Dopamine D4 receptor activation increases hippocampal gamma oscillations by enhancing synchronization of fast-spiking interneurons

Background

Gamma oscillations are electric activity patterns of the mammalian brain hypothesized to serve attention, sensory perception, working memory and memory encoding. They are disrupted or altered in schizophrenic patients with associated cognitive deficits, which persist in spite of treatment with antipsychotics. Because cognitive symptoms are a core feature of schizophrenia it is relevant to explore signaling pathways that potentially regulate gamma oscillations. Dopamine has been reported to decrease gamma oscillation power via D1-like receptors. Based on the expression pattern of D4 receptors (D4R) in hippocampus, and pharmacological effects of D4R ligands in animals, we hypothesize that they are in a position to regulate gamma oscillations as well.

Methodology/Principal Findings

To address this hypothesis we use rat hippocampal slices and kainate-induced gamma oscillations. Local field potential recordings as well as intracellular recordings of pyramidal cells, fast-spiking and non-fast-spiking interneurons were carried out. We show that D4R activation with the selective ligand PD168077 increases gamma oscillation power, which can be blocked by the D4R-specific antagonist L745,870 as well as by the antipsychotic drug Clozapine. Pyramidal cells did not exhibit changes in excitatory or inhibitory synaptic current amplitudes, but inhibitory currents became more coherent with the oscillations after application of PD168077. Fast-spiking, but not non-fast spiking, interneurons, increase their action potential phase-coupling and coherence with regard to ongoing gamma oscillations in response to D4R activation. Among several possible mechanisms we found that the NMDA receptor antagonist AP5 also blocks the D4R mediated increase in gamma oscillation power.

Conclusions/Significance

We conclude that D4R activation affects fast-spiking interneuron synchronization and thereby increases gamma power by an NMDA receptor-dependent mechanism. This suggests that converging deficits on fast-spiking interneurons may lead to decreased network function and thus aberrant gamma oscillations and cognitive decline in schizophrenia.     

Requirement of the TRPC1 cation channel in the generation of transient Ca2+ oscillations by the calcium-sensing receptor

The calcium-sensing receptor (CaR) is an allosteric protein that responds to extracellular Ca(2+) ([Ca(2+)](o)) and aromatic amino acids with the production of different patterns of oscillations in intracellular Ca(2+) concentration ([Ca(2+)](i)). An increase in [Ca(2+)](o) stimulates phospholipase C-mediated production of inositol 1,4,5-trisphosphate and causes sinusoidal oscillations in [Ca(2+)](i). Conversely, aromatic amino acid-induced CaR activation does not stimulate phospholipase C but engages an unidentified signaling mechanism that promotes transient oscillations in [Ca(2+)](i). We show here that the [Ca(2+)](i) oscillations stimulated by aromatic amino acids were selectively abolished by TRPC1 down-regulation using either a pool of small inhibitory RNAs (siRNAs) or two different individual siRNAs that targeted different coding regions of TRPC1. Furthermore, [Ca(2+)](i) oscillations stimulated by aromatic amino acids were also abolished by inhibition of TRPC1 function with an antibody that binds the pore region of the channel. We also show that aromatic amino acid-stimulated [Ca(2+)](i) oscillations can be prevented by protein kinase C (PKC) inhibitors or siRNA-mediated PKCalpha down-regulation and impaired by either calmodulin antagonists or by the expression of a dominant-negative calmodulin mutant. We propose a model for the generation of CaR-mediated transient [Ca(2+)](i) oscillations that integrates its stimulation by aromatic amino acids with TRPC1 regulation by PKC and calmodulin.     

Molecular Determinants of Allosteric Modulation at the M1 Muscarinic Acetylcholine Receptor

Benzylquinolone carboxylic acid (BQCA) is an unprecedented example of a selective positive allosteric modulator of acetylcholine at the M₁ muscarinic acetylcholine receptor (mAChR). To probe the structural basis underlying its selectivity, we utilized site-directed mutagenesis, analytical modeling, and molecular dynamics to delineate regions of the M₁ mAChR that govern modulator binding and transmission of cooperativity. We identified Tyr-85^2.64 in transmembrane domain 2 (TMII), Tyr-179 and Phe-182 in the second extracellular loop (ECL2), and Glu-397^7.32 and Trp-400^7.35 in TMVII as residues that contribute to the BQCA binding pocket at the M₁ mAChR, as well as to the transmission of cooperativity with the orthosteric agonist carbachol. As such, the BQCA binding pocket partially overlaps with the previously described “common” allosteric site in the extracellular vestibule of the M₁ mAChR, suggesting that its high subtype selectivity derives from either additional contacts outside this region or through a subtype-specific cooperativity mechanism. Mutation of amino acid residues that form the orthosteric binding pocket caused a loss of carbachol response that could be rescued by BQCA. Two of these residues (Leu-102^3.29 and Asp-105^3.32) were also identified as indirect contributors to the binding affinity of the modulator. This new insight into the structural basis of binding and function of BQCA can guide the design of new allosteric ligands with tailored pharmacological properties.     

