Journal of Pharmacology and Experimental Therapeutics NEUROPHARMACOLOGY

[NEUROPHARMACOLOGY] ADX47273 [S-(4-Fluoro-phenyl)-{3-[3-(4-fluoro-phenyl)-[1,2,4]-oxadiazol-5-yl]-piperidin-1-yl}-methanone]: A Novel Metabotropic Glutamate Receptor 5-Selective Positive Allosteric Modulator with Preclinical Antipsychotic-Like and Procognitive Activities

2008-11-12

Positive allosteric modulators (PAMs) of metabotropic glutamate receptor subtype 5 (mGlu5) enhance N-methyl-d-aspartate receptor function and may represent a novel approach for the treatment of schizophrenia. ADX47273 [S-(4-fluoro-phenyl)-{3-[3-(4-fluoro-phenyl)-[1,2,4]oxadiazol-5-yl]-piperidin-1-yl}-methanone], a recently identified potent and selective mGlu5 PAM, increased (9-fold) the response to threshold concentration of glutamate (50 nM) in fluorometric Ca²⁺ assays (EC₅₀ = 170 nM) in human embryonic kidney 293 cells expressing rat mGlu5. In the same system, ADX47273 dose-dependently shifted mGlu5 receptor glutamate response curve to the left (9-fold at 1 µM) and competed for binding of [³H]2-methyl-6-(phenylethynyl)pyridine (K_i = 4.3 µM), but not [³H]quisqualate. In vivo, ADX47273 increased extracellular signal-regulated kinase and cAMP-responsive element-binding protein phosphorylation in hippocampus and prefrontal cortex, both of which are critical for glutamate-mediated signal transduction mechanisms. In models sensitive to antipsychotic drug treatment, ADX47273 reduced rat-conditioned avoidance responding [minimal effective dose (MED) = 30 mg/kg i.p.] and decreased mouse apomorphine-induced climbing (MED = 100 mg/kg i.p.), with little effect on stereotypy or catalepsy. Furthermore, ADX47273 blocked phencyclidine, apomorphine, and amphetamine-induced locomotor activities (MED = 100 mg/kg i.p.) in mice and decreased extracellular levels of dopamine in the nucleus accumbens, but not in the striatum, in rats. In cognition models, ADX47273 increased novel object recognition (MED = 1 mg/kg i.p.) and reduced impulsivity in the five-choice serial reaction time test (MED = 10 mg/kg i.p.) in rats. Taken together, these effects are consistent with the hypothesis that allosteric potentiation of mGlu5 may provide a novel approach for development of antipsychotic and procognitive agents.

[NEUROPHARMACOLOGY] Nanomolar Concentrations of Pregnenolone Sulfate Enhance Striatal Dopamine Overflow in Vivo

Sadri-Vakili, G., Janis, G. C., Pierce, R. C., Gibbs, T. T., Farb, D. H. — 2008-11-12

The balance between GABA-mediated inhibitory and glutamate-mediated excitatory synaptic transmission represents a fundamental mechanism for controlling nervous system function, and modulators that can alter this balance may participate in the pathophysiology of neuropsychiatric disorders. Pregnenolone sulfate (PS) is a neuroactive steroid that can modulate the activity of ionotropic glutamate and GABA_A receptors either positively or negatively, depending upon the particular receptor subtype, and modulates synaptic transmission in a variety of experimental systems. To evaluate the modulatory effect of PS in vivo, we infused PS into rat striatum for 20 min via a microdialysis probe while monitoring local extracellular dopamine (DA) levels. The results demonstrate that PS at low nanomolar concentrations significantly increases extracellular DA levels. The PS-induced increase in extracellular DA is antagonized by the N-methyl-d-aspartate (NMDA) receptor antagonist, d-AP5 [d-(-)-2-amino-5-phosphonopentanoic acid], but not by the receptor antagonist, BD 1063 [1(-)[2-(3,4-dichlorophenyl)-ethyl]-4-methylpiperazine]. The results demonstrate that exogenous PS, at nanomolar concentrations, is able to increase DA overflow in the striatum through an NMDA receptor-mediated pathway.

[NEUROPHARMACOLOGY] Chronic Benzodiazepine Administration Potentiates High Voltage-Activated Calcium Currents in Hippocampal CA1 Neurons

2008-11-12

Signs of physical dependence as a consequence of long-term drug use and a moderate abuse liability limit benzodiazepine clinical usefulness. Growing evidence suggests a role for voltage-gated calcium channel (VGCC) regulation in mediating a range of chronic drug effects from drug withdrawal phenomena to dependence on a variety of drugs of abuse. High voltage-activated (HVA) calcium currents were measured in whole-cell recordings from acutely isolated hippocampal CA1 neurons after a 1-week flurazepam (FZP) treatment that results in withdrawal-anxiety. An ~1.8-fold increase in Ca²⁺ current density was detected immediately after and up to 2 days but not 3 or 4 days after drug withdrawal. Current density was unchanged after acute desalkyl-FZP treatment. A significant negative shift of the half-maximal potential of activation of HVA currents was also observed but steady-state inactivation remained unchanged. FZP and diazepam showed use- and concentration-dependent inhibition of Ca²⁺ currents in hippocampal cultured cells following depolarizing trains (FZP, IC₅₀ = 1.8 µM; diazepam, IC₅₀ = 36 µM), pointing to an additional mechanism by which benzodiazepines modulate HVA Ca²⁺ channels. Systemic preinjection of nimodipine (10 mg/kg), an L-type (L)-VGCC antagonist, prevented the benzodiazepine-induced increase in -amino-3-hydroxy-5-methylisoxasole-4-propionic acid receptor (AMPAR)-mediated miniature excitatory postsynaptic current in CA1 neurons 2 days after FZP withdrawal, suggesting that AMPAR potentiation, previously linked to withdrawal-anxiety may require enhanced L-VGCC-mediated Ca²⁺ influx. Taken together with prior work, these findings suggest that enhanced Ca²⁺ entry through HVA Ca²⁺ channels may contribute to hippocampal AMPAR plasticity and serve as a potential mechanism underlying benzodiazepine physical dependence.

