Functionally linked amygdala and prefrontal cortical regions are innervated by both single and double projecting cholinergic neurons

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. ### Competing Interest Statement The authors have declared no competing interest. * Aca : anterior commissure, anterior part ACo : anterior cortical amygdaloid nucleus Acp : anterior commissure, posterior part AHi : amygdalohippocampal area AHiAL : amygdalohippocampal area, anterolateral part AHiPM : amygdalohippocampal area, posteromedial part AI : agranular insular cortex APir : amygdalopiriform transition area BA : basal amygdala BAa : basal amygdala, anterior part BAp : basal amygdala, posterior part BFCNs : basal forebrain cholinergic neurons BLA : basolateral amygdala BMA : basomedial amygdala CeL : central amygdala, lateral division CeM : central amygdala, medial division Cg1 : cingulate cortex, area 1 Cg2 : cingulate cortex, area 2 ChAT : choline acetyltransferase DEn : dorsal endopiriform nucleus DI : dysgranular insular cortex DP : dorsal peduncular cortex dPFC : dorsal prefrontal cortex HDB : nucleus of the horizontal limb of the diagonal band IL : infralimbic cortex IPACL : interstitial nucleus of the posterior limb of the anterior commissure, lateral part IPACM : interstitial nucleus of the posterior limb of the anterior commissure, medial part LA : lateral amygdala LEnt : lateral entorhinal cortex LH : lateral hypothalamic area LO : lateral orbital cortex LPO : lateral preoptic area M1 : primary motor cortex M2 : secondary motor cortex MCPO : magnocellular preoptic nucleus MeA : medial amygdala, anterior part Mep : medial amygdala, posterior part MO : medial orbital cortex mPFC : medial prefrontal cortex MS : medial septal nucleus Pir : piriform cortex PL : prelimbic cortex PMCo : posteromedial cortical amygdaloid nucleus PS : parastrial nucleus S1 : primary somatosensory cortex SI : substantia innominata VAChT : vesicular acetylcholine transporter VGLUT3 : vesicular glutamate transporter 3 VO : ventral orbital cortex VP : ventral pallidum vPFC : ventral prefrontal cortex