Temporal dynamics of DA and 5-HT release evoked by cocaine and topographical organization of DR projectors

(A) Schematics of mouse preparation to record DA or 5-HT release in NAc and DS region. (B) Representative histological example of viral expression and fibre implantation in NAc and DS. (C) Schematic of recording timeline following i.p. injection of cocaine 15mg/kg. (D) Left: z-score group data (Mean ± SEM) of NAc (red) and DS (Blue) DA release evoked by cocaine. Right: Quantification of AUC after i.p. injection of cocaine (Two-sided Mann-Whitney test, ***P<0.001). (E) Left: z-score group data (Mean ± SEM) of NAc (red) and DS (Blue) 5-HT release evoked by cocaine. Right: Quantification of AUC after i.p. injection of cocaine (Two-sided Mann-Whitney test, P= 0.1310). (F) Schematic of retrobeads injection site used to map the topographical organization of DR projectors. (G) Representative examples of retrobeads injection site within the VTA, NAC and DS. (H) Proportion of cell (%) from VTA projectors (green), NAc projectors (red) and DS projectors on 4 different coronal planes.

Stimulation of VTA DA neurons increases striatal DA but not 5-HT signals.

(A) Left: Protocol of optogenetic stimulation used to assess DA and 5-HT release. Middle: Parameters of optogenetic stimulation consisted of 30 bursts at 2 Hz. Right: Each burst comprised five light stimulations of 10 ms each at 20 Hz every 500 ms. (B) Schematic of viral strategies used in DAT-Cre animals (n = 7) to simultaneously recorded DA release during optogenetic manipulation of VTA DA neurons. (C) z-score group data (Mean ± SEM) of NAc (red) and DS (blue) DA release evoked by optogenetic stimulation of VTA DA neurons. (D) Quantification of AUC during optogenetic stimulation (Two-sided Mann-Whitney test, ***P<0.001). (E, F) Left: Group average power spectral density plot (PSD, Mean ± SEM) in decibel (dB) of DA ΔF/F signal during stimulation (red for NAc and blue for DS) versus non-stimulation epochs (grey). Right: 2 Hz signal-to-noise ratio quantification (SNR) of biosensor (470) and isosbestic (405) signal during stimulation versus non stimulation period (NAc : Two-way repeated measures ANOVA: Channel effect, F(1,12) = 21.02, ***P<0.001; Stimulation effect, F(1,12) = 27.95, ***P<0.001; Channel vs Stimulation, F(1,12) = 24.64, ***P<0.001; Bonferroni post hoc analysis, ***P<0.001; DS: Two-way repeated measures ANOVA: Channel effect, F(1,12) = 191.7, ***P<0.001; Stimulation effect, F(1,12) = 19.21, ***P<0.001; Channel vs Stimulation, F(1,12) = 12.23, **P<0.01; Bonferroni post hoc analysis, ***P<0.001). (G) Schematic of viral strategies used in DAT-Cre animals (n = 10) to simultaneously recorded 5-HT release during optogenetic manipulation of VTA DA neurons. (H) z-score group data (Mean ± SEM) of NAc (red) and DS (Blue) 5-HT release evoked by optogenetic stimulation of VTA DA neurons. (I) Quantification of AUC during optogenetic stimulation (Two-sided Mann-Whitney test, P>0.05) (J,K) Left: Group average power spectral density plot (PSD, Mean ± SEM) in decibel (dB) of 5-HT ΔF/F signal during stimulation (red for NAc and blue for DS) versus non-stimulation epochs (grey). Right: 2 Hz signal-to-noise ratio quantification (SNR) of biosensor (470) and isosbestic (405) signal during stimulation versus non stimulation period (NAc: Two-way repeated measures ANOVA: Channel effect, F(1,18) = 31.57, ***P<0.001; Stimulation effect, F(1,18) = 6.88, **P<0.01; Channel vs Stimulation, F(1,18) = 8.231, **P<0.01; Bonferroni post hoc analysis, **P<0.01; DS: Two-way repeated measures ANOVA: Channel effect, F(1,18) = 14.43, **P<0.01; Stimulation effect, F(1,18) = 0.25, P>0.05; Channel vs Stimulation, F(1,18) = 0.050, P>0.05).

Stimulation of DRN 5-HT system increases 5-HT and synchronized DA signals in the striatum.

(A) Left: Protocol of optogenetic stimulation used to assess DA and 5-HT release. Middle: Parameters of optogenetic stimulation realized consisting of 30 bursts at 2 Hz. Right: Each burst comprised of 5 light stimulations of 10 ms each at 20 Hz every 500 ms. (B) Schematic of viral strategies used in SERT-Cre animals (n = 12) to simultaneously recorded 5-HT release during optogenetic manipulation of DRN serotonergic neurons. (C) z-score group data (Mean ± SEM) of NAc (red) and DS (Blue) 5-HT release evoked by optogenetic stimulation of DRN serotonergic neurons. (D) Quantification of AUC during optogenetic stimulation (Two-sided Mann-Whitney test, P = 0.0684). (E, F) Left: Group average power spectral density plot (PSD, Mean ± SEM) in decibel (dB) of 5-HT ΔF/F signal during stimulation (red for NAc and blue for DS) versus non-stimulation epochs (grey). Right: 2 Hz signal-to-noise ratio quantification (SNR) of biosensor (470) and isosbestic (405) signal during stimulation versus non stimulation period (NAc : Two-way repeated measures ANOVA: Channel effect, F(1,22) = 0.013, P>0.05; Stimulation effect, F(1,22) = 8.501, **P<0.01; Channel vs Stimulation, F(1,22) = 14.06, **P<0.01; Bonferroni post hoc analysis, ***P<0.001; DS: Two-way repeated measures ANOVA: Channel effect, F(1,22) = 16.70, ***P<0.001; Stimulation effect, F(1,22) = 12.98, **P<0.01; Channel vs Stimulation, F(1,22) = 19.34, ***P<0.01; Bonferroni post hoc analysis, ***P<0.001). (G) Schematic of viral strategies used in SERT-Cre animals (n = 10) to simultaneously record DA release during optogenetic manipulation of DRN serotonergic neurons. (H) z-score group data (Mean ± SEM) of NAc (red) and DS (Blue) DA release evoked by optogenetic stimulation of DRN serotonergic neurons. (I) Quantification of AUC during optogenetic stimulation (Two-sided Mann-Whitney test, P>0.05) (J,K) Left: Group average power spectral density plot (PSD, Mean ± SEM) in decibel (dB) of DA ΔF/F signal during stimulation (red for NAc and blue for DS) versus non-stimulation epochs (grey). Right: 2 Hz signal-to-noise ratio quantification (SNR) of biosensor (470) and isosbestic (405) signal during stimulation versus non stimulation period (NAc : Two-way repeated measures ANOVA: Channel effect, F(1,18) = 6.53, *P<0.05; Stimulation effect, F(1,18) = 7.758, *P<0.05; Channel vs Stimulation, F(1,18) = 12.07, **P<0.01; Bonferroni post hoc analysis, ***P<0.001; DS: Two-way repeated measures ANOVA: Channel effect, F(1,18) = 60.76, ***P<0.001; Stimulation effect, F(1,18) = 15.27, ***P<0.001; Channel vs Stimulation, F(1,18) = 13.57, **P<0.01; Bonferroni post hoc analysis, ***P<0.001).