[NEUROPHARMACOLOGY] Inhibition of Native and Recombinant Nicotinic Acetylcholine Receptors by the Myristoylated Alanine-Rich C Kinase Substrate Peptide

Gay, E. A., Klein, R. C., Melton, M. A., Blackshear, P. J., Yakel, J. L. — 2008-11-12

A variety of peptide ligands are known to inhibit the function of neuronal nicotinic acetylcholine receptors (nAChRs), including small toxins and brain-derived peptides such as β-amyloid_1–42 and synthetic apolipoproteinE peptides. The myristoylated alanine-rich C kinase substrate (MARCKS) protein is a major substrate of protein kinase C and is highly expressed in the developing and adult brain. The ability of a 25-amino acid synthetic MARCKS peptide, derived from the effector domain (ED), to modulate nAChR activity was tested. To determine the effects of the MARCKS ED peptide on nAChR function, receptors were expressed in Xenopus laevis oocytes, and two-electrode voltage-clamp experiments were performed. The MARCKS ED peptide completely inhibited acetylcholine (ACh)-evoked responses from 7 nAChRs in a dose-dependent manner, yielding an IC₅₀ value of 16 nM. Inhibition of ACh-induced responses was both activity- and voltage-independent. The MARCKS ED peptide was unable to block -bungarotoxin binding. A MARCKS ED peptide in which four serine residues were replaced with aspartate residues was unable to inhibit 7 nAChR-mediated currents. The MARCKS ED peptide inhibited ACh-induced 4β2 and 2β2 responses, although with decreased potency. The effects of the MARCKS ED peptide on native nAChRs were tested using acutely isolated rat hippocampal slices. In hippocampal interneurons, the MARCKS ED peptide was able to block native 7 nAChRs in a dose-dependent manner. The MARCKS ED peptide represents a novel antagonist of neuronal nAChRs that has considerable utility as a research tool.

[NEUROPHARMACOLOGY] Ethanol Decreases Purkinje Neuron Excitability by Increasing GABA Release in Rat Cerebellar Slices

Mameli, M., Botta, P., Zamudio, P. A., Zucca, S., Valenzuela, C. F. — 2008-11-12

Cerebellar Purkinje neurons (PNs) receive inhibitory GABAergic input from stellate and basket cells, which are located in the outer and inner portions of the molecular layer, respectively. Ethanol (EtOH) was recently shown to increase GABAergic transmission at PNs via a mechanism that involves enhanced calcium release from presynaptic internal stores (J Pharmacol Exp Ther 323:356–364, 2007). Here, we further characterized the effect of EtOH on GABA release and assessed its impact on PN excitability. Using whole-cell patch-clamp electrophysiological techniques in cerebellar vermis parasagittal slices, we found that EtOH acutely increases the frequency but not the amplitude or half-width of miniature and spontaneous inhibitory postsynaptic currents (IPSCs). EtOH significantly increased the amplitude and decreased the paired pulse ratio of IPSCs evoked by stimulation in the outer but not inner molecular layer. In current clamp, EtOH decreased both the amplitude of excitatory postsynaptic potentials evoked in PNs by granule cell axon stimulation and the number of action potentials triggered by these events; these effects depended on GABA_A receptor activation because they were not observed in presence of bicuculline. Loose-patch cell-attached PN recordings revealed that neither the spontaneous action potential firing frequency nor the coefficient of variation of the interspike interval was altered by acute EtOH exposure. These findings suggest that EtOH differentially affects GABAergic transmission at stellate cell- and basket cell-to-PN synapses and that it modulates PN firing triggered by granule cell axonal input. These effects could be in part responsible for the cerebellar impairments associated with acute EtOH intoxication.

[NEUROPHARMACOLOGY] Centrally Active Allosteric Potentiators of the M4 Muscarinic Acetylcholine Receptor Reverse Amphetamine-Induced Hyperlocomotor Activity in Rats

2008-11-12

Previous clinical and animal studies suggest that selective activators of M₁ and/or M₄ muscarinic acetylcholine receptors (mAChRs) have potential as novel therapeutic agents for treatment of schizophrenia and Alzheimer's disease. However, highly selective centrally penetrant activators of either M₁ or M₄ have not been available, making it impossible to determine the in vivo effects of selective activation of these receptors. We previously identified VU10010 [3-amino-N-(4-chlorobenzyl)-4, 6-dimethylthieno[2,3-b]pyridine-2-carboxamide] as a potent and selective allosteric potentiator of M₄ mAChRs. However, unfavorable physiochemical properties prevented use of this compound for in vivo studies. We now report that chemical optimization of VU10010 has afforded two centrally penetrant analogs, VU0152099 [3-amino-N-(benzo[d][1,3]dioxol-5-ylmethyl)-4,6-dimethylthieno[2,3-b]pyridine carboxamide] and VU0152100 [3-amino-N-(4-methoxybenzyl)-4,6-dimethylthieno[2,3-b]pyridine carboxamide], that are potent and selective positive allosteric modulators of M₄. VU0152099 and VU0152100 had no agonist activity but potentiated responses of M₄ to acetylcholine. Both compounds were devoid of activity at other mAChR subtypes or at a panel of other GPCRs. The improved physiochemical properties of VU0152099 and VU0152100 allowed in vivo dosing and evaluation of behavioral effects in rats. Interestingly, these selective allosteric potentiators of M₄ reverse amphetamine-induced hyperlocomotion in rats, a model that is sensitive to known antipsychotic agents and to nonselective mAChR agonists. This is consistent with the hypothesis that M₄ plays an important role in regulating midbrain dopaminergic activity and raises the possibility that positive allosteric modulation of M₄ may mimic some of the antipsychotic-like effects of less selective mAChR agonists.

