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Passive and active avoidance performance following small amygdaloid lesions in rats
Phystology and Behavtor Vol. 3, pp. 713-718. Pergamon Press, 1968. Printed m Great Britain
Passive and Active Avoidance Performance Following Small Amygdaloid Lesions in Rats E R N E S T D. K E M B L E 1, 2 A N D J A C K T. T A P P
Department of Psychology, Vanderbilt University, Nash ville, Tennessee (Received 22 M a r c h 1968) KEMBLE, E. D. AND J. T. TAPP. Passive and active avoidance performance following small amygdaloM lesion~ ill rat~. PHVSlOL.BEnAV. 3 (5) 713-718, 1968.--Following small bilateral arnygdaloid lesions or pyriform cortex lesions rats were tested for both passive and active avoidance. Damage to an area roughly corresponding to the basal amygdaloid nucleus was most consistently related to disruption of passive avoidance. Pyriform cortex lesions also impaired passive avoidance but this result was not replicated in a second experiment. Neither amygdaloid nor pyriform cortex lesions disrupted active avoidance.
Amygdalold lesions
Pyriform cortex lesions
Passive avoidance
BILATERAL ablation of the amygdaloid complex has been repeatedly shown to disrupt avoidance behavior in a variety of situations such as one- and two-way avoidance, passive avoidance and conditioned suppression [e.g. 3, 4, 10, 12]. Recently Ursin [14] has shown that passive and active avoidance behavior are separately organized within the amygdaloid complex of the cat. Destruction of the rostral half of the lateral amygdaloid nucleus was found to disrupt active but not passive avoidance behavior while lesions which included the stria terminalis or the medial amygdaloid nucleus produced deficits in passive avoidance performance, leaving active avoidance unimpaired. The delineation of such topographically distinct systems subserving different types of fear-motivated behavior is of considerable importance for a fuller understanding of amygdaloid function. Therefore it seemed of interest to examine the organization of these systems in an additional species in order to gain some impression of their generality. As Cowan, Raisman and Powell [1] have pointed out, however, amygdaloid lesions also necessarily include destruction of fibers that originate in the pyriform cortex and travel through or end in the amygdaloid complex. Consequently it also seemed necessary to provide some estimate of the role of these pyriform fibers. The need for such an assessment is underlined by recent studies of the projection pathways from the pyriform cortex [11] which show that the pyriform cortex contributes many, if not all, of the nerve fibers constituting the ventral amygdalofugal pathway in the rat. Moreover, Kaada, Rasmussen and Kveim [5] found passive avoidance behavior to be disrupted following lesions which included pyriform as well as neocortex. The present studies were designed (a) to localize the areas whose destruction produce disruption of passive and active avoidance performance by placing small lesions at various loci throughout the amygdaloid complex of the rat and (b) to
Active avoidance
Localization of funcUon
provide some assessment of the effects of pyriform cortex lesions on these two types of behavior. EXPERIMENT I METHODS
Subjects This experiment employed 125 male albino Holtzman rats 100-120 days of age when received. Postoperative mortality and failure to recuperate properly reduced this number to 86.
Apparatus Passive Avoidance. The apparatus was similar to one described by McCleary [9]. It consisted of an 8¼ x 14½ in. plywood chamber and a smaller (31 × 4 × 41 in.) reward chamber. The walls were painted flat gray and the top was clear Plexiglass. The floor of the larger chamber was of plywood painted flat gray while the floor of the reward chamber was covered by an aluminum plate. A small aluminum water cup (1 x t x ¼ in.) was inserted through a slot in the rear wall of the reward chamber. The rat completed a circuit when it stood on the aluminum plate with its mouth or tongue in contact with the water. Active avoidance. The apparatus was similar to one described by Lubar [8] that provides a one-way avoidance task which requires no handling of the animal between trials. It consisted of two identical boxes constructed of plywood (11t x 8 x 8 in.) painted flat black. The floors were aluminum rods spaced I in. apart. Each box had a hinged clear Plexiglass top. Both ends of each box were composed of vertically sliding doors. The aluminum rods comprising the floors were connected to a Triplett high voltage power source through a grid scrambler and selector switch. The floor of either box could be electrified by appropriately
1This article is based on a dissertation submitted in partial fulfilment of Ph.D. requirements at Vanderbilt University. ~Now at University of Minnesota, Morris. 713
714 setting a selector switch which delivered rapid intermittent shock pulses to the feet of the rat. Each box was separately attached to the power source so that the boxes could be moved about by the experimenter. The CS for the active avoidance task was provided by a low intensity tone of approximately 1,000 c/s and a 7.5 W light. The light and speaker were mounted on a stationary pedestal 23 in. high and 10 in. above the top of the box from which the rat began each trial.
Procedure Preoperative. Approximately 18 hr before surgery the rats were weighed and all food removed from the home cage. They were then randomly designated to receive: bilateral amygdaloid lesions (this group was subdivided into groups which received lesions to the rostro-laterai, rostro-medlal, caudo-lateral and caudo-medial portions of the amygdalold complex), pyriform cortex lesions, neocortical lesions, unilateral amygdaloid lesions or no lesion (operated control). Operative procedure. All operations were carried out under sodium pentobarbital anesthesia (50 mg/kg) and clean conditions. The rats designated to receive subcortical lesions were positioned in a stereotactlc instrument adjusted so that bregma was 1.0 mm higher than lambda. The skull was exposed through a midllne incision, burr holes drilled at the appropriate points and a 24 g hypodermic needle (insulated except for 1 mm at the tip) lowered to the desired depth. Lesions were produced by a Grass radio-frequency lesion maker. The cortical lesions were produced by heating the skull overlying the cortex with a glass insulated tungsten electrode connected to a Bantom Boyle thermocautery. Amygdaloid lesions. In order to consistently produce small bilaterally symmetrical lesions, a single large lesion on one side was paired with a smaller lesion of the contralateral side. The current used to produce the large lesion ranged from 3.0--4.0 mA for durations of 20-25 sec while 2.5-3.2 mA for 5-8 sec was used to produce small lesions. The following coordinates were used for the four amygdaloid subgroups using bregma, midsagittal suture and surface of the cortex as reference points: rostro-lateral, 1.3-1.5 mm posterior (P), 4.5--4.6 mm lateral (L), and 7.0-7.2 mm deep (D); rostromedial, 1.3-1.5 mm P, 3.7-3.9 mm L and 7.4-7.6 mm D; caudo-lateral, 2.0-2.4 mm P, 4.5-4.75 mm L and 8.0 mm D. Unilateral amygdaloid control rats sustained a single large lesion at these coordinates, but the electrode was simply lowered 6.0 mm on the contralateral side and withdrawn. Cortical lesions. After the skull was exposed through a midline incision, the temporal muscle was retracted from its attachment to the skull and the posterior attachment of the zygomatic arch to the brain case severed. Pyriform cortex lesions were produced by pressing the tungsten electrode tip against the skull, ventral to the level of attachment of the zygomatic arch, and heating the bone for five sec. Neocortical lesions were produced in exactly the same way except that the electrode tip was pressed against the skull dorsal to the level of attachment of the zygomatic arch. Half of the operated control rats received treatment identical to that described for the amygdaloid lesions except that the electrode was lowered and no lesion produced. The remaining half received the same treatment as cortical rats except that no lesions were produced. Following surgery Sulfanilamide powder was sprinkled over the wound surface, the incision closed with metal wound clips, and the edges of the incision covered with collodion.
