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

Figure 1.

Figure 1

Selective activation of basal forebrain (BF) cholinergic neurons by designer receptors exclusively activated by designer drugs (DREADD) in vivo and in vitro. (a) Coronal section shows the injection target (highlighted area) in ChAT-IRES-Cre transgenic mice. (b) Schematic representation of DIO-hM3Dq or hM4Di-mCherry-AAV vectors which are under the control of hSyn promoter. ITR, inverted terminal repeat; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element. (c) Microinjection of DIO-hM3Dq-mCherry-AAV into the BF of ChAT-IRES-Cre mice gave rise to the expression of hM3Dq and mCherry (red). (d) Humanized Renilla green fluorescent protein (hrGFP) immunolabeling indicates the location of cholinergic neurons. (e) Overlaid images of mCherry and hrGFP. (c1, d1, e1) Higher-power views of boxed region in (c–e), respectively. White arrows indicate selective expression of mCherry and hM3Dq receptors in cholinergic neurons (e1). (f) Representative image of mCherry (brown) and c-Fos (black) expression in the BF after injection of vehicle. (f1) Higher-power view of boxed region in (f), showing that most mCherry-expressing neurons did not express c-Fos (white arrows) after injection of vehicle. (g) Representative image of c-Fos in the BF after injection of 1 mg/kg clozapine-N-oxide (CNO). (g1) Higher-power view of boxed region in (g), showing that c-Fos was expressed in mCherry-expressing neurons after injection of 1 mg/kg CNO (black arrows). (h) Percentages of double-labeled neurons in mCherry-expressing neurons after treatment with vehicle or CNO, respectively (n=6 or 7). **P<0.01 vs vehicle, assessed by the non-paired, two-tailed Student's t-test. (i) IR-DIC and mCherry fluorescence image of recorded hM3Dq-mCherry-expressing neuron in BF slice. (j) Bath application of CNO induced firing in hM3Dq-mCherry-expressing neuron in the absence of TTX. (k) CNO produced depolarization in hM3Dq-mCherry-expressing neuron (bottom), but not neighboring control neuron (top), in the presence of TTX. (l) Mean membrane potential (MP) of hM3Dq-mCherry-expressing neurons before and after the application of 500 nM CNO (n=8 cells from six animals). **P<0.01 vs baseline, assessed by two-tailed paired Student's t-test.