Abstracts

Rationale: Group I metabotropic glutamate receptors (mGluR, consisting of mGluR1 or mGluR5 subunits) play roles in construction of the developing neocortex and controlling synaptic plasticity. Previous studies have shown that receptors formed from mGluR1 subunits are located postsynaptically and selectively activate low threshold spiking (LTS) interneurons within neocortex, to produce rhythmic firing that can also synchronize the activity of pyramidal neurons. We hypothesize that LTS interneurons are overactive within developmentally malformed cortex, creating increased columnar synchrony and increased probability of generating epileptiform activity. Here we have investigated the degree of modulation of interneurons by Group I mGluR agonists and antagonists in the rat freeze lesion model of microgyria. Methods: Using visualized whole cell patch clamp techniques, we have recorded spontaneous inhibitory postsynaptic currents (sIPSCs) in pyramidal cells in cortical layer V horizontal slices from P12-17 control and rats given transcranial freeze lesions on P1. The group I mGluR agonist, (S)-3,5-Dihydroxyphenylglycine hydrate (DHPG), the mGluR1 selective antagonist, 1-Amino-2,3-dihydro-1H-indene-1,5-dicarboxylic acid (AIDA), and the mGluR5 selective antagonist, 6-methyl-2-(phenylethynyl) pyridine hydrochloride (MPEP) were applied to the region of the recorded cell with a local perfusion system or included in the bath. DNQX and APV were included in the bath solution to limit activity to monosynaptic IPSCs. Standard Western Blotting detection was used to compare the amount of mGluR5 protein (normalized to β-actin) present in a 3X3 mm block of cortex adjacent to the malformation (paramicrogyral cortex, PMG). Anti-mGluR5 (AnaSpec, San Jose, CA) and anti-β-actin (Sigma-Aldrich) were diluted 1:2,000. Results: DHPG (10 μM) increased the IPSC frequency to a greater degree in PMG compared to unlesioned control cortex (354 ±71 versus 195 ±43% for 31 PMG versus 42 control cells, respectively, t-test, p <0.05). When the mGluR1 antagonist, AIDA was included in the bath (300 μM), DHPG failed to significantly change the IPSC frequency in control tissue. Under these same conditions, IPSC frequency was increased by 173 ±66% in PMG cortex (N=15). This increase was reversed by the mGluR5 antagonist, MPEP (10 μM). Since these results suggested that mGluR5 was present were it previously had been functionally absent, we used Western blots to detect the level of mGluR5. PMG cortex had 14% more mGluR5 present than control (100% ± 2.1% for controls; 113.8% ± 4.3% for PMG, p=0.03). Conclusions: These results suggest that a novel mGluR5 expression occurs on LTS interneurons in malformed cortex. This creates an additional avenue for activation of these cells that may then synchronize activity within individual cortical columns, and thereby increase the probability of epileptiform activity. Our results suggest that mGluR5 antagonists may be an effective anti-epileptogenic treatment for some patients with malformations. This work was supported by the Epilepsy Foundation and NIH grant NS054210.