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Comparison of thresholds for behavioral and electrical responses to cortical electrical stimulation in cats
EXPERIMENTAL
NEUROLOGY
Comparison
6,
315-331
(1962)
of Thresholds
Electrical
Responses
for
to Cortical
Stimulation WALTER Department
of Physiology,
and
Electrical
in Cats J.
FREEMAN
University Received
Behavioral
June
of California,
Berkeley,
California
27, 1962
Cats with stimulating and recording electrodes implanted in the prepyriform cortex were trained to press a bar for milk upon electrical stimulation of the lateral olfactory tract. Thresholds were determined for the cortical evoked potential and for a conditional response elicited by the same electrical stimulus. Threshold for the evoked potential, defined as stimulus voltage for minimum detectable responses, varied with method of detection. Threshold for electrical responses evoked by single shocks and recorded in an ink tracing was higher than that for responses evoked by trains of stimuh and recorded with an oscilloscope. Both exceeded threshold for responses evoked by low-frequency stimulation and recorded by means of an average response computer. Threshold for the bar-press response (after prolonged overtraining) corresponded on the average to the middle threshold, implying that the level of activity induced by stimulation had to approach or exceed the amount of synchronized spontaneous activity for a behavioral response to occur. Conditioning took place after orientation to the stimulus, which was achieved in this experiment by intense electrical stimulation just below threshold for seizure. Reduction of behavioral threshold to electrical threshold required additional training. Fully trained cats did not respond to externally generated fields of current grossly resembling prepyriform evoked activity, implying that large-scale extraneuronal currents (as compared with current distributions at the cellular level) were of no functional importance. Introduction
Electrical stimulation of the lateral olfactory tract through chronically implanted electrodes may be followed by a prepyriform cortical evoked potential, a stereotyped motor response by the animal, or a conditional response, each depending on stimulus parameters and the history of 1 This work was supported by a grant from the Foundation’s Fund for in Psychiatry (59-204). The assistance of Dr. Bernard Baird, Arthur Daily, Furman, and Richard Zipf is gratefully acknowledged. 315
Research Gershon
316
FREEMAN
stimulation. The purpose of this study was to compare thresholds these events. Methods
for
Bipolar stainless steel (#30) wire electrodes with tips 1.5 mm vertically apart were chronically implanted in six cats by methods previously described (4). In three cats, two pairs were placed 6 mm apart in the left and two in the right cortex. Each pair was suitable for either stimulation or recording. In three cats, four pairs were placed in the left cortex 2 to 4 mm apart, ranging from near the base of the olfactory bulb to the temporal portion of the prepyriform cortex. All placements were confirmed anatomically. In each case one electrode tip was in contact with the molecular layer or lateral olfactory tract and the other was at the base of the cortex. Seventeen of twenty-one placements aimed at the tract were in contact with it, and three were within 0.3 mm. Stimuli were delivered in bursts of pulses from a Tektronix 161 pulse generator through an isolation transformer, which also removed the d-c component. Pulse width (10 psec) was maintained at one-tenth chronaxie of the cortical evoked potential in order to establish wide variability in stimulus voltage by using as the scale of measurement the rising limb of the strength-duration curve. Pulse voltage was monitored with an ozcilloscope to within *3%. Pulse repetition rate was set to coincide either with the frequency of spontaneous activity or with maximum root-mean-square (r.m.s.) amplitude of evoked potential during short bursts of pulses. Burst duration was 0.4 set and burst repetition rate was 0.8 to 1.0 per sec. Records were made with a Grass 5P.S polygraph, a Tektronix preamplifier and oscilloscope and Dumont camera, and a Mnemotron CAT average response computer. Frequency-response curves for determination of optimal stimulus rate were constructed by a method previously described (5). Behavioral testing was carried out in a sound-resistant box made from an 8-ft3 refrigerator, which was fitted with a bar for the cats to press, a tube for delivering milk to the cats, and a signal light. Sufficient control of ambient noise was not achieved to permit quantitative study of the acquisition phases of learning. The cats were fed every 48 hours, thresholds being determined 1 to 2 hours prior to feeding, when response latencies for bar-pressing were usually less than 1 set for suprathreshold stimuli. Behavioral thresholds were determined numerous times, but those reported here were taken several months after establishment of the responses and after prolonged overtraining using surface-negative stimuli.
THRESHOLDS
317
For each site, intensity was raised in steps of about 10% of expected threshold allowing time for bar-pressing to occur between steps until both behavioral and electrical responses were observed. Surface-negative and surface-positive stimuli were used on alternate runs. The initial two trials were discarded, and thresholds were taken as the mean of the next three trials of each polarity. Results
Unconditional Responses. The effects of electrical stimuli on behavior were analyzed in terms of the threshold for single-shock evoked potential in an ink record for each stimulus site (using an alternate site for recording) rather than stimulus voltage, because this reduced the variability both between animals and between sites in each animal. Up to 6 X threshold no effect on behavior was seen in untrained cats. From 6 to 10 X threshold there was arrest and an orienting response (turning of the head and eyes in irregular fashion suggestive of search rather than specific forced movements, erection of the ears, and sniffing). These manifestations vanished upon sustained or oft-repeated stimulation. Above 10 to 15 X threshold, arrest became sustained, and after 5 to 20 set was followed by a seizure consisting of rhythmically repetitive blinking and retraction of the ears, lips and lower jaw at 2 to 3 per set, profuse salivation and lacrimation, occasionally urination, and rarely defecation or vomiting. On occasion extremely rapid locomotion occurred, not to be called flight since cowering and search for shelter were not in evidence. The duration of the seizure varied from a few seconds to more than a minute, during which the cat withdrew from the observer upon being touched or approached about the head and eyes but did not attack or vocalize. The seizure was followed by several minutes of abnormal behavior, which consisted either of rigid stance with repeated toneless vocalization, rolling on the floor by males or females, as in the female sexual afterreaction, hyperphagia if food was available, flight, unprovoked aggression, or excessively affectionate behavior such as kneading, purring, etc. The relation of postictal to preictal behavior was, unfortunately, not considered. In only one cat did such seizures terminate in tonic, then clonic convul,sions, and in this cat it was invariable. These seizures were accompanied by a repetitive spike pattern synchronous with the twitch of the muzzle, beginning at 4 to 6 per set and tapering to an irregular interval in some cats and a sustained, regular discharge at 2 or 3 per set in others (Fig. 1). The polarity of these
D k
THRESHOLDS
319
spikes was reversed between the electrodes, implying their origin in those cortical cells generating spontaneous activity, but in a relaxation rather than harmonic mode. Possible contributions of muscle or cable artifacts were excluded by the fact that similar facial and head movements induced by repeatedly threatening a blow to the muzzle were not accompanied by irregularities in the recordings. During the seizure and for several seconds thereafter both the “spontaneous” activity and evoked potential between spikes were diminished in amplitude-although neither was absentand during the abnormal behavior rose to relatively high amplitudes. Conditiomal Responses. The cats were trained to press a bar for milk upon stimulation either of the left (four cats) or right (two cats) prepyriform cortex, first by training (twenty trials daily for l-3 weeks) to press the bar in response to a signal light (on 15 set every 2-4 min, with milk available within 1 set of response on every trial), second by orienting using stimulation just below threshold for seizure, and third by conditioning to press the bar using somewhat lower intensities of electric stimulation (6-8 X threshold for the electrical response) as the conditional stimulus. Trains of stimuli were delivered for 1.5set at 45 per set in three cats and 30 per set in three cats every 2 to 4 min. During the early stages of training milk was delivered 1 set after the start of the electrical stimulus and during later stages conditionally upon each response. Criterion was set at nine correct responses out of ten consecutive trials. The cats established the response to electrical stimuli after twenty ‘trials daily for 2 to 3 weeks. Finally, the stimulus intensity was reduced until a stable threshold was reached, below which the animals consistently showed long latency, excessive bar-pressing between trials, or failure to respond altogether. This required another 2 to 3 weeks. After reduction in stimulus intensity, responses occurred to stimulus rates ranging from single shocks to 500 per set (the highest rate tested). They also followed stimuli at amplitudes and durations ranging from near threshold for the evoked potential up to threshold for seizure (Fig. 2). Stimulation at intensities near electrical threshold at sites in the ipsilateral cortex other than that used for initial training also elicited responses. Initially responses did not occur in three cats so tested to stimulation of the contralateral cortex, a finding of interest in view of the absence in these three cats (and in twelve other cats with bilaterally implanted electrodes) of a commissural evoked potential for the prepyriform cortex (4). High-
320
FREEMAN
amplitude stimuli, which were used to establish the absence of the barpressing response to contralateral cortical stimulation, evoked an orienting reaction indistinguishable from the preceding reaction to ipsilateral stimP-o-m-1.8 v.
