Acute and Repeated Administration of NLX-101, a Selective Serotonin-1A Receptor Biased Agonist, Reduces Audiogenic Seizures in Developing Fmr1 Knockout Mice.

Fragile XSyndrome (FXS) is a leading known genetic cause of Autism Spectrum Disorders (ASD) and intellectual disability. A consistent and debilitating phenotype of FXS is sensory hypersensitivity that manifests strongly in the auditory domain and may lead to delayed language and high anxiety. The mouse model of FXS, the Fmr1 KO mouse, also shows auditory hypersensitivity, an extreme form of which is seen as audiogenic seizures (AGS). The midbrain inferior colliculus (IC) is critically involved in generating audiogenic seizures and IC neurons are hyper-responsive to sounds in developing Fmr1 KO mice. Serotonin-1A receptor (5-HT1A) activation reduces IC activity. Therefore, we tested whether 5-HT1A activation is sufficient to reduce audiogenic seizures in Fmr1 KO mice. A selective and post-synaptic 5-HT1A receptor biased agonist, 3-Chloro-4-fluorophenyl-[4-fluoro-4-[[(5-methylpyrimidin-2-ylmethyl)amino]methyl]piperidin-1-yl] methanone (NLX-101, 0.6, 1.2, 1.8 or 2.4 mg/kg, i.p.) was administered to Fmr1 KO mice 15 min before seizure induction. Whereas the 0.6 mg/kg dose was ineffective in reducing seizures, the 1.2, 1.8 and 2.4 mg/kg doses of NLX-101 dramatically reduced seizures and increased mouse survival. Treatment with a combination of NLX-101 and 5-HT1A receptor antagonists prevented the protective effects of NLX-101, indicating that NLX-101 acts selectively through 5-HT1A receptors to reduce audiogenic seizures. NLX-101 (1.8 mg/kg) was still strongly effective in reducing seizures even after repeated administration over 5 days, suggesting an absence of tachyphylaxis to the effects of the compound. Together, these studies point to a promising treatment option targeting post-synaptic 5-HT1A receptors to reduce auditory hypersensitivity in FXS, and potentially across autism spectrum disorders.

NLX-112, a highly selective 5-HT1A receptor biased agonist, does not exhibit misuse potential in male rats or macaques.

NLX-112 (a.k.a. F13640 or befiradol) exhibits nanomolar affinity, exceptional selectivity and biased agonism at serotonin 5-HT1A receptors. NLX-112 displays robust analgesic activity in a number of rodent models of pain, and is currently developed as a treatment for l-DOPA-induced dyskinesia (LID) in Parkinson's disease (PD) patients. Noteworthy, PD patients can suffer from comorbid chronic pain, thus necessitating the use of analgesic drugs, such as opioids, which have potential for misuse. Additionally, dopamine agonists used to treat PD can produce cocaine-like effects in preclinical assays of misuse potential. The present study investigated whether NLX-112 possesses misuse potential of its own using two behavioural assays routinely used for this purpose: intracranial self-stimulation (ICSS) in rats, and cocaine discrimination in macaque monkeys. In rats, low doses of NLX-112 (0.03 and 0.1 mg/kg p.o.) did not alter ICSS frequency-rate curves, while higher doses (0.3 and 1.0 mg/kg) shifted the curve to the right and flattened it, i.e., reduced ICSS. As expected, cocaine (10 mg/kg i.p.) shifted the curve to the left, i.e., facilitated ICSS, but NLX-112 (0.03 and 0.1 mg/kg p.o.) did not further enhance cocaine-induced facilitation of ICSS. In monkeys trained to discriminate cocaine (0.4 mg/kg i.m.) from saline, NLX-112 (0.01-0.1 mg/kg p.o.) did not substitute for cocaine. Taken together, these results suggest that NLX-112, at doses displaying anti-dyskinetic activity in rat, marmoset and macaque models of LID, is free from misuse potential. From a translational perspective, this is a desirable property for a compound destined to be used in PD patients, who can suffer from comorbid chronic pain necessitating the use of potentially misused analgesic drugs.

