Decoupling of Sleep-Dependent Cortical and Hippocampal Interactions in a Neurodevelopmental Model of Schizophrenia

SummaryRhythmic neural network activity patterns are defining features of sleep, but interdependencies between limbic and cortical oscillations at different frequencies and their functional roles have not been fully resolved. This is particularly important given evidence linking abnormal sleep architecture and memory consolidation in psychiatric diseases. Using EEG, local field potential (LFP), and unit recordings in rats, we show that anteroposterior propagation of neocortical slow-waves coordinates timing of hippocampal ripples and prefrontal cortical spindles during NREM sleep. This coordination is selectively disrupted in a rat neurodevelopmental model of schizophrenia: fragmented NREM sleep and impaired slow-wave propagation in the model culminate in deficient ripple-spindle coordination and disrupted spike timing, potentially as a consequence of interneuronal abnormalities reflected by reduced parvalbumin expression. These data further define the interrelationships among slow-wave, spindle, and ripple events, indicating that sleep disturbances may be associated with state-dependent decoupling of hippocampal and cortical circuits in psychiatric diseases

Effect of subunit on allosteric modulation of ion channel function in stably expressed human recombinant -aminobutyric acidA receptors determined using 36Cl ion flux.

ABSTRACT Inhibitory ␥-aminobutyric acid (GABA) A receptors are subject to modulation at a variety of allosteric sites, with pharmacology dependent on receptor subunit combination. The influence of different ␣ subunits in combination with ␤3␥2s was examined in stably expressed human recombinant GABA A receptors by measuring 36 Cl influx through the ion channel pore. Muscimol and GABA exhibited similar maximal efficacy at each receptor subtype, although muscimol was more potent, with responses blocked by picrotoxin and bicuculline. Receptors containing the ␣3 subunit exhibited slightly lower potency. The comparative pharmacology of a range of benzodiazepine site ligands was examined, revealing a range of intrinsic efficacies at different receptor subtypes. Of the diazepam-sensitive GABA A receptors (␣1, ␣2, ␣3, ␣5), ␣5 showed the most divergence, being discriminated by zolpidem in terms of very low affinity, and CL218,872 and CGS9895 with different efficacies. Benzodiazepine potentiation at ␣3␤3␥2s with nonselective agonist chlordiazepoxide was greater than at ␣1, ␣2, or ␣5 (P Ͻ 0.001). The presence of an ␣4 subunit conferred a unique pharmacological profile. The partial agonist bretazenil was the most efficacious benzodiazepine, despite lower ␣4 affinity, and FG8205 displayed similar efficacy. Most striking were the lack of affinity/ efficacy for classical benzodiazepines and the relatively high efficacy of Ro15-1788 (53 Ϯ 12%), CGS8216 (56 Ϯ 6%), CGS9895 (65 Ϯ 6%), and the weak partial inverse agonist Ro15-4513 (87 Ϯ 5%). Each receptor subtype was modulated by pentobarbital, loreclezole, and 5␣-pregnan-3␣-ol-20-one, but the type of ␣ subunit influenced the level of potentiation. The maximal pentobarbital response was significantly greater at ␣4␤3␥2s (226 Ϯ 10% increase in the EC 20 response to GABA) than any other modulator. The rank order of potentiation for pregnanolone was ␣5 Ͼ ␣2 Ͼ ␣3 ϭ ␣4 Ͼ ␣1, for loreclezole ␣1 ϭ ␣2 ϭ ␣3 Ͼ ␣5 Ͼ ␣4, and for pentobarbital ␣4 ϭ ␣5 ϭ ␣2 Ͼ ␣1 ϭ ␣3

Prevalence and influence of cys407* Grm2 mutation in Hannover-derived Wistar rats:mGlu2 receptor loss links to alcohol intake, risk taking and emotional behaviour

