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Unilateral cortical spreading depression and conditioned eyeblink responses in the rabbit
EXPERIMENTAL
NEUROLOGY
Unilateral
Cortical
27, 34-45
Spreading
Eyeblink DAVID Institute
of Physiology,
(1970)
Depression
Responses ILIECIRIAN
Czechoslovak Received
November
Conditioned
in the Rabbit
1 AND JAN
Academy
and
of
BURES
Sciences,
Prague,
Czechoslovakia
14, 1969
Classically conditioned eyeblink reactions were elaborated in seven adult rabbits wing air puff to the right eye as UCS and electrical shock to left or right forelimb as CS. The CS-UCS interval was 400 msec, the intertrial interval 1 min. After the asymptomatic performance level had been reached, cortical spreading depression (CSD) was evoked by injecting %-4 p liters of 25% KC1 through implanted cannulae to the frontal or occipital pole of either hemisphere. Movement of the slow potential change (SPC) over the cortical surface was recorded with implanted Ag-AgCl electrodes and correlated with CR performance. CSD in the left hemisphere caused a complete suppression of the CR when the negative SPC invaded a cortical region at the level of bregma with a rostrocaudal extent of about 3 mm. The maximum impairment lasted for 3 min and was followed by gradual recovery completed within 8-10 min (occipital KC1 injection) or within 12 to 14 min (frontal KC1 injection). CSD in the right hemisphere decreased the CR performance from the initial 80-90s to 40% when it entered a cortical area slightly wider (about 6 mm) and more posteriorly located than in the left hemisphere. The results were independent of the side of CS application. Principles of CSD use in localisation studies are discussed. Introduction
Although cortical spreading depression (CSD) has been extensively used in behavioral studies to achieve functional elimination of neocortex (2) there are only a few papers in which the march of the slow potential change (SPC) has been directly correlated with the performance of the animal. Bar pressing was found to be impaired in rats (1) and rabbits (14) after the motor cortex had been invaded by SPC. In both speciesthe bar is usually operated by the head, by both forelimbs, or by the entire body. the cortical control of lever pressing being bilateral. Since each hemisphere may sustain bar pressing independently of the other (4), complete behavioral blockade can only be achieved by simultaneous elimination of the cortical areas in both cortices. The exact delimination of the critical regions I IBRO Travelling Fellow. Present address : Department of Medicine, University of Tasmania, Hobart, Australia. 34
of Physiology,
Faculty
CONDITIONED
EYEBLINR
.
35
RESPONSES
5
o RO
FIG. 1. Localization of the injecting cording electrodes (l-5) with respect in the left and right hemispheres are proportional to functional significance
cannulae (LF, RF, LO, RO) and of the reto the rabbit cranium (A). The critical areas indicated by stippling the density of which is (B).
by the fact that the SPC in the left and right hemissymmetrical and synchronous. In the present paper, the strictly lateralized conditioned eye blink response (8) is studied using the same technique with the aim to test the usefulness of CSD in localization experiments under simpler conditions.
is made uncertain pheres are never
completely
Methods Seven experimentally naive rabbits averaging 2.6-kg body wt (range 2-3.2) were used. Each animal was anesthetized with 50 mg/kg, ip of allobarbital (Spofa) prior to training and recording sessions.Metal cannulae (No. 30 hyperdermic needles, 14 mm in length) were placed over the frontal and occipital cortices of each hemisphere for injecting 25% KC1 (Fig.
1A).
These
cannulae
were perpendicular
to the cortical
surface,
flush with the under surface of the cranium. Electrodes for recording SPC in the cortical gray matter consisted of glass micropipets (0.2-0.3 mm-tip diameters) filled with 0.9% NaCl in agar gel. A length of 0.2-mm-diameter silver wire spiralled at one end was inserted into the broad end of the pipet and sealed in place with dental acrylic. The wires were soldered to a five pin mini-plug. A pair of these
36
MEGIRIAN
AND
BURES
electrodes was placed 5-8 mm from the midline in each hemisphere, one at the Ievel of the bregma and the other 6-9 mm posteriorly onto or just below the cortical surface. An indifferent electrode (silver screw 2.0 mm in diameter) was placed in the nasal bones in the midline. The assembly of cannulae and electrodes was anchored to the skull with dental acrylic. A diagram of their relative disposition with respect to bony landmarks is presented in Fig. 1A. A loop of thread sewn to the upper right eyelid was connected via a short length of wire to one end of a strain gauge for myographic recording of lid contraction. The other end of the strain gauge was fixed to a rigid U-shaped metal bar whose base was inserted into a stand molded from dental acrylic which was anchored to the frontal bones. A U-shaped tube, passed transversally through the anterior end of the dental acrylic base, channelled a puff of air (250 msec duration) onto the cornea of the right eye (the unconditioned stimulus) (UCS) . Feeble condenser discharges delivered via a pair of small safety pins to the skin of the forelimb comprised the conditioned stimulus (CS) . Forty-eight or more hours after recovery from anesthesia each rabbit was placed in a suitable restraining box. At l-min intervals the CS was delivered to one forelimb, followed 400 msec later by the onset of the unconditioned stimulus. The above interstimulus interval was found to be optimal by Frey and Ross (7). The presence of a conditioned response (CR) was scored positive when the blink preceded the onset of the unconditioned response. Rabbits under our experimental conditions consistently began scoring eight positive CR in ten paired stimuli after 240-300 training trials. Extinction did not manifest itself in 20 consecutive applications of the CS. If the CS was presented alone to a naive animal at 1-min intervals at strengths used to elaborate the CR, it failed to provoke eyelid contractions. The terms ipsilateral and contralateral are used to refer to myographically recorded eyelid contractions. Five channels of an electroencephalograph were used. Two channels with maximum filtering at the high end of the frequency response (time constant of 1 set) served to display the myogram, one channel at relatively low amplification and the other at high amplification. The SPC in cortical gray matter of each hemisphere, initially fed through a chopper (about S/set) was connected to two independent input channels. Two to 4 PIiters of 25% KC1 injected through a cannula triggered one wave of cortical spreading depression (CSD) . Two or more successive waves to a single injection were occasionally detected. A compensator circuit inserted between a recording electrode and the amplifier input served to adjust the cl-c level of the recording system. A fifth channel was used to signal presentation of the paired stimuli.