.Ol msec.
24&c
No Response
----~----~-~----~-
2.8 Y
150 mv.
-01 msec
20 c.p.s.
24/set.
Response c ,
No Response
FIG. 2. Electrical response to electrical stimulation below threshold (1.8 v), at threshold (2.1 v) and at 1.3 X threshold (2.8 v) in bursts indicated by the horizontal bars. Lowest tracing shows sinusoidal activity extrinsically generated at that frequency assumed by evoked cortical activity when driven to the same amplitude.
Following appropriate training, responsesregularly occurred to near electrical threshold delivered to either or both cortexes. The number of cats was too few to compare rates of learning for the two sides.
ulation. stimuli
THRESHOLDS
321
The conditional nature of the response was established by reversibly inhibiting it through extinction (suppression of the response by withholding the reward), satiation, and distraction with competing stimuli, and differential discrimination to respond to only one of two sites stimulated in the same cortex. Such results differed in no way qualita$vely from similar results using the signal light or a tone rather than the electrical stimulus. The period of strong electrical stimulation was an essential part of this training schedule. Attempts to train five cats (four of which were later trained by use of strong electrical stimuli, the fifth having died of an intercurrent lung infection) either to press a bar for milk or to avoid a puff of air delivered to the face within 1 set of the onset of electrical stimulation did not give conditional responses after twenty trials daily for 4 weeks for each cat, using stimuli about 2 X threshold for the electrical response as to conditional stimulus, but without first eliciting the orienting response. Electrical Responses. Threshold for the behavioral response was variable but clearly defined for each trial. Not so for the electrical response. In theory the onset of electrical activity with increasing stimulus intensity was the level at which any single cell discharged in response to the stimulus. Partly because of spontaneous activity (“noise”) and partly because of the unpredictability of the optimal location of the active site with respect to the recording electrode, the practical evaluation of threshold had to be in terms of detectability, and this varied with method of measurement. Four methods were used. The detection in ink tracings of potentials evoked by single shocks was very useful for initial selection of stimulus parameters, but it failed to take into account the relation between stimulus rate and response amplitude, and it was strongly affected by the level spontaneous activity. The second method consisted of applying short bursts of stimuli at different pulse rates to the tract and measuring the r.m.s. amplitude of the bursts of evoked potentials. The frequency-response curve (5,6) thus derived (Fig. 3) provided an estimate of the optimal stimulus frequency for evoking maximum response amplitude. Optimal frequency always increased with decreasing stimulus intensity and had to be set by extrapolation at threshold intensities (8). This method proved ineffectual after overtraining (7)) because the pulse rate for optimal amplitude no longer coincided with the frequency of spontaneous activity in these highly
322
FREEMAN
trained cats. This discrepancy in frequencies meant that the spontaneous activity could no longer be averaged out of the evoked potential by this method. Only when the frequency of the evoked activity equaled that of spontaneous activity-as it did in the naive state (f)-did the two activities add vectorially with cancellation of the latter. Cancellation
Average amplitude -
20
30
40
Number of Responses 12112
Stimulus voltage 3.0~.
Threshdd for: Polygraph
2.2 v.
F. R. C
1.8~.
C R 0
50
Stimulus Frequency in Pulses/ Second FIG. 3. Threshold for the evoked potential varied with the method of measurement. A comparison is shown for one cat of the stimulus voltages required for detection of potentials, evoked by single shocks and recorded in an ink tracing, evoked by trains of shocks and observed on an oscilloscope, and evoked by serial shocks and added on a computer. At the left are shown representative frequencyresponse curves constructed (5,6) to determine optimal stimulus rates for detecting evoked potentials by their integrated amplitudes.
depended on the fact that the phase of the evoked activity was locked to the recording period, whereas the phase of spontaneousactivity was not (9). Variations in r.m.s. amplitude of spontaneous activity, depending on responseoccurrence (Fig. 2) as well as on deprivation time, number of trials, respiratory rate, etc., exceeded the controlled increase in r.m.s. amplitude induced by electric stimulation near threshold.
323
THRESHOLDS
The third method diminished interference from spontaneous activity by display of bursts of evoked potentials on an oscilloscope screen in a stationary pattern, at an optimal pulse rate determined from the frequency-response curve for each site. Using step increases in stimulus voltage of about 10% of anticipated threshold, recognition by the observer of a stable pattern of potentials evoked by surface-negative stimuli was accompanied by a bar-pressing response by the cat (Fig. 4: r = 0.94, .
0
2
20-
0
l 0
3 .c,
IO-
2 ii I s
543-
m
‘j 2-
1! -c !i!
. .
I-
c I I
Threshold
Polarity: Surface-negative 0 Surface-positive Duration: IO Microseconds I I I 1 I 3 4 5 IO 20 l
.
c FIG. 4. of stimulus
..*
I 2
for Evoked Potential in Volts
Comparison of thresholds site and polarity.
for
bar-press
and
evoked
potential,
in
terms
p < 0.001). Reproducibility at low intensities was somewhat enhanced by virtue of the fact that the potentiometer winding of the stimulator gave incremental rather than continuous increases of the order of 0.05 volts. The coefficient of variation between the two thresholds-standard deviation between voltages -+ mean threshold for evoked potentials from nine runs-was 7.2 -+ 0.8 per cent for this particular set of conditions. This was within the range of magnitude of the step increases chosen for the comparison. Differences between sites and occasional changes in electrode impedance (8) causing shifts in threshold voltage did not affect the comparison, because behavioral and electrical thresholds were covariant.
