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The role of the olfactory system in avoidance learning and activity
Physiology and Behavior, Vot. 8, pp. 705-710, Brain Research Publications Inc., 1972. Printed in Great Britain.
The Role of the Olfactory System in Avoidance Learning and Activity' M I C H A E L H. S I E C K
Department of Psychology, University of California, Riverside, California, U.S.A. (Received 18 N o v e m b e r 1970) SIECK, M. H. The role of the olfactory system in avoidancelearningand activity. PHYSIOL.BEHAV.8 (4) 705-710, 1972.Male rats with bilateral ablations of the olfactory bulbs were tested on several activity measures and in passive and active avoidance situations. They were difficult to handle and more active in an open field than sham operates, were significantly poorer at passive avoidance acquisition, and significantly better at active avoidance. The results were discussed in terms of possible alterations in limbic function leading to hyperreactivity toward external stimulation. Olfactory system
Limbic system
Olfaction
Behavioral control mechanisms
SINCE KOLLIKER [7] first coined the term rhinencephalon (smell brain) to include most of the limbic system, the role of the olfactory system in limbic modulations of behavior has declined in interest. Despite research demonstIating extensive and relatively direct connections between the olfactory bulbs and such anterior limbic structures as the prepyriform cortex, amygdalar areas, medial forebrain bundle, and hypothalamus [5, 11, 12, 16], the olfactory system has been ignored as a possibly important contributor to the behavioral modulating activities of these antemior limbic structures. Nevertheless, some evidence suggests that olfactory system influences might play roles not commonly associated with olfactory discrimination per se. Wenzel and Salzman [15], and Wenzel, Albritton, Salzman, and Oberjat [14] demonstrated that pigeons with bilateral olfactory bulb ablations or nerve sections were inferior to controls in learning to respond in visual discrimination problems, but that they eventually attained similar performance levels. They also demonstrated that reactivity to sudden light or sound stimuli was greater in bulbectomized than normal birds, and from this suggested that discriminative performance may have been upset by too great an activation leading to an avoidance of the learning situation. Phillips [10] found that male hooded rats with bilateral bulb ablations were unable to acquire learning sets on different visual discrimination problems and even performed consistently poorer than normal rats given conflicting visual and olfactory cues. Unable to account for this in terms of any overt behavioral differences between the groups, Phillips noted that bulbectomized animals showed high variances in responding although they performed better than chance on all tasks. Douglas, Isaacson, and Moss [4] found that female rats with olfactory bulb ablations became hyperemotional when handled and were transiently poor at spontaneous alternation. They noted the similarity of their data to those obtained with
septally lesioned animals except that the home cage activity of bulbectomized rats was normal and the effects persisted longer than the classical septal syndrome. They also observed wide variability between animals: some were very emotional while others were quite normal, but they were unable to account for the differences according to lesion extent. Sex differences have also been reported. Douglas (reported in Phillips [10]) found that female hooded rats were made mote emotional by bulb lesions than male hooded rats. Phillips also noted that his lesioned males were not noticeably more emotional than control males. Hormonal differences may possibly account for this since several studies implicate the olfactory bulbs in pituitary control mechanisms for sex hormones [3], sexual behavior [6], and even thyroid activity [1]. In any case bulb ablations affect the behavior of both males and females but it is not clear how or why. The present study focuses on the relationship between the olfactory system and behavioral control in the less affected male rats in tasks requiring minimal or no olfactory input for their successful completion. To facilitate comparison with other studies, all of the tasks used here were selected from earlier experiments on limbic function.
Animals Animals were 60 naive male hooded rats from Simonsen Laboratories approximately two months old at the start of the experiments. The animals were individually caged in a temperature controlled room (23°C) on a 12hr reversed light--dark cycle. They were maintained on ad lib food and water throughout the experiments. Two experimental groups of 36 (Experiment 1) and 24 animals (Experiment 2) each were established; 12 animals in each group received bilateral olfactory bulb ablations (BULB), 12 served as sham operated controls (SHAM), and 12 animals in one received bilateral
XRcsearch supported in part by NIH Grant MH-15585. The services of Barry Gordon and Barbara Glover arc gratefully acknowledged. 705
706
SIECK
frontal pole ablations (FRONT). Two bulbectomized rats subsequently died so that final Ns consisted of 11 bulbectomized, 12 F R O N T S and 12 SHAMS for Experiment 1, and 11 BULBS and 12 SHAMS for Experiment 2.
Surgery All animals were injected IP with atropine sulfate (0.4mg/ kg) followed 15 min later by sodium pentobarbital (50 mg/kg). After being placed in the stereotaxic apparatus, scalp incisions were made and the periosteum overlying the skull was reflected. The skull above the olfactory bulbs was partially drilled through on the sham operates and was completely removed for the bulbectomy groups. Bulb removal was effected by suction under direct visual guidance. Care was taken to remove as much olfactory bulb as possible while avoiding damage to the frontal cortex. As a surgical control for anterior brain damage, the frontal poles were removed from 12 rats by suction under direct visual guidance after the skull over the area was removed. Deep lying olfactory structures were spared while removing as much as possible of the overlying tissue just posterior to the olfactory bulbs. At the completion of surgery, sterile gelfoam was packed into the opened skull to effect hemostasis, terramycin powder was sprinkled into the incision and the skin flaps were sutured closed. The latter two steps were also part of the sham operations. Bicillin (0.2 cc) was injected I M into all subjects and animals were returned to home cages for recovery.
Histology After completion of all testing, the rats were anesthetized with pentobarbital, perfused intracardially with 0.9% saline followed by 10% formalin, and decapitated. Most of the fascia and posterior skull were removed at once and the
IZ.O AP
10.4
11.6
10.0
remaining skull and brain were soaked in 10% formalin for one week before completion of skull removal. The amount of olfactory bulb removal was measured by comparisons to normal brains and the posterior extent of damage was measured by sections compared to the Pellegino and Cushman atlas of the rat brain [9]. Bulbectomies were essentially complete in all cases, i.e., all tissue anterior to A.P. 12.8 was removed in the least damaged brains and as far posterior as A.P. 12.1 in the greatest lesions. N o frontal pole damage occurred. Nine of the 12 frontally damaged animals showed essentially complete pole removal between A.P. 12.0 and 10.6. Total extents of the frontal lesions are apparent in Fig. 1.
