Fig. 2: Activation of A_2AR in the lateral septum (LS) augments spiking frequency of LS-A_2AR⁺ neurons with suppression of surrounding neurons.

a Biocytin (red) in the intracellular solution diffused into the cells on brain slices from A_2AR-Cre mice injected with rAAV2/9-hSyn-DIO-EYFP into LS during the in vitro electrophysiological recordings. The recorded A_2AR⁺ neurons (stained yellow) displayed long and numerous branches observed using confocal microscopy combined with 3D surface volume rendering. Scale bars: 1000/200/20 μm. LV, lateral ventricle; MS, medial septum; NAc, Nucleus Accumbens core; ST, striatum. b Representative image from a 300 µm slice after in vitro electrophysiology recording showing A_2AR⁺ neurons (green) co-immunostained with anti-GAD65 + 67 antibody (red) in the LS. Nuclei are stained with DAPI in blue. Scale bar: 100 μm. c Single cell RT-PCR analysis with total mRNA isolated from recorded cells showed that all EYFP-positive cells were GABAergic neurons. M, 1000 bp marker; A_2AR⁺, cell cytoplasm of LS-A_2AR⁺ neuron; H₂O, sterile double distilled water; solu, pipette solution. d Representative trace and statistical graph showing that the activation of A_2AR by CGS21680 (30 nM) increased the firing frequency of EYFP-positive neurons (n = 16 cells from 8 mice, Wilcoxon test, p = 0.00003, W = 136.0). e Representative trace and statistical graph showing that the effect of CGS21680 on EYFP-positive neurons was reversible after washout (Ctrl: CGS21680, n = 3 cells from 3 mice, Paired t test, p = 0.0309, t(2)=6.663; CGS21680: washout, n = 3 cells, 3 mice, Paired t test, p = 0.0238, t(2) = 6.364). f Left: Representative voltage-clamp recording showing the alterations of spontaneous inhibitory post-synaptic currents (sIPSC, recorded upon blockade of glutamatergic activity with 50 µM DL-AP₅ and 20 µM DNQX) in LS non-A_2AR⁺ neurons surrounding A_2AR⁺ neurons before and after the application of CGS21680 (30 nM). Right: Statistical graph showing that CGS21680 increased sIPSCs frequency (n = 6 cells from 4 mice, Paired Wilcoxon test, p = 0.0313, W = 21), without altering their amplitude (n = 6 cells from 4 mice, Paired t test, p = 0.6927, W = 21). DL-AP₅, DL-2-amino-5-phosphonopentanoic acid; DNQX, 6,7-dinitroquinoxaline-2,3-dione. *p < 0.05, ****p < 0.0001; n.s., no significant difference. Source data are provided as a Source Data file.