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Cerebral lateralization of olfactory-mediated affective processes in rats
53
Behavioural Brain Research, 40 (1990) 53-60 Elsevier BBR01089
Cerebral lateralization of olfactory-mediated affective processes in rats Robert Dantzer, Abdou Tazi and Rose-Marie Bluth6 INRA-INSERM, Bordeaux (France) (Received 4 October 1989) (Revised version received 4 April 1990) (Accepted 25 April 1990)
Key words: Cerebral lateralization; Emotion; Olfaction; Social recognition; Stress; Pituitary-adrenal hormone; Rat
To determine whether processing of information is lateralized in the brain of non-human mammalian species, rats that had undergone ablation of the left or right olfactory bulb were compared to sham-operated animals and to bilaterally bulbectomized animals in their response to emotionally positive or negative social odours. Left-bulbectomized rats were impaired in their behavioural reaction but not in their hormonal response to an odour from a stressed conspecific. They fully retained, however, their ability to recognize a nonstressed juvenile conspecific on the basis of its olfactory characteristics. These results suggest that hemispheric asymmetries develop in mammals not for recognition of emotional stimuli but for association of emotional experiences with appropriate adaptive behaviour.
INTRODUCTION
Studies using normal subjects and brainlesioned patients have demonstrated the existence of brain hemispheric asymmetries in the experience and expression of positive and negative affective states 5,2~. Hemispheric lateralization of function is not restricted to human beings and has also been observed in non-human species 1~. In particular, the behavioural consequence of unilateral neocortical ablations in rats are suggestive of hemispheric specialization in affective behavi o u r 3"6"7 but the exact nature of this bias and its consistency are still controversial ~3. Another way of investigating hemispheric specialization is to restrict sensory input to only one hemisphere in order to assess how this information is perceived,
decoded and responded to. Using this approach, left hemisphere dominance for the processing of visual information in birds 12,~8 and for the recognition of ultrasonic communication calls in mice 8 has recently been described. We decided to study lateralization of processing of olfactory information in rats since the olfactory cortex represents the principal cortical region in this species and social odours play an important role in its physiology and behaviour 2. Because rats have an incomplete nasal septum 1, occluding one nostril is not a satisfactory technique for asymmetrical olfactory stimulation. Therefore rats that had undergone left or right ablation of the olfactory bulb by aspiration were compared to sham-operated animals and to bilaterally bulbectomized animals in various testing
Correspondence: R. Dantzer, INRA-INSERM, Rue Camille St-Saens, F-33077 Bordeaux Cedex, France. 0166-4328/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
54 procedures that differed in the affective value of the olfactory signals emanating from conspecifics. Using this approach, we report here that rats have a left-hemisphere advantage in processing information only in the context of a danger that requires behavioural adjustments.
EXPERIMENT 1 - Effects of unilateral olfactory bulbectomy opt stress-induced analgesia
Rats that are injected in their hind paw with a formalin solution respond to the pain by repeatedly licking their injured paw and lifting it from the ground 1°. The latency with which they show this pain-motivated behaviour is delayed if they are exposed to a stressful condition such as the odour of a stressed conspecific 9. This form of stress-induced analgesia is dependent on endogenous opioids since it is blocked by pretreatment with an opiate antagonist. To test the possibility of an hemispheric specialization in the processing of emotionally negative olfactory information, we assessed whether formalin-injected rats still display stress-induced analgesia after they have been submitted to ablation of the right or the left olfactory bulb.
Animals and methods Male Wistar rats (Iffa-Credo, France), 3 months of age, were housed by groups of 4 - 5 in plastic cages with food and water ad libitum. Temperature and humidity of the animal colony room were regulated and an artificial dark-light cycle was imposed (light on from 6.00 to 20.00 h). Rats were submitted to unilateral bulbectomy, bilateral bulbectomy or sham surgery after craniotomy under chloral hydrate anesthesia. Bulbectomy was performed by aspiration and the cribriform plate was fully scraped. Great care was taken not to damage frontal lobes. The olfactory fossae were filled with sterile gelfoam before suturing. After 10 days recovery, animals were habituated to handling and placement in an observation chamber for 15 min during 7 consecutive days. Following this habituation period, they were sub-
mitted to an open-field lest in order to assess possible interference of the lesion with general reactivity. Rats were individually placed m a 1 x 1 x 0.4 m enclosure made of white laminated wood and lightened by a 100-W bulb placed 1.50 m above the center. The floor was divided in sixteen 0.25 x 0.25 m squares. Using a closed video circuit, an observer unaware of the treatment, scored during a 10-min session the number of grooming episodes, the number of rearings, the number of outer and inner squares entered and the number of defecations. On the day of test, rats were given a subcutaneous injection of 0.05 ml of a 15 ?0 formalin solution under the dorsal surface of their right hind paw and they were put back in their cage. Thirty rain later, they were placed in either a chamber that previously held an electrically shocked rat (1.3 mA, 0.1 s every s for 8 min) (stress) or a non-stressed conspecific (control) 9. Latency for the test rat to engage in the stereotypical formalin-induced paw licking was measured via a closed video circuit by an observer unaware of the treatment. If an animal never engaged in paw licking, it was given a latency score of 10 rain.