Apparatus Open field activity, passive and active avoidance acquisition, and home cage activity were measured. All tests except home cage activity were conducted in a quiet room, dimly illuminated with red light between 9 a.m. and noon. The home cage activity was measured in constant darkness. The open field was a white enameled 4 x 4 ft square wood box with walls 12 in. high. The floor was divided by black lines into sixteen 12-in. squares. The passive avoidance apparatus consisted of a 2 x 2 x 2 ft dark Plexiglas box over a grid floor with a centered 4 x 4 in. step down platform raised 3 in. above the grids. Intermittent shock could be applied to the grids through a shock scrambler set to deliver 150 V a.c. across a 220 K resistor (0.68 ma). A clock was activated when an animal was placed on the platform and stopped when the animal stepped down. Stepping down also triggered a 3-sec shock. Home cage activity was measured in 12 wooden living cages 9 in. square with wire mesh floors. Water and food were available ad lib from a bottle on one side and a supply of pellets on the floor. Four photo cell light units were
I1.0
10.0
D.6
9.2
FIG. 1. Areas of greatest (shaded) and least (dark) frontal pole lesions. (After sections in Pellegrino and Cushman Rat Atlas [9].)
OLFACTORY SYSTEM AND AVOIDANCE BEHAVIOR attached to each cage 1½ in. above the floor in the middle of each wall and beamed across each corner. Breaking any light beam activated an Esterline-Angus event recorder and a counter simultaneously. The apparatus was run continuously for the experiment's duration. The active avoidance apparatus was a two-way nondiscriminated shuttle box consisting of two identical dark Plexiglas boxes 9 × 9 × 9in. separated by a 3 × 3in. closed alley over a grid floor. Guillotine doors leading to the alley from either box started a buzzer and two timers when opened. Ten sec after starting, one timer activated a shock scrambler attached to the grids of the box and alley occupied by the animal. Shock level was set at 0.36 ma (80 v across 220 k). The other clock measured elapsed time until the animal moved to the opposite box. This timer and the buzzer-shock system were turned off when a light beam at the end of the alley was broken. A third timer then started measuring the I rain interval between trials.
707 EXPERIMENT 2
Procedures After 10 days of postoperative recovery, including 4 days of handling as in Experiment 1, the animals were tested in the passive avoidance situation described earlier. The acquisition cut-off was changed to 18 trials. There was no limit for extinction. The day after completion of passive avoidance training, the rats were run in the active avoidance apparatus. On Day 1 the doors were tied open and each animal explored the apparatus for 15 min. The following day, the rat was placed in one box with the doors shut and the first trial began when the doors were opened and the buzzer started. If the animal did not leave the start box within 10 sec, shock was applied to the grids. Moving from one box to the other before the 10-sec interval ended constituted successful avoidance. Each animal was given 20 trials a day. Animals were dropped if they had not reached a criterion of 8 out of 10 consecutive avoidances by Trial 180 (9 days).
EXPERIMENT 1
RESULTS
All animals were allowed 10 days of postoperative recovery before starting the experiment and were individually handled for 5 min a day on postoperative Days 7-10. Difficulty of handling, vocalization, and general activity were subjectively appraised during this time. All testing was done at approximately the same time of day for each animal. On postoperative Days 11 and 12, open field activity was measured for each animal during 5 min of free exploration. A count was recorded each time more than half of an animal's body crossed a line. Each animal was started from the same corner facing the field. Running order was reversed on the second day. The number of boluses was also recorded for each animal and the whole field was cleaned with water after each trial. Passive avoidance training started on Day 13. The rats were placed individually on the raised platform and allowed to explore the chamber for 5 min before being returned to the home cage. This procedure was repeated three times at hourly intervals. The next day a clock was activated when the rat was placed on the platform. When the animal put three feet on the grid, the clock stopped, and the floor was electrified for 3 see. After shock, the rat was allowed 10 sec of exploration before removal to the home cage. Three trials a day were given as above until a criterion of no-stepdown in 5 min was reached or a maximum of 12 trials (4 days) were run. Testing stopped as soon as the criterion was reached, and the rat was returned to its home cage and not tested again that day. The day following acquisition, extinction training began using the same procedure except that the grid was not electrified. Extinction was considered complete when an animal first stepped down in less than 5 min. Animals not reaching extinction after 12 trials (4 days) were removed to activity cages. The day following passive avoidance training, 6 BULBS and 6 SHAMS were placed in individual activity cages. Both cumulative activity and the pattern of activity in each 24-hr period were recorded. After being in constant darkness for 8 consecutive days animals were then returned to their home cages. The remaining 5 BULBS and 6 SHAMS were then tested in the same way. The 12 F R O N T S were not tested.
Results A. Handling, vocalization (subjective appraisals). Bulbecto-
Procedures
EXPERIMENT 1
mized animals were somewhat more difficult to remove from their home cages and were generally more active than sham operates or frontally lesioned animals when handled. A few were very difficult, escaping vigorously or attacking when approached. Vocalization differences followed the same pattern but at no time were any of the animals as unmanageable as freshly lesioned septal rats or female rats with olfactory bulb ablations (unpublished observations). If an animal was hyperreactive initially, this trait persisted throughout the experiment with only slight diminution, again unlike septal animals [2]. It is relevant that bulbectomized animals' responses to handling were so widely varied that many were undistinguishable from SHAMS. Histological analysis revealed little correlation between lesion extent and this phenomenon. B. Open field activity. ANOVAS for unequal Ns showed no activity differences for all animals on either day although the BULBS approached significance over SHAMS on Day 1. N o defecation differences occurred on either day. Since the frontally lesioned and s h a m operated groups were both controls for the bulbectomy operation and had similar scores on this measure, and since high variance was responsible for the low significance of the ANOVA, the F R O N T S and S H A M S were combined and compared to the BULBS as a single group. This test revealed that BULBS were significantly more active on both days (Day 1, t = 2.19, df=33,p < 0.025 ; Day 2, t = 1.81, p < 0.05). Table 1 summarizes the results. F o r all groups activity scores decreased from Day 1 to Day 2. C. Home cage activity. The BULB and S H A M groups did not differ on either total amount or pattern of activity during their 8 days in the photocell living cages (F = 0.80, d f = 62/7, n.s.). Figure 2 demonstrates daily activity similarities for the two groups. Diurnal activity patterns were inconsistent from rat to rat; i.e., someshowed characteristic bursts of activity at particular times of the day while others did not. All animals tended to be most active between 6 a.m. and n o o n - - t h e first six hr of
708
SIECK
Activity
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t
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"-" Y
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, ~
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i
, I
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FIG. 2. Total home cage activity recorded in constant darkness for sham operated controls and olfactory bulbectomized groups in Experiment 1.