Bell-shaped agonist activation of 5-HT1A receptor-coupled Gαi3 G-proteins: Receptor density-dependent switch in receptor signaling.

A previous study observed bell-shaped concentration-response isotherms for activation of Gαi3 G-protein subunits by high efficacy 5-HT1A receptor agonists in a Chinese hamster ovary (CHO) cell line expressing high levels of these receptors. This suggested that a signaling switch took place in that cell line (from Gαi3 to activation of other G-proteins) but it was unclear if such effects are observed for 5-HT1A receptors in other cellular environments. Here, using an antibody capture-based [35S]GTPγS binding assay for Gαi3 activation, we investigated whether efficacious 5-HT1A receptor agonists (5-HT, F13714, befiradol, NLX-101), prototypical agonists ((+) and (-)8-OH-DPAT), and partial agonist, antagonists, inverse agonists (pindolol, WAY100635, spiperone) produced similar effects on 5 cell lines expressing different levels of human 5-HT1A receptors. In membranes from cell lines (HeLa, C6-glia and CHO-low) expressing moderate receptor levels (between 1 and 4 pmol/mg of protein), 5-HT, F13714, befiradol and NLX-101 elicited classical sigmoid concentration-response isotherms. In contrast, in cell lines (CHO-high, HEK-293F) expressing high receptor levels (>9 pmol/mg) these agonists elicited bell-shaped concentration-response isotherms that peaked at nanomolar-range concentrations and then returned to baseline or below. Spiperone elicited inverse agonist inhibitory sigmoid isotherms in all membrane preparations while WAY100635 was mostly 'silent' for Gαi3 activation. The other compounds elicited diverse responses in the different cell lines suggesting that other factors, in addition to receptor expression levels, could be influencing Gαi3 activation. These data indicate that Gαi3 G-protein activation by 5-HT1A receptor ligands is highly dependent on receptor expression levels and on cellular background. Moreover, the induction of bell-shape concentration-response isotherms by 5-HT and other high-efficacy agonists is consistent with a switch in signaling to other G-protein-mediated signaling cascades, possibly elicited by receptor conformational changes.

NLX-112, a highly selective 5-HT1A receptor agonist, mediates analgesia and antidepressant-like activity in rats via spinal cord and prefrontal cortex 5-HT1A receptors, respectively.

NLX-112 (a.k.a. F13640 or befiradol) possesses marked activity in a variety of animal models of pain and of neuropsychiatric disorders; it exhibits nanomolar affinity, exceptional selectivity and high agonist efficacy at 5-hydroxytryptamine1A (5-HT1A) receptors. Although NLX-112 has been shown to activate 5-HT1A postsynaptic heteroreceptors in the prefrontal cortex (PFC), a brain region involved in the control of depressive states, the influence of NLX-112 on spinal cord 5-HT1A receptors (implicated in the control of pain) has not been described. Here we report on the ability, in rats, of NLX-112 to elicit analgesia in the intraplantar formalin model of nociceptive pain following intrathecal (i.t.) administration, and its ability to produce antidepressant-like activity in the forced swim test (FST) following in situ PFC microinjection. NLX-112, injected i.t. (L5-L6 region) induced analgesic effects in the formalin model of tonic nociceptive pain. At 20 µg, it almost abolished the effect of formalin on both the paw licking and paw elevation measures, and in both the early (0-5 min after formalin administration, reflecting acute pain) and the late (22.5-27.5 min, reflecting inflammatory pain) phases. The effects of NLX-112 (20 µg i.t.) were reversed by co-administration of 20 µg i.t. of the 5-HT1A receptor antagonist, WAY100635. Furthermore, the analgesic effects of systemically administered NLX-112 (0.63 mg/kg i.p.) were reversed by i.t. administration of WAY100635 (20 µg), most notably on paw licking. Finally, microinjection of NLX-112, bilaterally in the PFC, dose-dependently (MED 4 µg) and markedly reduced immobility in the FST (circa 90% reduction at 32 µg). In conclusion, the present data demonstrate that activation of spinal cord-located 5-HT1A receptors is sufficient for NLX-112 to mediate its analgesic effects in a rat model of tonic nociceptive pain. The data also highlight the involvement of PFC 5-HT1A receptors in the antidepressant-like activity of NLX-112 in the FST. Overall, the study suggests that highly selective and high efficacy 5-HT1A receptors agonists, such as NLX-112, could be useful to treat painful conditions associated with depressive states, through activation of different sub-populations of 5-HT1A receptors.

