The goal is to have each individual locate a cochlear nucleus unit 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 cochlear nucleus.

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).

A tracheotomy is performed. The skull is cleared of tissue and mounting screws are inserted. 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. The dura mater overlying the cerebellum is opened and the cerebellum' is aspirated to expose the region where the cochlear nucleus resides: just medial to the auditory nerve (which will not be visible). A 2% agar solution is placed over the CN (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.

• Make a sketch of where the electrode enters the CN. This will help you think about where the recording is made. Be especially careful to record all information about the depth of the electrode from the time it touches the surface of the CN. This event is noted as the electrode is advanced toward the surface through the agar by a sudden shift of 20 to 60 mv negative. Record the CF, PSTH response type and threshold of each unit encountered.
• Make a sketch later and indicate the unit(s) that you chose to analyze.

LOGIN: Username: neuro730

Password: (as given in class)

Acoustic Calibration

Micropipettes 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 one molar potassium chloride solution). The impedance (Z) measured at a low frequency (< I kHz) should be between 20 & 60 MW. Lower Z implies a larger tip and often longer recording times. High Z electrodes are better for recording from smaller neural elements.

In recording from the auditory nerve, both KCl and NaCl have been used as the electrolyte in the micropipette. It doesn't seem to make any difference. In recording from the cochlear nucleus could there be an effect of the contents of the electrode on the recording? E.g., there is always a possibility the one might puncture the cells- become intracellular thereby exposing the rather small volume of the cell to the electrolyte. If enough ions are leaked into the cytoplasm it is possible to alter the behavior of the cell. Enough chloride ions in the cell will alter the chloride reversal potential and cause normally negative gong IPSPs to flip- become positive going. Potassium acetate (as the electrolyte) is used to prevent this from happening--we won't worry about it. KCl electrodes have very good recording properties and remain stable for long periods of time.

The electrode will be advanced in ~1 mm steps using a remote controller. During the advance a sweep tone is played at a 70 dB SPL level in order to locate units, especially those that have low spontaneous activity. Note that it is very likely that ANFs will also be recorded in this procedure. They are more dense and sometimes more stable than the CN cells are. Be careful in interpreting your results.

• Note the criteria you used to differentiate between 1st and 2nd order neurons.

First start the DCP program. Type DCP.

Type EM INIT.

Then click on XP SER-the range sweep rate and level can 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 analysis.

Having determined the CF: collect an FTC (i.e. a frequency threshold curve).

Type EM TH. Default parameters will be set initially. You may need to change them especially for a noisy unit.

When done with the threshold program,

Collect: a RA (Response Area, use EM RA). Use a frequency range appropriate for your unit.

PSTH (short tone at CF, 250 reps, 50 ms every 200 ms=25% duty cycle). Use EM RCP

Rate level function: sequentially and in random order of SPL. Use EM RC.

Try a number of other data collection macros:

e.g. EM AM, amplitude modulation.

EM NRC, noise rate level function.

EM MRA or masked RA

EM MEOW for a cat vocalization

For the MRA you should first run an NRC or noise rate curve in order to determine what level of noise will produce 50% of the maximum response to noise. This value will then be used in the MRA procedure. The MRA is useful for mapping the extent of inhibitory sidebands.

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. We may share the data else 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.

Provide a brief description of the experiment: Intro, methods, results, and a brief discussion.

• Generate plots for your unit (s). (use RAP on the computers in Room 85).

The datafile is: N730L7

Specify the datafile with df N730l7 before running a macro.

• There are some analysis macros that are available

Short tones at CF: use n730pst.

Rate level function: use n730rlfc

(note: to run a macro in RAP, enter "em macro-name", e.g. "em n730pst")

• Analyze both the rate and temporal transfer functions for AM if you were able to collect them. How do they compare to ANF responses?

• What could explain the differences between the responses seen in the CN responses you recorded and ANF responses? Examining both temporal response patterns, response maps, isorate curves, etc ?

• If you have at least 10 repetitions for each 'point' in the response area for a unit, you can examine the PSTHs over the entire stimulus space. Type em PSTONLY.

• Provide graphs for all the data you collected, e.g., MRAs, AM, and comment on how these results indicate additional processing in the cochlear nucleus.

Note you can obtain spike rate plots for any data or sync coefficient plots using the commands: PL SP or PL SY after you select the proper data sequence using the ID # command. Plot any TH data with the command PL TH.