the normal cycle in the colony room. There were no discernible lesion effects on these patterns. D. Passive avoidance. Eight of the 10 BULBS and two each of 12 SHAMS and F R O N T S failed to reach acquisition criterion before attaining the 12-trial ceiling (B-S and B-F comparison p = 0.005, Fischer exact test). Of those entering extinction 0/2 BULBS, 5/10 SHAMS, and 7/10 F R O N T S failed to reach criterion. Because of the small number of BULBS involved, statistical tests were not run on these data. EXPERIMENT
2
If one assumes that rats utilize olfactory information to partially determine the relevance of any stimulus, loss of this input should lead to a partial failure in steering mechanisms for behavior control. Steering might consist of two interrelated mechanisms; channeling of ongoing behavior along appropriate lines based on stimulus discrimination, and starting and stopping different behavior sequences again based on some appropriate discrimination. Looking at these data as well as previous studies by Phillips [10J, Wenzel et aL [14, 15], and Douglas et al. [4] support for both of these ideas is presented.
Results .4. Handling, vocalization. The results were similar to those found in Experiment 1. The BULBS as a group were somewhat more difficult to handle but again wide individual differences were noted. B. Passive avoidance. Six of the 11 BULBS and all of the SHAMS reached criterion before the 18-trial acquisition limit (p = 0.004, Fischer exact test). Of those animals reaching criterion, the BULBS required significantly more trials (BULBS, M = 12.83; SHAMS, M----- 8.50; F----- 10.94, d r = 16/1, p < 0.005). There were no group differences in extinction (BULBS, M = 3.5; SHAMS, M = 3.2; F = 0.043, d f = 16/1). C. Active avoidance. Criterion was reached by 11 BULBS (M = 43.36 trials, SD = 14.75) and 10 SHAMS (M = 77.20, SD = 40.87). This difference was significant (F = 10.61, d f = 19/1, p -----0.004). DISCUSSION
The fact that on all measures frontally lesioned animals were unlike bulbectomized animals, although extensive damage to frontal areas just posterior to the olfactory bulbs occurred, supports the hypothesis that olfactory damage per se is responsible for effects observed. Although high variance on these measures makes interpretation difficult, data obtained from handling and open field work suggest that olfactory bulb lesions produce animals which are somewhat more reactive to novel stimulation than sham or frontally lesioned operates.
Channeling Phillips found no evidence for learning sets on visual discrimination problems and noted high variances in his operated animals' responses. Douglas et al. [4] noted high variability of emotionality in response to handling across the operated population of female rats, as was observed in the present study with males. Further, observations on individual animals' behavior in the passive avoidance situation in this experiment revealed that most animals which failed to reach criterion adopted relatively fixed strategies such as leaping toward the top of the chamber or jumping out onto the grid as soon as they were placed on the platform. That these strategies developed in response to shock, were never seen in SHAMS and persevered with little change throughout the experiments, suggests that BULBS did learn that they would be shocked but that they were unable to acquire an appropriate avoidance response. Even though it might be argued that this behavior is traceable to an arousability change or to some change in motor responsiveness such as an inability to withhold responding, the possibility exists that animals without olfactory system patency may lack appropriate feedback mechanisms to easily or correctly change behavior patterns once established. This may account for the high variability across animals observed by all authors; for the inability of operates to form learning sets (Phillips); and for the transient breakdown in spontaneous alternation after lesioning observed by Douglas et al.
OLFACTORY SYSTEM AND AVOIDANCE BEHAVIOR
709
Activation and Inhibition
The fact that the bulbectomized rats were typically more difficult to handle, were somewhat more active in the open field, took longer to learn passive avoidance, but did better at active avoidance can all be accounted for in terms of decreased freezing or motor inhibition, increased escape tendencies, increased emotionality, and so on. Further, the fact that home cage activity was similar for BULBS and SHAMS suggests that the effects were primarily situation dependent. Hence, in addition to possible changes in the channeling of behavior after lesions, changes in response thresholds or even ways of responding may occur. Since olfactory cues per se were not relevant in either the passive or active avoidance situations and were more or less constant in both, the idea that olfaction or patency of the olfactory system exerts tonic inhibitory action on other brain areas involved with motor control or activation must be considered. Wenzel et al. [14] concluded from their second study of visual discrimination and autonomic measures in pigeons that olfactory bulb or nerve lesions led to increased alerting responses, the intensity of which produced either repulsion or distraction and thus interfered with appropriate responses to stimuli. In their first study, Wenzel and Salzman [15] felt that decreased reactivity was involved in their results, and Phillips also entertained this hypothesis. However, the hypothesis that increased arousal led to inappropriate behavior also accounts for their data. Also, the facts that neither of those studies used negative reinforcement paradigms and that the Wenzel et aL studies were conducted on pigeons must be kept in mind. Unpublished work from this laboratory using positive reinforcement suggests that bulbectomized rats do indeed become hypoactive in some situations where normal animals do not. One approach to these different results suggests an interaction effect between task and lesion. More evidence for such an effect comes from an inspection of the data observed in the present studies. The bulbectomized population had consistently higher means and standard deviations than the sham operated group on all measures taken during open field testing and handling (Table 1), and much lower values during active avoidance testing. Passive
avoidance SDs were essentially equal when computed on all animals including those artificially constrained at the 12- or 18-trial limits (Experiment 1, BULBS, SD = 3.76, SHAMS, S D = 3.85; Experiment 2, BULBS, S D = 3.16, SHAMS, SD = 2.75), but undoubtedly would have shown greater variability for BULBS than SHAMS if no limit had been imposed. These data fit a hypothesis suggesting that BULBS have generally lower behavioral arousal thresholds than SHAMS. Thus, under low or moderate levels of external stimulation, BULBS would show greater exploratory approach activity while, at higher intensity levels, more stimulus or situation avoidance would be likely to occur. Increased escape tendencies at high stimulus intensity would then hinder passive avoidance acquisition while favoring active avoidance performance. Figure 3 summarizes this position in that the bulbectomized population is seen to be more reactive at given levels of external stimulation by a shift of the BULB curve to the left. This appears as greater activity during low levels of stimulation (open field) and greater escape at higher levels (passive and active avoidance). Also the curve predicts that some level of external stimulation will cause activity cessation in the BULBS before it is apparent in SHAMS. Data on this have been obtained in startle response experiments and Y-maze studies (Sieck and Gordon, in press). Nevertheless, all of the behavioral data provide only indirect evidence for this hypothesis. More direct tests will come from studies now underway on physiological responsiveness of bulbectomized and sham operated animals in various environments. In conclusion it seems clear that either olfactory stimulation or merely patency of the olfactory system is important in maintaining balance between activating and directing mechanisms in the rat brain. Results obtained from these experiments and deducible from others [4, 10, 14, 15] show marked similarities to studies involving damage to limbic structures such as the amygdala, septal region, hippocampal and frontal cortical zones [2, 8, 13]. Since the anatomical connections of the olfactory system to some of these areas are extensive and relatively direct, the possibility that olfaction is important to limbic function receives added support.
