Presynaptic 5-HT2A-mGlu2/3 Receptor-Receptor Crosstalk in the Prefrontal Cortex: Metamodulation of Glutamate Exocytosis.

The glutamatergic nerve endings of a rat prefrontal cortex (PFc) possess presynaptic 5-HT2A heteroreceptors and mGlu2/3 autoreceptors, whose activation inhibits glutamate exocytosis, and is measured as 15 mM KCl-evoked [3H]D-aspartate ([3H]D-asp) release (which mimics glutamate exocytosis). The concomitant activation of the two receptors nulls their inhibitory activities, whereas blockade of the 5-HT2A heteroreceptors with MDL11,939 (1 µM) strengthens the inhibitory effect elicited by the mGlu2/3 receptor agonist LY329268 (1 µM). 5-HT2A receptor antagonists (MDL11,939; ketanserin; trazodone) amplify the impact of low (3 nM) LY379268. Clozapine (0.1-10 µM) mimics the 5-HT2A agonist (±) DOI and inhibits the KCl-evoked [3H]D-asp overflow in a MDL11,939-dependent fashion, but does not modify the (±) DOI-induced effect. mGlu2 and 5-HT2A proteins do not co-immunoprecipitate from synaptosomal lysates, nor does the incubation of PFc synaptosomes with MDL11,939 (1 µM) or clozapine (10 µM) modify the insertion of mGlu2 subunits in synaptosomal plasma membranes. In conclusion, 5-HT2A and mGlu2/3 receptors colocalize, but do not physically associate, in PFc glutamatergic terminals, where they functionally interact in an antagonist-like fashion to control glutamate exocytosis. The mGlu2/3-5-HT2A metamodulation could be relevant to therapy for central neuropsychiatric disorders, including schizophrenia, but also unveil cellular events accounting for their development, which also influence the responsiveness to drugs regimens.

Microtiter plate quantification of mutant and wild-type huntingtin normalized to cell count.

Huntington's disease is caused by a gain-of-function neurotoxic mutation in normally neuroprotective huntingtin. Sensitive assays are required to discriminate mutant huntingtin from wild-type huntingtin. We have developed a normalized 384-plate assay for determination of mutant and wild-type huntingtin. Based on a single pipetting step, the sensitive assay uses two antibody pairs for simultaneous mutant and wild-type huntingtin time-resolved fluorescence resonance energy transfer detection combined with PicoGreen quantification of double-stranded DNA. The assay can be used for discovery of drugs reducing mutant huntingtin over wild-type huntingtin and for assessing the value of huntingtin as a disease progression marker, and it is adaptable to other proteins of interest.