Timothy Martin Piser

Cannabinoid Receptor Agonists Inhibit Glutamatergic Synaptic Transmission in Rat Hippocampal Cultures

Activation of cannabinoid receptors inhibits voltage-gated Ca2+ channels and activates K+ channels, reminiscent of other G-protein-coupled signaling pathways that produce presynaptic inhibition. We tested cannabinoid receptor agonists for effects on excitatory neurotransmission between cultured rat hippocampal neurons. Reducing the extracellular Mg2+ concentration to 0.1 mm elicited repetitive, transient increases in intracellular Ca2+ concentration ([Ca2+]i spikes) that resulted from bursts of action potentials, as measured by combined whole-cell current clamp and indo-1-based microfluorimetry. Pharmacological characterization indicated that the [Ca2+]i spikes required glutamatergic synaptic transmission. Cannabinoid receptor ligands inhibited stereoselectively the frequency of [Ca2+]i spiking in the rank order of potency: CP 54,939 > CP 55,940 > Win 55,212-2 > anandamide, with EC50 values of 0.36, 1.2, 2.7, and 71 nm, respectively. CP 55,940 was potent, but not efficacious, and reversed the inhibition produced by Win 55,212-2, indicating that it is a partial agonist. Inhibition of [Ca2+]i spiking by Win 55,212-2 was prevented by treatment of cultures with active, but not heat-treated, pertussis toxin. Win 55,212-2 (100 nm) inhibited stereoselectively CNQX-sensitive excitatory postsynaptic currents (EPSCs) elicited by presynaptic stimulation with an extracellular electrode, but did not affect the presynaptic action potential or currents elicited by direct application of kainate. Consistent with a presynaptic site of action, Win 55,212-2 increased both the number of response failures and the coefficient of variation of the evoked EPSCs. In contrast, cannabimimetics did not affect bicuculline-sensitive inhibitory postsynaptic currents. Thus, activation of cannabinoid receptors inhibits the presynaptic release of glutamate via an inhibitory G-protein.

Immediate and Sustained Improvements in Working Memory After Selective Stimulation of α7 Nicotinic Acetylcholine Receptors

BACKGROUND Nicotine improves cognition in humans and animal models of neuropsychiatric disorders. Here, we sought to establish whether selective stimulation of the neuronal nicotinic α7 receptor could improve spatial working memory in nonhuman primates. METHODS Beginning with an estimated dose range from rodent studies, the dose of the α7 agonist AZD0328 was titrated for a significant impact on working memory in rhesus macaques after acute administration. After training to stability on the spatial delayed response task, subjects were administered AZD0328 (1.6 ng/kg-.48 mg/kg; intramuscular) or vehicle 30 min before cognitive testing. AZD0328 (1 ng/kg-1.0 μg/kg; intramuscular) was then administered in a repeated, intermittent ascending dose regimen where each dose was given in two bouts for 4 days with a 1-week washout in between bouts, followed by 2-week washout. RESULTS Acute AZD0328 improved cognitive performance when the dose was titrated down to .0016 and .00048 mg/kg from a cognitively impairing dose of .48 mg/kg. In a subgroup, sustained enhancement of working memory was evident for 1 month or more after acute treatment. Immediate and sustained cognitive enhancement was also found during and after repeated administration of AZD0328 at .001 mg/kg. CONCLUSIONS These findings demonstrate that extremely low doses of a nicotinic α7 agonist can have profound acute and long-lasting beneficial consequences for cognition, dependent upon the integrity of dorsolateral prefrontal cortex. Thus, the α7 receptor might have a fundamental role in the neural circuitry of working memory and in the synaptic plasticity upon which it might depend.

Identification of short-acting κ-opioid receptor antagonists with anxiolytic-like activity.

