Clustering autism: using neuroanatomical differences in 26 mouse models to gain insight into the heterogeneity.

Autism is a heritable disorder, with over 250 associated genes identified to date, yet no single gene accounts for >1-2% of cases. The clinical presentation, behavioural symptoms, imaging and histopathology findings are strikingly heterogeneous. A more complete understanding of autism can be obtained by examining multiple genetic or behavioural mouse models of autism using magnetic resonance imaging (MRI)-based neuroanatomical phenotyping. Twenty-six different mouse models were examined and the consistently found abnormal brain regions across models were parieto-temporal lobe, cerebellar cortex, frontal lobe, hypothalamus and striatum. These models separated into three distinct clusters, two of which can be linked to the under and over-connectivity found in autism. These clusters also identified previously unknown connections between Nrxn1α, En2 and Fmr1; Nlgn3, BTBR and Slc6A4; and also between X monosomy and Mecp2. With no single treatment for autism found, clustering autism using neuroanatomy and identifying these strong connections may prove to be a crucial step in predicting treatment response.

Pathways to drug development for autism spectrum disorders.

Autism spectrum disorders (ASDs) are neurodevelopmental disorders whose prevalence has risen over the past two decades. Current drug treatments for ASDs and the related disorders--fragile X syndrome (FXS) and Rett syndrome--target specific symptoms but do not address the basic underlying etiologies. However, based partly on an improved understanding of the neurochemical underpinnings of FXS, pharmacotherapy for this syndrome has progressed to the point of clinical trials of several novel drug treatments. By contrast, our overall understanding of the neuropathophysiology of ASDs is still rudimentary. There is hope in the field that knowledge and experience gained in the study of fragile X and Rett syndromes may be applicable to the larger autism patient population. In this review, we discuss how recent advances in our understanding of the biochemistry and neuropathology of these disorders could lead to new more effective treatments for ASDs.

Altered glutamate and GABA release within thalamocortical circuitry in metabotropic glutamate receptor 4 knockout mice.

The metabotropic glutamate receptor 4 is highly expressed presynaptically on thalamocortical neurons that are involved in the pathogenesis of generalized absence seizures. Mutant mice devoid of metabotropic glutamate receptor 4 are completely resistant to absence seizures induced by low doses of GABA type A receptor antagonists. The purpose of this study was to test the hypothesis that there is altered glutamate and GABA release within thalamocortical circuitry in mice devoid of metabotropic glutamate receptor 4. Extracellular GABA and glutamate release were determined in ventrobasal thalamus, the nucleus reticularis thalami and laminae I-III, and IV-VI of cerebral cortex (laminae I-III of cerebral cortex, and laminae IV-VI of cerebral cortex) using in vivo microdialysis techniques on awake, free moving mice. A significant increase of both basal and K(+)-evoked glutamate release was detected in the ventrobasal thalamus, the nucleus reticularis thalami and laminae IV-VI of cerebral cortex of mice devoid of metabotropic glutamate receptor 4 mice. There also was a significant increase in both basal and K(+)-evoked GABA release in the mice devoid of metabotropic glutamate receptor 4, but a significant decrease of GABA release in laminae IV-VI of cerebral cortex. However, there was no alteration of either GABA or glutamate release in laminae I-III of cerebral cortex, cortical laminae that are not involved in absence seizures. These data indicate that deletion of the metabotropic glutamate receptor 4 gene results in a selective perturbation of glutamate and GABA release within the thalamocortical circuitry involved in the pathogenesis of absence seizures.

Increased seizure susceptibility in mice lacking metabotropic glutamate receptor 7.