Results The extent of the lesion was scored by visual inspection after killing of the animals at the end of the experiment. A score of 1 was given to lesion of the anterior part of the olfactory bulb, a score of 2 to medium size lesions and a score of 3 to full lesions. Left bulbectomized rats were less severely lesioned (mean score: 1.85) than right bulbectomized and bilaterally bulbectomized rats (2.78 and 2.71, respectively, F2.32 = 9.6, P < 0.01). Olfactory bulbectomy had no effect on reactivity to the open-field test (Table I). When responses to the stress odour were considered, however, a different picture emerged (Fig. 1). A 2-way ANOVA on licking latencies (4 treatments × 2 test conditions) showed a significant interaction ( F 3 , 4 1 = 2.91, P < 0.05). Post-hoc analysis of individual group means revealed that licking latencies were significantly higher in rats exposed to the stress odour than in rats exposed
55 TABLE I
Behavioural responses to the open-field test Each value gives the number of episodes ( + S.E.M.) observed during a 10-min session. Each group included 14 rats, except the bilateral bulbectomy group which included 7 rats.
Sham Rearings Outer squares Inner squares Grooming
39.2 120 22.8 5.92
+ 5.53 ___ 10.3 + 4.38 + 1.16
Left
Right
42.6+ 5.31 126 +_ 13.2 17.7 + 2.98 16.3 +- 1.07
42.7 118 23.6 5.92
to the control condition in the sham-operated group (P = 0.05, least significant difference test) and in the right bulbectomized animals (P < 0.05). However, this was not the case in bilaterally bulbectomized rats nor in left-bulbectomized rats. The important finding of this experiment is that left-bulbectomized rats behaved like bilaterallybulbectomized rats whereas right-bulbectomized rats did not differ from sham-operated animals. Although these data may be interpreted to suggest that left-bulbectomized rats were unable to perceive the emotional nature of the odour released by a stressed conspecific or to react to it, there are 2 potential biases. The first one is represented by the difference in the size of the left and fight lesions. The second one, perhaps more important, is represented by the side of formalin injection since all rats were injected in their right hind paw, i.e. contralateraUy from the side of the lesion in those unilateraUy-bulbectomized rats that did not engage in paw licking.
500 [''7 Control ~ Stress r*l >.-
Lu
L~ Z
Z
300 2oo r ~ lOO o SHAM
RIGHT LEFT
h
BIL
Fig. 1. Effects of unilateral or bilateral bulbectomy on latency of formalin-induced paw licking (mean _+ S.E.M.). *P < 0.05.
Bilateral +_5.40 + 8.65 +-4.20 + 1.13
52.0 93.4 12.3 5.80
+ 11.5 + 17.1 _+ 4.21 + 1.03
E X P E R I M E N T 2 - Effect of left bulbectomy on behavioural
and pituitary-adrenal responses to stress
To control the preceding biases and replicate our original findings, a partial replication of Experiment 1 was initiated. Formalin-induced behaviour was tested in independent groups of left-bulbectomized or sham-operated rats, after injection of formalin in their fight hind paw or in their left hind paw. In addition, in order to assess, whether the deficit displayed by left-bulbectomized rats concerns all aspects of the response to a stress odour or is restricted to its behavioural expression, the effects of left bulbectomy were assessed on the pituitary-adrenal response to the stress odour. Animals and methods Experimental conditions were similar to those of Experiment I except that only left-bulbectomized rats and sham-operated animals were tested. On the day of test, rats were randomly injected either in their fight hind paw or in their left hind paw and latency of paw licking was measured during a 10-min exposure to a chamber that previously held a non-stressed rat or to a chamber in which a donor rat had just been shocked. After a 1-week recovery period to allow for dissipation of the painful effects of formalin, leftbulbectomized and sham-operated rats were exposed either to the clean chamber or to the chamber containing the stress odour. By means of a closed video circuit, an observer unaware of the treatment measured the duration of sniffing,
30
grooming and rearing displayed by the rats in each chamber as well as the number of arbitrary sections entered during a 10-min test. Rats were killed by decapitation on the completion of the test, trunk blood was collected on EDTA and plasma was separated by centrifugation. Plasma corticosterone and A C T H levels were determined by radio-isotopic dilution methods~7.
I A B L E III
Effects of leflbulbeetomy on behavioural responses to a chamber containing the odour of a non-stressed (control) or a stressed con~pec(fic (stress) Each value represents the mean duration (_+ S.E.M.), in seconds, of the observed behavioural pattern, except for activity which was scored by counting the number of arbitrary squares entered. Each sham-operated group included 4 rats whereas each left bulbectomy group included I 0 rats.
Results
Visual inspection of left bulbectomy lesions revealed either medium size or large lesions (mean score = 2.5) in all animals. Paw licking latencies observed in each treatment group are presented in Table II. Shamoperated rats displayed an increased paw licking latency in response.to the stress odour (P < 0.05) whatever the side of formalin injection. This was not the case, however, in left-bulbectomized rats since they did not differ in their latency to engage in paw licking, whatever the condition of test and the side of injection. Table llI summarizes the behavioural responses to the chamber when rats were exposed back to the stress odour, but without any formalin injection in their hind paw. Sham-operated rats groomed less in response to the stress odour than in the presence of a non-stressful odour and this characteristic change was not seen in left bulbec-
Sham Control Grooming Sniffing Rearing Activity
Left bulbeetomy Stress
124+37 2 8 ± 7.3* 89 _+ 28 102 + 19 24 _+ 10 46 _+ 11 16+ 8.2 2 3 ± 5.1
Control
Stress
94-+ 9.3 98 + 45 57 ± 24 2 9 _ + 7.2
6 6 + 12 72 _+ 14 33 -+ 8.3 2 6 + 4.9
* P < 0.05 in comparison to the corresponding control value.
tomized rats (interaction term of the 2-way ANOVA on grooming duration: Fi,24 = 4.31, P < 0.05). Rearing, sniffing and activity score were not affected by the test condition nor the lesion. A 2-way ANOVA (lesion × test condition) on plasma A C T H levels revealed a significant effect of the test condition (FI,24 = 8.30, P < 0.01) (Table II). The same variation was observed with regard to plasma corticosterone levels (F1,24 = 5.37, P < 0.05).
