Cholinergic cells have been proposed to innervate simultaneously those cortical areas
that are mutually interconnected with each other. To test this hypothesis, we investigated
the cholinergic innervation of functionally linked amygdala and prefrontal cortical
regions. First, using tracing experiments, we determined that cholinergic cells located
in distinct basal forebrain (BF) areas projected to the different nuclei of the basolateral
amygdala (BLA). Specifically, cholinergic cells in the ventral pallidum/substantia
innominata (VP/SI) innervated the basal nucleus (BA), while the horizontal limb of
the diagonal band of Broca (HDB) projected to its basomedial nucleus (BMA). In addition,
cholinergic neurons in these two BF areas gave rise to overlapping innervation in
the medial prefrontal cortex (mPFC), yet their axons segregated in the dorsal and
ventral regions of the PFC. Using retrograde-anterograde viral tracing, we demonstrated
that a portion of mPFC-projecting cholinergic neurons also innervated the BLA, especially
the BA. By injecting retrograde tracers into the mPFC and BA, we found that 28% of
retrogradely labeled cholinergic cells were double labeled, which typically located
in the VP/SI. In addition, we found that vesicular glutamate transporter type 3 (VGLUT3)-expressing
neurons within the VP/SI were also cholinergic and projected to the mPFC and BA, implicating
that a part of the cholinergic afferents may release glutamate. In contrast, we uncovered
that GABA is unlikely to be a co-transmitter molecule in HDB and VP/SI cholinergic
neurons in adult mice. The dual innervation strategy, i.e., the existence of cholinergic
cell populations with single as well as simultaneous projections to the BLA and mPFC,
provides the possibility for both synchronous and independent control of the operation
in these cortical areas, a structural arrangement that may maximize computational
support for functionally linked regions. The presence of VGLUT3 in a portion of cholinergic
afferents suggests more complex functional effects of cholinergic system in cortical
structures.
