NEUROBIOLOGY LAB Spring 2005        

EXPERIMENT 4:

Profiles of the Sensory-evoked Potential

in the Forepaw Area of the Rat Somatosensory Neocortex.

 

ANESTHESIA and SURGICAL PREPARATION

            This lab will apply surgical procedures to record the response of the brain to sensory stimuli applied to the skin. All procedures conform to the animal care guidelines of the Public Health Services and the National Institutes of Health under the supervision of the UTD animal care and use committee, protocol #99-02.

 

Adult male rats (Spague-Dawley rats, 150-350 g body weight) will be deeply anesthetized with Urethane (intraperitoneal injections of 1.5g/kg body weight).  Approximately 15 min. following initial anesthesia, the rats will be tested for signs of arousal.  Only rats that do not respond to handling or tail pinch will be used.  Responses will be tested regularly at least once every 15 min. to ensure deep levels of surgical anesthesia.  If any responses are observed, additional anesthesia will be administered and the procedure will be delayed until no responses are observed.  Local anesthesia will be applied to all surgical wounds (subcutaneous injections of 2% Lidocaine).  The experiment will be immediately terminated by overdose of Urethane (3g/kg body weight) in the event of any signs of distress.

 

Following deep anesthesia, rats will be placed in a Kopf stereotaxic device (remember to secure tooth bar with nose clamp and level the skull) and the dorsal skull plates will be exposed by a midsagittal incision through the scalp.  Immediately following the incision the local anesthetic will be injected subcutaneously.  Then soft tissue will be cleared from the skull using cotton applicators, bone wax applied to stop oozing if necessary, and pencil marks will be placed on the skull centered at the intended penetration site: 4.0 mm directly lateral to bregma.  You should refer to the atlas (Paxino and Watson) to identify the structures under these penetration sites.

 

            Before proceeding with the DRILLING, check anesthesia level by looking for a response to tail pinch. If any response is observed, stop the procedure, inject supplemental anesthesia (0.5 ml 30% Urethane I.P.) and wait until no response can be elicited before proceeding. The instructor will be present at all times to monitor level of anesthesia. 

            Next drill a small square bone flap ( ~ 4 mm X 4 mm) centered around the marked penetration site. Remember no pressure should be needed to drill.  Tilt the drill and cut by pulling the drill away from the tip using the shaft to control depth.  Cut through the skull with several shallow lines until the bottom of the cut becomes moist.  When the bone flap is cut loose all the way around, gently lift away. This drilling can be done without any bleeding, but if bleeding occurs simply apply a cotton tip to it until the bleeding stops.  If you believe there is too much damage, drill a flap at the corresponding site on the other side. 

            After the flap is drilled, wash the skull with normal saline (0.9% NaCl) and gently dry with a KimWipe tissue.  Inspect the holes under the dissection scope to ensure the penetration site is not occluded by a thin layer of residual bone.  This may require gently touching the surface. 

 

Once you have removed the bone over the penetration site, CALL THE INSTRUCTOR. The instructor will drill two screw holes in the frontal bone on each side for the recording reference and ground.  After drilling these holes, the instructor will insert the screws with the gold pins attached and connect the system ground and the recording reference to the pins. The instructor will also attach a small screw to stimulate the forepaw contralateral to the recording site using tape to close the forepaw around the screw. The Grass SD9 Stimulator will be connected to the forepaw screw with the stimulation reference clipped to the scalp clamp. 

 

VERIFYING THE LOCATION OF THE FOREPAW AREA

            Before proceeding with ELECTRODE POSITIONING, check for deep anesthesia by looking for a response to tail pinch.  As always, apply a supplemental dose if necessary and wait until the rat is nonresponsive before proceeding.  The recording electrode will be positioned stereotaxically as precisely as possible using the manipulation towers.  Remember to straighten the electrode so it is as nearly vertical as possible (align with the vertical bar of the nose clamp).  Remember that to move in the anterior-posterior direction, the clamp on the rail must be loosened and this can cause the tower to tilt which may damage the electrode.  So, always raise the electrode at least 1 cm above the skull before moving in the anterior-posterior direction.

