Neurophysiological signals as potential translatable biomarkers for modulation of metabotropic glutamate 5 receptors.

The Group I metabotropic glutamate receptor subtype 5 (mGluR5) is widely distributed in the brain with dense expression in the cerebral cortex, hippocampus, and basal ganglia. These receptors have been implicated in psychiatric and neurological disorders such as schizophrenia, Fragile X syndrome, addiction, anxiety/depression, Parkinson's disease and neuropathic pain. The present study evaluated the effects of the mGluR5 negative allosteric modulators (NAMs) 4-difluoromethoxy-3-(pyridine-2-ylethynyl)phenyl)5H-pyrrolo[3,4-b]pyridine-6(7H)-yl methanone (GRN-529) and methyl (3aR,4S,7aR)-4-hydroxy-4-[(3-methylphenyl)ethynyl]octahydro-1H-indole-1-carboxylate (AFQ056) on polysomnographic (PSG) and quantitative electroencephalographic (qEEG) measures in freely moving rats. Furthermore, the anxiolytic profile of GRN-529 was characterized in anesthetized rats by measuring stimulation-induced hippocampal theta oscillation. The present findings demonstrate that inhibition of mGluR5 via its allosteric site profoundly modulates high-level neuronal network activities as indicated by changes in sleep-wake activity and power distribution of qEEG. Both GRN-529 and AFQ056 reduced the total time spent in rapid-eye movement with AFQ056 producing a significant increase in wakefulness at the highest dose tested. Additionally, qEEG revealed significant compound-induced increases in delta power concomitant with more subtle decreases in theta and alpha band power. Receptor occupancy (RO) studies revealed that GRN-529 and AFQ056 at all doses resulted in over 45% mGluR5 occupancy. Furthermore, GRN-529 dose-dependently decreased elicited hippocampal theta frequency, consistent with previous findings using clinically active anxiolytic compounds. The described changes in neurophysiological signals identified in freely moving rats may be considered suitable translational biomarkers for the clinical evaluation of mGluR5 NAMs.

Modulation of hippocampal theta oscillation by histamine H3 receptors.

Preclinical findings demonstrate procognitive actions of histamine 3 (H3) receptor antagonists/inverse agonists. Since a prominent role of neuronal network oscillations of the hippocampus, such as theta band oscillation, has been recognized in numerous cognitive functions, in the present study, the potential involvement of H3 receptors in modulation of hippocampal theta activity has been investigated using various recording paradigms. Systemic administration of the selective H3 receptor antagonists/inverse agonists, thioperamide and ciproxifan (0.1 mg/kg to 1 mg/kg i.v.), dose dependently increased hippocampal theta power, similarly to methylphenidate (0.1-1 mg/kg i.v.), in chloral hydrate anesthetized rats. When hippocampal theta oscillation was elicited by electrical brainstem (nucleus pontis oralis) stimulation, ciproxifan (1 mg/kg i.v.) augmented the power of stimulation-induced theta. In contrast, systemic administration of methylphenidate (1 mg/kg i.v.) did not modify elicited theta. To analyze the role of H3 receptors on stage- and behavior-dependent hippocampal theta activity, polysomnographic recordings were carried out together with field potential recordings at the hippocampal fissure in freely moving rats for 8 h during the light phase of the circadian cycle. Systemic administration of ciproxifan (3.0 mg/kg, i.p.) promoted wakefulness with a concomitant reduction in cortical delta power and augmented novelty-induced hippocampal theta activity. These findings provide evidence that H3 receptors play an important role in regulation of hippocampal theta oscillation, representing one of the probable mechanisms involved in histamine-induced modulation of higher brain functions, such as attention and learning.

Activation of alpha7 acetylcholine receptors augments stimulation-induced hippocampal theta oscillation.