TABLE II
Effects of left bulbectom7 on the latency of paw licking induced by formalin injection in the right hind paw (RH) or in the left one (LH) during a lO-min exposure to a chamber that previously held a non-stressed rat (control) or to a chamber in which a donor rat had just been shocked (stress), and on the pituitary-adrenal response to this situation but in the absence o f formalin injection
(1) Latency of paw licking (s) Control (n = 4) Stress (RH) (n = 5) Stress (LH) (n = 5) (2) Pituitary-adrenal response Plasma ACTH (pg/ml) Control (n = 7) Stress (n = 7) Plasma corticosterone (ng/ml) Control (n = 7) Stress (n = 7) * P < 0.05, **P < 0.01 compared to control values.
Sham
Left bulbectomy
222 + 115 528 + 57* 575 + 25*
363 _+ 121 414 ± 70 385 _+ 69
192 + 28.3 264 + 19.3"*
197 + 19.3 293 _+ 41.3'*
209 + 20 286 + 20*
225 + 31 283 + 34*
57 These findings replicate and extend those of Experiment I. Compared to sham-operated animals, left-bulbectomized rats bearing a medium or a full size lesion showed no interference of the stress odour with their recuperative behaviour. This characteristic was independent of the body side which was injured. In addition, in the absence of any body pain, they did not show the typical inhibition of grooming which was induced by presentation of the stress odour. However, they fully responded to the stress odour in terms of pituitary-adrenal hormones. These data suggest that left bulbectomized rats are still able to perceive and decode olfactory cues but that they are unable to behaviourally respond to them. However, before such a conclusion can be accepted, it is necessary to assess whether unilateral bulbectomy also affects the ability to behaviourally respond to olfactory information of different emotional valence.
EXPERIMENT
3 -
Effects of unilateral olfactory bulbectomy
on social recognition
Possible interference of unilateral olfactory bulbectomy with processing of emotionally positive olfactory information was assessed in a social recognition test 4"2°. Mature male rats confronted to a sexually immature rat display bouts of investigation of this social stimulus. The duration of investigation depends on familiarity with the stimulus animal. Since novel juveniles are investigated longer than familiar ones, the difference between social investigation times of the same stimulus animal presented at different intervals provides a convenient index of the memory for this individual. This form of memory is thought to be based on the olfactory characteristics of the juvenile since investigation of the stimulus animal is significantly reduced following preexposure to chemosensory stimuli (soiled bedding or urine) from this particuliar individual ~9. Animals and methods The subjects were Wistar male rats, 4.5 months of age. At their arrival in the laboratory 2 weeks before the start of the experiment, they were
housed in groups of 3-4 in transparent plastic cages and maintained on freely available food and water in a reversed dark-light cycle (lights on from 20.30 h to 08.30 h). Laboratory-reared juvenile Wistar male rats, 25-30 days of age, housed in groups of 10, were used as social stimuli. Experimental conditions were similar to those of Experiment 1. Rats were submitted to unilateral left (n = 4) or right (n = 5) bulbectomy, bilateral bulbectomy (n = 5) or sham surgery (n = 5). They were allowed a 2-week recovery period before being tested. The general procedure has already been fully described 4. Each test took place during the dark phase of the light-dark cycle and involved two presentations of a juvenile. Each rat was placed in an observation cage to which it had already been habituated. It was then presented with a juvenile for a 5-min session. The second exposure (recognition test) to the same or a different juvenile took place 10, 30 or 120 min later and lasted until 30 s without social investigation had elapsed or for a maximum of 5 min. Using a closed video circuit with an infra-red spot light, an observer unaware of the treatment, scored the duration of investigation (mainly anogenital sniffing) by typing pre-set keys on the keyboard of an Apple IIe computer, Results Visual inspection of the brains of lesioned animals following killing revealed a medium size lesion since the mean bulbectomy score was 2.0. The extent of the lesion was similar between groups ( F 2 , 1 1 = 0.26). When they were tested in an open field during the dark phase of the cycle, bilaterally bulbectomized rats displayed significantly less reari n g s (F3,15 = 7.81, P < 0.01) and exploration of outer squares (F3.~5 = 4.22, P < 0.05) than rats of other experimental groups (Table IV). Fig. 2 represents the duration of investigation on the second exposure test. The mean time spent investigating the juvenile on the occasion of the first exposure test is represented by the horizontal dotted line. Although there was a trend for lesioned animals to be less active than shamoperated animals, it was not significant (F3,~5 = 1.99). A 2-way ANOVA (4 experimental
5~ TABLE IV
Behavioural responses to the open-field test Each value corresponds to the number of episodes ( _+S.E.M.) in a 10-min session. Each experimental group included 5 animals except left bulbectomy (n = 4).
