From structure to clinic: Design of a muscarinic M1 receptor agonist with potential to treatment of Alzheimer's disease.

Current therapies for Alzheimer's disease seek to correct for defective cholinergic transmission by preventing the breakdown of acetylcholine through inhibition of acetylcholinesterase, these however have limited clinical efficacy. An alternative approach is to directly activate cholinergic receptors responsible for learning and memory. The M1-muscarinic acetylcholine (M1) receptor is the target of choice but has been hampered by adverse effects. Here we aimed to design the drug properties needed for a well-tolerated M1-agonist with the potential to alleviate cognitive loss by taking a stepwise translational approach from atomic structure, cell/tissue-based assays, evaluation in preclinical species, clinical safety testing, and finally establishing activity in memory centers in humans. Through this approach, we rationally designed the optimal properties, including selectivity and partial agonism, into HTL9936-a potential candidate for the treatment of memory loss in Alzheimer's disease. More broadly, this demonstrates a strategy for targeting difficult GPCR targets from structure to clinic.

Modulation of food consumption and sleep-wake cycle in mice by the neutral CB1 antagonist ABD459.

The brain endocannabinoid system is a potential target for the treatment of psychiatric and metabolic conditions. Here, a novel CB1 receptor antagonist (ABD459) was synthesized and assayed for pharmacological efficacy in vitro and for modulation of food consumption, vigilance staging and cortical electroencephalography in the mouse. ABD459 completely displaced the CB1 agonist CP99540 at a Ki of 8.6 nmol/l, and did not affect basal, but antagonized CP55940-induced GTPγS binding with a KB of 7.7 nmol/l. Acute ABD459 (3-20 mg/kg) reliably inhibited food consumption in nonfasted mice, without affecting motor activity. Active food seeking was reduced for 5-6 h postdrug, with no rebound after washout. Epidural recording of electroencephalogram confirmed that ABD459 (3 mg/kg) robustly reduced rapid eye movement (REM) sleep, with no alterations of wakefulness or non-REM sleep. Effects were strongest during 3 h postdrug, followed by a progressive washout period. The CB1 antagonist AM251 (3 mg/kg) and agonist WIN-55,212-2 (WIN-2: 3 mg/kg) also reduced REM, but variously affected other vigilance stages. WIN-2 caused a global suppression of normalized spectral power. AM251 and ABD459 lowered delta power and increased power in the theta band in the hippocampus, but not the prefrontal cortex. The neutral antagonist ABD459 thus showed a specific role of endocannabinoid release in attention and arousal, possibly through modulation of cholinergic activity.

Cannabinoids alter spontaneous firing, bursting, and cell synchrony of hippocampal principal cells.

Both natural and synthetic cannabinoid receptor (e.g., CB1) agonists such as Δ(9)-THC, WIN 55,212-2 (WIN-2), and HU-210 disrupt spatial cognition presumably through the inhibition of synchrony of hippocampal ensemble firing to task-related events. Although the CB1 receptor agonist CP 55,940 also disrupts the synchronous firing of hippocampal neurons, it does not seem to affect the average firing rate. This difference is not readily explained by the chemical structure and pharmacology of the different compounds thus warranting a more detailed examination into (i) how other cannabinoids affect the spontaneous firing, bursting, and cell synchrony of hippocampal principal cells located in CA3 and CA1 subfields, and (ii) whether these effects are indeed mediated through CB1 receptors, which will be explored by the selective antagonist AM-251. Male Long-Evans rats surgically implanted with multielectrode arrays to hippocampal CA3 and CA1 were anesthetized and principal cells discharging at 0.25-6.0 Hz were isolated and "tracked" following the systemic administration of Tween-80, Δ(9)-THC (1 or 3 mg/kg) or WIN-2 (1 mg/kg) or HU-210 (100 µg/kg), and 1.5 mg/kg AM-281. All cannabinoids except for 1 mg/kg Δ(9) -THC reliably reduced average firing rates and altered "burst" characteristics, which were reversible with AM-281 for Δ(9)-THC and WIN-2 but not for HU-210. In addition, all cannabinoids disrupted intrasubfield and intersubfield ensemble synchrony of pyramidal cells, which is an effect insensitive to AM-281 and thus unlikely to be CB1 mediated. We consider these cannabinoid effects on spike timing and firing/bursting of principal hippocampal neurons carried by CB1 and non-CB1 receptors to be physiological underpinnings of the cognitive impairments inherent to cannabinoid exposure.