CONDITIONED
nnnnnnnnnnnnnnn + _ + + +
EYEBLINK
-
-
37
RESPOKSES
-
-
_
+
-
+
+
2-L ,
1KC1 LF
3sec
4
Zmln
nnnnnnnnnnnnnn+ + + - + +
+ - + +
+
+
l
l-5 t ICI
.3sec
Ro 2mln
FIG. 2. myogram gram -, downward potential myogram.
I
Typical experiment with CSD in the left hemisphere. From top to bottom: - low amplification (closure of the eye - downward deflection) ; myohigh amplification; stimulus marker (upward deflection - forelimb shock, deflection-air puff onset) ; presence ( i- ) or absence (- ) of the CR ; slow recording. Calibration: 2 min and ICI my for the SPC; 3 set for the
FIG. 3. Typical as in Fig. 2.
experiment
with CSD in the right hemisphere. Other description
MEGIRIAN
AND
BURES
Results
Unilateral CSD and Impairment of the CR as a Function of Forelimb Stimulated. As illustrated in Fig. 2, prior to the injection of KCl, the myographic record shows that the application of the CS to the forelimb (upward deflection of signal marker) caused a CR to appear before onset of the unconditioned response (UCR) (downward deflection of signal marker). After 2 ,uliters of 25% KC1 were injected into the cortical grey matter, via the cannula over the frontal region of the contralateral hemisphere, CR disappeared with a latency of slightly less than 2 min when the wave of CSD passed the frontal recording electrode. Absent from that time onward, they reappeared about 8 min after the SPC passed the occipital recording lead (bipolar recording arrangement). This volume of injected KC1 generated SPC in the contralateral hemisphere only. An injection of an equal volume of 0.9% NaCl evoked no SPC. Figure 3 illustrates a sample of records from the same animal as presented in Fig. 2, before, during and after the passageof a wave of CSD in the ipsilateral hemisphere. Prior to the KC1 injection the CR was present, and as the wave of CSD swept the extent of this hemisphere only occasional CR were absent. This record also serves to illustrate that, when the experimenter’s presence to make the KC1 injection coincided with presentation of paired stimuli, the CR was sometimes absent even before the SPC was detected at the lead proximal to the injected site. Such distractions to the animal’s performance, which reduced CR scores, were taken into account in data analysis (see below). From these observations it would appear that the contralateral frontal area of the rabbit’s cerebral cortex subserves conditioned eyeblink responsesand that the ipsilateral hemisphere is partially contributory. Therefore, to study in detail and to quantify the degree and significance of impairment of the CR by unilateral CSD, experiments were carried out in a total of seven rabbits. One wave of CSD could be generated by a single injection of KCl; CS were applied first to one forelimb and then to the other. A total of 97 separate waves of CSD were evoked, 49 in the ipsilateral hemisphere and 48 in the contralateral one. The presence or absence of the CR was tabulated on cardboard strips upon which were also noted time of KC1 injections, (via which cannula and the passageof the maximum peak of the SPC) . When suitably arranged, summated scoresof CR minute by minute were computed. Scores were plotted on ordinate as a function of time and correlated with route of KC1 administration and resulting SPC. In a typical experiment with the CS applied to one forelimb, eight separate waves of CSD were generated, four in each hemisphere. Two waves, one at each of two recording sessionson separate days were provoked by KC1 injected via the frontal cannula and two waves by KC1
CONDITIONED
EYEBLINK
39
RESPONSES
injected via the occipital cannula over each hemisphere. The above procedure was repeated in the same animal but with the CS delivered to the other forelimb. On the analysis board, data were aligned in such a way that the maximum negative peak of the SPC was under the frontal recording lead in each hemisphere (frontal alignment). All data were analysed first with respect to which hemisphere was depressed but irrespective of the site of KC1 injection. Retaining frontal alignment on the analysis board, scores for impairment of the CR before, during, and after unilateral CSD were plotted at successive I-min intervals after the passage of the negative peak of the SPC under the respective leads. Figure 4 reveals that within 1 min after the SPC passed the contralateral frontal lead (Fig. 4A), CR were virtually absent, whereas they were reduced in frequency of occurrence by 50% within 2-3 min of passage of the SPC beneath the ipsilateral frontal lead (Fig. 4B). Moreover, inspection of Fig. 4 shows that the time course and degree of impairment of the CR was independent of which forelimb received the CS. In addition, recovery from impairment seemed slower during contralateral CSD than during ipsilateral CSD. Contralateral CSD and Site of KC1 Injection. Since the degree of impairment of the CR was not determined by which forelimb received the CS. data collection with frontal alignment was retained with attention directed toward which cannula, frontal or occipital, was used to administer the KC1 solution. Inspection of data on the analysis board suggested that with CSD in the contralateral hemisphere, recovery of the CR seemed
B % 100,
% ‘001
.
FIG. 4. The effect of left (A) or right (B) CSD on the average performance of the conditioned eyeblink reaction with CS applied to the left (dashed line) or right (full line) forelimb. Zero of the time axis corresponds to the negative maximum of SPC in the frontal electrodes. Note that the time course of CR impairment is independent from CS laterality.
10
MEGIRIAN
AND
BURES
to occur earlier when the wave was caused by KC1 delivery through the occipital cannula than when the injection was via the frontal one. Thus, as shown in Fig. SA, CR were totally abolished within 90 set after the SPC had passed the frontal leads, regardless of which direction the wave took : anterior to posterior (frontal injection) or posterior to anterior (occipital injection). However, during the recovery phase, CR reappeared earlier when KC1 was injected via the occipital cannula than when injected via the frontal cannula. Differences in recovery rates were statistically significant for this part of the curve. Recovery rate of the CR was slower after frontal than occipital injections, independently of the order of injections. Data aligned with respect to passage of the SPC under the contralateral occipital recording lead yielded Fig. 5B. First, as expected in this alignment, when KC1 was injected through the frontal cannula (dashed line and circles), CR were markedly impaired before SPC entered the occipital lead, i.e., between -1 and zero on the time axis. However, when the KC1 was delivered via the occipital cannula, impairment of CR occurred after SPC passed the occipital electrode, i.e., $2 min on the time axis. The declining portions of the respective curves were similar but were phase
A
KCI A I :: L-. c_-_-----__----_-----___ ------.I \’ 1
KCI
I3 n If i..,; r ______-__----_---_--------- i. r
FIG. 5. The effect of CSD evoked from the frontal (dashed line) or occipital (full line) cannulae in the left hemisphere on the average performance of the conditioned eyeblink. The upper curves show schematically the time of KC1 injection and the course of SPC in bipolar recording (frontal negativity upward). Zero of the time axis corresponds to the negative maximum of SPC in the frontal (A) or in the occipital (B) lead. Dots with the upward pointing arrows correspond to the impairment probably caused by distraction of the animal.