KEMBLE AND TAPP
Postoperattve care. The rats were placed In group cages (four animals per cage) for a period of 10-12 days. Dry food and water (containing sucrose and vitamin supplements) were available ad libitum during this recovery period. At the end of the recovery period the rats were returned to their home cages and received ad hbitum access to dry food and tap water for an additional two days. Approximately 24 hr before each animal was to begin passive avoidance training, all water was removed from the home cage. Dry food was available throughout the experiment. Passtve avotdance. The procedure employed in this phase of the experiment was similar to that employed by Kaada et al. [5]. After the rat had been deprived of water for 24 hr it was placed in the passive avoidance apparatus and allowed to drink from the aluminum dish for 15 mln. The rat was then returned to the home cage and received no additional water. This procedure was repeated for three days. On the fourth day the rat was placed in the passive avoidance apparatus once more with the water dish available. Ten sec after the rat began to drink a mild electric current (0.2 mA measured through the apparatus and 10,000 f~ resistance) was turned on and remained on for 20 rain. During this time interval two measures of passive avoidance were taken: the number of times the rat contacted the water and received a shock (contacts) and the number of times it entered the reward chamber without touching the water (entries). At the end of the 20 min test, the rat was returned to its home cage and given 30 mln ad libitum access to water. This schedule was followed for the remainder of the experiment. Active avoidance. At approximately the same time on the following day (approximately 1 hr before watering) each animal began active avoidance training. On each trial the box containing the rat was placed beneath the pedestal on which the speaker and hght were mounted. The second box was placed against the first so that the sliding doors of both boxes were directly opposite each other and approximately ½ in. apart. Simultaneously raising both doors closed a mlcroswltch connected to one of them. The closing of the mlcroswitch activated a 1,000 cps tone and the light, started a Cramer timer (1/100 sec) and a 5-sec time-delay switch. If the rat did not enter the second box within 5 sec, the timedelay switch closed and an intermittent current of 0.6 mA (measured through the apparatus and 10,000 f~ resistance) was delivered to the rat's feet. When the rat entered the second box, the experimenter closed a mlcroswitch which terminated the shock, tone, light, and timer and reset the 5 sec delay timer. The two sliding doors which separated the boxes were then lowered. During the intertrial interval (1 rain) the second box (now containing the animal) was moved back to a position under the pedestal to which the speaker and light were attached and the first box was moved to the former position of the second box. The rats received ten such trials per day until either ten consecutive avoidances were completed within a single day or until they had completed 100 training trials. Performance was measured by the number of errors (i.e. latencles of 5 sec or more) made in reaching acquisition criterion, and the mean response latency of the last five acquisition trials. Hlstologwal Procedures At the conclusion of active avoidance training the animals were perfused with isotonic saline followed by 10 per cent formalin solution while under deep pentobarbital anesthesia. After 1-3 days additional soaking in 10 per cent formalin,
AMYGDALOID LESIONS
715
the brains were removed, frozen, and sectioned in the frontal plane at 32 ~t. Every fourth and fifth section from the brains with subcortical lesions was saved. One series was stained for cells with cresyl violet, and the other series stained for myelin by the Weil method. Brains with cortical lesions were photographed from the sides and bottom, then frozen and sectioned in the frontal plane. Sections were retained at 0.5 mm intervals and stained with cresyl violet. These sections were used to determine the depth of the cortical lesions.
groups (F = 3.45, dr= 3/21, p < 0.05) and Newman-Keuls multiple comparisons [Winer, 15] indicated that the pyriform cortex group differed significantly from all other groups. (This was not true, however, if the data of the five rats with neocortical as well as pyriform cortex damage were included.) The unilateral amygdaloid, neocortical and operated control groups did not differ reliably among themselves and their data were pooled for further analyses.
TABLE 1 RESULTS
Histological examination revealed that 41 rats had sustained bilateral amygdaloid lesions. The lesions were found to vary both in their placement and extent, with damage to the internal capsule, caudate-putamen, claustrum and pyriform cortex variously included in some lesions. Nine rats sustained bilateral lesions to the pyriform cortex. Of the nine, however, five rats also sustained some damage to neocortex and were discarded. These lesions and a coronal section depicting the greatest subcortical encroachment found in the group are reconstructed in Fig. 1. The unilateral amygdaloid, neocortical and operated control groups were composed of seven animals each.
u28
~ 30
~= 9 6
FIG. 1. Reconstruction of pyriform cortex lesions (Experiment 1). Extent of bilateral lesion is represented by black area. Coronal section at lower right depicts the deepest lesion in this group.
