composition of GABA(A) receptors influences their biophysical and pharmacological properties, dictates neuronal location and the interaction with associated proteins, and markedly influences the impact of intracellular biochemistry. The focus has been on alpha and gamma subunits, with little attention given to beta subunits. Dentate gyrus granule cells (DGGCs) express all three beta subunit isoforms and exhibit both synaptic and extrasynaptic receptors that mediate 'phasic' and 'tonic' transmission, respectively. To investigate the subcellular distribution of the beta subunits we have utilized the patch-clamp technique to compare the properties of 'tonic' and miniature inhibitory postsynaptic currents (mIPSCs) recorded from DGGCs of hippocampal slices of P20-26 wild-type (WT), beta(2)(-/-), beta(2N265S) (etomidate-insensitive), alpha(1)(-/-) and delta(-/-) mice. Deletion of either the beta(2) or the delta subunit produced a significant reduction of the tonic current and attenuated the increase of this current induced by the delta subunit-preferring agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP). By contrast, mIPSCs were not influenced by deletion of these genes. Enhancement of the tonic current by the beta(2/3) subunit-selective agent etomidate was significantly reduced for DGGCs derived from beta(2N265S) mice, whereas this manipulation had no effect on the prolongation of mIPSCs produced by this anaesthetic. Collectively, these observations, together with previous studies on alpha(4)(-/-) mice, identify a population of extrasynaptic alpha(4)beta(2)delta receptors, whereas synaptic GABA(A) receptors appear to primarily incorporate the beta(3) subunit. A component of the tonic current is diazepam sensitive and is mediated by extrasynaptic receptors incorporating alpha(5) and gamma(2) subunits. Deletion of the beta(2) subunit had no effect on the diazepam-induced current and therefore these extrasynaptic receptors do not contain this subunit. The unambiguous identification of these distinct pools of synaptic and extrasynaptic GABA(A) receptors should aid our understanding of how they act in harmony, to regulate hippocampal signalling in health and disease.

Investigating the role of mGluR2 versus mGluR3 in antipsychotic-like effects, sleep-wake architecture and network oscillatory activity using novel Han Wistar rats lacking mGluR2 expression.

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.

Preclinical profile of a dopamine D1 potentiator suggests therapeutic utility in neurological and psychiatric disorders.

DETQ, an allosteric potentiator of the dopamine D1 receptor, was tested in therapeutic models that were known to respond to D1 agonists. Because of a species difference in affinity for DETQ, all rodent experiments used transgenic mice expressing the human D1 receptor (hD1 mice). When given alone, DETQ reversed the locomotor depression caused by a low dose of reserpine. DETQ also acted synergistically with L-DOPA to reverse the strong hypokinesia seen with a higher dose of reserpine. These results indicate potential as both monotherapy and adjunct treatment in Parkinson's disease. DETQ markedly increased release of both acetylcholine and histamine in the prefrontal cortex, and increased levels of histamine metabolites in the striatum. In the hippocampus, the combination of DETQ and the cholinesterase inhibitor rivastigmine increased ACh to a greater degree than either agent alone. DETQ also increased phosphorylation of the AMPA receptor (GluR1) and the transcription factor CREB in the striatum, consistent with enhanced synaptic plasticity. In the Y-maze, DETQ increased arm entries but (unlike a D1 agonist) did not reduce spontaneous alternation between arms at high doses. DETQ enhanced wakefulness in EEG studies in hD1 mice and decreased immobility in the forced-swim test, a model for antidepressant-like activity. In rhesus monkeys, DETQ increased spontaneous eye-blink rate, a measure that is known to be depressed in Parkinson's disease. Together, these results provide support for potential utility of D1 potentiators in the treatment of several neuropsychiatric disorders, including Parkinson's disease, Alzheimer's disease, cognitive impairment in schizophrenia, and major depressive disorder.

Alpha5GABAA receptors mediate the amnestic but not sedative-hypnotic effects of the general anesthetic etomidate.