324
FREEMAN
Outside these experimental conditions deviations between electrical and behavioral thresholds was observed. Repeated testing at one site, for example, resulted in lowering of threshold at that site for the behavioral, but not for the electrical response, so that in three cats after ten to twenty trials it was possible to demonstrate clear-cut behavioral responses in the absence of electrical responses detectable by this method. In two such cats, three recording sites were available, and it was ascertained that an evoked potential was discernible at none of them. The absolute difference between electrical and behavioral thresholds was small (e.g., 0.2-0.4 v). On the other hand elevation of behavioral above electrical threshold several minutes could be induced by 10 to 20 set of stimulation at 4 to 6 X threshold, after which cats would not respond to stimuli less than 20 to 40% above electrical threshold. This phenomenon was sufficiently variable as not to support ready quantitative treatment. Spontaneous elevations in behavioral threshold sometimes occurred abruptly, following repeated trials at or below electrical threshold accompanied by prolonged latency and excessive bar-pressing between trials. During additional trials behavioral threshold might then fall, but not as low as before the rise. On alternate runs thresholds were also determined for surface-positive stimuli at the same sites. A much poorer correlation was found (Y = 0.52; p < 0.02) with discrepancies occurring in either direction for individual sites. In all cases, electrical thresholds were higher, averaging 35% greater than for surface-negative stimuli. It must be noted that surfacenegative stimuli at 1.3 X threshold had ‘been delivered alinost daily for many weeks preceding these determinations to only one of four sites in each cat, and that surface-positive stimuli were rarely used at any site. It is uncertain whether additional training using the latter might have increased the correlation. The, existence of behavioral responses without detectable evoked potentials prompted use of the average response computer, by means of which the presence of the evoked potential was demonstrated in all cats at stimulus intensities below the behavioral threshold. To determine electrical threshold by this method a total of 128 single shock evoked potentials was averaged for one site in each cat (that habitually used for training purposes) at successively decreasing intensities. Electrical threshold by this method (1.1 -+ 0.6 v) averaged about SOY0 of behavioral thresholds for these six sites (2.1 t 0.8 v) .
325
THRESHOLDS
Some explanation was sought for the wide variation in electrical threshold between sites. A major determinant was proximity to the lateral olfactory tract (Fig. 5) ; twelve out of fourteen sites having surfacenegative thresholds 5 volts or less were found to lie in the tract, whereas
mm. 12 I
IO I I I
Lateral
8 I
I
6 I
I
4
Olfactory Tract \)
Thresholds: l l-5 volts A 5-10 volts 0 > IO volts
/’
2
Olf.
25
1 ‘( Bulb
,
23 21 19 17
0 q
FIG. 5. Anatomical to lateral olfactory on the same side.
5
A
Pyriform
location of tract. Bilateral
Lobe
stimulus (and recording) placements are shown at
sites with symmetrical
respect points
of eight sites with thresholds in excess of 5 volts, one was in the tract and two lay at its lateral margin. (Two sites were omitted from this tabulation because of defective solder connections in the plug attached to the skull.) One site at the edge of the tract with a relatively high threshold (6.5 v/O.01 msec) was found to lie in a glial scar about 1 mm
326
FREEMAN
.
in diameter; four sites showed focal demyelination in the tract on the side of the electrode posterolateral to the bulb. The average threshold for these five sites was not significantly greater than that for the remaining nine sites within the tract. Slight variation in area of tip exposure, separation of tips, etc., may have accounted for some of the re-, maining discrepancies, but this was not ascertained. Sinusoidal Current Stimulation of Cortex. A clear negative result was obtained from the attempt to elicit bar-pressing responses from three cats by applying an externally generated low-level signal to the prepyriform cortex using the same pairs of electrodes normally used for stimulation and recording. Each of these cats had four pairs of electrodes spaced 3 to 6 mm apart along the cortex. Sinusoidal currents from a signal ‘generator at frequencies ranging from 20 to 40 cycle/set were passed simultaneously between the tips of three out of four pairs at such intensities that the signal recorded from any one pair resembled intrinsically generated evoked activity (Fig. 2). From previous experiments in anesthetized cats it was known that currents from electrodes in the positions described would create a gross electric field in the basal forebrain closely resembling the prepyriform field except for its intracortical distribution. No responses occurred, whether bursts or steady trains of sine waves were applied, although the cats had previously learned to respond to a wide range of frequencies, intensities and sites of stimulation. Responses did occur if the current was increased 10 to 30 X, but only at amplitudes sufficiently high (OS1.5 v) to allow direct stimulation of the lateral olfactory tracL2 2 The averaged amplitudes of spontaneous activity resulting from the techniques used in this study reflected potential differences established by the flow of current from many cells across the resistance of the cortex. The question arose whether the changes in spontaneous amplitude might be ascribed separately to current changes or to transcortical resistance changes or both (1,2,10). To determine this a constant sinusoidal alternating current was passed between a pair of electrodes not normally used for recording but also situated in the cortex in the fashion described above, such that an exogenous field was superimposed on the prepyriform field with roughly the same spatial distribution (4). This signal was fixed at 400 or 1000 cycle/set, so that this and the spontaneous signal could be separated from each other by appropriate filters and separately integrated. The envelope of the highfrequency signal was also recorded concomitantly with the EEG. Because the current in the exogenous signal was constant (1.0 va r.m.s.), it was presumed that any major change in the magnitude of the extraneuronal impedance vector between the two recording electrodes would appear as a change in the
THRESHOLDS
327
Discussion
The use of electrical stimuli to afferent cortical pathways in place of sensory stimuli to the various receptors for instrumental conditioning circumvented problems of maintaining fixed levels of input to the brain in freely moving animals, as well as the uncertainty introduced by variability in function of sensory relay nuclei (12, 13, 14, 22). In place of these, other problems arose, principally those of confining the stimulus to the desired pathway, of locating the relevant electrical response and isolating it from background activity, and of estimating the extent to which the electrical stimulus might be homologous with exteroceptive stimulation. These problems and their proposed solutions for this cortex have been discussed (4, 6, 7, 8). The importance of electrode location was emphasized by the fact that placement of the stimulus cathode in the olfactory tract led to consistently lower thresholds for both electrical and behavioral responses in comparison with thresholds for anodal stimulation. That is, displacement of the cathode by 1.5 mm to the cortical junction with the internal white matter raised the threshold. So did removal of the stimulus from the tract, and by a much greater amount. The principal findings of the present study involved comparison of thresholds for behavioral and electrical responses to surface-negative stimuli. (a) Cats did not respond conditionally to an electrical stimulus to the afferent tract of the cortex until orientation to the stimulus had taken place; (b) after sufficient training threshold for behavioral response corresponded on the average to threshold for the electrical response in the cortex as detected without an averaging device; (c) with such a envelop or integrated amplitude of that signal. Changes in phase angle of the impedance vector were sought by displaying the exogenous signal from the electrodes on one axis of an oscilloscope and the output of the signal generator on the other, with measurement of the axes of the resulting ellipse. A Beckman 7360 EPUT meter was also used for phase measurements with somewhat better precision. Fluctuations in impedance were sought during bar-pressing for milk, lapping, and sitting or walking on a treadmill (9). None were found within 5~1% for magnitude and t0.1” for phase angle, despite concomitant changes in the amplitude of spontaneous activity recorded from the same electrodes of up to 240%. In view of this negative result, it was concluded that changes do not occur in the resistance across which cortical currents pass to give rise to detectable potentials. This excludes one possible mechanism accounting for EEG changes with behavior in this cortex, although it does not exclude the possibility of membrane conductance changes as the basis for EEG waves (1).
