Decreased striatal dopamine in group II metabotropic glutamate receptor (mGlu2/mGlu3) double knockout mice.

BACKGROUND: Group II metabotropic glutamate receptors (mGlu2 and mGlu3, encoded by Grm2 and Grm3) have been the focus of attention as treatment targets for a number of psychiatric conditions. Double knockout mice lacking mGlu2 and mGlu3 (mGlu2/3-/-) show a subtle behavioural phenotype, being hypoactive under basal conditions and in response to amphetamine, and with a spatial memory deficit that depends on the arousal properties of the task. The neurochemical correlates of this profile are unknown. Here, we measured tissue levels of dopamine, 5-HT, noradrenaline and their metabolites in the striatum and frontal cortex of mGlu2/3-/- double knockout mice, using high performance liquid chromatography. We also measured the same parameters in mGlu2-/- and mGlu3-/- single knockout mice. RESULTS: mGlu2/3-/-mice had reduced dopamine levels in the striatum but not in frontal cortex, compared to wild-types. In a separate cohort we replicated this deficit and, using tissue punches, found it was more prominent in the nucleus accumbens than in dorsolateral striatum. Noradrenaline, 5-HT and their metabolites were not altered in the striatum of mGlu2/3-/- mice, although the noradrenaline metabolite MHPG was increased in the cortex. In mGlu2-/- and mGlu3-/- single knockout mice we found no difference in any monoamine or metabolite, in either brain region, compared to their wild-type littermates. CONCLUSIONS: Group II metabotropic glutamate receptors impact upon striatal dopamine. The effect may contribute to the behavioural phenotype of mGlu2/3-/- mice. The lack of dopaminergic alterations in mGlu2-/- and mGlu3-/- single knockout mice reveals a degree of redundancy between the two receptors. The findings support the possibility that interactions between mGlu2/3 and dopamine may be relevant to the pathophysiology and therapy of schizophrenia and other disorders.

A novel series of benzimidazole NR2B-selective NMDA receptor antagonists.

A series of novel benzimidazoles are discussed as NR2B-selective N-methyl-d-aspartate (NMDA) receptor antagonists. High throughput screening (HTS) efforts identified a number of potent and selective NR2B antagonists such as 1. Exploration of the substituents around the core of this template identified a number of compounds with high potency for NR2B (pIC(50) >7) and good selectivity against the NR2A subunit (pIC(50) <4.3) as defined by FLIPR-Ca(2+) and radioligand binding studies. These agents offer potential for the development of therapeutics for a range of nervous system disorders including chronic pain, neurodegeneration, migraine and major depression.

Activation of α7 nicotinic acetylcholine receptors persistently enhances hippocampal synaptic transmission and prevents Aß-mediated inhibition of LTP in the rat hippocampus.

Nicotinic acetylcholine receptors mediate fast cholinergic modulation of glutamatergic transmission and synaptic plasticity. Here we investigated the effects of subtype selective activation of the α7 nicotinic acetylcholine receptors on hippocampal transmission and the inhibition of synaptic long-term potentiation by the Alzheimer's disease associated amyloid ß-protein (Aß). The α7 nicotinic acetylcholine receptor agonist "compound A" ((R)-N-(1-azabicyclo[2.2.2]oct-3-yl)(5-(2-pyridyl))thiophene-2-carboxamide) induced a rapid-onset persistent enhancement of synaptic transmission in the dentate gyrus in vitro. Consistent with a requirement for activation of α7 nicotinic acetylcholine receptors, the type II α7-selective positive allosteric modulator PheTQS ((3aR, 4S, 9bS)-4-(4-methylphenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide) potentiated, and the antagonist methyllycaconitine (MLA) prevented the persistent enhancement. Systemic injection of the agonist also induced a similar MLA-sensitive persistent enhancement of synaptic transmission in the CA1 area in vivo. Remarkably, although compound A did not affect control long-term potentiation (LTP) in vitro, it prevented the inhibition of LTP by Aß1-42 and this effect was inhibited by MLA. These findings strongly indicate that activation of α7 nicotinic acetylcholine receptors is sufficient to persistently enhance hippocampal synaptic transmission and to overcome the inhibition of LTP by Aß.