[NEUROPHARMACOLOGY] NS11394 [3'-[5-(1-Hydroxy-1-methyl-ethyl)-benzoimidazol-1-yl]-biphenyl-2-carbonitrile], a Unique Subtype-Selective GABAA Receptor Positive Allosteric Modulator: In Vitro Actions, Pharmacokinetic Properties and in Vivo Anxiolytic Efficacy

2008-11-12

The novel positive allosteric modulator NS11394 [3'-[5-(1-hydroxy-1-methyl-ethyl)-benzoimidazol-1-yl]-biphenyl-2-carbonitrile] possesses a functional selectivity profile at GABA_A receptors of ₅ > ₃ > ₂ > ₁ based on oocyte electrophysiology with human GABA_A receptors. Compared with other subtype-selective ligands, NS11394 is unique in having superior efficacy at GABA_A-₃ receptors while maintaining low efficacy at GABA_A-₁ receptors. NS11394 has an excellent pharmacokinetic profile, which correlates with pharmacodynamic endpoints (CNS receptor occupancy), yielding a high level of confidence in deriving in vivo conclusions anchored to an in vitro selectivity profile and allowing for translation to higher species. Specifically, we show that NS11394 is potent and highly effective in rodent anxiety models. The anxiolytic efficacy of NS11394 is most probably mediated through its high efficacy at GABA_A-₃ receptors, although a contributory role of GABA_A-₂ receptors cannot be excluded. Compared with benzodiazepines, NS11394 has a significantly reduced side effect profile in rat (sedation, ataxia, and ethanol interaction) and mouse (sedation), even at full CNS receptor occupancy. We attribute this benign side effect profile to very low efficacy of NS11394 at GABA_A-₁ receptors and an overall partial agonist profile across receptor subtypes. However, NS11394 impairs memory in both rats and mice, which is possibly attributable to its efficacy at GABA_A-₅ receptors, albeit activity at this receptor might be relevant to its antinociceptive effects (J Pharmacol Exp Ther 327:doi;10.1124/jpet.108.144, 2008). In conclusion, NS11394 has a unique subtype-selective GABA_A receptor profile and represents an excellent pharmacological tool to further our understanding on the relative contributions of GABA_A receptor subtypes in various therapeutic areas.

[NEUROPHARMACOLOGY] Comparison of the Novel Subtype-Selective GABAA Receptor-Positive Allosteric Modulator NS11394 [3'-[5-(1-Hydroxy-1-methyl-ethyl)-benzoimidazol-1-yl]-biphenyl-2-carbonitrile] with Diazepam, Zolpidem, Bretazenil, and Gaboxadol in Rat Models of Inflammatory and Neuropathic Pain

2008-11-12

Spinal administration of GABA_A receptor modulators, such as the benzodiazepine drug diazepam, partially alleviates neuropathic hypersensitivity that manifests as spontaneous pain, allodynia, and hyperalgesia. However, benzodiazepines are hindered by sedative impairments and other side effect issues occurring mainly as a consequence of binding to GABA_A receptors containing the ₁ subunit. Here, we report on the novel subtype-selective GABA_A receptor-positive modulator NS11394 [3'-[5-(1-hydroxy-1-methyl-ethyl)-benzoimidazol-1-yl]-biphenyl-2-carbonitrile], which possesses a functional efficacy selectivity profile of ₅ > ₃ > ₂ > ₁ at GABA_A subunit-containing receptors. Oral administration of NS11394 (1–30 mg/kg) to rats attenuated spontaneous nociceptive behaviors in response to hindpaw injection of formalin and capsaicin, effects that were blocked by the benzodiazepine site antagonist flumazenil. Ongoing inflammatory nociception, observed as hindpaw weight-bearing deficits after Freund's adjuvant injection, was also completely reversed by NS11394. Likewise, hindpaw mechanical allodynia was fully reversed by NS11394 in two rat models of peripheral neuropathic pain. Importantly, NS11394-mediated antinociception occurred at doses 20 to 40-fold lower than those inducing minor sedative or ataxic impairments. In contrast, putative antinociception associated with administration of either diazepam, zolpidem, or gaboxadol only occurred at doses producing intolerable side effects, whereas bretazenil was completely inactive despite minor influences on motoric function. In electrophysiological studies, NS11394 selectively attenuated spinal nociceptive reflexes and C-fiber-mediated wind-up in vitro pointing to involvement of a spinal site of action. The robust therapeutic window seen with NS11394 in animals suggests that compounds with this in vitro selectivity profile could have potential benefit in clinical treatment of pain in humans.

[NEUROPHARMACOLOGY] Antidepressants Targeting the Serotonin Reuptake Transporter Act via a Competitive Mechanism

2008-11-12

Although several antidepressants (including fluoxetine, imipramine, citalopram, venlafaxine, and duloxetine) are known to inhibit the serotonin transporter (SERT), whether or not these molecules compete with 5-hydroxytryptamine (serotonin) (5-HT) for binding to SERT has remained controversial. We have performed radioligand competition binding experiments and found that all data can be fitted via a simple competitive interaction model, using Cheng-Prusoff analysis (Biochem Pharmacol 22:3099–3108, 1973). Two different SERT-selective radioligands, [³H]N,N-dimethyl-2-(2-amino-4-cyanophenyl thio)-benzylamine (DASB) and [³H]S-citalopram, were used to probe competitive binding to recombinantly expressed human SERT or native SERT in rat cortical membranes. All the SERT inhibitors that we tested were able to inhibit [³H]DASB and [³H]S-citalopram binding in a concentration-dependent manner, with unity Hill coefficient. In accordance with the Cheng-Prusoff relationship for a competitive interaction, we observed that test compound concentrations associated with 50% maximal inhibition of radiotracer binding (IC₅₀) increased linearly with increasing radioligand concentration for all ligands: 5-HT, S-citalopram, R-citalopram, paroxetine, clomipramine, fluvoxamine, imipramine venlafaxine, duloxetine, indatraline, cocaine, and 2-β-carboxy-3-β-(4-iodophenyl)tropane. The equilibrium dissociation constant of 5-HT and SERT inhibitors were also derived using Scatchard analysis of the data set, and they were found to be comparable with the data obtained using the Cheng-Prusoff relationship. Our studies establish a reference framework that will contribute to ongoing efforts to understand ligand binding modes at SERT by demonstrating that 5-HT and the SERT inhibitors tested bind to the serotonin transporter in a competitive manner.