Memory encoding in hippocampal ensembles is negatively influenced by cannabinoid CB1 receptors.

It has previously been demonstrated that the detrimental effect on the performance of a delayed nonmatch to sample (DNMS) memory task by exogenously administered cannabinoid (CB1) receptor agonist, WIN 55212-2 (WIN), is reversed by the receptor antagonist rimonabant. In addition, rimonabant administered alone elevates DNMS performance, presumably through the suppression of negative modulation by released endocannabinoids during normal task performance. Other investigations have shown that rimonabant enhances encoding of DNMS task-relevant information on a trial-by-trial, delay-dependent basis. In this study, these reciprocal pharmacological actions were completely characterized by long-term, chronic intrahippocampal infusion of both agents (WIN and rimonabant) in successive 2-week intervals. Such long-term exposure allowed extraction and confirmation of task-related firing patterns, in which rimonabant reversed the effects of CB1 agonists. This information was then utilized to artificially impose the facilitatory effects of rimonabant and to reverse the effects of WIN on DNMS performance, by delivering multichannel electrical stimulation in the same firing patterns to the same hippocampal regions. Direct comparison of normal and WIN-injected subjects, in which rimonabant injections and ensemble firing facilitated performance, verified reversal of the modulation of hippocampal memory processes by CB1 receptor agonists, including released endocannabinoids.

Cannabinoid and cholinergic systems interact during performance of a short-term memory task in the rat.

It is now well established that cannabinoid agonists such as Δ(9)-tetrahydrocannabinol (THC), anandamide, and WIN 55,212-2 (WIN-2) produce potent and specific deficits in working memory (WM)/short-term memory (STM) tasks in rodents. Although mediated through activation of CB1 receptors located in memory-related brain regions such as the hippocampus and prefrontal cortex, these may, in part, be due to a reduction in acetylcholine release (i.e., cholinergic hypofunction). To determine the interaction between cannabinoid and cholinergic systems, we exposed rats treated with WIN-2 or cholinergic drugs to a hippocampal-dependent delayed nonmatch to sample (DNMS) task to study STM, and recorded hippocampal single-unit activity in vivo. WIN-2 induced significant deficits in DNMS performance and reduced the average firing and bursting rates of hippocampal principal cells through a CB1 receptor-mediated mechanism. Rivastigmine, an acetylcholinesterase inhibitor, reversed these STM deficits and normalized hippocampal discharge rates. Effects were specific to 1 mg/kg WIN-2 as rivastigmine failed to reverse the behavioral and physiological deficits that were observed in the presence of MK-801, an NMDA receptor antagonist. This supports the notion that cannabinoid-modulated cholinergic activity is a mechanism underlying the performance deficits in DNMS. Whether deficits are due to reduced nicotinic or muscarinic receptor activation, or both, awaits further analysis.

Recruitment of hippocampal neurons to encode behavioral events in the rat: alterations in cognitive demand and cannabinoid exposure.

Successful performance by rats of a delayed-nonmatch-to-sample (DNMS) task is hippocampal dependent. We have shown that neurons in hippocampus differentially encode task-relevant events. These responses are critical for correct DNMS performance and are diminished by exogenous cannabinoids. We therefore reasoned that hippocampal neural correlates of behavior are likely shaped during learning; however, to date, no work has examined these correlates during DNMS acquisition training. Consequently, the present study assessed the emergence of hippocampal neural encoding when (i) cognitive task demands were increased through prolongation of delay intervals between sample and nonmatch phase and (ii) when animals are under cannabinoid treatment and performance is compromised. Adult, male Long-Evans rats were trained to perform the DNMS task without delay and then implanted with multielectrode recording arrays directed to CA3 and CA1 subfields of the hippocampus. Following recovery, single units were isolated and animals divided into two treatment groups: vehicle or WIN 55,212-2 (WIN-2, 0.35 mg/kg). Ensemble firing was monitored during retraining in DNMS task at 0 s, and subsequently delay intervals were progressively increased to 1-10 s, 11-20 s, and 21-30 s when animals met criterion (80% correct) at each respective interval. Hippocampal CA3 and CA1 principal cells were isolated and recorded throughout treatment. Extension of the delay led to an increase in the number of task-correlated neurons in controls. This recruitment of novel cells was reduced/prevented in the presence of WIN-2 and was paralleled by impairment in acquisition learning at longer delay intervals. Moreover, WIN-2 suppressed hippocampal ensemble firing during the sample (encoding) but not nonmatch phase of the DNMS task across all delays. These cannabinoid-induced alterations in hippocampal neuronal activity may explain the observed deficits in DNMS performance.