Effects of the selective 5-HT(7) receptor antagonist SB-269970 in animal models of psychosis and cognition.

The 5-hydroxytryptamine7 (5-HT7) receptor is a G-protein coupled receptor for serotonin that has been implicated in the pathophysiology of psychiatric and neurological disorders including anxiety, depression and schizophrenia. A number of studies have attempted to evaluate the potential role of the 5-HT7 receptor in schizophrenia by utilising genetic or pharmacological tools but to date these have provided conflicting results. Here we investigate the effect of a selective 5-HT7 receptor antagonist, SB-269970, in in vivo psychosis and cognition models and relate efficacy to brain exposures of the compound. SB-269970 significantly attenuated amphetamine-induced rearing and circling in rats. A similar effect was observed in an N-methyl d-aspartic acid (NMDA) receptor antagonist driven psychosis model, where SB-269970 significantly reversed phencyclidine-induced hyperlocomotion, rearing and circling; although the effect was not as robust as with the 5-HT2a receptor antagonist positive control, MDL100,907. SB-269970 also attenuated a temporal deficit in novel object recognition (NOR), indicative of an improvement in recognition memory. Pharmacokinetic analysis of plasma and brain samples taken after behavioural testing confirmed that efficacy was achieved at doses and pre-treatment times where receptor occupancy was substantial. These findings highlight the anti-psychotic and pro-cognitive potential of 5-HT7 receptor antagonists and warrant further studies to explore their therapeutic potential in schizophrenia.

Fractionation of spatial memory in GRM2/3 (mGlu2/mGlu3) double knockout mice reveals a role for group II metabotropic glutamate receptors at the interface between arousal and cognition.

Group II metabotropic glutamate receptors (mGluR2 and mGluR3, encoded by GRM2 and GRM3) are implicated in hippocampal function and cognition, and in the pathophysiology and treatment of schizophrenia and other psychiatric disorders. However, pharmacological and behavioral studies with group II mGluR agonists and antagonists have produced complex results. Here, we studied hippocampus-dependent memory in GRM2/3 double knockout (GRM2/3(-/-)) mice in an iterative sequence of experiments. We found that they were impaired on appetitively motivated spatial reference and working memory tasks, and on a spatial novelty preference task that relies on animals' exploratory drive, but were unimpaired on aversively motivated spatial memory paradigms. GRM2/3(-/-) mice also performed normally on an appetitively motivated, non-spatial, visual discrimination task. These results likely reflect an interaction between GRM2/3 genotype and the arousal-inducing properties of the experimental paradigm. The deficit seen on appetitive and exploratory spatial memory tasks may be absent in aversive tasks because the latter induce higher levels of arousal, which rescue spatial learning. Consistent with an altered arousal-cognition relationship in GRM2/3(-/-) mice, injection stress worsened appetitively motivated, spatial working memory in wild-types, but enhanced performance in GRM2/3(-/-) mice. GRM2/3(-/-) mice were also hypoactive in response to amphetamine. This fractionation of hippocampus-dependent memory depending on the appetitive-aversive context is to our knowledge unique, and suggests a role for group II mGluRs at the interface of arousal and cognition. These arousal-dependent effects may explain apparently conflicting data from previous studies, and have translational relevance for the involvement of these receptors in schizophrenia and other disorders.

Transcription and pathway analysis of the superior temporal cortex and anterior prefrontal cortex in schizophrenia.

