The basal forebrain, an anatomically heterogeneous brain area containing multiple distinct subregions and neuronal populations, innervates many brain regions including the hippocampus (HIP), a key brain region responsible for learning and memory. Although recent studies have revealed that basal forebrain cholinergic neurons (BFCNs) are involved in olfactory associative learning and memory, the potential neural circuit is not clearly dissected yet. Here, using an anterograde monosynaptic tracing strategy, we revealed that BFCNs in different subregions projected to many brain areas, but with significant differentiations. Our rabies virus retrograde tracing results found that the dorsal HIP (dHIP) received heavy projections from the cholinergic neurons in the nucleus of the horizontal limb of the diagonal band (HDB), magnocellular preoptic nucleus (MCPO), and substantia innominate (SI) brain regions, which are known as the HMS complex (HMSc). Functionally, fiber photometry showed that cholinergic neurons in the HMSc were significantly activated in odor-cued go/no-go discrimination tasks. Moreover, specific depletion of the HMSc cholinergic neurons innervating the dHIP significantly decreased the performance accuracies in odor-cued go/no-go discrimination tasks. Taken together, these studies provided detailed information about the projections of different BFCN subpopulations and revealed that the HMSc-dHIP cholinergic circuit plays a crucial role in regulating olfactory associative learning.

Optogenetic manipulation is pivotal in basic neuroscience research and in studying neuropsychiatric disorders, enabling the activation or inhibition of neuronal populations with millisecond precision. However, this technique can induce artificial neuronal hypersynchronization that is rarely observed under physiological conditions. Given that epileptic seizures arise from abnormally synchronized neuronal discharges, the potential for optogenetic stimulation to trigger seizures and confound experimental outcomes warrants close examination. Here, using electrophysiological and behavioral recordings, we demonstrate that even single-trial optogenetic stimulation of CaMKII-positive neurons in the hippocampal CA1 region, anterior piriform cortex (APC), or lateral/medial entorhinal cortex (LEnt or MEnt) can induce seizure-like discharges and behaviors in adult male C57BL/6 mice. Repeated stimulation in the APC, LEnt, or MEnt elicited more severe seizure-like activity. Furthermore, stimulation protocols characterized by high power, long duration, high frequency, and medium pulse width were more prone to inducing such events. Additionally, we found that CA1 stimulation could impair subsequent contextual fear memory. These findings provide critical cautions and practical guidelines for the design and implementation of in vivo optogenetic experiments in neuroscience research.