Figure 1.

Cholinergic stimulation by ACh boosts spontaneous action potential firing in Hcrt+ neurons. A₁, Sketch of a brain slice showing Hcrt+ neurons (green cells in the red box) residing in the hypothalamus. A₂, Morphology of two Hcrt+ neurons shown in the fluorescent channel. A₃, Differential Interference Contrast video-microscopy showing the experimental paradigm of pressure application (puff) of drug onto the soma and proximal processes, while keeping the patch onto the neuron. Scale bar, 10 µm. B₁, Mechanical interference (puff of bath solution) frequently produces a temporary depression of spontaneous firing. B₂, In the presence of atropine (4 µM), application of ACh (1 mM) boosts action potential firing for tens of seconds. B₃, The same trace as in B₂, except on a different scale and filtered with Gaussian low pass 5 Hz. B₄, Injection of +10 pA of current into the Hcrt+ neuron notably increases the firing frequency. Red lines represent the timing and duration of ACh application or current injection. C, Mechanical interference does not affect firing (n = 4/11 cells), or results in a temporary depression of firing (n = 7/11 cells). D, Differential responses to the puff of ACh. In 7 out of 20 cells, firing was enhanced by ACh, 4 out of 20 cells were not affected, while 9 out of 20 cells were inhibited. E, With DHβE in the bath, ACh did not increase firing of any Hcrt+ neurons tested (n = 11/11 cells). F, With MLA in the bath, a puff of ACh boosted firing in 2 of 16 cells, had no effect in 9 of 16 cells, and decreased firing in 5 of 16 cells. The gray bar indicates the time duration of ACSF application; yellow bars indicate the time duration of ACh application. C−F, All experiments were conducted in the presence of atropine. G, The responses of Hcrt+ neurons to ACh in absence of atropine. In 8 out of 14 cells, firing was enhanced by ACh, in 1 out of 14 cells firing was unaffected, while in 5 out of 14 cells firing was inhibited.