The molecular basis of schizophrenia is poorly understood; however, different brain regions are believed to play distinct roles in disease symptomology. We have studied gene expression in the superior temporal cortex (Brodmann area 22; BA22), which may play a role in positive pathophysiology, and compared our results with data from the anterior prefrontal cortex (BA10), which shows evidence for a role in negative symptoms. Genome-wide mRNA expression was determined in the BA22 region in 23 schizophrenics and 19 controls and compared with a BA10 data set from the same subjects. After adjustments for confounding sources of variation, we carried out GeneGO pathway enrichment analysis in each region. Significant differences were seen in age-related transcriptional changes between the BA22 and the BA10 regions, 21.8% and 41.4% of disease-associated transcripts showing age association, respectively. After removing age associated changes from our data, we saw the highest enrichment in processes mediating cell adhesion, synaptic contact, cytoskeletal remodelling, and apoptosis in the BA22 region. For the BA10 region, we observed the strongest changes in reproductive signalling, tissue remodelling, and cell differentiation. Further exploratory analysis also identified potentially disease-relevant processes that were undetected in our more stringent primary analysis, including autophagy in the BA22 region and the amyloid process in the BA10 region. Collectively, our analysis suggests disruption of many common pathways and processes underpinning synaptic plasticity in both regions in schizophrenia, whereas individual regions emphasize changes in certain pathways that may help to highlight pathway-specific therapeutic opportunities to treat negative or positive symptoms of the disease.

Hippocampal mossy fiber long-term depression in Grm2/3 double knockout mice.

Group II metabotropic glutamate receptors (mGluR2, encoded by Grm2, and mGluR3, encoded by Grm3) are inhibitory autoreceptors that negatively modulate the adenylate cyclase signaling cascade. Within the hippocampus, mGluR2 is believed to play a key role in the induction of long-term depression (LTD) at mossy fiber-CA3 synapses. Here, we used Grm2/3 double knockout (dko) mice to investigate to what extent group II mGluRs are necessary for mossy fiber LTD. Surprisingly, we found that these mice displayed prominent mossy fiber LTD. However, the induction of this form of LTD was sensitive to the external Ca(2+) concentration. Mossy fiber LTD in Grm2/3 dko mice was indistinguishable from that in wild-type mice at 4 mM Ca(2+) , but largely absent at 2 mM external Ca(2+) . Mossy fiber LTD in Grm2/3 dko mice was not blocked by the N-methyl-D-aspartic acid (NMDA) receptor antagonist D-AP5, confirming that the observed response did not reflect NMDA receptor-dependent LTD in contaminating associational-commissural fibers, and enabling us to use the NMDA receptor-mediated EPSC to monitor mossy fiber LTD. Using whole-cell recordings, we demonstrated that LTD of the NMDA receptor-mediated EPSC in Grm2/3 dko mice was not affected by intracellular application of BAPTA and CsF to block postsynaptic Ca(2+) and G-protein-mediated effects. This presynaptic LTD was, however, blocked by the AMPA/kainate receptor antagonist, NBQX. Thus, an activity-dependent, external Ca(2+) concentration-sensitive form of mossy fiber LTD can be induced in Grm2/3 dko mice. Two mGluR antagonists also failed to block mossy fiber LTD under 4 mM conditions in wild-type mice, strengthening the conclusion that group II mGluRs are not obligatory for mossy fiber LTD.

ADAMTSL3 as a candidate gene for schizophrenia: gene sequencing and ultra-high density association analysis by imputation.

We previously reported an association with a putative functional variant in the ADAMTSL3 gene, just below genome-wide significance in a genome-wide association study of schizophrenia. As variants impacting the function of ADAMTSL3 (a disintegrin-like and metalloprotease domain with thrombospondin type I motifs-like-3) could illuminate a novel disease mechanism and a potentially specific target, we have used complementary approaches to further evaluate the association. We imputed genotypes and performed high density association analysis using data from the HapMap and 1000 genomes projects. To review all variants that could potentially cause the association, and to identify additional possible pathogenic rare variants, we sequenced ADAMTSL3 in 92 schizophrenics. A total of 71 ADAMTSL3 variants were identified by sequencing, many were also seen in the 1000 genomes data, but 26 were novel. None of the variants identified by re-sequencing was in strong linkage disequilibrium (LD) with the associated markers. Imputation analysis refined association between ADAMTSL3 and schizophrenia, and highlighted additional common variants with similar levels of association. We evaluated the functional consequences of all variants identified by sequencing, or showing direct or imputed association. The strongest evidence for function remained with the originally associated variant, rs950169, suggesting that this variant may be causal of the association. Rare variants were also identified with possible functional impact. Our study confirms ADAMTSL3 as a candidate for further investigation in schizophrenia, using the variants identified here. The utility of imputation analysis is demonstrated, and we recommend wider use of this method to re-evaluate the existing canon of suggestive schizophrenia associations.