TABLE 1 A. MEAN NUMBEROF LINES CRO~ED AND BOLUSESDROPPED BY BULBECTOMIZED~SHAMOPERATEDCONTROLAND FRONTALLYLESIONED RATSDURINGOPEN FIELDTESTSON TWO SUCCESSIVEDAYS. STANDARDDEVIATIONSAREALSOSHOWN.
Bulbs Shams Fronts
Activity
S.D.
139.55 90.00 101.67 F = 2.48 df = 32/2 (n.s.)
57.81 49.83 58.55
Day 1 Boluses 3.09 2.58 2.08 F = 0.438 (n.s.)
S.D.
Activity
S.D.
3.18 2.38 2.11
111.64 67.50 76.42 F = 1.66
77.93 50.57 52.61
Day 2 Boluses 2.27 2.08 2.67 F = 0.112
(n.s.)
(n.s.)
n. MEANS AS ABOVE FOR COMBINED SHAM OPERATES (S) AND FRONTALLY LESIONED (F) RATS.
S+F Bulbs
95.83 139.55 t = 2.19 dr= 33 p < 0.025
2.33 3.09 t = 0.82
71.96 111.64 t = 1.81
2.38 2.27 t = 0.09
(n.s.)
p < 0.05
(n.s.)
S.D. 3.20 2.84 3.17
710
SIECK
FIG. 3. Theoretical response curves correlating reactivity to stimulation with assumed arousal values of the tests involved in these experiments. The actual placements of the lines for open field, passive, and active avoidance are not critical except that they are assumed to be at approximately the areas of the curve indicated where they fit the data for the means and variances obtained (See Discussion).
REFERENCES 1. Balboni, G. C. Sur les modifications de radenohypophyse, de la thryoide et de l'ovaire apres ablation des bulbes olfactifs. Bull Ass. Anat., Paris, 52: 160-167, 1967. 2. Brady, J. V. and W. J. H. Nanta. Subcortical mechanisms in emotional behavior: affective changes following septal forebrain lesions in the albino rat. J. comp.physiol, Psychol. 46: 339-346, 1953. 3. Bruce, H. M. and D. V. M. Parrott, Role of olfactory sense in pregnancy block by strange males. Science 131: 1526, 1960. 4. Douglas, R. J., R. L. Isaacson and R. L. Moss. Olfactory lesions, emotionality and activity. Physiol. Behav. 4: 379-381, 1969. 5. Heimer, L. Synaptic distribution of centripetal and centrifugal nerve fibers in the olfactory system of the rat: An experimental anatomical study. J. Anat. 103: 413-432, 1968. 6. Heimer, L. and K. Larsson, Mating behavior of male rats after olfactory bulb lesions. Physiol. Behav. 2: 207-209, 1967. 7. Kolliker, A. Handbuch der Gewebelehre des Menschens. Leipzig: Wilhelm Englemann Press, 1896. 8. MeCleary, R. A. Response-modulating functions of the limbic system: initiation and suppression. In: Progress in Physiological Psychology, Vol. 1, edited by E. Stellar and J. M. Sprague. New York: Academic Press, 1966, pp. 210-272.
9. Pellegrino, L. J. and A. J. Cushman..4 Stereotaxic Atlas of the Rat Brain. New York: Appleton-Century-Crofts, 1967. 10. Phillips, D. S. Effects of olfactory bulb ablation on visual discrimination. Physiol. Behav. 5: 13-15, 1970. 11. Powell, T. P. S., W. M. Cowan and G. Raisman. Olfactory relationships of the diencephalon. Nature 199: 710-712, 1963. 12. Scott, J. W. and C. Pfalfmann. Olfactory input to the hypothalamus: electrophysiological evidence. Science 158: 15921594, 1969. 13. Thomas, G. T., G. Hostetter and D. J. Barker. Behavioral functions of the limbic system. In: Progress in Physiological Psychology, Vol. 2, edited by E. Stellar and J. M. Sprague. New York: Academic Press, 1968, pp. 230-311. 14. Wenzel, B. M., P. F. Albritton, A. Salzman and T. E. Oberjat. Behavioral changes in pigeons after olfactory nerve section or bulb ablation. In: Olfaction and Taste 111, edited by C. Pfaffmann. New York: Rockefeller University Press, 1969, pp. 278-287. 15. Wenzel, B. M. and A. Salzman. Olfactory bulb ablation or nerve section and pigeons' behavior in non-olfactory learning. Expl. Neurol. 22: 62-69, 1968. 16. White, L. E., Jr. Olfactory bulb projections of the rat. Anat. Rec. 152: 465-480, 1965.
