Neil Upton

The novel brain neuropeptide, orexin-A, modulates the sleep–wake cycle of rats

Orexin‐A is a novel neuropeptide initially isolated from hypothalamic extracts but now known to be present in fibres distributed throughout the rat CNS including many regions associated with sleep–wake regulation. The recognition of a particularly dense innervation of orexinergic nerves in the locus coeruleus, together with the observed increase in firing rate of locus coeruleus neurons following application of orexin‐A in vitro, further highlighted a potential involvement of the peptide in modulating the arousal state. The present study was undertaken to determine the effects of intracerebroventricularly (ICV) administered orexin‐A on the sleep–wake cycle of conscious rats using electroencephalographic and electromyographic recordings. When administered at the onset of the normal sleep period, orexin‐A (1, 10 or 30 μg/rat ICV) produced a dose‐dependent increase in the time rats spent awake during the second and third hours after dosing. The enhancement of arousal was accompanied by a marked reduction in paradoxical sleep and deep slow wave sleep at the highest dose. The latency to the first occurrence of paradoxical sleep was also prolonged. This overall profile of increased arousal and decreased paradoxical sleep is consistent with a high rate of firing of locus coeruleus neurons as would be expected to occur following ICV administration of orexin‐A. It is concluded that orexin‐A may play an important physiological role in regulating the sleep–wake state, a hypothesis considerably strengthened by the recently reported narcoleptic phenotype of prepro‐orexin (the precursor for orexin‐A) knockout mice.

Characterization of SB-269970-A, a selective 5-HT7 receptor antagonist

The novel 5‐HT7 receptor antagonist, SB‐269970‐A, potently displaced [3H]‐5‐CT from human 5‐HT7(a) (pKi 8.9±0.1) and 5‐HT7 receptors in guinea‐pig cortex (pKi 8.3±0.2). 5‐CT stimulated adenylyl cyclase activity in 5‐HT7(a)/HEK293 membranes (pEC50 7.5±0.1) and SB‐269970‐A (0.03–1 μM) inhibited the 5‐CT concentration‐response with no significant alteration in the maximal response. The pA2 (8.5±0.2) for SB‐269970‐A agreed well with the pKi determined from [3H]‐5‐CT binding studies. 5‐CT‐stimulated adenylyl cyclase activity in guinea‐pig hippocampal membranes (pEC50 of 8.4±0.2) was inhibited by SB‐269970‐A (0.3 μM) with a pKB (8.3±0.1) in good agreement with its antagonist potency at the human cloned 5‐HT7(a) receptor and its binding affinity at guinea‐pig cortical membranes. 5‐HT7 receptor mRNA was highly expressed in human hypothalamus, amygdala, thalamus, hippocampus and testis. SB‐269970‐A was CNS penetrant (steady‐state brain : blood ratio of ca. 0.83 : 1 in rats) but was rapidly cleared from the blood (CLb=ca. 140 ml min−1 kg−1). Following a single dose (3 mg kg−1) SB‐269970 was detectable in rat brain at 30 (87 nM) and 60 min (58 nM). In guinea‐pigs, brain levels averaged 31 and 51 nM respectively at 30 and 60 min after dosing, although the compound was undetectable in one of the three animals tested. 5‐CT (0.3 mg kg−1 i.p.) induced hypothermia in guinea‐pigs was blocked by SB‐269970‐A (ED50 2.96 mg kg−1 i.p.) and the non‐selective 5‐HT7 receptor antagonist metergoline (0.3–3 mg kg−1 s.c.), suggesting a role for 5‐HT7 receptor stimulation in 5‐CT induced hypothermia in guinea‐pigs. SB‐269970‐A (30 mg kg−1) administered at the start of the sleep period, significantly reduced time spent in Paradoxical Sleep (PS) during the first 3  h of EEG recording in conscious rats.

The 5-HT(6) receptor antagonist SB-271046 reverses scopolamine-disrupted consolidation of a passive avoidance task and ameliorates spatial task deficits in aged rats.

