Nisoxetine hydrochloride

Selective depletions of 5-HT in the OFC and amygdala were made using 5,7-DHT (9.92 mM; Fluka BioChemika, Sigma, Poole, United Kingdom) in saline/0.1% l-ascorbic acid. To protect noradrenaline (NA) and dopamine (DA) innervations, the solution also contained the NA uptake blocker, nisoxetine hydrochloride (50 mM; Sigma), and DA uptake blocker, GBR-12909 dihydrochloride (2.0 mM; Sigma). Injections were made at a rate of 0.05 μL/20 s through a glass cannula attached to a 2-μL Hamilton syringe (Precision Sampling Co., Baton Rouge, LA, USA). The coordinates and volumes of toxin administered are shown in Table 2. Sham-operated controls (2 amygdala and 2 OFC) underwent identical surgical procedures with the toxin omitted from the infusate. Details of the surgical procedure are described in Supplementary Methods.
Table 2

Stereotaxic coordinates for 5-HT depletions

Depletion area	Coordinates (mm)			Volume injected (μL)
	AP	LM	V
OFC	16.75	±2.5	0.7a	0.4
		±3.5	0.7a	0.4
	17.75	±2.0	0.7a	0.4
		±3.0	0.7a	0.4
	18.50	±2.0	0.7a	0.6
AMYGDALA	9.30	±5.6	4.0	0.5
			5.0	0.5

Coordinates are based on the interaural plane except for ^awhere the vertical was 0.7 mm above the base of the brain. AP, anterior–posterior; LM, lateral-medial; V, ventral.

³H-serotonin (80 Ci/mmol), ³H-norepinephrine (20 Ci/mmol), and ³H-dopamine (60 Ci/mmol) were purchased from M.G.P. (Zlín, Czech Republic). Forskolin was obtained from Scintila, s.r.o. (Jihlava, CZ). Paroxetine hydrochloride, citalopram hydrobromide, sertraline hydrochloride, fluoxetine hydrochloride, fluvoxamine maleate, venlafaxine hydrochloride, phenelzine sulfate salt, entacapone, GBR 12935 dihydrochloride, nisoxetine hydrochloride, hydrocortisone, and decynium-22 were purchased from Sigma-Aldrich (St. Louis, USA). Pierce™ BCA Protein Assay Kit was purchased from Thermo Fisher Scientific (Waltham, United States). All other chemicals were of analytical grade.

The immunotoxin aDBH was purchased from Advanced Targeting System (San Diego, CA, USA); nisoxetine hydrochloride and clonidine hydrochloride were from Tocris (Bristol, UK), quinpirole hydrochloride and raclopride tartrate were from Sigma-Aldrich. 2. A single, full effective dose of each drug was chosen, according to literature and our previous experience, to describe a qualitative rather than a quantitative effect (Nisoxetine: Chen and Reith, 1994 (link); Rowley et al., 2000 (link); Devoto et al., 2019 (link). Quinpirole: Devoto et al., 2001 (link); Marinelli et al., 2006 (link). Clonidine: Devoto et al., 2001 (link); Gobbi et al., 2001 (link). Raclopride: Wong et al., 2018 (link); Devoto et al., 2019 (link)). Drugs were dissolved in sterile distilled water, and IP or subcutaneously (SC) administered in a volume of 1 ml/kg body weight. Sub-groups of animals were treated by infusion through microdialysis probe with 20 µM clonidine in aCSF into the LC, or with 10 µM nisoxetine in aCSF into the mPFC.