Fig. 4: Acute ketamine and (2 R, 6 R)-HNK induced reduction of SVs recycling is mediated via A1R. | Molecular Psychiatry

Fig. 4: Acute ketamine and (2 R, 6 R)-HNK induced reduction of SVs recycling is mediated via A1R.

From: Ketamine decreases neuronally released glutamate via retrograde stimulation of presynaptic adenosine A1 receptors

Fig. 4

a Representative images and quantification of KCL-evoked syt1-L ab uptake in primary cortical neurons treated with 100 μM ketamine (30 min) without or with pre-treatment with A1R antagonist DPCPX (1 h; 2.5 μM). N = 34 CTRL cells; N = 36 ketamine; N = 36 DPCPX; N = 37 DPCPX/ketamine. Two-way ANOVA with Bonferroni’s Multiple Comparison Test, interaction F (1, 139) = 5.71 p = 0.018; ketamine F = 6.64 p = 0.011; DPCPX F = 75.65 p < 0.0001; ****p < 0.0001. b Representative images and quantification of KCL-evoked syt1-L ab uptake in primary cortical neurons treated with 100 μM (2 R, 6 R)-HNK (30 min) without or with pre-treatment with A1R antagonist DPCPX (1 h; 2.5 μM). N = 31 CTRL cells; N = 33 HNK; N = 35 DPCPX; N = 34 DPCPX/HNK. Two-way ANOVA with Bonferroni’s Multiple Comparison Test, interaction F (1, 129) = 19.93 p < 0.0001; HNK F = 12.03 p < 0.0007; DPCPX F = 62.99 p < 0.0001; **p < 0.01, ****p < 0.0001. c Representative images and quantification of P-S9-Synapsin immunofluorescence in CTRL cells or ketamine treated cells (100 μM; 30 min) without or with pre-treatment with DPCPX (1 h; 2.5 μM). Number of cells: CTRL N = 35; ketamine N = 37; DPCPX N = 36; DPCPX/ketamine N = 37. Two-way ANOVA with Bonferroni’s Multiple Comparison Test, interaction F (1, 141) = 1.88 p = 0.173; ketamine F = 7.17 p = 0.008; DPCPX F = 90.63 p < 0.0001; **p < 0.01, ****p < 0.0001. d KCL-evoked syt1-L ab uptake in primary neurons treated with 100 μM ketamine (30 min) in the absence or presence of an A1R agonist (CPA; 30 min; 2.5 μM). N = 35 CTRL cells; N = 40 ketamine; N = 31 CPA; N = 36 CPA/ketamine. Two-way ANOVA with Bonferroni’s Multiple Comparison Test, interaction F (1, 138) = 1.88 p < 0.0001; ketamine F = 0.25 p = 0.61; CPA F = 3.03 p = 0.084; ****p < 0.0001. e KCL-evoked syt1-L ab uptake in primary neurons treated with 100 μM (2 R, 6 R)-HNK (30 min) in the absence or presence of an A1R agonist (CPA; 30 min; 2.5 μM). N = 27 CTRL cells; N = 31HNK; N = 29 CPA; N = 33 CPA/HNK. Two-way ANOVA with Bonferroni’s Multiple Comparison Test, interaction F (1, 116) = 66.83 p < 0.0001; HNK F = 0.06 p = 0.81; CPA F = 1.42 p = 0.24; ****p < 0.0001. f Efficiency of A1R knockdown in primary cortical neurons assessed by Dansyl-NECA fluorescence upon 96 h of transfection using non-targeting (CTRLsiRNA) or Adora1 pool siRNA (A1RsiRNA). Student’s t test; ****p < 0.0001. g, h Absence of A1Rs completely occluded the effect of both ketamine and (2 R, 6 R)-HNK on KCL-evoked syt1-L ab uptake. Ketamine; CTRLsiRNA N = 30; CTRLsiRNA/ketamine N = 27; A1RsiRNA N = 27; A1RsiRNA/ketamine (N = 31). Two-way ANOVA with Bonferroni’s Multiple Comparison Test, interaction F (1, 111) = 6.47 p = 0.012; ketamine F = 21.60 p < 0.0001; A1RsiRNA F = 58.71 p < 0.0001; ***p < 0.001 ****p < 0.0001. (2 R, 6 R)-HNK; CTRLsiRNA N = 27; CTRLsiRNA/HNK N = 30; A1RsiRNA N = 29; A1RsiRNA/HNK N = 34 on KCL-evoked syt1-L ab uptake. Two -way ANOVA with Bonferroni’s Multiple Comparison Test, interaction F (1, 116) = 8.20 p = 0.005; HNK F = 12.24 p = 0.0007; A1RsiRNA F = 78.46 p < 0.0001; ***p < 0.001 ****p < 0.0001. Data originate from 3 independent cell culture experiments. Bars denote intensity values normalized to the mean intensity value in the control group ± SEM per each experiment.

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