To study the role of mGlu7 receptors (mGluR7), we used homologous recombination to generate mice lacking this metabotropic receptor subtype (mGluR7(-/-)). After the serendipitous discovery of a sensory stimulus-evoked epileptic phenotype, we tested two convulsant drugs, pentylenetetrazole (PTZ) and bicuculline. In animals aged 12 weeks and older, subthreshold doses of these drugs induced seizures in mGluR7(-/-), but not in mGluR7(+/-), mice. PTZ-induced seizures were inhibited by three standard anticonvulsant drugs, but not by the group III selective mGluR agonist (R,S)-4-phosphonophenylglycine (PPG). Consistent with the lack of signs of epileptic activity in the absence of specific stimuli, mGluR7(-/-) mice showed no major changes in synaptic properties in two slice preparations. However, slightly increased excitability was evident in hippocampal slices. In addition, there was slower recovery from frequency facilitation in cortical slices, suggesting a role for mGluR7 as a frequency-dependent regulator in presynaptic terminals. Our findings suggest that mGluR7 receptors have a unique role in regulating neuronal excitability and that these receptors may be a novel target for the development of anticonvulsant drugs.

Nature of the oligomers formed by muscarinic m2 acetylcholine receptors in Sf9 cells.

Wild-type, FLAG-tagged, and c-myc-tagged muscarinic m2 receptors extracted in digitonin-cholate from singly and co-infected Sf9 (Spodoptera frugiperda) cells were indistinguishable in their binding of [3H]quinuclidinylbenzilate, either before or after purification. The FLAG epitope was found to coimmunoprecipitate with the c-myc epitope when co-infected cells were solubilised in digitonin-cholate, n-dodecyl-beta-D-maltoside or Lubrol-PX. The degree of coprecipitation in digitonin-cholate was unaffected by preincubation of the extract for up to 60 min at 30 degrees C, with or without muscarinic receptor ligands; no coimmunoprecipitation occurred in mixed extracts from singly infected cells. As measured by [3H]quinuclidinylbenzilate, the efficiency of immunoprecipitation from co-infected cells was 87% of that from singly infected cells. The amount of receptor immunoprecipitated from the latter, as determined by densitometry, was 2.3-fold that expected from the loss of binding from the extract. The data suggest that at least some of the receptors were trimeric or larger and that oligomers neither formed nor dissociated under the conditions of the experiments. Also, some receptors appear to be non-functional or latent in digitonin-solubilised extracts.

Localization of the human mGluR4 gene within an epilepsy susceptibility locus(1).

The family of metabotropic glutamate receptors (mGluRs) consists of eight homologous G-protein coupled receptors. Several of the mGluRs, including the mGluR4 receptor subtype, are localized presynaptically; activation of this receptor induces an inhibition of neurotransmitter release from nerve terminals. Disruption of the mGluR4 gene in mice results in impaired motor and spatial learning, and alterations in seizure susceptibility. In this study, we have determined the structure of the human mGluR4 gene, as well as its chromosomal localization. A comparison of the gene structure of mGluR4 with the highly homologous mGluR6 receptor subtype reveals that both of the genes contain ten exons with similar exon/intron boundaries. A refined localization of mGluR4 was carried out by constructing a bacterial artificial chromosome clone contig of the region surrounding the gene. Thirteen sequence tagged sites (STSs) were identified within this contig. The gene was localized to chromosome 6 band p21.3 by fluorescence in situ hybridization (FISH). The mapping of the mGluR4 gene indicates that it is approximately 1 megabases centromeric of the major histocompatibility complex and 5 megabase from the GABA(B)R1 gene. The mGluR4 gene also falls within a susceptibility locus for juvenile myoclonic epilepsy suggesting a potential link to this form of epilepsy.

Pharmacological profiles of the metabotropic glutamate receptor ligands.

Metabotropic glutamate receptors (mGluRs) are a family of G-protein coupled receptors that are expressed in the central and peripheral nervous systems. The purpose of this study was to compare the ligand binding selectivity profiles of the mGluR agonist [(3)H]L-AP4 and the novel radiolabeled phenylglycine antagonist [(3)H]CPPG at all eight rat mGluR subtypes expressed in transfected human embryonic kidney cells. At a concentration of 30 nM [(3)H]L-AP4, no specific binding was detected in membranes expressing the group I receptors mGluR1a or mGluR5a, or in membranes expressing the group II mGluRs, mGluR2 and mGluR3. Among the group III mGluRs, specific [(3)H]L-AP4 binding was detected in cells expressing mGluR4a and mGluR8a but not in cells expressing mGluR6 or mGluR7a. The binding of [(3)H]CPPG showed an exceptional pattern of selectivity amongst the mGluR subtypes; at a concentration of 20 nM [(3)H]CPPG, a high level of specific binding was seen in membranes containing mGluR8a but not in any of the other mGluR subtypes. The affinity constant (K(D)) calculated for [(3)H]CPPG binding to mGluR8a was 183 nM. In competition experiments, the phosphono-substituted phenylglycine congeners including MPPG, (RS)-PPG, and unlabeled CPPG were the most potent inhibitors of [(3)H]CPPG binding while non-phosphonated compounds such as L-glutamate and MCPG were substantially less potent. These results demonstrate that [(3)H]L-AP4 and [(3)H]CPPG can be used as probes to selectively label group III mGluRs and that CPPG and related phenylglycine derivatives are useful for studying differences in the ligand recognition sites of highly homologous mGluRs.