NLX-112, a highly selective 5-HT1A receptor agonist: Effects on body temperature and plasma corticosterone levels in rats.

NLX-112 (a.k.a. F13640 or befiradol), exhibits nanomolar affinity, exceptional selectivity and high agonist efficacy at 5-hydroxytryptamine 5-HT1A receptors. It possesses marked activity in a variety of animal models of depression, pain and L-DOPA-induced dyskinesia. However, its influence on translational biomarkers of central 5-HT1A receptor activation has not been previously described. Here, we report on the activity, in rats, of NLX-112 to increase plasma corticosterone levels and produce hypothermia, two responses which are also elicited by 5-HT1A receptor agonists in humans. NLX-112 elicited dose-dependent hypothermia (minimal effective dose, MED: 0.31mg/kg p.o.) and also increased plasma corticosterone both by oral and intraperitoneal routes (MED: 0.63mg/kg in both cases). The increase in corticosterone induced by NLX-112 (0.63mg/kg p.o.) was abolished by co-administration of the selective 5-HT1A receptor antagonist, WAY100635. Additionally, NLX-112 also dose-dependently induced flat body posture, forepaw treading and lower lip retraction (MEDs 0.31-0.63mg/kg p.o.). The doses of NLX-112 which induce hypothermia or corticosterone release were similar to those inducing serotonergic behaviors but greater than those reported previously in models of therapeutic-like activity (range 0.04 to 0.16mg/kg). Overall, the present study provides information for clinical dose estimations of NLX-112 and suggests that therapeutic effects may occur at doses below those at which biomarker responses are observed.

Comparison of Ins(1,4,5)P3 receptors from rat cerebellum and bovine adrenal cortex.

Ins(1,4,5)P3 receptors in adrenal cortical and cerebellar membranes can be distinguished by their affinities for Ins(1,4,5)P3 as well as the potencies with which heparin and Mg2+ inhibit binding. We have found that the differences in Ins(1,4,5)P3 affinity and heparin inhibition are maintained upon receptor solubilization and purification. In contrast to this, heparin-agarose affinity purification of solubilized cerebellar receptors reduces the potency of Mg2+ inhibition to that in adrenal cortex. These results suggest that Ins(1,4,5)P3 receptors in adrenal cortex are structurally distinct from those in cerebellum. Monoclonal antibodies raised against C- and N-terminal regions of mouse cerebellar Ins(1,4,5)P3 receptors recognize 250-300-kDa proteins in both rat cerebellum and bovine adrenal cortex.

Activity of serotonin 5-HT(1A) receptor 'biased agonists' in rat models of Parkinson's disease and L-DOPA-induced dyskinesia.