Trace amine-associated receptor 1 agonism promotes wakefulness without impairment of cognition in Cynomolgus macaques.

Trace amine-associated receptor 1 (TAAR1) is a G-protein coupled receptor with affinity for the trace amines. TAAR1 agonists have pro-cognitive, antidepressant-, and antipsychotic-like properties in both rodents and non-human primates (NHPs). TAAR1 agonism also increases wakefulness and suppresses rapid-eye movement (REM) sleep in mice and rats and reduces cataplexy in two mouse models of narcolepsy. We investigated the effects of TAAR1 agonism in Cynomolgus macaques, a diurnal species that exhibits consolidated night-time sleep, and evaluated the effects of TAAR1 agonists on cognition using a working memory (WM) paradigm in this species. Adult male Cynomolgus macaques (n = 6) were surgically implanted to record the electroencephalogram (EEG), electromyogram, and locomotor activity (LMA) and the efficacy of the TAAR1 partial agonist RO5263397 (0.1,1,10 mg/kg, p.o.) on sleep/wake, EEG spectra, and LMA was determined. In a second experiment, the acute effects of RO5263397 (0.1,1,10 mg/kg, p.o.) were assessed on a delayed-match-to-sample test of WM in adult male macaques (n = 7). RO5263397 (10 mg/kg) administered at lights off, when sleep pressure was high, promoted wakefulness and reduced both REM and non-REM sleep without inducing hyperlocomotion. RO5263397 (10 mg/kg) also increased delta/theta activity during all vigilance states. RO5263397 had no effect on WM at either short (2 sec) or long (10 sec) delay intervals. The wake-enhancing and REM-suppressing effects of R05263397 shown here in a diurnal primate are consistent with previous results in nocturnal rodents. These effects and the associated alterations in EEG spectra occurred without inducing hyperlocomotion or affecting WM, encouraging further study of TAAR1 agonists as potential narcolepsy therapeutics.

Systemic Blockade of the CB1 Receptor Augments Hippocampal Gene Expression Involved in Synaptic Plasticity but Perturbs Hippocampus-Dependent Learning Task.

Chronic and acute agonism as well as acute antagonism of CB1 receptors reveal modulation of learning and memory during stable performance of a delayed-nonmatch-to-sample (DNMS) memory task. However, it remains unclear how chronic blockade of the CB1 receptor alters acquisition of the behavioral task. We examined the effects of chronic rimonabant exposure during DNMS task acquisition to determine if blockade of the CB1 receptor with the antagonist rimonabant enhanced acquisition of operant task. Long-Evans rats, trained in the DNMS task before imposition of the trial delay, were surgically implanted with osmotic mini pumps to administer rimonabant (1.0 mg/kg/day) or vehicle (dimethyl sulfoxide/Tween-80/Saline). Following surgical recovery, DNMS training was resumed with the imposition of gradually longer delays (1-30 sec). The number of days required to achieve stable performance with either increasing length of delay or reversal of task contingency was compared between vehicle and rimonabant-treated rats. Following the completion of DNMS training, animals were euthanized, and both hippocampi were harvested for gene expression assay analysis. Rimonabant treatment animals required more time to achieve stable DNMS performance than vehicle-treated controls. Quantitative real-time polymerase chain reaction analysis revealed that the expressions of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit, brain-derived neurotrophic factor, and synapsin 1 (Syn1) were significantly increased. These results are consistent with rimonabant increasing mRNAs for proteins associated with hippocampal synapse remodeling, but that those alterations did not necessarily accelerate the acquisition of an operant behavioral task that required learning new contingencies.

Electrophysiological characterization of sleep/wake, activity and the response to caffeine in adult cynomolgus macaques.