The Role of the Olfactory System in Avoidance Learning and Activity' M I C H A E L H. S I E C K
Department of Psychology, University of California, Riverside, California, U.S.A. (Received 18 N o v e m b e r 1970) SIECK, M. H. The role of the olfactory system in avoidancelearningand activity. PHYSIOL.BEHAV.8 (4) 705-710, 1972.Male rats with bilateral ablations of the olfactory bulbs were tested on several activity measures and in passive and active avoidance situations. They were difficult to handle and more active in an open field than sham operates, were significantly poorer at passive avoidance acquisition, and significantly better at active avoidance. The results were discussed in terms of possible alterations in limbic function leading to hyperreactivity toward external stimulation. Olfactory system
Limbic system
Olfaction
Behavioral control mechanisms
SINCE KOLLIKER [7] first coined the term rhinencephalon (smell brain) to include most of the limbic system, the role of the olfactory system in limbic modulations of behavior has declined in interest. Despite research demonstIating extensive and relatively direct connections between the olfactory bulbs and such anterior limbic structures as the prepyriform cortex, amygdalar areas, medial forebrain bundle, and hypothalamus [5, 11, 12, 16], the olfactory system has been ignored as a possibly important contributor to the behavioral modulating activities of these antemior limbic structures. Nevertheless, some evidence suggests that olfactory system influences might play roles not commonly associated with olfactory discrimination per se. Wenzel and Salzman [15], and Wenzel, Albritton, Salzman, and Oberjat [14] demonstrated that pigeons with bilateral olfactory bulb ablations or nerve sections were inferior to controls in learning to respond in visual discrimination problems, but that they eventually attained similar performance levels. They also demonstrated that reactivity to sudden light or sound stimuli was greater in bulbectomized than normal birds, and from this suggested that discriminative performance may have been upset by too great an activation leading to an avoidance of the learning situation. Phillips [10] found that male hooded rats with bilateral bulb ablations were unable to acquire learning sets on different visual discrimination problems and even performed consistently poorer than normal rats given conflicting visual and olfactory cues. Unable to account for this in terms of any overt behavioral differences between the groups, Phillips noted that bulbectomized animals showed high variances in responding although they performed better than chance on all tasks. Douglas, Isaacson, and Moss [4] found that female rats with olfactory bulb ablations became hyperemotional when handled and were transiently poor at spontaneous alternation. They noted the similarity of their data to those obtained with
septally lesioned animals except that the home cage activity of bulbectomized rats was normal and the effects persisted longer than the classical septal syndrome. They also observed wide variability between animals: some were very emotional while others were quite normal, but they were unable to account for the differences according to lesion extent. Sex differences have also been reported. Douglas (reported in Phillips [10]) found that female hooded rats were made mote emotional by bulb lesions than male hooded rats. Phillips also noted that his lesioned males were not noticeably more emotional than control males. Hormonal differences may possibly account for this since several studies implicate the olfactory bulbs in pituitary control mechanisms for sex hormones [3], sexual behavior [6], and even thyroid activity [1]. In any case bulb ablations affect the behavior of both males and females but it is not clear how or why. The present study focuses on the relationship between the olfactory system and behavioral control in the less affected male rats in tasks requiring minimal or no olfactory input for their successful completion. To facilitate comparison with other studies, all of the tasks used here were selected from earlier experiments on limbic function.
Animals Animals were 60 naive male hooded rats from Simonsen Laboratories approximately two months old at the start of the experiments. The animals were individually caged in a temperature controlled room (23°C) on a 12hr reversed light--dark cycle. They were maintained on ad lib food and water throughout the experiments. Two experimental groups of 36 (Experiment 1) and 24 animals (Experiment 2) each were established; 12 animals in each group received bilateral olfactory bulb ablations (BULB), 12 served as sham operated controls (SHAM), and 12 animals in one received bilateral
XRcsearch supported in part by NIH Grant MH-15585. The services of Barry Gordon and Barbara Glover arc gratefully acknowledged. 705
706
SIECK
frontal pole ablations (FRONT). Two bulbectomized rats subsequently died so that final Ns consisted of 11 bulbectomized, 12 F R O N T S and 12 SHAMS for Experiment 1, and 11 BULBS and 12 SHAMS for Experiment 2.
Surgery All animals were injected IP with atropine sulfate (0.4mg/ kg) followed 15 min later by sodium pentobarbital (50 mg/kg). After being placed in the stereotaxic apparatus, scalp incisions were made and the periosteum overlying the skull was reflected. The skull above the olfactory bulbs was partially drilled through on the sham operates and was completely removed for the bulbectomy groups. Bulb removal was effected by suction under direct visual guidance. Care was taken to remove as much olfactory bulb as possible while avoiding damage to the frontal cortex. As a surgical control for anterior brain damage, the frontal poles were removed from 12 rats by suction under direct visual guidance after the skull over the area was removed. Deep lying olfactory structures were spared while removing as much as possible of the overlying tissue just posterior to the olfactory bulbs. At the completion of surgery, sterile gelfoam was packed into the opened skull to effect hemostasis, terramycin powder was sprinkled into the incision and the skin flaps were sutured closed. The latter two steps were also part of the sham operations. Bicillin (0.2 cc) was injected I M into all subjects and animals were returned to home cages for recovery.
Histology After completion of all testing, the rats were anesthetized with pentobarbital, perfused intracardially with 0.9% saline followed by 10% formalin, and decapitated. Most of the fascia and posterior skull were removed at once and the
IZ.O AP
10.4
11.6
10.0
remaining skull and brain were soaked in 10% formalin for one week before completion of skull removal. The amount of olfactory bulb removal was measured by comparisons to normal brains and the posterior extent of damage was measured by sections compared to the Pellegino and Cushman atlas of the rat brain [9]. Bulbectomies were essentially complete in all cases, i.e., all tissue anterior to A.P. 12.8 was removed in the least damaged brains and as far posterior as A.P. 12.1 in the greatest lesions. N o frontal pole damage occurred. Nine of the 12 frontally damaged animals showed essentially complete pole removal between A.P. 12.0 and 10.6. Total extents of the frontal lesions are apparent in Fig. 1.
