M40

Cell lines

Nucleus tractus solitarius (NTS) neurons

Preparation Method

After the Sprague-Dawley neonatal rats was deeply anesthetized with halothane, a craniotomy was performed, and the forebrain was ablated at the caudal border of the pons by transection. The caudal brainstem and cervical spinal cord were isolated by dissection inmodified Krebs’ solution that contained 128.0mM NaCl, 3.0mM KCl, 0.5mM NaH₂PO₄, 1.5mM CaCl₂, 1.0mM MgSO₄, 21mM NaHCO₃, 1.0mM mannitol, 30.0mM glucose, and 10.0mM HEPES.

The stomach, connected to the esophagus with the vagus nerves linking it to the brainstem, was kept, and all the other internal organs were removed. The preparation was then isolated and pinned, with the dorsal surface up, on a layer of Sylgard resin in a recording chamber. An incision was made on the lateral surface of the stomach wall to minimize possible gastric vagal fiber damage. The stomach was opened, and its contents were removed. The stomach was then pinned down, and both mucosa and serosa were exposed to Krebs’ solution in the gastric compartment. The preparation was superfused with Krebs’ solution at 23±1℃. The bathing solution was aerated continuously with a mixture of 95% O₂ and 5% CO₂ and adjusted to pH 7.35 to 7.45.

Single tonic unitary discharges were recorded extracellularly in the medial subnucleus of the NTS by glass microelectrodes filled with 3M NaCl, with an impedance of 10 to 20 MΩ . A collision test was applied to identify orthodromic inputs to ensure that only second- or higher-order NTS neurons in the gastric vagal afferent system. Concentrations of galanin and M40 used in the experiment are 100nM. Each test compound was first dissolved in a small volume of Krebs’ solution. The concentrated solution was then applied to the gastric compartment. The final drug concentration in the gastric compartment was calculated based on the amount of concentrated solution and the total Krebs volume in the gastric compartment. Drug solution was applied 5min prior to any pharmacological observation to provide sufficient time for drug delivery to reach a steady-state level. After each observation, drug was washed out from the compartment. The NTS neuronal responses observed during pretrial or pretreatment (control) were compared with post-trial (washout) to confirm that brainstem neuronal activity returned to the control level after washout. .

Reaction Conditions

100nM; 5min

Applications

Animal models

Male specific pathogen-free Sprague-Dawley rats

Preparation Method

The animals were anaesthetized with urethane (1.1g/kg) a and immediately tracheotomized. The femoral artery was cannulated with a plastic catheter containing heparin (50IU/ml, 0.9% NaCl w/v) to record blood pressure and heart rate. and the animal was placed in a stereotaxic frame. The head was flexed 45°, the neck muscles were dissected with an electric knife to avoid bleeding and the atlanto-occipital membrane was exposed. The surgical procedure took 8-10min and the animals were allowed to stabilize for at least 30min. During the whole experiment, the body temperature was maintained at 37.5±0.5℃ by means of a thermostatic blanket.

To study if M40 alone exerts any effect on central 11 cardiovascular parameters, groups of rats: received intracisternal injections of M40 at doses of 1.0nmol.

Dosage form

1.0nmol; 50 min; intracisternal injection

Applications

References:
[1] YUAN C S, DEY L, XIE J T, et al. Gastric effects of galanin and its interaction with leptin on brainstem neuronal activity [J]. The Journal of pharmacology and experimental therapeutics, 2002, 301(2): 488-93.
[2] NARVáEZ J A, DíAZ-CABIALE Z, HEDLUND P B, et al. The galanin receptor antagonist M40 blocks the central cardiovascular actions of the galanin N-terminal fragment (1-15) [J]. Eur J Pharmacol, 2000, 399(2-3): 197-203.