Autism spectrum disorders are neurodevelopmental disorders characterized by two core symptoms; impaired social interactions and communication, and ritualistic or repetitive behaviors. Both epidemiological and biochemical evidence suggests that a subpopulation of autistics may be linked to immune perturbations that occurred during fetal development. These findings have given rise to an animal model, called the "maternal immune activation" model, whereby the offspring from female rodents who were subjected to an immune stimulus during early or mid-pregnancy are studied. Here, C57BL/6 mouse dams were treated mid-gestation with saline, lipopolysaccharide (LPS) to mimic a bacterial infection, or polyinosinic:polycytidylic acid (Poly IC) to mimic a viral infection. Autism-associated behaviors were examined in the adult offspring of the treated dams. Behavioral tests were conducted to assess motor activity, exploration in a novel environment, sociability, and repetitive behaviors, and data analyses were carried independently on male and female mice. We observed a main treatment effect whereby male offspring from Poly IC-treated dams showed reduced motor activity. In the marble burying test of repetitive behavior, male offspring but not female offspring from both LPS and Poly IC-treated mothers showed increased marble burying. Our findings indicate that offspring from mothers subjected to immune stimulation during gestation show a gender-specific increase in stereotyped repetitive behavior.
A kainic acid receptor was purified from Triton X-100/digitonin-solubilized frog brain membranes. The purification was carried out in two steps: ion exchange chromatography using DEAE-Sepharose CL-6B and affinity chromatography with domoic acid immobilized on Sepharose 4B. The specific binding activity of the affinity-purified receptor is 481-fold higher than that of the crude solubilized preparation and 1617-fold higher than that of the whole membrane fraction. Scatchard analyses of the affinity-purified receptor showed a curvilinear plot which fit a two-site model with dissociation constants of 5.5 and 34 nM and Bmax values of 1700 pmol/mg protein and 4400 pmol/mg protein for the high and low affinity components, respectively. The dissociation constants of the purified receptor are similar to those of the crude soluble preparation (4.8 and 39 nM). Inhibition constants for several kainic acid analogs were also similar for the purified and crude preparations. The active purified receptor migrated with a Mr = 570,000 on gel filtration analysis using Sepharose 6B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the affinity-purified receptor showed a single broad band with silver stain, migrating with a Mr = 48,000.
Abstract A homology model of the extracellular domain of the mGlu3 subtype of metabotropic glutamate (mGlu) receptor was generated and tested using site‐directed mutagenesis, a radioligand‐binding assay using the Group II selective agonist (2 S ,2′ R ,3′ R )‐2‐(2′,3′‐[ 3 H]dicarboxycyclopropyl) glycine ([ 3 H]DCG‐IV), and in a fluorescence‐based functional assay in live transiently transfected human embryonic kidney cells. Ten of the 12 mGlu3 mutants (R64A, R68A, Y150A, S151A, T174A, D194A, Y222A, R277A, D301A and K389) showed either no binding or a 90% or greater loss of specific [ 3 H]DCG‐IV binding. Several analogous mutations in mGlu2 supported the results obtained with mGlu3. These results demonstrate that the binding of [ 3 H]DCG‐IV to mGlu3 is exceptionally sensitive to mutagenesis‐induced perturbations. In silico docking of DCG‐IV into the agonist binding pocket of mGlu3 facilitated the interpretation the mutagenesis results. Tyrosines 150 and 222, and arginine 277 show close contacts with the third carboxylic acid group in DCG‐IV, which is not present in glutamate or (2 S ,1′ S ,2′ S )‐2‐(carboxycyclopropyl)glycine (L‐CCG‐I). Mutation of these three amino acids to alanine resulted in a near complete loss of receptor activation by DCG‐IV and retention of near wild‐type affinity for L‐CCG‐I. It is proposed that hydrogen bonding between this carboxylate and tyrosines 150 and 222 and arginine 277 provide a partial explanation for the high affinity and Group II selectivity of DCG‐IV. These findings define the essential features of the ligand‐binding pocket of mGlu3 and, together with other recent studies on mGlu receptors, provide new opportunities for structure‐based drug design.
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