3. M ATERIALS AND METHODS
3.2. Drugs and solutions
Prior to the experiments the three drugs under investigation were prepared: Alprazolam, α5IA-II and NS.A as well as the drugs used to model sensorimotor gating deficits:
methylphenidate, PCP and amphetamine.
3.2.1. Alprazolam
In the fear conditioning experiment (A1), the extended-tone fear conditioning experiment (B1) and the pre-pulse inhibition with PCP-deficit experiment (F1) the drug alprazolam was administered i.p. in dose volume of 2 ml/kg and at the different doses 0.4, 1.33 and 4 mg/kg. Consequently the following alprazolam concentrations were made: 0.2, 0.67 and 2 mg/(ml cremophor 5 %).
Reagents
Alprazolam, Cambrex Profarmaco, Italy
Cremophor RH40 (Polyoxyl 40 hydrogenated caster oil), BASF, Germany
Procedure
Alprazolam was dissolved in cremophor 5 %. In order to dissolve the drug the solution was standing on a 50 oC hot plate and stirred. To destroy any lumps the solution was thereafter treated with ultrasound (Sonifier) for 14 minutes and then stirred before the solution were diluted in cremophor 5 % to the different concentrations. The solutions were protected against light throughout the animal experiments by tinfoil.
3.2.2. α5IA-II
In the fear conditioning experiment (A2), the extended-tone fear conditioning experiment (B2) and the pre-pulse inhibition with PCP deficit experiment (F2) the drug α5IA-II was administered i.p. in dose volume of 2 ml/kg and at the different doses 1, 3 and 10 mg/kg. Consequently the following α5IA-II concentrations were made: 0.5, 1.5 and 5 mg/(ml tween 5 %). In the trace fear conditioning experiment (C) α5IA-II was administered i.p. in dose volume of 2 ml/kg and at the different doses 1 and 3 mg/kg.
Consequently the following α5IA-II concentrations were made: 0.5 and 1.5 mg/(ml tween 5 %). In the dose-response with PCP deficit experiment (H) α5IA-II was
administered i.p. in dose volume of 2 ml/kg and with the two possible doses: 30 and 10 mg/kg. Consequently the following α5IA-II concentrations were made: 15 and 5 mg/(ml tween 5 %).
Reagents
α5IA-II (C19H14N6O2), Blocona Company, India Tween (C58H114O26), Merck-Schuchardt, Germany
Ringer-klorid SAD (NaCl 0.9 %), Sygehusapotekerne i Danmark, Denmark
Procedure
α5IA-II was dissolved in pure tween. In order to dissolve the drug the solution was placed at a 50 oC hot plate and stirred. Hereafter tween and α5IA-II was diluted in NaCl 0.9 % in order to reach a tween 5 % solution, containing 5 mg/ml α5IA-II (1,5 mg/ml α5IA-II in the trace fear conditioning experiment and 15 mg/ml α5IA-II in the dose-response with PCP deficit experiment). To destroy any lumps the solution was treated with ultrasound (Sonifier) for a quarter of a time and then stirred before the solution was diluted in tween 5 % to the different concentrations. It was not possible to dissolve α5IA-II totally. To maintain the drug in a homologous suspension, the solutions at the concentrations of 5 and 15 mg/ml was constantly stirred and maintained at 50 oC hot plate throughout the animal experiments.
3.2.3. NS.A
In the fear conditioning experiment (A3), the extended-tone fear conditioning experiment (B3), the pulse inhibition with PCP deficit experiment (F3) and the pre-pulse inhibition with amphetamine deficit experiment (G) the drug NS.A was administered i.p. in dose volume of 2 ml/kg and at the different doses 0.3, 1 and 3 mg/kg. Consequently the following NS.A concentrations were made: 0.15, 0.5 and 1.5 mg/(ml tween 5 %).
Reagents
NS.A, Syngene, Blocona, India
Tween (C58H114O26), Merck-Schuchardt, Germany
Ringer-klorid SAD (NaCl 0.9 %), Sygehusapotekerne i Danmark, Denmark
Procedure
NS.A was dissolved in pure tween. In order to dissolve the drug the solution was placed on a 50 oC hot plate and stirred for 5 minutes. Hereafter tween and NS.A was diluted in NaCl 0.9 % in order to reach a tween 5 % solution, containing 3 mg/ml NS.A and stirred 5 minutes before the solution was diluted in tween 5 % to the different concentrations.
3.2.4. Methylphenidate
In the dose-response pre-pulse inhibition experiments (D1 and D2) the drug methylphenidate was administered s.c. in the scruff of the neck in dose volume of 10 ml/kg for mice (D1) and of 1 ml/kg for rats (D2). In both of the experiments methylphenidate was administered at the different doses 5, 10 and 20 mg/kg.
Consequently the following methylphenidate concentrations were made: 0.5, 1 and 2 mg/(ml NaCl 0.9 %) for the D1 experiment and 5, 10 and 20 mg/(ml NaCl 0.9 %) for the D2 experiment.
Reagents
Methylphenidate HCl, Orgamol The Swiss Fine Chemical Company, Switzerland Ringer-klorid SAD (NaCl 0.9 %), Sygehusapotekerne i Danmark, Denmark
Procedure
Methylphenidate was dissolved in NaCl 0.9 % and stirred. Hereafter the solution was diluted further in NaCl 0.9 % to the different concentrations.
3.2.5. PCP
Reagents
Phencyclidine HCl (PCP), NeuroSearch, Denmark
Ringer-klorid SAD (NaCl 0.9 %), Sygehusapotekerne i Danmark, Denmark
Procedure
In the dose-response pre-pulse inhibition experiments (72 hours pre-treatment time in experiment E1, 24 hours treatment time in experiment E2 and 10 minutes pre-treatment time in experiment E3) PCP was administered s.c. in the scruff of the neck in dose volume of 1 ml/kg and at the different doses 1.25, 2.5 and 5 mg/kg. Consequently the following PCP concentrations were made: 1.25, 2.5 and 5 mg/(ml NaCl 0.9 %).
PCP was dissolved in NaCl 0.9 % and stirred. Hereafter the solution was diluted further in NaCl 0.9 % to the different concentrations
Procedure
In the pre-pulse inhibitions with PCP deficit experiments (F1, F2 and F3) and dose-response with PCP deficit experiment (H) PCP was administered s.c. in the scruff of the neck in dose volume 1 ml/kg and at dose 2.5 mg/kg. Consequently the following PCP concentration was made at 2.5 mg/(ml NaCl 0.9 %). PCP was dissolved in NaCl 0.9 % and stirred.
3.2.6. Amphetamine
Reagents
S(+)-Methamphetamine HCl, Research Biochemicals International, USA Ringer-klorid SAD (NaCl 0.9 %), Sygehusapotekerne i Danmark, Denmark
Procedure
In the pre-pulse inhibition experiments with amphetamine deficit (G) amphetamine was administered s.c. in the scruff of the neck in dose volume 1 ml/kg and at dose 4 mg/kg.
Consequently the amphetamine concentration was made at 4 mg /(ml NaCl 0.9 %).
Amphetamine was dissolved in NaCl 0.9 % and stirred.