328
FREEMAN
device the evoked potential could be detected below level of the EEG whether or not accompanied by a and (d) behavioral responses did not occur during and around the cortex of an externally generated similar to the endogenous field.
the electrical “noise” behavioral response; establishment across electric field grossly
The interpretation of these data was limited by the fact that, although this cortex was regarded as a mosaic of cells relatively homogeneous in type, connectivity, and orientation, the element or unit of electrical activity was not identified, particularly in regard to whether the evoked potential of the component cells was fixed or graded in amplitude, monophasic or polyphasic, single or repetitive. Viewed en masse the cortex had a graded response nonlinearly proportional to stimulus input. Three points in this gradation were identified: the upper limit of amplitude to which the evoked potential could be carried by short bursts of intense stimulation just below threshold seizure; the lower limit of detectability, using an averaging device; and an intermediate point at which the amplitude of evoked activity was the same order of magnitude as that of spontaneous activity. Since the evoked potential had an inherent degree of synchrony probably not commensurate with that of spontaneous activity, comparison with the latter had to be in terms of amount of synchronized activity sufficient to give the rise to detectable waves rather than to total activity, which could not be measured. By comparison of the numbers of stimuli necessary to give summated initial surface-negative peaks of equal absolute magnitude for each of the three critical intensities, it was estimated roughly that activation of the cortex sufficient to be detected without averaging required utilization of eight to twelve times the electrogenic potentiality of the cortex manifested at the minimum detectable level, and in turn represented utilization of about 3 to 5% of the total electrogenic potentiality of the cortex. The correlation of thresholds implied that evoked activity of the cortex sufficient to lead to a behavioral response had to approach or exceed the amount of synchronized spontaneous activity. This may have represented the existence of a requirement for exciting enough cells to fill a pre-existing spatial pattern of excitability in the cortex prior to “recognition.” This could be viewed instead as the establishment by synchronization of a signal: noise ratio (3) sufficient for the brain as well as the observer to detect the event without recourse to storage and averaging over time periods longer than the duration of an evoked po-
THRESHOLDS
329
tential or the phosphorescence of the oscilloscope screen. Such sychrony would be important if the “noise” had the same frequency, wave form, and average amplitude as the signal. Certainly the essential event cannot be regarded as the establishment of a detectable field of extracellular current, since although this tends to occur at the same level, it need not, and the artificial generation of such a field had no effect on *behavior. The work of Lashley, Chow and Semmes (15)) Sperry (20)) and others has discredited the notion that such fields might play a significant role in brain function, and Tasaki, Polley and Orrego (21) have provided adequate theoretical justification for rejecting it. In any case, systematic comparison of electrical and behavioral thresholds clearly will require measurements of the prevailing level of spontaneous activity, particularly those components having the same wave form and frequency as the evoked potential. The correlation between threshold and proximity of site to the lateral olfactory tract can in part be ascribed to the fact that the afferent fibers are larger in diameter prior to ramifying over the molecular layer and must therefore have a lower threshold to electrical stimulation. The requirement for some minimal quantity of neural activity (apart from its spatio-temporal patterning) may be more important; the tract provides access to a far greater proportion of the cortex than any site off the tract but within the cortex. In this respect the prepyriform response appears to differ from the “direct cortical response” of neocortex, which is based on volume electrical activation of divergent terminals or sparsely distrihuted horizontal collaterals, and to resemble more the “sensory response” based on localized exteroceptive stimulation in the anesthetized state, which is well localized in neocortex for both unit and graded activity. The difference in electrical polarity of the response can obviously be ascribed to the direction of approach of the afferent fibers. The necessity for an orienting response preceding formation of a conditional reflex is well documented (17)) and its elicitation by highintensity stimuli (cf. 16) can be comprehended in terms of the general requirement that novel stimuli be more intense and persistent than familiar stimuli for recognition to occur (18). This tells nothing about mechanism. Electrical stimulation may provide a context for experimental study of orienting complementary to that generally used (its somatic and visceral manifestations), e.g., the cellular nature of the cortical excitability change thus wrought, the corticosubcortical system by which it normally occurs,
330
FREEMAN
perhaps involving the medial olfactory tract, the meansby which intense stimulation circumvents or activates that system, and its relation to thalamic or reticular stimulation (11) or to establishment of a “dominant focus” by strychnine or d-c polarization of the cortex (19). The basis for this opportunity is measurementof the cellular responseof the brain at the port of stimulus entry, so that thresholds for the responsesof the brain and of the whole animal can be measured separately. References
R. T. KADO, and J. Dm~o. 1962. Impedance measurements in brain tissue of animals using microvolt signals. Exptl. Neural. 6: 47-66. 2. BROWN, G. W. 1957. Impedance variation within the lateral hypothalamus of the cat. Physiologist 1: 15. 3. COMMUNICATIONS BIOPHYSICS GROUP and SIEBERT, W. M. 1959. “Processing Neuroelectric Data.” Research Laboratory of Electronics, Mass. Inst. of Tech, Tech. Rep. 351, Technology Press, Cambridge. 4. FREEMAN, W. J. 1959. Distribution in time and space of prepyriform electrical activity. J. Neurophysiol. 22: 644-665. 5. FREEMAN, W. J. 1961. Q meter for measuring frequency specificity of cortica1 reactivity to electrical stimulation. J. Appl. Physiol. 16: 750-751. 6. FREEMAN, W. J. 1%2a. Linear approximation of prepyriform evoked potential in cats. Exptl. Neurol. 6: 477-499. 7. FREEMAN, W. J. 1962b. Phasic and long-term excitability changes in prepyriform cortex of cats. Exptl. Neural. 5: 500-518. 8. FREEMAN, W. J. 1962~. Alterations of prepyriform evoked potential with changes in stimulus intensity. Exptl. Neural. 6: 70-84. 9. FREEMAN, W. J. 1963. Amplitude and excitability changes of prepiriform cortex related to work performance by cats. (To be published.) 10. FREYGANG, W. H. JR., and W. M. LANDAU. 1955. Some relations between resistivity and electrical activity in the cerebral cortex of the cat. J. Cell. 1.
ADEY,
Camp.
W.
R.,
Physiol
FUSTER,
J.
45:
377-392.
1958. Effects of stimulation of brain stem on tachistoscopic perception. Science 127: 150. 12. HERNANDEZ-PEON, R., A. LAVIN, C. ALCOCER-CUARON,and J. P. MARCELIN. 1960. Electrical activity of the olfactory bulb during wakefulness and sleep. Electroencephalog. and Clin. Neurophysiol. 12: 41-58. 13. KERR, D. I.’ B. 1960. Properties of the olfactory efferent system. Australian J. Exptl. Biol. Med. Sci. 38: 29-36. 14. KERR, ‘D. I. B., and K.-E. HAGBARTH. 1955. An investigation of olfactory centrifugal fiber system. J. Neurophysiol. 16: 362-374. 15. LASHLEY, K. S., K. L. CHOW, and J. SEMMES. 1960. An examination of the electrical field theory of cerebral integration. Psychol. Revs. 57: 345-361. 16. NEILSON, H. C., R. W. DOTY, and L. T. RUTLEDGE. 1958. Motivational and perceptual aspects of subcortical stimulation in cats. Am. J. Physiol. 194: 427-432. 11.
THRESHOLDS 17. 18. 19.
20.
21.
22.
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PAVLOV, I. P. 1927. “Conditioned reflexes.” G. V. Anrep [tr. and ed.]. Oxford Univ. Press, London. ROSENZWEXG, M. R., and L. POSTMAN:’ 1927. Frequency of usage and the perception of words. Science 127: 263-266. RUSINOV, V. S. 1958. Electrophysiological investigation of foci of stationary excitation in the central nervous system. Pavlov J. Higher Nervous Activity 8: 444-451. SPERRY, R. W. 1947. Cerebral regulation of motor coordination in monkeys following multiple transection of sensori-motor cortex. J. NeurophysioZ. 10: 275-294. TASAKI, I., E. H. POLLEY, and F. ORREGO. 1954. Action potentials from individual elements in cat geniculate and striate cortex. J. Neurophysiol. 17: 454-474. YAMAMOTO, C., and K. IWAMA. 1961. Arousal reaction of the olfactory bulb. Japan. 1. Physiol. 11: 335-345.
