Rats were anesthetized with an intraperitoneal injection of chloral hydrate (300 mg kg−1, i.p.), and secured in a stereotaxic apparatus (SN-2N, Narishige Group, Tokyo, Japan). One stainless steel 23-gauge cannula with two tubes was implanted into the NTS. The stereotaxic coordinates of the NTS were 13.9 mm caudal to bregma, 0.5 mm lateral to the midline, and 7.8 mm below the surface of the skull27 . The cannula was fixed with three screws and dental acrylic resin, and the obstructor (30 gauge) was inserted. A prophylactic dose of penicillin was administered intramuscularly at the end of surgery. All rats underwent a recovery period of 5 to 7 days before the start of the experiments. The experiments were done in accordance with the Principles of Laboratory Animal Care (NIH Publication No. 85-23) and were approved by the Institutional Animal Care Committee of Xi’an Jiaotong University.
Sn 2n
Manufactured by Narishige
Sourced in Japan
The SN-2N is a stereotaxic instrument designed for precise and controlled animal experimentation. It features a sturdy frame, adjustable head holder, and precise manipulator arms to facilitate accurate positioning of experimental subjects. The SN-2N enables researchers to conduct a variety of neuroscience and behavioral studies while maintaining a high level of experimental control and reproducibility.
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7 protocols using sn 2n
1
Cannulation of the Nucleus Tractus Solitarius in Rats
2
Unilateral 6-OHDA Lesion in Rat Model
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The unilateral 6-OHDA lesions of the MFB in rats were carried out as previously described (Carvalho et al., 2013 (link)). Rats were anesthetized with sodium pentobarbital (40 mg/kg, i.p.) followed by the injection of desipramine (25 mg/kg, i.p.) to protect noradrenergic neurons. Then the rats were placed on a stereotaxic frame (SN-2N; Narishige, Tokyo, Japan) and 6-OHDA (12 μg/4 μl) was injected into the left MFB (AP –4.4 mm, ML –1.2 mm, DV –7.8 mm; Paxinos and Watson, 1998 ). After the injection, the glass pipette was left in place for 5 min to allow diffusion. Two weeks later, rats were subcutaneously injected with apomorphine (0.05 mg/kg, s.c.) to verify the effectiveness of 6-OHDA lesion (Wang et al., 2009 (link)) and those showing more than 20 contralateral turns per 5 min were selected for further experiments. All rats used in the following experiments turned consistently toward right of >30 turns per 5 min.
3
Unilateral Parkinson's Rat Model
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The method to cause unilateral SNc damage was the same as described previously (Fan et al., 2011). In brief, the rats were anesthetized with 4% chloral hydrate (400 mg/kg, intraperitoneally) and pretreated with desipramine (Sigma-Aldrich, St. Louis, MO, USA), dissolved in normal saline, 25 mg/kg, intraperitoneally, to protect norepinephrine neurons from 6-OHDA toxicity. Subsequently, the rats were fixed on a stereotaxic frame (SN-2N, Narishige, Tokyo, Japan). 6-OHDA (Sigma-Aldrich; dissolved in saline containing 0.01% ascorbic acid, 8 µg/4 µL) was injected into the right SNc (anteroposterior – 5.0–5.2 mm, lateral 1.9–2.0 mm, dorsal 7.1–7.2 mm; Paxinos and Watson, 2005). The injection was made at a rate of 0.5 µL/min using a glass micropipette connected to a 10 µL microsyringe. At 1 week after the operation, the rats were injected subcutaneously with apomorphine 0.05 mg/kg body weight (the apomorphine, Sigma-Aldrich, was dissolved in saline containing 0.01% ascorbic acid). Only rats that rotated to the unlesioned side more than 20 times every 5 minutes were selected for the subsequent experiment.