Rearings Outer squares Inner squares Grooming
Sham
Left
Right
Bilateral
48.4+ 4.0 116 _+ 24.3 24.2 _+ 8.13 28.3 _+ 7.0
53.0_+ 3.9 188 _+ 32.7 21.0 _+ 7.31 ~2.2 _+ 22.6
45.6_+ 9.8 108 _+ 25.0 20.0 _+ 5.11 51.3 _+ 25.2
13.2 41.0 4.40 10.9
_+ 5.6* _+ 31.6" _+ 2.20 + 3.7
* P < 0.05 in comparison to the sham values.
groups x 4 test conditions) on the duration of investigation of the juvenile on the second exposure revealed a significant group x condition interaction (F9,45 = 2.71, P = 0.01). In all experimental groups except the bilaterally bulbectomized animals, the duration of investigation on the second exposure was shorter when the same juvenile was presented again after a 10- or 30-min interval than when it was presented 120 min later or when a new juvenile was presented at the 30-min interval (P < 0.01 by the method of con-
10 i
tx'sa
~" 140
~ U
Z 120 ~oo
It-
IEI 30 ~ Same juvenile (ran) 120 r 30 Differen! juvenile
~
z i
60 4O
_
0 ~ 2o o SHAM
RIGHT
LEFT
BILATERAL
BULB~TOMY
Fig. 2. Effects of unilateral or bilateral bulbectomy on social recognition. The vertical axis represents the mean duration of investigation, in seconds, during the second exposure test to the same or a different juvenile than the one presented earlier. The horizontal dotted line represents the mean duration of investigation during the first exposure to the juvenile. * P < 0.05; **P < 0.01 compared to the duration of exploration of the same juvenile, measured after an interval of 10 or 30 min.
trasts for all groups except bilaterally bulbectomized animals for which P > 0.10). These results confirm that recognition is based on olfactory information. Moreover, they indicate that the processing of olfactory cues emanating from nonstressed conspecifics is not lateralized. DISCUSSION
There is a wealth of clinical and experimental evidence suggesting that in the human brain the right hemisphere is dominant over the left one in the decoding of negative emotional informationS'2L This bias already exists in infancy and is not restricted to auditory-visual cues since neonates tasting a bitter citric acid solution have been found to show greater right frontal activation 16. In the present experiment, we used unilaterally bulbectomized rats to study the biological origin of this hemispheric asymmetry in emotional experiences. Although the olfactory system projects both to the ipsilateral and to the contralateral hemisphere via the anterior commissure, it has already been demonstrated in rats that memories for conditioned olfactory preferences are stored unilaterally on the ipsilateral side and that the role of the anterior commissure is to provide bilateral access to these unilaterally represented memories 14,~s. This means that left bulbectomized rats process olfactory information with their right hemisphere. Since they do not respond behaviourally to a stress odour in contrast to right bulbectomized rats which use their left hemisphere for
59 doing so, the present findings reveal a left hemisphere advantage in the processing of olfactory stimuli. This lateralization is restricted to stressful stimuli since it does not affect social recognition of a juvenile conspecific. In addition, the left hemisphere bias affects only the coupling of perception to action but not its impact on bodily processes since the pituitary-adrenal component of the response to a stress odour was not altered by left bulbectomy. There have been other reports of differential hemispheric processing of sensory information but only for different components of the behavioural response to an identical sensory input or in relation to different early experiences. In the first case, 6-day-old pups which have still immature commissural systems, were trained to associate an odour presented to only one nostril with intraoral infusions of milk. Conditioned orientation toward this odour and conditioned preferences were found to be represented only in the hemisphere ipsilateral to the side of olfactory stimulation, whereas odour-induced behavioural activation was not lateralized ~4. This phenomenon is not restricted to immature brains and is influenced by previous experience, since processing of ultrasonic communication calls by the left hemisphere in female mice was found to occur only in females with maternal experience but not in naive females 8. The results of the present experiments add a new dimension to our understanding of the functional aspect of hemispheric specialization in non-human species. They show that the basic processes involved in identification and recognition of olfactory social stimuli are not lateralized. Additionally, lateralization is not critically dependent on the emotional content of the processed information. Neural processing of negative emotional stimuli appears to be lateralized in the left hemisphere only when perception of these stimuli is coupled to a highly integrated adaptive behavioural response. These data should be considered as a possible basis of lateralization of emotions in man.
ACKNOWLEDGEMENTS
Many thanks are due to Anne Colas for her help in running the behavioural experiments and to Dr. Pierre Morm6de for hormonal assays. REFERENCES 1 Bennett, M.H., A reversible nasal block for the rat, Physiol. Behav., 7 (1971) 269-270. 2 Brown, R.E., Mammalian social odors: a critical review, Adv. Study Behav., 10 (1979) 104-163. 3 Crowne, D.P., Richardson, C.M. and Dawson, K.A., Lateralization of emotionality in right parietal cortex of the rat, Behav. Neurosci., 101 (1987) 134-138. 4 Dantzer, R., Bluth6, R.M., Koob, G.F. and Le Moal, M., Modulation of social memory in male rats by neurohypophyseal peptides, Psychopharmacology, 91 (1987) 363-368. 5 Davidson, R.J., Hemispheric asymmetry and emotion. In K.R. Scherer and P. Ekman (Eds.), Approaches to Emotion, Erlbaum, Hillsdale, NJ, 1984, pp. 39-57. 6 Denenberg, V.H., General systems, theory, brain organization, and early experiences, Am. J. Physiol. (Regul. Integr. Comp. Physiol.), 7 (1980) R3-R13. 7 Denenberg, V.H., Garbanati, J., Sherman, G., Yutzey, D.A. and Kaplan, R., Infantile stimulation induces brain lateralization in rats, Science, 201 (1978) 1150-1152. 8 Ehret, G., Left hemisphere advantage in the mouse brain for recognizing ultrasonic communication calls, Nature, 325 (1987) 249-251. 9 Fanselow, M.S., Odors released by stressed rats produce opioid analgesia in unstressed rats, Behav. Neurosci., 99 (1985) 589-592. 10 Fanselow, M.S. and Baackes, M.P., Conditioned fearinduced opiate analgesia on the formalin test: evidence for two aversive motivational systems, Learn. Motiv. 13 (1982) 200-221. 11 Glick, S.D (Ed.), Cerebral Lateralization in Nonhuman Species, Academic Press, Orlando, FL, 1985. 12 Gunturkun, O., and Kesch, S., Visual lateralization during feeding in pigeons, Behav. Neurosci., 101 (1987) 433-435. 13 Kolb, B., MacKintosh, A., Whishaw, I.Q. and Sutherland, R.J., Evidence for anatomical but not functional asymmetry in the hemidecorticate rat, Behav. Neurosci., 98 (1984) 44-58. 14 Kucharski, D. and Hall, W.G., New routes to early memories, Science, 238 (1987) 786-788. 15 Kucharski, D., Johanson, I.B. and Hall, W.G., Unilateral olfactory conditioning in 6-day-old rat pups, Behav. Neural Biol., 46 (1986) 472-490.