Selective activation of mGlu4 metabotropic glutamate receptors is protective against excitotoxic neuronal death.

Activation of group III metabotropic glutamate receptors (mGluR4, mGluR6, mGluR7, and mGluR8) has been established to be neuroprotective in vitro and in vivo. To disclose the identity of the receptor subtype(s) that exert(s) the protective effect, we have used group III agonists in combination with mGluR4 subtype-deficient mice (-/-). In cortical cultures prepared from wild-type (+/+) mice and exposed to a toxic pulse of NMDA, the selective group III agonist (+)-4-phosphonophenylglycine [(+)-PPG] reversed excitotoxicity with an EC(50) value of 4.9 microm, whereas its enantiomer (-)-PPG was inactive. This correlated closely with the potency of (+)-PPG in activating recombinant mGluR4a. In cortical neurons from -/- mice, (+)-PPG showed no protection against the NMDA insult up to 300 microm, whereas group I/II mGluR ligands still retained their protective activity. Classical group III agonists (l-2-amino-4-phosphonobutyrate and l-serine-O-phosphate) were also substantially neuroprotective against NMDA toxicity in +/+ and heterozygous (+/-) cultures but were inactive in -/- cultures. Interestingly, -/- cultures were more vulnerable to low concentrations of NMDA and showed higher extracellular glutamate levels compared with +/+ cultures. We have also examined neurodegeneration induced by intrastriatal infusion of NMDA in wild-type or mGluR4-deficient mice. Low doses of (R,S)-PPG (10 nmol/0.5 microl) substantially reduced NMDA toxicity in +/+ mice but were ineffective in -/- mice. Higher doses of (R,S)-PPG were neuroprotective in both strains of animals. Finally, microdialysis studies showed that intrastriatal infusion of NMDA increased extracellular glutamate levels to a greater extent in -/- than in +/+ mice, supporting the hypothesis that the mGluR4 subtype is necessary for the maintenance of the homeostasis of extracellular glutamate levels.

Constraints on proper folding of the amino terminal domains of group III metabotropic glutamate receptors.

The glutamate binding site of the G-protein coupled metabotropic glutamate receptors (mGluRs) is contained within the large extracellular amino terminal domain (ATD) of the receptor. In this study, we examined the ligand binding properties and cellular dispositions of the membrane-bound mGluR4 and mGluR8 subtypes of mGluRs, and a series of truncated versions of these receptors. Truncation of the ATDs of mGluR4 and mGluR8 40 amino acids upstream of the first transmembrane domain produced soluble proteins that were secreted into the cell culture media of transfected human embryonic kidney cells. The soluble receptors retained ligand binding capabilities. Additional constructs of the ATDs of mGluR4 and mGluR8 were assessed for their ability to bind the agonist [(3)H]L-AP4 and for secretion from cells. A shorter mGluR4 construct truncated 98 amino acids upstream from the first transmembrane domain failed to bind [(3)H]L-AP4, while the analogous mGluR8 construct displayed a low level of binding. Unlike the full-length receptors, which were expressed on the cell surface, or the soluble constructs which were secreted, the shorter constructs were primarily associated with intracellular membranes. These observations suggest that the cysteine-rich region may be important for efficient secretion, but not absolutely obligatory for ligand binding. Surprisingly, longer constructs encoding the entire ATDs of mGluR4 and mGluR8 failed to bind ligand and were localized intracellularly. Together, these findings demonstrate that there are strict limitations on the proper folding of truncated versions of the ATDs of mGluR4 and mGluR8. Specifically, all of the leucine-isoleucine-valine binding protein homology region, and part of the cysteine-rich region is required for optimal secretion in a soluble form that retains ligand binding activity.