The highly potent and selective 5-HT6 receptor antagonist SB-271046 [5-chloro-N-(4-methoxy-3-piperazin-1-yl-phenyl)-3-methyl-2-benzothiophenesulfonamide] has previously been demonstrated to improve retention significantly in a spatial water maze paradigm in adult rats. However, SB-271046 did not have any effect on task acquisition. As these apparently contradictory findings may be reconciled by a prime influence of SB-271046 on memory consolidation, the ability of this compound to reverse the discrete temporal action of a cholinergic antagonist in the 6-h period following passive avoidance training was investigated. SB-271046, given orally, by gavage, 30 min prior to training Wistar rats in a step-through, light–dark passive avoidance task, was found to reverse significantly the amnesia produced by administering scopolamine (0.8 mg/kg, intraperitoneal) in the 6-h post-training period. The effect was dose-dependent over a range of 3–20 mg/kg. Further, we investigated the cognition-enhancing effects of chronic SB-271046 administration (10 or 20 mg/kg/day; 40 days) on the acquisition and consolidation of a water maze spatial learning task in a population of 20-month-old Wistar rats with age-related learning deficits. Drug treatment progressively and significantly decreased platform swim angle and escape latencies over the five sequential trials on four consecutive daily sessions compared to vehicle-treated controls. SB-271046 also improved task recall as measured by significant increases in the searching of the target quadrant on post-training days 1 and 3, when the animals would have been substantially drug-free. This significant improvement of task recall suggests SB-271046, in addition to inducing symptomatic cognition-enhancing actions, also attenuates age-related decline in neural function.

Characterization of SB-271046 : A potent, selective and orally active 5-HT6 receptor antagonist

SB‐271046, potently displaced [3H]‐LSD and [125I]‐SB‐258585 from human 5‐HT6 receptors recombinantly expressed in HeLa cells in vitro (pKi 8.92 and 9.09 respectively). SB‐271046 also displaced [125I]‐SB‐258585 from human caudate putamen and rat and pig striatum membranes (pKi 8.81, 9.02 and 8.55 respectively). SB‐271046 was over 200 fold selective for the 5‐HT6 receptor vs 55 other receptors, binding sites and ion channels. In functional studies on human 5‐HT6 receptors SB‐271046 competitively antagonized 5‐HT‐induced stimulation of adenylyl cyclase activity with a pA2 of 8.71. SB‐271046 produced an increase in seizure threshold over a wide‐dose range in the rat maximal electroshock seizure threshold (MEST) test, with a minimum effective dose of 0.1 mg kg−1 p.o. and maximum effect at 4 h post‐dose. The level of anticonvulsant activity achieved correlated well with the blood concentrations of SB‐271046 (EC50 of 0.16 μM) and brain concentrations of 0.01–0.04 μM at Cmax. These data, together with the observed anticonvulsant activity of other selective 5‐HT6 receptor antagonists, SB‐258510 (10 mg kg−1, 2–6 h pre‐test) and Ro 04‐6790 (1–30 mg kg−1, 1 h pre‐test), in the rat MEST test, suggest that the anticonvulsant properties of SB‐271046 are likely to be mediated by 5‐HT6 receptors. Overall, these studies demonstrate that SB‐271046 is a potent and selective 5‐HT6 receptor antagonist and is orally active in the rat MEST test. SB‐271046 represents a valuable tool for evaluating the in vivo central function of 5‐HT6 receptors.

GABAB receptor isoforms GBR1a and GBR1b, appear to be associated with pre- and post-synaptic elements respectively in rat and human cerebellum

Metabotropic γ‐aminobutyric acid (GABA) receptors, GABAB, are coupled through G‐proteins to K+ and Ca2+ channels in neuronal membranes. Cloning of the GABAB receptor has not uncovered receptor subtypes, but demonstrated two isoforms, designated GBR1a and GBR1b, which differ in their N terminal regions. In the rodent cerebellum GABAB receptors are localized to a greater extent in the molecular layer, and are reported to exist on granule cell parallel fibre terminals and Purkinje cell (PC) dendrites, which may represent pre‐ and post‐synaptic receptors. The objective of this study was to localize the mRNA splice variants, GBR1a and GBR1b for GABAB receptors in rat cerebellum, for comparison with the localization in human cerebellum using in situ hybridization. Receptor autoradiography was performed utilizing [3H]‐CGP62349 to localize GABAB receptors in rat and human cerebellum. Radioactively labelled oligonucleotide probes were used to localize GBR1a and GBR1b, and by dipping slides in photographic emulsion, silver grain images were obtained for quantification at the cellular level. Binding of 0.5 nM [3H]‐CGP62349 demonstrated significantly higher binding to GABAB receptors in the molecular layer than the granule cell (GC) layer of rat cerebellum (molecular layer binding 200±11% of GC layer; P<0.0001). GBR1a mRNA expression was found to be predominantly in the GC layer (PC layer grains 6±6% of GC layer grains; P<0.05), and GBR1b expression predominantly in PCs (PC layer grains 818±14% of GC layer grains; P<0.0001). The differential distribution of GBR1a and GBR1b mRNA splice variants for GABAB receptors suggests a possible association of GBR1a and GBR1b with pre‐ and post‐synaptic elements respectively.