[NEUROPHARMACOLOGY] Kinetic and Thermodynamic Assessment of Binding of Serotonin Transporter Inhibitors

2008-11-12

Several serotonin reuptake inhibitors are in clinical use for treatment of depression and anxiety disorders. However, to date, reported pharmacological differentiation of these ligands has focused mainly on their equilibrium binding affinities for the serotonin transporter. This study takes a new look at antidepressant binding modes using radioligand binding assays with [³H]S-citalopram to determine equilibrium and kinetic rate constants across multiple temperatures. The observed dissociation rate constants at 26°C fall into a narrow range for all molecules. Conversely, association rate constants generally decreased with increasing equilibrium binding affinities. Consistent with this, the measured activation energy for S-citalopram association was relatively large (19.5 kcal · mol^-1), suggesting conformational change upon ligand binding. For most of the drugs, including citalopram, the enthalpy (H^O) and entropy (-TS^O) contributions to reaction energetics were determined by van't Hoff analyses to be roughly equivalent (25–75% G^O) and to correlate (positively for enthalpy) with the polar surface area of the drug. However, the binding of the drug fluvoxamine was predominantly entropically driven. When these data are considered in the context of the physicochemical properties of these ligands, two distinct binding modes can be proposed. The citalopram-type binding mode probably uses a polar binding pocket that allows charged or polar interactions between ligand and receptor with comparatively small loss in enthalpy due to dehydration. The fluvoxamine-type binding mode is fueled by energy released upon burying hydrophobic ligand moieties into a binding pocket that is flexible enough to suffer minimal loss in entropy from conformational constraint.

[NEUROPHARMACOLOGY] Inhibition of Calpain Prevents N-Methyl-D-aspartate-Induced Degeneration of the Nucleus Basalis and Associated Behavioral Dysfunction

2008-10-17

N-Methyl-d-aspartate (NMDA) receptor-mediated excitotoxicity is thought to underlie a variety of neurological disorders, and inhibition of either the NMDA receptor itself, or molecules of the intracellular cascade, may attenuate neurodegeneration in these diseases. Calpain, a calcium-dependent cysteine protease, has been identified as part of such an NMDA receptor-induced excitotoxic signaling pathway. The present study addressed the question of whether inhibition of calpain can prevent neuronal cell death and associated behavioral deficits in a disease-relevant animal model, which is based on excitotoxic lesions of the cholinergic nucleus basalis magnocellularis of Meynert. Excitotoxic lesions of the nucleus basalis with NMDA induced a markedly impaired performance in the novel object recognition test. Treatment with the calpain inhibitor, N-(1-benzyl-2-carbamoyl-2-oxoethyl)-2-[E-2-(4-diethlyaminomethylphenyl) ethen-1-yl]benzamide (A-705253), dose-dependently prevented the behavioral deficit. Subsequent analysis of choline acetyltransferase in the cortical mantle of the lesioned animals revealed that application of A-705253 dose-dependently and significantly attenuated cholinergic neurodegeneration. Calpain inhibition also significantly diminished the accompanying gliosis, as determined by immunohistochemical analysis of microglia activation. Finally, inhibition of calpain by A-705253 and the peptidic calpain inhibitor N-acetyl-Leu-Leu-Nle-CHO did not impair long-term potentiation in hippocampal slices, indicating that calpain inhibition interrupts NMDA excitotoxicity pathways without interfering with NMDA receptor-mediated signaling involved in cognition. We conclude that inhibition of calpains may represent a valuable strategy for the prevention of excitotoxicity-induced neuronal decline without interfering with the physiological neuronal functions associated with learning and memory processes. Thus, calpain inhibition may be a promising and novel approach for the treatment of various neurodegenerative disorders.

[NEUROPHARMACOLOGY] Sustained Inhibition of Neurotransmitter Release from Nontransient Receptor Potential Vanilloid Type 1-Expressing Primary Afferents by {micro}-Opioid Receptor Activation-Enkephalin in the Spinal Cord

Zhou, H.-Y., Chen, S.-R., Chen, H., Pan, H.-L. — 2008-10-17

Removing transient receptor potential vanilloid type 1 (TRPV1)-expressing primary afferent neurons reduces presynaptic µ-opioid receptors but potentiates opioid analgesia. However, the sites and underlying cellular mechanisms for this paradoxical effect remain uncertain. In this study, we determined the presynaptic and postsynaptic effects of the µ-opioid receptor agonist [d-Ala²,N-Me-Phe⁴,Gly-ol⁵]-enkephalin (DAMGO) using whole-cell patch-clamp recordings of lamina II neurons in rat spinal cord slices. Treatment with the ultrapotent TRPV1 agonist resiniferotoxin (RTX) eliminated TRPV1-expressing dorsal root ganglion neurons and their central terminals in the spinal dorsal horn and significantly reduced the basal amplitude of glutamatergic excitatory postsynaptic currents (EPSCs) evoked from primary afferents. Although RTX treatment did not significantly alter the concentration-response effect of DAMGO on evoked monosynaptic and polysynaptic EPSCs, it causes a profound long-lasting inhibitory effect of DAMGO on evoked EPSCs. Subsequent naloxone treatment did not reverse the prolonged inhibitory effect of DAMGO on evoked EPSCs. Furthermore, brief application of DAMGO produced a sustained inhibition of miniature EPSCs in RTX-treated rats. However, the concentration response and the duration of the effects of DAMGO on G protein-coupled inwardly rectifying K⁺ currents in lamina II neurons were not significantly different between vehicle- and RTX-treated groups. These data suggest that stimulation of µ-opioid receptors on non-TRPV1 afferent terminals causes extended inhibition of neurotransmitter release to spinal dorsal horn neurons. The differential effect of µ-opioid receptor agonists on different phenotypes of primary afferents provides a cellular basis to explain why the analgesic action of opioids on mechanonociception is prolonged when TRPV1-expressing primary afferents are removed.