Immunocytochemical localization of the metabotropic glutamate receptor mGluR4a in the piriform cortex of the rat.

This study evaluates the localization of the metabotropic glutamate receptor mGluR4a in the piriform cortex of rats using preembedding immunocytochemical methods. At the light microscopic level, punctate labeling was evident in layers Ia and Ib of the piriform cortex, and immunolabeled fibers were present in layers II and III. Following bilateral destruction of the olfactory bulb, the density of labeled puncta in layer Ia decreased. These results suggest that the receptor is present on the terminals of the lateral olfactory tract (LOT). Electron microscopic evaluation of layers Ia and Ib revealed that mGluR4a was localized in synaptic terminals in layers Ia and Ib. The terminals had clear, round synaptic vesicles and terminated on asymmetric synapses on dendritic spines and shafts. There was also immunolabeling of some dendritic profiles in layers Ia and Ib that were postsynaptic to unlabeled presynaptic terminals. These observations suggest that mGluR4a is present on presynaptic terminals in the layers of the piriform cortex that receive LOT and associational synapses. This is the same area in which previous studies have revealed the presence of mGluR7 and mGluR8, suggesting that all three receptors may be colocalized.

Inhibition of microtubule formation by metabotropic glutamate receptors.

Activation of glutamate receptors is known to alter the biophysical state of the cytoskeleton of neurons in the developing brain. In this study, we examined the ability of G protein-coupled metabotropic glutamate receptors (mGluRs) to inhibit the formation of processes induced by the expression of the microtubule-associated protein MAP2c. The infection of insect MG-1 cells with a recombinant baculovirus (BV) encoding MAP2c induced the formation of fine filamentous processes. The binding of MAPs to tubulin promotes tubulin polymerization and the formation of microtubules. Co-infection with BVs for the phosphoinositide (PI)-linked mGluR1a or mGluR1b receptor subtypes inhibited the formation of processes induced by MAP2c, whereas co-infection with BVs encoding the mGluR4a or mGluR4b subtypes that couple to adenylyl cyclase did not inhibit the formation of processes. The biochemical pathways responsible for producing the inhibitory effect of mGluR1 were investigated. Inhibitors of protein kinase C, calcium/calmodulin-dependent kinase, and protein tyrosine kinases did not block the inhibitory effect of mGluR1a. The calcium chelator BAPTA and the calcium depletor thapsigargin also did not affect the ability of mGluR1a to inhibit process formation. In contrast, inhibitors of phospholipase C reversed the effect of mGluR1 on process formation, suggesting that one or more metabolites in the PI pathway were responsible for the inhibitory effect. These findings indicate that PIs generated by activation of mGluRs inhibit the binding of MAPs to tubulin and reduce tubulin polymerization and microtubule stability.

Probing the ligand-binding domain of the mGluR4 subtype of metabotropic glutamate receptor.

Metabotropic glutamate receptors (mGluRs) are G-protein-coupled glutamate receptors that subserve a number of diverse functions in the central nervous system. The large extracellular amino-terminal domains (ATDs) of mGluRs are homologous to the periplasmic binding proteins in bacteria. In this study, a region in the ATD of the mGluR4 subtype of mGluR postulated to contain the ligand-binding pocket was explored by site-directed mutagenesis using a molecular model of the tertiary structure of the ATD as a guiding tool. Although the conversion of Arg(78), Ser(159), or Thr(182) to Ala did not affect the level of protein expression or cell-surface expression, all three mutations severely impaired the ability of the receptor to bind the agonist L-[(3)H]amino-4-phosphonobutyric acid. Mutation of other residues within or in close proximity to the proposed binding pocket produced either no effect (Ser(157) and Ser(160)) or a relatively modest effect (Ser(181)) on ligand affinity compared with the Arg(78), Ser(159), and Thr(182) mutations. Based on these experimental findings, together with information obtained from the model in which the glutamate analog L-serine O-phosphate (L-SOP) was "docked" into the binding pocket, we suggest that the hydroxyl groups on the side chains of Ser(159) and Thr(182) of mGluR4 form hydrogen bonds with the alpha-carboxyl and alpha-amino groups on L-SOP, respectively, whereas Arg(78) forms an electrostatic interaction with the acidic side chains of L-SOP or glutamate. The conservation of Arg(78), Ser(159), and Thr(182) in all members of the mGluR family indicates that these amino acids may be fundamental recognition motifs for the binding of agonists to this class of receptors.