The muscarinic M(4) receptor is the functionally predominant subtype in rat and mouse striatum as demonstrated using [(35)S] GTPγS binding.

We have used selective muscarinic receptor antagonists and M(2) and M(4) receptor knockout (KO) mouse tissue to define the functional muscarinic acetylcholine receptor populations in rodent striatum. [(3)H] NMS binding studies in rat and mouse striatum demonstrated that approximately 30% of muscarinic acetylcholine receptors expressed are M(1) receptors. Radioligand binding studies suggest that the remaining muscarinic acetylcholine receptor population is largely M(4) with small levels of M(2). In agreement, carbachol-induced GTPγS binding studies in M(2) and M(4) receptor KO mouse striatum implicated the M(4) receptor as the predominant functional receptor subtype. Based on these data we have developed a novel, native tissue M(4) receptor [(35)S] GTPγS binding assay. Pharmacological assessment of M(4) receptor agonist and positive 3modulators revealed clear differences in the potencies observed in a human recombinant CHO-M(4) receptor [(35)S] GTPγS binding assay as compared to the native tissue [(35)S] GTPγS binding assay. These differences are believed to reflect differences in receptor reserve between the assay systems as well as differences in compound pharmacology (relative contribution of compound affinity and efficacy to observed potency). These studies have demonstrated the importance of understanding the pharmacology of test compounds in a native environment when predicting in vivo response.

Adenoviral-mediated Cre expression effectively suppresses GlyT1 binding in the thalamic area of GlyT1 conditional knock-out mice.

To properly understand the function of genes of neurological interest, in vivo manipulation in the adult is essential, particularly when the target gene is involved in brain development. Moreover, since the physiological effects of target protein may be region-specific, targeting a distinct brain region could be required to dissect these effects in specific brain locations. Infection of somatic tissues of transgenic mice bearing loxP-flanked gene sequences with a viral vector expressing Cre recombinase provides a means of allowing flexible spatio-temporal control of target gene expression. Viral vector-mediated Cre expression could be used to mediate localized gene modulation in a specific brain region. In the present study this technology was applied to the glycine transporter type-1 (GlyT1) protein which is responsible for the uptake of synaptic glycine in the forebrain and has been implicated as a therapeutic target for the treatment of schizophrenia. Since GlyT1 is widely expressed in glial cells, we employed an adenoviral-based vector (Ad5) to deliver Cre protein, due to the preferentially transduction of glial cells by adenoviral vectors in rodent brain. We show significant reduced GlyT1 binding specifically in the thalamic area of conditional GlyT1 (GlyT1c) transgenic mice injected with Ad5-Cre virus, as measured by GlyT1 autoradiography. In conclusion, we demonstrated the validity of viral vector-mediated delivery of Cre to loxP targeted transgenic mice as a novel strategy to investigate target gene function in selected subregions of the adult brain, which provides a valuable technique to investigate gene function both in normal physiology and in disease models.

Identification of novel alpha7 nAChR positive allosteric modulators with the use of pharmacophore in silico screening methods.

The pharmacophore model of in house potent and selective alpha7 nAChR positive allosteric modulators is reported. The model was used to fish out commercially-available compounds from corporate 3D databases. As a result, novel alpha7 positive modulator chemotypes were identified. A rat full PK profile of a representative compound is also described.

Glutamate-dopamine crosstalk in the rat prefrontal cortex is modulated by Alpha7 nicotinic receptors and potentiated by PNU-120596.

The aim of this study was to explore the modulation by alpha7 nicotinic receptors (nAChRs) of dopamine and glutamate release in the rat prefrontal cortex where these receptors are implicated in attentional processes and are therapeutic targets for cognitive deficits. The presence of presynaptic alpha7 nAChRs on glutamate terminals is supported by the ability of the subtype-selective agonist Compound A to evoke [(3)H]D-aspartate release from synaptosomes: This response was potentiated by the selective allosteric potentiator PNU-120596 and blocked by alphabungarotoxin. Compound A also evoked dopamine overflow in the prefrontal cortex in vivo, and this was potentiated by PNU-120596. alpha7 nAChR-evoked [(3)H]dopamine release from tissue prisms in vitro was blocked by antagonists of NMDA and AMPA receptors. These data are consistent with a model in which alpha7 nAChRs present on glutamate terminals increase glutamate release that (1) contributes to presynaptic facilitation and synaptic plasticity and (2) co-ordinately enhances dopamine release from neighbouring boutons.