Effects of Pan- and Subtype-Selective N-Methyl-d-aspartate Receptor Antagonists on Cortical Spreading Depression in the Rat: Therapeutic Potential for Migraine

Spreading depression (SD) has long been associated with the underlying pathophysiology of migraine. Evidence that the N-methyl-d-aspartate (NMDA) glutamate receptor (NMDA-R) is implicated in the generation and propagation of SD has itself been available for more than 15 years. However, to date, there are no reports of NMDA-R antagonists being developed for migraine therapy. In this study, an uncompetitive, pan-NMDA-R blocker, memantine, approved for clinical use, and two antagonists with selectivity for NMDA-R containing the NR2B subunit, (1S,2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol (CP-101,606) and (±)-(R*,S*)-α-(4-hydroxyphenyl)-β-methyl-4-(phenylmethyl)-1-piperidine propanol (Ro 25-6981), were investigated to assess their protective effects against SD in the rat. Under isoflurane anesthesia, d.c. potential and the related cortical blood flow and partial pressure of O2 (pO2) were recorded simultaneously at separate cortical sites. Drugs (1, 3, and 10 mg/kg i.p.) were given 1 h or 30 min before KCl application to the brain surface. Core temperature and arterial pCO2,pO2, and pH measurements confirmed physiological stability. KCl induced 7.7 ± 1.8 (mean ± S.D.) SD events with d.c. amplitude of 14.9 ± 2.8 mV. Memantine and CP-101,606 dose-dependently decreased SD event number (to 2.0 ± 1.8 and 2.3 ± 2.9, respectively) and SD amplitude at doses relevant for therapeutic use. Ro 25-6981 also decreased SD events significantly, but less effectively (to 4.5 ± 1.6), without affecting amplitude. These results indicate that NR2B-containing NMDA receptors are key mediators of SD, and as such, memantine- and NR2B-selective antagonists may be useful new therapeutic agents for the treatment of migraine and other SD-related disorders (e.g., stroke and brain injury). Whether chronic, rather than acute, treatment may improve their efficacy remains to be determined.

Differentiation of potent μ- and κ-opiate agonists using heat and pressure antinociceptive profiles and combined potency analysis

Abstract The antinociceptive profiles of strong μ- and κ-opiate agonists were determined in the tail clip, hot plate (55°C) and tail immersion (48°C and 55°C) tests in mice. All μ- and κ-agonists produced steep dose-response lines in all the tests. Using a combined analysis of potencies for pressure against heat noxia it was possible to diferentiate between μ- and κ-agonists and this paralleled findings in other laboratories using isolated tissue preparations. It was also concluded that qualitative differences between noxia were responsible for the observed differentiation since potency differences for μ- and κ-agonists between tests were maintained even after the intensity of heat and pressure noxia were equated. It was also proposed that use of the combined tail clip and immersion tests may be useful for rapid behavioural classification of novel synthetic μ- and κ-opiate analgesics.

Cortical spreading depression produces increased cGMP levels in cortex and brain stem that is inhibited by tonabersat (SB-220453) but not sumatriptan

Migraine headache is proposed to be mediated by nitric oxide (NO). Suitable mechanisms for eliciting increases in brain NO concentration in migraineurs have not yet been identified, although, animal models highlight cortical spreading depression (CSD) as a potential candidate. These studies have focused on CSD-associated NO release at highly acute time points (min-hours) and have not employed markers of NO metabolism with direct clinical application e.g. cGMP. The current study evaluated changes in plasma cGMP concentrations 3 h, 24 h and 3 days post-CSD and compared these to cortical and brainstem cGMP concentrations at 3 days. Moreover, this study also examined the effect of sumatriptan, a clinically effective antimigraine agent, and tonabersat (SB-220453) a potential novel antimigraine agent, on any observed changes in cGMP. Following pre-treatment with vehicle (n=3), sumatriptan (300 microg kg(-1) i.v, n=3) or tonabersat (SB-220453 10 mg kg(-1) i.p., n=3), CSD was evoked in anaesthetised rats by a 6-min KCl application to the parietal cortex. In the vehicle-treated group a median of eight depolarisations, were observed. Sumatriptan had no effect on the number of depolarisations, whereas tonabersat significantly reduced the number of events (median=2). No depolarisation events were observed throughout the recording period in the sham group. Following KCl application plasma cGMP concentrations were reduced up to 24 h post-CSD, but not significantly different from sham animals at 3 days. CSD in vehicle-treated animals produced a highly significant elevation in cGMP concentration in the brain stem 3 days after application of KCl. cGMP concentration increased 2.3-fold from 68+/-8 fmol/mg in sham animals (n=3) to 158+/-28 fmol/mg in the vehicle group. This increase in brain stem cGMP was abolished by tonabersat pre-treatment but not by sumatriptan.

