Willeys Methods

A large, robust Sears milling head was mounted vertically on a heavy steel plate bracket. The jaws of the milling head were used to grip an aluminum plate measuring 7 • 8 • 5/8 in. thick. Mounted on the plate was a 6-in.-diameter, angle-indexed disk supporting a vertical, V2-in.-diameter, 11.5 in. stainless steel rod. The disk and rod were free to rotate together in the plate to select . . A dc servomotor and an angle-sensing potentiometer were attached to the milling head to generate known tilts of angle $ at programmed rates. The motor was controlled by a closed-loop servo system.

An Arenivaga colony was maintained in the laboratory; the original animals were obtained from Dr. Hartman. A living animal was mounted ventral-side down on the disk using Cenco tackiwax, and dissected to expose its head. The head was then immobilized with a wax collar, and a trapezoidal window was cut in the frons. The antennae were removed and the antennal nerves severed. The protocerebrum was exposed, and kept moist with a special saline solution.

Extracellular nerve recording was done using etched, glass-coated, 0.01-in.-diameter, 70% Pt/30% Ir microelectrodes. Electrodes were similar to those used by Northrop and Guignon (1970). An electrode was spot-welded into the end of a No. 26 metal hypodermic needle, the tip was then electrolytically etched to a ne point in a cyanide bath, then insulated with solder glass down to the tip. The bare tip was coated with ne platinum black to lo wer electrode impedance and reduce noise. The hypodermic needle and microelectrode were held by a micromanipulator attached to the stainless steel post in the center of the rotatable disk. A ne Ag|AgCl wire electrode was placed in the moist interior of the head as a reference electrode. Once the microelectrode penetrated the outer membrane of the protocerebrum, it was slowly advanced until a candidate positional unit was found. Once a candidate PCP unit was located, the experimenter veri ed that it w as purely positional. If the unit were purely PCP, air puffs from a medicine dropper directed at the cerci would not affect the ring rate of the unit, nor would an ON-OFF visual stimulus, or loud low-frequency sounds; tilting the animal would, however.

A mixture of mineral oil and white vaseline was then infused around the pro-tocerebrum to prevent drying and to give electrical insulation. When the head angle . was changed, the disk, rod, animal, and microelectrode rotated as a unit. Tilt angle $ was controlled electrically by the servosystem. The entire system was extremely robust, and weighed over 50 kg. It was especially important that the relative position of the animal's brain and the electrode tip not change during selection of . and tilting ^ used for all of Willey's preparations was 45°. Tilts were done at 3.2°/s.

An exposed microelectrode tip was typically a 45° cone, about 3 ^m high. Thus, in dense nerve tissue such as the protocerebrum, it picked up the external potential changes caused by several neurons ring in its proximity. A relevant protocerebral positional unit (PPU) was not always the largest unit potential recorded. Thus, Willey used a pulse-height window circuit (Northrop and Grossman, 1974) to select the desired PP unit. If another, non-PP unit had an amplitude close to that of a desired PP unit, the window was useless, and a new recording site had to be found.

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