Glutamate hyperexcitability and seizure-like activity throughout the brain and spinal cord upon relief from chronic glutamate receptor blockade in culture.

Cortical structures such as the hippocampus and cerebral cortex are considered to be particularly susceptible to seizure and epileptiform electrical activity and, as such, are the focus of intense investigation relative to hyperexcitability. To determine whether parallel glutamate-mediated hyperexcitability and seizure-like activity in the rat can be generated by neurons irrespective of their origin within the CNS, we maintained cells from the spinal cord,hippocampus, olfactory bulb, striatum, hypothalamus, and cortex in the long-term presence of glutamate receptor antagonists 2-amino-5-phosphonovalerate and 6-cyano-7-nitroquinoxaline-2-3-dione. After removal of chronic (three to 11 weeks) glutamate receptor block, whole-cell patch-clamp recordings from current-clamped neurons (n = 94) revealed an immediate increase in large excitatory postsynaptic potentials and a depolarization of 20-35 mV that was often sustained for recording periods lasting 5 min (54% of 66 neurons from all six areas). The intense activity was not seen in age-matched control neurons not subjected to chronic glutamate receptor block. Selective blockade of ionotropic glutamate receptors showed that the hyperexcitability was due to an enhanced response through both AMPA/kainate and N-methyl-D-aspartate receptors. Relief from chronic glutamate receptor block also increased inhibitory activity, as revealed by an increase in inhibitory postsynaptic currents while neurons were voltage-clamped at -25 mV. These inhibitory postsynaptic currents could be blocked with bicuculline, indicating that they were mediated by an enhanced GABA release. This enhanced GABA activity reduced, but did not eliminate, the glutamate-mediated hyperactivity, shown by an increase in both intracellular Ca2+ and excitatory electrical activity when bicuculline was added. When the glutamate receptor block was removed, cells (n > 1000) from all six regions showed exaggerated Ca2+ activity, characterized by abnormally high increases in intracellular Ca2+, rising from basal levels of 50-100 nM up to 150-1600 nM. Cd2+ eliminated the hyperexcitability by blocking Ca2+ channels, and reducing excitatory transmitter release and response. Fura-2 digital imaging revealed Ca2+ oscillations with periods ranging from 4 to 60 s. Ca2+ peaks in oscillations in oscillations were synchronized among most neurons recorded simultaneously. That synchronization was dependent on a mechanism involving voltage-dependent Na+ channels was demonstrated with experiments with tetrodotoxin that blocked Ca2+ rises and synchronous cellular behavior. Removal of the glutamate receptor antagonists resulted in the glutamate-mediated death of 44% of the cells after 23 days of chronic block and 82% cell death after 40 days of chronic block. Nimodipine substantially reduced cell death, indicating that one mechanism responsible for the enhanced cell death after relief from chronic glutamate receptor block was increased intracellular Ca2+ entry through L-type voltage-gated calcium channels. These data indicate that glutamate is released by neurons from all areas studied, including the spinal cord. Sufficient amounts of glutamate can be released from axon terminals from all areas to cause cell hippocampal and cortical neurons, but also by neurons from any of the brain regions tested after chronic deprivation of glutamate receptor stimulation during development. This hyperexcitability is mediated by glutamatergic mechanisms independent of the specific excitatory connections existing in vivo. The epileptiform activity of neurons from one region is indistinguishable from that of another in culture, underlining the importance of synaptic connections in vivo that define the responses characteristic of neurons from different brain regions.