Benserazide hcl

Rats were anesthetized with chloral hydrate (400 mg kg⁻¹, i.p.). Thirty minutes before surgery, rats received an i.p. injection of desipramine hydrochloride (25 mg kg⁻¹; Sigma) in order to prevent noradrenergic terminals from taking up 6-OHDA65 (link). To selectively deplete meso-cortical dopamine innervation to the M1, 8 μg of 6-OHDA (Sigma, St Louis, MO, USA) dissolved in 2 μl of sterile 0.9% saline and 0.02% ascorbic acid (or 2 μl of 0.9% saline for sham injection) was injected into bilateral M1 forelimb territory at a rate of 1 μl min⁻¹ respectively. The micro-syringe for injection stayed in the brain for 15 min in order to prevent backflow of solution. For levodopa (L-DOPA)-treated group, 6-OHDA lesioned or sham-operated animals received intraperitoneal injection of L-DOPA (Sigma) 30 min before behaviour training. L-DOPA was given at a dose of 15 mg kg⁻¹ dissolved in vehicle solution (saline with 0.1% ascorbic acid), combined with the peripheral decarboxylase inhibitor benserazide-HCl (Sigma) at a dose of 15 mg kg⁻¹.

To create the 6-OHDA lesion, rats were anesthetized with sodium pentobarbital (60 mg/kg) and then immobilized in a stereotaxic frame to target the unilateral SNpc (coordinates AP-5.2, ML-2.1, DV-8.0) relative to bregma. A single 6-OHDA (5 μg/μL, diluted in normal saline containing 0.02% ascorbic acid; Sigma) solution was injected over 3 min with an infusion rate of 1 μL/min, followed by 3 min of equilibrium before retracting the needle. All rats were allowed to a 21-day recovery period before experiment start. Then, intraperitoneal injection of L-DOPA [L-DOPA methyl ester HCl (5 mg/kg) combined with benserazide HCl (2.5 mg/kg) diluted in 0.9% saline; Sigma] and intragastrical administration of RES (20 mg/kg, diluted in 0.1% carboxymethylcellulose sodium aqueous solution; Sigma) were performed daily.

Starting after a 4-week recovery period, animals with a 6-OHDA lesion that met the inclusion criterion of three or fewer forelimb adjusting steps received drug treatments on five consecutive days/week (Mon–Fri), for two or three weeks. In week 1, all rats received a daily vehicle injection (10% Cremophor EL in 0.9% saline, 2 mL/kg, i.p.; Sigma-Aldrich), followed 30 min later by the L-DOPA (LD) injection (5 mg/kg, i.p., 2 mL/kg; Alfa Aesar, Tewksbury, MA, USA; coadministered with 12.5 mg/kg benserazide HCl; Sigma-Aldrich). In week 2, one cohort received the same treatment of vehicle + L-DOPA as in week 1 (6/Veh/LD; n = 8), and a second cohort received a treatment of vilazodone HCl (VIL) (10 mg/kg, i.p.; Cayman Chemical, Ann Arbor, MI, USA; in 10% Cremophor EL), followed 30 min later by L-DOPA (6/VIL/LD; n = 14).
To assess a potential role for 5-HTr_1A in mediating the actions of vilazodone, a third cohort of 6-OHDA-infused rats received vehicle + L-DOPA (6/Veh/LD) in week 1, vilazodone + L-DOPA (6/VIL/LD) in week 2, and in week 3, they received the selective 5-HTr_1A antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl-cyclo- hexanecarboxamide, 2Z-butenedioate (WAY-100635 (WAY); 0.5 mg/kg, i.p.; Cayman Chemical) 5 min prior to the vilazodone treatment (6/W/VIL/LD; n = 11), in a within-subject design.

Rats were randomly assigned to different treatments. The 6-OHDA-infused rats with stepping deficits were treated with either L-DOPA (LD) (5 mg/kg, i.p., 2 mL/kg; Alfa Aesar, Tewksbury, MA, USA; plus 12.5 mg/kg benserazide HCl, Sigma-Aldrich) or vehicle, 30 min after receiving an injection of vilazodone HCl (VIL) (10 mg/kg, i.p., 2 mL/kg [52 (link)]; Cayman Chemical, Ann Arbor, MI, USA; in 10% Cremophor EL in saline, Sigma-Aldrich) or vehicle (Cremophor) (groups 6-OHDA/VIL/LD, n = 8; 6-OHDA/Veh/LD, n = 6; 6-OHDA/VIL/Veh, n = 8; 6-OHDA/Veh/Veh, n = 8). The sham lesion group received repeated injections of vehicle (Sham/Veh/Veh; n = 8). Animals received these drug treatments once daily on 5 days (Mon–Fri) for two weeks. In week 3, rats were treated on 3 days. Following the last injection, the rat was placed in an open-field apparatus (43 × 43 cm), and turning behavior was recorded with a video camera for 40 min. Rats were killed 1 h after the final treatment.

L-dopa (Sigma Aldrich: 12 mg/kg s.c. on alternate days) was given in combination with benserazide HCL (Sigma Aldrich 12 mg/kg). Administration commenced 5 weeks prior to cell transplantation and continued for 12 weeks post transplantation. Exendin-4 (Tocris Bioscience, Bristol, UK) was administered intra-peritoneally i.p. at a dose of 0.5 µg/kg twice daily [24 (link)] while liraglutide (Novo Nordisk, Victoza^®, Bagsværd, Denmark) was given once daily at dose of 100 µg/kg [76 (link)], doses were consistent with previous studies showing neuroprotection. Both drugs were started one day prior to cell transplantation and continued for 12 weeks. In the lesion only group of set 1, the Exendin-4 was started 3 weeks post lesion and continued for 16 weeks.

In order to test the effects of a single injection of L-DOPA on motor-skilled reaching and grasping abilities, the staircase test was performed on two consecutive days (16 weeks post-grafting). L-DOPA methyl ester (10 mg/kg, Sigma-Aldrich, Saint Louis, MO, USA) and the peripheral DOPA decarboxylase inhibitor benserazide-HCl (10 mg/kg, Sigma-Aldrich) were freshly dissolved in 0.9% saline immediately prior to use. On the first day, the mice were tested on the staircase for 10 min in order to measure motor performances 16 weeks after transplantation. On the second day, the mice were injected with drugs (0.1 mL/10 g body weight, i.p.) 30 min prior to placing them on the staircase for a 10 min session in order to cover the period of maximal drug effect. During the analysis of the skilled reaching abilities, one lesioned and two intranigral-grafted mice were excluded from the count because they did not demonstrate entry into the corridor of the device.