 

            The first step in stereotaxic positioning is to ZERO YOUR COORDINATE SYSTEM.  We will be using the skull landmark bregma for the anterior-posterior zero coordinate.  A teflon-coated stainless steel wire (50 mm diameter) will be used to record from the forepaw area of the cortex.  Position the electrode directly over bregma.  Then lift the electrode at least 1 cm and move the electrode lateral 4.0 millimeters to the penetration coordinate.  Now lower the electrode to the surface of the brain while looking under the dissection scope until it just touches the surface.  Then lift the electrode.  If bone is in the way, carefully drill it away without damaging the electrode.   Puncture the dura mater at the site where the electrode contacts it using a dura pick prepared by bending the tip of a hypodermic needle.  Finally, lower the electrode until it just touches the brain where the dura is removed and note the dorsal-ventral measurement on the vertical arm of the tower.

 

            Before proceeding with ELECTRODE INSERTION,  check anesthesia level and apply anesthesia if necessary as described above. Turn on the amplifier (AC 100X; 0.1-10k Hz filters) and connect the output to the oscilloscope.  You should observe a varying electrical signal recording from the surface.  This signal is the cortical electroencephalogram (EEG).

 

After checking that the stimulator toggle trigger is in the off position, turn on the stimulator and adjust the duration to 0.05 ms, the delay to 5 ms and the voltage to 5 V.  Be sure that the pretrigger output of the stimulator is connected to the EXT trigger input of the oscilloscope.  Trigger a single stimulus while looking at the rat for any signs of reflexive movement evoked by the stimulus.  The oscilloscope trigger level may need to be adjusted to capture the EEG response in store mode.  Continue to trigger a stimulus about once every 2-3 seconds while gradually increasing the voltage.  You should see a deflection in the EEG corresponding to the neural response of the cortex under the recording electrode.  Adjust the oscilloscope vertical amplifiers and time base for an optimal sweep display.

 

            Before proceeding with ELECTRODE INSERTION,  check anesthesia level and apply anesthesia if necessary as described above.  Ask the instructor to check the surface EEG response to forepaw stimulation. 

 

PART I: Plot the input/output response of the somatosensory-evoked potential.

Record the responses at the surface of the cortex to a range of stimulus voltages from subthreshold to maximal and make a plot superimposing this range of responses.

FOR YOUR LAB PAPER:

(1)  Include the plot recorded at the surface of five superimposed responses from threshold to maximum.

(2)  Include a graph showing this I/O response plotting peak amplitudes as a function of stimulus intensity (there should be more than five points in this graph including at least two maximal responses).

 

PART II: Plot the DEPTH PROFILE of the somatosensory-evoked potential.

Adjust the stimulator to give a half-maximal response.  Begin a new plot of the surface recording. Then lower the recording electrode by 0.2 mm below the surface of the cortex.  Repeat the stimulation and superimpose a plot of the depth response on the surface response.  Repeat this procedure at 0.2mm steps and superimpose the plots to demonstrate how the response changes as you go deeper and deeper.  The resulting plot of superimposed responses recorded at all depths is called the depth profile. If the response is generated at this site in cortex, then the surface and the depth responses should be opposite in polarity (i.e. the surface response should involve a positive peak and the depth response should flip over to become a negative peak).  Why does this confirm the local origin of the neural response?

FOR YOUR LAB PAPER:

(1)  Include the plot of superimposed responses recorded at each depth.

(2)  Include a graph showing the peak response amplitude as a function of depth.

 

PART III: Plot the input/output response at the depth of maximum negativity.

Position the electrode at the depth of maximum negativity which you located with your depth profile.  What can you say is located at this depth of maximum negativity? Record the responses to a range of stimulus voltages from subthreshold to maximal and make a plot superimposing this range of responses. 

FOR YOUR LAB PAPER:

(1)  Include the plot recorded at the depth of maximal negativity of five superimposed responses from threshold to maximum.

(2)  Include a graph showing this I/O response plotting peak amplitudes as a function of stimulus intensity (there should be more than five points in this graph including at least two maximal responses).