CONDITIONED
EYEBLINK
41
RESPOKSES
Kc11 ?.
B
10 . 20, 0'
-1
.,..,.,......,.,...( 0
4
0
12 min
Ob-1....‘..‘...........( 0 c a
12 min
FIG. 6. The effect of CSD evoked from the frontal (dashed line) or occipital (full line) cannulae in the right hemisphere on the average performance of the conditioned eyeblink. Other description as in Fig. 5.
shifted by 3 min. which is in agreement with the average distance between leads in this hemisphere. As in Fig. 5A (frontal alignment), the recovery was more rapid when KC1 injections were made via the occipital can&a than via the frontal one. Although statistically significant only for the upper one-third of the recovery curves, they had similar recovery rates as shown in Fig. 5A in spite of the 3-min. phase shift bringing now the recovery portions of the respective curves closer together. Ipsilateral CSD and Site of KCI Injection. As shown in Fig. 6A, impaitment of the CR during ipsilateral CSD was more gradual and less pronounced (Figs. 2 and 3.) than during contralateral CSD. Recovery of the CR was significantly faster after occipital than after frontal injections. This is in keeping with comparable observations during contralateral CSD. However, the less striking degree and duration of impairment with CSD in the ipsilateral hemisphere made estimation of phase shifting difficult. Figure 6B shows the samegeneral tendencies for impairment of the CR as noted for this alignment during contralateral CSD but not to the same degree. The curve for depression of the CR as a consequenceof the SPC sweeping over the ipsilateral hemisphere following KC1 injection via the frontal cannula was displaced, as expected, to the left of the curve for OCcipital injections. Howeve;, the declining part of the respective curves was quite irregular due to wide dispersion of results and occurrence of “negative CRs”, i.e., distraction of animal to perform due to experimenter’s pres-
4.2
MEGIRIAN
AND
BURES
ence for KC1 injection. Therefore, rather than attempting to assess the phase shift based on difference in the similar rates of impaired CRs in a given time interval, the time difference between peak of impairment seemed the better choice. Thus, after frontal injection peak impairment was reached at zero on the time axis, whereas after occipital injection peak impairment occurred at -1-3. Taking into account the geometry of the recording electrodes and the time shifts of impairment evoked from occipital and frontal cannuiae, tine approximate cortical area, the invasion of which corresponds with observed impairments, can be reliably estimated. A conduction velocity of 3 mm/min is presumed. The CSD triggered from the frontal cannula caused maximum impairment only when reaching a point 6 mm caudal to the frontal lead, whereas when triggered from the occipital cannula the maximum impairment occurred when the resulting wave of CSD reached a point about 3 mm rostra1 to the frontal lead. The area delimited by this rostra1 and caudal dimension is the area participating in the performance of the conditioned eyeblink response. The findings suggest that the ipsilateral cortical area subserving the CR was greater in its anterior-posterior extent than in the contralateral hemisphere. The ipsilateral focus tends to be located a little more posteriorly than the contralateral one as diagrammed in Fig. 1B. Discussion
The experimental findings reveal that conditioned eyeblink responses are chiefly mediated by the frontal region of the rabbit’s neocortex contralateral to the eye receiving the unconditioned stimulus when the CS is applied to either forelimb. Contributions by the ipsilateral hemisphere, mainly the frontoparietal area, though not as marked, are significant. Therefore, the side to which the UCS is directed, rather than the side to which CS is applied, determines which hemisphere preferentially subserves cortical control of eyeblinking. In view of the marked lateral position of the eyes and of the unilateral character of the UCR in the rabbit (8) such finding is not unexpected. The more pronounced degree of impairment and longer recovery following contralateral CSD by comparison with ipsilateral CSD provides a quantitative measure of the relative degree of contribution each hemisphere makes to the elaboration of the CR. It was observed, however, that rate of recovery of the CR during CSD is a function of which cannula is used to elicite CSD. The tardiness with which the CR recovers after KC1 injection via the frontal cannula is probably due to several factors. Not only does the KC1 set in operation the mechanism of CSD, but it also probably exerts a direct depolarizing action over a part of the cortical area essential for the development of the CR.
CONDITIONED
EYEBLINK
RESPONSES
13
The main purpose of this study is to show how the CSD technique may be used for topographical localization of cortical functions. The front of the CSD wave separates with considerable accuracy the depressed and functional cortex. Since the performance is proportional to the intactness of the critical region, we can assume that the extent of the cortical focus in the direction normal to the CSD wavefront is determined by the time between the onset and culmination of impairment in the average curves. Reliability of estimations based on mean CR incidence is better the closer the point used for synchronization of individual recordings is to the focus. Thus, the extent of the focus can be estimated from Fig. 5 as less or more than 6 mm. according to whether the passage of the slow potential under the frontal or occipital electrode is used for the alignment of data. As the same individual curves are displaced in both cases, it is obvious that with occipital alignment the random differences in spreading rate and electrode localization increase the dispersion and decrease the steepness of the onset of behavioral impairment. Of course, it must be also taken into account that with a 1-min intertrial interval, each individual measurement has an inherent error of a half-minute, which increases the dispersion of average data. When full depression develops during 2 tnin, the focus is certainly less than 6 mm wide, but its extent can also be less than 3 mm. This possibility is indicated by Fig. 4A where full impairment of CR to ipsilateral CS develops during 1 min. The localization of the focus with respect to the electrode position can be obtained in a similar way. As follows from Fig. SA, CR are still unimpaired when SPC is 3 mm from the frontal electrode but the impairment is complete when it arrives 3 mm beyond this point. Similar estimation can be made from Fig. 5B, but greater dispersion makes the result less accurate. The 3-min time shift between the descendent portions of the two curves indicates that they would overlap in a point located approximately 5 mm rostrally from the occipital electrode. This point corresponds to the center of the focus and its position is in good agreement with that based on frontal alignment of data. As the results are independent of the direction of CSD march, it can be concluded that the remaining cortex contributes little to the performance of the conditioned eyeblink, which can probably be maintained by a small island of cortical tissue in the fashion described for somesthetic discrimination in the cat ( 13 ). Such results indicate that Lashley’s (9) law of mass action does not apply to local CRs of the above type. The less complete impairment due to right CSD (Fig. 6) can be interpreted in a similar way. The focus seems to be slightly larger (3 min from onset to maximum) and the l-min phase shift between the SPC waves in Fig. 6A indicates that the center of the focus lies l-2 mm caudally from the frontal recording electrode.