NEUROLOGY
Comparison
6,
315-331
(1962)
of Thresholds
Electrical
Responses
for
to Cortical
Stimulation WALTER Department
of Physiology,
and
Electrical
in Cats J.
FREEMAN
University Received
Behavioral
June
of California,
Berkeley,
California
27, 1962
Cats with stimulating and recording electrodes implanted in the prepyriform cortex were trained to press a bar for milk upon electrical stimulation of the lateral olfactory tract. Thresholds were determined for the cortical evoked potential and for a conditional response elicited by the same electrical stimulus. Threshold for the evoked potential, defined as stimulus voltage for minimum detectable responses, varied with method of detection. Threshold for electrical responses evoked by single shocks and recorded in an ink tracing was higher than that for responses evoked by trains of stimuh and recorded with an oscilloscope. Both exceeded threshold for responses evoked by low-frequency stimulation and recorded by means of an average response computer. Threshold for the bar-press response (after prolonged overtraining) corresponded on the average to the middle threshold, implying that the level of activity induced by stimulation had to approach or exceed the amount of synchronized spontaneous activity for a behavioral response to occur. Conditioning took place after orientation to the stimulus, which was achieved in this experiment by intense electrical stimulation just below threshold for seizure. Reduction of behavioral threshold to electrical threshold required additional training. Fully trained cats did not respond to externally generated fields of current grossly resembling prepyriform evoked activity, implying that large-scale extraneuronal currents (as compared with current distributions at the cellular level) were of no functional importance. Introduction
Electrical stimulation of the lateral olfactory tract through chronically implanted electrodes may be followed by a prepyriform cortical evoked potential, a stereotyped motor response by the animal, or a conditional response, each depending on stimulus parameters and the history of 1 This work was supported by a grant from the Foundation’s Fund for in Psychiatry (59-204). The assistance of Dr. Bernard Baird, Arthur Daily, Furman, and Richard Zipf is gratefully acknowledged. 315
Research Gershon
316
FREEMAN
stimulation. The purpose of this study was to compare thresholds these events. Methods
for
Bipolar stainless steel (#30) wire electrodes with tips 1.5 mm vertically apart were chronically implanted in six cats by methods previously described (4). In three cats, two pairs were placed 6 mm apart in the left and two in the right cortex. Each pair was suitable for either stimulation or recording. In three cats, four pairs were placed in the left cortex 2 to 4 mm apart, ranging from near the base of the olfactory bulb to the temporal portion of the prepyriform cortex. All placements were confirmed anatomically. In each case one electrode tip was in contact with the molecular layer or lateral olfactory tract and the other was at the base of the cortex. Seventeen of twenty-one placements aimed at the tract were in contact with it, and three were within 0.3 mm. Stimuli were delivered in bursts of pulses from a Tektronix 161 pulse generator through an isolation transformer, which also removed the d-c component. Pulse width (10 psec) was maintained at one-tenth chronaxie of the cortical evoked potential in order to establish wide variability in stimulus voltage by using as the scale of measurement the rising limb of the strength-duration curve. Pulse voltage was monitored with an ozcilloscope to within *3%. Pulse repetition rate was set to coincide either with the frequency of spontaneous activity or with maximum root-mean-square (r.m.s.) amplitude of evoked potential during short bursts of pulses. Burst duration was 0.4 set and burst repetition rate was 0.8 to 1.0 per sec. Records were made with a Grass 5P.S polygraph, a Tektronix preamplifier and oscilloscope and Dumont camera, and a Mnemotron CAT average response computer. Frequency-response curves for determination of optimal stimulus rate were constructed by a method previously described (5). Behavioral testing was carried out in a sound-resistant box made from an 8-ft3 refrigerator, which was fitted with a bar for the cats to press, a tube for delivering milk to the cats, and a signal light. Sufficient control of ambient noise was not achieved to permit quantitative study of the acquisition phases of learning. The cats were fed every 48 hours, thresholds being determined 1 to 2 hours prior to feeding, when response latencies for bar-pressing were usually less than 1 set for suprathreshold stimuli. Behavioral thresholds were determined numerous times, but those reported here were taken several months after establishment of the responses and after prolonged overtraining using surface-negative stimuli.
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317
For each site, intensity was raised in steps of about 10% of expected threshold allowing time for bar-pressing to occur between steps until both behavioral and electrical responses were observed. Surface-negative and surface-positive stimuli were used on alternate runs. The initial two trials were discarded, and thresholds were taken as the mean of the next three trials of each polarity. Results
Unconditional Responses. The effects of electrical stimuli on behavior were analyzed in terms of the threshold for single-shock evoked potential in an ink record for each stimulus site (using an alternate site for recording) rather than stimulus voltage, because this reduced the variability both between animals and between sites in each animal. Up to 6 X threshold no effect on behavior was seen in untrained cats. From 6 to 10 X threshold there was arrest and an orienting response (turning of the head and eyes in irregular fashion suggestive of search rather than specific forced movements, erection of the ears, and sniffing). These manifestations vanished upon sustained or oft-repeated stimulation. Above 10 to 15 X threshold, arrest became sustained, and after 5 to 20 set was followed by a seizure consisting of rhythmically repetitive blinking and retraction of the ears, lips and lower jaw at 2 to 3 per set, profuse salivation and lacrimation, occasionally urination, and rarely defecation or vomiting. On occasion extremely rapid locomotion occurred, not to be called flight since cowering and search for shelter were not in evidence. The duration of the seizure varied from a few seconds to more than a minute, during which the cat withdrew from the observer upon being touched or approached about the head and eyes but did not attack or vocalize. The seizure was followed by several minutes of abnormal behavior, which consisted either of rigid stance with repeated toneless vocalization, rolling on the floor by males or females, as in the female sexual afterreaction, hyperphagia if food was available, flight, unprovoked aggression, or excessively affectionate behavior such as kneading, purring, etc. The relation of postictal to preictal behavior was, unfortunately, not considered. In only one cat did such seizures terminate in tonic, then clonic convul,sions, and in this cat it was invariable. These seizures were accompanied by a repetitive spike pattern synchronous with the twitch of the muzzle, beginning at 4 to 6 per set and tapering to an irregular interval in some cats and a sustained, regular discharge at 2 or 3 per set in others (Fig. 1). The polarity of these
D k
THRESHOLDS
319
spikes was reversed between the electrodes, implying their origin in those cortical cells generating spontaneous activity, but in a relaxation rather than harmonic mode. Possible contributions of muscle or cable artifacts were excluded by the fact that similar facial and head movements induced by repeatedly threatening a blow to the muzzle were not accompanied by irregularities in the recordings. During the seizure and for several seconds thereafter both the “spontaneous” activity and evoked potential between spikes were diminished in amplitude-although neither was absentand during the abnormal behavior rose to relatively high amplitudes. Conditiomal Responses. The cats were trained to press a bar for milk upon stimulation either of the left (four cats) or right (two cats) prepyriform cortex, first by training (twenty trials daily for l-3 weeks) to press the bar in response to a signal light (on 15 set every 2-4 min, with milk available within 1 set of response on every trial), second by orienting using stimulation just below threshold for seizure, and third by conditioning to press the bar using somewhat lower intensities of electric stimulation (6-8 X threshold for the electrical response) as the conditional stimulus. Trains of stimuli were delivered for 1.5set at 45 per set in three cats and 30 per set in three cats every 2 to 4 min. During the early stages of training milk was delivered 1 set after the start of the electrical stimulus and during later stages conditionally upon each response. Criterion was set at nine correct responses out of ten consecutive trials. The cats established the response to electrical stimuli after twenty ‘trials daily for 2 to 3 weeks. Finally, the stimulus intensity was reduced until a stable threshold was reached, below which the animals consistently showed long latency, excessive bar-pressing between trials, or failure to respond altogether. This required another 2 to 3 weeks. After reduction in stimulus intensity, responses occurred to stimulus rates ranging from single shocks to 500 per set (the highest rate tested). They also followed stimuli at amplitudes and durations ranging from near threshold for the evoked potential up to threshold for seizure (Fig. 2). Stimulation at intensities near electrical threshold at sites in the ipsilateral cortex other than that used for initial training also elicited responses. Initially responses did not occur in three cats so tested to stimulation of the contralateral cortex, a finding of interest in view of the absence in these three cats (and in twelve other cats with bilaterally implanted electrodes) of a commissural evoked potential for the prepyriform cortex (4). High-
320
FREEMAN
amplitude stimuli, which were used to establish the absence of the barpressing response to contralateral cortical stimulation, evoked an orienting reaction indistinguishable from the preceding reaction to ipsilateral stimP-o-m-1.8 v.
