Electronic Supplementary Materials s2

Supplementary Information

Blockade of 5-HT1A receptors by (±)-pindolol potentiates cortical 5-HT outflow, but not antidepressant-like activity of paroxetine: microdialysis and behavioral approaches in 5-HT1A receptor knockout mice

Jean-Philippe Guilloux 1, Denis J.P. David1, Bruno P. Guiard1, Franck Chenu2, Christelle Repérant1, Miklos Toth3, Michel Bourin2, Alain M. Gardier1,*

1 Laboratoire de Neuropharmacologie EA 3544, Faculté de Pharmacie, Université Paris-Sud, 92296 Châtenay-Malabry cedex, France.

2 Department of Pharmacology, Weill Medical College of Cornell University, New York, New York 10021, USA.

3 Laboratoire Neurobiologie de l'anxiété et de la dépression EA3256, Faculté de Médecine, Université de Nantes, 44035 Nantes, France.

* Correspondence: Alain M. GARDIER, Laboratoire de Neuropharmacologie EA 3544, Tour D1, 2ème étage, Faculté de Pharmacie, Université Paris-Sud, 5 rue J-B. Clément, 92296 Châtenay-Malabry cedex, France. Tel: +33.1.46.83.54.16. Fax: +33.1.46.83.53.55 e-mail: .

Figure 8 : Effects of local intra-raphé infusion of (±)-pindolol on cortical [5-HT]ext levels in 5-HT1A +/+ and 5-HT1A -/- mice.

In the frontal cortex, (a) in 5-HT1A +/+ and 5-HT1A -/-: Data are means ± SEM of cortical [5-HT]ext expressed as percentages of basal values. WT 5-HT1A +/+ (white items) and KO 5-HT1A -/- (black items) mice received either aCSF + (±)-pindolol (100 µM) (£,¢) or aCSF alone ( , ) perfused during the 0-60 min period via the microdialysis probe implanted in the Dorsal Raphé Nucleus. (b) Data are area under the curve (AUC; mean ± SEM) values calculated for the amount of 5-HT outflow measured during the 0-180 min post-treatment period in the frontal cortex of 5-HT1A +/+ (empty bars) and 5-HT1A -/- (full bars) mice, and expressed as percentages of baseline (n=5-6 mice per group).

In the Dorsal Raphé Nucleus, (c) in 5-HT1A +/+ and 5-HT1A -/-: Data are means ± SEM of [5-HT]ext in the dorsal raphé nucleus expressed as percentages of basal values. 5-HT1A +/+ (white items) and 5-HT1A -/- (black items) mice received either aCSF + (±)-pindolol (100 µM) (£,¢) or aCSF alone ( , ) perfused during the 0-60 min period via the microdialysis probe implanted in the Dorsal Raphé Nucleus. (d) Data are area under the curve (AUC; mean ± SEM) values calculated for the amount of 5-HT outflow measured during the 0-180 min post-treatment period in the dorsal raphé nucleus of 5-HT1A +/+ (empty bars) and 5-HT1A -/- (full bars) mice, and expressed as percentages of baseline (n=5-6 mice per group).

A two-way ANOVA (treatment×genotype) on AUC values revealed no significant effects of genotype factor [F(1,18)=0.34; P=0.57] or treatment factor [F(1,18)=1.76; P=0.20].

When perfused locally in the Dorsal Raphé Nucleus, (±)-pindolol (100 µM) had an effect neither on local (i.e., the dorsal raphé nucleus) nor on cortical dialysate 5-HT levels in both 5-HT1A +/+ and 5-HT1A -/- mice.

Figure 9 : Effect of the systemic administration of (±)-pindolol (s.c.) or WAY 100635 - combined with paroxetine (4 mg/kg, i.p.) on cortical [5-HT]ext in wild type and 5-HT1A knockout mice.

Data are expressed as means ± SEM of cortical [5-HT]ext (percentages of basal values). (a) 5-HT1A +/+ (£) and 5-HT1A -/- mice (¢) received paroxetine 4 mg/kg, i.p. (left arrow), then one hour later, (±)-pindolol (10 mg/kg; s.c.) (right arrow). Time course of the effects of paroxetine 4 mg/kg with (±)-pindolol at dosages of 1 and 5 mg/kg in 5-HT1A +/+ and 5-HT1A -/- mice are not shown. (b) 5-HT1A +/+ (£) and 5-HT1A -/- mice (¢) received paroxetine 4 mg/kg (left arrow), then one hour later, the 5-HT1A receptor antagonist WAY-100635 (0.5 mg/kg; s.c.) (right arrow).

Figure 10 : Effects of local intra-raphé infusion of either (±)-pindolol or WAY-100635 on [5-HT]ext levels in the frontal cortex and Dorsal Raphé Nucleus following systemic paroxetine administration in 5-HT1A +/+and 5-HT1A -/- mice.

In the frontal cortex, (a) in 5-HT1A +/+, (b) in 5-HT1A -/- : Data are expressed as means ± SEM of cortical [5-HT]ext ( percentages of basal values). Mice received either the vehicle ( , ) or paroxetine 4 mg/kg, i.p. (¯,¿) (left arrow), then one hour later, (±)-pindolol (100 µM, r,p) or WAY-100635 (100 µM, £,¢) perfused during the 60-120 min period via the microdialysis probe implanted in the Dorsal Raphé Nucleus.

In the Dorsal Raphé Nucleus, (c) in 5-HT1A +/+, (d) in 5-HT1A -/- mice: Data are means ± SEM of [5-HT]ext in the dorsal ( , )or paroxetine 4 mg/kg, i.p. (¯,¿) (left arrow), then one hour later, (±)-pindolol (100 µM, r,p) or WAY-100635 (100 µM, £,¢) were perfused locally during the 60-120 min period via the microdialysis probe implanted in the Dorsal Raphé Nucleus.

Figure 11: Effects of the administration of (±)-pindolol or WAY-100635 on 5-HT1A +/+ and -/- mice’behaviour in the forced swimming test.

Results are means ± SEM of the immobility time (in seconds). 5-HT1A +/+ and -/- received either the vehicle, (±)-pindolol (10 mg/kg, s.c.) or WAY-100635 (1 mg/kg, s.c.). Statistical analyses were carried out using a two-way ANOVA followed by Fisher PLSD: no significant effect of either pindolol or WAY-100635 was observed.