Serotonin 5-HT1A receptor agonists reduce L-DOPA-induced dyskinesia (LID) in animal models of Parkinson's disease (PD). Here, we compared the effects of novel 5-HT1A receptor 'biased agonists' on LID in hemiparkinsonian rats. F13714 preferentially activates pre-synaptic 5-HT1A autoreceptors. F15599 preferentially activates cortical postsynaptic 5-HT1A heteroreceptors. The partial agonist, tandospirone, does not differentiate these receptor subpopulations. The drugs were also tested on rotational behavior, rotarod and cylinder test for evaluation of locomotor activity, motor coordination and forelimb akinesia. Finally, the effects of F13714 and F15599 on 5-HT, DA, glutamate, and GABA release were investigated by microdialysis. F13714 abolished L-DOPA-induced AIMs even at very low doses (0.02-0.04 mg/kg). This effect was reversed by the selective 5-HT1A receptor antagonist, WAY100635. F13714 also elicited ipsilateral rotations (which were blocked by WAY100635) and potentiated the rotational activity of a sub-threshold dose of L-DOPA (2 mg/kg). F13714 profoundly inhibited striatal 5-HT release on both sides of the brain, and slightly increased DA release on the intact side. F15599 inhibited the L-DOPA-induced AIMs only at a dose (0.16 mg/kg) that reduced 5-HT release. Tandospirone produced a modest attenuation of peak AIMs severity and did not elicit rotations. F13714, F15599 and tandospirone did not modify the action of L-DOPA in the cylinder test but impaired rotarod performance at the highest doses tested. Targeting 5-HT1A receptors with selective biased agonists exerts distinct effects in the rat model of PD and LID. Preferential activation of 5-HT1A autoreceptors could potentially translate to superior antidyskinetic and L-DOPA dose-sparing effects in PD patients.

NLX-112, a novel 5-HT1A receptor agonist for the treatment of L-DOPA-induced dyskinesia: Behavioral and neurochemical profile in rat.

L-DOPA is the gold-standard treatment for Parkinson's disease (PD), but induces troublesome dyskinesia after prolonged treatment. This is associated with the 'false neurotransmitter' conversion of L-DOPA to dopamine by serotonin neurons projecting from the raphe to the dorsal striatum. Reducing their activity by targeting pre-synaptic 5-HT1A receptors should thus be an attractive therapeutic strategy, but previous 5-HT1A agonists have yielded disappointing results. Here, we describe the activity of a novel, highly selective and potent 5-HT1A agonist, NLX-112 (also known as befiradol or F13640) in rat models relevant to PD and its associated affective disorders. NLX-112 (0.16 mg/kg, i.p.) potently and completely reversed haloperidol-induced catalepsy in intact rats and abolished L-DOPA-induced Abnormal Involuntary Movements (AIMs) in hemiparkinsonian rats, an effect that was reversed by the selective 5-HT1A antagonist, WAY100635. In microdialysis experiments, NLX-112 profoundly decreased striatal 5-HT extracellular levels, indicative of inhibition of serotonergic function. NLX-112 also blunted the L-DOPA-induced surge in dopamine levels on the lesioned side of the brain, an action that likely underlies its anti-dyskinetic effects. NLX-112 (0.16 mg/kg, i.p.) robustly induced rotations in hemiparkinsonian rats, suggesting that it has a motor facilitatory effect. Rotations were abolished by WAY100635 and were ipsilateral to the lesioned side, suggesting a predominant stimulation of the dopamine system on the non-lesioned side of the brain. NLX-112 also efficaciously reduced immobility time in the forced swim test (75% reduction at 0.16 mg/kg, i.p.) and eliminated stress-induced ultrasonic vocalization at 0.08 mg/kg, i.p., effects consistent with potential antidepressant- and anxiolytic-like properties. In other tests, NLX-112 (0.01-0.16 mg/kg, i.p.) did not impair the ability of L-DOPA to rescue forepaw akinesia in the cylinder test but decreased rotarod performance, probably due to induction of flat body posture and forepaw treading which are typical of 5-HT1A agonists upon acute administration. However, upon repeated administration of NLX-112 (0.63 mg/kg, i.p., twice a day), flat body posture and forepaw treading subsided within 4 days of treatment. Taken together, these observations suggest that NLX-112 could exhibit a novel therapeutic profile, combining robust anti-dyskinetic properties without impairing the therapeutic properties of L-DOPA, and with additional beneficial effects on non-motor (affective) symptoms.