.Ol msec.
24&c
No Response
----~----~-~----~-
2.8 Y
150 mv.
-01 msec
20 c.p.s.
24/set.
Response c ,
No Response
FIG. 2. Electrical response to electrical stimulation below threshold (1.8 v), at threshold (2.1 v) and at 1.3 X threshold (2.8 v) in bursts indicated by the horizontal bars. Lowest tracing shows sinusoidal activity extrinsically generated at that frequency assumed by evoked cortical activity when driven to the same amplitude.
Following appropriate training, responsesregularly occurred to near electrical threshold delivered to either or both cortexes. The number of cats was too few to compare rates of learning for the two sides.
ulation. stimuli
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321
The conditional nature of the response was established by reversibly inhibiting it through extinction (suppression of the response by withholding the reward), satiation, and distraction with competing stimuli, and differential discrimination to respond to only one of two sites stimulated in the same cortex. Such results differed in no way qualita$vely from similar results using the signal light or a tone rather than the electrical stimulus. The period of strong electrical stimulation was an essential part of this training schedule. Attempts to train five cats (four of which were later trained by use of strong electrical stimuli, the fifth having died of an intercurrent lung infection) either to press a bar for milk or to avoid a puff of air delivered to the face within 1 set of the onset of electrical stimulation did not give conditional responses after twenty trials daily for 4 weeks for each cat, using stimuli about 2 X threshold for the electrical response as to conditional stimulus, but without first eliciting the orienting response. Electrical Responses. Threshold for the behavioral response was variable but clearly defined for each trial. Not so for the electrical response. In theory the onset of electrical activity with increasing stimulus intensity was the level at which any single cell discharged in response to the stimulus. Partly because of spontaneous activity (“noise”) and partly because of the unpredictability of the optimal location of the active site with respect to the recording electrode, the practical evaluation of threshold had to be in terms of detectability, and this varied with method of measurement. Four methods were used. The detection in ink tracings of potentials evoked by single shocks was very useful for initial selection of stimulus parameters, but it failed to take into account the relation between stimulus rate and response amplitude, and it was strongly affected by the level spontaneous activity. The second method consisted of applying short bursts of stimuli at different pulse rates to the tract and measuring the r.m.s. amplitude of the bursts of evoked potentials. The frequency-response curve (5,6) thus derived (Fig. 3) provided an estimate of the optimal stimulus frequency for evoking maximum response amplitude. Optimal frequency always increased with decreasing stimulus intensity and had to be set by extrapolation at threshold intensities (8). This method proved ineffectual after overtraining (7)) because the pulse rate for optimal amplitude no longer coincided with the frequency of spontaneous activity in these highly
322
FREEMAN
trained cats. This discrepancy in frequencies meant that the spontaneous activity could no longer be averaged out of the evoked potential by this method. Only when the frequency of the evoked activity equaled that of spontaneous activity-as it did in the naive state (f)-did the two activities add vectorially with cancellation of the latter. Cancellation
Average amplitude -
20
30
40
Number of Responses 12112
Stimulus voltage 3.0~.
Threshdd for: Polygraph
2.2 v.
F. R. C
1.8~.
C R 0
50
Stimulus Frequency in Pulses/ Second FIG. 3. Threshold for the evoked potential varied with the method of measurement. A comparison is shown for one cat of the stimulus voltages required for detection of potentials, evoked by single shocks and recorded in an ink tracing, evoked by trains of shocks and observed on an oscilloscope, and evoked by serial shocks and added on a computer. At the left are shown representative frequencyresponse curves constructed (5,6) to determine optimal stimulus rates for detecting evoked potentials by their integrated amplitudes.
depended on the fact that the phase of the evoked activity was locked to the recording period, whereas the phase of spontaneousactivity was not (9). Variations in r.m.s. amplitude of spontaneous activity, depending on responseoccurrence (Fig. 2) as well as on deprivation time, number of trials, respiratory rate, etc., exceeded the controlled increase in r.m.s. amplitude induced by electric stimulation near threshold.
323
THRESHOLDS
The third method diminished interference from spontaneous activity by display of bursts of evoked potentials on an oscilloscope screen in a stationary pattern, at an optimal pulse rate determined from the frequency-response curve for each site. Using step increases in stimulus voltage of about 10% of anticipated threshold, recognition by the observer of a stable pattern of potentials evoked by surface-negative stimuli was accompanied by a bar-pressing response by the cat (Fig. 4: r = 0.94, .
0
2
20-
0
l 0
3 .c,
IO-
2 ii I s
543-
m
‘j 2-
1! -c !i!
. .
I-
c I I
Threshold
Polarity: Surface-negative 0 Surface-positive Duration: IO Microseconds I I I 1 I 3 4 5 IO 20 l
.
c FIG. 4. of stimulus
..*
I 2
for Evoked Potential in Volts
Comparison of thresholds site and polarity.
for
bar-press
and
evoked
potential,
in
terms
p < 0.001). Reproducibility at low intensities was somewhat enhanced by virtue of the fact that the potentiometer winding of the stimulator gave incremental rather than continuous increases of the order of 0.05 volts. The coefficient of variation between the two thresholds-standard deviation between voltages -+ mean threshold for evoked potentials from nine runs-was 7.2 -+ 0.8 per cent for this particular set of conditions. This was within the range of magnitude of the step increases chosen for the comparison. Differences between sites and occasional changes in electrode impedance (8) causing shifts in threshold voltage did not affect the comparison, because behavioral and electrical thresholds were covariant.