44
MEGIRIAN
AND
BURES
Since CSD spreads concentrically from the point of KC1 injection it also affects areas localized laterally from the arbitrary line connecting the electrodes. The onset of impairment coinciding with the penetration of SPC into a given region (e.g., corresponding to the placement of the recording electrode) indicates that the focus lies on the perimeter of a circle with the center in the point of injection and with a radius corresponding to the injection-recording distance. When the behavioral impairment coincides wit-h the SPC developing in a cortical area even for CSD waves evoked from :he opposite directions, then this area corresponds to the point of contact between the two hypothetical circles and thus necessarily lies on the connecting line of their centers. Foci located more laterally can be obtained by finding the intersection of the two circles centered around the frontal and occipital injection points. To summarize, the contralateral focus lies slightly anteriorly to bregma with a rostrocaudal extent of about 3 mm. The ipsilateral cortical focus lies somewhat more posteriorly then the contralateral one and has a rostrocaudal span of about 6 mm. The regions deliminated in this way correspond to somatosensory and motor areas established in the rabbit by the evoked potential technique (5, 6, 16). Stippled areas in Fig. 1B denote the critical zones with respect to bony landmarks and their relative densities indicate their relative functional importance. Although the critical regions in the left and right hemisphere overlap to a considerable extent, they have different functional significance. The CRs impairment caused by left CSD is evidently due to elimination of motor cortex, which is an indispensable output link of the conditioned reaction. On the contrary, contribution of the right hemisphere is not so essential. The observed impairment is probably due to unilateral elimination of the somatosensory cortex and thus to partial blockade of CS transmission (this assumesbilateral projection of the CS and participation of callosal fibers in CS convergence onto the left motor cortex). It is also possible, however, that the impairment is due to changes occurring in the left hemisphere or in subcortical centers while CSD moves across the right hemisphere. Such changes might be due to elimination of transcallosal tonic influences, to the interfering effects of the transcallosally mediated discharge accompanying the CSD wavefront, or to general distraction caused by reticular activation (3)) abortive seizures, or by the sensory consequencesof functional decortication. The conditioned eyeblink used in our experiments can be considered a classically conditioned reaction, since with appropriate position of the air jet, the lid movement does not prevent the air stream from reaching the cornea. Classically conditioned vegetative reactions were shown to persist during bilateral CSD in rats (IO) and according to Ross and Russell (12 j they differ from instrumental reactions by being nonlateralized at the
CONDITIONED
EYEBLINK
RESPONSES
45
cortical level. The present data indicate that the degree of cortical participation and lateralization depends on the character of the unconditioned reaction rather than on the conditioning paradigm. This conclusion is in good agreement with the experimental evidence demonstrating suppression of the classically conditioned eyehlink reactions during bilateral CSD in the rabbit ( 11) and in the cat ( 15). References 1. BURES, J., and 0. BURE~OVA, 1960. The use of Leao’s spreading cortical depression in research on conditioned reflexes. El~~cfroc~tcephalogr. Clitz. Nruroph>lsiol. 13 : 359-376. 2. BURES, J., and 0, BURESOVA, 1968. Applications rkcentes de la dCpression envahissante corticale i l’&tude du comportment. .-irfual. Ncrrroplz~~siol. 8 : 161178. 3. BL-RES, J., 0. BURESOVA, and E. FIFKOV~, 1961. The effect of cortical and hippocampal spreading depression on activity of bulbopontine reticular units in the rat. Arch. Ital. Biol. 99: 23-32. 4. BURES, J., 0. BURESOVA, E. FIFKOV~~, J. OLDS, M. OLDS, and R. P. TRAVIS, 1961. Spreading depres,sion and subcortical drive centers. Physiol. Bolmrrosloe~. 10 : 321-331. 5. CAZARD, P., and P. BUSER, 1963. Reponses sensorielles recueillies au niveau du cortex moteur chez le Lapin. Elrctroencephalogr. C&L. Newophysiol. 15 : 403412. 6. CHANG, H. T., 1955. Cortical responses to stimulation of medullary pyramid in rabbit. J. Nettroplz~lsiol. 18 : 332-352. 7. FREY, P. W., and L. E. Ross, 1968. Clas,sical conditioning of the rabbit eyelid response as a function of interstimulus interval. J. Colrrp. Ph)viol. Psgchol. 65 : 24U50. 8. GORMEZANO, I. 1966. Classical conditioning, pp. 385320. 1~ “Experimental Methods and Instrumentation in Psychology.” J. B. SIDOVSKI [ed.], McGrawHill, New York. 9. LASHLEY, K. S. 1929. “Brain Mechanisms and Intelligence.” University of Chicago Press, Chicago. 10. MOGENSON, G. J., and R. J. PETERSOK, 1966. Effects of spreading cortical depression on cardiac and somatomotor conditioned responses. Cax. J. Physiol. Pharmacol. 44: 39-45. 11. PAPSDORF, J. D., D. LONGMAX, and I. GORMEZANO, 1965. Spreading depression: effects of applying KC1 to the dura of the rabbit on the conditioned nictitating membrane response. PS~~CICOIG. Sci. 2: 125-126. 12. ROSS, R. B., and I. S. RUSSELL, 1967. Subcortical storage of classical conditioning. L~atzwc, 214: Zl&Zll. 13. SPERRY, R. W. 1959. Preservation of high-order function in isolated somatic cortex in callosum-,sectioned cat. J. Nearophgsiol. 22 : 78-87. 14. SUZ~IZ, H., and K. UNEOKA, 1966. Effect of cortical spreading depression on operant behavior in the rabbit. Pltysiol. Behav. 1: 301-304. 15. WOODY, C. D., and G. BROZEK. 1969. Conditioned eye blink in the cat: evoked responses of short latency. Brailr Rrs. 12: 257-260. 16. WOOLSEY. C. N. 1947. Pattern of sensory representation in the cerebral cortex. Fed. Proc. 6: 437-441.