Passive avoidance. Imtial comparisons were carried out among the three control groups and the pyriform cortex group. As can be seen in the upper portion of Table 1, the pyriform cortex group made many more contacts with the electrified water dish than did the control groups. Analysis of variance revealed a statistically reliable difference among
PERFORMANCE OF AMYGDALOID PYRIFORM AND CONTROL IN PASSIVE AVOIDANCE
Group Control Groups Amygdaloid Neocortical Operated Control Pyriform Cortex Amygdaloid Basolateral Corticomedlal
GROUPS
N
Mean Contacts
Range
21 7 7 7 4 41 13 13
14.2 18.0 14.9 9.5 43.7 34.9 46.2 24.2
5-51 8-32 5-51 5-21 10-80 2-183 5-183 2-98
A series of preliminary analyses were carried out to determine whether the size of the lesion or inclusion of nonamygdaloid structures such as internal capsule and caudateputamen were related to deficits in passive avoidance performance. I n no case was there a suggestion that these factors had contributed in any obvious way to passiveavoidance deficits. Comparisons were next carried out among rats which had sustained bilateral amygdaloid lesions (regardless of its locus or extent), pyriform and control animals. The results of this comparison are included in Table 1. It can be seen that both the amygdaloid lesions taken as a whole, and the pyriform cortex lesions disrupted passive avoidance. Analysis of variance indicated a significant difference among groups. (F = 3.33, df = 2/63, p < 0.05). Both the amygdaloid (t = 2.64, df= 61, p < 0.05) and pyriform (t = 5.02, dr= 24, p < 0.01) groups differed reliably from the pooled control groups but not from each other (t = 0.8). Although none of the rats in the present experiment had lesions which were entirely restricted to the medial nucleus, it was possible to form a group of rats whose lesions were largely restricted to the corticomedial group of nuclei (N = 13] and a second group whose lesions were almost entirely confined to the basolateral division (N = 13). Four representative lesions from the basolateral and corticomedial groups are presented in Figs. 2 and 3. A single section is shown for each rat which depicts the largest bilateral extent of the lesion. If the medial nucleus is essential for adequate passive avoidance performance in the rat, as it is in the cat, the corticomedial group should be deficient when compared with either the basolateral or control groups. This comparison is presented in the lower portion of Table 1. Although corticomedial lesions did seem to impair passive avoidance performance to some extent, the basolateral lesions produced an even more severe deficit. Analysis of variance indicated that these groups differed significantly among themselves (F = 4.16, df = 2/44, p < 0.05). The basolateral group
716
KEMBLE AND TAPP
30 # 93
J
'~ 9 5
i
59
32
[~INO
[hi=illI # IOI
~ IIO
FIG. 2. Four typical corticomedial amygdaloid lesions. One plate is shown for each rat depicting the greatest extent of bilateral damage. (plates from K6nig & Klippel, 1963).
34
OVERLAP
OVERLAP OF TWO LESIONS
, OVERLAP OF THREE LESIONS
I
56
OVERLAP OF FOUR OR MORE LESIONS
FIG. 4. Coronal sections depicting overlap of bilateral amydaloid lesions of the most severely disrupted rats (passive avoidance) in Experiment 1. (plates redrawn from Kbnlg & Khppel 1963). #7
#14 /['"
24
#46
FIG. 3. Four typical basolateral amygdaloid lesions. One plate is shown for each rat depicting the greatest extent of bilateral damage. (plates from KOnig & Klippel, 1963).
differed significantly from the pooled control groups ( t 2.37, d f = 33, p < 0.05). The basolateral and corticomedial groups did not, however, differ reliably from each other, (t : 1.01, d f - - 25). In an attempt to localize the area responsible for these deficits more precisely, the animals that were most severely impaired in this task were next selected (i.e. those rats who were two or more standard deviations above the mean of the pooled control rats) and their amygdalold lesions superimposed. A composite reconstruction of these 12 lesions is shown in Fig. 4. Although damage to every amygdaloid nucleus was noted in this group, an area including the medial and lateral portions of the basal nucleus seems to have been most consistently included in the lesions (11 of the 12 rats). The central (7 rats) and cortical (6 rats) nuclei were also frequently included in the lesions while the lateral and medial nuclei were least frequently damaged. This examination thus confirms the impression that damage to the basolateral division most consistently produces passive avoidance decrements in the rat and further suggests that damage in the area of the basal nucleus is most consistently related to this disruption. The number of approaches toward the electrified water dish (entries) failed to discriminate among groups on any of the analyses.
Active avoidance. Two measures of active avoidance performance were recorded in this experiment; errors to criterion and the mean latency of the last five acquisition trials. Errors to criterion did not reveal any reliable differences among groups in any of the analyses employed. The mean latency for the last five acquisition trials was found to be significantly longer for amygdaloid than control rats (F : 3.19, d f - - 2/59, p < 0.05). When amygdaloid lesions which included damage to the internal capsule or caudate-putamen were excluded from the analysis, however, no deficit could be shown. These results are shown in Table 2. TABLE 2 COMPARISON Ok AVOIDANCE LATENCY AMONG CONTROL CONDITIONS, AMYGDALOID LESIONS AND AMYGDALOID LESIONS WHICH INCLUDE DAMAGE TO THE INTERNAL CAPSULE AND/OR CAUDATEPUTAMEN
Group Pooled Controls Amygdaloid Lesions Amygdalold Lesions (plus internal capsule and/or caudate-putamen)
N
Mean Latency
Range
21 24 17
2.35 sec 2.29 sec 2.82 sec
0.694.67 sec 0.96~.10 sec 1.40-5.67sec
Thus amygdaloid lesions per ~e could not be shown to produce any detectable impairment on the active avoidance task. EXPERIMENT II Although the results of Experiment I suggest that pyriform cortex lesions disrupt passive avoidance, the number of rats
AMYGDALOID LESIONS
717
that sustained such lesions was small. Since this finding is of some importance in delineating passive avoidance areas of the amygdaloid complex, it was felt that a second group of rats should be tested in passive avoidance following pyriform cortex lesions. N o active avoidance test was given in this experiment.
I
3
4
METHOD
Subjects Subjects were 12 male albino Holtzman rats weighing 275-300 g when received. Six rats sustained pyriform cortex lesions, three rats received neocortical lesions, and the remaining three were simply anesthetized.
5
9
I0
FIG. 5. Reconstruction of pyriform cortex lesions in Experiment 11.
Apparatus The apparatus used in this experiment was identical to that described for passive avoidance in Experiment I.
TABLE 3 PERFORMANCE OF PYRIFORM A N D CONTROL GROUPS IN PASSIVE AVOIDANCE
Surgery A slightly different surgical approach to the pyriform cortex was employed in the present experiment. Rather than sever the attachment of the temporal muscle at the top of the skull, the zygomatic arch was approached and severed through an incision placed slightly dorsal to the zygomatic arch and through the temporal muscle. This permitted suturing of the incision and resulted in more rapid postoperative recovery. Otherwise, the lesions were produced in the same way as Experiment I. Following surgery the rats in the present experiment were individually, rather than grouphoused.