In the septohippocampal formation alpha7 nicotinic receptors (alpha7 nAChRs) are predominantly expressed by neurons well positioned to modulate hippocampal theta oscillation, such as GABAergic interneurons in the hippocampus, and by both GABAergic and cholinergic septal neurons. In the present experiments, we evaluated the efficacy of the recently developed selective alpha7 nAChR agonist PNU-282987 on hippocampal theta oscillation in anaesthetized rats. This compound shows high affinity for the rat alpha7 nAChRs (Ki = 26 nM) but a negligible activity at other nAChRs. Systemic administration of PNU-282987 significantly enhanced the power (by 40%) of hippocampal theta oscillation induced by electrical stimulation of the brainstem reticular formation. In contrast, the amnesic and muscarinic receptor antagonist scopolamine significantly decreased the power (by 68%) of the stimulation-induced theta oscillation. Given the connection between hippocampal theta oscillation and cognitive processes, it is proposed that precognitive actions of alpha7 nAChR agonists could be mediated, at least in part, by modulation of hippocampal oscillatory activity.

Functional consequences of posttranslational isomerization of Ser46 in a calcium channel toxin.

The venom of the funnel-web spider Agelenopsis aperta contains several peptides that paralyze prey by blocking voltage-sensitive calcium channels. Two peptides, omega-Aga-IVB (IVB) and omega-Aga-IVC (IVC), have identical amino acid sequences, yet have opposite absolute configurations at serine 46. These toxins had similar selectivities for blocking voltage-sensitive calcium channel subtypes but different potencies for blocking P-type voltage-sensitive calcium channels in rat cerebellar Purkinje cells as well as calcium-45 influx into rat brain synaptosomes. An enzyme purified from venom converts IVC to IVB by isomerizing serine 46, which is present in the carboxyl-terminal tail, from the L to the D configuration. Unlike the carboxyl terminus of IVC, that of IVB was resistant to the major venom protease. These results show enzymatic activities in A. aperta venom being used in an unprecedented strategy for coproduction of necessary neurotoxins that possess enhanced stability and potency.

Activity and distribution of binding sites in brain of a nonpeptide substance P (NK1) receptor antagonist.

CP-96,345, a nonpeptide substance P antagonist, is selective for the tachykinin NK1 receptor. The compound binds to a single population of sites in guinea pig brain and potently inhibits substance P-induced excitation of locus ceruleus neurons. CP-96,345 should be a useful tool for studying the action of substance P in the central nervous system.

Spectral analysis of the electroencephalogram in the developing rat.

Power spectral measures of the EEG obtained from the frontal cortex and hippocampal formation during different vigilance states in the developing rat have been computed and compared. The most significant ontogenetic changes were observed in the hippocampal power spectra obtained during the vigilance state of REM sleep. These spectral analyses have revealed in the hippocampus: (1) a significant increase in the frequency at which the peak power occurs in the theta-frequency (4-11 Hz) band from 14 to 45 days of age; (2) a decrease in the quality factor of the peak from 14 to 45 days of age; (3) a decrease in the relative power co-ordinate for the center of spectral mass associated with the 0-4-Hz frequency band coupled with an increase in the frequency coordinate of the 4-11-Hz frequency band from 14 to 45 days of age, and; (4) a significant decrease in the average percent relative power associated with the 0-4-Hz frequency band from 14 to 22 days of age. For the EEG obtained from the frontal cortex, the major findings of note were: (1) a dominant contribution of relative power in the 0-4-Hz frequency band which was observed at every age and during every vigilance state tested, and; (2) a significant increase in the average percent relative power associated with this band at 18, 22, and 45 days of age. The results of this study provide a quantitative description of the electroencephalographic (EEG) ontogeny of the hippocampal formation and the frontal cortex in the rat. These ontogenetic changes in EEG activity relate closely to development of the internal circuitry and synaptic maturation in the hippocampal formation and frontal cortex.

Effect of protein malnutrition on hippocampal kindling: electrographic and behavioral measures.

Rats born to dams fed either a 6% (malnourished) or a 25% (control) casein diet during gestation and lactation and maintained on the diet of the dam after weaning were tested for electrographic and behavioral responses to electrically induced kindling of the CA1 field of the hippocampus beginning at 44 days of age. Animals in the 6% diet group had a significantly lower threshold to afterdischarge (AD), a significantly faster spread of AD activity to distal recording sites, significantly longer average duration of AD activity at all recording sites and a markedly altered behavioral progression toward seizure activity compared to control animals. These findings indicate that prenatal protein malnutrition results in hippocampal dysfunction as evidenced by both the electrographic and behavioral correlates of the kindling process. The data presented suggest that prenatal proteins malnutrition alters the response of hippocampal CA1 pyramids to electrical stimulation and that this alteration results in marked changes to both the electrographic and behavioral correlates of kindling.