The paired activation of the two components of the muscarinic M3 receptor dimer is required for induction of ERK1/2 phosphorylation

Muscarinic M(3) receptors stimulate ERK1/2, the mitogen-activated protein kinase pathway. A mutant of the muscarinic M(3) receptor in which most of the third intracellular (i3) loop had been deleted (M(3)-short) completely lost the ability to stimulate the ERK1/2 phosphorylation in COS-7 cells. This loss was evident despite the fact that the receptor was able to couple efficiently to the phospholipase C second messenger pathway. In co-transfected cells, M(3)-short greatly reduced the ability of M(3) to activate ERK1/2. In another set of experiments we tested the ability of a mutant M(3)/M(2)(16aa) receptor, in which the first 16 amino acids of the i3 loop of the M(3) receptor were replaced with the corresponding segment of the muscarinic M(2) receptor to stimulate ERK1/2 phosphorylation. This mutant is not coupled to Galpha(q), but it is weakly coupled to Galpha(i). Despite its coupling modification this receptor was able to stimulate ERK1/2 phosphorylation. Again, M(3)-short greatly reduced the ability of M(3)/M(2)(16aa) to activate ERK1/2 in co-transfected cells. Similar results were obtained in stable-transfected Chinese hamster ovary (CHO) cells lines. In CHO M(3) cells carbachol induced a biphasic increase of ERK1/2 phosphorylation; a first increase at doses as low as 0.1 microm and a second increase starting from 10 microm. In CHO M(3)-short and in double-transfected CHO M(3)/M(3)-short cells we observed only the lower doses increase of ERK1/2 phosphorylation; no further increase was observed up to 1 mm carbachol. This suggests that in double-transfected CHO cells M(3)-short prevents the effect of the higher doses of carbachol on the M(3) receptor. In a final experiment we tested the ability of co-transfected chimeric alpha(2)/M(3) and M(3)/alpha(2) receptors to activate the ERK1/2 pathway. When given alone, carbachol and, to a lesser extent, clonidine, stimulated the coupling of the co-transfected chimeric receptors to the phospholipase C second messenger pathway, but they were unable to stimulate ERK1/2 phosphorylation. On the contrary, a strong stimulation of ERK1/2 phosphorylation was observed when the two agonists were given together despite the fact that the overall increase in phosphatidylinositol hydrolysis was not dissimilar from that observed in cells treated with carbachol alone. Our data suggest that the activation of the ERK1/2 pathway requires the coincident activation of the two components of a receptor dimer.     

c-di-GMP induction of Dictyostelium cell death requires the polyketide DIF-1

Cell death in the model organism Dictyostelium, as studied in monolayers in vitro, can be induced by the polyketide DIF-1 or by the cyclical dinucleotide c-di-GMP. c-di-GMP, a universal bacterial second messenger, can trigger innate immunity in bacterially infected animal cells and is involved in developmental cell death in Dictyostelium. We show here that c-di-GMP was not sufficient to induce cell death in Dictyostelium cell monolayers. Unexpectedly, it also required the DIF-1 polyketide. The latter could be exogenous, as revealed by a telling synergy between c-di-GMP and DIF-1. The required DIF-1 polyketide could also be endogenous, as shown by the inability of c-di-GMP to induce cell death in Dictyostelium HMX44A cells and DH1 cells upon pharmacological or genetic inhibition of DIF-1 biosynthesis. In these cases, c-di-GMP–induced cell death was rescued by complementation with exogenous DIF-1. Taken together, these results demonstrated that c-di-GMP could trigger cell death in Dictyostelium only in the presence of the DIF-1 polyketide or its metabolites. This identified another element of control to this cell death and perhaps also to c-di-GMP effects in other situations and organisms.     

Hippocampal deletion of BDNF gene attenuates gamma oscillations in area CA1 by up-regulating 5-HT3 receptor

Background

Pyramidal neurons in the hippocampal area CA3 express high levels of BDNF, but how this BDNF contributes to oscillatory properties of hippocampus is unknown.

