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Reaction to olfactory stimuli in odor-exposed rats
BEHAVIORAL AND NEURAL BIOLOGY
32, 7%88 (1981)
Reaction to Olfactory Stimuli in Odor-Exposed Rats TORDIS DALLAND
Department of Physiological Psychology, University of Bergen, Arstadveien 21, 5000 Bergen, Norway KJELL
B. DOVING
Institute of Zoophysiology, University of Oslo, Blindern, Oslo 3, Norway Rat litters were isolated from Day 14 and kept in exposing cages with clean air blown into the cages from above. Parallel with exposing rats to clean air, other animals were exposed to acetophenone or 4-methylvaleric acid for various periods of time. Histological examination showed the appearance of selective degeneration of mitral cells in the olfactory bulb. The behavioral reaction in a modified passive avoidance situation was tested using the exposing odors as conditioning stimuli. The results showed that the concentrations at which the rats lost the avoidance reaction were similar for the control rats and the rats which had been exposed to odors. Exposure to an odor did not change the concentration at which the avoidance reaction to this or to a different odor was lost. Odor exposure sufficient to induce selective degeneration in the olfactory bulb of rats did not significantly alter their reaction to olfactory stimuli. W h e n y o u n g r a t s , 14 d a y s o l d , a r e e x p o s e d to an e n v i r o n m e n t w h e r e o n e o d o r is in e x c e s s , m o r p h o l o g i c a l c h a n g e s a p p e a r in t h e cells o f t h e o l f a c t o r y b u l b ( D e v i n g & P i n c h i n g , 1973). T h i s p h e n o m e n o n h a s b e e n c a l l e d s e l e c t i v e d e g e n e r a t i o n , d u e to t h e g r e a t r e s e m b l a n c e s b e t w e e n t h e m o r p h o l o g i c a l c h a n g e s in b u l b a r cells o f o d o r - e x p o s e d a n i m a l s a n d t h o s e changes appearing after removal of the olfactory mucosa after which the s e c o n d a r y n e u r o n e s in t h e b u l b s h o w signs o f t r a n s n e u r a l d e g e n e r a t i o n ( P i n c h i n g & P o w e l l , 1971a). T h e p a t t e r n o f s e l e c t i v e d e g e n e r a t i o n is d i f f e r e n t for d i f f e r e n t o d o r s , t h u s i n d i c a t i n g a s p a t i a l r e p r e s e n t a t i o n o f some parameters of the quality of the olfactory stimulus (Pinching & D e v i n g , 1974). E l e c t r o p h y s i o l o g i c a l s t u d i e s o f t h e e x p o s e d r a t s i n d i c a t e f u n c t i o n a l d i f f e r e n c e s b e t w e e n cells f r o m t h e n o r m a l a n d d e g e n e r a t e d a r e a s . C e l l s o f t h e d e g e n e r a t e d a r e a t e n d e d to h a v e a s p o n t a n e o u s a c t i v i t y l o w e r t h a n t h a t o f cells o f t h e n o r m a l a r e a , m o r e o v e r cells o f t h e d e g e n e r a t e d a r e a w e r e l e s s f r e q u e n t l y i n h i b i t e d a n d l e s s r e s p o n s i v e to o d o r 79 0163-1047/81/050079-10502.00/0 Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
80
DALLANDAND DOVING
stimulation than cells of the normal area (Oakley, Dcving, & Pinching, 1980). There has been one previous study published reporting behavioral effects of olfactory exposure in rats. Laing and Panhuber (1978) showed that rats exposed to acetophenone had a sensitivity for cyclohexanone lower than that of normal rats or rats exposed to cyclohexanone. The animals exposed to cyclohexanone did not perform substantially differently from normal rats in the acuity tests for acetophenone, cyclohexanone, and heptanol. The olfactory acuity in their experiments was measured by the mean correct response levels over 4 consecutive days to one concentration and therefore habituation might have been a factor influencing the results. For acetophenone the concentration used was given as 3 × 10-4 ppM. In the present experiment we studied the possible behavioral differences between odor-exposed and control rats. Rats were exposed to acetophenone or 4-methylvaleric acid for various periods of time. Later, the responses to these two substances were tested in a modified passive avoidance situation, using olfactory conditioning stimuli (CS). The results demonstrated that odor exposure sufficient to induce selective degeneration patterns in the olfactory bulb of the rats did not significantly alter their reaction to olfactory stimuli. MATERIAL AND METHODS
