Figure 2
From: Activation of basal forebrain purinergic P2 receptors promotes wakefulness in mice
The P2R agonist, ATP-γ-S, excites basal forebrain (BF) cortically-projecting wake-active cholinergic and GABAergic neurons and increases local glutamate release. (a–c) Identification and responses of BF cholinergic (Ach+), and two types of large-sized GABAergic (large Ih GABA+ and small Ih GABA+) to the P2R agonist ATP-γ-S in vitro. Left: Black-and-white fluorescent and infra-red-differential interference contrast (IR-DIC) images of the recorded neurons in slices prepared from GAD67-GFP knock-in mice. In these mice, GFP is selectively located in GABAergic neurons, with cholinergic neurons being GFP negative. Scale bar: 25 µm. Middle: Characteristic voltage responses of cholinergic and cortically-projecting GABAergic neurons in response to hyperpolarizing and depolarizing current injection. Cholinergic neurons exhibit a delayed return to baseline following hyperpolarizing current pulses due to an A-type potassium current (asterisk, A) whereas GABAergic neurons demonstrate a depolarizing sag during hyperpolarizing current steps due to activation of the hyperpolarization-activated-inward current (Ih, black arrow, b and c). We previously characterized these neurons in detail27,31. Right: All three types of neurons were depolarized by ATP-γ-S in the presence of tetrodotoxin (TTX). (d) Summary of BF neuronal voltage responses to 100 µM ATP-γ-S in control (500 nM TTX, n = 17) and marked attenuation of the response in the presence of a P2XR antagonist (500 nM TTX + 30 µM PPADS, n = 16), or in the presence of a cocktail of glutamate & GABAA receptor antagonists (500 nM TTX + 20 µM CNQX + 50 µM D-AP5 + 10 µM GABAzine. n = 8) tested using a one-way ANOVA with Bonferroni post-hoc test. ***p < 0.001. (e and f) In vivo hourly measurements of extracellular glutamate concentration and average glutamate concentrations with ACSF/1 mM ATP-γ-S infused during the 3 h recovery period after sleep deprivation, showing that ATP-γ-S increases extracellular glutamate concentrations in the BF. RS1/2/3: recovery sleep hour 1/2/3. n = 6. Paired-t-test. *p < 0.05.