Most preclinical sleep studies are conducted in nocturnal rodents that have fragmented sleep in comparison to humans who are primarily diurnal, typically with a consolidated sleep period. Consequently, we sought to define basal sleep characteristics, sleep/wake architecture and electroencephalographic (EEG) activity in a diurnal non-human primate (NHP) to evaluate the utility of this species for pharmacological manipulation of the sleep/wake cycle. Adult, 9-11 y.o. male cynomolgus macaques (n = 6) were implanted with telemetry transmitters to record EEG and electromyogram (EMG) activity and Acticals to assess locomotor activity under baseline conditions and following injections either with vehicle or the caffeine (CAF; 10 mg/kg, i.m.) prior to the 12 h dark phase. EEG/EMG recordings (12-36 h in duration) were analyzed for sleep/wake states and EEG spectral composition. Macaques exhibited a sleep state distribution and architecture similar to previous NHP and human sleep studies. Acute administration of CAF prior to light offset enhanced wakefulness nearly 4-fold during the dark phase with consequent reductions in both NREM and REM sleep, decreased slow wave activity during wakefulness, and increased higher EEG frequency activity during NREM sleep. Despite the large increase in wakefulness and profound reduction in sleep during the dark phase, no sleep rebound was observed during the 24 h light and dark phases following caffeine administration. Cynomolgus macaques show sleep characteristics, EEG spectral structure, and respond to CAF in a similar manner to humans. Consequently, monitoring EEG/EMG by telemetry in this species may be useful both for basic sleep/wake studies and for pre-clinical assessments of drug-induced effects on sleep/wake.

Alterations in High-Frequency Neuronal Oscillations in a Cynomolgus Macaque Test of Sustained Attention Following NMDA Receptor Antagonism.

A growing body of evidence indicates that neuronal oscillations in the gamma frequency range (30-80 Hz) are disturbed in schizophrenic patients during cognitive processes and may represent an endophenotype of the disease. N-methyl-D-aspartate (NMDA) receptor antagonists have been used experimentally to induce schizophrenia-like symptoms including cognitive deficits in animals and humans. Here we characterized neuronal oscillations and event-related potentials (ERPs) in Cynomolgus macaques fully trained to perform a continuous performance test (CPT) in the presence and absence of the NMDA antagonist phencyclidine (PCP). Macaques (n=8) were trained to touch 'target' stimuli and ignore 'distractor' stimuli presented randomly on a touchscreen. Subsequently, all subjects were implanted with epidural EEG electrodes over frontal (FC) and parietal cortices (PC) and later tested under vehicle (saline, i.m.) or acute PCP (0.1-0.3 mg/kg, i.m.) conditions. Compared with vehicle treatment, PCP produced a significant dose-dependent decrease in CPT performance accuracy and increased reaction times. Furthermore, PCP elevated the amplitudes of 'low' (30-50 Hz) and 'high' (51-80 Hz) gamma oscillations in FC and PC around target presentations for all correct responses. The CPT accuracy was inversely correlated with the gamma band amplitude in the presence of PCP. Additionally, PCP delayed the N100 peak latency in FC, and prolonged and suppressed the cognitively relevant P300 component of mean ERPs in FC and PC, respectively. The NMDA receptor antagonist-induced alteration in neuronal oscillations and ERPs may contribute to the observed cognitive deficits in macaques, and enhance our understanding of EEG recordings as a translatable biomarker.

Closing the loop for memory prosthesis: detecting the role of hippocampal neural ensembles using nonlinear models.

A major factor involved in providing closed loop feedback for control of neural function is to understand how neural ensembles encode online information critical to the final behavioral endpoint. This issue was directly assessed in rats performing a short-term delay memory task in which successful encoding of task information is dependent upon specific spatio-temporal firing patterns recorded from ensembles of CA3 and CA1 hippocampal neurons. Such patterns, extracted by a specially designed nonlinear multi-input multi-output (MIMO) nonlinear mathematical model, were used to predict successful performance online via a closed loop paradigm which regulated trial difficulty (time of retention) as a function of the "strength" of stimulus encoding. The significance of the MIMO model as a neural prosthesis has been demonstrated by substituting trains of electrical stimulation pulses to mimic these same ensemble firing patterns. This feature was used repeatedly to vary "normal" encoding as a means of understanding how neural ensembles can be "tuned" to mimic the inherent process of selecting codes of different strength and functional specificity. The capacity to enhance and tune hippocampal encoding via MIMO model detection and insertion of critical ensemble firing patterns shown here provides the basis for possible extension to other disrupted brain circuitry.