Subjects. Male Wistar rats of about 14 days of age had a mean weight of 27 g when put into the exposing cages. The rats were given standard rat food pellets and water ad lib. After exposure the rats were housed in the animal room in standard laboratory cages for two, and kept in these throughout the testing period until the time of sacrifice for histological examination. Food was available ad lib during the testing period. The exposure conditions of the individual rats were not known to the experimenter at the time of testing. The exposure and survival times of the experimental animals are given in Table 1. Group I: Six rats were exposed for 50 days and sacrificed at the end of the exposure period for histological examination. Group II: Twelve rats were exposed for 33 days and transferred to laboratory cages. Testing started 40 days later.They were sacrificed for histological examination 3 weeks after end of testing. Data from one control rat were discarded due to inconsistent licking. Group III: Nine rats were exposed for 230 days. One rat from each exposure condition was tested 200 days after end of exposure. All rats in this group were sacrificed for histological examination 150 days after testing ended. Odor exposure. Rats were raised in six cylindrical Lucite cages 20 cm in diameter and 20 cm high, with closed tops. The cages were placed on a coarse grid, and the droppings were removed twice a day. Fresh air was
81
ODOR REACTIONS IN RATS TABLE 1 Survival Times and Weights of Experimental Animals Tested
Group I Group II Group III
Sacrificed
Number
Exposure (days)
Number
Age period (days)
Age (days)
Mean weight
6 12 9
50 33 230
0 13 3
-73- 98 437-503
50 120 656
178 340 410
Note. In each group an equal number of rats was exposed to acetophenone, 4-methylvaleric acid, and control conditions (no odor). At the start of the experiments the mean weight of the animals was 27 g.
blown through charcoal filters and molecular sieves and reached the cages from above. The flow of air was monitored with venturimeters and varied b e t w e e n 400 and 500 crrf s-1 through each cage. Substances added to the cages were introduced into the airstream from a glass bottle, the content of which was weighed before and after the exposure to m e a s u r e the mean concentration of substances in the air stream. In two cages a c e t o p h e n o n e was added at concentrations of 2.0 x 10-~ and 1.2 x 10-7 M. In two cages with control animals no odor was added. Testing apparatus. Reactions to an olfactory CS were tested in a G r a s o n Stadler Skinner box, using a Grason Stadler drinkometer and electromechanical p r o g r a m m i n g equipment. The inside of the b o x was lit by a 15-W red lamp, and a fan secured air circulation and provided a masking noise. A p e r s p e x cylinder, 3 cm in diameter, was attached to the outside of one wall. The cylinder was a continuation of a flow olfact o r m e t e r (Dcving & Schieldrop, 1975) through which the airflow was kept at 300 ml sec -~ . An external fan created a slight negative pressure in the cage. The metal spout of the water bottle was inserted into the cylinder, 6 c m a b o v e the floor of the Skinner box. The rat had to push its nose through a hole in the wall and the p e r s p e x cylinder from the olfact o m e t e r in order to reach the spout and was thus under continuous e x p o s u r e to the odorized air stream when drinking. The conditioned stimulus (CS) was a 20-sec presentation of either a c e t o p h e n o n e or 4-methylvaleric acid given by activating an infusion pump, injecting saturated o d o r v a p o r s into the olfactometer. The speed of the m o t o r could be varied in discrete levels and gave the dilution steps, 5, 2, and 1 in powers of ten. The actual concentrations of a c e t o p h e n o n e and 4-methylvaleric acid are given in Tables 2 and 3. The unconditioned stimulus (UCS) was a 1-sec scrambled shock of 0.4 m A administered through the grid floor of the box. The U C S was given immediately following CS offset. There was a delay of m a x i m u m 2 sec from CS on- and offset until the odor reached the w a t e r spout. The U C S
82
DALLAND AND D~VING
would therefore overlap with the last part of the CS. CS, UCS, and each contact the rat made with the water spout were recorded by a pen recorder, running at a speed of 18 cm/min. Testing procedure. The rats were water deprived for 2 ~ hr preceding each daily test session. They were first given 7 days of pretraining, consisting of 15 min access to the water bottle. Air was flowing through the olfactometer but no CS or UCS was given. By the end of this period, all rats were drinking at a relatively constant rate. Modified Passive Avoidance training: Each rat was tested with one of the two odors. CS and UCS presentations were administered manually. CS was only given while the rat was drinking, and never until it had been drinking continuously for at least 15 sec. On the first 2 training days, UCS followed each CS, no matter how the rat responded. During later sessions, UCS was not given if the rat responded to the CS with an avoidance, i.e., stopped drinking within 6 sec after CS onset, without resuming contact with the spout for more than 3 sec for the remaining CS period. The number of CS presentations varied for each 15 min test period, depending on the consistency of the drinking response. Training started with the strongest CS (level 1). After five consecutive avoidances (not necessarily in the same session), or eight avoidances in a sequence of l0 CS presentations, CS was changed to the next, lower level and training continued. When no more than four avoidance responses occurred during 20 