The Activity of Single Cells in the Midbrain and Hypothalamus of the Cat during Affective Defense Behavior
Methods Page 3

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Experimental procedure

Several electrode guides were implanted over the hypothalamus and midbrain on the skull of the cat during a sterhe operation conducted while the cat was anesthetized with sodium pentobarbital. The electrode itself was not introduced until the day of recording, at least several days after the operation.

The electrode was advanced by turning the sleeve in which it was mounted, which caused it to screw down the threads of the guide. One twentieth of a revolution advanced the electrode 40 μ. The electrode was secured in one position by tightening a set screw on the sleeve.

Attack trials were conducted approximately every 100 μ advance of the electrode, whether or not a single cell had been located, which made it possible to find and test cells with no spontaneous activity. An attack trial consisted of the following sequence: a ten second baseline period was recorded during which the experimenter did not move or make any sound ; the door to the attacking cat was opened; the attacking cat was grasped by the collar; electrical stimulation was begun; as soon as the attacking cat began to hiss, the partition was opened and the attacking cat was moved towards the recording cat; the experimenter pressed the hand switch to mark each hiss or striking motion by the recording cat; then the attacking cat was withdrawn and the partition closed. It was often possible to discontinue the use of brain stimulation after the first few tria1s of a session since the cats would hiss and strike at each other whenever the partition was opened and they were moved near each other.

When a cell was located it was tested by the following manipulations of the cat. First an attack trial was carried out as described above. Then a trial of control manipulations was carried out including a tell second baseline period, presentation of ten clicks and ten flashes, lifting of the cat off the floor and dropping it again, pulling the cat's foreleg and releasing it so that the cat could pull it back, and pinching the cat's tail. The tail pinch was usually mild enough that the cat did not attack the experimenter, because attack would have confounded the results of tail pinch with those of affective defense, but it was strong enough to elicit a withdrawal response. Each manipulation was marked by a simultaneous press of the handswitch. Following the control manipulations the cat was given at least one more attack trial, and if the cell seemed to be related to the affective defense behavior additional trials of attack and control manipulations were conducted as long as recording could be maintained from the cell.

Other control manipulations were sometimes applied On separate trials. These included additional auditory stimuli as described above, moving the hand across the visual field, rotating or lifting the cat's head, grasping the paw, and stroking the cat's back. If the approach of the experimenter towards the cage and the opening of the door to the attack cat seemed to affect the cell's firing rate, this procedure was also marked by the hand switch.

After all recording from a cell was completed the area surrounding that cell was electrically stimulated and the intensity of stimulation was increased up to 0.8 mA or until the cat made a behavioral response. At certain points along each electrode track a DC current was passed to aid in histological localization.

At the conclusion of an experiment the cat was anesthetized and perfused with saline and 10% formalin. The brain was frozen, sectioned, and stained in the following manner: every second section was stained with Prussian blue to show up the marker lesions and then counterstained with neutral red or basic fuchsin; other sections were stained with cresyl violet or Weil techniques. Localization of cells was accomplished by interpolation between Prussian blue markers and recorded on the appropriate brain sections in the atlases of Jasper and Ajmone-Marsan (9) for the hypothalamus and Reinoso-Suarez (11) for the midbrain.

Criteria for viable single cells

Final data were based only on recordings from single cells which showed no signs of injury and which were held through at least one instance of affective defense and then returned to the original baseline firing rate.

Recordings were not analyzed unless the spikes of a single cell (as judged by consistent amplitude, interspike interval, and waveform monitored on the oscilloscope) were clearly larger than multiple unit background activity.

Recordings were also not analyzed if the spikes had abnormal waveform (notching or positive-negative waveform), abnormal frequencies (injury discharges or progressive and irreversible changes in baseline firing rate), or varying amplitude (change in spatial relationship between cell and electrode).

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