A fundamental objective of anesthesia research is to identify the receptors and brain regions that mediate the various behavioral components of the anesthetic state, including amnesia, immobility, and unconsciousness. Using complementary in vivo and in vitro approaches, we found that GABAA receptors that contain the alpha5 subunit (alpha5GABAARs) play a critical role in amnesia caused by the prototypic intravenous anesthetic etomidate. Whole-cell recordings from hippocampal pyramidal neurons showed that etomidate markedly increased a tonic inhibitory conductance generated by alpha5GABAARs, whereas synaptic transmission was only slightly enhanced. Long-term potentiation (LTP) of field EPSPs recorded in CA1 stratum radiatum was reduced by etomidate in wild-type (WT) but not alpha5 null mutant (alpha5-/-) mice. In addition, etomidate impaired memory performance of WT but not alpha5-/- mice for spatial and nonspatial hippocampal-dependent learning tasks. The brain concentration of etomidate associated with memory impairment in vivo was comparable with that which increased the tonic inhibitory conductance and blocked LTP in vitro. The alpha5-/- mice did not exhibit a generalized resistance to etomidate, in that the sedative-hypnotic effects measured with the rotarod, loss of righting reflex, and spontaneous motor activity were similar in WT and alpha5-/- mice. Deletion of the alpha5 subunit of the GABAARs reduced the amnestic but not the sedative-hypnotic properties of etomidate. Thus, the amnestic and sedative-hypnotic properties of etomidate can be dissociated on the basis of GABAAR subtype pharmacology.

Treating insomnia: Current and investigational pharmacological approaches.

Chronic insomnia affects a significant proportion of young adult and elderly populations. Treatment strategies should alleviate nighttime symptoms, the feeling of nonrestorative sleep, and impaired daytime function. Current pharmacological approaches focus primarily on GABA, the major inhibitory neurotransmitter in the central nervous system. Benzodiazepine receptor agonists (BzRA) have been a mainstay of pharmacotherapy; the classical benzodiazepines and non-benzodiazepines share a similar mode of action and allosterically enhance inhibitory chloride currents through the GABA(A) receptor, a ligand-gated protein comprising 5 subunits pseudosymmetrically arranged around a core anion channel. Variations in GABA(A) receptor subunit composition confer unique pharmacological, biophysical, and electrophysiological properties on each receptor subtype. Classical benzodiazepines bind non-selectively to GABA(A) receptors containing a gamma2 subunit, whereas non-benzodiazepine hypnotics bind with higher relative affinity to alpha1-containing receptors. The non-benzodiazepine compounds generally represent an improvement over benzodiazepines as a result of improved binding selectivity and pharmacokinetic profiles. However, the enduring potential for amnestic effects, next day residual sedation, and abuse and physical dependence, particularly at higher doses, underscores the need for new treatment strategies. Novel pharmacotherapies in development act on systems believed to be specifically involved in the regulation of the sleep-wake cycle. The recently approved melatonin receptor agonist, ramelteon, targets circadian mechanisms. Gaboxadol, an investigational treatment and a selective extrasynaptic GABA(A) receptor agonist (SEGA), targets GABA(A) receptors containing a delta subunit, which are located outside the synaptic junctions of thalamic and cortical neurons thought to play an important regulatory role in the onset, maintenance, and depth of the sleep process.

Differential contribution of GABA(A) receptor subtypes to the anticonvulsant efficacy of benzodiazepine site ligands.