Allosteric modulators of NR2B-containing NMDA receptors: molecular mechanisms and therapeutic potential.

N-methyl-D-aspartate receptors (NMDARs) are ion channels gated by glutamate, the major excitatory neurotransmitter in the mammalian central nervous system (CNS). They are widespread in the CNS and are involved in numerous physiological and pathological processes including synaptic plasticity, chronic pain and psychosis. Aberrant NMDAR activity also plays an important role in the neuronal loss associated with ischaemic insults and major degenerative disorders including Parkinson's and Alzheimer's disease. Agents that target and alter NMDAR function may, thus, have therapeutic benefit. Interestingly, NMDARs are endowed with multiple extracellular regulatory sites that recognize ions or small molecule ligands, some of which are likely to regulate receptor function in vivo. These allosteric sites, which differ from agonist-binding and channel-permeation sites, provide means to modulate, either positively or negatively, NMDAR activity. The present review focuses on allosteric modulation of NMDARs containing the NR2B subunit. Indeed, the NR2B subunit confers a particularly rich pharmacology with distinct recognition sites for exogenous and endogenous allosteric ligands. Moreover, NR2B-containing receptors, compared with other NMDAR subtypes, appear to contribute preferentially to pathological processes linked to overexcitation of glutamatergic pathways. The actions of extracellular H+, Mg2+, Zn2+, of polyamines and neurosteroids, and of the synthetic compounds ifenprodil and derivatives ('prodils') are presented. Particular emphasis is put upon the structural determinants and molecular mechanisms that underlie the effects exerted by these agents. A better understanding of how NR2B-containing NMDARs (and NMDARs in general) operate and how they can be modulated should help define new strategies to counteract the deleterious effects of dysregulated NMDAR activity.

Evaluation of expression and function of the H+/myo-inositol transporter HMIT.

BACKGROUND: The phosphoinositide (PIns) signalling pathway regulates a series of neuronal processes, such as neurotransmitter release, that are thought to be altered in mood disorders. Furthermore, mood-stabilising drugs have been shown to inhibit key enzymes that regulate PIns production and alter neuronal growth cone morphology in an inositol-reversible manner. Here, we describe analyses of expression and function of the recently identified H+/myo-inositol transporter (HMIT) investigated as a potential regulator of PIns signalling. RESULTS: We show that HMIT is primarily a neuronal transporter widely expressed in the rat and human brain, with particularly high levels in the hippocampus and cortex, as shown by immunohistochemistry. The transporter is localised at the Golgi apparatus in primary cultured neurones. No HMIT-mediated electrophysiological responses were detected in rat brain neurones or slices; in addition, inositol transport and homeostasis were unaffected in HMIT targeted null-mutant mice. CONCLUSION: Together, these data do not support a role for HMIT as a neuronal plasma membrane inositol transporter, as previously proposed. However, we observed that HMIT can transport inositol triphosphate, indicating unanticipated intracellular functions for this transporter that may be relevant to mood control.

Electrophysiological characterisation of the actions of kynurenic acid at ligand-gated ion channels.