CS presentations, or no avoidances occurred in 8 consecutive presentations, testing on this level was discontinued. The rat was then given another few CS presentations of the previous (higher) level, to test for the presence of the CR. Testing continued for 15 days. On Days 5 and 10 of training " d u m m y " sessions were given to test for possible nonolfactory cues for avoidance, e.g., the sound of the injection motor. No CS or UCS was given in these sessions, but otherwise the procedure was exactly as on the other training days. Six rats from group II, i.e., two from each exposure condition and two control rats, were tested with each odor, i.e., acetophenone and 4-methylvaleric acid. Three rats from group III, one from each exposure condition, were exposed for 6 months. After exposure, they were further kept in an ordinary animal room for eight months before being tested with acetophenone according to the procedure described above. Statistics. Because of the small number of subjects in each testing condition, statistical treatment was based on data from individual rats, using a binomial test (Siegel, 1956). For each rat the number of spout contacts (licks) were counted from the pen recordings for each CS period, and for a period of 15 sec before each CS onset. A reaction to the CS ("avoidance") was defined as a decrease of 20% or more in licks during CS compared to the pre-CS period (disregarding the criteria used during training to decide whether or not to administer the UCS). The validity of
ODOR REACTIONS IN RATS
83
this criterion for a response to CS is supported by the results from the two " d u m m y " sessions. When licks during " C S " and " p r e - C S " on these sessions are calculated there is a tendency for the number of licks during '~CS" to be higher than during " p r e - C S , " but the difference is not significant by binomial test. As avoidance training progressed, all rats improved the consistency of their performance. This meant that on criterion days (Tables 2 and 3) all avoidance responses were in fact at least 35%, and usually more, below pre-CS values. For each CS level, a binomial test was used to assess whether the number of times a rat decreased its licking rate at least 20% during CS, relative to the total number of CS presentations, was statistically above chance. A clear response was defined as seven consecutive avoidance reactions to the CS (p = .008, binomial test), or as eight avoidance reactions in a series of 9 consecutive CS presentations (p = .02, binomial test). A loss of response was defined as absence of avoidance reactions to 7 consecutive CS presentations ( p = .008, binomial test), or to only one avoidance reaction in a series of 10 CS presentations (p = .011, binomial test). Histology. The animals were anesthetized and perfused with buffered saline and fixed with a formaldehyde-glutaraldehyde mixture (Pinching & Powell, 1971b). Blocks of the olfactory bulb were dehydrated and embedded in A r a l d i t e - E p o n . After these had polymerized, 2-/~m sections were cut in several coroneal planes for each bulb and stained with the method used by Richardson, Jarret, and Finke (1960). RESULTS
Behavioral Testing All rats developed an avoidance reaction to the strongest CS within 3 days. There were no differences among rats from the three exposure conditions in the speed with which they learned the avoidance response. No differences in reactions of the individual rats to the olfactory stimuli were observed during testing. Rats from the three exposure conditions did not differ in the number of sessions necessary to reach a CS level low enough to give a loss of avoidance reaction. The results for the nine rats tested with acetophenone are shown in Table 2. All the rats had a clear response to level 3 (7.2 x 10-,~ M), irrespective of previous exposure condition. Eight of the rats lost the response at level 4 (3.6 x 10-" M). Of these, seven met the strict criterion for a loss of avoidance response, while one rat (previously exposed to acetophenone) showed inconsistent responses for a long time, neither meeting the criterion for an avoidance response nor for a loss of response. One of the control rats also had a long period of inconsistent response at level 4. H o w e v e r , on the last day of testing, this rat attained the criterion
84
DALLAND AND D~VING TABLE 2 Results fron Testing with A c e t o p h e n o n e E x p o s u r e level
Subjects
Clear r e s p o n s e s
R e s p o n s e s lost
3 4 3
4 not tested 4
3 3 3
4 4 4
3 3 3
4 4 4b
Controls 1 2 3~ Acetophenone exposed 4 5 6° 4-Methylvaleric acid e x p o s e d 7 8 9a
Note. Level 3 c o r r e s p o n d s to 7.2 x 10- ~ M and level 4 to 3.6 x 10-~ M concentrations of a c e t o p h e n o n e in the airstream. Rats from e x p o s e d group III tested after 6 m o n t h s e x p o s u r e and surviving 8 m o n t h s before histologieal examination. b This rat lost the clear r e s p o n s e at this level, but showed inconsistent r e s p o n s e that did not meet the stricter criterion for a loss of response.