60 16 Leventhal, H. and Tomarken, A.J., Emotion: today's problems, Annu. Rev. Psychol., 37 (1986) 565-610. 17 Morm6de, P., The vasopressin receptor antagonist dPTyr(Me)AVP does not prevent stress-induced ACTH and corticosterone release, Nature, 302 (1983) 345-346. 18 Rogers, L.J., Light experience and asymmetry of brain function in chickens, Nature, 297 (1982) 223-225. 19 Sawyer, T.F., Hengehold, A.K. and Perez, W.A., Chemo-
sensory and hormonal mediation of social memory in male rats, Behav. Neurosci., 98 (1984) 908-913. 20 Thor, D.H. and Holloway, W.R., Social memory of the male laboratory rat, J. Comp. Physiol. Ps;vchol., 96 (1982) 1000-1006. 21 Tucker, D.M., Lateral brain function, emotion, and conceptualization, Psychol. Bull., 89 (1981) 19-46.
Behavioural Brain Research, 40 (1990) 53-60 Elsevier BBR01089
Cerebral lateralization of olfactory-mediated affective processes in rats Robert Dantzer, Abdou Tazi and Rose-Marie Bluth6 INRA-INSERM, Bordeaux (France) (Received 4 October 1989) (Revised version received 4 April 1990) (Accepted 25 April 1990)
Key words: Cerebral lateralization; Emotion; Olfaction; Social recognition; Stress; Pituitary-adrenal hormone; Rat
To determine whether processing of information is lateralized in the brain of non-human mammalian species, rats that had undergone ablation of the left or right olfactory bulb were compared to sham-operated animals and to bilaterally bulbectomized animals in their response to emotionally positive or negative social odours. Left-bulbectomized rats were impaired in their behavioural reaction but not in their hormonal response to an odour from a stressed conspecific. They fully retained, however, their ability to recognize a nonstressed juvenile conspecific on the basis of its olfactory characteristics. These results suggest that hemispheric asymmetries develop in mammals not for recognition of emotional stimuli but for association of emotional experiences with appropriate adaptive behaviour.
INTRODUCTION
Studies using normal subjects and brainlesioned patients have demonstrated the existence of brain hemispheric asymmetries in the experience and expression of positive and negative affective states 5,2~. Hemispheric lateralization of function is not restricted to human beings and has also been observed in non-human species 1~. In particular, the behavioural consequence of unilateral neocortical ablations in rats are suggestive of hemispheric specialization in affective behavi o u r 3"6"7 but the exact nature of this bias and its consistency are still controversial ~3. Another way of investigating hemispheric specialization is to restrict sensory input to only one hemisphere in order to assess how this information is perceived,
decoded and responded to. Using this approach, left hemisphere dominance for the processing of visual information in birds 12,~8 and for the recognition of ultrasonic communication calls in mice 8 has recently been described. We decided to study lateralization of processing of olfactory information in rats since the olfactory cortex represents the principal cortical region in this species and social odours play an important role in its physiology and behaviour 2. Because rats have an incomplete nasal septum 1, occluding one nostril is not a satisfactory technique for asymmetrical olfactory stimulation. Therefore rats that had undergone left or right ablation of the olfactory bulb by aspiration were compared to sham-operated animals and to bilaterally bulbectomized animals in various testing
Correspondence: R. Dantzer, INRA-INSERM, Rue Camille St-Saens, F-33077 Bordeaux Cedex, France. 0166-4328/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
54 procedures that differed in the affective value of the olfactory signals emanating from conspecifics. Using this approach, we report here that rats have a left-hemisphere advantage in processing information only in the context of a danger that requires behavioural adjustments.
EXPERIMENT 1 - Effects of unilateral olfactory bulbectomy opt stress-induced analgesia
Rats that are injected in their hind paw with a formalin solution respond to the pain by repeatedly licking their injured paw and lifting it from the ground 1°. The latency with which they show this pain-motivated behaviour is delayed if they are exposed to a stressful condition such as the odour of a stressed conspecific 9. This form of stress-induced analgesia is dependent on endogenous opioids since it is blocked by pretreatment with an opiate antagonist. To test the possibility of an hemispheric specialization in the processing of emotionally negative olfactory information, we assessed whether formalin-injected rats still display stress-induced analgesia after they have been submitted to ablation of the right or the left olfactory bulb.