Metabotropic glutamate receptor involvement in models of acute and persistent pain: prospects for the development of novel analgesics.

The excitatory amino acid glutamate plays a major role in nociceptive processing. Ionotropic and metabotropic glutamate receptors are expressed in relevant areas of the brain, spinal cord and periphery that are involved in pain sensation and transmission. Activation of mGlu receptors along the pain neuraxis can result in either pronociceptive or antinociceptive behaviors depending on the subtype of mGluR and its location. The data published to date most strongly support the idea that mGlu1 antagonists might act as broad-spectrum analgesics. Several studies pointing to a functional upregulation of mGlu2/3 in chronic pain models suggest that agonists of these receptors might also be effective analgesics in certain conditions, most notably inflammation-induced hyperalgesia and allodynia. The expression of mGluRs throughout the pain neuraxis and the differing roles of the mGluRs in each of these regions makes it difficult to predict the efficacy of mGluR ligands based on in vitro or local administration studies. Potent, systemically active compounds that show mGluR subtype selectivity will be critical to undertake more detailed analyses in animal models of pain.

Role of metabotropic glutamate receptor subtype 5 (mGluR5) in the maintenance of cold hypersensitivity following a peripheral mononeuropathy in the rat.

The present series of experiments were designed to examine the contribution of metabotropic glutamate receptor subtype 5 (mGluR5) to neuropathic pain by determining the effects of the selective mGluR5 antagonist MPEP (2-methyl-6-(phenylethynyl)-pyridine) on neuropathy-induced cold hypersensitivity. Unilateral chronic constriction injury (CCI) to the sciatic nerve in rats produced an increase in the number of hind paw withdrawals from a cold surface (4 +/- 2 degrees C) which was dose-dependently inhibited by systemic (i.p.) injection of MPEP (ID(50) = 11.3 mg/kg). In vivo brain mGluR5 receptor occupancy following systemic (i.p.) MPEP revealed that >90% occupancy is required for behavioral efficacy. Intracerebroventricular (i.c.v.) injection of MPEP dose-dependently inhibited CCI-induced cold hypersensitivity (ID(50) = 123.5 nmol), while microinjection of MPEP directly into the rostral ventromedial medulla (RVM) potently inhibited this hypersensitivity (ID(50) = 1.3 pmol). A role for mGluR5 in the RVM was further supported by the observation that intra-RVM injection of the mGluR5 agonist CHPG (10 nmol; 2-chloro-5-hydroxyphenylglycine) produced cold hypersensitivity in naïve rats that was blocked by pretreatment with intra-RVM MPEP (3 nmol). Intrathecal (500 nmol; i.t.) or intraplantar (300 nmol; i.pl.) injection of MPEP was ineffective in reversing CCI-induced cold hypersensitivity. These results demonstrate that mGluR5 contributes to cold hypersensitivity following peripheral neuropathy exclusively at supraspinal sites in the CNS. Additionally, mGluR5 in the RVM significantly contributes to the maintenance of cold hypersensitivity, likely via activation of descending nociceptive facilitatory systems.

Metabotropic glutamate receptor 5 antagonist-induced stimulation of hypothalamic-pituitary-adrenal axis activity: interaction with serotonergic systems.

The mGluR5 antagonist 2-methyl-6-(phenylethynyl) pyridine (MPEP) produces anxiolytic or antidepressant effects in several rodent models through incompletely described mechanisms. Anxiolytics and antidepressants share several neuroendocrine features, including acute activation of the hypothalamic-pituitary-adrenal (HPA)-axis, desensitization of neuroendocrine responses with repeated dosing, and desensitization of the HPA axis to 5-HT1A agonist stimulation. We characterized these neuroendocrine parameters in rats treated systemically with MPEP and compared them to those induced by the anxiolytic buspirone. Acutely, MPEP dose-dependently (0.1-10 mg/kg i.p.) increased plasma corticosterone concentrations. These responses were blocked by 50% with the 5-HT1A antagonist WAY100635. The corticosterone responses to both 3 mg/kg MPEP and buspirone were decreased by 80% after 5 days of twice-daily injections. Repeated injection with MPEP decreased HPA-axis sensitivity to buspirone challenge by 75%. This desensitization was not associated with changes in mGluR5 or 5-HT1A receptor binding properties, expression of G-protein subunits coupled to these receptors, or in 5-HT-stimulated binding of [(3)H]-GTPgammaS to membranes. We conclude that MPEP acutely disinhibits the HPA axis, in part through uncharacterized changes in serotonergic signaling. Desensitization of 5-HT1A responses after repeated MPEP administration may indicate that, like other anxiolytics and antidepressants, plasticity in 5-HT signal transduction pathways has occurred.