for a significant avoidance response at this level. The criterion for avoidance r e s p o n s e w a s met on the last day of testing, and this rat was therefore not tested on level 5. The results for rats tested with 4-methyl-valeric acid are s h o w n in Table 3. Data for o n e control rat were discarded due to inconsistent licking TABLE 3 Results from Testing with 4-Methylvaleric Acid E x p o s u r e level Subjects Controls 10 Acetophenone exposed 11 12 4-Methylvaleric acid 13 14
Clear r e s p o n s e s
R e s p o n s e s lost
5
6
5 6
6 not tested
5 5
6 6a
Note. Level 5 c o r r e s p o n d s to a concentration of 4-methylvaleric acid of 1.1 x 10-7 M, level 6 to 4.2 x 10- 8 M. a This rat lost the clear r e s p o n s e at this level, but s h o w e d inconsistent r e s p o n s e that did not meet the stricter criterion for a loss o f response.
ODOR REACTIONS IN RATS
85
throughout the experiment. The remaining five rats responded at CS level 5 (1.1 x 10-7 M). Of these, three rats--one from each exposure condition--met the strict criterion for a loss of avoidance responding, while one rat (previously exposed to 4-methylvaleric acid) responded inconsistently for a long period. One rat (previously exposed to acetophenone) gave a significant response to level 6 on the last day of testing, after a long period of inconsistent response at this level.
Histology The sections of rat bulbs were examined with respect to the extent of degeneration of the mitral cell categorizing them as normal, with light cytoplasm and nucleus, degenerated with dark cytoplasm but pale nucleus, single-hatched in the drawings, and heavy degenerated cells where both cytoplasm and nucleus were dark. The patterns of degeneration of the mitral cells were drawn and some examples of these patterns are given in Fig. 1. Microscopic inspection confirmed grossly the previous findings as to the pattern and extent of selective degeneration. Within the bulbs of rats from groups II and III with long survival time there was a tendency for the patterns to be less precise than those found in rats from group I. In the rats with long survival time there were few mitral cells that could be characterized as heavily degenerated. The control rats from group I showed mitral cells with a slightly darker cytoplasm throughout the circumference of the bulb. In the control rats of groups II and III the degenerated mitral cells were found in the mediodorsal aspect of the bulb and also in patches of the lateral part of the bulb. The pattern of degenerated mitral cells in rats exposed to acetophenone and 4-methylvaleric acid was found to vary with the exposure time and the survival. Examples of sections from the three groups are shown in Fig. 1. In rats exposed to acetophenone a region of normal cells was regularly found in the dorsomedial part of the bulb. In rats exposed to 4-methylvaleric acid there were also variations in the pattern of selectivity of degenerated mitral cells. Generally the dorsal and lateroventral parts of the bulb showed normal cells while the more medial and ventral areas showed various degrees of degenerated cells. In some rats the granular cells showed conspicuous signs of degeneration. DISCUSSION Rats that were sacrificed for histological examination immediately after the exposure to acetophenone and 4-methylvaleric acid showed clear shrinkage and cytoplasmic darkening of mitral cells in distinct patterns. Rats that were left for behavioral experiments had a variable odor experience after leaving the exposure cages. In these anmals the selective degeneration of mitral cells was less precise with regard to both the
86
DALLAND AND DOVING Control
Acetophenone
4- Me- valeric acid
Group~ I
Group H
! FIG. 1. Coronal sections of the middle part of the olfactory bulb showing the monolayer of mitral cell bodies. In the Group I1 control an outline is given of the olfactory bulb circumference to indicate the position of the mitral cell layer in the olfactory bulb. In this section the dorsal D, ventral V, medial M, and lateral L regions are indicated. All sections are oriented in the same way. Clear regions indicate the appearance of normal light-colored mitral cells, hatched areas indicate presence of mitral cell bodies with dark cytoplasm. Black regions mark areas where the mitral cells had a shrunken appearance, dark cytoplasm, and dark nucleus. c h a n g e s in m o r p h o l o g y o f t h e i n d i v i d u a l cells a n d t h e p a t t e r n o f s e l e c t i v e degeneration. T h e v a r i a t i o n s f o u n d in t h e m o r p h o l o g i c a l f e a t u r e s o f t h e m i t r a l cells most probably reflect the changing environment that rats have experie n c e d a f t e r e x p o s u r e . T h u s e v e n if s e l e c t i v e d e g e n e r a t i o n p e r s i s t s a f t e r considerable survival time after exposure, the patterns might lose their precision. These results confirm the observation by Laing and Panhuber
ODOR REACTIONS IN RATS
87