Procedures Procedures were identical with those described for passive avoidance in Experiment I with one exception; a current of 0.3 m A (rather than 0.2 mA) was employed for the passive avoidance test.
Histology Upon completion of passive avoidance testing, the rats were perfused with isotonic saline followed by 10 per cent formalin solution. The extent and placement of the lesions were plotted with the aid of a proportional divider. The brains were then sectioned and stained as described in Experiment I. RESULTS
The pyriform cortex lesions produced in the present study are depicted in Fig. 5. It can be seen that all six rats sustained bilateral pyriform cortex lesions which included no bilateral neocortical damage. Moreover, no damage was noted to any subcortical structures. The performance of the pyriform, neocortical and sham groups on the passive avoidance test is presented in Table 2. It can be seen that the performance of all three groups was quite similar with no suggestion of a deficit among the pyriform cortex rats. (F = 0.52, d f = 2/9). DISCUSSION
In his research with the cat Ursin [14] found passive avoidance deficits to be most closely related to damage in the medial amygdaloid nucleus. Such localization does not
Group
N
Mean Contacts
Range
Pyriform Cortex Nee,cortex Sham
6 3 3
8.8 9.7 6.6
4-15 5-10 6-15
seem to be indicated for the rat in the present experiments. Although there was some suggestion that corticomedial lesions interfered with the passive avoidance, the results of Experiment 1 indicate that destruction of an area roughly corresponding to the medial and lateral portions of the basal nucleus is most consistently associated with severe disruption of passive avoidance. Moreover, among the severely impaired animals of this experiment damage to the medial nucleus was least frequently included in the lesions. This finding is consistent with results of two other experiments. Pellegrino [10] found the passive avoidance of an electrified water spout to be more severely impaired following basolateral than corticomedial amygdaloid lesions in rats. Thompson and Schwartzbaum [13] found that lesions within a region including the cortical and basal amygdaloid nuclei of the rat to be most consistently related to disruption of conditioned suppression. These results point to the same region of the amygdaloid complex as the present results and, certainly, away from the medial nucleus. Moreover, these studies suggest that this localization does not change markedly with minor procedural variations. The passive avoidance deficit which followed pyriform cortex lesions in Experiment 1 raises the possibility that the pyriform cortex and ventral amygdalofugal pathway are also involved in passive avoidance. Such a finding might help to reconcile the difference in localization between cat and rat. In view of the failure to replicate this finding in Experiment II, however, this suggestion must remain tentative. It is possible that the slightly higher shock level used in Experiment II obscured this deficit. One other possibility is that the difference in localization reflects a species difference in the amygdaloid organization of passive avoidance. Although there seems to be no major differences in the anatomical organization of the amygdaloid complex of the two species, it is possible that the difference
718
KEMBLE AND TAPP
lies m the more complex and less obvious connections such as the detailed pattern of intraamygdaloid connections. The failure to demonstrate active avoidance deficits in Experiment I contrasts with the findings of Horvath [4] and Ursm [14] for the cat and Robinson [12] for the rat. One possible explanation is suggested by the results of Horvath [4] who found a more serious interference with two-way (shuttle box) than with one-way avoidance training following amygdaioid lesions. Therefore, it might be suspected that the easier one-way task used in Experiment I masked deficits that a two-way task would have revealed. It might also be suggested that active avoidance is more diffusely organized m the amygdaloid complex of the rat than it is in the cat. Thus, the lesions of Experiment I might not have disrupted
active avoidance because they were too small. In subsequent experiments, however, neither small nor extensive amygdaloid lesions disrupted a two-way avoidance response. Other experimenters seem to have experienced a simdar difficulty. Dicara [2] found that rats with amydaloid lesions were not impaired in the acqmsition of a lever-press shock avoidance response. Although King [6] found that amydaloid lesions in rats produced somewhat longer latencies during the acquisition of a two-way avoidance response, he could detect no difference in the number of errors. It would seem that the exact conditions necessary to demonstrate active avoidance deficits in amygdaloid rats are not fully understood. A series of experiments are in progress which will attempt to identify these conditions.
REFERENCES I. Cowan, W. M., G. Raisman and T. P. S. Powell. The connexions of the amygdala. J. neurol, neurosurg. Psychiat. 28: 137-151, 1965. 2. Dicara, L. V. Effect of amygdaloid lesions on avoidance learning in the rat. Psychonom. Sci. 4: 279-280, 1966. 3. Goddard, G. V. Functions of the Amygdala. Psychol. Bull. 62: 89-109, 1964. 4. Horvath, F. E. Effects of the basolateral amygdalectomy on three types of avoidance behavior m cats. J. comp. physiol. Psychol. 56: 380-389, 1963. 5. Kaada, B. R., E. W. Rasmussen and O. Kveim. Impaired acquisition of passive avoidance behavior by subcallosal, septal, hypothalamic, and insular lesions. J. eomp. physiol. Psyehol. 55: 661-670, 1962. 6. King, F. A. Effects of septal and amygdaloid lesions on emotional behavior and conditioned avoidance responses in the rat. J. nerv. ment. Dis. 126: 57-63, 1958. 7. K~,nig, J. F. R. and R. A. Klippel. The Rat Brain. Baltimore Wilhams and Wilkins, 1963. 8. Lubar, J. F. Effect of medial cortical lesions on the avoidance behavior of the cat. J. comp. physiol. Psychol. 58: 38-46, 1964.
9. McCleary, R. A. Response specificity in the behavioral effects of limbic system lesions in the cat. J. comp. physiol. Psychol. 54: 605-613, 1961. 10. Pellegrino, L. Effects of amygdaloid lesions on passive avoidance, paper presented at Eastern Psychological Association convention, 1966. 11. Powell, T. P. S., W. M. Cowan and G. Raisman. The central olfactory connexions. J. Anat. 99: 791-813, 1965. 12. Robinson, E. Effect of amygdalectomy on fear-motivated behavior in rats. J. comp. physiol. Psychol. 56: 814-820, 1963. 13. Thompson, J. B. and J. S. Schwartzbaum. Discrimination behavior and conditioned suppression (CER) following localized lesions in the amygdala and putamen. Psychol. Rep. 15: (Mon. Supple. 4-V15) 587-606, 1964. 14. Ursln, H. Effect of amygdaloid lesions on avoidance behavior and visual discrimination in cats. Expl Neurol. 11: 298-317, 1965. 15. Winer, B. J. Statistical Prtnciples in Experimental Destgn, New York: McGraw-Hill, 1962, 85-89.
