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Ventromedial hypothalamus: Fear conditioning and passive avoidance in rats
Physiology & Behavior, Vol. 16, pp. 91-95. Pergamon Press and
Brain Research Publ., 1976. Printed in the U.S.A.
BRIEF COMMUNICATION Ventromedial Hypothalamus: Fear Conditioning and Passive Avoidance in Rats 1 FRANCIS C. COLPAERT 2'3 AND PIET R. WlEPKEMA
Zoological Laboratory, University o f Groningen (Haren ), The Netherlands (Received 16 September 1974) COLPAERT, F. C. AND P. R. WIEPKEMA. The ventromedial hypothalamus: fear conditioning and passive avoidance in rats. PHYSIOL. BEHAV. 16(1) 91-95, 1976. - Lesions in the ventromedial hypothalamus are found to induce deficient fear acquisition in rats. This deficit parsimoniously explains a secondary, i.e. passive avoidance, deficit and predicts other behavioral disturbances. Ventromedial hypothalamus
Fear
Passiveavoidance
Lesions
situations (i.e. footshock) and avoidance responding. Therefore, by direct observation, a continuous recording was made of all ongoing behavior performed by the animal submitted to the test procedure.
THE ventromedial hypothalamus (VMH) is considered to be involved in a so-called response suppressor system [9]. Chemical or lesion-produced inactivation of this hypothalamic area results in the enhanced occurrence or persistence of punished behavior [6, 8, 9]. The passive avoidance deficit, which is conceived as a response inhibitory deficit [ 10], together with the heightened reactivity to footshock [ 15 ] is proposed [ 16] as an explanation of improved active conditioned avoidance responding (ACAR) in a two-way shuttle box. The latter p h e n o m e n o n indeed has been revealed in rats following VMH lesioning [5,16] or cholinergic blockade [4]. These conceptions of VMH function in behavior make it clear that the investigation of the possible role of the VMH in behavior should also be concerned with fear conditioning, since the latter mechanism is expected to be intimately involved in response suppression. One problem raised by these studies is, whether the establishment of conditioned aversive reactions and/or the suppressant effect of aversive conditioning upon punished behavior is disturbed by VMH lesions in rats. If VMH lesioned rats show a decreased fear conditioning without demonstrating response suppression, then it would be likely that the passive avoidance deficit and the improved ACAR following VMH lesions would be explained by deficient fear. The purpose of the present study bears on fear conditioning and passive avoidance in VMH lesioned rats. A second target is to search for alternative behavioral measurements of fear. Preliminary studies in normal rats suggested that, among other behavioral categories, scanning (attention posture) is related in a particular way to aversive
METHOD
Animals All animals were housed individually in commercial plastic cages and had food and water available ad lib. Room temperature was held constant at 21°C; ventilation provided background masking noise. The light-dark cycle, and consequently the diurnal activity rhythm of the animals was reversed, the lights going off at 8 a.m. and on at 8 p.m.; observations were done during the period between 8 a.m. and 4 p.m. A total number of 42 adult female Wistar rats was used; 21 animals were assigned to the normal control group, the others to the VMH lesioned group.
Procedu re Surgery. Stereotaxically directed bilateral electrolytical lesions in the VMH were produced in all rats assigned to the VMH lesioned group. Under ether anaesthesia, a unipolar 0.4 mm stainless steel electrode, insulated except for the cross-sectional area at the tip, was implanted at the following VMH coordinates: AP = 5.8; H = - 3 . 2 ; L = 0.7 [3]. A 1.5 rnA anodal current was passed for 8 sec between the implanted electrode and a cathode attached on the
The authors are much indebted to Christine L~ert for helpful suggestions to the preparation of the manuscript. 2Supported by a grant from the European Training Program in Brain and Behavior Research. 3Requests for reprints should be sent to F. C. Colpaert. Present address: Department of Pharmacology, Janssen Pharmaceutica, B-2340 Beerse, Belgium. 91
92
COLPAERT AND WlEPKEMA
exposed neck muscle. The implant was then withdrawn and the procedure repeated on the contralateral side of the brain. Histology. Following decapitation and fixation in 10% buffered neutral Formalin, the brains were dehydrated, cleaned and embedded in paraffin. Sections in the frontal plane were cut at 10 u thickness through the anteriorposterior extent of the lesion. Luxol was used for fiber staining, and cresyl violet for cell staining [7].
marized in Table 1. It follows that on the first day, normal rats defecate more than VMH lesioned ones. Shock administration (Test Day 2) induces an increase to a level similar for both groups. This high level is maintained on the third day by the intact animals but not by the VMH lesioned ones. However, conditioning effects are still present on this third day in VMH rats, though to a smaller extent as compared with control rats.
EXPERIMENT 1
MEDIAN VALUES AND 95% CONFIDENCE LIMITS AND STATISTICAL EVALUATIONOF RESULTS WITH REGARDTO DEFECATION DATA.
The first experiment is designed to test the acquisition and retention of a fear response established by electrical footshock [ 14].
TABLE !