Pharmacological elevation of anandamide impairs short-term memory by altering the neurophysiology in the hippocampus.

In rodents, many exogenous cannabinoid agonists including Δ(9)-THC and WIN55,212-2 (WIN-2) have been shown to impair short-term memory (STM) by inhibition of hippocampal neuronal assemblies. However, the mechanisms by which endocannabinoids such as anandamide and 2-arachidonyl glycerol (2-AG) modulate STM processes are not well understood. Here the effects of anandamide on performance of a Delayed-Non-Match-to-Sample (DNMS) task (i.e. STM task) and concomitant hippocampal ensemble activity were assessed following administration of either URB597 (0.3, 3.0 mg/kg), an inhibitor of the Fatty Acid Amide Hydrolase (FAAH), AM404 (1.5, 10.0 mg/kg), a putative anandamide uptake/FAAH inhibitor, or R-methanandamide (3.0, 10.0 mg/kg), a stable analog of anandamide. Principal cells from hippocampal CA3/CA1 were recorded extracellularly by multi-electrode arrays in Long-Evans rats during DNMS task (1-30 s delays) performance and tracked throughout drug administration and recovery. Both R-methanandamide and URB597 caused dose- and delay-dependent deficits in DNMS performance with suppression of hippocampal ensemble activity during the encoding (sample) phase. R-methanandamide-induced effects were not reversed by capsaicin excluding a contribution of TRPV-1 receptors. AM404 produced subtle deficits at longer delay intervals but did not alter hippocampal neuronal activity during task-specific events. Collectively, these data indicate that endocannabinoid levels affect performance in a STM task and their pharmacological elevation beyond normal concentrations is detrimental also for the underlying physiological responses. They also highlight a specific window of memory processing, i.e. encoding, which is sensitive to cannabinoid modulation.

A Pilot Study into the Effects of the CB1 Cannabinoid Receptor Agonist WIN55,212-2 or the Antagonist/Inverse Agonist AM251 on Sleep in Rats.

The plant cannabinoid Δ(9)-tetrahydrocannabinol and the endocannabinoid anandamide increase the amount of sleep via a CB1 receptor mediated mechanism. Here, we explored the use of a novel electroencephalogram (EEG) recording device based on wireless EEG microchip technology (Neurologger) in freely-moving rats, and its utility in experiments of cannabinoids-induced alterations of EEG/vigilance stages. EEG was recorded through epidural electrodes placed above pre-frontal and parietal cortex (overlaying the dorsal hippocampus). As cannabinoids, we acutely administered the full synthetic CB1 receptor agonist, WIN55,212-2 (1 mg/kg), and the antagonist/inverse agonist, AM251 (2 mg/kg), either alone or together through the intraperitoneal route. WIN55,212-2 increased the total amount of NREM sleep and the length of each NREM bout, but this was unlikely due to CB1 receptor activation since it was not prevented by AM251. However, WIN55,212-2 also lowered overall EEG spectral power especially in theta and alpha frequency bands during wakefulness and NREM sleep, and this effect was reversed by AM251. The antagonist/inverse agonist caused no sleep alterations by itself and moderately increased spectral power in Theta, alpha and beta frequency bands during NREM sleep when administered on its own. Implications of endocannabinoid modulation of the sleep-wake cycle and its possible interactions with other transmitter systems are considered.

Tracking the changes of hippocampal population nonlinear dynamics in rats learning a memory-dependent task.

Neurobiological processes associated with learning are known to be highly nonlinear, dynamical, and time-varying. Characterizing the time-varying functional input-output properties of neural systems is a critical step to understand the neurobiological basis of learning. In this paper, we present a study on tracking of the changes of neural dynamics in rat hippocampus during learning of a memory-dependent delayed nonmatch-to-sample (DNMS) task. The rats were first trained to perform the DNMS task without a delay between the sample and response events. After reaching a performance level, they were subjected to the DNMS task with variable delays with a 5s mean duration. Spike trains were recorded from hippocampal CA3 (input) and CA1 (output) regions during all training sessions and constitute the input-output data for modeling. We applied the time-varying Generalized Laguerre-Volterra Model to study the changes of the CA3-CA1 nonlinear dynamics using these data. Result showed significant changes in the Volterra kernels after the introduction of delays. This result suggests that the CA3-CA1 nonlinear dynamics established in the initial training sessions underwent a functional reorganization as animals were learning to perform the task that now requires delays.