(1978) who found that the degeneration in animals exposed to acetophenone or cyclohexanone for 4 months followed by 5 months of exposure to normal rat and laboratory odors was less extensive and less severe than that directly following 2 months of exposure. These observations point to the importance of keeping the experimental animals in exposure conditions during the behavioral studies. The results of the present study did not seem to be influenced by these variations in the pattern of selective degeneration. Behavioral results showed that all rats had similar reactions to the different odors. The presence of obvious morphological changes in the bulb did not strongly affect the olfactory behavior in our test situation. The rats were still able to respond to the odor that they had been exposed to and also reacted to other odors. In our test situation we could not detect any differences in the behavior of the rats which could be related to their previous odor experience that had led to morphological changes in the relay cells of the bulb. There was, however, some individual variation between the rats in the exact level at which the avoidance response disappeared. This is to be expected considering that discrete steps of stimulation were used and that the testing procedure depended on motivation levels and learning and generalization abilities as well as on sensory capacity. However, the variation may also reflect true individual differences in lower threshold. In each odor condition, the majority of rats lost the avoidance response at the same CS level. It seems likely that for these rats the threshold was between 3.6 and 7.2 x 10-~ M for acetophenone and between 1.1 and 4.2 x 10-8 M for 4-methylvaleric acid. However, in each condition, one rat finally made a clear response at the lower of these levels, after a long period of inconsistent responding. In addition, in each condition one of the rats that lost the clear avoidance response responded inconsistently for a long period without meeting the strict criterion for an absence of reaction to that level. Inconsistent response is a typical reaction to stimulation near threshold level. For these rats, therefore, the conclusion may be that the lower threshold was very close to the last presented CS level. The aim of the present experiment was not to define the absolute threshold for the two odor substances used, but to investigate possible behavioral differences between odor-exposed and control rats. The concentrations at which the rats ceased to show avoidance reactions are not necessarily identical to the thresholds for these odors tested by other methods. The fact that the rats in the present experiment were water deprived and were drinking at the time of CS presentation might have caused them to stop responding at a stimulation level that would have given a response under different circumstances. In conclusion, no effect on the ability to use an olfactory CS as a cue to avoid shock was found to result from early exposure to the same odor, or
88
DALLAND AND D~VING
to an odor that gives a different degenerative pattern. Loss of response occurred at approximately the same odor level for all groups. The term "selective degeneration" was adopted by Dcving and Pinching (1973) because the morphological changes in the mitral cells following odor exposure were similar to those found upon severing the olfactory nerve (Pinching & Powell, 1971a). It was stressed that the term selective degeneration did not necessarily imply cell death. Electrophysiological experiments made on odor-exposed rats (Oakley, Dcving, & Pinching, unpublished observation) show that mitral cells in the degenerated areas reacted to odors. The behavioral results in the present experiment are another indication that the morphological changes do not represent a loss of function in the olfactory pathways. REFERENCES Dcving, K. B., & Pinching, A. J. (1973). Selective degeneration of neurones in the olfactory bulb following prolonged odour exposure. Brain Research, 52, 115-129. Dcving, K. B., & Schieldrop, B. (1975). An apparatus based on turbulent mixing for delivery of odorous stimuli. Chemical Senses and Flavor, 1, 371-374. Laing, D. G., & Panhuber, H. (1978). Neuronal and behavioral changes in rats following continuous exposure to an odour. Journal of Comparative Physiology, 124, 259-265. Oakley, B., Dcving, K. B., & Pinching, A. J. (1980). Physiological effects of chronic odour exposure on rat olfactory bulb neurones. In preparation. Pinching, A. J., & Dcving, K. B. (1974). Selective degeneration in the rat olfactory bulb following exposure to different odours. Brain Research, 82, 195-204. Pinching, A. J., & Powell, T. P. S. (1971). Ultrastructural features of transneuronal cell degeneration in the olfactory system. Journal of Cell Science, 8, 253-287. (a) Pinching, A. J., & Powell, T. P. S. (1971). The neuron types of the glomenalar layer of the olfactory bulb. Journal of Cell Science, 9, 305-345. (b) Richardson, K. C., Jarett, L., & Finke, E. H. (1960). Embedding in epoxy resins for ultrathin sectioning in electron microscopy. Stain Technology, 35, 313-323. Siegel, S. (1956). Nonparametric Statistics for Behavioural Sciences. New York: McGraw-Hill.