4
Ibotenic Acid Lesions in Rat Mediodorsal Thalamus
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The rats were anesthetized with 4% chloral hydrate (400 mg/kg, intraperitoneally) and fixed on the stereotaxic frame (SN-2N, Narishige, Tokyo, Japan). The three-dimensional coordinates of MD were determined: anteroposterior −2.8 mm, lateral 0.5−0.6 mm, dorsal 5.3 mm (Paxinos and Watson, 2005). The 1 µL microsyringe (Hamilton, Reno, NV, USA), closely connected with the glass microelectrode, was used to slowly position the needle at the determined coordinates. Ibotenic acid (Sigma-Aldrich) was dissolved in PBS (10 µg/µL) and 0.3 µL was injected into MD at a rate of 0.1 µL/min. All rats were given 80 thousand units of penicillin, subcutaneously, before and after operations to prevent infection. For the groups with combined SNc and MD lesions, one group had Ibotenic acid injected into the MD 1 week after SNc lesions, the other group 3 weeks after (Figure 1
5
Neonatal Ventral Hippocampal Lesion Model
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Seven-day-old male pups were randomly assigned to either the sham or NVHL groups. The pups were anesthetized via hypothermia by placing the animal at a 0°C chamber in a refrigerator for 10–12 min. The anesthesia was considered complete, when the distal limbs were no longer pink and no spontaneous limb movement was observed. Thereafter, the pups were fixed on a custom-made platform attached to a stereotaxic apparatus (SN-2N, Narishige, Japan). An incision was made into the skin to expose the cranium, and two small holes (diameter: approximately 1 mm) were drilled into the skull bone. A steel tube was implanted bilaterally into the ventral hippocampus. The coordinates used were AP −3.0 mm, ML ±3.5 mm, and DV −5.0 mm. Ibotenic acid (3 μg in 0.3 μL, Sigma, St Louis, MO, USA), dissolved in 0.15 M phosphate buffer saline (PBS, pH = 7.4) or vehicle (sham), was infused into the ventral hippocampus through the implanted tube at a flow rate of 0.1 μL/min.
Rats were weaned on postnatal day 25 and moved to standard plastic cages (two per cage) on postnatal day 49. To reduce stress, rats were handled daily until the commencement of the electrophysiological recording experiments. Three days before the experiments, rats were moved again, in order to be housed separately in single-occupancy Plexiglas cages.
Rats were weaned on postnatal day 25 and moved to standard plastic cages (two per cage) on postnatal day 49. To reduce stress, rats were handled daily until the commencement of the electrophysiological recording experiments. Three days before the experiments, rats were moved again, in order to be housed separately in single-occupancy Plexiglas cages.
6
Stereotaxic Implantation of DMH Cannulas
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Rats were anesthetized with pentobarbital sodium (50mg/kg, intraperitoneally, i.p.) and secured on a stereotaxic apparatus (SN-2N, Narishige Group, Tokyo, Japan). The unilateral guide cannulas (23 gauge) were implanted into the DMH. The stereotaxic coordinates of the NTS were determined according to the atlas of Paxinos and Watson Lam (Lam et al. 2010; Walton & Paxions. 2007 ). The detailed settings were 0.5mm lateral to the midline, 2.8mm posterior to bregma, and 6.6mm ventral from the skull surface. The tips of the cannulas were placed 2mm above the DMH. The cannulas were fixed to the cranium using dental acrylic resin and jeweler screws. 30-Gauge metal obturators were used to fill the cannulas during the intervals of experiments between tests. All rats were injected with penicillin (20,000units, i.p.) during the first three postoperative days to prevent infection and to recover for at least 7 days before starting the experiments.
7
Aldosterone Microinjection in Rat NTS
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Aldosterone was purchased from Sigma and dissolved in a solution of DMSO in H2O (at a volume ratio of 1:100). The same concentration of DMSO was used in the control group as a vehicle. A 0.1 μl volume of drugs was injected into the bilateral NTS. A final concentration of 5 ng/0.1 μl Aldosterone was used for microinjection, as previously reported (Brailoiu et al., 2013 (link); Formenti et al., 2013 (link); Francis et al., 2001 (link)). The following instruments were used in this study: a 1‐μl microinjector (Hamilton), a stereotaxic apparatus (SN‐2N, Narishige), a concentric electrode (CEA 200, MicroProbes), a lesion‐making device (53500, Ugo Basile), metabolism/feeding‐drinking cages, part of a feeding‐drinking‐activity analyzer (41800111213, UGO), and a small animal anesthetic machine (RWD).
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