To block A1 cholinergic synapses shortly before neurophysiological recordings, 100 nl of solution was injected into ACtx 500 μm below the brain surface at a speed of 10 nl/min using a motorized injector (Stoelting Co.). The solution contained 10 μM of atropine sulfate, 1 μM of dihydro-b-erythroidine hydrobromide, and 150 μM methyllycaconitine citrate dissolved in artificial cerebrospinal fluid. For the control experiments, 100 nl of artificial cerebrospinal fluid was injected. The injection pipette was removed 5 minutes following the injection and the animal was transferred to the head-fixed recording rig. Recordings began once the mouse exhibited normal treadmill running and grooming behavior.
Motorized injector
Manufactured by Stoelting
Sourced in United States
The Motorized Injector is a laboratory instrument designed for precise and controlled injection of samples or reagents. It features a motorized mechanism that allows for accurate and repeatable delivery of liquids. The core function of this product is to facilitate consistent and reliable sample introduction in various analytical procedures.
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Lab products found in correlation
Hamilton syringe, by Stoelting (2 mentions)
Stereotactic frame, by Harvard Apparatus (2 mentions)
Fk 506 rapamycin, by LC Laboratories (1 mentions)
Hardset antifade mounting medium with dapi, by Vector Laboratories (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit or anti mouse secondary antibody, by Thermo Fisher Scientific (1 mentions)
Alexa 546, by Thermo Fisher Scientific (1 mentions)
Telmisartan, by Merck Group (1 mentions)
Candesartan, by AstraZeneca (1 mentions)
Rapamycin, by LC Laboratories (1 mentions)
Fk506, by LC Laboratories (1 mentions)
10 protocols using motorized injector
1
Blocking Cholinergic Synapses in Mouse Cortex
2
Selective Lesioning of NA Neurons
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As in our previous studies (30 (link),31 (link)), we used a highly specific approach to lesion NA neurons projecting to the anterior vlBNST. The lesioning agent (DSAP) is a conjugate of a mouse monocloncal antibody to dopamine beta-hydroxylase (DbH) and the ribosome-inactivating protein, saporin [Anti-DbH-SAP; Advanced Targeting Systems, Inc.]. Rats (N = 25) were anesthetized by inhalation of isoflurane (1–3% in oxygen) and placed into a stereotaxic apparatus. The anterior vlBNST (located 0.35mm posterior, 2.3mm lateral, and 7.4mm ventral to bregma) was targeted using a 10-degree angle from vertical to avoid passing through the lateral ventricle and dorsal BNST. DSAP was diluted to 40ng/100nl in 0.15M NaCl vehicle containing 0.05% CTB, which does not interfere with DSAP lesion efficacy (30 (link)). DSAP (N= 11 rats) or CTB vehicle alone (N= 10 rats) was delivered (200nl bilaterally) into the avlBNST by pressure injection (20nl/min for 10 min) using a Hamilton syringe driven by a motorized injector (Stoelting Co.). The syringe was left in place for 10 minutes after each injection. Rats were allowed to recover from surgery and were tested for passive avoidance retention at least two weeks later, which is sufficient for effective lesioning (30 (link),31 (link),40 (link)–42 (link)).
3
Intracerebral Transplantation of SPIO-labeled mESCs
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All animal procedures were performed under an approved protocol from our Institutional Animal Care and Use Committee (IACUC). Three 3-week-old male BALB/c mice (Harlan Laboratories, IN, USA) were anesthetized using 2% isoflurane, were shaved, and then immobilized in a stereotactic frame (Harvard Apparatus, MA, USA). SPIO-labeled Luc-mESCs (5 × 104 cells in 2 μL of volume) were injected into the brain using a 31-gauge needle and a motorized injector (Stoelting Co., IL, USA) at a rate of 0.5 μL/min and the following coordinates: anterior–posterior (AP) = 0 mm, medial–lateral (ML) = 2.0 mm, and dorsal–ventral (DV) = 1.5 mm. The needle was carefully withdrawn 2 minutes after the end of the injection to minimize backflow. Animals were immunosuppressed by intraperitoneal daily administration of a cocktail of (FK-506) + (rapamycin) (1 mg/kg each; LC Laboratories, Woburn, Massachusetts), beginning 3 days before cell transplantation, and then daily until sacrifice.
4
Retrograde Tracing of Cholinergic Neurons
5
Ang II Effects on Brain Cholesterol
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A group of adult Sprague Dawley rats was injected in the third ventricle with a single dose of 5 μg of Ang II in 3 μL sterile saline or sterile saline alone as a control group to study the effects of Ang II on brain cholesterol levels. The Ang II-injected dose was determined based on our previous studies [15 (link),38 (link)].