32, 7%88 (1981)
Reaction to Olfactory Stimuli in Odor-Exposed Rats TORDIS DALLAND
Department of Physiological Psychology, University of Bergen, Arstadveien 21, 5000 Bergen, Norway KJELL
B. DOVING
Institute of Zoophysiology, University of Oslo, Blindern, Oslo 3, Norway Rat litters were isolated from Day 14 and kept in exposing cages with clean air blown into the cages from above. Parallel with exposing rats to clean air, other animals were exposed to acetophenone or 4-methylvaleric acid for various periods of time. Histological examination showed the appearance of selective degeneration of mitral cells in the olfactory bulb. The behavioral reaction in a modified passive avoidance situation was tested using the exposing odors as conditioning stimuli. The results showed that the concentrations at which the rats lost the avoidance reaction were similar for the control rats and the rats which had been exposed to odors. Exposure to an odor did not change the concentration at which the avoidance reaction to this or to a different odor was lost. Odor exposure sufficient to induce selective degeneration in the olfactory bulb of rats did not significantly alter their reaction to olfactory stimuli. W h e n y o u n g r a t s , 14 d a y s o l d , a r e e x p o s e d to an e n v i r o n m e n t w h e r e o n e o d o r is in e x c e s s , m o r p h o l o g i c a l c h a n g e s a p p e a r in t h e cells o f t h e o l f a c t o r y b u l b ( D e v i n g & P i n c h i n g , 1973). T h i s p h e n o m e n o n h a s b e e n c a l l e d s e l e c t i v e d e g e n e r a t i o n , d u e to t h e g r e a t r e s e m b l a n c e s b e t w e e n t h e m o r p h o l o g i c a l c h a n g e s in b u l b a r cells o f o d o r - e x p o s e d a n i m a l s a n d t h o s e changes appearing after removal of the olfactory mucosa after which the s e c o n d a r y n e u r o n e s in t h e b u l b s h o w signs o f t r a n s n e u r a l d e g e n e r a t i o n ( P i n c h i n g & P o w e l l , 1971a). T h e p a t t e r n o f s e l e c t i v e d e g e n e r a t i o n is d i f f e r e n t for d i f f e r e n t o d o r s , t h u s i n d i c a t i n g a s p a t i a l r e p r e s e n t a t i o n o f some parameters of the quality of the olfactory stimulus (Pinching & D e v i n g , 1974). E l e c t r o p h y s i o l o g i c a l s t u d i e s o f t h e e x p o s e d r a t s i n d i c a t e f u n c t i o n a l d i f f e r e n c e s b e t w e e n cells f r o m t h e n o r m a l a n d d e g e n e r a t e d a r e a s . C e l l s o f t h e d e g e n e r a t e d a r e a t e n d e d to h a v e a s p o n t a n e o u s a c t i v i t y l o w e r t h a n t h a t o f cells o f t h e n o r m a l a r e a , m o r e o v e r cells o f t h e d e g e n e r a t e d a r e a w e r e l e s s f r e q u e n t l y i n h i b i t e d a n d l e s s r e s p o n s i v e to o d o r 79 0163-1047/81/050079-10502.00/0 Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
80
DALLANDAND DOVING
stimulation than cells of the normal area (Oakley, Dcving, & Pinching, 1980). There has been one previous study published reporting behavioral effects of olfactory exposure in rats. Laing and Panhuber (1978) showed that rats exposed to acetophenone had a sensitivity for cyclohexanone lower than that of normal rats or rats exposed to cyclohexanone. The animals exposed to cyclohexanone did not perform substantially differently from normal rats in the acuity tests for acetophenone, cyclohexanone, and heptanol. The olfactory acuity in their experiments was measured by the mean correct response levels over 4 consecutive days to one concentration and therefore habituation might have been a factor influencing the results. For acetophenone the concentration used was given as 3 × 10-4 ppM. In the present experiment we studied the possible behavioral differences between odor-exposed and control rats. Rats were exposed to acetophenone or 4-methylvaleric acid for various periods of time. Later, the responses to these two substances were tested in a modified passive avoidance situation, using olfactory conditioning stimuli (CS). The results demonstrated that odor exposure sufficient to induce selective degeneration patterns in the olfactory bulb of the rats did not significantly alter their reaction to olfactory stimuli. MATERIAL AND METHODS
