Chronopharmacological studies of ketamine in normal and NMDA epsilon1 receptor knockout mice.

BACKGROUND: The effectiveness and toxicity of many drugs depends on the dosing-time schedule, relative to the circadian rhythms of biochemical, physiological, and behavioural processes. Previous studies have found chronopharmacology of ketamine, which is a N-methyl-d-aspartate (NMDA) receptor antagonist. The in vivo contribution of the NMDA receptor epsilon1 subunit (NR2A) in this effect is unclear. METHODS: In the present study, daily variations in the hypnotic effect of ketamine were determined in wild-type mice and NMDA epsilon1 knockout (KO) mice. RESULTS: The effect of ketamine had a definite daily variation in wild-type mice. No significant difference in blood concentration was observed at different dosing times (10:00 and 22:00). In NMDA receptor epsilon1 KO mice, the hypnotic effect of ketamine was weaker than in wild-type mice and there was no dependence on the time of administration. Significant pharmacokinetic differences were not observed between wild-type and KO mice. CONCLUSIONS: The enhanced hypnotic effect in the active phase of the circadian cycle is likely a result of changes with the time of day in the susceptibility of the central nervous system to ketamine. Knockout of the NMDA receptor epsilon1 subunit gene markedly reduced the effect of ketamine, and eliminated the time-dependent sensitivity to ketamine.

Auditory triggered mental imagery of shape involves visual association areas in early blind humans.

Previous neuroimaging studies identified a large network of cortical areas involved in visual imagery in the human brain, which includes occipitotemporal and visual associative areas. Here we test whether the same processes can be elicited by tactile and auditory experiences in subjects who became blind early in life. Using positron emission tomography, regional cerebral blood flow was assessed in six right-handed early blind and six age-matched control volunteers during three conditions: resting state, passive listening to noise sounds, and mental imagery task (imagery of object shape) triggered by the sound of familiar objects. Activation foci were found in occipitotemporal and visual association areas, particularly in the left fusiform gyrus (Brodmann areas 19-37), during mental imagery of shape by both groups. Since shape imagery by early blind subjects does involve similar visual structures as controls at an adult age, it indicates their developmental crossmodal reorganization to allow perceptual representation in the absence of vision.

Roles of the glutamate receptor epsilon2 and delta2 subunits in the potentiation and prepulse inhibition of the acoustic startle reflex.

We examined the regulation of the acoustic startle response in mutant mice of the N-methyl-D-aspartate (NMDA)- and delta-subtypes of the glutamate receptor (GluR) channel, which play important roles in neural plasticity in the forebrain and the cerebellum, respectively. Heterozygous mutant mice with reduced GluRepsilon2 subunits of the NMDA receptor showed strongly enhanced startle responses to acoustic stimuli. On the other hand, heterozygous and homozygous mutation of the other NMDA receptor GluRepsilon subunits exerted no, or only small effects on acoustic startle responses. The threshold of the auditory brainstem response of the GluRepsilon2-mutant mice was comparable to that of the wild-type littermates. The primary circuit of the acoustic startle response is a relatively simple oligosynaptic pathway located in the lower brainstem, whilst the expression of GluRepsilon2 is restricted to the forebrain. We thus suggest that the NMDA receptor GluRepsilon2 subunit plays a role in the regulation of the startle reflex. Ablation of the cerebellar Purkinje cell-specific delta2 subunit of the GluR channel exerted little effect on the acoustic startle response but resulted in the enhancement of prepulse inhibition of the reflex. Because inhibition of the acoustic startle response by a weak prepulse is a measure of sensorimotor gating, the process by which an organism filters sensory information, these observations indicate the involvement of the cerebellum in the modulation of sensorimotor gating.

Hyperfunction of dopaminergic and serotonergic neuronal systems in mice lacking the NMDA receptor epsilon1 subunit.