Medians
Apparatus and Training Procedure The testing box, 70 x 30 x 30 cm high, was divided into 2 equal sized compartments by an aluminum vertically sliding door. One compartment (A) was black painted and not illuminated, the other (B) was of aluminum color and illuminated by a 25 W bulb. The front wall and top were opaque. The box was fitted with a stainless steel grid floor consisting of 1.4 mm rods 15 mm apart. On Test Day 1 (preshock test) the door was raised 7 cm, providing an opening between the two compartments. Each animal was placed individually in the black compartment and allowed to explore the entire box for 5 rain. On Test Day 2 the door was closed, and the animal was introduced into the black compartment; a 1.0 mA and 2 sec long scrambled shock was delivered every 13 sec during a 5 rain session. On Test Day 3 (postshock test) the procedure of Day 1 was repeated. The 3 test sessions were given on consecutive days. After each session, the number of fecal boli produced by each animal was counted. During the entire 5 min sessions the animal's behavior was continuously observed and recorded, according to the following behavior categories: (1) scanning and attention posture (the rat standing motionless with a normal palpebral aperture and without sniffing); (2) locomotion; (3) sniffing; (4) grooming; (5) presence in compartment B. Registration was done with a multiple event marker and recorded on an Esterline-Angus Eventrecorder. This procedure allowed recall of the onset and offset of each individual behavioral bout with an accuracy of + 0.5 sec. The experiment started on the seventh day after surgery.
Results Histology. The neural damage in the rats (n = 17) with VMH lesions was quantitatively defined by an area circumscribed by the following coordinates: AP = 5.2 to 6.4; H = - 2 to - 3 . 5 ; L = 0.0 to 2.0 [2]. Some lesions (n = 5) formed one continuous cavity bilaterally involving the nucleus ventromedialis hypothalami (NVMH) and extending into the third ventricle. In some animals, the ventral part of the nucleus dorsomedialis was either unilaterally (n = 2) or bilaterally (n = 2) affected by the lesion. Other lesions were bilaterally confined to the NVMH; in 3 instances only partial destruction of the NVMH was observed on one side of the brain. Thus, in each rat at least partial bilateral damage to the NVMH was observed; no clearcut relationship between lesion size and behavioral measurements could be established. Behavioral. The data regarding defecation are sum-
NI: 2 (0-4) N2: 6 (3-7) N3: 6 (3-8) VMH 1: 0 (0-2) VMH2:7 ( 3 - 1 0 ) VMH3:0 ( 0 - 4 )
Statistics Comparison NI-N2 N1-N3 N2-N3 VMH 1-VMH2 VMHI-VMH3 VMH2-VMH3 NI-VMH1 N2-VMH2 N3-VMH3
p <0.01 <0.01 >0.05 <0.01 <0.02 <0.01 <0.05 >0.05 <0.05
Symbols: N, normal control group; VMH, lesioned group; 1, 2 and 3 refer to consecutive test days; p, pertains to two-tailed probability. Table 2 presents median values and statistical evaluation of differences as to the total time spent in B (illuminated compartment), as well as to the frequency and mean duration of visits to this compartment. The latency to return to A for the first time after having gone to B, is also shown. This provides a more sensitive measure of fear than the speed with which the rat leaves the box [ 1 ]. On the first day VMH lesioned rats spent more time in B, and visit B more frequently than normals. The mean duration of these visits as well as the latency to return to A however does not differ from the normal control values. It can be inferred that both groups demonstrate a preference for the dark compartment, this trend however being less appreciable in VMH lesioned than in normal rats. On Day 3, normal rats spent more time in B, and stay there for longer periods than on Day 1. The number of visits is also significantly reduced, while the latency of return to A is dramatically increased. All these differences are significant at the 0.01 level. Except for the total time spent in B, which is not significantly altered, similar changes were revealed in VMH rats (p<0.05). Consequently, the preference for the dark compartment is considered to be reversed in normal, but left virtually unaffected in VMH lesioned rats. Thus, although both groups do show conditioning effects, the extent of these effects is significantly larger in normal than in VMH lesioned rats. Figure I shows median values of the time spent on 4 behavioral categories during 3 consecutive test days. The following main tendencies were revealed. (a) On the first Test Day the lesioned animals perform less scanning, but more locomotion and sniffing than the normal ones. (b) The shock session (Test Day 2) causes a gross behavioral change which is characterized by the enhancement of
VMH LESIONS AND F E A R
93 TABLE 2
MEDIAN VALUES AND 95% CONFIDENCE LIMITS, AND STATISTICALEVALUATIONOF RESULTS WITHREGARDTO PRESENCEIN COMPARTMENTSA AND B (TIMEIN SEC)
N1 N3 VMH1 VMH3
Total time in B
Frequency to B
Mean duration--B
Latency returtr--A
46 (38-61) 246 (229-287)t 105 (71-125)~ 103 (68-159)
4 (4-6) 2 (1-4)t 7 (5-8)$ 5 (3-6)*
9 (8-13) 111 (49-287)t 13 (10-20) 18 (14-26)*
26 (16-38) 213 (68-300)t 20 (14-26) 24 (14-114)*