Subjects. Male Wistar rats of about 14 days of age had a mean weight of 27 g when put into the exposing cages. The rats were given standard rat food pellets and water ad lib. After exposure the rats were housed in the animal room in standard laboratory cages for two, and kept in these throughout the testing period until the time of sacrifice for histological examination. Food was available ad lib during the testing period. The exposure conditions of the individual rats were not known to the experimenter at the time of testing. The exposure and survival times of the experimental animals are given in Table 1. Group I: Six rats were exposed for 50 days and sacrificed at the end of the exposure period for histological examination. Group II: Twelve rats were exposed for 33 days and transferred to laboratory cages. Testing started 40 days later.They were sacrificed for histological examination 3 weeks after end of testing. Data from one control rat were discarded due to inconsistent licking. Group III: Nine rats were exposed for 230 days. One rat from each exposure condition was tested 200 days after end of exposure. All rats in this group were sacrificed for histological examination 150 days after testing ended. Odor exposure. Rats were raised in six cylindrical Lucite cages 20 cm in diameter and 20 cm high, with closed tops. The cages were placed on a coarse grid, and the droppings were removed twice a day. Fresh air was
81
ODOR REACTIONS IN RATS TABLE 1 Survival Times and Weights of Experimental Animals Tested
Group I Group II Group III
Sacrificed
Number
Exposure (days)
Number
Age period (days)
Age (days)
Mean weight
6 12 9
50 33 230
0 13 3
-73- 98 437-503
50 120 656
178 340 410
Note. In each group an equal number of rats was exposed to acetophenone, 4-methylvaleric acid, and control conditions (no odor). At the start of the experiments the mean weight of the animals was 27 g.
blown through charcoal filters and molecular sieves and reached the cages from above. The flow of air was monitored with venturimeters and varied b e t w e e n 400 and 500 crrf s-1 through each cage. Substances added to the cages were introduced into the airstream from a glass bottle, the content of which was weighed before and after the exposure to m e a s u r e the mean concentration of substances in the air stream. In two cages a c e t o p h e n o n e was added at concentrations of 2.0 x 10-~ and 1.2 x 10-7 M. In two cages with control animals no odor was added. Testing apparatus. Reactions to an olfactory CS were tested in a G r a s o n Stadler Skinner box, using a Grason Stadler drinkometer and electromechanical p r o g r a m m i n g equipment. The inside of the b o x was lit by a 15-W red lamp, and a fan secured air circulation and provided a masking noise. A p e r s p e x cylinder, 3 cm in diameter, was attached to the outside of one wall. The cylinder was a continuation of a flow olfact o r m e t e r (Dcving & Schieldrop, 1975) through which the airflow was kept at 300 ml sec -~ . An external fan created a slight negative pressure in the cage. The metal spout of the water bottle was inserted into the cylinder, 6 c m a b o v e the floor of the Skinner box. The rat had to push its nose through a hole in the wall and the p e r s p e x cylinder from the olfact o m e t e r in order to reach the spout and was thus under continuous e x p o s u r e to the odorized air stream when drinking. The conditioned stimulus (CS) was a 20-sec presentation of either a c e t o p h e n o n e or 4-methylvaleric acid given by activating an infusion pump, injecting saturated o d o r v a p o r s into the olfactometer. The speed of the m o t o r could be varied in discrete levels and gave the dilution steps, 5, 2, and 1 in powers of ten. The actual concentrations of a c e t o p h e n o n e and 4-methylvaleric acid are given in Tables 2 and 3. The unconditioned stimulus (UCS) was a 1-sec scrambled shock of 0.4 m A administered through the grid floor of the box. The U C S was given immediately following CS offset. There was a delay of m a x i m u m 2 sec from CS on- and offset until the odor reached the w a t e r spout. The U C S
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DALLAND AND D~VING
would therefore overlap with the last part of the CS. CS, UCS, and each contact the rat made with the water spout were recorded by a pen recorder, running at a speed of 18 cm/min. Testing procedure. The rats were water deprived for 2 ~ hr preceding each daily test session. They were first given 7 days of pretraining, consisting of 15 min access to the water bottle. Air was flowing through the olfactometer but no CS or UCS was given. By the end of this period, all rats were drinking at a relatively constant rate. Modified Passive Avoidance training: Each rat was tested with one of the two odors. CS and UCS presentations were administered manually. CS was only given while the rat was drinking, and never until it had been drinking continuously for at least 15 sec. On the first 2 training days, UCS followed each CS, no matter how the rat responded. During later sessions, UCS was not given if the rat responded to the CS with an avoidance, i.e., stopped drinking within 6 sec after CS onset, without resuming contact with the spout for more than 3 sec for the remaining CS period. The number of CS presentations varied for each 15 min test period, depending on the consistency of the drinking response. Training started with the strongest CS (level 1). After five consecutive avoidances (not necessarily in the same session), or eight avoidances in a sequence of l0 CS presentations, CS was changed to the next, lower level and training continued. When no more than four avoidance responses occurred