WIN55,212-2 induced deficits in spatial learning are mediated by cholinergic hypofunction.

Cannabinoids acting on CB(1) receptors induce learning and memory impairments. However, the identification of novel non-CB(1) receptors which are insensitive to the psychoactive ingredient of marijuana, Delta(9)-tetrahydrocannabinol (Delta(9)-THC) but sensitive to synthetic cannabinoids such as WIN55,212-2 (WIN-2) or endocannabinoids like anandamide lead us to question whether WIN-2 induced learning and memory deficits are indeed mediated by CB(1) receptor activation. Given the relative paucity of receptor subtype specific antagonists, a way forward would be to determine the transmitter systems, which are modulated by the respective cannabinoids. This study set out to evaluate this proposition by determination of the effects of WIN-2 on acquisition of spatial reference memory using the water maze in rats. Particular weight was given to performance in trial 1 of each daily session as an index of between-session long-term memory, and in trial 4 as an index of within-session short-term memory. Intraperitoneal (i.p.) administration of WIN-2 (1 mg/kg and 3 mg/kg) prior to training impaired long-term, but not short-term memory. This deficit was not reversed by the CB(1) antagonists/inverse agonists Rimonabant (3mg/kg i.p.) and AM281 (0.5 mg/kg i.p.), but recovered in the presence of the cholinesterase inhibitor rivastigmine (1 mg/kg). Reversal by rivastigmine was specific to WIN-2, as it failed to reverse MK801 (0.08 mg/kg) induced learning impairments. Collectively, these data suggest that in this spatial reference memory task WIN-2 causes a reduction in cholinergic activation, possibly through a non-CB(1)-like mechanism, which affects long-term but not short-term spatial memory.

Changes of hippocampal CA3-CA1 population nonlinear dynamics across different training sessions in rats performing a memory-dependent task.

Delayed-nonmatch-to-sample (DNMS) task is memory-dependent. Hippocampal CA3 and CA1 cells were shown to be encoding the required spatial and temporal information to complete this task. In order to identify possible changes in neural population nonlinear dynamics during learning of the DNMS task, we have first modeled the input-output transformation of spike trains across brain subregions from learning animals using a multiple-input, multiple-output (MIMO) nonlinear dynamic model. The feedforward and feedback kernels describing the relations between hippocampal CA3 and CA1 subregions have shown significant changes at different training sessions.

Short-term memory is modulated by the spontaneous release of endocannabinoids: evidence from hippocampal population codes.

Population codes derived from ensembles of hippocampal neurons were assessed to determine whether endocannabinoids were active when rats performed a delayed-nonmatch-to-sample (DNMS) short-term memory task. Multivariate discriminant analyses of the firing patterns of ensembles of CA1 and CA3 hippocampal neurons extracted representations of information encoded at the time of the sample response (SmR codes) during individual DNMS trials. The 'strength' or distinctiveness of trial-specific SmR codes in normal sessions was compared with sessions in which either rimonabant, the well-characterized cannabinoid CB1 receptor antagonist, or WIN 55212-2 (WIN-2), a cannabinoid CB1 receptor agonist, were administered. Results show that performance on trials with delay intervals longer than 10 s was facilitated by rimonabant (2.0 mg/kg) owing to a significantly increased frequency of trials with stronger SmR codes. In contrast, WIN-2 (0.35 mg/kg) suppressed the strength of SmR codes necessary to perform trials with delays greater than 10 s. The positive influence of rimonabant on performance indicated that the action of endocannabinoids was to reduce SmR code strength, resulting in trials that were at risk for errors if the delay exceeded 10 s. Thus endocannabinoids, like exogenously administered cannabinoids, reduced hippocampal encoding necessary to perform long-delay trials. The findings therefore indicate a direct relationship between the actions of endocannabinoids on hippocampal processes and the ability to encode information into short-term memory.