To better understand the effects of the tryptophan metabolite kynurenic acid (kynA) in the brain, we characterised its actions at five ligand-gated ion channels: NMDA, AMPA, GABA(A), glycine and alpha7 nicotinic acetylcholine receptors. Using whole-cell patch-clamp recordings, we found that kynA was a more potent antagonist at human NR1a/NR2A compared with NR1a/NR2B receptors (IC(50): 158 muM and 681 muM, respectively; in 30 muM glycine). KynA inhibited AMPA-evoked currents to a similar degree in cultured hippocampal neurons and a human GluR2(flip/unedited) cell line (IC(50): 433 and 596 muM, respectively) and at higher concentrations, kynA also inhibited the strychnine-sensitive glycine receptor ( approximately 35% inhibition by 3 mM kynA). Interestingly, kynA inhibited the peak amplitude (IC(50): 2.9 mM for 10 muM GABA) and slowed the decay kinetics of GABA-evoked currents in cultured neurons. In contrast, we found that kynA (1-3 mM) had no effect on ACh-evoked, methyllycaconitine (MLA)-sensitive currents in a human alpha7 nicotinic receptor (nAChR) cell line, rat hippocampal neurons in primary culture or CA1 stratum radiatum interneurons in rat brain slices. However, DMSO (>1%) did inhibit alpha7 nAChR-mediated currents. In conclusion, kynA is an antagonist at NMDA, AMPA and glycine receptors and a modulator of GABA(A) receptors, but we find no evidence for any effect of kynA at the alpha7 nAChR.

Effects of N-acetylaspartylglutamate (NAAG) at group II mGluRs and NMDAR.

A group II metabotropic glutamate receptor (mGluR) agonist was recently reported to be clinically efficacious against symptoms of schizophrenia [Patil, S.T., Zhang, L., Martenyi, F., Lowe, S.L., Jackson, K.A., Andreev, B.V., Avedisova, A.S., Bardenstein, L.M., Gurovich, I.Y., Morozova, M.A., Mosolov, S.N., Neznanov, N.G., Reznik, A.M., Smulevich, A.B., Tochilov, V.A., Johnson, B.G., Monn, J.A., Schoepp, D.D., 2007. Activation of mGlu2/3 receptors as a new approach to treat schizophrenia: a randomized phase 2 clinical trial. Nature Med 13, 1102-1107]. The endogenous neuropeptide N-acetylaspartylglutamate (NAAG) has been described as an agonist at mGluR2 and mGluR3 [Wroblewska, B., Wroblewski, J.T., Pshenichkin, S., Surin, A., Sullivan, S.E., Neale, J.H., 1997. N-acetylaspartylglutamate selectively activates mGluR3 receptors in transfected cells. J. Neurochem. 69, 174-181; Cartmell, J., Adam, G., Chaboz, S., Henningsen, R., Kemp, J.A., Klingelschmidt, A., Metzler, V., Monsma, F., Schaffhauser, H., Wichmann, J., Mutel, V., 1998. Characterization of [3H]-(2S,2'R,3'R)-2-(2',3'-dicarboxy-cyclopropyl)glycine ([3H]-DCG IV) binding to metabotropic mGlu2 receptor-transfected cell membranes. Br. J. Pharmacol. 123, 497-504] and is degraded by the enzyme glutamate carboxypeptidase II (also known as N-acetyl-alpha-linked acidic dipeptidase or NAALADase). Hence, elevating the concentration of endogenous NAAG by inhibition of NAALADase represents a potential strategy for the treatment of schizophrenia via group II mGluR activation. We therefore investigated the activity of NAAG at both rat native and human recombinant mGluRs. We found that NAAG had no effect on synaptic transmission at the medial perforant pathway inputs to the rat dentate gyrus which is known to be sensitive to group II mGluR activation. We proceeded to examine the effects of NAAG at human recombinant mGluR2 and mGluR3 in a cellular G protein-activated K+ channel electrophysiology assay. Furthermore, due to discrepancies in the literature concerning the activity of NAAG at the N-methyl-d-aspartate receptor [NMDAR; Westbrook, G.L., Mayer, M.L., Namboodiri, M.A., Neale, J.H., 1986. High concentrations of N-acetylaspartylglutamate (NAAG) selectively activate NMDA receptors on mouse spinal cord neurons in cell culture. J. Neurosci. 6, 3385-3392; Losi, G., Vicini, S., Neale, J., 2004. NAAG fails to antagonize synaptic and extrasynaptic NMDA receptors in cerebellar granule neurons. Neuropharmacology 46, 490-496], we also tested NAAG at NMDARs in rat hippocampal neurons in culture. We found that a purified NAAG preparation had no effect at mGluR2, mGluR3 or NMDAR. Taken together, these findings do not support a rationale for targeting NAALADase and increasing extracellular NAAG levels as a therapeutic strategy for the treatment of schizophrenia.

alpha7 and non-alpha7 nicotinic acetylcholine receptors modulate dopamine release in vitro and in vivo in the rat prefrontal cortex.