The κ-opioid receptor plays a central role in mediating the response to stressful life events. Inhibiting κ-opioid receptor signaling is proposed as a mechanism for treating stress-related conditions such as depression and anxiety. Preclinical testing consistently confirms that disruption of κ-opioid signaling is efficacious in animal models of mood disorders. However, concerns about the feasibility of developing antagonists into drugs stem from an unusual pharmacodynamic property of prototypic κ-opioid receptor-selective antagonists; they inhibit receptor signaling for weeks to months after a single dose. Several fundamental questions include - is it possible to identify short-acting antagonists; is long-lasting inhibition necessary for efficacy; and is it safe to develop long-acting antagonists in the clinic. Here, we test representative compounds (AZ-ECPC, AZ-MTAB, and LY-DMPF) from three new chemical series of κ-opioid receptor ligands for long-lasting inhibition. Each compound dose-dependently reversed κ-opioid agonist-induced diuresis. However, unlike the prototypic antagonist, nBNI, which fully inhibited evoked diuresis for at least four weeks, the new compounds showed no inhibition after one week. The two compounds with greater potency and selectivity were tested in prenatally-stressed rats on the elevated plus maze, an exploration-based model of anxiety. Spontaneous exploration of open arms in the elevated plus maze was suppressed by prenatal stress and restored with both compounds. These findings indicate that persistent inhibition is not an inherent property of κ-opioid-selective antagonists and that post-stress dosing with transient inhibitors can be effective in a mood disorder model. This further supports κ-opioid receptor as a promising target for developing novel psychiatric medications.

Linking the cytokine and neurocircuitry hypotheses of depression: a translational framework for discovery and development of novel anti-depressants.

Recent studies suggest a model of depression that links the cytokine hypothesis from the field of psychoneuroimmunology with the neurocircuitry hypothesis derived from burgeoning insight into neurophysiological changes observed in depressed patients. According to the neurocircuitry hypothesis of depression, failure of homeostatic synaptic plasticity in cortical-striatal-limbic nodes of a distributed network of neural circuits involving the sub-genual anterior cingulate cortex is responsible for core symptoms of depression: loss of interest or pleasure (anhedonia) and depressed mood (sadness). According to the cytokine hypothesis of depression, inflammatory cytokines act on neural circuits to evoke the behavioral and physiological changes observed in depression. Synthesis of these hypotheses implicates cytokines released during injury, infection, illness, or psychological stress as a cause of dysregulated synaptic plasticity in cortical-striatal-limbic circuits implicated in depression. These neural circuits process affective and reward-based information for optimal cost-benefit decision-making, a function that may link cytokine-evoked changes in synaptic plasticity to translatable measures of specific behavioral impairments observed in depressed patients. This viewpoint outlines evidence linking the cytokine and neurocircuitry hypotheses of depression to offer a translational model of major depressive disorder suitable for novel drug discovery and development.

In vitro metabolism of α7 neuronal nicotinic receptor agonist AZD0328 and enzyme identification for its N-oxide metabolite

AZD0328 was pharmacologically characterized as a α7 neuronal nicotinic receptor agonist intended for treatment of Alzheimer′s disease. In vitro AZD0328 cross species metabolite profile and enzyme identification for its N-oxide metabolite were evaluated in this study. AZD0328 was very stable in the human hepatocyte incubation, whereas extensively metabolized in rat, dog and guinea pig hepatocyte incubations. The N-oxidation metabolite (M6) was the only metabolite detected in human hepatocyte incubations, and it also appeared to be the major in vitro metabolic pathway in a number of preclinical species. In addition, N-glucuronide metabolite of AZD0328 was observed in human liver microsomes. Other metabolic pathways in the preclinical species include hydroxylation in azabicyclo octane or furopyridine part of the molecule. Pyridine N-methylation of AZD0328 (M2) was identified as a dog specific metabolite, not observed in human or other preclinical species. Multiple enzymes including CYP2D6, CYP3A4/5, FMO1 and FMO3 catalyzed AZD0328 metabolism. The potential for AZD0328 to be inhibited clinically by co-administered drugs or genetic polymorphism is relative low.

Microplate gel-filtration method for radioligand-binding assays

A thin-layer gel-filtration chromatographic method has been developed in a 96-well format to separate free and protein-bound ligand in radioligand-binding assays. The mobile phase in the gel-filtration plate is removed via quick centrifugation before samples are applied. Protein-bound ligand is recovered via centrifugation into another 96-well plate for radioactivity measurements. The method exhibits excellent recovery of protein-ligand complexes and less opportunity for dissociation of the complexes since it eliminates major dilution effects from the mobile phase of a column and from elution steps in conventional gel-filtration chromatography. It offers other advantages: simple, rapid, inexpensive, quantitative, and able to handle a large number of samples as required in drug discovery and clinical settings. This microplate gel-filtration method was optimized in studies of receptor-ligand interactions using estrogen receptors as examples and can be used in other radioligand-binding assays.