Methodology/Principal Findings

Here we examined carbachol-induced gamma oscillations in hippocampal slices lacking BDNF gene in the area CA3. The power of oscillations was reduced in the hippocampal area CA1, which coincided with increases in the expression and activity of 5-HT3 receptor. Pharmacological block of this receptor partially restored power of gamma oscillations in slices from KO mice, but had no effect in slices from WT mice.

Conclusion/Significance

These data suggest that BDNF facilitates gamma oscillations in the hippocampus by attenuating signaling through 5-HT3 receptor. Thus, BDNF modulates hippocampal oscillations through serotonergic system.     

Molecular Mechanisms of Bitopic Ligand Engagement with the M1 Muscarinic Acetylcholine Receptor

TBPB and 77-LH-28-1 are selective agonists of the M₁ muscarinic acetylcholine receptor (mAChR) that may gain their selectivity through a bitopic mechanism, interacting concomitantly with the orthosteric site and part of an allosteric site. The current study combined site-directed mutagenesis, analytical pharmacology,and molecular modeling to gain further insights into the structural basis underlying binding and signaling by these agonists. Mutations within the orthosteric binding site caused similar reductions in affinity and signaling efficacy for both selective and prototypical orthosteric ligands. In contrast, the mutation of residues within transmembrane helix (TM) 2 and the second extracellular loop (ECL2) discriminated between the different classes of ligand. In particular, ECL2 appears to be involved in the selective binding of bitopic ligands and in coordinating biased agonism between intracellular calcium mobilization and ERK1/2 phosphorylation. Molecular modeling of the interaction between TBPB and the M₁ mAChR revealed a binding pose predicted to extend from the orthosteric site up toward a putative allosteric site bordered by TM2, TM3, and TM7, thus consistent with a bitopic mode of binding. Overall, these findings provide valuable structural and mechanistic insights into bitopic ligand actions and receptor activation and support a role for ECL2 in dictating the active states that can be adopted by a G protein-coupled receptor. This may enable greater selective ligand design and development for mAChRs and facilitate improved identification of bitopic ligands.     

A detailed anatomical and mathematical model of the hippocampal formation for the generation of sharp-wave ripples and theta-nested gamma oscillations

The mechanisms underlying the broad variety of oscillatory rhythms measured in the hippocampus during the sleep-wake cycle are not yet fully understood. In this article, we propose a computational model of the hippocampal formation based on a realistic topology and synaptic connectivity, and we analyze the effect of different changes on the network, namely the variation of synaptic conductances, the variations of the CAN channel conductance and the variation of inputs. By using a detailed simulation of intracerebral recordings, we show that this is able to reproduce both the theta-nested gamma oscillations that are seen in awake brains and the sharp-wave ripple complexes measured during slow-wave sleep. The results of our simulations support the idea that the functional connectivity of the hippocampus, modulated by the sleep-wake variations in Acetylcholine concentration, is a key factor in controlling its rhythms.     

The regulation of M1 muscarinic acetylcholine receptor desensitization by synaptic activity in cultured hippocampal neurons

To better understand metabotropic/ionotropic integration in neurons we have examined the regulation of M1 muscarinic acetylcholine (mACh) receptor signalling in mature (> 14 days in vitro), synaptically-active hippocampal neurons in culture. Using a protocol where neurons are exposed to an EC(50) concentration of the muscarinic agonist methacholine (MCh) prior to (R1), and following (R2) a desensitizing pulse of a high concentration of this agonist, we have found that the reduction in M(1) mACh receptor responsiveness is decreased in quiescent (+tetrodotoxin) neurons and increased when synaptic activity is enhanced by blocking GABA(A) receptors with picrotoxin. The picrotoxin-mediated effect on M1 mACh receptor responsiveness was completely prevented by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor blockade. Inhibition of endogenous G protein-coupled receptor kinase 2 by transfection with the non-G(q/11)alpha-binding, catalytically-inactive (D110A,K220R)G protein-coupled receptor kinase 2 mutant, decreased the extent of M1 mACh receptor desensitization under all conditions. Pharmacological inhibition of protein kinase C (PKC) activity, or chronic phorbol ester-induced PKC down-regulation had no effect on agonist-mediated receptor desensitization in quiescent or spontaneously synaptically active neurons, but significantly decreased the extent of receptor desensitization in picrotoxin-treated neurons. MCh stimulated the translocation of diacylglycerol- sensitive eGFP-PKCepsilon, but not Ca2+/diacylglycerol-sensitive eGFP-PKCbetaII in both the absence, and presence of tetrodotoxin. Under these conditions, MCh-stimulated eGFP-myristoylated, alanine-rich C kinase substrate translocation was dependent on PKC activity, but not Ca2+/calmodulin. In contrast, picrotoxin-driven translocation of myristoylated, alanine-rich C kinase substrate was accompanied by translocation of PKCbetaII, but not PKCepsilon, and was dependent on PKC and Ca2+/calmodulin. Taken together these data suggest that the level of synaptic activity may determine the different kinases recruited to regulate M1 mACh receptor desensitization in neurons.     