2-Methyl-6-(phenylethynyl)-pyridine (MPEP), a potent, selective and systemically active mGlu5 receptor antagonist.

In the present paper we describe 2-methyl-6-(phenylethynyl)-pyridine (MPEP) as a potent, selective and systemically active antagonist for the metabotropic glutamate receptor subtype 5 (mGlu5). At the human mGlu5a receptor expressed in recombinant cells, MPEP completely inhibited quisqualate-stimulated phosphoinositide (PI) hydrolysis with an IC50 value of 36 nM while having no agonist or antagonist activities at cells expressing the human mGlu1b receptor at concentrations up to 30 microM. When tested at group II and III receptors, MPEP did not show agonist or antagonist activity at 100 microM on human mGlu2, -3, -4a, -7b, and -8a receptors nor at 10 microM on the human mGlu6 receptor. Electrophysiological recordings in Xenopus laevis oocytes demonstrated no significant effect at 100 microM on human NMDA (NMDA1A/2A), rat AMPA (Glu3-(flop)) and human kainate (Glu6-(IYQ)) receptor subtypes nor at 10 microM on the human NMDA1A/2B receptor. In rat neonatal brain slices, MPEP inhibited DHPG-stimulated PI hydrolysis with a potency and selectivity similar to that observed on human mGlu receptors. Furthermore, in extracellular recordings in the CA1 area of the hippocampus in anesthetized rats, the microiontophoretic application of DHPG induced neuronal firing that was blocked when MPEP was administered by iontophoretic or intravenous routes. Excitations induced by microiontophoretic application of AMPA were not affected.

Effects of ketamine and N-methyl-D-aspartate on glutamate and dopamine release in the rat prefrontal cortex: modulation by a group II selective metabotropic glutamate receptor agonist LY379268.

Previous studies have shown that the metabotropic glutamate receptor (mGluR)2/3 agonist LY354740 attenuated glutamate release in medial prefrontal cortex (mPFC) induced by the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist phencyclidine. In the present study we examined the effects of the more potent mGluR2/3 selective agonist LY379268 on ketamine-evoked glutamate and dopamine (DA) release in mPFC of male rats. Subjects were implanted with a unilateral microdialysis probe in the mPFC and were tested 12-24 h after implantation. Ketamine (18 mg/kg, s.c.) evoked a significant release of glutamate and DA, although the glutamate response was slower in onset compared with DA. Pretreatment with either systemic (3 mg/kg s.c.) or local (1 microM, in the probe) LY379268 blocked ketamine-evoked glutamate, but not DA, release. When applied directly to the mPFC via the dialysis probe, ketamine (1 mM in the probe) had no effect on glutamate release but did significantly enhance the release of DA. Application of NMDA (500 microM in the probe), on the other hand, decreased DA while increasing glutamate release. The effect of NMDA on evoking glutamate release was blocked by systemic but not local administration of LY379268. These findings indicate that systemic ketamine increases both glutamate and DA release in mPFC and that the effect on glutamate can be blocked by stimulating mPFC group II mGluR receptors. Local ketamine, on the other hand, does not increase glutamate but does increase DA release. This suggests that ketamine acts outside of the mPFC to enhance glutamate, but within the mPFC to enhance DA release. The origin of the ketamine effect on mPFC glutamate is currently not known.