[NEUROPHARMACOLOGY] Dynamics of A{beta} Turnover and Deposition in Different {beta}-Amyloid Precursor Protein Transgenic Mouse Models Following {gamma}-Secretase Inhibition

2008-10-17

Human β-amyloid precursor protein (APP) transgenic mice are commonly used to test potential therapeutics for Alzheimer's disease. We have characterized the dynamics of β-amyloid (Aβ) generation and deposition following -secretase inhibition with compound LY-411575 [N²-[(2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl]-N¹-[(7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl]-l-alaninamide]. Kinetic studies in preplaque mice distinguished a detergent-soluble Aβ pool in brain with rapid turnover (half-lives for Aβ40 and Aβ42 were 0.7 and 1.7 h) and a much more stable, less soluble pool. Aβ in cerebrospinal fluid (CSF) reflected the changes in the soluble brain Aβ pool, whereas plasma Aβ turned over more rapidly. In brain, APP C-terminal fragments (CTF) accumulated differentially. The half-lives for -secretase degradation were estimated as 0.4 and 0.1 h for C99 and C83, respectively. Three different APP transgenic lines responded very similarly to -secretase inhibition regardless of the familial Alzheimer's disease mutations in APP. Amyloid deposition started with Aβ42, whereas Aβ38 and Aβ40 continued to turn over. Chronic -secretase inhibition lowered amyloid plaque formation to a different degree in different brain regions of the same mice. The extent was inversely related to the initial amyloid load in the region analyzed. No evidence for plaque removal below baseline was obtained. -Secretase inhibition led to a redistribution of intracellular Aβ and an elevation of CTFs in neuronal fibers. In CSF, Aβ showed a similar turnover as in preplaque animals demonstrating its suitability as marker of newly generated, soluble Aβ in plaque-bearing brain. This study supports the use of APP transgenic mice as translational models to characterize Aβ-lowering therapeutics.

[NEUROPHARMACOLOGY] {sigma}-1 Receptor Modulation of Acid-Sensing Ion Channel a (ASIC1a) and ASIC1a-Induced Ca2+ Influx in Rat Cortical Neurons

2008-10-17

Acid-sensing ion channels (ASICs) are proton-gated cation channels found in peripheral and central nervous system neurons. The ASIC1a subtype, which has high Ca²⁺ permeability, is activated by ischemia-induced acidosis and contributes to the neuronal loss that accompanies ischemic stroke. Our laboratory has shown that activation of receptors depresses ion channel activity and [Ca²⁺]_i dysregulation during ischemia, which enhances neuronal survival. Whole-cell patch-clamp electrophysiology and fluorometric Ca²⁺ imaging were used to determine whether receptors regulate the function of ASIC in cultured rat cortical neurons. Bath application of the selective ASIC1a blocker, psalmotoxin1, decreased proton-evoked [Ca²⁺]_i transients and peak membrane currents, suggesting the presence of homomeric ASIC1a channels. The pan-selective -1/-2 receptor agonists, 1,3-di-o-tolyl-guanidine (100 µM) and opipramol (10 µM), reversibly decreased acid-induced elevations in [Ca²⁺]_i and membrane currents. Pharmacological experiments using receptor-subtype-specific agonists demonstrated that -1, but not -2, receptors inhibit ASIC1a-induced Ca²⁺ elevations. These results were confirmed using the irreversible receptor antagonist metaphit (50 µM) and the selective -1 antagonist BD1063 (10 nM), which obtunded the inhibitory effects of the -1 agonist, carbetapentane. Activation of ASIC1a was shown to stimulate downstream Ca²⁺ influx pathways, specifically N-methyl-d-aspartate and (±)--amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainate receptors and voltage-gated Ca²⁺ channels. These subsequent Ca²⁺ influxes were also inhibited upon activation of -1 receptors. These findings demonstrate that -1 receptor stimulation inhibits ASIC1a-mediated membrane currents and consequent intracellular Ca²⁺ accumulation. The ability to control ionic imbalances and Ca²⁺ dysregulation evoked by ASIC1a activation makes receptors an attractive target for ischemic stroke therapy.

[NEUROPHARMACOLOGY] {alpha}Conotoxin Arenatus IB[V11l,V16D] Is a Potent and Selective Antagonist at Rat and Human Native {alpha}7 Nicotinic Acetylcholine Receptors

