Real-time Analysis of Gut-brain Neural Communication: Cortex wide Calcium Dynamics in Response to Intestinal Glucose Stimulation

Our research scope reveals gut-brain neural communications, especially its role in food preference. Experiments involved the vagus nerve have often treated as a single bundle, but recent research have revealed that it selects properties and organ specificity. So I think investigating that its statistics, you know, will attract attention in the future.

This method of attaching catheter to the gut is suitable at low cost, less invasive, and easier than the method proposed earlier. Traditionally, catheter attachment to the gut was done by suturing. Here we mitigated the surgical damage to mice by substituting suturing with cyanoacrylate glue attachment.

In our laboratory, we are exploring the mechanism of how physical and psychological stress affect gut-brain neural communication in mice. To begin, use scissors to cut the silicon tube to a precise length of seven centimeters. Using cyanoacrylate glue, fix diminutive plastic beads, approximately three millimeters from the end of the silicon tube.

Excise the apex of a 23-gauge needle and cut 1.5 centimeters from the needle tip. Insert the cut section of the needle on the opposite side of the bead into the silicon tube. Using pliers, excise one centimeter from the needle tip.

Attach the modified 23-gauge injection needle to a 2.5-milliliter syringe. Sheath a 15-centimeter silicon tube over the 23-gauge injection needle. Cut the injection needle 1.5 centimeters from the tip and attach it to the silicon tube.

To begin, place the anesthetized mouse supine on the surgical table, aligning its mouth proximate to the inhalation apparatus. Using adhesive tape, secure the mouse's oral cavity, front legs, and back legs, to the surgical table. Apply depilatory cream to remove hair from the upper left abdomen.

Make a 1.5-centimeter skin incision on the right side of the abdomen and five millimeters below the xiphoid process. Then create a 1.5-centimeter incision in the abdominal wall at the same location as the initial skin incision. Gently move the left hepatic lobe laterally with blunt-end forceps to expose the stomach.

Now, lift the stomach and gently remove it through the incision. Using a scissor, create a diminutive perforation in the pyloric antrum. Introduce the end of the catheter with a bead into the perforation.

After confirming the firm attachment of the catheter to the stomach, carefully reposition the stomach to its original position. Suture the abdominal wall, allowing the catheter to exit externally. Then close the skin incision in a manner analogous to the abdominal closure.

Clean the operated region with chlorhexidine gluconate solution and place the mouse in a sanitized cage. To begin, secure the anesthetized mouse onto a stereotaxic platform using auxiliary ear bars to mitigate the effects of pulsation and respiration. Using an electric shaver or hair removal cream, carefully remove the hair from the scalp.

Disinfect the scalp's surface with 0.1 to 0.5%chlorhexidine gluconate solution. Apply a local anesthetic gel to the scalp and wait for five to 10 minutes. Next, using scissors, make a straight cut from the back of the head to the forehead.

Use clips to pull back any excess skin exposing the skull. Remove the connective tissue of the periosteum with a cotton swab. Immediately apply the acrylic cement to the skull and wait for five minutes for the cement to dry.

Move the mouse under a fluorescence stereo microscope. For imaging, use a wideband blue fluorescence filter set in combination with a mercury light source. Next, remove the catheter needle from the end of the catheter.

Purge any residual content within the mouse-side catheter using approximately 0.03 milliliters of saline. Then remove the catheter from the mouse. Aspirate the appropriate dose of 10%glucose solution into the syringe and attach it to the catheter.

In the imaging software, check the camera recognition and set the frame rate to 10 hertz. Set the resolution to 512 x 512 pixels and depth to 16 bits. Click on the recording process button and acquire spontaneous data for 50 seconds.

Finally, with a gradual infusion of the glucose solution, record the mouse's physiological state data. Spontaneous neural activity revealed random calcium oscillations throughout the cortex. Temporal fluorescence intensity changes showed calcium oscillations following a burst suppression pattern.

Glucose injection showed significant changes in cortical calcium dynamics within four to eight seconds after the completion of glucose administration with immediate activation in the secondary motor cortex. However, no changes were observed following water administration. Compared to water administration, substantial changes in fluorescence intensity ratios were observed in the secondary motor cortex region following glucose injection.

Activation levels in different cortical regions post-injection showed significant differences only in the secondary motor cortex region.