NEUROLOGY
Unilateral
Cortical
27, 34-45
Spreading
Eyeblink DAVID Institute
of Physiology,
(1970)
Depression
Responses ILIECIRIAN
Czechoslovak Received
November
Conditioned
in the Rabbit
1 AND JAN
Academy
and
of
BURES
Sciences,
Prague,
Czechoslovakia
14, 1969
Classically conditioned eyeblink reactions were elaborated in seven adult rabbits wing air puff to the right eye as UCS and electrical shock to left or right forelimb as CS. The CS-UCS interval was 400 msec, the intertrial interval 1 min. After the asymptomatic performance level had been reached, cortical spreading depression (CSD) was evoked by injecting %-4 p liters of 25% KC1 through implanted cannulae to the frontal or occipital pole of either hemisphere. Movement of the slow potential change (SPC) over the cortical surface was recorded with implanted Ag-AgCl electrodes and correlated with CR performance. CSD in the left hemisphere caused a complete suppression of the CR when the negative SPC invaded a cortical region at the level of bregma with a rostrocaudal extent of about 3 mm. The maximum impairment lasted for 3 min and was followed by gradual recovery completed within 8-10 min (occipital KC1 injection) or within 12 to 14 min (frontal KC1 injection). CSD in the right hemisphere decreased the CR performance from the initial 80-90s to 40% when it entered a cortical area slightly wider (about 6 mm) and more posteriorly located than in the left hemisphere. The results were independent of the side of CS application. Principles of CSD use in localisation studies are discussed. Introduction
Although cortical spreading depression (CSD) has been extensively used in behavioral studies to achieve functional elimination of neocortex (2) there are only a few papers in which the march of the slow potential change (SPC) has been directly correlated with the performance of the animal. Bar pressing was found to be impaired in rats (1) and rabbits (14) after the motor cortex had been invaded by SPC. In both speciesthe bar is usually operated by the head, by both forelimbs, or by the entire body. the cortical control of lever pressing being bilateral. Since each hemisphere may sustain bar pressing independently of the other (4), complete behavioral blockade can only be achieved by simultaneous elimination of the cortical areas in both cortices. The exact delimination of the critical regions I IBRO Travelling Fellow. Present address : Department of Medicine, University of Tasmania, Hobart, Australia. 34
of Physiology,
Faculty
CONDITIONED
EYEBLINR
.
35
RESPONSES
5
o RO
FIG. 1. Localization of the injecting cording electrodes (l-5) with respect in the left and right hemispheres are proportional to functional significance
cannulae (LF, RF, LO, RO) and of the reto the rabbit cranium (A). The critical areas indicated by stippling the density of which is (B).
by the fact that the SPC in the left and right hemissymmetrical and synchronous. In the present paper, the strictly lateralized conditioned eye blink response (8) is studied using the same technique with the aim to test the usefulness of CSD in localization experiments under simpler conditions.
is made uncertain pheres are never
completely
Methods Seven experimentally naive rabbits averaging 2.6-kg body wt (range 2-3.2) were used. Each animal was anesthetized with 50 mg/kg, ip of allobarbital (Spofa) prior to training and recording sessions.Metal cannulae (No. 30 hyperdermic needles, 14 mm in length) were placed over the frontal and occipital cortices of each hemisphere for injecting 25% KC1 (Fig.
1A).
These
cannulae
were perpendicular
to the cortical
surface,
flush with the under surface of the cranium. Electrodes for recording SPC in the cortical gray matter consisted of glass micropipets (0.2-0.3 mm-tip diameters) filled with 0.9% NaCl in agar gel. A length of 0.2-mm-diameter silver wire spiralled at one end was inserted into the broad end of the pipet and sealed in place with dental acrylic. The wires were soldered to a five pin mini-plug. A pair of these
36
MEGIRIAN
AND
BURES
electrodes was placed 5-8 mm from the midline in each hemisphere, one at the Ievel of the bregma and the other 6-9 mm posteriorly onto or just below the cortical surface. An indifferent electrode (silver screw 2.0 mm in diameter) was placed in the nasal bones in the midline. The assembly of cannulae and electrodes was anchored to the skull with dental acrylic. A diagram of their relative disposition with respect to bony landmarks is presented in Fig. 1A. A loop of thread sewn to the upper right eyelid was connected via a short length of wire to one end of a strain gauge for myographic recording of lid contraction. The other end of the strain gauge was fixed to a rigid U-shaped metal bar whose base was inserted into a stand molded from dental acrylic which was anchored to the frontal bones. A U-shaped tube, passed transversally through the anterior end of the dental acrylic base, channelled a puff of air (250 msec duration) onto the cornea of the right eye (the unconditioned stimulus) (UCS) . Feeble condenser discharges delivered via a pair of small safety pins to the skin of the forelimb comprised the conditioned stimulus (CS) . Forty-eight or more hours after recovery from anesthesia each rabbit was placed in a suitable restraining box. At l-min intervals the CS was delivered to one forelimb, followed 400 msec later by the onset of the unconditioned stimulus. The above interstimulus interval was found to be optimal by Frey and Ross (7). The presence of a conditioned response (CR) was scored positive when the blink preceded the onset of the unconditioned response. Rabbits under our experimental conditions consistently began scoring eight positive CR in ten paired stimuli after 240-300 training trials. Extinction did not manifest itself in 20 consecutive applications of the CS. If the CS was presented alone to a naive animal at 1-min intervals at strengths used to elaborate the CR, it failed to provoke eyelid contractions. The terms ipsilateral and contralateral are used to refer to myographically recorded eyelid contractions. Five channels of an electroencephalograph were used. Two channels with maximum filtering at the high end of the frequency response (time constant of 1 set) served to display the myogram, one channel at relatively low amplification and the other at high amplification. The SPC in cortical gray matter of each hemisphere, initially fed through a chopper (about S/set) was connected to two independent input channels. Two to 4 PIiters of 25% KC1 injected through a cannula triggered one wave of cortical spreading depression (CSD) . Two or more successive waves to a single injection were occasionally detected. A compensator circuit inserted between a recording electrode and the amplifier input served to adjust the cl-c level of the recording system. A fifth channel was used to signal presentation of the paired stimuli.