*(p<0.05) and t (p<0.01) refer to the significance of differences between conditions (Days 1 and 3) for the same group (comparisons N1-N3 and VMH1-VMH3). $ (p <0.01) refers to the significance of differences between groups for the same conditions (comparison N1-VMH1). Other symbols as in Table 1. EXPERIMENT 2 scanning and a decrease of locomotion, sniffing and grooming. This behavioral pattern is most marked in the The second experiment constitutes a test for both fear normal control group. (c) In normal rats this behavioral • conditioning and passive avoidance, in that the animals are change, except for grooming, is continued on the third day, required to refrain from an ongoing behavioral response as is revealed by the comparison N1-N3. The comparison (i.e. locomotion) in order to avoid punishment (i.e. electric N2-N3 further indicates that locomotion is still more footshock). suppressed, whereas sniffing and grooming are partly recovered. The high level of scanning remains unchanged. In Apparatus and Training Procedure VMH lesioned rats too, the comparison VMH1-VMH3 evidences the persistance of most conditioning effects. The apparatus consisted of a 65 x 30 x 30 cm high Comparing VMH2-VMH3 reveals the partial recovery o f aluminum box, fitted with a stainless steel grid floor of 1.4 grooming, whereas other behavioral categories remain mm rods 15 mm apart. The floor was connected to a essentially unaltered. scrambled shock source which provided a 1.05 mA shock
300]
DAY 1 300-
~
NORMAL CONTROL
F~VMH
30o]
DAY 2
DAY 3
LESIONED
250-
z 200¢J z
2so1
2so1
20O I
20° I
C--- I50-
m 100-
50-
SCAN
LOCO
SNIF
GROO
SCAN
day 3 vs day 2
:
...2...3w.ayl
: : :
LOCO
:
SNIF
: *
GRO0
: **
SCAN
: :
LOCO
SNiF
~
~
: **
:
GRO0 !
*
FIG. 1. Median total time spent on scanning (SCAN), locomotion (LOCO), sniff'mg (SNIF) and grooming (GROO), and 95% confidence limits. For explanation of symbols referring to statistical evaluation, see Table 1.
94
COLPAERT AND WIEPKEMA
during 1 sec immediately after the onset of a locomotion response; shock delivery was manually controlled by the experimenter. On Day 1 the animal was introduced into the testing box for 4 min, during which all locomotion responses were punished. A locomotion response was defined by the animal moving in the horizontal plane for an estimated distance of 5 cm. On the following day (postshock test) the animals were retested once again during 4 min without shock. On the first day, only locomotion counts were recorded. On the second day, the same locomotion recording was made, and in addition the behavioral repertoire as described above was observed and recorded on the Esterline Eventrecorder. Day 1 of this experiment coincided with the seventeenth day after surgery.
Fig. 2a
•
NORMAL
0
VMH LESIONED
% 100 90 80-
~R T '\", I \ 'XI ,,
7060" 50-
Results
" ',1
40-
The results represented for the first day of Experiment 2 (Fig. 2) pertain to the frequency of locomotion responses. The frequency of responses during the second, third and fourth min was expressed as a percent of locomotion frequency observed during the first minute; median percent change per group for each minute are shown in Fig. 2a. VMH lesioned rats show a significantly higher frequency of locomotion as compared to the normal group (p<0.01), and consequently received a higher number of shocks (MedianN = 27; MedianvM H = 41). From a further analysis (Fig. 2b), it follows that the only significant decrease between consecutive minutes occurs between the first and the second one, both in normal and VMH lesioned rats. Despite this apparent similarity between both groups, it should be noted that the overall percent decrease between the first and the fourth minute in normal rats reliably exceeds that in VMH lesioned ones. The results of the behavioral recordings made on the second day of Experiment 2 can be summarized as follows: the VMH lesioned rats showed significantly less scanning (p<0.001) and more sniffing (p<0.001) than the normal control animals did; grooming was virtually abolished in both groups, and there was no difference (p>0.05) between groups. The VMH lesioned group spent more time in locomotion than the control group (/9<0.001), whereas the frequency of the locomotion response (as compared to the first-day-performance) was reduced in the control (p<0.001) but not in the VMH lesioned group (p>0.05). DISCUSSION
The results of Experiment 1 support two important assumptions. First, it is indicated that VMH lesioned rats show less fear conditioning effects than normal rats. This conclusion is based on the data concerning fecal production, visits to compartment B, and first return to compartment A. Second, it is revealed that, as was already suggested by preliminary studies, some particular behavioral changes are induced by shock and maintained through conditioning, these changes being intimately related to other more commonly recognized measurements of fear conditioning. On the first day the VMH lesioned rats defecate less and spent less time on scanning and more on locomotion and sniffing than normal rats do. They also spent more time in compartment B. Moreover since it is known that light has some aversive significance in rats [3] it may be concluded that VMH rats exhibit less fear behavior than normals in a
CONTROL
\\
30-
\\ 20-
\\
100 I
F---I 2
.___i
F--4 3
/. MIN.
Fig. 2b
COMPARISON
N
VMH
N-VMH
1 - 2 rain.
.00031
.0069
.11
2-3
.039
.72
.73
.15
.25
.70
.00L/.