NMDA receptors, an ionotropic subtype of glutamate receptors (GluRs) forming high Ca(2+)-permeable cation channels, are composed by assembly of the GluRzeta subunit (NR1) with any one of four GluRepsilon subunits (GluRepsilon1-4; NR2A-D). In the present study, we investigated neuronal functions in mice lacking the GluRepsilon1 subunit. GluRepsilon1 mutant mice exhibited a malfunction of NMDA receptors, as evidenced by alterations of [(3)H]MK-801 binding as well as (45)Ca(2+) uptake through the NMDA receptors. A postmortem brain analysis revealed that both dopamine and serotonin metabolism were increased in the frontal cortex and striatum of GluRepsilon1 mutant mice. The NMDA-stimulated [(3)H]dopamine release from the striatum was increased, whereas [(3)H]GABA release was markedly diminished in GluRepsilon1 mutant mice. When (+)bicuculline, a GABA(A) receptor antagonist, was added to the superfusion buffer, NMDA-stimulated [(3)H]dopamine release was significantly increased in wild-type, but not in the mutant mice. GluRepsilon1 mutant mice exhibited an increased spontaneous locomotor activity in a novel environment and an impairment of latent learning in a water-finding task. Hyperlocomotion in GluRepsilon1 mutant mice was attenuated by treatment with haloperidol and risperidone, both of which are clinically used antipsychotic drugs, at doses that had no effect in wild-type mice. These findings provide evidence that NMDA receptors are involved in the regulation of behavior through the modulation of dopaminergic and serotonergic neuronal systems. In addition, our findings suggest that GluRepsilon1 mutant mice are useful as an animal model of psychosis that is associated with NMDA receptor malfunction and hyperfunction of dopaminergic and serotonergic neuronal systems.

Characterization of nociceptin/orphanin FQ-induced pain responses in conscious mice: neonatal capsaicin treatment and N-methyl-D-aspartate receptor GluRepsilon subunit knockout mice.

Activation of primary afferent C fibers gives rise to spinal release of substance P and glutamate, and these mediators facilitate the cascade of nociceptive processing. We recently showed that intrathecal administration of nociceptin or orphanin FQ (hereafter called nociceptin) induced hyperalgesia to noxious thermal stimuli and allodynia to innocuous tactile stimuli applied to conscious mice. In the present study, we designed experiments to elucidate the pathways and mediators of nociceptin-evoked pain responses. Neonatal capsaicin treatment eliminated the induction of hyperalgesia and allodynia by nociceptin. Whereas this treatment markedly reduced the content of substance P in the spinal cord, it did not affect the nociceptin content or the expression of nociceptin receptors and GluRvarepsilon and GluRzeta subunits of N-methyl-D-aspartate receptors in it. The substance P antagonists CP96,345 and CP99,994 blocked the nociceptin-induced hyperalgesia, but not the allodynia. In contrast, the nociceptin-evoked allodynia, but not hyperalgesia, disappeared in N-methyl-D-aspartate receptor GluRvarepsilon1 subunit knockout mice. Both nociceptin-evoked hyperalgesia and allodynia were attenuated by morphine in a dose-dependent manner. Taken together, these results demonstrate that capsaicin-sensitive primary afferent fibers are involved not only in thermal hyperalgesia but also in tactile allodynia induced by nociceptin, but in different pathways; the former is mediated by substance P and the latter is mediated by glutamate through the N-methyl-D-aspartate receptor comprising the GluRvarepsilon1 subunit.

Cerebellar activation during ataxic gait in olivopontocerebellar atrophy: a PET study.

OBJECTIVE: To investigate the possible abnormal regional brain metabolism during ataxic gait in olivopontocerebellar atrophy (OPCA), and to evaluate the response of the cerebellar subregions to instability during bipedal gait. MATERIAL AND METHODS: On 9 patients with OPCA in early phase and on 10 age-matched normal subjects, we performed positron emission tomography (PET) with 2-[18F]fluoro-2-deoxy-D-glucose (FDG) under two different conditions: supine resting and 30 min treadmill walking. RESULTS: Both in normals and in patients with OPCA, the FDG uptake in the walking state (Uwalk) was significantly greater than that in the resting state (Urest) in the pyramis, declive-folium-tuber and culmen of the cerebellar vermis, and in the thalamus. In the patients, the Uwalk was also significantly greater than the Urest in the posterior lobe of cerebellar hemisphere and in the pons and midbrain. In the pyramis, the activation ratio (= Uwalk/Urest) of the patients was significantly lower than that of the normals. CONCLUSIONS: We considered that these findings reflect the pathophysiology of ataxic gait in OPCA patients and the compensatory mechanism for the instability during ataxic gait.

Citron, a Rho-target, interacts with PSD-95/SAP-90 at glutamatergic synapses in the thalamus.

