C5776

Noradrenaline-bitartrate(10 nM - 100 μM; I9278, Sigma), the α₁-AR antagonist prazosine (5 µM; P7791, Sigma), the α₂-AR antagonist yohimbine (5 µM; Y3125, Sigma), the α_2A-AR antagonist BRL 44408 (10 μM, C5776, Sigma), the α_2B-AR antagonist ARC 239, the α_1A-AR antagonist WB 4101, the α_1C, D-AR antagonist CEC (1 nM – 1 µM, Q102, Sigma) and the α_1A-AR agonist A61603 (100 nM; A5861, Sigma) were added to the normal aCSF.
To reduce glutamatergic and GABAergic synaptic input to the recorded neurons, 10 µM CNQX (6-cyano-7-nitroquinoxaline-2,3-dione, C127, Sigma-Aldrich), 50 µM DL-AP5 (DL-2-amino-5-phosphonopentanoic acid, BN0086, Biotrend), and 100 µM PTX (picrotoxin, P1675, Sigma Aldrich) was added to the extracellular saline.

Neurotransmitter release was evoked by either bath application of a high concentration of potassium or electrical stimulation. Potassium-evoked release was performed by perfusing tissue with high K⁺ (100 mM, replacing an equimolar amount of NaCl) aCSF for 1 min once a stable 30 s baseline recording had been obtained. Electrically-evoked release was performed with a bipolar stimulating electrode placed close to the carbon fiber recording electrode within the dorsal telencephalon. Current pulses were generated by the acquisition software and applied via a stimulus isolator (Iso-Flex; AMP Instruments). The tissue was allowed to recover for a minimum of either 5 min (electrical stimulation) or 30 min (for high K⁺ stimulation) between stimulations. In some experiments we added drugs targeting neurotransmitter systems to the aCSF: 10 μM GBR 12909 (selective DA reuptake inhibitor; Sigma Aldrich D052); or 10 μM cocaine hydrochloride (DA, NA and 5-HT reuptake inhibitor; Sigma Aldrich C5776). GBR 12909 was first dissolved in DMSO with gentle warming before being directly added to the aCSF. Cocaine was made into a stock solution in water before being added to aCSF. Drugs were not perfused onto tissue until at least 3 stable baseline recordings had been obtained.

Cocaine sensitisation experiments were carried out over five successive days (starting at 10am), treating four animals simultaneously. Each day, mice were weighed and then allowed to habituate for 20 min in the open field. After 20 min, they were each given IP injections of cocaine (Sigma–Aldrich, C5776) at 20 mg/kg or 15 mg/kg, in a solution of 4 mg/ml. Their horizontal locomotor activity was recorded in the open field for 60 min with EthoVision XT. Two LPD-exposed mice administered with 20 mg/kg cocaine exhibiting outlier locomotor behaviour were excluded from the analysis.