324
FREEMAN
Outside these experimental conditions deviations between electrical and behavioral thresholds was observed. Repeated testing at one site, for example, resulted in lowering of threshold at that site for the behavioral, but not for the electrical response, so that in three cats after ten to twenty trials it was possible to demonstrate clear-cut behavioral responses in the absence of electrical responses detectable by this method. In two such cats, three recording sites were available, and it was ascertained that an evoked potential was discernible at none of them. The absolute difference between electrical and behavioral thresholds was small (e.g., 0.2-0.4 v). On the other hand elevation of behavioral above electrical threshold several minutes could be induced by 10 to 20 set of stimulation at 4 to 6 X threshold, after which cats would not respond to stimuli less than 20 to 40% above electrical threshold. This phenomenon was sufficiently variable as not to support ready quantitative treatment. Spontaneous elevations in behavioral threshold sometimes occurred abruptly, following repeated trials at or below electrical threshold accompanied by prolonged latency and excessive bar-pressing between trials. During additional trials behavioral threshold might then fall, but not as low as before the rise. On alternate runs thresholds were also determined for surface-positive stimuli at the same sites. A much poorer correlation was found (Y = 0.52; p < 0.02) with discrepancies occurring in either direction for individual sites. In all cases, electrical thresholds were higher, averaging 35% greater than for surface-negative stimuli. It must be noted that surfacenegative stimuli at 1.3 X threshold had ‘been delivered alinost daily for many weeks preceding these determinations to only one of four sites in each cat, and that surface-positive stimuli were rarely used at any site. It is uncertain whether additional training using the latter might have increased the correlation. The, existence of behavioral responses without detectable evoked potentials prompted use of the average response computer, by means of which the presence of the evoked potential was demonstrated in all cats at stimulus intensities below the behavioral threshold. To determine electrical threshold by this method a total of 128 single shock evoked potentials was averaged for one site in each cat (that habitually used for training purposes) at successively decreasing intensities. Electrical threshold by this method (1.1 -+ 0.6 v) averaged about SOY0 of behavioral thresholds for these six sites (2.1 t 0.8 v) .
325
THRESHOLDS
Some explanation was sought for the wide variation in electrical threshold between sites. A major determinant was proximity to the lateral olfactory tract (Fig. 5) ; twelve out of fourteen sites having surfacenegative thresholds 5 volts or less were found to lie in the tract, whereas
mm. 12 I
IO I I I
Lateral
8 I
I
6 I
I
4
Olfactory Tract \)
Thresholds: l l-5 volts A 5-10 volts 0 > IO volts
/’
2
Olf.
25
1 ‘( Bulb
,
23 21 19 17
0 q
FIG. 5. Anatomical to lateral olfactory on the same side.
5
A
Pyriform
location of tract. Bilateral
Lobe
stimulus (and recording) placements are shown at
sites with symmetrical
respect points
of eight sites with thresholds in excess of 5 volts, one was in the tract and two lay at its lateral margin. (Two sites were omitted from this tabulation because of defective solder connections in the plug attached to the skull.) One site at the edge of the tract with a relatively high threshold (6.5 v/O.01 msec) was found to lie in a glial scar about 1 mm
326
FREEMAN
.
in diameter; four sites showed focal demyelination in the tract on the side of the electrode posterolateral to the bulb. The average threshold for these five sites was not significantly greater than that for the remaining nine sites within the tract. Slight variation in area of tip exposure, separation of tips, etc., may have accounted for some of the re-, maining discrepancies, but this was not ascertained. Sinusoidal Current Stimulation of Cortex. A clear negative result was obtained from the attempt to elicit bar-pressing responses from three cats by applying an externally generated low-level signal to the prepyriform cortex using the same pairs of electrodes normally used for stimulation and recording. Each of these cats had four pairs of electrodes spaced 3 to 6 mm apart along the cortex. Sinusoidal currents from a signal ‘generator at frequencies ranging from 20 to 40 cycle/set were passed simultaneously between the tips of three out of four pairs at such intensities that the signal recorded from any one pair resembled intrinsically generated evoked activity (Fig. 2). From previous experiments in anesthetized cats it was known that currents from electrodes in the positions described would create a gross electric field in the basal forebrain closely resembling the prepyriform field except for its intracortical distribution. No responses occurred, whether bursts or steady trains of sine waves were applied, although the cats had previously learned to respond to a wide range of frequencies, intensities and sites of stimulation. Responses did occur if the current was increased 10 to 30 X, but only at amplitudes sufficiently high (OS1.5 v) to allow direct stimulation of the lateral olfactory tracL2 2 The averaged amplitudes of spontaneous activity resulting from the techniques used in this study reflected potential differences established by the flow of current from many cells across the resistance of the cortex. The question arose whether the changes in spontaneous amplitude might be ascribed separately to current changes or to transcortical resistance changes or both (1,2,10). To determine this a constant sinusoidal alternating current was passed between a pair of electrodes not normally used for recording but also situated in the cortex in the fashion described above, such that an exogenous field was superimposed on the prepyriform field with roughly the same spatial distribution (4). This signal was fixed at 400 or 1000 cycle/set, so that this and the spontaneous signal could be separated from each other by appropriate filters and separately integrated. The envelope of the highfrequency signal was also recorded concomitantly with the EEG. Because the current in the exogenous signal was constant (1.0 va r.m.s.), it was presumed that any major change in the magnitude of the extraneuronal impedance vector between the two recording electrodes would appear as a change in the
THRESHOLDS
327
Discussion
The use of electrical stimuli to afferent cortical pathways in place of sensory stimuli to the various receptors for instrumental conditioning circumvented problems of maintaining fixed levels of input to the brain in freely moving animals, as well as the uncertainty introduced by variability in function of sensory relay nuclei (12, 13, 14, 22). In place of these, other problems arose, principally those of confining the stimulus to the desired pathway, of locating the relevant electrical response and isolating it from background activity, and of estimating the extent to which the electrical stimulus might be homologous with exteroceptive stimulation. These problems and their proposed solutions for this cortex have been discussed (4, 6, 7, 8). The importance of electrode location was emphasized by the fact that placement of the stimulus cathode in the olfactory tract led to consistently lower thresholds for both electrical and behavioral responses in comparison with thresholds for anodal stimulation. That is, displacement of the cathode by 1.5 mm to the cortical junction with the internal white matter raised the threshold. So did removal of the stimulus from the tract, and by a much greater amount. The principal findings of the present study involved comparison of thresholds for behavioral and electrical responses to surface-negative stimuli. (a) Cats did not respond conditionally to an electrical stimulus to the afferent tract of the cortex until orientation to the stimulus had taken place; (b) after sufficient training threshold for behavioral response corresponded on the average to threshold for the electrical response in the cortex as detected without an averaging device; (c) with such a envelop or integrated amplitude of that signal. Changes in phase angle of the impedance vector were sought by displaying the exogenous signal from the electrodes on one axis of an oscilloscope and the output of the signal generator on the other, with measurement of the axes of the resulting ellipse. A Beckman 7360 EPUT meter was also used for phase measurements with somewhat better precision. Fluctuations in impedance were sought during bar-pressing for milk, lapping, and sitting or walking on a treadmill (9). None were found within 5~1% for magnitude and t0.1” for phase angle, despite concomitant changes in the amplitude of spontaneous activity recorded from the same electrodes of up to 240%. In view of this negative result, it was concluded that changes do not occur in the resistance across which cortical currents pass to give rise to detectable potentials. This excludes one possible mechanism accounting for EEG changes with behavior in this cortex, although it does not exclude the possibility of membrane conductance changes as the basis for EEG waves (1).