Non-selective benzodiazepines, such as diazepam, interact with equivalent affinity and agonist efficacy at GABA(A) receptors containing either an alpha1, alpha2, alpha3 or alpha5 subunit. However, which of these particular subtypes are responsible for the anticonvulsant effects of diazepam remains uncertain. In the present study, we examined the ability of diazepam to reduce pentylenetetrazoLe (PTZ)-induced and maximal electroshock (MES)-induced seizures in mice containing point mutations in single (alpha1H101R, alpha2H101R or alpha5H105R) or multiple (alpha125H-->R) alpha subunits that render the resulting GABA(A) receptors diazepam-insensitive. Furthermore, the anticonvulsant properties of diazepam, the alpha1- and alpha3-selective compounds zolpidem and TP003, respectively, and the alpha2/alpha3 preferring compound TP13 were studied against PTZ-induced seizures. In the transgenic mice, no single subtype was responsible for the anticonvulsant effects of diazepam in either the PTZ or MES assay and neither the alpha3 nor alpha5 subtypes appeared to confer anticonvulsant activity. Moreover, whereas the alpha1 and alpha2 subtypes played a modest role with respect to the PTZ assay, they had a negligible role in the MES assay. With respect to subtype-selective compounds, zolpidem and TP003 had much reduced anticonvulsant efficacy relative to diazepam in both the PTZ and MES assays whereas TP13 had high anticonvulsant efficacy in the PTZ but not the MES assay. Taken together, these data not only indicate a role for alpha2-containing GABA(A) receptors in mediating PTZ and MES anticonvulsant activity but also suggest that efficacy at more than one subtype is required and that these subtypes act synergistically.

Gaboxadol--a new awakening in sleep.

Drugs that enhance synaptic gamma-aminobutyric acid (GABA)ergic neurotransmission are widely utilized in the clinical setting. Barbiturates and benzodiazepine receptor agonists, for example, both potentiate an inhibitory chloride conductance through GABA-gated channels, and thereby achieve their sedative-hypnotic effects. The primary locus of action of these agents, and indeed most neuroactive drugs, is the postsynaptic junction. By contrast, gaboxadol, a selective extrasynaptic GABA receptor agonist and late-stage investigational treatment for insomnia, acts on a unique delta-containing GABAA receptor subtype found exclusively outside of the synapse. Although the mechanistic details of extrasynaptic neurotransmission remain to be fully established, it is now clear that these receptors demonstrate unique pharmacological, biophysical and electrophysiological properties. Importantly, the delta-containing GABAA receptor subtype activated by gaboxadol is highly expressed in the thalamus, where it might behave as a 'gain control' (independently controlling the strength of signals) in the corticothalamic pathways that govern sleep-relevant neuronal oscillations. This unique mechanism has contributed to our increased understanding of sleep mechanisms, and targeting of this system offers potential advantages over existing insomnia treatments.

Polypharmacological in Silico Bioactivity Profiling and Experimental Validation Uncovers Sedative-Hypnotic Effects of Approved and Experimental Drugs in Rat.

In this work, we describe the computational ("in silico") mode-of-action analysis of CNS-active drugs, which is taking both multiple simultaneous hypotheses as well as sets of protein targets for each mode-of-action into account, and which was followed by successful prospective in vitro and in vivo validation. Using sleep-related phenotypic readouts describing both efficacy and side effects for 491 compounds tested in rat, we defined an "optimal" (desirable) sleeping pattern. Compounds were subjected to in silico target prediction (which was experimentally confirmed for 21 out of 28 cases), followed by the utilization of decision trees for deriving polypharmacological bioactivity profiles. We demonstrated that predicted bioactivities improved classification performance compared to using only structural information. Moreover, DrugBank molecules were processed via the same pipeline, and compounds in many cases not annotated as sedative-hypnotic (alcaftadine, benzatropine, palonosetron, ecopipam, cyproheptadine, sertindole, and clopenthixol) were prospectively validated in vivo. Alcaftadine, ecopipam cyproheptadine, and clopenthixol were found to promote sleep as predicted, benzatropine showed only a small increase in NREM sleep, whereas sertindole promoted wakefulness. To our knowledge, the sedative-hypnotic effects of alcaftadine and ecopipam have not been previously discussed in the literature. The method described extends previous single-target, single-mode-of-action models and is applicable across disease areas.

Phase-amplitude coupled persistent theta and gamma oscillations in rat primary motor cortex in vitro.