CONDITIONED
nnnnnnnnnnnnnnn + _ + + +
EYEBLINK
-
-
37
RESPOKSES
-
-
_
+
-
+
+
2-L ,
1KC1 LF
3sec
4
Zmln
nnnnnnnnnnnnnn+ + + - + +
+ - + +
+
+
l
l-5 t ICI
.3sec
Ro 2mln
FIG. 2. myogram gram -, downward potential myogram.
I
Typical experiment with CSD in the left hemisphere. From top to bottom: - low amplification (closure of the eye - downward deflection) ; myohigh amplification; stimulus marker (upward deflection - forelimb shock, deflection-air puff onset) ; presence ( i- ) or absence (- ) of the CR ; slow recording. Calibration: 2 min and ICI my for the SPC; 3 set for the
FIG. 3. Typical as in Fig. 2.
experiment
with CSD in the right hemisphere. Other description
MEGIRIAN
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Results
Unilateral CSD and Impairment of the CR as a Function of Forelimb Stimulated. As illustrated in Fig. 2, prior to the injection of KCl, the myographic record shows that the application of the CS to the forelimb (upward deflection of signal marker) caused a CR to appear before onset of the unconditioned response (UCR) (downward deflection of signal marker). After 2 ,uliters of 25% KC1 were injected into the cortical grey matter, via the cannula over the frontal region of the contralateral hemisphere, CR disappeared with a latency of slightly less than 2 min when the wave of CSD passed the frontal recording electrode. Absent from that time onward, they reappeared about 8 min after the SPC passed the occipital recording lead (bipolar recording arrangement). This volume of injected KC1 generated SPC in the contralateral hemisphere only. An injection of an equal volume of 0.9% NaCl evoked no SPC. Figure 3 illustrates a sample of records from the same animal as presented in Fig. 2, before, during and after the passageof a wave of CSD in the ipsilateral hemisphere. Prior to the KC1 injection the CR was present, and as the wave of CSD swept the extent of this hemisphere only occasional CR were absent. This record also serves to illustrate that, when the experimenter’s presence to make the KC1 injection coincided with presentation of paired stimuli, the CR was sometimes absent even before the SPC was detected at the lead proximal to the injected site. Such distractions to the animal’s performance, which reduced CR scores, were taken into account in data analysis (see below). From these observations it would appear that the contralateral frontal area of the rabbit’s cerebral cortex subserves conditioned eyeblink responsesand that the ipsilateral hemisphere is partially contributory. Therefore, to study in detail and to quantify the degree and significance of impairment of the CR by unilateral CSD, experiments were carried out in a total of seven rabbits. One wave of CSD could be generated by a single injection of KCl; CS were applied first to one forelimb and then to the other. A total of 97 separate waves of CSD were evoked, 49 in the ipsilateral hemisphere and 48 in the contralateral one. The presence or absence of the CR was tabulated on cardboard strips upon which were also noted time of KC1 injections, (via which cannula and the passageof the maximum peak of the SPC) . When suitably arranged, summated scoresof CR minute by minute were computed. Scores were plotted on ordinate as a function of time and correlated with route of KC1 administration and resulting SPC. In a typical experiment with the CS applied to one forelimb, eight separate waves of CSD were generated, four in each hemisphere. Two waves, one at each of two recording sessionson separate days were provoked by KC1 injected via the frontal cannula and two waves by KC1
CONDITIONED
EYEBLINK
39
RESPONSES
injected via the occipital cannula over each hemisphere. The above procedure was repeated in the same animal but with the CS delivered to the other forelimb. On the analysis board, data were aligned in such a way that the maximum negative peak of the SPC was under the frontal recording lead in each hemisphere (frontal alignment). All data were analysed first with respect to which hemisphere was depressed but irrespective of the site of KC1 injection. Retaining frontal alignment on the analysis board, scores for impairment of the CR before, during, and after unilateral CSD were plotted at successive I-min intervals after the passage of the negative peak of the SPC under the respective leads. Figure 4 reveals that within 1 min after the SPC passed the contralateral frontal lead (Fig. 4A), CR were virtually absent, whereas they were reduced in frequency of occurrence by 50% within 2-3 min of passage of the SPC beneath the ipsilateral frontal lead (Fig. 4B). Moreover, inspection of Fig. 4 shows that the time course and degree of impairment of the CR was independent of which forelimb received the CS. In addition, recovery from impairment seemed slower during contralateral CSD than during ipsilateral CSD. Contralateral CSD and Site of KC1 Injection. Since the degree of impairment of the CR was not determined by which forelimb received the CS. data collection with frontal alignment was retained with attention directed toward which cannula, frontal or occipital, was used to administer the KC1 solution. Inspection of data on the analysis board suggested that with CSD in the contralateral hemisphere, recovery of the CR seemed
B % 100,
% ‘001
.
FIG. 4. The effect of left (A) or right (B) CSD on the average performance of the conditioned eyeblink reaction with CS applied to the left (dashed line) or right (full line) forelimb. Zero of the time axis corresponds to the negative maximum of SPC in the frontal electrodes. Note that the time course of CR impairment is independent from CS laterality.
10
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to occur earlier when the wave was caused by KC1 delivery through the occipital cannula than when the injection was via the frontal one. Thus, as shown in Fig. SA, CR were totally abolished within 90 set after the SPC had passed the frontal leads, regardless of which direction the wave took : anterior to posterior (frontal injection) or posterior to anterior (occipital injection). However, during the recovery phase, CR reappeared earlier when KC1 was injected via the occipital cannula than when injected via the frontal cannula. Differences in recovery rates were statistically significant for this part of the curve. Recovery rate of the CR was slower after frontal than occipital injections, independently of the order of injections. Data aligned with respect to passage of the SPC under the contralateral occipital recording lead yielded Fig. 5B. First, as expected in this alignment, when KC1 was injected through the frontal cannula (dashed line and circles), CR were markedly impaired before SPC entered the occipital lead, i.e., between -1 and zero on the time axis. However, when the KC1 was delivered via the occipital cannula, impairment of CR occurred after SPC passed the occipital electrode, i.e., $2 min on the time axis. The declining portions of the respective curves were similar but were phase
A
KCI A I :: L-. c_-_-----__----_-----___ ------.I \’ 1
KCI
I3 n If i..,; r ______-__----_---_--------- i. r
FIG. 5. The effect of CSD evoked from the frontal (dashed line) or occipital (full line) cannulae in the left hemisphere on the average performance of the conditioned eyeblink. The upper curves show schematically the time of KC1 injection and the course of SPC in bipolar recording (frontal negativity upward). Zero of the time axis corresponds to the negative maximum of SPC in the frontal (A) or in the occipital (B) lead. Dots with the upward pointing arrows correspond to the impairment probably caused by distraction of the animal.