2008-10-17

A recently developed -conotoxin, -conotoxin Arenatus IB-[V11l,V16D] (-CtxArIB[V11L,V16D]), is a potent and selective competitive antagonist at rat recombinant 7 nicotinic acetylcholine receptors (nAChRs), making it an attractive probe for this receptor subtype. 7 nAChRs are potential therapeutic targets that are widely expressed in both neuronal and non-neuronal tissues, where they are implicated in a variety of functions. In this study, we evaluate this toxin at rat and human native nAChRs. Functional 7 nAChR responses were evoked by choline plus the allosteric potentiator PNU-120596 [1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea] in rat PC12 cells and human SH-SY5Y cells loaded with calcium indicators. -CtxArIB[V11L,V16D] specifically inhibited 7 nAChR-mediated increases in Ca²⁺ in PC12 cells. Responses to other stimuli, 5-I-A-85380 [5-iodo-3-(2(S)-azetidinylmethoxy)pyridine dihydrochloride], nicotine, or KCl, that did not activate 7 nAChRs were unaffected. Human 7 nAChRs were also sensitive to -CtxArIB[V11L, V16D]; acetylcholine-evoked currents in Xenopus laevis oocytes expressing human 7 nAChRs were inhibited by -CtxArIB[V11L,V16D] (IC₅₀, 3.4 nM) in a slowly reversible manner, with full recovery taking 15 min. This is consistent with the time course of recovery from blockade of rat 7 nAChRs in PC12 cells. -CtxArIB[V11L,V16D] inhibited human native 7 nAChRs in SHSY5Y cells, activated by either choline or AR-R17779 [(2)-spiro[1-azabicyclo[2.2.2]octane-3,59-oxazolidin]-29-one] plus PNU-120596. Rat brain 7 nAChRs contribute to dopamine release from striatal minces; -CtxArIB[V11L,V16D] (300 nM) selectively inhibited choline-evoked dopamine release without affecting responses evoked by nicotine that activates heteromeric nAChRs. This study establishes that -CtxArIB[V11L,V16D] selectively inhibits human and rat native 7 nAChRs with comparable potency, making this a potentially useful antagonist for investigating 7 nAChR functions.

[NEUROPHARMACOLOGY] N-Arachidonyl Maleimide Potentiates the Pharmacological and Biochemical Effects of the Endocannabinoid 2-Arachidonylglycerol through Inhibition of Monoacylglycerol Lipase

2008-10-17

Inhibition of the metabolism of the endocannabinoids, anandamide (AEA) and 2-arachidonyl glycerol (2-AG), by their primary metabolic enzymes, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively, has the potential to increase understanding of the physiological functions of the endocannabinoid system. To date, selective inhibitors of FAAH, but not MAGL, have been developed. The purpose of this study was to determine the selectivity and efficacy of N-arachidonyl maleimide (NAM), a putative MAGL inhibitor, for modulation of the effects of 2-AG. Our results showed that NAM unmasked 2-AG activity in a tetrad of in vivo tests sensitive to the effects of cannabinoids in mice. The efficacy of 2-AG (and AEA) to produce hypothermia was reduced compared with ⁹-tetrahydrocannabinol; however, 2-AG differed from AEA by its lower efficacy for catalepsy. All tetrad effects were partially CB₁ receptor-mediated because they were attenuated (but not eliminated) by SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-H-pyrazole-3-carboxamide HCl] and in CB₁^-/- mice. In vitro, NAM increased endogenous levels of 2-AG in the brain. Furthermore, NAM raised the potency of 2-AG, but not AEA, in agonist-stimulated guanosine 5'-O-(3-[³⁵S]thio)triphosphate binding assay, a measure of G-protein activation. These results suggest that NAM is an MAGL inhibitor with in vivo and in vitro efficacy. NAM and other MAGL inhibitors are valuable tools to elucidate the biological functions of 2-AG and to examine the consequences of dysregulation of this endocannabinoid. In addition, NAM's unmasking of 2-AG effects that are only partially reversed by SR141716A offers support for the existence of non-CB₁, non-CB₂ cannabinoid receptors.

[NEUROPHARMACOLOGY] The Effects of Methylphenidate on Knockin Mice with a Methylphenidate-Resistant Dopamine Transporter

Tilley, M. R., Gu, H. H. — 2008-10-17

Methylphenidate (Ritalin) is one of the most commonly abused prescription drugs. It is a psychostimulant that inhibits the dopamine and norepinephrine transporters with high affinity. In mice, methylphenidate stimulates locomotor activity, is self-administered, and produces conditioned place preference, typical properties of an addictive drug. We have generated a knockin mouse line bearing a mutant dopamine transporter that is approximately 80-fold less sensitive to cocaine inhibition than wild type. It is interesting to note that this mutant is also almost 50-fold less sensitive to methylphenidate inhibition, suggesting similarities in the binding site for cocaine and methylphenidate. Because methylphenidate is not effective at inhibiting the mutant dopamine transporter, we hypothesized that it would not stimulate locomotor activity or produce reward in the knockin mice. In these knockin mice, doses up to 40 mg/kg methylphenidate either inhibit or fail to stimulate locomotor activity and do not produce conditioned place preference. Doses up to 40 mg/kg methylphenidate also fail to produce stereotypy in the knockin mice. Nisoxetine and desipramine, selective norepinephrine transporter inhibitors, also reduce locomotor activity in wild-type and knockin mice. These results indicate that enhanced dopaminergic neurotransmission is required for methylphenidate's stimulating and rewarding effects. In addition, we observed that drugs enhancing noradrenergic neurotransmission inhibit locomotor activity in mice, which is consistent with the notion that methylphenidate's ability to inhibit the norepinephrine transporter may contribute to its efficacy in treating attention deficit hyperactivity disorder.

[NEUROPHARMACOLOGY] Antidepressant-Like Pharmacological Profile of a Novel Triple Reuptake Inhibitor, (1S,2S)-3-(Methylamino)-2-(naphthalen-2-yl)-1-phenylpropan-1-ol (PRC200-SS)