Nicotine enhances attentional and working memory aspects of executive function in the prefrontal cortex (PFC) where dopamine plays a major role. Here, we have determined the nicotinic acetylcholine receptor (nAChR) subtypes that can modulate dopamine release in rat PFC using subtype-selective drugs. Nicotine and 5-Iodo-A-85380 (beta2* selective) elicited [(3)H]dopamine release from both PFC and striatal prisms in vitro and dopamine overflow from medial PFC in vivo. Blockade by dihydro-beta-erythroidine supports the participation of beta2* nAChRs. However, insensitivity of nicotine-evoked [(3)H]dopamine release to alpha-conotoxin-MII in PFC prisms suggests no involvement of alpha6beta2* nAChRs, in contrast to the striatum, and this distinction is supported by immunoprecipitation of nAChR subunits from these tissues. The alpha7 nAChR-selective agonists choline and Compound A also promoted dopamine release from PFC in vitro and in vivo, and their effects were enhanced by the alpha7 nAChR-selective allosteric potentiator PNU-120596 and blocked by specific antagonists. DNQX and MK801 inhibited [(3)H]dopamine release evoked by choline and PNU-120596, suggesting crosstalk between alpha7 nAChRs, glutamate and dopamine in the PFC. In vivo, systemic (but not local) administration of PNU-120596, in the absence of agonist, facilitated dopamine overflow in the medial PFC, consistent with the activation of extracortical alpha7 nAChRs by endogenous acetylcholine or choline. These data establish that both beta2* and alpha7 nAChRs can modulate dopamine release in the PFC in vitro and in vivo. Through their distinct actions on dopamine release, these nAChR subtypes could contribute to executive function, making them specific therapeutic targets for conditions such as schizophrenia and attention deficit hyperactivity disorder.

Investigation of G72 (DAOA) expression in the human brain.

BACKGROUND: Polymorphisms at the G72/G30 locus on chromosome 13q have been associated with schizophrenia or bipolar disorder in more than ten independent studies. Even though the genetic findings are very robust, the physiological role of the predicted G72 protein has thus far not been resolved. Initial reports suggested G72 as an activator of D-amino acid oxidase (DAO), supporting the glutamate dysfunction hypothesis of schizophrenia. However, these findings have subsequently not been reproduced and reports of endogenous human G72 mRNA and protein expression are extremely limited. In order to better understand the function of this putative schizophrenia susceptibility gene, we attempted to demonstrate G72 mRNA and protein expression in relevant human brain regions. METHODS: The expression of G72 mRNA was studied by northern blotting and semi-quantitative SYBR-Green and Taqman RT-PCR. Protein expression in human tissue lysates was investigated by western blotting using two custom-made specific anti-G72 peptide antibodies. An in-depth in silico analysis of the G72/G30 locus was performed in order to try and identify motifs or regulatory elements that provide insight to G72 mRNA expression and transcript stability. RESULTS: Despite using highly sensitive techniques, we failed to identify significant levels of G72 mRNA in a variety of human tissues (e.g. adult brain, amygdala, caudate nucleus, fetal brain, spinal cord and testis) human cell lines or schizophrenia/control post mortem BA10 samples. Furthermore, using western blotting in combination with sensitive detection methods, we were also unable to detect G72 protein in a number of human brain regions (including cerebellum and amygdala), spinal cord or testis. A detailed in silico analysis provides several lines of evidence that support the apparent low or absent expression of G72. CONCLUSION: Our results suggest that native G72 protein is not normally present in the tissues that we analysed in this study. We also conclude that the lack of demonstrable G72 expression in relevant brain regions does not support a role for G72 in modulation of DAO activity and the pathology of schizophrenia via a DAO-mediated mechanism. In silico analysis suggests that G72 is not robustly expressed and that the transcript is potentially labile. Further studies are required to understand the significance of the G72/30 locus to schizophrenia.

