Fig 1.
Expression of opsins in ChAT-Cre/Ai32(ChR2-YFP) and ChAT-Cre/Ai35(Arch-GFP) mice.
(A) Opsin (anti-GFP) expression and corresponding anti-ChAT immunohistochemistry in posterior basal forebrain from example ChAT-Cre/Ai32(ChR2-YFP) (left) and ChAT-Cre/Ai35(Arch-GFP) (right) mice. Widefield images of 50 μm-thick coronal sections. Asterisks indicate locations of ChAT+ somata. (B) Quantification of cell counts in basal forebrain, showing penetrance of opsin expression in the cholinergic population in basal forebrain (% of ChAT+ neurons expressing opsin, bars represent mean ± SEM) and lack of opsin expression in non-cholinergic neurons (% of opsin-expressing neurons also ChAT+). (C) Opsin (anti-GFP) expression in visual cortex from example ChAT-Cre/Ai32(ChR2-YFP) and ChAT-Cre/Ai35(Arch-GFP) mice. Confocal maximum intensity projections of 6 μm-thick optical sections. Inset: background intensity in wild-type tissue, acquired under identical imaging conditions. (D) Example of ectopic expression in a ChAT-Cre/Ai35(Arch-GFP) mouse. Widefield images of a 100 μm-thick coronal section.
Fig 2.
Depolarization of cholinergic neurons from ChAT-Cre/Ai32(ChR2-YFP) mice.
Whole-cell recordings from ChR2-YFP-labeled neurons in nucleus basalis in acute slices from ChAT-Cre/Ai32(ChR2-YFP) mice, illustrating the effects of blue illumination (bar). Dashed horizontal lines denote 0 mV. (A) Upper panel: 300 ms, 20 μW/mm2 illumination, resting membrane potential -46 mV. Lower panel: 1 ms, 20 mW/mm2 illumination, resting membrane potential -46 mV. (B) Trains of 20 stimuli (each 1 ms, 20 mW/mm2) at 5, 10 and 20 Hz. Resting membrane potentials -46 mV, -46 mV and -47 mV, respectively. (C) Number of spikes per stimulus during trains of stimuli, as a function of stimulus rate. Each point represents mean ± SEM for 7 neurons.
Fig 3.
Hyperpolarization of cholinergic neurons from ChAT-Cre/Ai35(Arch-GFP) mice.
(A) Whole-cell recordings from Arch-GFP-labeled neurons in nucleus basalis in acute slices from Arch-ChR2 mice, illustrating the effects of 300 ms white illumination (bar) at intensities of 1.2, 1.7, 2.2 and 3.7 mW/mm2. Dashed horizontal lines denote 0 mV. Resting membrane potentials -51, -51, -52 and -52 mV. (B) Steady-state hyperpolarization and membrane time constant during white illumination from the recording illustrated in panel A. Arrowheads indicate example traces in panel A. (C and D) Inhibition of spiking by white illumination. Spiking was evoked by DC (C) or 2Hz (D) current injection to depolarize cell beyond spike threshold. Spikes were eliminated by 7 mW/mm2 white illumination (bar) for 1 second (C) or for 20 ms (D). Initial membrane potential (during DC current injection) was -33 mV in panel C and resting membrane potential was -49 mV in panel D.
Table 1.
Basal forebrain cell counts.
Fig 4.
Cholinergic cell densities in basal forebrain.
Cell densities of ChAT-positive neurons in basal forebrain from ChAT-Cre, ChAT-Cre/Ai32(ChR2-YFP), ChAT-Cre/Ai35(Arch-GFP) and C57BL/6J (WT) mice. Each bar represents mean ± SEM cell density from 3 mice.
Table 2.
Membrane properties of cholinergic neurons.
Fig 5.
Spike characteristics of cholinergic neurons in ChAT-Cre/Ai32(ChR2-YFP) and ChAT-Cre/Ai35(Arch-GFP) mice.
(A) Whole-cell recordings from a ChR2-YFP-labeled neuron in nucleus basalis in an acute slice, illustrating the spiking pattern (upper panel) and after-spike potentials (lower panel) when spikes were evoked by somatic current injections (upper panel 300 ms, 50 pA; lower panel 1 ms, 500 pA current). Dashed horizontal lines denote 0 mV. Resting membrane potentials were -50 mV and -51 mV for upper and lower recordings, respectively. (B) Mean ± SEM spiking frequency as a function of current injected at the somata of 10 cholinergic neurons from ChAT-Cre/Ai32(ChR2-YFP) mice. Grey line: mean spike rates for cholinergic neurons from wild-type mice, from Hedrick & Waters (2010). (C) Whole-cell recordings from an Arch-GFP-labeled neuron in nucleus basalis in an acute slice, illustrating the spiking pattern (upper panel) and after-spike potentials (lower panel) when spikes were evoked by somatic current injections (upper panel 300 ms, 150 pA; lower panel 1 ms, 2000 pA current). Dashed horizontal lines denote 0 mV. Resting membrane potentials were -53 mV and -52 mV for upper and lower recordings, respectively. (D) Mean ± SEM spiking frequency as a function of current injected at the somata of 9 cholinergic neurons from ChAT-Cre/Ai35(Arch-GFP) mice. Grey line: mean spike rates for cholinergic neurons from wild-type mice, from Hedrick & Waters (2010).
Fig 6.
Comparison of visual discrimination by wild-type, ChAT-Cre/Ai32(ChR2-YFP) and ChAT-Cre/Ai35(Arch-GFP) mice.
(A) Schematic summary of the visual discrimination task. The visual object moved along the horizon from left to right at a rate which was proportional to the mouse's running speed. (B) Running speed for two trials, one with a rewarded and one with an unrewarded object, each at 100% contrast. The left (medial) and right (temporal) edge of the monitor corresponded to distances of 0 and 1920 pixels. The reward window is illustrated as a grey bar. (C) Psychometric curves from a single behavioral session, describing performance (stop probability) as a function of object contrast for rewarded and unrewarded objects. (D) Comparison of performance (d') for wild-type, ChAT-Ai32 and ChAT-Ai35 mice. Points denote mean ± SEM for 2 wild-type, 4 ChAT-Ai32 and 7 ChAT-Ai35 mice. (E) Mean ± SEM running speed (throughout the behavioral session, including stationary periods) for 4 wild-type, 9 ChAT-Ai32 and 12 ChAT-Ai35 mice. (F) Proportion of mice that learned the visual discrimination task during 6 weeks of training. Total of 4 wild-type, 9 ChAT-Ai32 and 12 ChAT-Ai35 mice. (G) Mean ± SEM training time (one session per day) required for mice to reach criterion performance. Total of 3 wild-type, 6 ChAT-Ai32 and 8 ChAT-Ai35 mice.