Passive and Active Avoidance Performance Following Small Amygdaloid Lesions in Rats E R N E S T D. K E M B L E 1, 2 A N D J A C K T. T A P P
Department of Psychology, Vanderbilt University, Nash ville, Tennessee (Received 22 M a r c h 1968) KEMBLE, E. D. AND J. T. TAPP. Passive and active avoidance performance following small amygdaloM lesion~ ill rat~. PHVSlOL.BEnAV. 3 (5) 713-718, 1968.--Following small bilateral arnygdaloid lesions or pyriform cortex lesions rats were tested for both passive and active avoidance. Damage to an area roughly corresponding to the basal amygdaloid nucleus was most consistently related to disruption of passive avoidance. Pyriform cortex lesions also impaired passive avoidance but this result was not replicated in a second experiment. Neither amygdaloid nor pyriform cortex lesions disrupted active avoidance.
Amygdalold lesions
Pyriform cortex lesions
Passive avoidance
BILATERAL ablation of the amygdaloid complex has been repeatedly shown to disrupt avoidance behavior in a variety of situations such as one- and two-way avoidance, passive avoidance and conditioned suppression [e.g. 3, 4, 10, 12]. Recently Ursin [14] has shown that passive and active avoidance behavior are separately organized within the amygdaloid complex of the cat. Destruction of the rostral half of the lateral amygdaloid nucleus was found to disrupt active but not passive avoidance behavior while lesions which included the stria terminalis or the medial amygdaloid nucleus produced deficits in passive avoidance performance, leaving active avoidance unimpaired. The delineation of such topographically distinct systems subserving different types of fear-motivated behavior is of considerable importance for a fuller understanding of amygdaloid function. Therefore it seemed of interest to examine the organization of these systems in an additional species in order to gain some impression of their generality. As Cowan, Raisman and Powell [1] have pointed out, however, amygdaloid lesions also necessarily include destruction of fibers that originate in the pyriform cortex and travel through or end in the amygdaloid complex. Consequently it also seemed necessary to provide some estimate of the role of these pyriform fibers. The need for such an assessment is underlined by recent studies of the projection pathways from the pyriform cortex [11] which show that the pyriform cortex contributes many, if not all, of the nerve fibers constituting the ventral amygdalofugal pathway in the rat. Moreover, Kaada, Rasmussen and Kveim [5] found passive avoidance behavior to be disrupted following lesions which included pyriform as well as neocortex. The present studies were designed (a) to localize the areas whose destruction produce disruption of passive and active avoidance performance by placing small lesions at various loci throughout the amygdaloid complex of the rat and (b) to
Active avoidance
Localization of funcUon
provide some assessment of the effects of pyriform cortex lesions on these two types of behavior. EXPERIMENT I METHODS
Subjects This experiment employed 125 male albino Holtzman rats 100-120 days of age when received. Postoperative mortality and failure to recuperate properly reduced this number to 86.
Apparatus Passive Avoidance. The apparatus was similar to one described by McCleary [9]. It consisted of an 8¼ x 14½ in. plywood chamber and a smaller (31 × 4 × 41 in.) reward chamber. The walls were painted flat gray and the top was clear Plexiglass. The floor of the larger chamber was of plywood painted flat gray while the floor of the reward chamber was covered by an aluminum plate. A small aluminum water cup (1 x t x ¼ in.) was inserted through a slot in the rear wall of the reward chamber. The rat completed a circuit when it stood on the aluminum plate with its mouth or tongue in contact with the water. Active avoidance. The apparatus was similar to one described by Lubar [8] that provides a one-way avoidance task which requires no handling of the animal between trials. It consisted of two identical boxes constructed of plywood (11t x 8 x 8 in.) painted flat black. The floors were aluminum rods spaced I in. apart. Each box had a hinged clear Plexiglass top. Both ends of each box were composed of vertically sliding doors. The aluminum rods comprising the floors were connected to a Triplett high voltage power source through a grid scrambler and selector switch. The floor of either box could be electrified by appropriately
1This article is based on a dissertation submitted in partial fulfilment of Ph.D. requirements at Vanderbilt University. ~Now at University of Minnesota, Morris. 713
714 setting a selector switch which delivered rapid intermittent shock pulses to the feet of the rat. Each box was separately attached to the power source so that the boxes could be moved about by the experimenter. The CS for the active avoidance task was provided by a low intensity tone of approximately 1,000 c/s and a 7.5 W light. The light and speaker were mounted on a stationary pedestal 23 in. high and 10 in. above the top of the box from which the rat began each trial.
Procedure Preoperative. Approximately 18 hr before surgery the rats were weighed and all food removed from the home cage. They were then randomly designated to receive: bilateral amygdaloid lesions (this group was subdivided into groups which received lesions to the rostro-laterai, rostro-medlal, caudo-lateral and caudo-medial portions of the amygdalold complex), pyriform cortex lesions, neocortical lesions, unilateral amygdaloid lesions or no lesion (operated control). Operative procedure. All operations were carried out under sodium pentobarbital anesthesia (50 mg/kg) and clean conditions. The rats designated to receive subcortical lesions were positioned in a stereotactlc instrument adjusted so that bregma was 1.0 mm higher than lambda. The skull was exposed through a midllne incision, burr holes drilled at the appropriate points and a 24 g hypodermic needle (insulated except for 1 mm at the tip) lowered to the desired depth. Lesions were produced by a Grass radio-frequency lesion maker. The cortical lesions were produced by heating the skull overlying the cortex with a glass insulated tungsten electrode connected to a Bantom Boyle thermocautery. Amygdaloid lesions. In order to consistently produce small bilaterally symmetrical lesions, a single large lesion on one side was paired with a smaller lesion of the contralateral side. The current used to produce the large lesion ranged from 3.0--4.0 mA for durations of 20-25 sec while 2.5-3.2 mA for 5-8 sec was used to produce small lesions. The following coordinates were used for the four amygdaloid subgroups using bregma, midsagittal suture and surface of the cortex as reference points: rostro-lateral, 1.3-1.5 mm posterior (P), 4.5--4.6 mm lateral (L), and 7.0-7.2 mm deep (D); rostromedial, 1.3-1.5 mm P, 3.7-3.9 mm L and 7.4-7.6 mm D; caudo-lateral, 2.0-2.4 mm P, 4.5-4.75 mm L and 8.0 mm D. Unilateral amygdaloid control rats sustained a single large lesion at these coordinates, but the electrode was simply lowered 6.0 mm on the contralateral side and withdrawn. Cortical lesions. After the skull was exposed through a midline incision, the temporal muscle was retracted from its attachment to the skull and the posterior attachment of the zygomatic arch to the brain case severed. Pyriform cortex lesions were produced by pressing the tungsten electrode tip against the skull, ventral to the level of attachment of the zygomatic arch, and heating the bone for five sec. Neocortical lesions were produced in exactly the same way except that the electrode tip was pressed against the skull dorsal to the level of attachment of the zygomatic arch. Half of the operated control rats received treatment identical to that described for the amygdaloid lesions except that the electrode was lowered and no lesion produced. The remaining half received the same treatment as cortical rats except that no lesions were produced. Following surgery Sulfanilamide powder was sprinkled over the wound surface, the incision closed with metal wound clips, and the edges of the incision covered with collodion.