Animals and methods Male Wistar rats (Iffa-Credo, France), 3 months of age, were housed by groups of 4 - 5 in plastic cages with food and water ad libitum. Temperature and humidity of the animal colony room were regulated and an artificial dark-light cycle was imposed (light on from 6.00 to 20.00 h). Rats were submitted to unilateral bulbectomy, bilateral bulbectomy or sham surgery after craniotomy under chloral hydrate anesthesia. Bulbectomy was performed by aspiration and the cribriform plate was fully scraped. Great care was taken not to damage frontal lobes. The olfactory fossae were filled with sterile gelfoam before suturing. After 10 days recovery, animals were habituated to handling and placement in an observation chamber for 15 min during 7 consecutive days. Following this habituation period, they were sub-
mitted to an open-field lest in order to assess possible interference of the lesion with general reactivity. Rats were individually placed m a 1 x 1 x 0.4 m enclosure made of white laminated wood and lightened by a 100-W bulb placed 1.50 m above the center. The floor was divided in sixteen 0.25 x 0.25 m squares. Using a closed video circuit, an observer unaware of the treatment, scored during a 10-min session the number of grooming episodes, the number of rearings, the number of outer and inner squares entered and the number of defecations. On the day of test, rats were given a subcutaneous injection of 0.05 ml of a 15 ?0 formalin solution under the dorsal surface of their right hind paw and they were put back in their cage. Thirty rain later, they were placed in either a chamber that previously held an electrically shocked rat (1.3 mA, 0.1 s every s for 8 min) (stress) or a non-stressed conspecific (control) 9. Latency for the test rat to engage in the stereotypical formalin-induced paw licking was measured via a closed video circuit by an observer unaware of the treatment. If an animal never engaged in paw licking, it was given a latency score of 10 rain.
Results The extent of the lesion was scored by visual inspection after killing of the animals at the end of the experiment. A score of 1 was given to lesion of the anterior part of the olfactory bulb, a score of 2 to medium size lesions and a score of 3 to full lesions. Left bulbectomized rats were less severely lesioned (mean score: 1.85) than right bulbectomized and bilaterally bulbectomized rats (2.78 and 2.71, respectively, F2.32 = 9.6, P < 0.01). Olfactory bulbectomy had no effect on reactivity to the open-field test (Table I). When responses to the stress odour were considered, however, a different picture emerged (Fig. 1). A 2-way ANOVA on licking latencies (4 treatments × 2 test conditions) showed a significant interaction ( F 3 , 4 1 = 2.91, P < 0.05). Post-hoc analysis of individual group means revealed that licking latencies were significantly higher in rats exposed to the stress odour than in rats exposed
55 TABLE I
Behavioural responses to the open-field test Each value gives the number of episodes ( + S.E.M.) observed during a 10-min session. Each group included 14 rats, except the bilateral bulbectomy group which included 7 rats.
Sham Rearings Outer squares Inner squares Grooming
39.2 120 22.8 5.92
+ 5.53 ___ 10.3 + 4.38 + 1.16
Left
Right
42.6+ 5.31 126 +_ 13.2 17.7 + 2.98 16.3 +- 1.07
42.7 118 23.6 5.92
to the control condition in the sham-operated group (P = 0.05, least significant difference test) and in the right bulbectomized animals (P < 0.05). However, this was not the case in bilaterally bulbectomized rats nor in left-bulbectomized rats. The important finding of this experiment is that left-bulbectomized rats behaved like bilaterallybulbectomized rats whereas right-bulbectomized rats did not differ from sham-operated animals. Although these data may be interpreted to suggest that left-bulbectomized rats were unable to perceive the emotional nature of the odour released by a stressed conspecific or to react to it, there are 2 potential biases. The first one is represented by the difference in the size of the left and fight lesions. The second one, perhaps more important, is represented by the side of formalin injection since all rats were injected in their right hind paw, i.e. contralateraUy from the side of the lesion in those unilateraUy-bulbectomized rats that did not engage in paw licking.
500 [''7 Control ~ Stress r*l >.-
Lu
L~ Z
Z
300 2oo r ~ lOO o SHAM
RIGHT LEFT
h
BIL
Fig. 1. Effects of unilateral or bilateral bulbectomy on latency of formalin-induced paw licking (mean _+ S.E.M.). *P < 0.05.
Bilateral +_5.40 + 8.65 +-4.20 + 1.13
52.0 93.4 12.3 5.80
+ 11.5 + 17.1 _+ 4.21 + 1.03
E X P E R I M E N T 2 - Effect of left bulbectomy on behavioural
and pituitary-adrenal responses to stress
To control the preceding biases and replicate our original findings, a partial replication of Experiment 1 was initiated. Formalin-induced behaviour was tested in independent groups of left-bulbectomized or sham-operated rats, after injection of formalin in their fight hind paw or in their left hind paw. In addition, in order to assess, whether the deficit displayed by left-bulbectomized rats concerns all aspects of the response to a stress odour or is restricted to its behavioural expression, the effects of left bulbectomy were assessed on the pituitary-adrenal response to the stress odour. Animals and methods Experimental conditions were similar to those of Experiment I except that only left-bulbectomized rats and sham-operated animals were tested. On the day of test, rats were randomly injected either in their fight hind paw or in their left hind paw and latency of paw licking was measured during a 10-min exposure to a chamber that previously held a non-stressed rat or to a chamber in which a donor rat had just been shocked. After a 1-week recovery period to allow for dissipation of the painful effects of formalin, leftbulbectomized and sham-operated rats were exposed either to the clean chamber or to the chamber containing the stress odour. By means of a closed video circuit, an observer unaware of the treatment measured the duration of sniffing,
30
grooming and rearing displayed by the rats in each chamber as well as the number of arbitrary sections entered during a 10-min test. Rats were killed by decapitation on the completion of the test, trunk blood was collected on EDTA and plasma was separated by centrifugation. Plasma corticosterone and A C T H levels were determined by radio-isotopic dilution methods~7.