CONDITIONED
EYEBLINK
41
RESPOKSES
Kc11 ?.
B
10 . 20, 0'
-1
.,..,.,......,.,...( 0
4
0
12 min
Ob-1....‘..‘...........( 0 c a
12 min
FIG. 6. The effect of CSD evoked from the frontal (dashed line) or occipital (full line) cannulae in the right hemisphere on the average performance of the conditioned eyeblink. Other description as in Fig. 5.
shifted by 3 min. which is in agreement with the average distance between leads in this hemisphere. As in Fig. 5A (frontal alignment), the recovery was more rapid when KC1 injections were made via the occipital can&a than via the frontal one. Although statistically significant only for the upper one-third of the recovery curves, they had similar recovery rates as shown in Fig. 5A in spite of the 3-min. phase shift bringing now the recovery portions of the respective curves closer together. Ipsilateral CSD and Site of KCI Injection. As shown in Fig. 6A, impaitment of the CR during ipsilateral CSD was more gradual and less pronounced (Figs. 2 and 3.) than during contralateral CSD. Recovery of the CR was significantly faster after occipital than after frontal injections. This is in keeping with comparable observations during contralateral CSD. However, the less striking degree and duration of impairment with CSD in the ipsilateral hemisphere made estimation of phase shifting difficult. Figure 6B shows the samegeneral tendencies for impairment of the CR as noted for this alignment during contralateral CSD but not to the same degree. The curve for depression of the CR as a consequenceof the SPC sweeping over the ipsilateral hemisphere following KC1 injection via the frontal cannula was displaced, as expected, to the left of the curve for OCcipital injections. Howeve;, the declining part of the respective curves was quite irregular due to wide dispersion of results and occurrence of “negative CRs”, i.e., distraction of animal to perform due to experimenter’s pres-
4.2
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ence for KC1 injection. Therefore, rather than attempting to assess the phase shift based on difference in the similar rates of impaired CRs in a given time interval, the time difference between peak of impairment seemed the better choice. Thus, after frontal injection peak impairment was reached at zero on the time axis, whereas after occipital injection peak impairment occurred at -1-3. Taking into account the geometry of the recording electrodes and the time shifts of impairment evoked from occipital and frontal cannuiae, tine approximate cortical area, the invasion of which corresponds with observed impairments, can be reliably estimated. A conduction velocity of 3 mm/min is presumed. The CSD triggered from the frontal cannula caused maximum impairment only when reaching a point 6 mm caudal to the frontal lead, whereas when triggered from the occipital cannula the maximum impairment occurred when the resulting wave of CSD reached a point about 3 mm rostra1 to the frontal lead. The area delimited by this rostra1 and caudal dimension is the area participating in the performance of the conditioned eyeblink response. The findings suggest that the ipsilateral cortical area subserving the CR was greater in its anterior-posterior extent than in the contralateral hemisphere. The ipsilateral focus tends to be located a little more posteriorly than the contralateral one as diagrammed in Fig. 1B. Discussion
The experimental findings reveal that conditioned eyeblink responses are chiefly mediated by the frontal region of the rabbit’s neocortex contralateral to the eye receiving the unconditioned stimulus when the CS is applied to either forelimb. Contributions by the ipsilateral hemisphere, mainly the frontoparietal area, though not as marked, are significant. Therefore, the side to which the UCS is directed, rather than the side to which CS is applied, determines which hemisphere preferentially subserves cortical control of eyeblinking. In view of the marked lateral position of the eyes and of the unilateral character of the UCR in the rabbit (8) such finding is not unexpected. The more pronounced degree of impairment and longer recovery following contralateral CSD by comparison with ipsilateral CSD provides a quantitative measure of the relative degree of contribution each hemisphere makes to the elaboration of the CR. It was observed, however, that rate of recovery of the CR during CSD is a function of which cannula is used to elicite CSD. The tardiness with which the CR recovers after KC1 injection via the frontal cannula is probably due to several factors. Not only does the KC1 set in operation the mechanism of CSD, but it also probably exerts a direct depolarizing action over a part of the cortical area essential for the development of the CR.
CONDITIONED
EYEBLINK
RESPONSES
13
The main purpose of this study is to show how the CSD technique may be used for topographical localization of cortical functions. The front of the CSD wave separates with considerable accuracy the depressed and functional cortex. Since the performance is proportional to the intactness of the critical region, we can assume that the extent of the cortical focus in the direction normal to the CSD wavefront is determined by the time between the onset and culmination of impairment in the average curves. Reliability of estimations based on mean CR incidence is better the closer the point used for synchronization of individual recordings is to the focus. Thus, the extent of the focus can be estimated from Fig. 5 as less or more than 6 mm. according to whether the passage of the slow potential under the frontal or occipital electrode is used for the alignment of data. As the same individual curves are displaced in both cases, it is obvious that with occipital alignment the random differences in spreading rate and electrode localization increase the dispersion and decrease the steepness of the onset of behavioral impairment. Of course, it must be also taken into account that with a 1-min intertrial interval, each individual measurement has an inherent error of a half-minute, which increases the dispersion of average data. When full depression develops during 2 tnin, the focus is certainly less than 6 mm wide, but its extent can also be less than 3 mm. This possibility is indicated by Fig. 4A where full impairment of CR to ipsilateral CS develops during 1 min. The localization of the focus with respect to the electrode position can be obtained in a similar way. As follows from Fig. SA, CR are still unimpaired when SPC is 3 mm from the frontal electrode but the impairment is complete when it arrives 3 mm beyond this point. Similar estimation can be made from Fig. 5B, but greater dispersion makes the result less accurate. The 3-min time shift between the descendent portions of the two curves indicates that they would overlap in a point located approximately 5 mm rostrally from the occipital electrode. This point corresponds to the center of the focus and its position is in good agreement with that based on frontal alignment of data. As the results are independent of the direction of CSD march, it can be concluded that the remaining cortex contributes little to the performance of the conditioned eyeblink, which can probably be maintained by a small island of cortical tissue in the fashion described for somesthetic discrimination in the cat ( 13 ). Such results indicate that Lashley’s (9) law of mass action does not apply to local CRs of the above type. The less complete impairment due to right CSD (Fig. 6) can be interpreted in a similar way. The focus seems to be slightly larger (3 min from onset to maximum) and the l-min phase shift between the SPC waves in Fig. 6A indicates that the center of the focus lies l-2 mm caudally from the frontal recording electrode.