2008-10-17

Due to the putative involvement of dopaminergic circuits in depression, triple reuptake inhibitors are being developed as a new class of antidepressant, which is hypothesized to produce a more rapid onset and better efficacy than current antidepressants selective for serotonin or norepinephrine neurotransmission. (1S,2S)-3-(Methylamino)-2-(naphthalen-2-yl)-1-phenylpropan-1-ol (PRC200-SS), a new triple reuptake inhibitor, potently bound to the human serotonin, norepinephrine, and dopamine transporters with K_d values of 2.3, 0.63, and 18 nM, respectively. Inhibition of serotonin, norepinephrine, and dopamine uptake by PRC200-SS was also shown in cells expressing the corresponding transporter (K_i values of 2.1, 1.5, and 61 nM, respectively). In vivo, PRC200-SS dose-dependently decreased immobility in the forced-swim test in rats and in the tail-suspension test in mice, models predictive of antidepressant activity, with effects comparable with imipramine. These results in the behavioral models did not seem to result from the stimulation of locomotor activity. Consistent with the in vitro data and behavioral effects, peripheral administration of PRC200-SS (5 and 10 mg/kg i.p.) significantly increased extracellular levels of serotonin and norepinephrine in the medial prefrontal cortex, and of serotonin and dopamine in the core of nucleus accumbens, with reduction of levels of 3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid compared with levels for saline control. Furthermore, PRC200-SS self-administration, which was used as a marker of abuse liability, was not observed with rats. Therefore, it seems that PRC200-SS may represent a novel triple reuptake inhibitor and possess antidepressant activity.

[NEUROPHARMACOLOGY] MDMA (3,4-Methylenedioxymethamphetamine)-Mediated Distortion of Somatosensory Signal Transmission and Neurotransmitter Efflux in the Ventral Posterior Medial Thalamus

Starr, M. A., Page, M. E., Waterhouse, B. D. — 2008-09-19

MDMA (3,4-methylenedioxymethamphetamine, Ecstasy) is reported to enhance tactile sensory perception, an effect that is believed to contribute to its popularity as a recreational drug. To date, no literature exists that addresses the neurophysiological mechanisms underlying the effects of MDMA on somatosensation. However, MDMA interactions with the serotonin transporter protein (SERT) are well known. The rat trigeminal somatosensory system has been studied extensively and receives serotonergic afferents from the dorsal raphe nucleus. Given that these fibers express SERT, they should be vulnerable to MDMA-induced effects. We found that short-term low-dose MDMA administration (3 mg/kg i.p.) led to a significant increase in 5-hydroxytryptamine (5-HT) efflux in the ventral posterior medial (VPM) thalamus, the main relay along the lemniscal portion of the rodent trigeminal somatosensory pathway. We further evaluated the potential for MDMA to modulate whisker-evoked discharge (WED) of individual neurons in this region. After surgically implanting stainless steel 8-wire multichannel electrode bundles, we recorded spike train activity from single cells of halothane-anesthetized rats while mechanically activating the whisker pathway. We found that short-term low-dose MDMA (3 mg/kg i.p.) increased the spontaneous firing rate but reduced the magnitude and duration of WED in individual VPM thalamic neurons. It is noteworthy that the time course of drug action on neuronal firing patterns was generally consistent with increased 5-HT efflux as shown from our microdialysis studies. Based on these results, we propose the working hypothesis that MDMA may "distort" rather than enhance tactile experiences in humans, in part, by disrupting normal spike firing patterns through somatosensory thalamic relay circuits.

[NEUROPHARMACOLOGY] Paraquat Exposure Reduces Nicotinic Receptor-Evoked Dopamine Release in Monkey Striatum

2008-09-19

Paraquat, an herbicide widely used in the agricultural industry, has been associated with lung, liver, and kidney toxicity in humans. In addition, it is linked to an increased risk of Parkinson's disease. For this reason, we had previously investigated the effects of paraquat in mice and showed that it influenced striatal nicotinic receptor (nAChR) expression but not nAChR-mediated dopaminergic function. Because nonhuman primates are evolutionarily closer to humans and may better model the effects of pesticide exposure in man, we examined the effects of paraquat on striatal nAChR function and expression in monkeys. Monkeys were administered saline or paraquat once weekly for 6 weeks, after which nAChR levels and receptor-evoked [³H]dopamine ([³H]DA) release were measured in the striatum. The functional studies showed that paraquat exposure attenuated dopamine (DA) release evoked by 3/6β2^* (nAChR that is composed of the 3 or 6 subunits, and β2; the asterisk indicates the possible presence of additional subunits) nAChRs, a subtype present only on striatal dopaminergic terminals, with no decline in release mediated by 4β2^* (nAChR containing 4 and β2 subunits, but not 3 or 6) nAChRs, present on both DA terminals and striatal neurons. Paraquat treatment decreased 4β2^* but not 3/6β2^* nAChR expression. The differential effects of paraquat on nAChR expression and receptor-evoked [³H]DA release emphasize the importance of evaluating changes in functional measures. The finding that paraquat treatment has a negative impact on striatal nAChR-mediated dopaminergic activity in monkeys but not mice indicates the need for determining the effects of pesticides in higher species.

[NEUROPHARMACOLOGY] The Effects of Herkinorin, the First {micro}-Selective Ligand from a Salvinorin A-Derived Scaffold, in a Neuroendocrine Biomarker Assay in Nonhuman Primates

2008-09-19

Herkinorin is the first µ-opioid receptor-selective ligand from the salvinorin A diterpenoid scaffold. Herkinorin has relative µ > > binding selectivity, and it can act as an agonist at both µ- and -receptors, in vitro. These studies were the first in vivo evaluation of the effects of herkinorin in nonhuman primates, using prolactin release, a neuroendocrine biomarker assay that is responsive to both µ- and -agonists, as well as to compounds with limited ability to cross the blood-brain barrier. In cumulative dosing studies (0.01–0.32 mg/kg i.v.), herkinorin produced only small effects in gonadally intact males (n = 4), but a more robust effect in females (n = 4). Time course studies with herkinorin (0.32 mg/kg) confirmed this greater effectiveness in females and revealed a fast onset after i.v. administration (e.g., by 5–15 min). Antagonism experiments with different doses of nalmefene (0.01 and 0.1 mg/kg) caused dose-dependent and complete prevention of the effect of herkinorin in females. This is consistent with a principal µ-agonist effect of herkinorin, with likely partial contribution by -agonist effects. The peripherally selective antagonist quaternary naltrexone (1 mg/kg s.c.) caused approximately 70% reduction in the peak effect of herkinorin (0.32 mg/kg) in females, indicating that this effect of herkinorin is prominently mediated outside the blood-brain barrier.

