Abstracts

Rationale: In humans and in animal models, mutations in genes that repress the mechanistic target of rapamycin (mTOR) signaling cascade result in hyperactivation of the pathway and are associated with autism, intellectual disability and epilepsy. Recent data suggest this is the case even when these are somatic mutations that affect only limited populations of neurons. Although altered brain morphology is a hallmark of known mTOR-related neurological diseases in humans, recent studies in humans and in animal models show that disease phenotypes and abnormal synaptic transmission in these neurons can precede, or occur in the absence of, morphological changes. This suggests that synaptic alterations contribute to, or may even cause, the neurological phenotypes. In vivo, many effects of mTOR hyperactivation have been reported. However, it is difficult to assess which of these effects are directly due to abnormal mTOR signaling and which are secondary or activity-dependent changes due to altered brain activity such as epilepsy. In addition, in more intact preparations, such as acute brain slices, the difficulty of directly measuring presynaptic function means that electrophysiological studies have been limited to postsynaptic descriptions of glutamatergic neurons. Methods: In order to investigate both presynaptic and postsynaptic changes caused by mTOR hyperactivation, as well as the interactions between excitatory and inhibitory neurons, we developed a two-neuron culture system in which one of the two neurons lacked the phosphatase and tensin homolog on chromosome ten (Pten) gene, a known repressor of mTOR signaling. We then performed voltage clamp recordings from neuron pairs consisting of either one glutamatergic and one GABAergic neuron, or two glutamatergic neurons. Results: We found that loss of Pten in glutamatergic neurons was sufficient to increase both glutamatergic and GABAergic synaptic input to the mutant neuron, but not the output of the mutant neuron in response to low frequency stimulation. Further analysis showed that the enhancement of synaptic input was due to a combination of increased synapse formation and increased quantal amplitude. Surprisingly, high frequency stimulation of Pten-/- neurons caused a strong facilitation of the EPSC when the postsynaptic neuron was glutamatergic, but not when it was GABAergic, while control pairs showed mild depression regardless of the postsynaptic target. Conclusions: While the postsynaptic effects of Pten loss result in a parallel increase in excitation and inhibition, the presynaptic effects may shift the balance toward excitation during periods of high activity. Thus alterged target-dependent short term plasticity may contribute to network hyperexcitability and the development of epilepsy.