The Input-Output Relationship of the Cholinergic Basal Forebrain

Abstract

Basal forebrain cholinergic neurons influence cortical state, plasticity, learning, and attention. They collectively innervate the entire cerebral cortex, differentially controlling acetylcholine efflux across different cortical areas and timescales. Such control might be achieved by differential inputs driving separable cholinergic outputs, although no input-output relationship on a brain-wide level has ever been demonstrated. Here, we identify input neurons to cholinergic cells projecting to specific cortical regions by infecting cholinergic axon terminals with a monosynaptically restricted viral tracer. This approach revealed several circuit motifs, such as central amygdala neurons synapsing onto basolateral amygdala-projecting cholinergic neurons or strong somatosensory cortical input to motor cortex-projecting cholinergic neurons. The presence of input cells in the parasympathetic midbrain nuclei contacting frontally projecting cholinergic neurons suggest that the network regulating the inner eye muscles are additionally regulating cortical state via acetylcholine efflux. This dataset enables future circuit-level experiments to identify drivers of known cortical cholinergic functions.

Keywords: ChAT-cre; acetylcholine; basal forebrain; basalocortical; cholinergic; connectome; monosynaptic; rabies; rat; transgenic.

Figure 1. Monosynaptic viral tracing

(A) Cre-dependent helper viruses limit starter cells to the cholinergic cell type. Virus placed at cortical terminals retrogradely labels a subset of cholinergic cells that project to the cortical injection site (figure based on Wall et al., 2010). (B) 12 subjects received helper virus across 5 basal forebrain sites, and (C) received retrograde virus in a single BF projection target, whether in motor cortex (red), medial prefrontal cortex (green), orbitofrontal cortex (blue) or amygdala (yellow)(figure based on Paxinos and Watson, 2007). (D) Cells are visualized by the presence of mCherry (helper virus in cholinergic cells), GFP (afferent cells, monosynaptic inputs to starter cells) or both fluorophores (cholinergic starter cells).

Figure 2. Topography of starter cells

Cholinergic cells capable of spreading virus monosynaptically (mCherry + / GFP +) in 12 subjects following retrograde viral injection in M1/M2 (red), mPFC (green), VO/LO (blue), or amygdala (yellow). Starter cells from individual cases (labeled with differently shaped symbols) were warped into a common template brain. ac, anterior commissure; BLA, anterior basolateral amygdala; f, fornix; GP, globus pallidus; HDB, horizontal diagonal band; ic, internal capsule; lo, lateral olfactory tract; LV, lateral ventricle; mt, mammillothalamic tract; MS/VDB, medial septum/ vertical diagonal band; opt, optic tract; sm, stria medullaris; SI/EA, substantia innominata/ extended amygdala.

Figure 3. Distribution of inputs across all brain regions

Percent of labeled inputs to cholinergic starter cells across all brain regions is shown per subject (individual bars clustered in groups of three). Regions of sparse input (where no injection group averaged > 0.5%) are not shown. See also Figures S3-S4 and Table S2. M1/M2, motor cortex; mPFC, medial prefrontal cortex; VO/LO, ventral/lateral orbitofrontal cortices.

Figure 4. Distribution of striatal input cells

There is a topography of afferents originating across different levels (rows) in the CPu when comparing three subjects with different rabies injection sites: the amygdala (left column), motor cortex (middle), or ventral orbitofrontal cortex (right column). Various contours delineate cytoarchitectonic areas where cells appear. BFc, basal forebrain cholinergic cells; BMP, posterior basomedial amygdala; M1/M2, primary/secondary motor cortex; VO, ventral orbitofrontal cortex.

Figure 5. Input cells from cortex

In each pair of panels, GFP appears on DAPI (blue, left) and thionin Nissl (violet, right). (A) S1 cells synapsing onto M1/M2-targeted ChAT. (B) Labeled cell in the medial entorhinal cortex (mEC) synapsing onto mPFC-targeted ChAT. (C) Endopiriform cell synapsing onto mPFC-targeted ChAT. (D) Retrosplenial cell synapsing onto mPFC-targeted ChAT. (E, F, I) CA1 cells synapsing onto mPFC-targeted ChAT. (G) vOFC cell synapsing onto vOFC-targeted ChAT. (H) CA3 cell synapsing onto mPFC-targeted ChAT. Scale 100 μM. cc, corpus callosum; cg, cingulate gyrus; CPu, caudate putamen; DG, dentate gyrus; En, endopiriform nucleus; fi, fimbria; or, oriens; PaS, parasubiculum; Pir, piriform cortex; rad, radiatum; S1, primary somatosensory cortex; VO/vOFC, ventral orbitofrontal cortex.

Figure 6. Input cells from amygdala

In each pair of panels, GFP appears on DAPI (blue, left) and thionin Nissl (violet, right). (A) AA cells synapsing onto mPFC-targeted ChAT. (B) BMP cells synapsing onto mPFC-targeted ChAT. (C) CeC cells synapsing onto BMP-targeted ChAT. (D) ACo cells synapsing onto mPFC-targeted ChAT. (E) CeL cells synapsing onto M1/M2-targeted ChAT. (F) AHiPM cell synapsing onto M1/M2-targeted ChAT. Scale 100 μM. AA, anterior amygdaloid area; ACo, anterior cortical amygdaloid nucleus; AHiPM, posteromedial amygdalohippocampal area; BLA, anterior basolateral amygdala; CA3, hippocampal field CA3; Ce, central nucleus, amygdala; CeC, central nucleus, amygdala, capsular part; CeL, central nucleus, amygdala, lateral part; cp, cerebral peduncle; CPu, caudate putamen; cst, commissural stria terminalis; En, endopiriform nucleus; I, intercalated nucleus, amygdala; La, lateral amygdala; LOT, nucleus of the lateral olfactory tract; LV, lateral ventricle; opt, optic tract.

Figure 7. Input cells from brainstem

(A) GFP on DAPI background shows inputs in RIP, synapsing onto mPFC-targeted ChAT cells. (A′) The same cells from panel a on thionin Nissl (violet). (B) GFP on DAPI background shows inputs in dorsal raphe, synapsing onto M1/M2-targeted ChAT cells. (B′) The same cells from panel b on thionin Nissl (violet). (C, D) GFP inputs in VTA on Nissl (violet) background synapsing onto mPFC-targeted or M1/M2-targeted ChAT cells, respectively. (E) GFP inputs in nucleus Darkschewitsch (Dk) projecting to mPFC-targeted ChAT cells. (E′) The same cells from panel e on thionin Nissl (violet). (F) Macro view of GFP inputs in PAG and PPT synapsing onto M1/M2-targeted ChAT cells. Scale 200 μM. 3N, oculomotor nucleus; 7, facial nucleus; g7, facial nerve; Gi, gigantocellular reticular nucleus; IP, interpeduncular nucleus; isRt, isthmic reticular formation; MB, mammillary body; MG, medial geniculate nucleus; ml, medial lemniscus; mRt, mesencephalic reticular formation; PAG, periaqueductal gray; Po, posterior thalamic nuclei; PPT, pedunculopontine tegmentum; PT, pretectal nucleus; RIP, raphe interpositus; SNC, substantia nigra pars compacta; SNR, substantia nigra pars reticulata; VTA, ventral tegmental area.