Proteins of the membrane-associated guanylate kinase family play an important role in the anchoring and clustering of neurotransmitter receptors in the postsynaptic density (PSD) at many central synapses. However, relatively little is known about how these multifunctional scaffold proteins might provide a privileged site for activity- and cell type-dependent specification of the postsynaptic signaling machinery. Rho signaling pathway has classically been implicated in mechanisms of axonal outgrowth, dendrogenesis, and cell migration during neural development, but its contribution remains unclear at the synapses in the mature CNS. Here, we present evidence that Citron, a Rho-effector in the brain, is enriched in the PSD fraction and interacts with PSD-95/synapse-associated protein (SAP)-90 both in vivo and in vitro. Citron colocalization with PSD-95 occurred, not exclusively but certainly, at glutamatergic synapses in a limited set of neurons, such as the thalamic excitatory neurons; Citron expression, however, could not be detected in the principal neurons of the hippocampus and the cerebellum in the adult mouse brain. In a heterologous system, Citron was shown to form a heteromeric complex not only with PSD-95 but also with NMDA receptors. Thus, Citron-PSD-95/SAP-90 interaction may provide a region- and cell type-specific link between the Rho signaling cascade and the synaptic NMDA receptor complex.

Hereditary spastic paraplegia with a thin corpus callosum and thalamic involvement in Japan.

The authors examined two Japanese siblings with a recessive hereditary spastic paraplegia (HSP) with dementia and a thin corpus callosum. Both showed thalamic glucose hypometabolism on PET. Recessive HSP with a thin corpus callosum is a rare disorder, with less than 20 reported patients, that may be a Japanese subtype of HSP.

Rings of negatively charged amino acids determine the acetylcholine receptor channel conductance.

The structure-function relationship of the nicotinic acetylcholine receptor (AChR) has been effectively studied by the combination of complementary DNA manipulation and single-channel current analysis. Previous work with chimaeras between the Torpedo californica and bovine AChR delta-subunits has shown that the region comprising the hydrophobic segment M2 and its vicinity contains an important determinant of the rate of ion transport through the AChR channel. It has also been suggested that this region is responsible for the reduction in channel conductance caused by divalent cations and that segment M2 contributes to the binding site of noncompetitive antagonists. To identify those amino acid residues that interact with permeating ions, we have introduced various point mutations into the Torpedo AChR subunit cDNAs to alter the net charge of the charged or glutamine residues around the proposed transmembrane segments. The single-channel conductance properties of these AChR mutants expressed in Xenopus laevis oocytes indicate that three clusters of negatively charged and glutamine residues neighbouring segment M2 of the alpha-, beta-, gamma- and delta-subunits, probably forming three anionic rings, are major determinants of the rate of ion transport.

Selective coupling with K+ currents of muscarinic acetylcholine receptor subtypes in NG108-15 cells.

The primary structures of two muscarinic acetylcholine receptor (mAChR) species, designated as mAChR I and mAChR II, have been elucidated by cloning and sequence analysis of DNAs complementary to the porcine cerebral and cardiac messenger RNAs, respectively. mAChR I and mAChR II expressed in Xenopus oocytes differ from each other both in acetylcholine-induced response and in antagonist binding properties. These results, together with the differential tissue location of the two mAChR mRNAs, have indicated that pharmacologically distinguishable subtypes of the mAChR represent distinct gene products. The primary structures of two additional mammalian mAChR species, designated as mAChR III and mAChR IV, have subsequently been deduced from the nucleotide sequences of the cloned cDNAs or genomic DNAs. We report here that mAChR I and mAChR III expressed in NG108-15 neuroblastoma-glioma hybrid cells, but not mAChR II and mAChR IV, efficiently mediate phosphoinositide hydrolysis, activation of a Ca2+-dependent K+ current and inhibition of the M-current, a voltage-dependent K+ current sensitive to muscarinic agonists.

Molecular distinction between muscarinic acetylcholine receptor subtypes.

The muscarinic acetylcholine receptor (mAChR) mediates various cellular responses, including inhibition of adenylate cyclase, breakdown of phosphoinositides and modulation of potassium channels, through the action of guanine nucleotide-binding regulatory proteins. Pharmacologically distinguishable forms of the mAChR occur in different tissues and have provisionally been classified into M1 (I), M2 cardiac (II) and M2 glandular (III) subtypes on the basis of their difference in apparent affinity for antagonists. In an attempt to elucidate the molecular basis of the functional heterogeneity of the mAChR, we have cloned and sequenced DNAs complementary to porcine cerebral and cardiac messenger RNAs encoding mAChRs and have thereby deduced the primary structures of the receptor proteins. We report here that the messenger RNA generated by transcription of the cardiac complementary DNA directs the formation of a functional mAChR in Xenopus oocytes and that this mAChR differs from the mAChR formed by expression of the cerebral cDNA both in acetylcholine (ACh)-induced response and in antagonist binding properties. Our results provide evidence indicating that the mAChR encoded by the cerebral cDNA (designated as mAChR I) and the mAChR encoded by the cardiac cDNA (mAChR II) are of the M1 (I) and the M2 cardiac (II) subtype, respectively.