Improper usage of general anesthesia may cause fatal damage to the central nervous system. Therefore, its safety becomes a major concern. In order to reveal the changing patterns of neuronal activity during the period of increasing anesthetic depth, the present paper investigated the changes of excitability and signal conduction of neurons in the rat hippocampus during deep anesthesia till brain death with urethane. By using the techniques of microelectrode array recording and electrical stimulation, we recorded population spikes (PS) in the pyramidal layer of hippocampal CA1 region that were evoked either by orthodromic stimulation of the Schaffer collateral or by antidromic stimulation of the alveus. The amplitude and latency of PS were used as indices to evaluate the changes of neuronal activity. The results showed that as the urethane concentration in the plasma increased, the amplitudes of PS decreased and the latencies of PS increased, indicating that the urethane suppressed the neuronal excitability as well as the synaptic transmission and axon conduction. Particularly, there was a turning point that divided the whole decline period of neuronal activity into two distinct stages: slow stage and fast stage. The catastrophic decline of fast stage resulted in brain death rapidly. In addition, the urethane concentration in plasma, rather than the duration of anesthesia, might cause the appearance of turning point. However, the prolonged duration of slow stage caused by slower injection rate of urethane could also induce more damage to neuronal functions. These results provide important information for the safe application of narcotics in both animal experiments and clinic usage.