In vivo, theta (4-7 Hz) and gamma (30-80 Hz) neuronal network oscillations are known to coexist and display phase-amplitude coupling (PAC). However, in vitro, these oscillations have for many years been studied in isolation. Using an improved brain slice preparation technique we have, using co-application of carbachol (10 µM) and kainic acid (150 nM), elicited simultaneous theta (6.6 ± 0.1 Hz) and gamma (36.6 ± 0.4 Hz) oscillations in rodent primary motor cortex (M1). Each oscillation showed greatest power in layer V. Using a variety of time series analyses we detected significant cross-frequency coupling in 74% of slice preparations. Differences were observed in the pharmacological profile of each oscillation. Thus, gamma oscillations were reduced by the GABAA receptor antagonists, gabazine (250 nM and 2 µM), and picrotoxin (50 µM) and augmented by AMPA receptor antagonism with SYM2206 (20 µM). In contrast, theta oscillatory power was increased by gabazine, picrotoxin and SYM2206. GABAB receptor blockade with CGP55845 (5 µM) increased both theta and gamma power, and similar effects were seen with diazepam, zolpidem, MK801 and a series of metabotropic glutamate receptor antagonists. Oscillatory activity at both frequencies was reduced by the gap junction blocker carbenoxolone (200 µM) and by atropine (5 µM). These data show theta and gamma oscillations in layer V of rat M1 in vitro are cross-frequency coupled, and are mechanistically distinct. The development of an in vitro model of phase-amplitude coupled oscillations will facilitate further mechanistic investigation of the generation and modulation of coupled activity in mammalian cortex.

Electroencephalographic, cognitive, and neurochemical effects of LY3130481 (CERC-611), a selective antagonist of TARP-γ8-associated AMPA receptors.

6-[(1S)-1-[1-[5-(2-hydroxyethoxy)-2-pyridyl]pyrazol-3-yl]ethyl]-3H-1,3-benzothiazol-2-one (LY3130481 or CERC-611) is a selective antagonist of AMPA receptors containing transmembrane AMPA receptor regulatory protein (TARP) γ-8. This molecule has been characterized as a potent and efficacious anticonvulsant in an array of acute and chronic epilepsy models in rodents. The present set of experiments was designed to assess the effects of LY3130481 on the electroencephelogram (EEG), cognitive function, and neurochemical outflow. LY3130481 disrupted food-maintained responding in rats and spontaneous alternation in a Y-maze in mice. In rat fear conditioning, LY3130481 caused a deficit in trace (hippocampal-dependent), but not in delay fear conditioning. Although these effects on cognitive performances were observed, the known cognitive-impairing anticonvulsant, topiramate, did not always produce deficits under these assay conditions. LY3130481 produced modest increases in wake times in rats. In addition, LY3130481 was able to attenuate some impairing effects of standard antiepileptic drugs. The motor-impairing effects of the lacosamide were attenuated by LY3130481 as was the decrease in non-rapid-eye movement sleep induced by carbamazepine. Evaluation of the effect of LY3130481 on neurotransmitter and metabolite efflux in the rat medial prefrontal cortex, using in vivo microdialysis, revealed significant increases in the pro-cognitive and wake-promoting neurotransmitters, histamine and acetylcholine, as well as in serotonin, telemethylhistamine, 5-HIAA, HVA and MHPG. LY3130481 thus presents a novel behavioral profile that will have to be evaluated in patients to fully appreciate its implications for therapeutics. LY3130481 is currently under clinical development as CERC-611 as an antiepileptic.

TaiNi: Maximizing research output whilst improving animals' welfare in neurophysiology experiments.

Understanding brain function at the cell and circuit level requires representation of neuronal activity through multiple recording sites and at high sampling rates. Traditional tethered recording systems restrict movement and limit the environments suitable for testing, while existing wireless technology is still too heavy for extended recording in mice. Here we tested TaiNi, a novel ultra-lightweight (<2 g) low power wireless system allowing 72-hours of recording from 16 channels sampled at ~19.5 KHz (9.7 KHz bandwidth). We captured local field potentials and action-potentials while mice engaged in unrestricted behaviour in a variety of environments and while performing tasks. Data was synchronized to behaviour with sub-second precision. Comparisons with a state-of-the-art wireless system demonstrated a significant improvement in behaviour owing to reduced weight. Parallel recordings with a tethered system revealed similar spike detection and clustering. TaiNi represents a significant advance in both animal welfare in electrophysiological experiments, and the scope for continuously recording large amounts of data from small animals.