Lab 6. Recording from the auditory nerve.

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.

Methods:

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 airpassage. 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 which 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: neuro730
Password: (as given in class)

Acoustic Calibration

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.

• For what reason might this be an important consideration in recording from the auditory nerve?

The electrode will be advanced in ~1um steps using a remote controller. During the advance a sweep tone 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.

Then click on XP SER-the range sweep rate, level, duration of the sweep, etc. can all be set. 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.

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.

Type EM TH. .....The same information can be obtained from a response field (area) using short tones as stimuli.

Collect: a 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. AM. EM AM.

(masked RA & speech?)

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. The data can be shared if not everyone is successful in the data collection. It is possible to try on an alternate day. It is also possible to leave open the evening to come back and finish up. This is preferable since fewer animals are used this way.

Generate plots for your unit (s). (use $RAP). You have to define the data file. DF fname

Select the data sequence e.g. ID 3-2.

There are some analysis macros that are prepared.

Short tones at CF: use EM n730pst.

Rate level function: use EM n730rlf

• Compare the FTC to an IRC.
• Is there any difference between the randomized rate curve and the monotonically increasing level curve?
• Superimpose the two on the same graph.
• Should you have expected any difference?
• What factors play a role in adaptation in an auditory nerve fiber?
• What other measures of temporal/spectral processing did you/could you try?