328
FREEMAN
device the evoked potential could be detected below level of the EEG whether or not accompanied by a and (d) behavioral responses did not occur during and around the cortex of an externally generated similar to the endogenous field.
the electrical “noise” behavioral response; establishment across electric field grossly
The interpretation of these data was limited by the fact that, although this cortex was regarded as a mosaic of cells relatively homogeneous in type, connectivity, and orientation, the element or unit of electrical activity was not identified, particularly in regard to whether the evoked potential of the component cells was fixed or graded in amplitude, monophasic or polyphasic, single or repetitive. Viewed en masse the cortex had a graded response nonlinearly proportional to stimulus input. Three points in this gradation were identified: the upper limit of amplitude to which the evoked potential could be carried by short bursts of intense stimulation just below threshold seizure; the lower limit of detectability, using an averaging device; and an intermediate point at which the amplitude of evoked activity was the same order of magnitude as that of spontaneous activity. Since the evoked potential had an inherent degree of synchrony probably not commensurate with that of spontaneous activity, comparison with the latter had to be in terms of amount of synchronized activity sufficient to give the rise to detectable waves rather than to total activity, which could not be measured. By comparison of the numbers of stimuli necessary to give summated initial surface-negative peaks of equal absolute magnitude for each of the three critical intensities, it was estimated roughly that activation of the cortex sufficient to be detected without averaging required utilization of eight to twelve times the electrogenic potentiality of the cortex manifested at the minimum detectable level, and in turn represented utilization of about 3 to 5% of the total electrogenic potentiality of the cortex. The correlation of thresholds implied that evoked activity of the cortex sufficient to lead to a behavioral response had to approach or exceed the amount of synchronized spontaneous activity. This may have represented the existence of a requirement for exciting enough cells to fill a pre-existing spatial pattern of excitability in the cortex prior to “recognition.” This could be viewed instead as the establishment by synchronization of a signal: noise ratio (3) sufficient for the brain as well as the observer to detect the event without recourse to storage and averaging over time periods longer than the duration of an evoked po-
THRESHOLDS
329
tential or the phosphorescence of the oscilloscope screen. Such sychrony would be important if the “noise” had the same frequency, wave form, and average amplitude as the signal. Certainly the essential event cannot be regarded as the establishment of a detectable field of extracellular current, since although this tends to occur at the same level, it need not, and the artificial generation of such a field had no effect on *behavior. The work of Lashley, Chow and Semmes (15)) Sperry (20)) and others has discredited the notion that such fields might play a significant role in brain function, and Tasaki, Polley and Orrego (21) have provided adequate theoretical justification for rejecting it. In any case, systematic comparison of electrical and behavioral thresholds clearly will require measurements of the prevailing level of spontaneous activity, particularly those components having the same wave form and frequency as the evoked potential. The correlation between threshold and proximity of site to the lateral olfactory tract can in part be ascribed to the fact that the afferent fibers are larger in diameter prior to ramifying over the molecular layer and must therefore have a lower threshold to electrical stimulation. The requirement for some minimal quantity of neural activity (apart from its spatio-temporal patterning) may be more important; the tract provides access to a far greater proportion of the cortex than any site off the tract but within the cortex. In this respect the prepyriform response appears to differ from the “direct cortical response” of neocortex, which is based on volume electrical activation of divergent terminals or sparsely distrihuted horizontal collaterals, and to resemble more the “sensory response” based on localized exteroceptive stimulation in the anesthetized state, which is well localized in neocortex for both unit and graded activity. The difference in electrical polarity of the response can obviously be ascribed to the direction of approach of the afferent fibers. The necessity for an orienting response preceding formation of a conditional reflex is well documented (17)) and its elicitation by highintensity stimuli (cf. 16) can be comprehended in terms of the general requirement that novel stimuli be more intense and persistent than familiar stimuli for recognition to occur (18). This tells nothing about mechanism. Electrical stimulation may provide a context for experimental study of orienting complementary to that generally used (its somatic and visceral manifestations), e.g., the cellular nature of the cortical excitability change thus wrought, the corticosubcortical system by which it normally occurs,
330
FREEMAN
perhaps involving the medial olfactory tract, the meansby which intense stimulation circumvents or activates that system, and its relation to thalamic or reticular stimulation (11) or to establishment of a “dominant focus” by strychnine or d-c polarization of the cortex (19). The basis for this opportunity is measurementof the cellular responseof the brain at the port of stimulus entry, so that thresholds for the responsesof the brain and of the whole animal can be measured separately. References
R. T. KADO, and J. Dm~o. 1962. Impedance measurements in brain tissue of animals using microvolt signals. Exptl. Neural. 6: 47-66. 2. BROWN, G. W. 1957. Impedance variation within the lateral hypothalamus of the cat. Physiologist 1: 15. 3. COMMUNICATIONS BIOPHYSICS GROUP and SIEBERT, W. M. 1959. “Processing Neuroelectric Data.” Research Laboratory of Electronics, Mass. Inst. of Tech, Tech. Rep. 351, Technology Press, Cambridge. 4. FREEMAN, W. J. 1959. Distribution in time and space of prepyriform electrical activity. J. Neurophysiol. 22: 644-665. 5. FREEMAN, W. J. 1961. Q meter for measuring frequency specificity of cortica1 reactivity to electrical stimulation. J. Appl. Physiol. 16: 750-751. 6. FREEMAN, W. J. 1%2a. Linear approximation of prepyriform evoked potential in cats. Exptl. Neurol. 6: 477-499. 7. FREEMAN, W. J. 1962b. Phasic and long-term excitability changes in prepyriform cortex of cats. Exptl. Neural. 5: 500-518. 8. FREEMAN, W. J. 1962~. Alterations of prepyriform evoked potential with changes in stimulus intensity. Exptl. Neural. 6: 70-84. 9. FREEMAN, W. J. 1963. Amplitude and excitability changes of prepiriform cortex related to work performance by cats. (To be published.) 10. FREYGANG, W. H. JR., and W. M. LANDAU. 1955. Some relations between resistivity and electrical activity in the cerebral cortex of the cat. J. Cell. 1.
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1958. Effects of stimulation of brain stem on tachistoscopic perception. Science 127: 150. 12. HERNANDEZ-PEON, R., A. LAVIN, C. ALCOCER-CUARON,and J. P. MARCELIN. 1960. Electrical activity of the olfactory bulb during wakefulness and sleep. Electroencephalog. and Clin. Neurophysiol. 12: 41-58. 13. KERR, D. I.’ B. 1960. Properties of the olfactory efferent system. Australian J. Exptl. Biol. Med. Sci. 38: 29-36. 14. KERR, ‘D. I. B., and K.-E. HAGBARTH. 1955. An investigation of olfactory centrifugal fiber system. J. Neurophysiol. 16: 362-374. 15. LASHLEY, K. S., K. L. CHOW, and J. SEMMES. 1960. An examination of the electrical field theory of cerebral integration. Psychol. Revs. 57: 345-361. 16. NEILSON, H. C., R. W. DOTY, and L. T. RUTLEDGE. 1958. Motivational and perceptual aspects of subcortical stimulation in cats. Am. J. Physiol. 194: 427-432. 11.
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PAVLOV, I. P. 1927. “Conditioned reflexes.” G. V. Anrep [tr. and ed.]. Oxford Univ. Press, London. ROSENZWEXG, M. R., and L. POSTMAN:’ 1927. Frequency of usage and the perception of words. Science 127: 263-266. RUSINOV, V. S. 1958. Electrophysiological investigation of foci of stationary excitation in the central nervous system. Pavlov J. Higher Nervous Activity 8: 444-451. SPERRY, R. W. 1947. Cerebral regulation of motor coordination in monkeys following multiple transection of sensori-motor cortex. J. NeurophysioZ. 10: 275-294. TASAKI, I., E. H. POLLEY, and F. ORREGO. 1954. Action potentials from individual elements in cat geniculate and striate cortex. J. Neurophysiol. 17: 454-474. YAMAMOTO, C., and K. IWAMA. 1961. Arousal reaction of the olfactory bulb. Japan. 1. Physiol. 11: 335-345.