Lithium-induced decrease in spontaneous Ca2+ oscillations in single GH3 rat pituitary cells.

1. Measurement of [Ca2+]i in single rat pituitary GH3 cells by dynamic single cell imaging techniques demonstrated that under basal conditions there is a large variation in the temporal pattern of [Ca2+]i signalling between individual cells ranging from high frequency asynchronous oscillations to quiescence. 2. We have reported previously that treatment of GH3 cells with 1 mM Li+ (a concentration used therapeutically in the treatment of manic depression) for 7 days reduces basal and thyrotrophin-releasing hormone (TRH)-stimulated levels of mass inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. In the present study, we show that this is associated with a reduction in the number of cells exhibiting basal Ca2+ oscillations over a sampling period of 60 s, whereas the maximum amplitude of oscillations is unaffected. 3. The pattern of [Ca2+]i responses to the agonist TRH varied considerably between individual cells, making quantitation of the responses difficult; however, data obtained from measurements made on a population of cells showed that increases in peak [Ca2+]i induced by high concentrations of TRH were reduced in cells treated with 1 mM Li+ for 7 days relative to control cells. 4. The sensitivity of the phosphoinositide pathway to [Ca2+]i was investigated by loading GH3 cells with BAPTA/AM at a concentration sufficient to lower 'basal' [Ca2+]i in a population of cells and to inhibit agonist-stimulated increases in [Ca2+]i. Under these conditions, basal and TRH-stimulated mass Ins(1,4,5)P3 levels were unaffected. 5. These results demonstrate that a 7-day Li+ treatment leads to an alteration in Ca2+ signalling, in particular by reducing the number of cells exhibiting high frequency Ca2+ oscillations under basal conditions. The significance of these results to the clinical effectiveness of Li+ in the treatment of manic depression is discussed.

Selective antagonism of opioid-induced ventilatory depression by an ampakine molecule in humans without loss of opioid analgesia.

Ventilatory depression is a significant risk associated with the use of opioids. We assessed whether opioid-induced ventilatory depression can be selectively antagonized by an ampakine without reduction of analgesia. In 16 healthy men, after a single oral dose of 1,500 mg of the ampakine CX717, a target concentration of 100 ng/ml alfentanil decreased the respiratory frequency by only 2.9 +/- 33.4% as compared with 25.6 +/- 27.9% during placebo coadministration (P < 0.01).Blood oxygenation and the ventilatory response to hypercapnic challenge also showed significantly smaller decreases with CX717 than with placebo. In contrast, CX717 did not affect alfentanil-induced analgesia in either electrical or heat-based experimental models of pain. Both ventilatory depression and analgesia were reversed with 1.6 mg of naloxone. These results support the use of ampakines as selective antidotes in humans to counter opioid-induced ventilatory depression without affecting opioid-mediated analgesia.

Chronic lithium treatment inhibits basal and agonist-stimulated responses in rat cerebral cortex and GH3 pituitary cells.

Li+ is used clinically in the management of bipolar-disordered (manic-depressive) illness, but the mechanism of its clinical efficacy remains unclear. Li+ inhibits the metabolism of certain inositol phosphates, leading to a decreased cycling of inositol that may be sufficient to reduce phosphoinositide metabolism. We have tested this hypothesis in slices of rat cerebral cortex and in rat pituitary GH3 cells grown in the presence of low extracellular inositol. We show that basal and stimulated mass levels of inositol-1,4,5-trisphosphate were reduced in rat cerebral cortex and in GH3 cells after chronic, but not acute, treatment with a therapeutic concentration of Li+. In GH3 cells chronic treatment with Li+ also decreased basal levels of intracellular Ca2+ and secretion of prolactin, effects that were prevented by the presence of myo-inositol. Agonist-stimulated mobilization of Ca2+ and prolactin release were also reduced in Li(+)-treated cells. These findings show that chronic perturbation of the phosphoinositide pathway by Li+ is sufficient to reduce basal and agonist-stimulated cellular responses, an action that may underlie its effectiveness in the alleviation of affective disorders.

