Fig. 3: Reduced NMDA-receptor activity does not impair N2O-induced L5 activation in vivo. | Nature Communications

Fig. 3: Reduced NMDA-receptor activity does not impair N2O-induced L5 activation in vivo.

From: Nitrous oxide activates layer 5 prefrontal neurons via SK2 channel inhibition for antidepressant effect

Fig. 3: Reduced NMDA-receptor activity does not impair N2O-induced L5 activation in vivo.The alt text for this image may have been generated using AI.

a Top, timeline of L5 imaging (teal box) before, following local (L-) drug application (red cloud), and during N2O exposure (blue shaded region). Middle, cartoon and representative coronal section of Cg1 region after pressure application of MK801 mixed with Rhodamine 6 G. Scale bar, 500 μm. Created in BioRender. Cichon, J. (2025) https://BioRender.com/i08h748. Bottom, Representative two-photon images of peak GCaMP6 signal from Cg1 L5 neurons under room air conditions, L-MK801, N2O. b Representative GCaMP6 traces of L5 neurons under room air conditions, L-MK801 (top—high concentration/100 μM; n = 103 from 3 mice; middle—low concentration/10 μM; n = 202 from 4 mice), or L-MK801 + CNQX (lower traces; each drug at 100 μM mixed; n = 96 from 3 mice) followed by N2O exposure. c Representative GCaMP6 traces of L5 neurons from mice infected with siRNA specific to GRIN1 (top; n = 165 from 4 mice) and scramble-siRNA (bottom; n = 116 from 4 mice) under room air, N2O, following N2O. d Summary of individual L5 responses (from b, c) normalized to its baseline captured under room air conditions (Kruskal-Wallis (513): P = 4.3 × 10−103 followed by Dunn’s multiple comparisons, L-MK801 100 μM, P = 1.5 × 10−11; L-MK801 10 μM, P = 1.8 × 10−22; L-APV, P = 1.3 x 10−6; L-MK801 + CNQX, P = 2.0 x 10−23; GRIN1-siRNA, P = 3.8 x 10−18; Scramble siRNA, P = 5.0 x 10−15). Cells/animals per condition listed in (b, c). e L5 imaging of CORT mice exposed different drug sequences. Top, room air, L-ketamine (100 μM; n = 243 from 4 mice), followed by N2O. Middle 2 traces, room air, N2O exposure, followed by systemic ketamine (10 mg/kg i.p. injection; n = 88 neurons from 3 mice) and its reverse order (ketamine then N2O; n = 140 neurons from 4 mice). Bottom, room air → N2O → systemic ketamine → N2O re-exposure (n = 104 neurons from 3 mice). f Summary of L5 responses from e (Kruskal-Wallis (135): P = 2.5 x 10−25 followed by Dunn’s multiple comparisons, L-ketamine → N2O, P = 4.6 × 10−4; N2O → ketamine, P = 5.1 x 10−13; ketamine → N2O, P > 0.99; N2O → ketamine (comparison underlined) → N2O, P = 0.005; N2O → ketamine → N2O, P > 0.99). Cells/animals per condition listed in (e). g N2O-induced activity was negatively correlated with ketamine-induced activity in L5 neurons (two-sided Pearson correlation: N2O → ketamine, r = -0.83, P = 1.8 × 10−49; ketamine → N2O, r = -0.24, P = 0.004). Error bars show s.e.m.

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