.003
rain.
3 - 1 . rain. t-/.
min.
< 00005
FIG. 2. (a) Median frequency and 95% confidence limits of locomotion on 4 consecutive minutes as the percent of locomotion frequency on the first minute. (b) Statistical testing of progressive percent decrease of locomotion frequency over 4 consecutive minutes. Results are expressed as the two-tailed probability that the various listed comparisons were the same. Symbols as in Table l. situation that represents a novel environment, as is the case on the first day. The latter conclusion is consistent with some earlier findings [5,11] showing that VMH lesioned rats eat reliably sooner in a novel environment than normal rats do. The results of Experiment 2 confirm earlier reports [8,9] regarding passive avoidance in VMH lesioned rats. However, the extention of this finding to the experimental conditions presently described is relevant. Indeed, from the present results it follows that the passive avoidance deficit in VMH lesioned rats is independent from any possible interference with food or water motivation that may be enhanced by this type of brain lesion [ 11 ]. Thus, despite the increased reactivity of VMH lesioned rats to footshock [15], a passive avoidance deficit is evidenced in VMH lesioned rats, using a shock as the US. These findings, as well as the results of Experiment 1 lent support to the conclusion that the fear conditioning deficit, rather than a response inhibitory deficit [9] more
VMH LESIONS AND F E A R
95
parsimoniously explains the passive avoidance deficit following VMH lesions. The fact that the behavioral pattern, found to be related to fear conditioning, occurs in the passive avoidance experiment too, further supports this conclusion. However, this deficit in fear conditioning, and consequently the passive avoidance deficit, does not represent a total loss of fear. Indeed, the VMH animals, although to a smaller degree, show some clear conditioning effects both in the first and second experiment. It is obvious that fear conditioning plays a very prominent adaptive role in behavior, particularly in spon-
taneous social behavior. In the view of the present experiments on fear conditioning in VMH lesioned rats, it can be hypothesised that these animals may also show some abnormalities in social behavior, and particularly in these social interactions which to a certain degree are controlled by fear. It is further suggested that the measurement o f scanning, locomotion, sniffing and grooming may possibly constitute an alternative approach to the distinction between response inhibition and fear deficit as explanations of experimentally induced behavioral changes.
REFERENCES 1. Blanchard, R. J. and D. C. Blanchard. Escape and avoidance responses to a fear eliciting situation. Psychon. Sci. 13: 19-20, 1968. 2. De Groot, J. The rat forebrain in stereotaxic coordinates. Proc. K. Ned. Akad. Wet., C. 52: 1-40, 1959. 3. Desiderato, O. Generalization of acquired fear as a function of CS intensity and number of acquisition trials. J. exp. Psychol. 67: 41-47, 1964. 4. Grossman, S. P. The VMH: A center for affective reactions, satiety, or both? Physiol. Behav. 1: 1-10, 1966. 5. Grossman, S. P. Aggression, avoidance, and reaction to novel environments in female rats with ventromedial hypothalamic lesions. Z comp. physiol. Psychol. 78: 274-283, 1972. 6. Kaada, B. R., E. W. Rasmussen and O. Kveim. Impaired acquisition of passive avoidance behavior by subcallosal, septal, hypothalamic and insular lesions in the rat. J. comp. physiol. Psychol. 55: 661-670, 1962. 7. Kliiver, H. and E. Barrera. A method for the combined staining of cells and fibers in the nervous system. J. Neuropath. exp. Neurol. 12: 400-403, 1953. 8. Margules, D. L. and L. Stein. Neuroleptics vs tranquillizers: Evidence from animal behavior studies of mode and site of action. In: Neuropsycho-pharmacology, edited by H. Bull, J. O. Cole, P. Deniker, H. Hippius and P. B. Bradley. New York: Excerpta Medica Foundation, 1966.
9. Margules, D. L. and L. Stein. Cholinergic synapses in the ventromedial hypothalamus for the suppression of operant behavior by punishment and satiety. J. comp. physiol. Psychol. 67: 327-335, 1969. 10. McCleary, R. A. Response specificity in the behavioral effects of limbic system lesions in the cat. J. comp. physiol. Psychol. 54: 605-613, 1961. 11. Sclafani, A. and S. P. Grossman. Reactivity of hypeiphagic and normal rats to quinine and electric shock. Z comp. physiol. Psychol. 74: 157-166, 1971. 12. Siegel, S. Nonparametric Statistics for the Behavioral Sciences. New York: McGraw-Hill, 1956. 13. Singh, D. Comparison of behavioral deficits caused by lesions in septal and ventromedial hypothalamic areas of female rats. J. comp. physioL PsychoL 84: 370-379, 1973. 14. Slotniek, B. M. and D. L. Brown. Fear conditioning and passive avoidance in mice with septal lesions. Physiol. Behav. 5: 1255-1259, 1970. 15. Turner, S. G., J. A. Sechzer and R. A. Liebelt. Sensitivity to electrical shock after ventromedial hypothalamic lesions. Expl Neurol. 19: 236-244, 1967. 16. Weisman, R. N. and L. W. Hamilton. Two-way avoidance responding following VMH lesions: Effects of varying shock intensity. Physiol. Behav. 9: 243-246, 1972.