AbstractModulation of metabotropic glutamate 2 (mGlu2) receptor function has huge potential for treating psychiatric and neurological diseases. Development of drugs acting on mGlu2 receptors depends on the development and use of translatable animal models of disease. We report here a stop codon mutation at cysteine 407 in Grm2 (cys407*) that is common in some Wistar rats. Therefore, researchers in this field need to be aware of strains with this mutation. Our genotypic survey found widespread prevalence of the mutation in commercial Wistar strains, particularly those known as Han Wistar. Such Han Wistar rats are ideal for research into the separate roles of mGlu2 and mGlu3 receptors in CNS function. Previous investigations, unknowingly using such mGlu2 receptor-lacking rats, provide insights into the role of mGlu2 receptors in behaviour. The Grm2 mutant rats, which dominate some selectively bred lines, display characteristics of altered emotionality, impulsivity and risk-related behaviours and increased voluntary alcohol intake compared with their mGlu2 receptor-competent counterparts. In addition, the data further emphasize the potential therapeutic role of mGlu2 receptors in psychiatric and neurological disease, and indicate novel methods of studying the role of mGlu2 and mGlu3 receptors

Phase-amplitude coupled persistent theta and gamma oscillations in rat primary motor cortex in vitro

In vivo, theta (4-7 Hz) and gamma (30-80 Hz) neuronal network oscillations are known to coexist and display phase-amplitude coupling (PAC). However, in vitro, these oscillations have for many years been studied in isolation. Using an improved brain slice preparation technique we have, using co-application of carbachol (10 μM) and kainic acid (150 nM), elicited simultaneous theta (6.6 ± 0.1 Hz) and gamma (36.6 ± 0.4 Hz) oscillations in rodent primary motor cortex (M1). Each oscillation showed greatest power in layer V. Using a variety of time series analyses we detected significant cross-frequency coupling 74% of slice preparations. Differences were observed in the pharmacological profile of each oscillation. Thus, gamma oscillations were reduced by the GABAA receptor antagonists, gabazine (250 nM and 2 μM), and picrotoxin (50 μM) and augmented by AMPA receptor antagonism with SYM2206 (20 μM). In contrast, theta oscillatory power was increased by gabazine, picrotoxin and SYM2206. GABAB receptor blockade with CGP55845 (5 μM) increased both theta and gamma power, and similar effects were seen with diazepam, zolpidem, MK801 and a series of metabotropic glutamate receptor antagonists. Oscillatory activity at both frequencies was reduced by the gap junction blocker carbenoxolone (200 μM) and by atropine (5 μM). These data show theta and gamma oscillations in layer V of rat M1 in vitro are cross-frequency coupled, and are mechanistically distinct. The development of an in vitro model of phase-amplitude coupled oscillations will facilitate further mechanistic investigation of the generation and modulation of coupled activity in mammalian cortex

Key Amino Acids in the ␥ Subunit of the ␥-Aminobutyric Acid A Receptor that Determine Ligand Binding and Modulation at the Benzodiazepine Site