I A B L E III
Effects of leflbulbeetomy on behavioural responses to a chamber containing the odour of a non-stressed (control) or a stressed con~pec(fic (stress) Each value represents the mean duration (_+ S.E.M.), in seconds, of the observed behavioural pattern, except for activity which was scored by counting the number of arbitrary squares entered. Each sham-operated group included 4 rats whereas each left bulbectomy group included I 0 rats.
Results
Visual inspection of left bulbectomy lesions revealed either medium size or large lesions (mean score = 2.5) in all animals. Paw licking latencies observed in each treatment group are presented in Table II. Shamoperated rats displayed an increased paw licking latency in response.to the stress odour (P < 0.05) whatever the side of formalin injection. This was not the case, however, in left-bulbectomized rats since they did not differ in their latency to engage in paw licking, whatever the condition of test and the side of injection. Table llI summarizes the behavioural responses to the chamber when rats were exposed back to the stress odour, but without any formalin injection in their hind paw. Sham-operated rats groomed less in response to the stress odour than in the presence of a non-stressful odour and this characteristic change was not seen in left bulbec-
Sham Control Grooming Sniffing Rearing Activity
Left bulbeetomy Stress
124+37 2 8 ± 7.3* 89 _+ 28 102 + 19 24 _+ 10 46 _+ 11 16+ 8.2 2 3 ± 5.1
Control
Stress
94-+ 9.3 98 + 45 57 ± 24 2 9 _ + 7.2
6 6 + 12 72 _+ 14 33 -+ 8.3 2 6 + 4.9
* P < 0.05 in comparison to the corresponding control value.
tomized rats (interaction term of the 2-way ANOVA on grooming duration: Fi,24 = 4.31, P < 0.05). Rearing, sniffing and activity score were not affected by the test condition nor the lesion. A 2-way ANOVA (lesion × test condition) on plasma A C T H levels revealed a significant effect of the test condition (FI,24 = 8.30, P < 0.01) (Table II). The same variation was observed with regard to plasma corticosterone levels (F1,24 = 5.37, P < 0.05).
TABLE II
Effects of left bulbectom7 on the latency of paw licking induced by formalin injection in the right hind paw (RH) or in the left one (LH) during a lO-min exposure to a chamber that previously held a non-stressed rat (control) or to a chamber in which a donor rat had just been shocked (stress), and on the pituitary-adrenal response to this situation but in the absence o f formalin injection
(1) Latency of paw licking (s) Control (n = 4) Stress (RH) (n = 5) Stress (LH) (n = 5) (2) Pituitary-adrenal response Plasma ACTH (pg/ml) Control (n = 7) Stress (n = 7) Plasma corticosterone (ng/ml) Control (n = 7) Stress (n = 7) * P < 0.05, **P < 0.01 compared to control values.
Sham
Left bulbectomy
222 + 115 528 + 57* 575 + 25*
363 _+ 121 414 ± 70 385 _+ 69
192 + 28.3 264 + 19.3"*
197 + 19.3 293 _+ 41.3'*
209 + 20 286 + 20*
225 + 31 283 + 34*
57 These findings replicate and extend those of Experiment I. Compared to sham-operated animals, left-bulbectomized rats bearing a medium or a full size lesion showed no interference of the stress odour with their recuperative behaviour. This characteristic was independent of the body side which was injured. In addition, in the absence of any body pain, they did not show the typical inhibition of grooming which was induced by presentation of the stress odour. However, they fully responded to the stress odour in terms of pituitary-adrenal hormones. These data suggest that left bulbectomized rats are still able to perceive and decode olfactory cues but that they are unable to behaviourally respond to them. However, before such a conclusion can be accepted, it is necessary to assess whether unilateral bulbectomy also affects the ability to behaviourally respond to olfactory information of different emotional valence.
EXPERIMENT
3 -
Effects of unilateral olfactory bulbectomy
on social recognition
Possible interference of unilateral olfactory bulbectomy with processing of emotionally positive olfactory information was assessed in a social recognition test 4"2°. Mature male rats confronted to a sexually immature rat display bouts of investigation of this social stimulus. The duration of investigation depends on familiarity with the stimulus animal. Since novel juveniles are investigated longer than familiar ones, the difference between social investigation times of the same stimulus animal presented at different intervals provides a convenient index of the memory for this individual. This form of memory is thought to be based on the olfactory characteristics of the juvenile since investigation of the stimulus animal is significantly reduced following preexposure to chemosensory stimuli (soiled bedding or urine) from this particuliar individual ~9. Animals and methods The subjects were Wistar male rats, 4.5 months of age. At their arrival in the laboratory 2 weeks before the start of the experiment, they were
housed in groups of 3-4 in transparent plastic cages and maintained on freely available food and water in a reversed dark-light cycle (lights on from 20.30 h to 08.30 h). Laboratory-reared juvenile Wistar male rats, 25-30 days of age, housed in groups of 10, were used as social stimuli. Experimental conditions were similar to those of Experiment 1. Rats were submitted to unilateral left (n = 4) or right (n = 5) bulbectomy, bilateral bulbectomy (n = 5) or sham surgery (n = 5). They were allowed a 2-week recovery period before being tested. The general procedure has already been fully described 4. Each test took place during the dark phase of the light-dark cycle and involved two presentations of a juvenile. Each rat was placed in an observation cage to which it had already been habituated. It was then presented with a juvenile for a 5-min session. The second exposure (recognition test) to the same or a different juvenile took place 10, 30 or 120 min later and lasted until 30 s without social investigation had elapsed or for a maximum of 5 min. Using a closed video circuit with an infra-red spot light, an observer unaware of the treatment, scored the duration of investigation (mainly anogenital sniffing) by typing pre-set keys on the keyboard of an Apple IIe computer, Results Visual inspection of the brains of lesioned animals following killing revealed a medium size lesion since the mean bulbectomy score was 2.0. The extent of the lesion was similar between groups ( F 2 , 1 1 = 0.26). When they were tested in an open field during the dark phase of the cycle, bilaterally bulbectomized rats displayed significantly less reari n g s (F3,15 = 7.81, P < 0.01) and exploration of outer squares (F3.~5 = 4.22, P < 0.05) than rats of other experimental groups (Table IV). Fig. 2 represents the duration of investigation on the second exposure test. The mean time spent investigating the juvenile on the occasion of the first exposure test is represented by the horizontal dotted line. Although there was a trend for lesioned animals to be less active than shamoperated animals, it was not significant (F3,~5 = 1.99). A 2-way ANOVA (4 experimental
5~ TABLE IV
Behavioural responses to the open-field test Each value corresponds to the number of episodes ( _+S.E.M.) in a 10-min session. Each experimental group included 5 animals except left bulbectomy (n = 4).