Brain Research Publ., 1976. Printed in the U.S.A.
BRIEF COMMUNICATION Ventromedial Hypothalamus: Fear Conditioning and Passive Avoidance in Rats 1 FRANCIS C. COLPAERT 2'3 AND PIET R. WlEPKEMA
Zoological Laboratory, University o f Groningen (Haren ), The Netherlands (Received 16 September 1974) COLPAERT, F. C. AND P. R. WIEPKEMA. The ventromedial hypothalamus: fear conditioning and passive avoidance in rats. PHYSIOL. BEHAV. 16(1) 91-95, 1976. - Lesions in the ventromedial hypothalamus are found to induce deficient fear acquisition in rats. This deficit parsimoniously explains a secondary, i.e. passive avoidance, deficit and predicts other behavioral disturbances. Ventromedial hypothalamus
Fear
Passiveavoidance
Lesions
situations (i.e. footshock) and avoidance responding. Therefore, by direct observation, a continuous recording was made of all ongoing behavior performed by the animal submitted to the test procedure.
THE ventromedial hypothalamus (VMH) is considered to be involved in a so-called response suppressor system [9]. Chemical or lesion-produced inactivation of this hypothalamic area results in the enhanced occurrence or persistence of punished behavior [6, 8, 9]. The passive avoidance deficit, which is conceived as a response inhibitory deficit [ 10], together with the heightened reactivity to footshock [ 15 ] is proposed [ 16] as an explanation of improved active conditioned avoidance responding (ACAR) in a two-way shuttle box. The latter p h e n o m e n o n indeed has been revealed in rats following VMH lesioning [5,16] or cholinergic blockade [4]. These conceptions of VMH function in behavior make it clear that the investigation of the possible role of the VMH in behavior should also be concerned with fear conditioning, since the latter mechanism is expected to be intimately involved in response suppression. One problem raised by these studies is, whether the establishment of conditioned aversive reactions and/or the suppressant effect of aversive conditioning upon punished behavior is disturbed by VMH lesions in rats. If VMH lesioned rats show a decreased fear conditioning without demonstrating response suppression, then it would be likely that the passive avoidance deficit and the improved ACAR following VMH lesions would be explained by deficient fear. The purpose of the present study bears on fear conditioning and passive avoidance in VMH lesioned rats. A second target is to search for alternative behavioral measurements of fear. Preliminary studies in normal rats suggested that, among other behavioral categories, scanning (attention posture) is related in a particular way to aversive
METHOD
Animals All animals were housed individually in commercial plastic cages and had food and water available ad lib. Room temperature was held constant at 21°C; ventilation provided background masking noise. The light-dark cycle, and consequently the diurnal activity rhythm of the animals was reversed, the lights going off at 8 a.m. and on at 8 p.m.; observations were done during the period between 8 a.m. and 4 p.m. A total number of 42 adult female Wistar rats was used; 21 animals were assigned to the normal control group, the others to the VMH lesioned group.
Procedu re Surgery. Stereotaxically directed bilateral electrolytical lesions in the VMH were produced in all rats assigned to the VMH lesioned group. Under ether anaesthesia, a unipolar 0.4 mm stainless steel electrode, insulated except for the cross-sectional area at the tip, was implanted at the following VMH coordinates: AP = 5.8; H = - 3 . 2 ; L = 0.7 [3]. A 1.5 rnA anodal current was passed for 8 sec between the implanted electrode and a cathode attached on the
The authors are much indebted to Christine L~ert for helpful suggestions to the preparation of the manuscript. 2Supported by a grant from the European Training Program in Brain and Behavior Research. 3Requests for reprints should be sent to F. C. Colpaert. Present address: Department of Pharmacology, Janssen Pharmaceutica, B-2340 Beerse, Belgium. 91
92
COLPAERT AND WlEPKEMA
exposed neck muscle. The implant was then withdrawn and the procedure repeated on the contralateral side of the brain. Histology. Following decapitation and fixation in 10% buffered neutral Formalin, the brains were dehydrated, cleaned and embedded in paraffin. Sections in the frontal plane were cut at 10 u thickness through the anteriorposterior extent of the lesion. Luxol was used for fiber staining, and cresyl violet for cell staining [7].
marized in Table 1. It follows that on the first day, normal rats defecate more than VMH lesioned ones. Shock administration (Test Day 2) induces an increase to a level similar for both groups. This high level is maintained on the third day by the intact animals but not by the VMH lesioned ones. However, conditioning effects are still present on this third day in VMH rats, though to a smaller extent as compared with control rats.
EXPERIMENT 1
MEDIAN VALUES AND 95% CONFIDENCE LIMITS AND STATISTICAL EVALUATIONOF RESULTS WITH REGARDTO DEFECATION DATA.
The first experiment is designed to test the acquisition and retention of a fear response established by electrical footshock [ 14].
TABLE !