Apparatus Open field activity, passive and active avoidance acquisition, and home cage activity were measured. All tests except home cage activity were conducted in a quiet room, dimly illuminated with red light between 9 a.m. and noon. The home cage activity was measured in constant darkness. The open field was a white enameled 4 x 4 ft square wood box with walls 12 in. high. The floor was divided by black lines into sixteen 12-in. squares. The passive avoidance apparatus consisted of a 2 x 2 x 2 ft dark Plexiglas box over a grid floor with a centered 4 x 4 in. step down platform raised 3 in. above the grids. Intermittent shock could be applied to the grids through a shock scrambler set to deliver 150 V a.c. across a 220 K resistor (0.68 ma). A clock was activated when an animal was placed on the platform and stopped when the animal stepped down. Stepping down also triggered a 3-sec shock. Home cage activity was measured in 12 wooden living cages 9 in. square with wire mesh floors. Water and food were available ad lib from a bottle on one side and a supply of pellets on the floor. Four photo cell light units were
I1.0
10.0
D.6
9.2
FIG. 1. Areas of greatest (shaded) and least (dark) frontal pole lesions. (After sections in Pellegrino and Cushman Rat Atlas [9].)
OLFACTORY SYSTEM AND AVOIDANCE BEHAVIOR attached to each cage 1½ in. above the floor in the middle of each wall and beamed across each corner. Breaking any light beam activated an Esterline-Angus event recorder and a counter simultaneously. The apparatus was run continuously for the experiment's duration. The active avoidance apparatus was a two-way nondiscriminated shuttle box consisting of two identical dark Plexiglas boxes 9 × 9 × 9in. separated by a 3 × 3in. closed alley over a grid floor. Guillotine doors leading to the alley from either box started a buzzer and two timers when opened. Ten sec after starting, one timer activated a shock scrambler attached to the grids of the box and alley occupied by the animal. Shock level was set at 0.36 ma (80 v across 220 k). The other clock measured elapsed time until the animal moved to the opposite box. This timer and the buzzer-shock system were turned off when a light beam at the end of the alley was broken. A third timer then started measuring the I rain interval between trials.
707 EXPERIMENT 2
Procedures After 10 days of postoperative recovery, including 4 days of handling as in Experiment 1, the animals were tested in the passive avoidance situation described earlier. The acquisition cut-off was changed to 18 trials. There was no limit for extinction. The day after completion of passive avoidance training, the rats were run in the active avoidance apparatus. On Day 1 the doors were tied open and each animal explored the apparatus for 15 min. The following day, the rat was placed in one box with the doors shut and the first trial began when the doors were opened and the buzzer started. If the animal did not leave the start box within 10 sec, shock was applied to the grids. Moving from one box to the other before the 10-sec interval ended constituted successful avoidance. Each animal was given 20 trials a day. Animals were dropped if they had not reached a criterion of 8 out of 10 consecutive avoidances by Trial 180 (9 days).
EXPERIMENT 1
RESULTS
All animals were allowed 10 days of postoperative recovery before starting the experiment and were individually handled for 5 min a day on postoperative Days 7-10. Difficulty of handling, vocalization, and general activity were subjectively appraised during this time. All testing was done at approximately the same time of day for each animal. On postoperative Days 11 and 12, open field activity was measured for each animal during 5 min of free exploration. A count was recorded each time more than half of an animal's body crossed a line. Each animal was started from the same corner facing the field. Running order was reversed on the second day. The number of boluses was also recorded for each animal and the whole field was cleaned with water after each trial. Passive avoidance training started on Day 13. The rats were placed individually on the raised platform and allowed to explore the chamber for 5 min before being returned to the home cage. This procedure was repeated three times at hourly intervals. The next day a clock was activated when the rat was placed on the platform. When the animal put three feet on the grid, the clock stopped, and the floor was electrified for 3 see. After shock, the rat was allowed 10 sec of exploration before removal to the home cage. Three trials a day were given as above until a criterion of no-stepdown in 5 min was reached or a maximum of 12 trials (4 days) were run. Testing stopped as soon as the criterion was reached, and the rat was returned to its home cage and not tested again that day. The day following acquisition, extinction training began using the same procedure except that the grid was not electrified. Extinction was considered complete when an animal first stepped down in less than 5 min. Animals not reaching extinction after 12 trials (4 days) were removed to activity cages. The day following passive avoidance training, 6 BULBS and 6 SHAMS were placed in individual activity cages. Both cumulative activity and the pattern of activity in each 24-hr period were recorded. After being in constant darkness for 8 consecutive days animals were then returned to their home cages. The remaining 5 BULBS and 6 SHAMS were then tested in the same way. The 12 F R O N T S were not tested.
Results A. Handling, vocalization (subjective appraisals). Bulbecto-
Procedures
EXPERIMENT 1
mized animals were somewhat more difficult to remove from their home cages and were generally more active than sham operates or frontally lesioned animals when handled. A few were very difficult, escaping vigorously or attacking when approached. Vocalization differences followed the same pattern but at no time were any of the animals as unmanageable as freshly lesioned septal rats or female rats with olfactory bulb ablations (unpublished observations). If an animal was hyperreactive initially, this trait persisted throughout the experiment with only slight diminution, again unlike septal animals [2]. It is relevant that bulbectomized animals' responses to handling were so widely varied that many were undistinguishable from SHAMS. Histological analysis revealed little correlation between lesion extent and this phenomenon. B. Open field activity. ANOVAS for unequal Ns showed no activity differences for all animals on either day although the BULBS approached significance over SHAMS on Day 1. N o defecation differences occurred on either day. Since the frontally lesioned and s h a m operated groups were both controls for the bulbectomy operation and had similar scores on this measure, and since high variance was responsible for the low significance of the ANOVA, the F R O N T S and S H A M S were combined and compared to the BULBS as a single group. This test revealed that BULBS were significantly more active on both days (Day 1, t = 2.19, df=33,p < 0.025 ; Day 2, t = 1.81, p < 0.05). Table 1 summarizes the results. F o r all groups activity scores decreased from Day 1 to Day 2. C. Home cage activity. The BULB and S H A M groups did not differ on either total amount or pattern of activity during their 8 days in the photocell living cages (F = 0.80, d f = 62/7, n.s.). Figure 2 demonstrates daily activity similarities for the two groups. Diurnal activity patterns were inconsistent from rat to rat; i.e., someshowed characteristic bursts of activity at particular times of the day while others did not. All animals tended to be most active between 6 a.m. and n o o n - - t h e first six hr of
708
SIECK
Activity
~
~
/
¢=v,M ¢ n o a u ~
t
',
,'\/ ~ II
"-" Y
\
iI
, ~
\
i
, I
',,/ I
/.lut.,ss
I
FIG. 2. Total home cage activity recorded in constant darkness for sham operated controls and olfactory bulbectomized groups in Experiment 1.