Developmental expression of the group III metabotropic glutamate receptor mGluR4a in the medial nucleus of the trapezoid body of the rat.

A preembedding immunocytochemical method for light microscopy was used to study the postnatal development of expression of the group III metabotropic glutamate receptor mGluR4a in the medial nucleus of the trapezoid body (MNTB) of the rat. Immunoreactivity for mGluR4a was localized in axonal endings wrapping the principal globular neurons in MNTB, known as calyces of Held. The percentage of calyces of Held immunoreactive for mGluR4a increased progressively from postnatal day 3 (PND3), showing the highest density of labeled calyces by PND9. From this postnatal age on, a gradual reduction in the number of mGluR4a-immunopositive calyces of Held was observed, reaching the lowest level of labeled profiles in adult tissue. The developmental expression of mGluR4a in calyces of Held correlates well with previous studies in young animals showing a modulation of synaptic neurotransmission by group III mGluRs in these giant excitatory synapses made on MNTB principal neurons. All these observations together suggest that the expression of mGluR4a mainly between PND7 and PND12 might be relevant to the maturation and modulation of synaptic transmission at the calyces of Held.

Ligand binding to the amino-terminal domain of the mGluR4 subtype of metabotropic glutamate receptor.

The metabotropic glutamate receptor (mGluR) 4 subtype of metabotropic glutamate receptor is a presynaptic receptor that modulates neurotransmitter release. We have characterized the properties of a truncated, epitope-tagged construct containing part of the extracellular amino-terminal domain of mGluR4. The truncated receptor was secreted into the cell culture medium of transfected human embryonic kidney cells. The oligomeric structure of the soluble truncated receptor was assessed by gel electrophoresis. In the presence of high concentrations of a reducing agent, the truncated receptor migrated as a monomer; at lower concentrations of the reducing agent, only higher molecular weight oligomers were observed. Competition binding experiments using the radiolabeled agonist [3H]L-2-amino-4-phosphonobutyric acid revealed that the rank order of potency of metabotropic ligands at the truncated receptor was similar to that of the full-length membrane-bound receptor. However, the truncated receptor displayed higher affinities for agonists and lower affinities for antagonists compared with the full-length receptor. Deglycosylation produced a shift in the relative molecular weight of the soluble protein from Mr = 71,000 to Mr = 63,000; deglycosylation had no effect on the binding of [3H]L-2-amino-4-phosphonobutyric acid, indicating that the asparagine-linked carbohydrates are not necessary for agonist binding. These results demonstrate that although the primary determinants of ligand binding to mGluR4 are contained within the first 548 amino acids of the receptor, additional amino acids located downstream of this region may influence the affinity of ligands for the binding site.

Contribution of metabotropic glutamate receptor mGluR4 to L-2-[3H]amino-4-phosphonobutyrate binding in mouse brain.

The binding of L-2-[3H]amino-4-phosphonobutyrate ([3H]L-AP4) was examined in brain sections of wild-type mice and mice lacking the mGluR4 subtype of metabotropic glutamate receptors (mGluRs). Very high relative densities of [3H]L-AP4 binding were observed in the molecular layer of the cerebellar cortex, the nucleus basalis, the outer layer of the superior colliculus, and the substantia nigra. In mGluR4 knock-out mice, very low levels of binding were observed in these regions. The moderate levels of binding observed with wild-type mice in the molecular layer of the hippocampal dentate gyrus and in the thalamus were absent in mGluR4 knock-out mice. In contrast, the moderate levels observed in most of the cerebral cortex, caudate putamen, and globus pallidus were not different in mGluR4 knock-out mice compared with wild-type. In these regions, mGluR8 is likely to be labeled by [3H]L-AP4 because mGluR8 is expressed in such brain regions and, like mGluR4, has high affinity for L-AP4. We conclude that mGluR4 contributes substantially to the high-affinity binding site for [3H]L-AP4 in several regions of mouse brain, including cerebellar cortex, nucleus basalis, thalamus, superior colliculus, substantia nigra, and hippocampal dentate gyrus.

