Ultrastructural quantification of glutamate receptors at excitatory synapses in hippocampus of synapsin I+II double knock-out mice.

Previous findings, mainly in in vitro systems, have shown that the density of vesicles and the synaptic efficacy at excitatory synapses are reduced in the absence of synapsins, despite the fact that transgenic mice lacking synapsins develop an epileptic phenotype. Here we study glutamate receptors by quantitative immunoblotting and by quantitative electron microscopic postembedding immunocytochemistry in hippocampus of perfusion fixed control wild type and double knock-out mice lacking synapsins I and II. In wild type hippocampus the densities of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunits were higher (indicated for glutamate receptor subunit 1, highly significant for glutamate receptor subunits 2/3) in mossy fiber-to-cornu ammonis 3 pyramidal cell synapses than in the Schaffer collateral/commissural-to-cornu ammonis 1 pyramidal cell synapses, the two synapse categories that carry the main excitatory throughput of the hippocampus. The opposite was true for N-methyl-D-aspartate receptors. The difference in localization of glutamate receptor subunit 1 receptor subunits was increased in the double knock-out mice while there was no change in the overall expression of the glutamate receptors in hippocampus as shown by quantitative Western blotting. The increased level of glutamate receptor subunit 1 at the mossy fiber-to-cornu ammonis 3 pyramidal cell synapse may result in alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors with reduced proportions of glutamate receptor subunit 2, and hence increased Ca2+ influx, which could cause increased excitability despite of impaired synaptic function (cf. [Krestel HE, Shimshek DR, Jensen V, Nevian T, Kim J, Geng Y, Bast T, Depaulis A, Schonig K, Schwenk F, Bujard H, Hvalby O, Sprengel R, Seeburg PH (2004) A genetic switch for epilepsy in adult mice. J Neurosci 24:10568-10578]), possibly underlying the seizure proneness in the synapsin double knock-out mice. In addition, the tendency to increased predominance of N-methyl-d-aspartate receptors at the main type of excitatory synapse onto cornu ammonis 1 pyramidal cells might contribute to the seizure susceptibility of the synapsin deficient mice. The results showed no significant changes in the proportion of 'silent' Schaffer collateral/commissural synapses lacking alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors or in the synaptic membrane size, indicating that plasticity involving these parameters is not preferentially triggered due to lack of synapsins.