the normal cycle in the colony room. There were no discernible lesion effects on these patterns. D. Passive avoidance. Eight of the 10 BULBS and two each of 12 SHAMS and F R O N T S failed to reach acquisition criterion before attaining the 12-trial ceiling (B-S and B-F comparison p = 0.005, Fischer exact test). Of those entering extinction 0/2 BULBS, 5/10 SHAMS, and 7/10 F R O N T S failed to reach criterion. Because of the small number of BULBS involved, statistical tests were not run on these data. EXPERIMENT
2
If one assumes that rats utilize olfactory information to partially determine the relevance of any stimulus, loss of this input should lead to a partial failure in steering mechanisms for behavior control. Steering might consist of two interrelated mechanisms; channeling of ongoing behavior along appropriate lines based on stimulus discrimination, and starting and stopping different behavior sequences again based on some appropriate discrimination. Looking at these data as well as previous studies by Phillips [10J, Wenzel et aL [14, 15], and Douglas et al. [4] support for both of these ideas is presented.
Results .4. Handling, vocalization. The results were similar to those found in Experiment 1. The BULBS as a group were somewhat more difficult to handle but again wide individual differences were noted. B. Passive avoidance. Six of the 11 BULBS and all of the SHAMS reached criterion before the 18-trial acquisition limit (p = 0.004, Fischer exact test). Of those animals reaching criterion, the BULBS required significantly more trials (BULBS, M = 12.83; SHAMS, M----- 8.50; F----- 10.94, d r = 16/1, p < 0.005). There were no group differences in extinction (BULBS, M = 3.5; SHAMS, M = 3.2; F = 0.043, d f = 16/1). C. Active avoidance. Criterion was reached by 11 BULBS (M = 43.36 trials, SD = 14.75) and 10 SHAMS (M = 77.20, SD = 40.87). This difference was significant (F = 10.61, d f = 19/1, p -----0.004). DISCUSSION
The fact that on all measures frontally lesioned animals were unlike bulbectomized animals, although extensive damage to frontal areas just posterior to the olfactory bulbs occurred, supports the hypothesis that olfactory damage per se is responsible for effects observed. Although high variance on these measures makes interpretation difficult, data obtained from handling and open field work suggest that olfactory bulb lesions produce animals which are somewhat more reactive to novel stimulation than sham or frontally lesioned operates.
Channeling Phillips found no evidence for learning sets on visual discrimination problems and noted high variances in his operated animals' responses. Douglas et al. [4] noted high variability of emotionality in response to handling across the operated population of female rats, as was observed in the present study with males. Further, observations on individual animals' behavior in the passive avoidance situation in this experiment revealed that most animals which failed to reach criterion adopted relatively fixed strategies such as leaping toward the top of the chamber or jumping out onto the grid as soon as they were placed on the platform. That these strategies developed in response to shock, were never seen in SHAMS and persevered with little change throughout the experiments, suggests that BULBS did learn that they would be shocked but that they were unable to acquire an appropriate avoidance response. Even though it might be argued that this behavior is traceable to an arousability change or to some change in motor responsiveness such as an inability to withhold responding, the possibility exists that animals without olfactory system patency may lack appropriate feedback mechanisms to easily or correctly change behavior patterns once established. This may account for the high variability across animals observed by all authors; for the inability of operates to form learning sets (Phillips); and for the transient breakdown in spontaneous alternation after lesioning observed by Douglas et al.
OLFACTORY SYSTEM AND AVOIDANCE BEHAVIOR
709
Activation and Inhibition
The fact that the bulbectomized rats were typically more difficult to handle, were somewhat more active in the open field, took longer to learn passive avoidance, but did better at active avoidance can all be accounted for in terms of decreased freezing or motor inhibition, increased escape tendencies, increased emotionality, and so on. Further, the fact that home cage activity was similar for BULBS and SHAMS suggests that the effects were primarily situation dependent. Hence, in addition to possible changes in the channeling of behavior after lesions, changes in response thresholds or even ways of responding may occur. Since olfactory cues per se were not relevant in either the passive or active avoidance situations and were more or less constant in both, the idea that olfaction or patency of the olfactory system exerts tonic inhibitory action on other brain areas involved with motor control or activation must be considered. Wenzel et al. [14] concluded from their second study of visual discrimination and autonomic measures in pigeons that olfactory bulb or nerve lesions led to increased alerting responses, the intensity of which produced either repulsion or distraction and thus interfered with appropriate responses to stimuli. In their first study, Wenzel and Salzman [15] felt that decreased reactivity was involved in their results, and Phillips also entertained this hypothesis. However, the hypothesis that increased arousal led to inappropriate behavior also accounts for their data. Also, the facts that neither of those studies used negative reinforcement paradigms and that the Wenzel et aL studies were conducted on pigeons must be kept in mind. Unpublished work from this laboratory using positive reinforcement suggests that bulbectomized rats do indeed become hypoactive in some situations where normal animals do not. One approach to these different results suggests an interaction effect between task and lesion. More evidence for such an effect comes from an inspection of the data observed in the present studies. The bulbectomized population had consistently higher means and standard deviations than the sham operated group on all measures taken during open field testing and handling (Table 1), and much lower values during active avoidance testing. Passive
avoidance SDs were essentially equal when computed on all animals including those artificially constrained at the 12- or 18-trial limits (Experiment 1, BULBS, SD = 3.76, SHAMS, S D = 3.85; Experiment 2, BULBS, S D = 3.16, SHAMS, SD = 2.75), but undoubtedly would have shown greater variability for BULBS than SHAMS if no limit had been imposed. These data fit a hypothesis suggesting that BULBS have generally lower behavioral arousal thresholds than SHAMS. Thus, under low or moderate levels of external stimulation, BULBS would show greater exploratory approach activity while, at higher intensity levels, more stimulus or situation avoidance would be likely to occur. Increased escape tendencies at high stimulus intensity would then hinder passive avoidance acquisition while favoring active avoidance performance. Figure 3 summarizes this position in that the bulbectomized population is seen to be more reactive at given levels of external stimulation by a shift of the BULB curve to the left. This appears as greater activity during low levels of stimulation (open field) and greater escape at higher levels (passive and active avoidance). Also the curve predicts that some level of external stimulation will cause activity cessation in the BULBS before it is apparent in SHAMS. Data on this have been obtained in startle response experiments and Y-maze studies (Sieck and Gordon, in press). Nevertheless, all of the behavioral data provide only indirect evidence for this hypothesis. More direct tests will come from studies now underway on physiological responsiveness of bulbectomized and sham operated animals in various environments. In conclusion it seems clear that either olfactory stimulation or merely patency of the olfactory system is important in maintaining balance between activating and directing mechanisms in the rat brain. Results obtained from these experiments and deducible from others [4, 10, 14, 15] show marked similarities to studies involving damage to limbic structures such as the amygdala, septal region, hippocampal and frontal cortical zones [2, 8, 13]. Since the anatomical connections of the olfactory system to some of these areas are extensive and relatively direct, the possibility that olfaction is important to limbic function receives added support.