SUMMARY Pharmacological analyses of ␥-aminobutyric acid A (GABA A ) receptor subtypes have suggested that both the ␣ and ␥ subunits, but not the ␤ subunit, contribute to the benzodiazepine binding site. We took advantage of the different pharmacological properties conferred by the inclusion of different ␥ subunits in the receptor macromolecule to identify amino acids ␥2Phe77 and ␥2Met130 as key determinants of the benzodiazepine binding site. ␥2Phe77 was required for high affinity binding of the benzodiazepine site ligands flumazenil, CL218,872, and methyl-␤-carboline-3-carboxylate but not flunitrazepam. This amino acid was, however, required for allosteric modulation by flunitrazepam, as well as other benzodiazepine site ligands. In contrast, ␥2Met130 was required for high affinity binding of flunitrazepam, clonazepam, and triazolam but not flumazenil, CL218,872, or methyl-␤-carboline-3-carboxylate and did not affect benzodiazepine efficacy. Introduction of the phenylalanine and methionine into the appropriate positions of ␥1 was not sufficient to confer high affinity for the benzodiazepine site ligand zolpidem. These data show that ␥2Phe77 and ␥2Met130 are necessary for high affinity binding of a number of benzodiazepine site ligands. Although most previous studies have focused on the contribution of the ␣ subunit, we demonstrated a critical role for the ␥ subunit at the benzodiazepine binding site, indicating that this modulatory site is located at the interface of these two subunits. Furthermore, ␥2Phe77 is homologous to ␣1Phe64, which has been previously shown to be a key determinant of the GABA binding site, suggesting a conservation of motifs between different ligand binding sites on the GABA A receptor. The GABA A receptor, a member of the ligand-gated ion channel family, mediates synaptic inhibition through the gating of chloride ions, resulting in hyperpolarization of the cell membrane. It is the site of action of a number of pharmacological agents, including BZs, barbiturates, and anesthetics. The hetero-oligomeric receptor is formed from the coassembly of five different subunit classes [␣, ␤, ␥, ␦ (1, 2), and ⑀ (3, 4)] in a presumed pentameric arrangement (5, 6) to yield a family of receptor subtypes. It is the heterogeneity within these subunits that provides the molecular basis for the differences in pharmacology of receptor subtypes (7). Classic BZ pharmacology is exhibited by receptors containing a ␥2 subunit in combination with an ␣ and a ␤ subunit (8). The affinity of BZ ligands for the receptor is dependent on the ␣ subunit isoform, and hence compounds such as CL218,872 and zolpidem have higher affinity for ␣1␤n␥2 (n ϭ 1, 2, or 3) receptors than for other ␣ subunit-containing receptors (9, 10), and flunitrazepam and diazepam (11, 12) have very low affinity (Ͼ10 M) for ␣4␤n␥2 and ␣6␤n␥2. Mutagenesis studies have identified two amino acids on the ␣ subunit as contributing to the BZ binding sit

Allosteric modulators affect the efficacy of partial agonists for recombinant GABA(A) receptors

1. Different α subunits of human γ-aminobutyric acid type A (GABA(A)) receptors were transiently expressed together with β(3) and γ(2) subunits in Xenopus oocytes to examine the interactions of various GABA(A) agonists and representative allosteric modulators. Chloride currents elicited by agonists were measured using two electrode voltage clamp electrophysiology. 2. Where compounds behaved as full agonists, i.e. GABA on all subtypes and 4,5,6,7-tetrahydroisoxazolo [5,4-c]pyridin-3-ol (THIP) on α2β(3)γ(2) GABA(A) receptors, agonist concentration-response curves were shifted to the left by the benzodiazepine full agonist chlordiazepoxide and the anticonvulsant loreclezole, or to the right by the inverse agonist 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylic acid methyl ester (DMCM), with no effect on the maximal currents (I(max)). 3. In contrast, maximal responses for different partial GABA(A) agonists on all benzodiazepine-sensitive α(x)β(3)γ(2) GABA(A) receptors were enhanced by chlordiazepoxide. I(max) values for piperidine-4-sulphonic acid (P4S) on α(1)β(3)γ(2), THIP on α(3)β(3)γ(2), and 5-(4-piperidyl)isothiazol-3-ol (thio-4-PIOL) on α(2)β(3)γ(2) and α(5)β(3)γ(2) GABA(A) receptors were increased by chlordiazepoxide, while that for P4S on α(1)β(3)γ(2) receptors was decreased by DMCM. 4. The I(max) values for partial agonists were also enhanced by pentobarbitone, the neurosteroid allopregnanolone and loreclezole irrespective of receptor subtype or the nature of the partial agonist. 5. In the light of models of ligand-gated ion channel receptor activation we suggest two possible mechanisms of action for the effects of allosteric modulators on partial agonist receptor activation: either selective modulation of agonist affinity for the open/closed state, or direct modulation of the gating process itself