Altered hippocampal expression of glutamate receptors and transporters in GRM2 and GRM3 knockout mice.

Group II metabotropic glutamate receptors (mGluR2 and mGluR3, also called mGlu2 and mGlu3, encoded by GRM2 and GRM3, respectively) are therapeutic targets for several psychiatric disorders. GRM3 may also be a schizophrenia susceptibility gene. mGluR2-/- and mGluR3-/- mice provide the only unequivocal means to differentiate between these receptors, yet interpretation of in vivo findings may be complicated by secondary effects on expression of other genes. To address this issue, we examined the expression of NMDA receptor subunits (NR1, NR2A, NR2B) and glutamate transporters (EAAT1-3), as well as the remaining group II mGluR, in the hippocampus of mGluR2-/- and mGluR3-/- mice, compared with wild-type controls. mGluR2 mRNA was increased in mGluR3-/- mice, and vice versa. NR2A mRNA was increased in both knockout mice. EAAT1 (GLAST) mRNA and protein, and EAAT2 (GLT-1) protein, were reduced in mGluR3-/- mice, whereas EAAT3 (EAAC1) mRNA was decreased in mGluR2-/- mice. Transcripts for NR1 and NR2B were unchanged. The findings show a compensatory upregulation of the remaining group II metabotropic glutamate receptor in the knockout mice. Upregulation of NR2A expression suggests modified NMDA receptor signaling in mGluR2-/- and mGluR3-/- mice, and downregulation of glutamate transporter expression suggests a response to altered synaptic glutamate levels. The results show a mutual interplay between mGluR2 and mGluR3, and also provide a context in which to interpret behavioral and electrophysiological results in these mice.

Presynaptic alpha 7- and beta 2-containing nicotinic acetylcholine receptors modulate excitatory amino acid release from rat prefrontal cortex nerve terminals via distinct cellular mechanisms.

Nicotine can enhance working memory and attention. Activation of both alpha7 and beta2(*) nicotinic acetylcholine receptors (nAChRs) in the prefrontal cortex (PFC) has been implicated in these processes. The ability of presynaptic nAChRs to modulate neurotransmitter release, notably glutamate release, is postulated to contribute to nicotine's effects. We have examined the cellular mechanisms underlying alpha7 and beta2(*) nAChR-mediated [(3)H]d-aspartate release from the PFC in vitro. Using the alpha7 and beta2(*) nAChR-selective agonists (R)-N-(1-azabicyclo[2.2.2]-oct-3-yl)(5-(2-pyridyl)thiophene-2-carboxamide) (compound A) and 5-iodo-3-(2(S)-azetidinylmethoxy)pyridine (5-iodo-A-85380), respectively, in conjunction with inhibitors of voltage-operated Ca(2+) channels (VOCCs) and intracellular Ca(2+) stores, we show that [(3)H]d-aspartate release evoked by activation of beta2(*) nAChRs occurs via VOCCs. In contrast, alpha7 nAChR-evoked release was unaffected by VOCC blockers but was abolished by modulators of Ca(2+) stores, including ryanodine. The alpha7 nAChR ligand alpha-bungarotoxin and ryanodine receptors were colocalized to a subpopulation of PFC synaptosomes. Compound A-evoked [(3)H]d-aspartate release was also blocked by mitogen-activated protein kinase kinase 1 inhibitors, implicating extracellular signal-regulated kinase (ERK)1/2 in alpha7 nAChR-evoked exocytosis. Western blotting confirmed that compound A, but not 5-iodo-A-85380, application increased ERK2 phosphorylation in PFC synaptosomes, and this was dependent on ryanodine-sensitive stores. Compound A also promoted synapsin-1 phosphorylation at ERK1/2-dependent sites, in a ryanodine-sensitive manner. Thus, beta2(*) and alpha7 nAChR subtypes in the PFC mediate [(3)H]d-aspartate release via distinct mechanisms as a result of their differential coupling to VOCCs and Ca(2+)-induced Ca(2+) release (CICR), respectively. The ability of alpha7 nAChRs to promote the phosphorylation of presynaptic ERK2 and synapsin-1, downstream of CICR, provides a potential mechanism for presynaptic facilitation in the PFC.