44
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Since CSD spreads concentrically from the point of KC1 injection it also affects areas localized laterally from the arbitrary line connecting the electrodes. The onset of impairment coinciding with the penetration of SPC into a given region (e.g., corresponding to the placement of the recording electrode) indicates that the focus lies on the perimeter of a circle with the center in the point of injection and with a radius corresponding to the injection-recording distance. When the behavioral impairment coincides wit-h the SPC developing in a cortical area even for CSD waves evoked from :he opposite directions, then this area corresponds to the point of contact between the two hypothetical circles and thus necessarily lies on the connecting line of their centers. Foci located more laterally can be obtained by finding the intersection of the two circles centered around the frontal and occipital injection points. To summarize, the contralateral focus lies slightly anteriorly to bregma with a rostrocaudal extent of about 3 mm. The ipsilateral cortical focus lies somewhat more posteriorly then the contralateral one and has a rostrocaudal span of about 6 mm. The regions deliminated in this way correspond to somatosensory and motor areas established in the rabbit by the evoked potential technique (5, 6, 16). Stippled areas in Fig. 1B denote the critical zones with respect to bony landmarks and their relative densities indicate their relative functional importance. Although the critical regions in the left and right hemisphere overlap to a considerable extent, they have different functional significance. The CRs impairment caused by left CSD is evidently due to elimination of motor cortex, which is an indispensable output link of the conditioned reaction. On the contrary, contribution of the right hemisphere is not so essential. The observed impairment is probably due to unilateral elimination of the somatosensory cortex and thus to partial blockade of CS transmission (this assumesbilateral projection of the CS and participation of callosal fibers in CS convergence onto the left motor cortex). It is also possible, however, that the impairment is due to changes occurring in the left hemisphere or in subcortical centers while CSD moves across the right hemisphere. Such changes might be due to elimination of transcallosal tonic influences, to the interfering effects of the transcallosally mediated discharge accompanying the CSD wavefront, or to general distraction caused by reticular activation (3)) abortive seizures, or by the sensory consequencesof functional decortication. The conditioned eyeblink used in our experiments can be considered a classically conditioned reaction, since with appropriate position of the air jet, the lid movement does not prevent the air stream from reaching the cornea. Classically conditioned vegetative reactions were shown to persist during bilateral CSD in rats (IO) and according to Ross and Russell (12 j they differ from instrumental reactions by being nonlateralized at the
CONDITIONED
EYEBLINK
RESPONSES
45
cortical level. The present data indicate that the degree of cortical participation and lateralization depends on the character of the unconditioned reaction rather than on the conditioning paradigm. This conclusion is in good agreement with the experimental evidence demonstrating suppression of the classically conditioned eyehlink reactions during bilateral CSD in the rabbit ( 11) and in the cat ( 15). References 1. BURES, J., and 0. BURE~OVA, 1960. The use of Leao’s spreading cortical depression in research on conditioned reflexes. El~~cfroc~tcephalogr. Clitz. Nruroph>lsiol. 13 : 359-376. 2. BURES, J., and 0, BURESOVA, 1968. Applications rkcentes de la dCpression envahissante corticale i l’&tude du comportment. .-irfual. Ncrrroplz~~siol. 8 : 161178. 3. BL-RES, J., 0. BURESOVA, and E. FIFKOV~, 1961. The effect of cortical and hippocampal spreading depression on activity of bulbopontine reticular units in the rat. Arch. Ital. Biol. 99: 23-32. 4. BURES, J., 0. BURESOVA, E. FIFKOV~~, J. OLDS, M. OLDS, and R. P. TRAVIS, 1961. Spreading depres,sion and subcortical drive centers. Physiol. Bolmrrosloe~. 10 : 321-331. 5. CAZARD, P., and P. BUSER, 1963. Reponses sensorielles recueillies au niveau du cortex moteur chez le Lapin. Elrctroencephalogr. C&L. Newophysiol. 15 : 403412. 6. CHANG, H. T., 1955. Cortical responses to stimulation of medullary pyramid in rabbit. J. Nettroplz~lsiol. 18 : 332-352. 7. FREY, P. W., and L. E. Ross, 1968. Clas,sical conditioning of the rabbit eyelid response as a function of interstimulus interval. J. Colrrp. Ph)viol. Psgchol. 65 : 24U50. 8. GORMEZANO, I. 1966. Classical conditioning, pp. 385320. 1~ “Experimental Methods and Instrumentation in Psychology.” J. B. SIDOVSKI [ed.], McGrawHill, New York. 9. LASHLEY, K. S. 1929. “Brain Mechanisms and Intelligence.” University of Chicago Press, Chicago. 10. MOGENSON, G. J., and R. J. PETERSOK, 1966. Effects of spreading cortical depression on cardiac and somatomotor conditioned responses. Cax. J. Physiol. Pharmacol. 44: 39-45. 11. PAPSDORF, J. D., D. LONGMAX, and I. GORMEZANO, 1965. Spreading depression: effects of applying KC1 to the dura of the rabbit on the conditioned nictitating membrane response. PS~~CICOIG. Sci. 2: 125-126. 12. ROSS, R. B., and I. S. RUSSELL, 1967. Subcortical storage of classical conditioning. L~atzwc, 214: Zl&Zll. 13. SPERRY, R. W. 1959. Preservation of high-order function in isolated somatic cortex in callosum-,sectioned cat. J. Nearophgsiol. 22 : 78-87. 14. SUZ~IZ, H., and K. UNEOKA, 1966. Effect of cortical spreading depression on operant behavior in the rabbit. Pltysiol. Behav. 1: 301-304. 15. WOODY, C. D., and G. BROZEK. 1969. Conditioned eye blink in the cat: evoked responses of short latency. Brailr Rrs. 12: 257-260. 16. WOOLSEY. C. N. 1947. Pattern of sensory representation in the cerebral cortex. Fed. Proc. 6: 437-441.