Medians
Apparatus and Training Procedure The testing box, 70 x 30 x 30 cm high, was divided into 2 equal sized compartments by an aluminum vertically sliding door. One compartment (A) was black painted and not illuminated, the other (B) was of aluminum color and illuminated by a 25 W bulb. The front wall and top were opaque. The box was fitted with a stainless steel grid floor consisting of 1.4 mm rods 15 mm apart. On Test Day 1 (preshock test) the door was raised 7 cm, providing an opening between the two compartments. Each animal was placed individually in the black compartment and allowed to explore the entire box for 5 rain. On Test Day 2 the door was closed, and the animal was introduced into the black compartment; a 1.0 mA and 2 sec long scrambled shock was delivered every 13 sec during a 5 rain session. On Test Day 3 (postshock test) the procedure of Day 1 was repeated. The 3 test sessions were given on consecutive days. After each session, the number of fecal boli produced by each animal was counted. During the entire 5 min sessions the animal's behavior was continuously observed and recorded, according to the following behavior categories: (1) scanning and attention posture (the rat standing motionless with a normal palpebral aperture and without sniffing); (2) locomotion; (3) sniffing; (4) grooming; (5) presence in compartment B. Registration was done with a multiple event marker and recorded on an Esterline-Angus Eventrecorder. This procedure allowed recall of the onset and offset of each individual behavioral bout with an accuracy of + 0.5 sec. The experiment started on the seventh day after surgery.
Results Histology. The neural damage in the rats (n = 17) with VMH lesions was quantitatively defined by an area circumscribed by the following coordinates: AP = 5.2 to 6.4; H = - 2 to - 3 . 5 ; L = 0.0 to 2.0 [2]. Some lesions (n = 5) formed one continuous cavity bilaterally involving the nucleus ventromedialis hypothalami (NVMH) and extending into the third ventricle. In some animals, the ventral part of the nucleus dorsomedialis was either unilaterally (n = 2) or bilaterally (n = 2) affected by the lesion. Other lesions were bilaterally confined to the NVMH; in 3 instances only partial destruction of the NVMH was observed on one side of the brain. Thus, in each rat at least partial bilateral damage to the NVMH was observed; no clearcut relationship between lesion size and behavioral measurements could be established. Behavioral. The data regarding defecation are sum-
NI: 2 (0-4) N2: 6 (3-7) N3: 6 (3-8) VMH 1: 0 (0-2) VMH2:7 ( 3 - 1 0 ) VMH3:0 ( 0 - 4 )
Statistics Comparison NI-N2 N1-N3 N2-N3 VMH 1-VMH2 VMHI-VMH3 VMH2-VMH3 NI-VMH1 N2-VMH2 N3-VMH3
p <0.01 <0.01 >0.05 <0.01 <0.02 <0.01 <0.05 >0.05 <0.05
Symbols: N, normal control group; VMH, lesioned group; 1, 2 and 3 refer to consecutive test days; p, pertains to two-tailed probability. Table 2 presents median values and statistical evaluation of differences as to the total time spent in B (illuminated compartment), as well as to the frequency and mean duration of visits to this compartment. The latency to return to A for the first time after having gone to B, is also shown. This provides a more sensitive measure of fear than the speed with which the rat leaves the box [ 1 ]. On the first day VMH lesioned rats spent more time in B, and visit B more frequently than normals. The mean duration of these visits as well as the latency to return to A however does not differ from the normal control values. It can be inferred that both groups demonstrate a preference for the dark compartment, this trend however being less appreciable in VMH lesioned than in normal rats. On Day 3, normal rats spent more time in B, and stay there for longer periods than on Day 1. The number of visits is also significantly reduced, while the latency of return to A is dramatically increased. All these differences are significant at the 0.01 level. Except for the total time spent in B, which is not significantly altered, similar changes were revealed in VMH rats (p<0.05). Consequently, the preference for the dark compartment is considered to be reversed in normal, but left virtually unaffected in VMH lesioned rats. Thus, although both groups do show conditioning effects, the extent of these effects is significantly larger in normal than in VMH lesioned rats. Figure I shows median values of the time spent on 4 behavioral categories during 3 consecutive test days. The following main tendencies were revealed. (a) On the first Test Day the lesioned animals perform less scanning, but more locomotion and sniffing than the normal ones. (b) The shock session (Test Day 2) causes a gross behavioral change which is characterized by the enhancement of
VMH LESIONS AND F E A R
93 TABLE 2
MEDIAN VALUES AND 95% CONFIDENCE LIMITS, AND STATISTICALEVALUATIONOF RESULTS WITHREGARDTO PRESENCEIN COMPARTMENTSA AND B (TIMEIN SEC)
N1 N3 VMH1 VMH3
Total time in B
Frequency to B
Mean duration--B
Latency returtr--A
46 (38-61) 246 (229-287)t 105 (71-125)~ 103 (68-159)
4 (4-6) 2 (1-4)t 7 (5-8)$ 5 (3-6)*
9 (8-13) 111 (49-287)t 13 (10-20) 18 (14-26)*
26 (16-38) 213 (68-300)t 20 (14-26) 24 (14-114)*
