The basal forebrain cholinergic innervation of the medial prefrontal cortex (mPFC) is crucial for cognitive performance. However, little is known about the organization of connectivity between the basal forebrain and the mPFC in the mouse. Using focal virus injections inducing Cre-dependent enhanced yellow fluorescent protein expression in ChAT–IRES–Cre mice, we tested the hypothesis that there is a topographic mapping between the basal forebrain cholinergic neurons and their axonal projections to the mPFC. We found that ascending cholinergic fibers to the mPFC follow four pathways and that cholinergic neurons take these routes depending on their location in the basal forebrain. In addition, a general mapping pattern was observed in which the position of cholinergic neurons measured along a rostral to caudal extent in the basal forebrain correlated with a ventral to dorsal and a rostral to caudal shift of cholinergic fiber distribution in mPFC. Finally, we found that neurons in the rostral and caudal parts of the basal forebrain differentially innervate the superficial and deep layers of the ventral regions of the mPFC. Thus, a frontocaudal organization of the cholinergic system exists in which distinct mPFC areas and cortical layers are targeted depending on the location of the cholinergic neuron in the basal forebrain.

Neuroanatomical tracing methods remain fundamental for elucidating the complexity of brain circuits. During the past decades, the technical arsenal at our disposal has been greatly enriched, with a steady supply of fresh arrivals. This paper provides a landscape view of classical and modern tools for tract-tracing purposes. Focus is placed on methods that have gone viral, i.e., became most widespread used and fully reliable. To keep an historical perspective, we start by reviewing one-dimensional, standalone transport-tracing tools; these including today’s two most favorite anterograde neuroanatomical tracers such asPhaseolus vulgaris-leucoagglutinin and biotinylated dextran amine. Next, emphasis is placed on several classical tools widely used for retrograde neuroanatomical tracing purposes, where Fluoro-Gold in our opinion represents the best example. Furthermore, it is worth noting that multi-dimensional paradigms can be designed by combining different tracers or by applying a given tracer together with detecting one or more neurochemical substances, as illustrated here with several examples. Finally, it is without any doubt that we are currently witnessing the unstoppable and spectacular rise of modern molecular-genetic techniques based on the use of modified viruses as delivery vehicles for genetic material, therefore, pushing the tract-tracing field forward into a new era. In summary, here, we aim to provide neuroscientists with the advice and background required when facing a choice on which neuroanatomical tracer—or combination thereof—might be best suited for addressing a given experimental design.

We characterized presubicular neurons giving rise to bilateral projections to the medial entorhinal cortex (MEA) of the rat. Retrograde labeling of presubiculo–entorhinal projections with horseradish peroxidase and subsequent GABA immunocytochemistry revealed that 20–30% of the ipsilaterally projecting neurons are GABAergic. No GABAergic projections to the contralateral MEA were observed. GABAergic projection neurons were observed only in the dorsal part of the presubiculum, which, when taking into account the topography of presubicular projections to MEA, indicates that only the dorsal part of MEA receives GABAergic input. The GABAergic projection neurons constitute ∼30-40% of all GABAergic neurons present in the superficial layers of the dorsal presubiculum. Using double-label fluorescent retrograde tracing, we found that the ipsilateral and contralateral presubiculo–entorhinal projections originate from different populations of neurons. Anterograde labeling of presubiculo–entorhinal projections and electron microscopical analysis of labeled terminals substantiated the presence of a restricted GABAergic presubiculo–entorhinal projection. A small fraction of afferents to only ipsilateral dorsal MEA formed symmetrical synapses with dendritic shafts. No symmetrical synapses on spines were noted. Most afferents to the dorsal part of ipsilateral MEA, as well as all afferents to the remaining ipsilateral and contralateral MEA, formed asymmetrical synapses with both spines and dendritic shafts in an almost equal ratio. Thus, we conclude that the majority of the presubiculo–entorhinal projections exert an excitatory effect on both principal neurons and interneurons. The projections from the dorsal part of the presubiculum comprise a small inhibitory component that originates from GABAergic neurons and targets entorhinal interneurons.