Altered spatial learning and memory in mice lacking the mGluR4 subtype of metabotropic glutamate receptor.

The glutamate analog, L-2-amino-4-phosphonobutyric acid (L-AP4) is a selective agonist for several members of the metabotropic glutamate receptor (mGluR) family. Activation of presynaptic mGluRs by L-AP4 causes a suppression of synaptic transmission in the central nervous system. In this study, the role of 1 subtype of mGluR in the nervous system was investigated by analyzing mutant mice lacking the L-AP4-sensitive receptor, mGluR4. Experiments designed to probe hippocampal function showed no impairments in acquisition of spatial learning in the water maze task. However, in a spatial reversal learning task, the mutant mice exhibited significantly accelerated learning performance. Furthermore, in a probe trial administered 6 weeks posttraining, these mice showed impaired spatial accuracy. The results suggest that the mutant mice differed in their ability to learn and integrate new spatial information into previously formed memory traces and that their use of stored spatial information also was altered. Thus, the presynaptically expressed mGluR4 plays a role in the processing of spatial information.

The activation of glutamate receptors by kainic acid and domoic acid.

The neurotoxins kainic acid and domoic acid are potent agonists at the kainate and alphaamino-5-methyl-3-hydroxyisoxazolone-4-propionate (AMPA) subclasses of ionotropic glutamate receptors. Although it is well established that AMPA receptors mediate fast excitatory synaptic transmission at most excitatory synapses in the central nervous system, the role of the high affinity kainate receptors in synaptic transmission and neurotoxicity is not entirely clear. Kainate and domoate differ from the natural transmitter, L-glutamate, in their mode of activation of glutamate receptors; glutamate elicits rapidly desensitizing responses while the two neurotoxins elicit non-desensitizing or slowly desensitizing responses at AMPA receptors and some kainate receptors. The inability to produce desensitizing currents and the high affinity for AMPA and kainate receptors are undoubtedly important factors in kainate and domoate-mediated neurotoxicity. Mutagenesis studies on cloned glutamate receptors have provided insight into the molecular mechanisms responsible for these unique properties of kainate and domoate.

Down-regulation of mGluR5 by antisense deoxynucleotides alters pharmacological responses to applications of ACPD in the rat hippocampus.

Metabotropic glutamate receptors are thought to be important regulators of synaptic transmission and plasticity in the hippocampus. The metabotropic glutamate receptor subtype mGluR5 is expressed in hippocampal pyramidal neurons but its function remains unknown due to the lack of selective pharmacological blockers. We inhibited the synthesis of mGluR5 with antisense oligonucleotides injected into the hippocampus in vivo. The functional effects of altered mGluR5 expression were measured electrophysiologically in the CA1 region of the hippocampus during applications of the metabotropic agonist 1S,3R-ACPD (50 microM) to hippocampal slices from injected animals. The results show a concomitant reduction of the mGluR5 receptor protein and physiological effects in the hippocampus. The major effect found in the antisensetreated animals was the lack of an excitatory action normally produced by 1S,3R-ACPD. Another effect attributed to metabotropic glutamate receptors, depression of synaptic transmission, had a more rapid onset, but unchanged magnitude, while long-term potentiation remained unchanged. The specificity and effectiveness of the antisense treatment were confirmed using mismatched oligonucleotides and immunoblotting. We conclude that the metabotropic glutamate receptor subtype mGluR5 plays a major role in the regulation of cell excitability in the hippocampus without directly affecting synaptic transmission or long-term potentiation. Moreover, in vivo applications of antisense deoxynucleotides are a useful approach in studies of neurotransmitter receptor subtypes.