*(p<0.05) and t (p<0.01) refer to the significance of differences between conditions (Days 1 and 3) for the same group (comparisons N1-N3 and VMH1-VMH3). $ (p <0.01) refers to the significance of differences between groups for the same conditions (comparison N1-VMH1). Other symbols as in Table 1. EXPERIMENT 2 scanning and a decrease of locomotion, sniffing and grooming. This behavioral pattern is most marked in the The second experiment constitutes a test for both fear normal control group. (c) In normal rats this behavioral • conditioning and passive avoidance, in that the animals are change, except for grooming, is continued on the third day, required to refrain from an ongoing behavioral response as is revealed by the comparison N1-N3. The comparison (i.e. locomotion) in order to avoid punishment (i.e. electric N2-N3 further indicates that locomotion is still more footshock). suppressed, whereas sniffing and grooming are partly recovered. The high level of scanning remains unchanged. In Apparatus and Training Procedure VMH lesioned rats too, the comparison VMH1-VMH3 evidences the persistance of most conditioning effects. The apparatus consisted of a 65 x 30 x 30 cm high Comparing VMH2-VMH3 reveals the partial recovery o f aluminum box, fitted with a stainless steel grid floor of 1.4 grooming, whereas other behavioral categories remain mm rods 15 mm apart. The floor was connected to a essentially unaltered. scrambled shock source which provided a 1.05 mA shock
300]
DAY 1 300-
~
NORMAL CONTROL
F~VMH
30o]
DAY 2
DAY 3
LESIONED
250-
z 200¢J z
2so1
2so1
20O I
20° I
C--- I50-
m 100-
50-
SCAN
LOCO
SNIF
GROO
SCAN
day 3 vs day 2
:
...2...3w.ayl
: : :
LOCO
:
SNIF
: *
GRO0
: **
SCAN
: :
LOCO
SNiF
~
~
: **
:
GRO0 !
*
FIG. 1. Median total time spent on scanning (SCAN), locomotion (LOCO), sniff'mg (SNIF) and grooming (GROO), and 95% confidence limits. For explanation of symbols referring to statistical evaluation, see Table 1.
94
COLPAERT AND WIEPKEMA
during 1 sec immediately after the onset of a locomotion response; shock delivery was manually controlled by the experimenter. On Day 1 the animal was introduced into the testing box for 4 min, during which all locomotion responses were punished. A locomotion response was defined by the animal moving in the horizontal plane for an estimated distance of 5 cm. On the following day (postshock test) the animals were retested once again during 4 min without shock. On the first day, only locomotion counts were recorded. On the second day, the same locomotion recording was made, and in addition the behavioral repertoire as described above was observed and recorded on the Esterline Eventrecorder. Day 1 of this experiment coincided with the seventeenth day after surgery.
Fig. 2a
•
NORMAL
0
VMH LESIONED
% 100 90 80-
~R T '\", I \ 'XI ,,
7060" 50-
Results
" ',1
40-
The results represented for the first day of Experiment 2 (Fig. 2) pertain to the frequency of locomotion responses. The frequency of responses during the second, third and fourth min was expressed as a percent of locomotion frequency observed during the first minute; median percent change per group for each minute are shown in Fig. 2a. VMH lesioned rats show a significantly higher frequency of locomotion as compared to the normal group (p<0.01), and consequently received a higher number of shocks (MedianN = 27; MedianvM H = 41). From a further analysis (Fig. 2b), it follows that the only significant decrease between consecutive minutes occurs between the first and the second one, both in normal and VMH lesioned rats. Despite this apparent similarity between both groups, it should be noted that the overall percent decrease between the first and the fourth minute in normal rats reliably exceeds that in VMH lesioned ones. The results of the behavioral recordings made on the second day of Experiment 2 can be summarized as follows: the VMH lesioned rats showed significantly less scanning (p<0.001) and more sniffing (p<0.001) than the normal control animals did; grooming was virtually abolished in both groups, and there was no difference (p>0.05) between groups. The VMH lesioned group spent more time in locomotion than the control group (/9<0.001), whereas the frequency of the locomotion response (as compared to the first-day-performance) was reduced in the control (p<0.001) but not in the VMH lesioned group (p>0.05). DISCUSSION
The results of Experiment 1 support two important assumptions. First, it is indicated that VMH lesioned rats show less fear conditioning effects than normal rats. This conclusion is based on the data concerning fecal production, visits to compartment B, and first return to compartment A. Second, it is revealed that, as was already suggested by preliminary studies, some particular behavioral changes are induced by shock and maintained through conditioning, these changes being intimately related to other more commonly recognized measurements of fear conditioning. On the first day the VMH lesioned rats defecate less and spent less time on scanning and more on locomotion and sniffing than normal rats do. They also spent more time in compartment B. Moreover since it is known that light has some aversive significance in rats [3] it may be concluded that VMH rats exhibit less fear behavior than normals in a
CONTROL
\\
30-
\\ 20-
\\
100 I
F---I 2
.___i
F--4 3
/. MIN.
Fig. 2b
COMPARISON
N
VMH
N-VMH
1 - 2 rain.
.00031
.0069
.11
2-3
.039
.72
.73
.15
.25
.70
.00L/.
.003
rain.
3 - 1 . rain. t-/.
min.