PTS1-independent targeting of isocitrate lyase to peroxisomes requires the PTS1 receptor Pex5p

The targeting of castor bean isocitrate lyase to peroxisomes was studied by expression in the heterologous host Saccharomyces cerevisae from which the endogenous ICL1 gene had been removed by gene disruption. Peroxisomal import of ICL was dependent upon the PTS1 receptor Pex5p and was lost by deletion of the last three amino acids, Ala-Arg-Met. However, removal of an additional 16 amino acids restored the ability of this truncated ICL to be targeted to peroxisomes and this import activity, like that of the full-length protein, was dependent upon Pex5p. The ability of peptides corresponding to the carboxyl terminal ends of wild-type and Δ3 and Δ19 mutants of ICL to interact with the PTS1-binding portion of Pex5p from humans, plants and yeast was determined using the yeast two-hybrid system. The peptide corresponding to wild-type ICL interacted with all three Pex5p proteins to differing extents, but neither mutant could interact with Pex5p from any species. Thus, ICL can be targeted to peroxisomes in a Pex5p-dependent but PTS1-independent fashion. These results help to clarify the contradictory published data about the requirement of the PTS1 signal for ICL targeting.     

Elongation Factor 1A Family Regulates the Recycling of the M4 Muscarinic Acetylcholine Receptor

In this study, we tested the hypothesis that the elongation 1A (eEF1A) family regulates the cell surface density of the M4 subtype of the muscarinic acetylcholine receptors (mAChR) following agonist-induced internalization. Here, we show that mouse brains lacking eEF1A2 have no detectable changes in M4 expression or localization. We, however, did discover that eEF1A1, the other eEF1A isoform, is expressed in adult neurons contrary to previous reports. This novel finding suggested that the lack of change in M4 expression and distribution in brains lacking eEF1A2 might be due to compensatory effects of eEF1A1. Supporting this theory, we demonstrate that the overexpression of either eEF1A1 or eEF1A2 inhibits M4 recovery to the cell surface after agonist-induced internalization in PC12 cells. Furthermore, eEF1A1 or eEF1A2 had no effect on the recovery of the M1 subtype in PC12 cells. These results demonstrate the novel ability of the eEF1A family to specifically regulate the M4 mAChR.     

Characterization of the cysteinyl-containing peptides of the gamma subunit of coupling factor 1

The cysteinyl peptides of the gamma subunit of chloroplast coupling factor 1 (CF1) have been analyzed by high performance liquid chromatography. Analysis of the reduced enzyme alkylated with 4-vinylpyridine showed that the gamma subunit contains four cysteinyl residues. Two of these residues are involved in a disulfide linkage in CF1 either in solution or bound to washed thylakoid membranes. Two free sulfhydryls, one that is readily attacked by alkylating reagents and another that is less reactive, were also detected. Each of these four cysteinyl residues is present in a separate tryptic peptide derived from the gamma subunit. These results show that 4-vinylpyridine is an excellent reagent for the analysis of cysteinyl-containing peptides and support our analyses of the roles of cysteinyl residues in the gamma subunit in ATP synthesis and hydrolysis.     

PTS1-independent targeting of isocitrate lyase to peroxisomes requires the PTS1 receptor Pex5p

The targeting of castor bean isocitrate lyase to peroxisomes was studied by expression in the heterologous host Saccharomyces cerevisae from which the endogenous ICL1 gene had been removed by gene disruption. Peroxisomal import of ICL was dependent upon the PTS1 receptor Pex5p and was lost by deletion of the last three amino acids, Ala-Arg-Met. However, removal of an additional 16 amino acids restored the ability of this truncated ICL to be targeted to peroxisomes and this import activity, like that of the full-length protein, was dependent upon Pex5p. The ability of peptides corresponding to the carboxyl terminal ends of wild-type and Delta 3 and Delta 19 mutants of ICL to interact with the PTS1-binding portion of Pex5p from humans, plants and yeast was determined using the yeast two-hybrid system. The peptide corresponding to wild-type ICL interacted with all three Pex5p proteins to differing extents, but neither mutant could interact with Pex5p from any species. Thus, ICL can be targeted to peroxisomes in a Pex5p-dependent but PTS1-independent fashion. These results help to clarify the contradictory published data about the requirement of the PTS1 signal for ICL targeting.