Anatomical evidence for a ponto-septal pathway via the nucleus incertus in the rat

Hippocampal theta activity is involved in sensory-motor integration and constitutes a functional basis for mnemonic functions. The medial septum-diagonal band of Broca (MS/DBv) is a key structure as pacemaker of the oscillation. In addition, some brainstem reticular structures are crucial for the activation of MS/DBv. Specifically, the nucleus reticularis pontis oralis (RPO) is considered the most effective pontine site for eliciting theta rhythm. Nevertheless, its connection with the MS/DBv is not direct. A previous study by our group pointed out that the nucleus incertus (NI) could be considered as a relay in this multisynaptic pathway. From this study, the stimulation of RPO increased the discharge rate of NI neurons in anesthetized rats and the lesion of the NI suppressed the RPO-elicited hippocampal theta. Those findings suggested a projection from RPO to NI, although the existing literature did not support this hypothesis. In order to clarify the dichotomy between the anatomical and the electrophysiological data, we performed a set of tracing studies. Anterograde tracer injections into RPO showed a profuse projection to NI. This connection was confirmed by retrograde tracer injections into NI. Injections of retrograde tracer in MS/DBv confirmed the intense NI-MS/DBv projection. Furthermore, simultaneous injections of anterograde and retrograde tracers into RPO and MS/DBv respectively resulted in a high-correlated pattern of terminal-like fibers over labeled somata in the NI. This study provides the first anatomical evidence of a ponto-septal pathway via the NI that contributes to generation and modulation the hippocampal theta activity.