The metabolites released by gut microbiota produce complex effects on the host. This protocol presents a method to interrogate the role of gut metabolites in the mouse brain and behavior. This technique provides a precise, meticulous, and validated method to deliver gut metabolites into a brain in freely moving mice.
We provide a comprehensive and physiological best methodology for insight into gut brain axis research. Place the mouse onto the stereotaxic frame and fix the mouse incisors on the incisor bar in the stereotaxic incisor holder. Cover the nose with the nose cone mask.
Then evaluate the nociception of the mouse via the top pinch reflex, and ensure a constant breathing rate before incising the surgical site. Once trimmed fur on the head is removed by the adhesive tape, subcutaneously inject the analgesic ketoprofen to relieve pain and apply eye ointment. Next, fix the head by inserting the pointed ear bar in the ear canal.
Tighten the nose clamp on the incisor holder and press the head gently to ensure that the head is fixed. Use 3 alternating scrubs of chlorhexidine swab to disinfect the scalp. Now, use dissecting scalpel to make an incision of less than one centimeter in the scalp in an anterior posterior manner.
After opening the incision, wipe the skull with a cotton swab held by micro dissecting forceps. Based on the stereotaxic coordinates, identify and label the position of the right lateral ventricle as described in the manuscript. Next, drill a hole through the skull at the labeled site using a stereotaxic drill, and later, drill two to four more holes in the skull.
Wipe the skull with lidocaine cotton swab and mount 2 to 4 stainless screws on the holes. Place the commercial guide cannula on the stereotaxic cannula holder and disinfect with the glass speed sterilizer at 150 degrees Celsius. Then, move the stereotaxic cannula holder to the hole drilled for the lateral ventricle and slowly insert the commercial guide cannula into the hole until the desired depth is attained.
Now apply 10 microliters of n-Butyl cyanoacrylate adhesive to fix the commercial guide cannula in the drilled hole and wait for 3 to 4 minutes. Release the commercial guide cannula gently from the stereotaxic cannula holder and apply dental acrylic to the incised scalp to fix the commercial guide cannula and wait for at least 5 minutes. Implant the commercial dummy into the commercial guide cannula.
Then, place the mouse into a new cage with a heating pad underneath for recovery from anesthesia and continuously observe until the mouse fully awakens. Mount the microliter syringe, containing 10 microliters of distilled water, on the microinjection pump. Using an insulin syringe, fill the polyethylene tube with distilled water and connect the microliter syringe to the hanging polyethylene tube.
Turn on the microinjection controller and press display all channels to access the command screen. Next, press configuration and set volume target to 9, 800 nanoliters with delivery rate of 100 nanoliters per second. Then press direction to switch to the infuse mode and press run to infuse 9, 800 nanoliters of distilled water from the polyethylene tube connected to the microliter syringe.
Now, place the injector into a vial containing the mineral oil, press configuration and set the volume target to 3, 000 nanoliters with delivery rate to 100 nanoliters per second. For withdrawing 3, 000 nanoliters of mineral oil, press direction to switch to the withdraw mode and then press run. Disassemble the polyethylene tube from the microliter syringe needle.
To spit out 3, 000 nanoliters of distilled water from the microliter syringe needle, press direction to switch to the infuse mode and press run. Then insert the microliter syringe back into the polyethylene tube. Place the injector into a vial containing SCFAs.
Press configuration and set volume target to 9, 500 nanoliters with delivery rate to 100 nanoliters per second. Withdraw 9, 500 nanoliters of SCFAs by pressing direction to switch to the withdraw mode and press run. Label the oil SCFAs phase to validate whether the SCFAs are successfully infused.
Press configuration and set the desired volume target with delivery rate to 7 nanoliters per second. Press direction to switch to the infuse mode. Press run to infuse the microliter syringe forward until the liquid comes out at the front end of the commercial injector before inserting the injector into the cannula for SCFAs injection.
After anesthetizing the mouse, insert the commercial injector into the commercial guide cannula. Once the mouse is recovered from anesthesia, place it in a novel cage and allow it to explore for 35 minutes freely. Infuse SCFAs using a delivery rate of 7 nanoliters per second for a target volume of 2, 100 nanoliters in the first 5 minutes.
Then press direction to the infuse mode and press run. After behavioral testing, remove the commercial injector from the commercial guide cannula. Infuse 2, 100 nanoliters of the neutral tracer with the delivery rate of 7 nanoliters per second to verify the infusion site.
Image the fluorescent signal in the infusion site using a fluorescence microscope. Both ACSF and SCFAs-infused mice traveled in a novel cage for a total of 35 minutes. No difference was observed in the locomotor activity in the novel cage between infusion of SCFAs and ACSF.
The fluorescent dye was detected in the lateral ventricle and the surrounding regions of the mouse brain when infused with a commercial guide cannula. Similarly, infusion through the stainless steel guide cannula leads to the detection of signals in the lateral ventricle and the surrounding regions of the mouse brain. Combining this procedure with circuit based neural technologies will allow scientists to gain insight into the circuit based control of the brain and behavior contributed by gut metabolites.
This technique paves the way for the researchers to explore the role of microbiota and metabolites in the intricate gut brain axis.