BRL 46470A: a highly potent, selective and long acting 5-HT3 receptor antagonist with anxiolytic-like properties.

The novel 5-HT3 antagonist, BRL 46470A (endo-N-(8-methyl-8-azabicyclo [3.2.1]oct-3-yl)2,3-dihydro-3,3 dimethyl-indole-1-carboxamide, hydrochloride), has been investigated in a series of in vitro and in vivo tests, including the effect of the drug in models of anxiolysis. In classical tests for 5-HT3 receptor antagonism, BRL 46470A was shown to antagonise 5-HT3 mediated responses in the guinea-pig ileum [pA2 8.3±0.5, slope 0.98±0.20, mean±SEM (5)], the rabbit isolated heart (pA2 10.1±0.1, slope 1.2±0.2,n=5) and the rat Bezold-Jarisch model (ID50 0.7 µg/kg IV±0.1,n=8), with a long duration of action (>3 h). BRL 46470A selectively displaced [3H]-BRL 43694 from 5-HT3 binding sites in rat brain membranes (Ki 0.32 nM±0.04,n=4) without displacing (at concentrations greater than 1 µM) a wide variety of ligands binding to other neurotransmitter receptors, opioid receptors and to neurotransmitter gated ion channel complexes. In vivo, BRL 46470A showed anxiolytic-like activity in two animal models predictive of antianxiety effects-elevated X-maze and social interaction in rats. In both models, BRL 46470A showed significant activity over a wide dose-range following both oral (0.0001–0.1 mg/kg PO) and systemic administration. The unique level of potency of BRL 46470A was apparent in the rat social interaction test and was shown to be 100 fold more potent than the 5-HT3 receptor antagonist ondansetron, with no evidence of a bell-shaped dose response curve over 4 orders of magnitude. BRL 46470A (0.0001 and 0.01 mg/kg SC) also reduced the anxiogenic effects of m-CPP (1-(3-chlorophenyl) piperazine) in the rat elevated X-maze. BRL 46470A is therefore a chemically novel potent and selective 5-HT3 receptor antagonist with anxiolytic potential and a long duration of action in animal studies.

Tonabersat (SB-220453) a novel benzopyran with anticonvulsant properties attenuates trigeminal nerve-induced neurovascular reflexes.

The effects of tonabersat (SB‐220453) were evaluated on trigeminal nerve ganglion stimulation‐induced sensory‐autonomic neurovascular reflexes in the anaesthetized cat. Comparisons were made to intravenous administration of carabersat (SB‐204269), and to valproate, gabapentin and lamotrigine following intraduodenal administration. There were no effects on resting blood pressure, heart rate, carotid blood flow or carotid vascular resistance for any compound evaluated. Trigeminal nerve ganglion stimulation increased carotid blood flow by 65% and reduced vascular resistance by 41% with minimal effect on blood pressure (<10%) and no effect on heart rate. Intravenous infusion of tonabersat or carabersat (both 3.4 μmol h−1) produced time related reductions in stimulation‐induced responses with a maximal inhibition (relative to control) of 30±7% (n=4), at 240 min for tonabersat and 33±4% (n=3) at 180 min for carabersat. Tonabersat (11.5 μmol h−1) produced a similar inhibitory effect (32±9%, n=4) after 120 min of infusion. Following intraduodenal administration of tonabersat, the maximal inhibition of nerve stimulation‐induced responses was 55±4% at 120 min (n=4) for tonabersat 10 mg kg−1, and 24±2% after 180 min for 1 mg kg−1 (n=4). Intraduodenal administration of sodium valproate (10 or 100 mg kg−1 n=4/group) had no effect on neurovascular reflexes. Maximal inhibition of nerve ganglion‐stimulated reductions in carotid vascular resistance were observed at 150 min for lamotrigine (50 mg kg−1, 52±12%, n=4) and gabapentin (100 mg kg−1, 17±13%, n=3). Lamotrigine 10 mg kg−1 produced 22±11% (n=3) inhibition after 180 min. These data demonstrate blockade of trigeminal parasympathetic reflexes with tonabersat, carabersat and other anticonvulsants. These agents may therefore have therapeutic benefit in conditions where this type of reflex is evident.