The MEA 4-AP spinal slice preparation described to you provides a platform to study the connectivity of dorsal horn circuits and how these networks interact during spinal sensory processing. The methodology presented enables dorsal horn circuit activity to be investigated at a macroscopic regional level of resolution. This preparation can be used as a rapid screening tool to assess the ability of compounds to disrupt signaling in spinal sensory circuits.
This method helps bridge the gap in our understanding of how the activity of specific cell types and small micro circuits influence large populations of neurons to subsequently shape the output of the dorsal horn behavior responses and ultimately the pain experience. To begin, fill the MEA well with horse serum for 30 minutes. After removing the serum, thoroughly rinse the MEA five times with distilled water until the distilled water becomes non-foamy.
Then, fill the well with artificial CSF. After decapitating the anesthetized mouse, remove the skin over the abdominal region by making a small cut in the skin at the level of the hips. Then, pull the skin on either side of the cut rostrally until all the skin is removed from the top of the rib cage to the top of the pelvis, both ventrally and dorsally.
After placing the body on ice, use a ventral approach to expose the vertebral column by removing the viscera and cutting through the ribs laterally to the sternum. Remove the ventral rib cage, both scapulae, and the lower limbs and pelvis. Transfer the vertebral column and rib preparation to a dissecting bath containing ice cold sucrose artificial CSF.
Pin all four corners of the preparation by placing pins through the lower back muscles and the attached upper ribs. Remove all muscle and connective tissue overlying the ventral surface of the vertebrae with the rongeurs and identify the vertebral region over the lumbo-sacral enlargement, which lies approximately beneath the T12-L2 vertebral bodies. Remove a vertebral body caudal to the lumbo-sacral enlargement region to access the spinal cord sitting on the vertebral canal.
Using curved spring scissors, cut through the vertebral pedicles bilaterally while lifting and pulling the vertebral body rostrally to separate the ventral and dorsal aspects of the vertebrae and expose the spinal cord. Once the vertebral bodies are removed to reveal the lumbo-sacral enlargement, carefully clear the remaining roots that anchor the spinal cord with spring scissors until the cord floats free. Isolate the spinal cord with rostral and caudal cuts above and below the lumbo-sacral enlargement to make the target region of the cord float free.
For transverse slices, lift and place the lumbo-sacral segment by an attached route on a precut polystyrene block with a shallow channel cut in the center. Then, use cyanoacrylate adhesive to attach the block and cord to the sectioning platform and place it in the cutting bath containing ice cold sucrose artificial CSF slurry. Once the 300 micrometers thick slices are obtained, transfer the slices to an air interface incubation chamber containing oxygenated artificial CSF and allow the slices to equilibrate for one hour at room temperature.
To begin recording dorsal horn activity, transfer the slice from the incubator to the MEA well using a large tip pasteur pipette filled with artificial CSF and add additional artificial CSF. Use a fine short hair paint brush to position the slice over the 60 electrode recording array. Then, place a weighted neck over the tissue to hold it in place and promote good contact with MEA electrodes.
Place the MEA in the recording head stage. Check the position of the tissue over the electrodes using an inverted microscope to confirm that as many electrodes as possible are under the superficial DH.Ensure that at least two to six electrodes do not contact the slice. After connecting the camera to the device, take a reference image of the slice relative to the MEA for use during the analysis.
Then, press start DAQ in the recording software and confirm that all electrodes receive a clear signal. Next, attach the perfusion inlet and outlet lines to the MEA well filled with artificial CSF and turn the perfusion system on. Check the flow rate and ensure that the outflow is sufficient to prevent overflow of the superfusate.
After equilibrating the tissue for five minutes, record the raw baseline data for five minutes. Move the perfusion inlet line from artificial CSF to a 4-aminopyridine solution and wait for 12 minutes for the 4-aminopyridine induced rhythmic activity to reach steady state. Then, record five minutes of 4-aminopyridine induced activity and be prepared for the subsequent recordings to test the drugs or check the stability of 4-aminopyridine.
Following each recording session, rinse the lines with artificial CSF. After removing the MEA from the head stage and removing the net and the tissue from the MEA well, rinse the MEA and net with artificial CSF and repeat the whole process with a new slice. Open the analysis software and load the pre-made analysis layout.
Open the file of interest and deselect the reference electrode and any electrodes deemed to be excessively noisy. Set the time window for analysis. Then, move to the cross channel filter tab.
Select the complex reference and reference electrodes based on the image taken and notes made during the experiment and press Explore before continuing. Move to the EAP filter tab and apply a second order high-pass Butterworth filter to remove LFP activity. Move to the LFP filter tab and apply a second order band pass Butterworth filter to remove EAP activity.
In the EAP detector tab, ensure to select auto threshold, tick the rising and falling edge boxes, and set the dead time to three milliseconds. To set positive and negative thresholds, inspect the data by returning to the raw data analyzer screen, moving the time marker, returning to the EAP detector tab, and pressing explore. Repeat the process until the set detection threshold captures EAPs without capturing noise or non-physiological activity.
In the LFP detector tab, ensure to select the manual threshold, tick the rising and falling edge boxes, and set the dead time to three milliseconds. Set threshold for one electrode and, once satisfied, select apply to all and then press start analysis. The bath application of the voltage gated sodium channel antagonist tetrodotoxin abolished both EAP and LFP activity, confirming the spike dependency of these signals.
The stability of 4-aminopyridine induced activity parameters was characterized for EAP or LFP. All activity characteristics plus the coincidence of activity for LFPs were stable based on the similarity of 4-aminopyridine induced activity at 12 minutes after 4-aminopyridine application and 15 minutes later. The feature's EAPs or LFPs were characterized by an increase in the number of coincident events detected across multiple electrodes.
The number of linked adjacent electrodes, and the strength of linkages between adjacent electrodes and LFPs following 4-aminopyridine stimulation and then relative stability. The slice orientation is an important consideration for in vitro preparations. 4-aminopyridine stimulation induced a similar rhythmic activity in the SDH regardless of slice orientation.
Finally, to shed further light on the relevance of 4-aminopyridine activation of the DH networks, an elevated potassium artificial CSF solution was bath applied and shown to evoke a similar DH response to 4-aminopyridine stimulation. Key steps in the protocol are in the tissue preparation and placement of sections, ensuring the tissue is healthy through careful, but timely, dissection, as well as delicate optimal positioning of the tissue on the MEA will aid in obtaining quality results.