Spectral analysis of neocortical and hippocampal EEG in the protein malnourished rat.

In these studies, power spectral analysis techniques were utilized to quantify changes in the cortical and hippocampal EEG obtained from rats reared on either an 8% or 25% casein diet as they mature from 14 to 22 days of age. Analyses of the power spectral data obtained from the frontal cortex of rats reared on the 25% casein diet during this preweaning period (14-22 days) indicated increases in the power in the 0.5-3.5 c/sec frequency band primarily during slow-wave sleep. Prenatal protein malnutrition (8% casein diet) significantly reduced the low frequency power content in the cortical EEG obtained during slow-wave sleep at 18 and 22 days of age. Analyses of power spectral data obtained from the hippocampal EEG during REM sleep also revealed developmental and diet-related differences. The frequency at which the peak power occurs in the theta band (4-11 c/sec) was found to increase normally in a linear fashion from 14 to 22 days of age (from 4.4 to 5.5 c/sec) in rats reared on the 25% casein diet. In addition, power in the 4-7 c/sec band significantly increased during this preweaning period. Prenatal protein malnutrition significantly slowed the development of theta frequency and produced higher values of power in the component of theta rhythm. Finally, vigilance profiles showed that rats normally progress from 14 days of age when REM sleep dominates their vigilance profile, to 22 days of age when they spend significantly more time in slow-wave sleep. Prenatal protein malnutrition retards this normal developmental sequence by significantly reducing the REM sleep time at 14 and 18 days of age and increasing the time spend in aroused waking at both these ages. However, at the time of weaning, i.e., at 22 days of age, these differences in sleep-waking behavior were no longer observed to be significant.

Structural characteristics of prostaglandin synthetase from sheep vesicular gland.

Prostaglandin synthetase contains both oxygenase and peroxidase activity and catalyzes the first step of prostaglandin synthesis. Aspirin (acetylsalicylic acid) inhibits oxygenase activity by acetylating a serine residue of the enzyme. In the current study, we have investigated the subunit structure of this complex enzyme and the stoichiometry of aspirin-mediated acetylation of the enzyme. The enzyme was purified to near homogeneity in both active and aspirin-acetylated forms. The purified protein was analyzed for enzymatic activity, [3H]acetate content following treatment with [acetyl-3H]aspirin, NH2-terminal sequence, and amino acid composition. The results show first, that the enzyme can be purified to near homogeneity in an active form; second, that the enzyme consists of a single polypeptide chain (molecular weight 72,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis) with a unique NH2-terminal sequence (Ala-Asp-Pro-Gly-Ala-Pro-Ala-Pro-Val-Asn-Pro-Met-Gly-); and third, that aspirin inhibits the enzyme by transfer of one acetate per enzyme monomer. Therefore, the two distinct enzymatic activities, oxygenation and peroxidation, are present in a single polypeptide chain. Experiments with a cross-linking agent indicate that in nonionic detergent the enzyme is a dimer of two identical subunits.

Acetylation of the NH2-terminal serine of prostaglandin synthetase by aspirin.

Aspirin (acetylsalicylic acid) inhibits prostaglandin synthesis by acetylating an active site portion of the enzyme, prostaglandin synthetase. In the current study, the site of acetylation has been demonstrated to be a seryl residue at the NH2 terminus of the enzyme. Purified [3H]acetyl enzyme was prepared from seminal vesicle homogenates treated with [acetyl-3H]aspirin. The [3H]acetate to protein bond was stable to hydroxylamine, indicating an N-acetyl linkage. The [3H]acetyl enzyme was fragmented sequentially with cyanogen bromide, trypsin, and pronase. The 3H material isolated from the pronase digest was identified as N-acetylserine. This finding indicates that the oxygenase portion of prostaglandin synthetase has an NH2-terminal serine which is involved in enzymatic activity and is susceptible to acetylation by aspirin.