< 00005
FIG. 2. (a) Median frequency and 95% confidence limits of locomotion on 4 consecutive minutes as the percent of locomotion frequency on the first minute. (b) Statistical testing of progressive percent decrease of locomotion frequency over 4 consecutive minutes. Results are expressed as the two-tailed probability that the various listed comparisons were the same. Symbols as in Table l. situation that represents a novel environment, as is the case on the first day. The latter conclusion is consistent with some earlier findings [5,11] showing that VMH lesioned rats eat reliably sooner in a novel environment than normal rats do. The results of Experiment 2 confirm earlier reports [8,9] regarding passive avoidance in VMH lesioned rats. However, the extention of this finding to the experimental conditions presently described is relevant. Indeed, from the present results it follows that the passive avoidance deficit in VMH lesioned rats is independent from any possible interference with food or water motivation that may be enhanced by this type of brain lesion [ 11 ]. Thus, despite the increased reactivity of VMH lesioned rats to footshock [15], a passive avoidance deficit is evidenced in VMH lesioned rats, using a shock as the US. These findings, as well as the results of Experiment 1 lent support to the conclusion that the fear conditioning deficit, rather than a response inhibitory deficit [9] more
VMH LESIONS AND F E A R
95
parsimoniously explains the passive avoidance deficit following VMH lesions. The fact that the behavioral pattern, found to be related to fear conditioning, occurs in the passive avoidance experiment too, further supports this conclusion. However, this deficit in fear conditioning, and consequently the passive avoidance deficit, does not represent a total loss of fear. Indeed, the VMH animals, although to a smaller degree, show some clear conditioning effects both in the first and second experiment. It is obvious that fear conditioning plays a very prominent adaptive role in behavior, particularly in spon-
taneous social behavior. In the view of the present experiments on fear conditioning in VMH lesioned rats, it can be hypothesised that these animals may also show some abnormalities in social behavior, and particularly in these social interactions which to a certain degree are controlled by fear. It is further suggested that the measurement o f scanning, locomotion, sniffing and grooming may possibly constitute an alternative approach to the distinction between response inhibition and fear deficit as explanations of experimentally induced behavioral changes.
REFERENCES 1. Blanchard, R. J. and D. C. Blanchard. Escape and avoidance responses to a fear eliciting situation. Psychon. Sci. 13: 19-20, 1968. 2. De Groot, J. The rat forebrain in stereotaxic coordinates. Proc. K. Ned. Akad. Wet., C. 52: 1-40, 1959. 3. Desiderato, O. Generalization of acquired fear as a function of CS intensity and number of acquisition trials. J. exp. Psychol. 67: 41-47, 1964. 4. Grossman, S. P. The VMH: A center for affective reactions, satiety, or both? Physiol. Behav. 1: 1-10, 1966. 5. Grossman, S. P. Aggression, avoidance, and reaction to novel environments in female rats with ventromedial hypothalamic lesions. Z comp. physiol. Psychol. 78: 274-283, 1972. 6. Kaada, B. R., E. W. Rasmussen and O. Kveim. Impaired acquisition of passive avoidance behavior by subcallosal, septal, hypothalamic and insular lesions in the rat. J. comp. physiol. Psychol. 55: 661-670, 1962. 7. Kliiver, H. and E. Barrera. A method for the combined staining of cells and fibers in the nervous system. J. Neuropath. exp. Neurol. 12: 400-403, 1953. 8. Margules, D. L. and L. Stein. Neuroleptics vs tranquillizers: Evidence from animal behavior studies of mode and site of action. In: Neuropsycho-pharmacology, edited by H. Bull, J. O. Cole, P. Deniker, H. Hippius and P. B. Bradley. New York: Excerpta Medica Foundation, 1966.
9. Margules, D. L. and L. Stein. Cholinergic synapses in the ventromedial hypothalamus for the suppression of operant behavior by punishment and satiety. J. comp. physiol. Psychol. 67: 327-335, 1969. 10. McCleary, R. A. Response specificity in the behavioral effects of limbic system lesions in the cat. J. comp. physiol. Psychol. 54: 605-613, 1961. 11. Sclafani, A. and S. P. Grossman. Reactivity of hypeiphagic and normal rats to quinine and electric shock. Z comp. physiol. Psychol. 74: 157-166, 1971. 12. Siegel, S. Nonparametric Statistics for the Behavioral Sciences. New York: McGraw-Hill, 1956. 13. Singh, D. Comparison of behavioral deficits caused by lesions in septal and ventromedial hypothalamic areas of female rats. J. comp. physioL PsychoL 84: 370-379, 1973. 14. Slotniek, B. M. and D. L. Brown. Fear conditioning and passive avoidance in mice with septal lesions. Physiol. Behav. 5: 1255-1259, 1970. 15. Turner, S. G., J. A. Sechzer and R. A. Liebelt. Sensitivity to electrical shock after ventromedial hypothalamic lesions. Expl Neurol. 19: 236-244, 1967. 16. Weisman, R. N. and L. W. Hamilton. Two-way avoidance responding following VMH lesions: Effects of varying shock intensity. Physiol. Behav. 9: 243-246, 1972.