KEMBLE AND TAPP
Postoperattve care. The rats were placed In group cages (four animals per cage) for a period of 10-12 days. Dry food and water (containing sucrose and vitamin supplements) were available ad libitum during this recovery period. At the end of the recovery period the rats were returned to their home cages and received ad hbitum access to dry food and tap water for an additional two days. Approximately 24 hr before each animal was to begin passive avoidance training, all water was removed from the home cage. Dry food was available throughout the experiment. Passtve avotdance. The procedure employed in this phase of the experiment was similar to that employed by Kaada et al. [5]. After the rat had been deprived of water for 24 hr it was placed in the passive avoidance apparatus and allowed to drink from the aluminum dish for 15 mln. The rat was then returned to the home cage and received no additional water. This procedure was repeated for three days. On the fourth day the rat was placed in the passive avoidance apparatus once more with the water dish available. Ten sec after the rat began to drink a mild electric current (0.2 mA measured through the apparatus and 10,000 f~ resistance) was turned on and remained on for 20 rain. During this time interval two measures of passive avoidance were taken: the number of times the rat contacted the water and received a shock (contacts) and the number of times it entered the reward chamber without touching the water (entries). At the end of the 20 min test, the rat was returned to its home cage and given 30 mln ad libitum access to water. This schedule was followed for the remainder of the experiment. Active avoidance. At approximately the same time on the following day (approximately 1 hr before watering) each animal began active avoidance training. On each trial the box containing the rat was placed beneath the pedestal on which the speaker and hght were mounted. The second box was placed against the first so that the sliding doors of both boxes were directly opposite each other and approximately ½ in. apart. Simultaneously raising both doors closed a mlcroswltch connected to one of them. The closing of the mlcroswitch activated a 1,000 cps tone and the light, started a Cramer timer (1/100 sec) and a 5-sec time-delay switch. If the rat did not enter the second box within 5 sec, the timedelay switch closed and an intermittent current of 0.6 mA (measured through the apparatus and 10,000 f~ resistance) was delivered to the rat's feet. When the rat entered the second box, the experimenter closed a mlcroswitch which terminated the shock, tone, light, and timer and reset the 5 sec delay timer. The two sliding doors which separated the boxes were then lowered. During the intertrial interval (1 rain) the second box (now containing the animal) was moved back to a position under the pedestal to which the speaker and light were attached and the first box was moved to the former position of the second box. The rats received ten such trials per day until either ten consecutive avoidances were completed within a single day or until they had completed 100 training trials. Performance was measured by the number of errors (i.e. latencles of 5 sec or more) made in reaching acquisition criterion, and the mean response latency of the last five acquisition trials. Hlstologwal Procedures At the conclusion of active avoidance training the animals were perfused with isotonic saline followed by 10 per cent formalin solution while under deep pentobarbital anesthesia. After 1-3 days additional soaking in 10 per cent formalin,
AMYGDALOID LESIONS
715
the brains were removed, frozen, and sectioned in the frontal plane at 32 ~t. Every fourth and fifth section from the brains with subcortical lesions was saved. One series was stained for cells with cresyl violet, and the other series stained for myelin by the Weil method. Brains with cortical lesions were photographed from the sides and bottom, then frozen and sectioned in the frontal plane. Sections were retained at 0.5 mm intervals and stained with cresyl violet. These sections were used to determine the depth of the cortical lesions.
groups (F = 3.45, dr= 3/21, p < 0.05) and Newman-Keuls multiple comparisons [Winer, 15] indicated that the pyriform cortex group differed significantly from all other groups. (This was not true, however, if the data of the five rats with neocortical as well as pyriform cortex damage were included.) The unilateral amygdaloid, neocortical and operated control groups did not differ reliably among themselves and their data were pooled for further analyses.
TABLE 1 RESULTS
Histological examination revealed that 41 rats had sustained bilateral amygdaloid lesions. The lesions were found to vary both in their placement and extent, with damage to the internal capsule, caudate-putamen, claustrum and pyriform cortex variously included in some lesions. Nine rats sustained bilateral lesions to the pyriform cortex. Of the nine, however, five rats also sustained some damage to neocortex and were discarded. These lesions and a coronal section depicting the greatest subcortical encroachment found in the group are reconstructed in Fig. 1. The unilateral amygdaloid, neocortical and operated control groups were composed of seven animals each.
u28
~ 30
~= 9 6
FIG. 1. Reconstruction of pyriform cortex lesions (Experiment 1). Extent of bilateral lesion is represented by black area. Coronal section at lower right depicts the deepest lesion in this group.
