Glutamate and GABA are two major fast neurotransmitters (excitatory and inhibitory, respectively) in the CNS, including the hypothalamus. They play a key role in the control of excitation/inhibition balance and determine the activity and excitability of neurons in many neuronal circuits. Using neuronal cultures, whole-cell recording, Ca(2+) imaging, and Northern blots, we studied the compensatory regulation of neuronal activity during a prolonged decrease in glutamate excitation. We report here that after a chronic (6-17 d) blockade of ionotropic glutamate receptors, neurons in hypothalamic cultures revealed excitatory electrical and Ca(2+) synaptic activity, which was not elicited in the control cultures that were not subjected to glutamate blockade. This activity was suppressed with acetylcholine (ACh) receptor antagonists and was potentiated by eserine, an inhibitor of acetylcholinesterase, suggesting its cholinergic nature. The upregulation of ACh receptors and the contribution of ACh to the control of the excitation/inhibition balance in cultures after a prolonged decrease in glutamate activity were also demonstrated. Enhanced ACh transmission was also found in chronically blocked cerebellar but not cortical cultures, suggesting the region-specific character of glutamate-ACh interactions in the brain. We believe that in the absence of glutamate excitation in the hypothalamus in vitro, ACh, a neurotransmitter normally exhibiting only weak activity in the hypothalamus, becomes the major excitatory neurotransmitter and supports the excitation/inhibition balance. The increase in excitatory ACh transmission during a decrease in glutamate excitation may represent a novel form of neuronal plasticity that regulates activity and excitability of neurons during the glutamate/GABA imbalance.