[NEUROPHARMACOLOGY] Methylphenidate-Induced Increases in Vesicular Dopamine Sequestration and Dopamine Release in the Striatum: The Role of Muscarinic and Dopamine D2 Receptors

2008-09-19

Methylphenidate (MPD) administration alters the subcellular distribution of vesicular monoamine transporter-2 (VMAT-2)-containing vesicles in rat striatum. This report reveals previously undescribed pharmacological features of MPD by elucidating its receptor-mediated effects on VMAT-2-containing vesicles that cofractionate with synaptosomal membranes after osmotic lysis (referred to herein as membrane-associated vesicles) and on striatal dopamine (DA) release. MPD administration increased DA transport into, and decreased the VMAT-2 immunoreactivity of, the membrane-associated vesicle subcellular fraction. These effects were mimicked by the D2 receptor agonist quinpirole and blocked by the D2 receptor antagonist eticlopride. Both MPD and quinpirole increased vesicular DA content. However, MPD increased, whereas quinpirole decreased, K⁺-stimulated DA release from striatal suspensions. Like MPD, the muscarinic receptor agonist, oxotremorine, increased K⁺-stimulated DA release. Both eticlopride and the muscarinic receptor antagonist scopolamine blocked MPD-induced increases in K⁺-stimulated DA release, whereas the N-methyl-d-aspartate receptor antagonist (-)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) was without effect. This suggests that D2 receptors mediate both the MPD-induced redistribution of vesicles away from synaptosomal membranes and the MPD-induced up-regulation of vesicles remaining at the membrane. This results in a redistribution of DA within the striatum from the cytoplasm into vesicles, leading to increased DA release. However, D2 receptor activation alone is not sufficient to mediate the MPD-induced increases in striatal DA release because muscarinic receptor activation is also required. These novel findings provide insight into the mechanism of action of MPD, regulation of DA sequestration/release, and treatment of disorders affecting DA disposition, including attention-deficit hyperactivity disorder, substance abuse, and Parkinson's disease.

[NEUROPHARMACOLOGY] Alterations in Fos-Related Antigen 2 and {sigma}1 Receptor Gene and Protein Expression Are Associated with the Development of Cocaine-Induced Behavioral Sensitization: Time Course and Regional Distribution Studies

Liu, Y., Matsumoto, R. R. — 2008-09-19

Repeated exposure to cocaine results in neuroadaptations that can alter the way the brain responds to subsequent stimuli. Earlier studies demonstrated that acute administration of cocaine up-regulates the immediate-early gene fos-related antigen 2 (fra-2) followed by a later up-regulation of ₁ receptor gene and protein levels in brain regions involved in addiction and reward. To test whether such alterations could have long-term consequences on behavior, the present study was undertaken. Using a cocaine-induced behavioral sensitization model coupled with gene and protein expression studies in mice, the results show that cocaine induces the expression of fra-2, which leads to a progressive increase in ₁ receptor gene and protein expression over a period of days. This progressive increase in ₁ expression corresponds to the steady increase in the locomotor response to repeated cocaine administration in mice. The cocaine-induced changes in fra-2 and ₁ receptor gene and protein expression occur in brain regions that subserve drug abuse, such as the cortex, striatum, and hippocampus, but not the cerebellum. Moreover, the prototypic ₁ receptor antagonist 1-[2-(3,4-dichloropheny)ethyl]-4-methylpiperazine (BD1063) significantly attenuates both the molecular adaptations and behavioral sensitization induced by cocaine. These data suggest that repeated exposure to cocaine elicits alterations in fra-2 and ₁ receptor-mediated mechanisms, which ultimately manifest as altered behavioral responses to cocaine.

[NEUROPHARMACOLOGY] Purinergic Type 2 Receptors at GABAergic Synapses on Ventral Tegmental Area Dopamine Neurons Are Targets for Ethanol Action

Xiao, C., Zhou, C., Li, K., Davies, D. L., Ye, J. H. — 2008-09-19

The current study investigated whether ethanol alters ATP activation of purinergic type 2 receptors (P2Rs) in the ventral tegmental area (VTA). The VTA is a key region of the brain that has been implicated in the development of alcohol addiction. We investigated the effects of ATP and ethanol on spontaneous inhibitory postsynaptic currents (sIPSCs) and the spontaneous firings in the VTA dopaminergic neurons, obtained using an enzyme-free procedure. These neurons preserved some functional GABA-releasing terminals after isolation. We found that ATP (1–200 µM) either increased or decreased the frequency of sIPSCs and the activity of VTA dopaminergic neurons. The effects of ATP on sIPSC frequency inversely correlated with its effects on dopaminergic neuron activity. The ATP-induced changes in sIPSC frequency were blocked by tetrodotoxin (a sodium channel blocker) and by suramin (a nonselective P2R antagonist). Furthermore, ,β-methylene ATP, a selective P2X₁ and P2X₃ receptor agonist, increased sIPSC frequency, whereas adenosine 5'-[β-thio]diphosphate, a preferential agonist of P2Y receptors, decreased sIPSC frequency. In experiments testing the effects of ethanol (10 and 40 mM) on sIPSCs, we found that ethanol significantly attenuated ATP-induced increase and enhanced ATP-induced decrease in sIPSC frequency. Taken together, the results demonstrate that multiple subtypes of P2Rs exist on GABA-releasing terminals that make synapses on VTA dopaminergic neurons. It seems that ATP increases sIPSC frequency involving P2X₁ and/or P2X₃ receptors, and ATP decreases sIPSC frequency involving P2YRs. These findings are also consistent with the notion that P2Rs at GABA-releasing terminals on VTA dopaminergic neurons are important targets for ethanol action.