Passive avoidance. Imtial comparisons were carried out among the three control groups and the pyriform cortex group. As can be seen in the upper portion of Table 1, the pyriform cortex group made many more contacts with the electrified water dish than did the control groups. Analysis of variance revealed a statistically reliable difference among
PERFORMANCE OF AMYGDALOID PYRIFORM AND CONTROL IN PASSIVE AVOIDANCE
Group Control Groups Amygdaloid Neocortical Operated Control Pyriform Cortex Amygdaloid Basolateral Corticomedlal
GROUPS
N
Mean Contacts
Range
21 7 7 7 4 41 13 13
14.2 18.0 14.9 9.5 43.7 34.9 46.2 24.2
5-51 8-32 5-51 5-21 10-80 2-183 5-183 2-98
A series of preliminary analyses were carried out to determine whether the size of the lesion or inclusion of nonamygdaloid structures such as internal capsule and caudateputamen were related to deficits in passive avoidance performance. I n no case was there a suggestion that these factors had contributed in any obvious way to passiveavoidance deficits. Comparisons were next carried out among rats which had sustained bilateral amygdaloid lesions (regardless of its locus or extent), pyriform and control animals. The results of this comparison are included in Table 1. It can be seen that both the amygdaloid lesions taken as a whole, and the pyriform cortex lesions disrupted passive avoidance. Analysis of variance indicated a significant difference among groups. (F = 3.33, df = 2/63, p < 0.05). Both the amygdaloid (t = 2.64, df= 61, p < 0.05) and pyriform (t = 5.02, dr= 24, p < 0.01) groups differed reliably from the pooled control groups but not from each other (t = 0.8). Although none of the rats in the present experiment had lesions which were entirely restricted to the medial nucleus, it was possible to form a group of rats whose lesions were largely restricted to the corticomedial group of nuclei (N = 13] and a second group whose lesions were almost entirely confined to the basolateral division (N = 13). Four representative lesions from the basolateral and corticomedial groups are presented in Figs. 2 and 3. A single section is shown for each rat which depicts the largest bilateral extent of the lesion. If the medial nucleus is essential for adequate passive avoidance performance in the rat, as it is in the cat, the corticomedial group should be deficient when compared with either the basolateral or control groups. This comparison is presented in the lower portion of Table 1. Although corticomedial lesions did seem to impair passive avoidance performance to some extent, the basolateral lesions produced an even more severe deficit. Analysis of variance indicated that these groups differed significantly among themselves (F = 4.16, df = 2/44, p < 0.05). The basolateral group
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30 # 93
J
'~ 9 5
i
59
32
[~INO
[hi=illI # IOI
~ IIO
FIG. 2. Four typical corticomedial amygdaloid lesions. One plate is shown for each rat depicting the greatest extent of bilateral damage. (plates from K6nig & Klippel, 1963).
34
OVERLAP
OVERLAP OF TWO LESIONS
, OVERLAP OF THREE LESIONS
I
56
OVERLAP OF FOUR OR MORE LESIONS
FIG. 4. Coronal sections depicting overlap of bilateral amydaloid lesions of the most severely disrupted rats (passive avoidance) in Experiment 1. (plates redrawn from Kbnlg & Khppel 1963). #7
#14 /['"
24
#46
FIG. 3. Four typical basolateral amygdaloid lesions. One plate is shown for each rat depicting the greatest extent of bilateral damage. (plates from KOnig & Klippel, 1963).
differed significantly from the pooled control groups ( t 2.37, d f = 33, p < 0.05). The basolateral and corticomedial groups did not, however, differ reliably from each other, (t : 1.01, d f - - 25). In an attempt to localize the area responsible for these deficits more precisely, the animals that were most severely impaired in this task were next selected (i.e. those rats who were two or more standard deviations above the mean of the pooled control rats) and their amygdalold lesions superimposed. A composite reconstruction of these 12 lesions is shown in Fig. 4. Although damage to every amygdaloid nucleus was noted in this group, an area including the medial and lateral portions of the basal nucleus seems to have been most consistently included in the lesions (11 of the 12 rats). The central (7 rats) and cortical (6 rats) nuclei were also frequently included in the lesions while the lateral and medial nuclei were least frequently damaged. This examination thus confirms the impression that damage to the basolateral division most consistently produces passive avoidance decrements in the rat and further suggests that damage in the area of the basal nucleus is most consistently related to this disruption. The number of approaches toward the electrified water dish (entries) failed to discriminate among groups on any of the analyses.
Active avoidance. Two measures of active avoidance performance were recorded in this experiment; errors to criterion and the mean latency of the last five acquisition trials. Errors to criterion did not reveal any reliable differences among groups in any of the analyses employed. The mean latency for the last five acquisition trials was found to be significantly longer for amygdaloid than control rats (F : 3.19, d f - - 2/59, p < 0.05). When amygdaloid lesions which included damage to the internal capsule or caudate-putamen were excluded from the analysis, however, no deficit could be shown. These results are shown in Table 2. TABLE 2 COMPARISON Ok AVOIDANCE LATENCY AMONG CONTROL CONDITIONS, AMYGDALOID LESIONS AND AMYGDALOID LESIONS WHICH INCLUDE DAMAGE TO THE INTERNAL CAPSULE AND/OR CAUDATEPUTAMEN
Group Pooled Controls Amygdaloid Lesions Amygdalold Lesions (plus internal capsule and/or caudate-putamen)
N
Mean Latency
Range
21 24 17
2.35 sec 2.29 sec 2.82 sec
0.694.67 sec 0.96~.10 sec 1.40-5.67sec
Thus amygdaloid lesions per ~e could not be shown to produce any detectable impairment on the active avoidance task. EXPERIMENT II Although the results of Experiment I suggest that pyriform cortex lesions disrupt passive avoidance, the number of rats
AMYGDALOID LESIONS
717
that sustained such lesions was small. Since this finding is of some importance in delineating passive avoidance areas of the amygdaloid complex, it was felt that a second group of rats should be tested in passive avoidance following pyriform cortex lesions. N o active avoidance test was given in this experiment.
I
3
4
METHOD
Subjects Subjects were 12 male albino Holtzman rats weighing 275-300 g when received. Six rats sustained pyriform cortex lesions, three rats received neocortical lesions, and the remaining three were simply anesthetized.
5
9
I0
FIG. 5. Reconstruction of pyriform cortex lesions in Experiment 11.
Apparatus The apparatus used in this experiment was identical to that described for passive avoidance in Experiment I.
TABLE 3 PERFORMANCE OF PYRIFORM A N D CONTROL GROUPS IN PASSIVE AVOIDANCE
Surgery A slightly different surgical approach to the pyriform cortex was employed in the present experiment. Rather than sever the attachment of the temporal muscle at the top of the skull, the zygomatic arch was approached and severed through an incision placed slightly dorsal to the zygomatic arch and through the temporal muscle. This permitted suturing of the incision and resulted in more rapid postoperative recovery. Otherwise, the lesions were produced in the same way as Experiment I. Following surgery the rats in the present experiment were individually, rather than grouphoused.
Procedures Procedures were identical with those described for passive avoidance in Experiment I with one exception; a current of 0.3 m A (rather than 0.2 mA) was employed for the passive avoidance test.
