This protocol describes techniques that can be utilized by small animal physiologists to elucidate the role of neuropeptides and other hormonal factors that regulate the digestive and or excretory system. These techniques allow for measurements of micro size biological samples that would otherwise not be possible using techniques designed specifically for larger animal models, including, for example, rodents and teleus. This method can provide insight into the endocrine regulation of the gut, including transporting epithelial, as well as smooth muscle associated with the gastrointestinal tract in insects and in similarly sized non-insect species.
Demonstrating the insect renal tubal procedures will be Farwa Sajadi, a PhD candidate from my laboratory. Additionally, a former graduate student from my lab Aryan Lajevardi will be demonstrating the hindgut focused protocols. Begin by preparing the Ramsay and contraction assays dishes for the experiments.
Use pipette and fill wells with up to 20 microliters of solution. For unstimulated controls, fill the wells with 20 microliters of Aedes saline Schneider's medium. Once all wells are filled, pour hydrated mineral oil into the assay dish until the wells and Minutien pins are submerged.
After immersing the tubule in the well, pick up the proximal end of the tubule with forceps, remove it from the bathing droplet, and wrap the end around the pin. Wrap the tubule around the pin twice keeping the length of tubule remaining in the bathing droplet consistent with the other tubules. To make a sodium selective microelectrode, use a one milliliter syringe to backfill the electrode with 100 millimolar sodium chloride.
Ensure the backfill solution fills until the tip of the electrode. If air bubbles appear, gently flick the microelectrode or remove the solution and refill. Dip a 10 microliter pipette tip into the sodium selective ionophore solution.
Line up the electrode perpendicular to the pipette, then place a gloved finger over the bottom of the tip to create pressure and expel a small drop of ionophore. Carefully touch the drop of ionophore to the microelectrode tip, making sure not to break it. Fill a small beaker halfway with 100 millimolar sodium chloride and place some modeling clay onto the inside at the top of the beaker.
After the ionophore has been taken up, place the electrode tip down onto the wall of the beaker, letting the tips within the sodium chloride. Keep ISME in the beaker until it is ready to use. To make the ISME reference electrode, backfill an electrode with 500 millimolar potassium chloride and store it in a beaker.
Coat the electrode tips using a solution of approximately 3.5%polyvinyl chloride, dissolved in tetrahydrofuran, to avoid displacement of the ionophore when submerged in paraffin oil. To calibrate the sodium electrode, place 10 microliter droplets of sodium chloride standard concentrations onto the edge of the Ramsey dish with the incubated Malpighian tubules. Place the standard droplets two centimeters apart with the higher concentration on top.
Insert both the reference electrode and ion selective electrode over the chloride silver wires and fasten them securely using electrode holders that are attached to micro manipulators. Navigate both the electrodes toward the 200 millimolar sodium chloride droplet using the micro manipulators, ensuring that the electro tips do not touch the bottom of the dish. Turn on the electrometer to start recording and allow the reading to stabilize.
Record the reading and continue to the next standard. After acquiring measurements of the fluid secretion rate, carefully move the reference and ion selective electrodes into the secreted droplet using the micro manipulators. Turn on the recording and allow the reading to stabilize, then record.
Turn on the IPA-2 Ion/Polarographic Amplifier, the light microscope, and the computers. Place the sodium selective microelectrode on a holder consisting of a silver chloride wire and attach it into the female connector jack. Remove one reference electrode from the beaker with the potassium chloride, placing one finger at one end and tilting the glass capillary towards this finger to prevent the agar from falling out.
Carefully place one end into the holder, ensuring that no bubbles form. If there is a bubble, remove the reference electrode, refill the holder with three molar potassium chloride and repeat. Place the electrode holder into the female connector jack.
Following calibration, dissect the organ. Place the poly-L-lysine dish with the dissected sample on the microscope stage and insert the tip of the reference electrode inside the saline. Submerge the tip of the ion selective microelectrode in the saline, taking care not to break the tip.
Use the manual adjustment knobs to adjust the microelectrode position while looking under the light microscope. Adjust the vertical position of the microelectrode such that its tip is on the same plane as the organ or tissue, then turn the motor switch to enable. Using the computer arrow keys, move the microelectrode horizontally to a position three millimeters away from the tissue to measure background recordings.
When ready, begin recording by pressing F5, obtain five measurements of background activity. Move the microelectrode tip close to the tissue taking care not pierce the organ. Reduce the key hit sensitivity to place the microelectrode tip two micrometers directly to the right, perpendicular to the tissue.
Obtain three recordings at site along the rectal pad to identify the site of greatest ion activity, then obtain baseline saline measurements at the site displaying greatest activity. To record hindgut contractions, fill one of the wells in the dish with a known volume of Aedes saline. Following dissection, carefully transfer the dissected hindgut attached to the mid gut into a well in another dish, making sure not to pinch the ileum.
Submerge the gut in the saline inside the well and place Minutien pins into the mid gut and rectum. The ileum should not be under tension and spontaneous contractions originating at the pyloric valve at the anterior ileum should be observed. Connect the video camera to the stereoscopic microscope.
Then place the dish containing the dissected organ under the microscope and record a video for two minutes. Application of DH31 against unstimulated Malpighian tubules results in a significant increase in fluids secretion rate, confirming its role as a diuretic hormone in Aedes mosquitoes. When tubules are treated with AedaeCAPA-1, a reduction in secretion rate is observed in DH31 stimulated Malpighian tubules.
Ion selective electrodes were used to measure sodium concentrations in the secreted droplets. Treatment of DH31 on the MTs had no effect on the sodium concentration in the secreted droplet. However, with the application of AedaeCAPA-1, the sodium concentration in the secreted fluid was significantly increased.
Additionally, compared with unstimulated controls, DH31 led to a significantly greater sodium transport rate, whereas AedaeCAPA-1 abolished this increase in DH31 stimulated tubules. The SIET was used to assess changes in sodium transport along the rectal pad epithelial of adult female mosquitoes. A leucokinin analog was used to examine changes in sodium absorption, which resulted in a four-fold decrease in sodium absorption compared to saline control.
To assess the role of a neuropeptide pyrokinin2 on ileal motility, a Rhodnius prolixus analog was used, which was previously shown to activate the A.aegypti PK2 receptor enriched in the mosquito ileum. Relative to baseline levels, PK2 significantly inhibits ileal contractions. When performing a Ramsay assay with Malpighian tubules and measuring ion or fluid secretion rates, it is imperative that tubules aren't damaged during dissection or during the transfer into the assay dish.
Following the Ramsay assay, specific membrane transporters can be targeted either pharmacologically or molecularly using reverse genetic techniques to discern their specific contribution to transepithelial transport of solutes in the simple epithelium.