Are there subtypes of the inositol 1,4,5-trisphosphate receptor?

We have compared the properties of the [3H]Ins(1,4,5)P3-binding sites from a number of tissues in an attempt to determine if heterogeneity exists within the Ins(1,4,5)P3-receptor family. The binding of Ins(1,4,5)P3 was characterized in detail by using membranes prepared from human uterine smooth muscle and bovine adrenal cortex. Ins(1,4,5)P3 exhibited an approx. 5 times greater affinity for the binding site in adrenal cortex (KD = 9.81 +/- 1.92 nM) compared with uterine smooth muscle (KD = 37.1 +/- 1.8 nM). The binding was dependent on pH in both tissues, with a maximum at pH 8.3; at this pH various inositol phosphates and nucleotides competed for the binding sites with similar potencies on both tissues. However, the binding of Ins(1,4,5)P3 to the uterine smooth-muscle membranes was Ca2(+)-sensitive, whereas that to the bovine adrenal cortex was not; furthermore, heparin displaced the binding of Ins(1,4,5)P3 in the uterus with an IC50 value (concn. of displacer giving 50% inhibition of specific binding) of 3.9 micrograms/ml (2.5, 6.4; lower, upper range), compared with a value of 22 (13, 30) micrograms/ml in adrenal cortex. In view of the ability of Ins(1,4,5)P3 and heparin to distinguish between these binding sites, their effect on other tissues was examined. Ins(1,4,5)P3 showed a similar affinity for receptors located in the bovine cerebellum to those in the bovine adrenal cortex, but heparin displaced Ins(1,4,5)P3 binding with a 5-fold greater affinity from the cerebellum. Ins(1,4,5)P3 had a 2-fold greater affinity for its receptor with human platelets, as compared with human uterus, but heparin was unable to distinguish between these sites. In guinea-pig ileum, Ins(1,4,5)P3 displayed a similar affinity for the receptors in the longitudinal muscle compared with the circular muscle, but heparin could distinguish between these sites. These data show that small differences exist between tissues, but no clear picture is apparent. It is possible that these results reflect tissue-dependent factors such as phosphorylation, the presence of calmedin etc., rather than the presence of receptor subtypes or species difference.

Reduced stress-induced hyperthermia in mGluR5 knockout mice.

It hs been suggested that metabotropic glutamate receptor subtype 5 (mGluR5) play a role in the expression of anxiety, based on anxiolytic-like effects of the selective mGluR5 antagonist MPEP (2-methyl-6-(phenylethynyl)pyridine) in rodent models of anxiety, including stress-induced hyperthermia (SIH). To examine the suggested role of mGlu5 receptors in the expression of anxiety, we examined the stress response in mice lacking mGluR5 in several variations of the SIH procedure. In this paradigm, stress causes a mild increase in body temperature that can be blocked by known anxiolytic agents. Three procedures were employed: classical SIH using rectal-probe measurement of body temperature, and radiotelemetric measurement of body temperature in response to either saline injection or to the introduction of an intruder into the home cage. In all three procedures the mGluR5-knockout mice displayed a significant attenuation of the hyperthermic response to stress compared to littermate wild-type control mice. To confirm that our observations were likely to be due to the absence of mGluR5 in the knockout mice we also tested the effect of the recently described selective mGluR5 antagonist MTEP (3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine) in both the wild-type and mGluR5 knockout mice. Administration of MTEP in the wild-type mice, but not the mGluR5 knockout mice, attenuated SIH. That the mGluR5 knockout mice displayed an anxiolytic-like phenotype and that the mGluR5 antagonist, MTEP, showed a anxiolytic-like effect only in mice possessing mGluR5 further supports the suggestion that mGluR5 antagonists may be useful in the treatment of anxiety.