Rearings Outer squares Inner squares Grooming
Sham
Left
Right
Bilateral
48.4+ 4.0 116 _+ 24.3 24.2 _+ 8.13 28.3 _+ 7.0
53.0_+ 3.9 188 _+ 32.7 21.0 _+ 7.31 ~2.2 _+ 22.6
45.6_+ 9.8 108 _+ 25.0 20.0 _+ 5.11 51.3 _+ 25.2
13.2 41.0 4.40 10.9
_+ 5.6* _+ 31.6" _+ 2.20 + 3.7
* P < 0.05 in comparison to the sham values.
groups x 4 test conditions) on the duration of investigation of the juvenile on the second exposure revealed a significant group x condition interaction (F9,45 = 2.71, P = 0.01). In all experimental groups except the bilaterally bulbectomized animals, the duration of investigation on the second exposure was shorter when the same juvenile was presented again after a 10- or 30-min interval than when it was presented 120 min later or when a new juvenile was presented at the 30-min interval (P < 0.01 by the method of con-
10 i
tx'sa
~" 140
~ U
Z 120 ~oo
It-
IEI 30 ~ Same juvenile (ran) 120 r 30 Differen! juvenile
~
z i
60 4O
_
0 ~ 2o o SHAM
RIGHT
LEFT
BILATERAL
BULB~TOMY
Fig. 2. Effects of unilateral or bilateral bulbectomy on social recognition. The vertical axis represents the mean duration of investigation, in seconds, during the second exposure test to the same or a different juvenile than the one presented earlier. The horizontal dotted line represents the mean duration of investigation during the first exposure to the juvenile. * P < 0.05; **P < 0.01 compared to the duration of exploration of the same juvenile, measured after an interval of 10 or 30 min.
trasts for all groups except bilaterally bulbectomized animals for which P > 0.10). These results confirm that recognition is based on olfactory information. Moreover, they indicate that the processing of olfactory cues emanating from nonstressed conspecifics is not lateralized. DISCUSSION
There is a wealth of clinical and experimental evidence suggesting that in the human brain the right hemisphere is dominant over the left one in the decoding of negative emotional informationS'2L This bias already exists in infancy and is not restricted to auditory-visual cues since neonates tasting a bitter citric acid solution have been found to show greater right frontal activation 16. In the present experiment, we used unilaterally bulbectomized rats to study the biological origin of this hemispheric asymmetry in emotional experiences. Although the olfactory system projects both to the ipsilateral and to the contralateral hemisphere via the anterior commissure, it has already been demonstrated in rats that memories for conditioned olfactory preferences are stored unilaterally on the ipsilateral side and that the role of the anterior commissure is to provide bilateral access to these unilaterally represented memories 14,~s. This means that left bulbectomized rats process olfactory information with their right hemisphere. Since they do not respond behaviourally to a stress odour in contrast to right bulbectomized rats which use their left hemisphere for
59 doing so, the present findings reveal a left hemisphere advantage in the processing of olfactory stimuli. This lateralization is restricted to stressful stimuli since it does not affect social recognition of a juvenile conspecific. In addition, the left hemisphere bias affects only the coupling of perception to action but not its impact on bodily processes since the pituitary-adrenal component of the response to a stress odour was not altered by left bulbectomy. There have been other reports of differential hemispheric processing of sensory information but only for different components of the behavioural response to an identical sensory input or in relation to different early experiences. In the first case, 6-day-old pups which have still immature commissural systems, were trained to associate an odour presented to only one nostril with intraoral infusions of milk. Conditioned orientation toward this odour and conditioned preferences were found to be represented only in the hemisphere ipsilateral to the side of olfactory stimulation, whereas odour-induced behavioural activation was not lateralized ~4. This phenomenon is not restricted to immature brains and is influenced by previous experience, since processing of ultrasonic communication calls by the left hemisphere in female mice was found to occur only in females with maternal experience but not in naive females 8. The results of the present experiments add a new dimension to our understanding of the functional aspect of hemispheric specialization in non-human species. They show that the basic processes involved in identification and recognition of olfactory social stimuli are not lateralized. Additionally, lateralization is not critically dependent on the emotional content of the processed information. Neural processing of negative emotional stimuli appears to be lateralized in the left hemisphere only when perception of these stimuli is coupled to a highly integrated adaptive behavioural response. These data should be considered as a possible basis of lateralization of emotions in man.
ACKNOWLEDGEMENTS
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