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. 2016 Feb 17;41(8):2133–2146. doi: 10.1038/npp.2016.13

Figure 5.

Figure 5

The projection of basal forebrain (BF) cholinergic neurons and the c-Fos expression after activating BF cholinergic neurons by hM3Dq receptors. Cre-dependent expression of humanized Renilla green fluorescent protein (hrGFP) in BF cholinergic neurons (a–d). (a) Coronal section showing unilateral injection target of DIO-hrGFP-AAV (HDB/SI/MCPO, delimited area). (b) Microinjection of DIO-hrGFP-AAV into ChAT-IRES-Cre mice resulted in the expression of hrGFP in the BF. (c) mCherry immunolabeling indicates location of cholinergic neurons in the BF. (d) Overlaid images of hrGFP and mCherry indicate selective expression of hrGFP in BF cholinergic soma (white arrows). (e) Coronal section shows the projection site in cortex from BF cholinergic neurons. Square in (e) delimits the cortical area shown in (f). (f) Microinjection of DIO-hrGFP-AAV into the BF resulted in highly dense expression of hrGFP in the ipsilateral secondary motor cortex (M2) and cingulate cortex (Cg). (g) Schematics show the ventrolateral preoptic nucleus (VLPO, high light area). (h) Microinjection of DIO-hrGFP-AAV into the BF resulted in sparse expression of hrGFP in VLPO. (f1, h1) Higher power view of boxed region in (f, h); white arrows indicate projecting fibers from BF cholinergic neurons. acp, anterior commissure posterior; aca, anterior commissure anterior. Representative photomicrographs of c-Fos expression in M2/Cg (i, j) and VLPO (l, m) of ChAT-IRES-Cre mice expressing hM3Dq receptors after administration of vehicle or 1 mg/kg clozapine-N-oxide (CNO). (i1, j1, l1, m1) Higher power view of boxed region in (i, j, l, m), respectively; black arrows indicates the c-Fos positive neurons. Mean numbers of c-Fos-positive neurons in M2/Cg (k) and VLPO (n) after activating of BF cholinergic neurons by CNO treatment (n=8). *P<0.05, **P<0.01 vs vehicle injection, assessed by the non-paired, two-tailed Student's t-test.