Planar multi electrode arrays are used to record from the UL nasal organ. The VNO is removed from the mouse such that the neuro epithelium remains intact. First, the VNO capsule is removed from the mouse.
Then the neuro epithelium is isolated from the bony capsule, blood vessel and basal lamina. The tissue is placed in the MEA with the dendritic knob side up. Once this setup is complete, we use a robotic arm to administer liquid stimuli to the tissue.
Hi, I'm Hannah Arnon from the laboratory of Tim Wholly in the Department of Anatomy and Neurobiology at Washington University School of Medicine. I'm Cheyenne Fu, also from the Holy lab. Today we'll show you a procedure for multi electrode array recording of the V nasal organ.
We use this procedure in our lab to study the sensory properties or receptor neurons in the accessory atory system. So let's get started. Electrical recordings are obtained with a planar multi electrode array or MEA with 10 micrometer flat titanium nitride electrodes insulated with silicon nitride.
60 electrodes are arranged in two fields consisting of a five by six grid with center to center spacing of 30 micrometers. Using an MEA 10 60 amplifier, the electrical signals are amplified. 1000 fold signals are required at 10 kilohertz.
Using a data acquisition card and custom data acquisition software, the tissue is held down on the MEA by a nylon mesh, which is attached to a custom insert, which fits snugly inside the culture. To bring of the MEA, now that we've seen the meas, let's see how to do the VNO dissection. Prior to performing the dissection, fill the MEA well with bovine serum albumin or BSA next, rinse the MEA using warm bubbled ringer solution and place it on ice.
To prepare for the dissection, fill two 60 by 15 millimeter Petri dishes with bubbled ringer solution and place them on ice. Also, fill two sill guard coated Petri dishes of the same size with ringer solution and place them on ice. Chill an additional 50 milliliters of bubbled ringers for use during the dissection.
Begin with a sacrificed mouse by making bilateral incisions from the side of the mouth running parallel to the jawline to the back of the head. Join the two cuts along the back of the neck and remove the dorsal part of the head, leaving the lower jaw attached to the mouse using fine scissors. Cut between the tissue and either side of the upper jawbone to separate the tissue from the bone.
Next, use number three forceps to peel off the palate tissue, exposing the nasal cavity using small scissors or a scalpel cut between and on either side of the upper two front teeth to free the vulnerable nasal capsule from the bone using number three forceps. Pick up the vulnerable nasal capsule by the flat bone and place it in the Petri dish on ice. The preceding steps through immersion of the VNO in chilled ringers should be performed in less than three minutes.
To prevent tissue death, place the dish under a stereo microscope illuminated with reflected light. Use number three forceps to remove the bony capsule from one VNO at a time. Use one hand to stabilize the bony capsule by gripping from the coddle end and the other hand to peel the bone away from the VNO taking care not to damage the VNO.
Once the bone is removed, transfer the v and o to a cigar coated Petri dish. Use the remaining dish for the other VNO. Switch the illumination to using transmitted light using micro scissors.
Or number five forceps. Separate the VNO from the blood vessel by cutting along the edge of the VNO. At this point, you will have the neural sheet completely separated from the blood vessel.
Place the VNO with the internal side up using number three forceps. Anchor the VO to the S guard at a corner of the tissue using a small fragment of a Teflon coated razor blade held with a clamp. Peel the neural epithelium from the basal lamina holding the blade at a shallow angle less than 30 degrees from the dish surface.
Works best. Using a glass pipette. Transfer the piece of neural epithelium from the dish to the MEA position the VNO on top of the electrode field with the dendritic side up.
Remove excess ringer solution with a pipette and then place the mesh tissue holder into the well of the array to hold the vno in place. Fill the well with chilled ringer solution with dissection complete. Let's see how to record an To begin a recording session, place the MEA in an MEA 10 60 amplifier and position the heater probe directly above the tissue to confirm proper heater.
Probe placement stimulate with ringer solution containing 50 millimolar potassium for two seconds. Adjust the positioning to minimize the latency of the neural response to the potassium solution. The tissue requires continuous superfusion, which is achieved by supplying ringer solution from an HPLC pump.
Super fuse the tissue with heated ringer solution for 45 minutes to one hour prior to recording to allow the tissue to acclimate. Stimuli are delivered using the Gilson two 15 liquid handler robotic arm. They're presented by performing injections into an HPLC valve and switching between flow through and the loop Under the control of Gilson software, the robotic arm delivers the stimuli to the profusion line while constant flow rate and pressure are maintained.
The state of the HPLC valve is represented by an electrical voltage, which is recorded by the acquisition computer as an extra channel. To ensure synchronization with neuronal activity, electrical signals are saved to disc using custom recording software. Now we will show you some representative results of VNO multi electrode array.Recording.
This procedure yields large amounts of data due to the large number of electrodes, and the preparation typically remains stable for around six hours. Here is an example of 400 milliseconds of data from one half of the MEA. This procedure is useful in measuring the response of the sensory neurons to ligands shown here is the response to 100 fold diluted female mouse urine recorded from a single electrode.
The urine was delivered for 10 seconds as indicated by the black bar. Note, the increase in firing following stimulus presentation. We've just shown you how to perform multi electro array recordings on the mouse VNO when performing this procedure.
It's important to remember to be patient. So that's it. Thanks for your watching and good luck with your experiments.