The surgical procedure was similar to that performed in 6-OHDA lesions. The stereotaxic coordinates were −0.8 mm posterior to bregma and −6.5 mm ventral to the skull at the midline in the flat skull position. The tooth bar was set at 0 mm. The solution was injected using a 5 µL Hamilton syringe coupled to a motorized injector (Stoelting, Wood Dale, IL, USA), at a rate of 0.5 µL/min, and the needle was left in situ 2 min after injection. After 48 h, rats were stunned with carbon dioxide and killed via decapitation. Ventral mesencephalon and striatum brain areas were dissected for the measurements of cholesterol levels.
The surgical procedure was similar to that performed in 6-OHDA lesions. The stereotaxic coordinates were −0.8 mm posterior to bregma and −6.5 mm ventral to the skull at the midline in the flat skull position. The tooth bar was set at 0 mm. The solution was injected using a 5 µL Hamilton syringe coupled to a motorized injector (Stoelting, Wood Dale, IL, USA), at a rate of 0.5 µL/min, and the needle was left in situ 2 min after injection. After 48 h, rats were stunned with carbon dioxide and killed via decapitation. Ventral mesencephalon and striatum brain areas were dissected for the measurements of cholesterol levels.
6
Evaluating Nigral Neuroinflammation in Rats
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Rats received unilateral intra-substantia nigra injection of saline or MG132 (9.5 ng) under general anesthesia (0.8–1.5% isoflurane in 30% oxygen with air). The stereotaxic coordinates for substantia nigra were laternal +2.0 mm, antero-posterior from the bregma point -5.3 mm and dorsi-ventral +7.8 mm [24 (link)]. The solution was injected into the substantia nigra with 10 μl Hamilton syringe coupled to a motorized injector (Stoelting, Wood Dale, IL) at a rate of 0.2 μl/min and the needle was left in situ for at least 5 min after injection. After surgery, the rats were kept in the same cages for recovery. Rats expressing infection signs were excluded from the study. The rats were sacrificed using overdose isoflurane at 24 h after MG-132 injection. For double immunofluorescent labelling studies, the substantia nigra sections were stained with two primary antibodies, mouse anti- TH (1:1000) and rabbit anti-HO-1 (1:500), then with Alexa 488 (excitation 495 nm, emission 519 nm) or Alexa 546 (excitation 556 nm, emission 573 nm)-conjugated goat anti-rabbit or anti-mouse secondary antibody (1:500; Invitrogen, CA, USA). DAPI (1 μg/ml) was used to stain nucleus and the fluorescent images were obtained using a confocal microscope (model SP5 LAS; Leica, Heidelberg, Germany).
7
Stereotaxic Adeno-Associated Virus Delivery for Parkinson's Disease
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Injections were performed in the right mesencephalon dorsal to the nigra to minimize possible traumatic damage. Stereotaxic coordinates were − 5.4 mm anterior to bregma, − 1.9 mm right of midline, and − 7.0 mm ventral to the dura; tooth bar was at − 2.3 mm [23 ]. The viral vectors were injected using a 5-μl Hamilton syringe coupled to a motorized injector (Stoelting, Wood Dale, IL). Injections were accomplished in pulses of 0.5 μl/min (2 μl for the AAV9-WT α-syn-injected group and 1 μl for the AAV9-A53T α-syn-injected group), and once completed, the microsyringe was left in place for an additional time of 10 min before withdrawal, to avoid viral vector reflux through the injection tract. Groups of animals received oral treatment with the AT1 receptor antagonists candesartan (groups A2 and B2 1 mg/kg/day; AstraZeneca, Madrid, Spain) or telmisartan (groups B2 and B3; 1 mg/kg/day; Sigma, St. Louis, MO) from 2 weeks before AAV9 injection until they were sacrificed. The doses were selected on the basis of the results of our previous studies [24 (link), 25 (link)]. The powered drug was administered orally mixed with “Nocilla” hazelnut cream (Nutrexpa, Barcelona, Spain). Animals in control groups were given “Nocilla” hazelnut cream only.
8
Alpha-synuclein Pathology in ApoE Mice
9
Stereotactic Injection of ASO NEAT1 in Mice
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Mice were anesthetized with ketamine 10% (100 mg/kg body weight) and xylazine 2% (10 mg/kg body weight) in 0.9% NaCl (max. injection volume 10 ml/kg body weight) and then fixed in a stereotactic frame on a warming plate under deep anesthesia. After application of an eye ointment, antiseptic treatment, skin incision, and drill hole trepanation (diameter: 0.6 mm), injections were made at x (1.5 mm from the sagittal suture), y (− 2.0 mm from the bregma), and z (2.5 mm depth) via a motorized injector (Stoelting Wood Dale, IL, USA). ASO NEAT1 (2 nmol, 2 μl) or ASO scramble (2 nmol, 2 μl) was slowly injected over 5 min using a 10 μl Hamilton cannula (outer diameter 240 μm; injection rate: 400 nl/min) once a week for 2 consecutive weeks before MCAO surgery (thus two injections in total). After the injection, the cannula remained in the tissue for 4 min to prevent reflux through the injection channel. The stereotactic injection paradigms essentially followed a protocol described by Jin et al. [17 (link)].
10
Transplantation of SPIO-Labeled mESCs in Mice
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