Physiology 735-lab 5
Recording from the auditory nerve
Oct. 29 report due Nov. 4
Check: http://www.physiology.wisc.edu/phys735/labs/ for the latest.
See the lab orientation on the WEB.
The goal is to have each individual locate a nerve fiber and collect a FTC, response area, short tone at CF, rate curve (with and without randomization). There will be 'Macros' that perform the necessary initialization of the equipment.
The animal will be prepared and the electrode positioned above the auditory nerve.
A chinchilla, (average weight 0.4 kg) is anesthetized with sodium pentobarbital at a dose rate of 75 mg/kg. Booster doses are 0.2 cc's and administered whenever the animal responds to a paw pinch (you are to check every 15min).
Surgery. A tracheotomy is performed to minimize the chance that fluids will obstruct the air passage. The skull is cleared of tissue and mounting screws are inserted and cemented in. The ear is removed and an opening is made in the bulla to insert a vent tube. The skull overlying the cerebellum is removed. This is a narrow region between the bullas that are very large in the chinchilla. The dura mater overlying the cerebellum is opened and the cerebellum is aspirated to expose the region where the auditory nerve inserts in the temporal bone. The nerve is not visible without retracting the brain stem. Small cotton pellets are used as wedges between the skull and the brain stem to stretch the nerve. Only a very small extent will be visible. A 2% agar solution is placed over the nerve (depth of covering ~1 mm). This serves to stabilize the region-i.e. reduce brain pulsations. A further step in stabilizing the brain is to mount a plastic chamber on the skull. This chamber will be filled with warmed mineral oil (37 C) and covered with glass. This forms a hydraulic seal that replaces the seal of the skull itself.
LOGIN: username: phys735
Select your group id (a or b)
Run NEUCAL: 100 - 20,000Hz in 100Hz steps.
Plot calibration curve. Be sure to save the curve in the file for data collection.
Micropipetes are manufactured from 1 mm borosilicate glass tubes using any of the modern microelectrode pullers, e.g., Kopf, Sutter, etc.
The glass is drawn to a tip diameter between 0.1 and 0.2 mm. It is filled with a 1 MKCl solution (i.e., a 1 molar potassium chloride solution). The impedance (Z) measured at a low frequency (< 1kHz) should be between 20 & 60 M W. Lower Z implies a larger tip and often yields longer recording times. High-Z electrodes are better for intracellular recording but are more likely to damage the neuron/axon due to their sharpness.
The electrode will be advanced in ~1um steps using a remote controller. During the advance a sweep tone (FM) is played at 70 dB SPL in order to locate ANFs, especially those that have low spontaneous activity. That is, the firing in response to the tone lets the presence of an ANF be known to the experimenter.
First start the DCP program. Type $DCP.
Type EM INIT. *this initializes some parameters for the data collection
Then click on XP SER-the frequency range, sweep rate, level, duration of the sweep, etc. can all be set. A sweep range of 100-30000Hz with a 1 sec rise/fall time is a good starting point. The on-line display of discharge rate vs frequency aids in determining the CF of the unit along with the range of frequencies that the unit responds to. This is useful for setting the frequency and intensity range for subsequent data collection. You can plot the sweep response.
Spikes are discriminated using the trigger circuit of the oscilloscope. You have to set this level… so as to be certain that noise isn’t generating false triggers that will be timed and entered in the data base as spike firings. The oscilloscope has to be watched constantly. The gain of the scope can be adjusted as needed.
Name all the data sets: Unit no.-sequence no., e.g., 3-4 is the fourth sequence for the third unit encountered.
Having determined the CF: collect a FTC (i.e. a frequency threshold curve). The normal criteria is 1 additional spike during the presentation of a 50ms tone pip. This can fail for high-spon ANFs.
Type EM TH. .....The same information can be obtained from a response field (area) using short tones as stimuli.
Collect: a response area $RA. Click on EM RA.
PSTH (short tone at CF, tone repeated 250 times.) Click EM RCP.
Rate level function: sequentially and random presentation. Click EM RC.
You could try a number of other data collection macros:
e.g. amplitude modulation. EM AM.
Try to collect as much data as you can in the three-hour period with each member of a group trying to record the data from one fiber. It is possible that during a 3-hour period the recordings will not be completed. However, the preparation should be there beyond three hours so there is flexibility. If need be, the data can be shared if not everyone is successful in the data collection. It is also possible to leave open the evening to come back and finish up.
Report: Introduction, Methods, Results, Discussion.
Generate plots for your unit (s). (use $RAP).
You have to define the data file. DF file_name.
Select the data sequence e.g. ID 3-2.
There are some analysis macros that are prepared.
Short tones at CF: use EM p735pst.
Rate level function: use EM p735rlf
1) Plot Spike_rate vs frequency type pl sp;
2) Plot sync coefficient vs frequency (with a ‘mask’ on. Set ry mask .001). What does this do?
3) Set xv 2 and plot spikes. This results in a set of rate-level curves. Then reset the x-variable: xv 1