NEUROBIOLOGY LAB Spring 2005

EXPERIMENT 5: Hippocampal Long-Term Potentiation (LTP)

in the in vivo Rat Preparation.

IN VIVO ANESTHETIZED ACUTE SURGICAL PREPARATION

This lab will apply acute surgical procedures to prepare anesthetized rats for intracranial stimulation and recording of hippocampal responses. 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 ( 0.05 g Urethane I.P.) 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 (>3 g/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 sites:

For recording: 4.0 mm posterior to bregma; 2.5 mm lateral to midsagittal.

For stimulating: 8.0 mm posterior to bregma; 4.8 mm lateral to midsagittal.

You should refer to the axis (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 no response is observed, inject the supplemental dose of anesthesia and proceed. If any response is observed, stop the procedure, inject supplemental anesthesia and wait until no response can be elicited before proceeding. Next drill small square bone flaps around the marked penetration sites. Remember no pressure should be needed. 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 all the way around, gently pry loose and lift away. This 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 holes on the other side. After the holes are drilled, wash the skull with normal saline (0.9% NaCl) and dry. 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 with the dura pick.

In addition to the holes over the penetration sites for recording and stimulation, three screw holes will be drilled; one in the frontal bone for the preparation ground; one anterior and lateral to the recording site in the parietal bone (about 4 mm directly lateral to bregma) for the recording reference; and another caudal to the stimulation site in the occipital bone for the simulating reference. After drilling these screw holes insert the screws.

Before proceeding with ELECTRODE POSITIONING, check for deep anesthesia by looking for a response to tail pinch. As always, apply the supplemental dose and wait until the rat is non-responsive before proceeding. The recording and stimulating electrodes will be positioned stereotaxically as precisely as possible using the manipulation towers. Remember to straighten the electrodes so they are as nearly vertical as possible (align them 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 electrodes. 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. Position the electrode (position the stimulating electrode first) directly over bregma and note the anterior-posterior measurement on the rail. Then lift the electrode at least 1 cm, move the tower posterior to the penetration coordinate (4.0mm for recording; 8.0 mm for stimulating) and position the electrode directly over the midline and note the medio-lateral measurement on the cross arm of the tower (from above). Move the electrode lateral to the penetration coordinate (2.5 mm for recording; 4.8mm for stimulating). 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 barbing 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. Repeat for both electrodes.

Before proceeding with ELECTRODE INSERTION, check anesthesia level and apply anesthesia if necessary as described above. Connect the system ground to the screw in the frontal bone; the recording electrode reference to the screw near the recording site; and the stimulating reference to the posterior screw. Turn on the amplifier (AC 100X; 0.1-10k Hz filters) and connect the output to both an oscilloscope and an audio monitor. The stimulator should be set to 0.5 ms pulse duration, 2 ms delay, 0.2 pulses per second and a voltage of 5 volts.

First, SLOWLY lower the recording electrode while listening to the audio monitor and watching the electrical activity of single units on the oscilloscope. Note the depth where intense, storm-like activity is observed. This should be near 1.8 mm below the surface (see atlas). Continue to slowly lower the electrode until rain-like activity is observed (about 2.6 mm below the surface).

Second, turn the simulator to REPEAT and adjust the oscilloscope to observe evoked potentials (ext. trigger from stimulator, 2 ms / horizontal division, 50 mV / vertical division). SLOWLY lower the stimulating electrode while watching the response on the oscilloscope. At some point the response should grow above .5 mV amplitude. If you go below 5 mm and still find no response, check your position and call an assistant. The optimal response will be a positive-going field EPSP with a population spike of at least 1 mV. To get this optimal response it may be necessary to slightly adjust your recording depth. The response to look just like that in Bliss and Lomo (1973) or McNaughton et al. (1978).

OBJECTIVES:

The overall objective of this experiment is to replicate the pioneering findings of Bliss and Lomo, 1973. This involves measuring the difference in the hippocampal response before and after a brief period of intense activation. This robust form of synaptic plasticity is an excellent candidate for the neurobiological basis of long-term memory storage.

1. PRE-TREATMENT INPUT-OUTPUT ANALYSIS: Upon obtaining an optimal evoked hippocampal response that remains stable for at least ten minutes, perform a stimulus/response (Input/Output) analysis. Plot the response waveforms evoked across a range of stimulus intensities from below threshold to just above saturation. Note the voltage intensity of the stimulus at the threshold for the field EPSP and also at the threshold for the population spike. From these plots you will measure the amplitudes of the EPSP and spike as shown below. Always use the same latency, T, to measure the EPSP.

Plot these measures as a function of the stimulus intensity. Also, since the spike is triggered by the EPSP, you should plot the spike amplitude as a function of EPSP amplitude. These plots will characterize the input-output properties of this neural system.

2. PRE-TETANUS BASELINE: Adjust the stimulus intensity to the point that evokes a 1mV population spike. Superimpose five plots of the responses to this stimulus delivered once every two minutes. If the response does not stay very stable during this baseline period, readjust the stimulus intensity if necessary and repeat the baseline. Measure the EPSP and spike amplitude from the oscilloscope on each of these five responses so they may be plotted as a function of time. You will continue this time series of measurements after the train throughout steps 4 & 5. You will need to work together to get these two measures, write them in your notebook, plot the waveform and keep accurate timing. You should practice before starting because you can not turn back after the train.

3. TETANIC STIMULUS TRAIN: After obtaining a stable baseline, turn off the stimulator. Adjust the number of pulses per second to 100. Then, manually switch the stimulus to repeat for as close to 1 second as possible. Repeat this five times, about once every three seconds. Then readjust the number of pulses per second to 0.1.

4. POST-TETANIC POTENTIATION: Superimpose plots and note measures of the responses evoked once every minute for the first ten minutes. THIS WILL REQUIRE COORDINATION.

5. LONG-TERM POTENTIATION: Superimpose plots and note measures of responses evoked once every five minutes for the next 30 minutes.

6. POST-TREATMENT INPUT-OUTPUT ANALYSIS: Repeat the stimulus/response procedure described in step one using the same stimulus intensities.

As figures for your laboratory report, you should include at least the THREE plots from the above (the before and after I/O series and the baseline/post-tetanic time series) and TWO graphs:

input-output curves before versus after tetanus.

the time course of the EPSP and spike amplitudes across the experiment.

REFERENCES

Bliss, T. and T. Lomo (1973) Long-lasting potentiation of synaptic transmission in the dentate area of the anesthetized rabbit following stimulation of the perforant path. J. Physiol. (Lond.) 232:331-356.

McNaughton, B.L., R.M. Douglas and G.V. Goddard (1978) Synaptic enhancement in fascia dentata: Cooperativity among coactive afferents. Brain Res. 157:277-293.