Histology Upon completion of passive avoidance testing, the rats were perfused with isotonic saline followed by 10 per cent formalin solution. The extent and placement of the lesions were plotted with the aid of a proportional divider. The brains were then sectioned and stained as described in Experiment I. RESULTS
The pyriform cortex lesions produced in the present study are depicted in Fig. 5. It can be seen that all six rats sustained bilateral pyriform cortex lesions which included no bilateral neocortical damage. Moreover, no damage was noted to any subcortical structures. The performance of the pyriform, neocortical and sham groups on the passive avoidance test is presented in Table 2. It can be seen that the performance of all three groups was quite similar with no suggestion of a deficit among the pyriform cortex rats. (F = 0.52, d f = 2/9). DISCUSSION
In his research with the cat Ursin [14] found passive avoidance deficits to be most closely related to damage in the medial amygdaloid nucleus. Such localization does not
Group
N
Mean Contacts
Range
Pyriform Cortex Nee,cortex Sham
6 3 3
8.8 9.7 6.6
4-15 5-10 6-15
seem to be indicated for the rat in the present experiments. Although there was some suggestion that corticomedial lesions interfered with the passive avoidance, the results of Experiment 1 indicate that destruction of an area roughly corresponding to the medial and lateral portions of the basal nucleus is most consistently associated with severe disruption of passive avoidance. Moreover, among the severely impaired animals of this experiment damage to the medial nucleus was least frequently included in the lesions. This finding is consistent with results of two other experiments. Pellegrino [10] found the passive avoidance of an electrified water spout to be more severely impaired following basolateral than corticomedial amygdaloid lesions in rats. Thompson and Schwartzbaum [13] found that lesions within a region including the cortical and basal amygdaloid nuclei of the rat to be most consistently related to disruption of conditioned suppression. These results point to the same region of the amygdaloid complex as the present results and, certainly, away from the medial nucleus. Moreover, these studies suggest that this localization does not change markedly with minor procedural variations. The passive avoidance deficit which followed pyriform cortex lesions in Experiment 1 raises the possibility that the pyriform cortex and ventral amygdalofugal pathway are also involved in passive avoidance. Such a finding might help to reconcile the difference in localization between cat and rat. In view of the failure to replicate this finding in Experiment II, however, this suggestion must remain tentative. It is possible that the slightly higher shock level used in Experiment II obscured this deficit. One other possibility is that the difference in localization reflects a species difference in the amygdaloid organization of passive avoidance. Although there seems to be no major differences in the anatomical organization of the amygdaloid complex of the two species, it is possible that the difference
718
KEMBLE AND TAPP
lies m the more complex and less obvious connections such as the detailed pattern of intraamygdaloid connections. The failure to demonstrate active avoidance deficits in Experiment I contrasts with the findings of Horvath [4] and Ursm [14] for the cat and Robinson [12] for the rat. One possible explanation is suggested by the results of Horvath [4] who found a more serious interference with two-way (shuttle box) than with one-way avoidance training following amygdaioid lesions. Therefore, it might be suspected that the easier one-way task used in Experiment I masked deficits that a two-way task would have revealed. It might also be suggested that active avoidance is more diffusely organized m the amygdaloid complex of the rat than it is in the cat. Thus, the lesions of Experiment I might not have disrupted
active avoidance because they were too small. In subsequent experiments, however, neither small nor extensive amygdaloid lesions disrupted a two-way avoidance response. Other experimenters seem to have experienced a simdar difficulty. Dicara [2] found that rats with amydaloid lesions were not impaired in the acqmsition of a lever-press shock avoidance response. Although King [6] found that amydaloid lesions in rats produced somewhat longer latencies during the acquisition of a two-way avoidance response, he could detect no difference in the number of errors. It would seem that the exact conditions necessary to demonstrate active avoidance deficits in amygdaloid rats are not fully understood. A series of experiments are in progress which will attempt to identify these conditions.
REFERENCES I. Cowan, W. M., G. Raisman and T. P. S. Powell. The connexions of the amygdala. J. neurol, neurosurg. Psychiat. 28: 137-151, 1965. 2. Dicara, L. V. Effect of amygdaloid lesions on avoidance learning in the rat. Psychonom. Sci. 4: 279-280, 1966. 3. Goddard, G. V. Functions of the Amygdala. Psychol. Bull. 62: 89-109, 1964. 4. Horvath, F. E. Effects of the basolateral amygdalectomy on three types of avoidance behavior m cats. J. comp. physiol. Psychol. 56: 380-389, 1963. 5. Kaada, B. R., E. W. Rasmussen and O. Kveim. Impaired acquisition of passive avoidance behavior by subcallosal, septal, hypothalamic, and insular lesions. J. eomp. physiol. Psyehol. 55: 661-670, 1962. 6. King, F. A. Effects of septal and amygdaloid lesions on emotional behavior and conditioned avoidance responses in the rat. J. nerv. ment. Dis. 126: 57-63, 1958. 7. K~,nig, J. F. R. and R. A. Klippel. The Rat Brain. Baltimore Wilhams and Wilkins, 1963. 8. Lubar, J. F. Effect of medial cortical lesions on the avoidance behavior of the cat. J. comp. physiol. Psychol. 58: 38-46, 1964.
9. McCleary, R. A. Response specificity in the behavioral effects of limbic system lesions in the cat. J. comp. physiol. Psychol. 54: 605-613, 1961. 10. Pellegrino, L. Effects of amygdaloid lesions on passive avoidance, paper presented at Eastern Psychological Association convention, 1966. 11. Powell, T. P. S., W. M. Cowan and G. Raisman. The central olfactory connexions. J. Anat. 99: 791-813, 1965. 12. Robinson, E. Effect of amygdalectomy on fear-motivated behavior in rats. J. comp. physiol. Psychol. 56: 814-820, 1963. 13. Thompson, J. B. and J. S. Schwartzbaum. Discrimination behavior and conditioned suppression (CER) following localized lesions in the amygdala and putamen. Psychol. Rep. 15: (Mon. Supple. 4-V15) 587-606, 1964. 14. Ursln, H. Effect of amygdaloid lesions on avoidance behavior and visual discrimination in cats. Expl Neurol. 11: 298-317, 1965. 15. Winer, B. J. Statistical Prtnciples in Experimental Destgn, New York: McGraw-Hill, 1962, 85-89.