TABLE 1 A. MEAN NUMBEROF LINES CRO~ED AND BOLUSESDROPPED BY BULBECTOMIZED~SHAMOPERATEDCONTROLAND FRONTALLYLESIONED RATSDURINGOPEN FIELDTESTSON TWO SUCCESSIVEDAYS. STANDARDDEVIATIONSAREALSOSHOWN.
Bulbs Shams Fronts
Activity
S.D.
139.55 90.00 101.67 F = 2.48 df = 32/2 (n.s.)
57.81 49.83 58.55
Day 1 Boluses 3.09 2.58 2.08 F = 0.438 (n.s.)
S.D.
Activity
S.D.
3.18 2.38 2.11
111.64 67.50 76.42 F = 1.66
77.93 50.57 52.61
Day 2 Boluses 2.27 2.08 2.67 F = 0.112
(n.s.)
(n.s.)
n. MEANS AS ABOVE FOR COMBINED SHAM OPERATES (S) AND FRONTALLY LESIONED (F) RATS.
S+F Bulbs
95.83 139.55 t = 2.19 dr= 33 p < 0.025
2.33 3.09 t = 0.82
71.96 111.64 t = 1.81
2.38 2.27 t = 0.09
(n.s.)
p < 0.05
(n.s.)
S.D. 3.20 2.84 3.17
710
SIECK
FIG. 3. Theoretical response curves correlating reactivity to stimulation with assumed arousal values of the tests involved in these experiments. The actual placements of the lines for open field, passive, and active avoidance are not critical except that they are assumed to be at approximately the areas of the curve indicated where they fit the data for the means and variances obtained (See Discussion).
REFERENCES 1. Balboni, G. C. Sur les modifications de radenohypophyse, de la thryoide et de l'ovaire apres ablation des bulbes olfactifs. Bull Ass. Anat., Paris, 52: 160-167, 1967. 2. Brady, J. V. and W. J. H. Nanta. Subcortical mechanisms in emotional behavior: affective changes following septal forebrain lesions in the albino rat. J. comp.physiol, Psychol. 46: 339-346, 1953. 3. Bruce, H. M. and D. V. M. Parrott, Role of olfactory sense in pregnancy block by strange males. Science 131: 1526, 1960. 4. Douglas, R. J., R. L. Isaacson and R. L. Moss. Olfactory lesions, emotionality and activity. Physiol. Behav. 4: 379-381, 1969. 5. Heimer, L. Synaptic distribution of centripetal and centrifugal nerve fibers in the olfactory system of the rat: An experimental anatomical study. J. Anat. 103: 413-432, 1968. 6. Heimer, L. and K. Larsson, Mating behavior of male rats after olfactory bulb lesions. Physiol. Behav. 2: 207-209, 1967. 7. Kolliker, A. Handbuch der Gewebelehre des Menschens. Leipzig: Wilhelm Englemann Press, 1896. 8. MeCleary, R. A. Response-modulating functions of the limbic system: initiation and suppression. In: Progress in Physiological Psychology, Vol. 1, edited by E. Stellar and J. M. Sprague. New York: Academic Press, 1966, pp. 210-272.
9. Pellegrino, L. J. and A. J. Cushman..4 Stereotaxic Atlas of the Rat Brain. New York: Appleton-Century-Crofts, 1967. 10. Phillips, D. S. Effects of olfactory bulb ablation on visual discrimination. Physiol. Behav. 5: 13-15, 1970. 11. Powell, T. P. S., W. M. Cowan and G. Raisman. Olfactory relationships of the diencephalon. Nature 199: 710-712, 1963. 12. Scott, J. W. and C. Pfalfmann. Olfactory input to the hypothalamus: electrophysiological evidence. Science 158: 15921594, 1969. 13. Thomas, G. T., G. Hostetter and D. J. Barker. Behavioral functions of the limbic system. In: Progress in Physiological Psychology, Vol. 2, edited by E. Stellar and J. M. Sprague. New York: Academic Press, 1968, pp. 230-311. 14. Wenzel, B. M., P. F. Albritton, A. Salzman and T. E. Oberjat. Behavioral changes in pigeons after olfactory nerve section or bulb ablation. In: Olfaction and Taste 111, edited by C. Pfaffmann. New York: Rockefeller University Press, 1969, pp. 278-287. 15. Wenzel, B. M. and A. Salzman. Olfactory bulb ablation or nerve section and pigeons' behavior in non-olfactory learning. Expl. Neurol. 22: 62-69, 1968. 16. White, L. E., Jr. Olfactory bulb projections of the rat. Anat. Rec. 152: 465-480, 1965.