during 20 CS presentations, or no avoidances occurred in 8 consecutive presentations, testing on this level was discontinued. The rat was then given another few CS presentations of the previous (higher) level, to test for the presence of the CR. Testing continued for 15 days. On Days 5 and 10 of training " d u m m y " sessions were given to test for possible nonolfactory cues for avoidance, e.g., the sound of the injection motor. No CS or UCS was given in these sessions, but otherwise the procedure was exactly as on the other training days. Six rats from group II, i.e., two from each exposure condition and two control rats, were tested with each odor, i.e., acetophenone and 4-methylvaleric acid. Three rats from group III, one from each exposure condition, were exposed for 6 months. After exposure, they were further kept in an ordinary animal room for eight months before being tested with acetophenone according to the procedure described above. Statistics. Because of the small number of subjects in each testing condition, statistical treatment was based on data from individual rats, using a binomial test (Siegel, 1956). For each rat the number of spout contacts (licks) were counted from the pen recordings for each CS period, and for a period of 15 sec before each CS onset. A reaction to the CS ("avoidance") was defined as a decrease of 20% or more in licks during CS compared to the pre-CS period (disregarding the criteria used during training to decide whether or not to administer the UCS). The validity of
ODOR REACTIONS IN RATS
83
this criterion for a response to CS is supported by the results from the two " d u m m y " sessions. When licks during " C S " and " p r e - C S " on these sessions are calculated there is a tendency for the number of licks during '~CS" to be higher than during " p r e - C S , " but the difference is not significant by binomial test. As avoidance training progressed, all rats improved the consistency of their performance. This meant that on criterion days (Tables 2 and 3) all avoidance responses were in fact at least 35%, and usually more, below pre-CS values. For each CS level, a binomial test was used to assess whether the number of times a rat decreased its licking rate at least 20% during CS, relative to the total number of CS presentations, was statistically above chance. A clear response was defined as seven consecutive avoidance reactions to the CS (p = .008, binomial test), or as eight avoidance reactions in a series of 9 consecutive CS presentations (p = .02, binomial test). A loss of response was defined as absence of avoidance reactions to 7 consecutive CS presentations ( p = .008, binomial test), or to only one avoidance reaction in a series of 10 CS presentations (p = .011, binomial test). Histology. The animals were anesthetized and perfused with buffered saline and fixed with a formaldehyde-glutaraldehyde mixture (Pinching & Powell, 1971b). Blocks of the olfactory bulb were dehydrated and embedded in A r a l d i t e - E p o n . After these had polymerized, 2-/~m sections were cut in several coroneal planes for each bulb and stained with the method used by Richardson, Jarret, and Finke (1960). RESULTS
Behavioral Testing All rats developed an avoidance reaction to the strongest CS within 3 days. There were no differences among rats from the three exposure conditions in the speed with which they learned the avoidance response. No differences in reactions of the individual rats to the olfactory stimuli were observed during testing. Rats from the three exposure conditions did not differ in the number of sessions necessary to reach a CS level low enough to give a loss of avoidance reaction. The results for the nine rats tested with acetophenone are shown in Table 2. All the rats had a clear response to level 3 (7.2 x 10-,~ M), irrespective of previous exposure condition. Eight of the rats lost the response at level 4 (3.6 x 10-" M). Of these, seven met the strict criterion for a loss of avoidance response, while one rat (previously exposed to acetophenone) showed inconsistent responses for a long time, neither meeting the criterion for an avoidance response nor for a loss of response. One of the control rats also had a long period of inconsistent response at level 4. H o w e v e r , on the last day of testing, this rat attained the criterion
84
DALLAND AND D~VING TABLE 2 Results fron Testing with A c e t o p h e n o n e E x p o s u r e level
Subjects
Clear r e s p o n s e s
R e s p o n s e s lost
3 4 3
4 not tested 4
3 3 3
4 4 4
3 3 3
4 4 4b
Controls 1 2 3~ Acetophenone exposed 4 5 6° 4-Methylvaleric acid e x p o s e d 7 8 9a
Note. Level 3 c o r r e s p o n d s to 7.2 x 10- ~ M and level 4 to 3.6 x 10-~ M concentrations of a c e t o p h e n o n e in the airstream. Rats from e x p o s e d group III tested after 6 m o n t h s e x p o s u r e and surviving 8 m o n t h s before histologieal examination. b This rat lost the clear r e s p o n s e at this level, but showed inconsistent r e s p o n s e that did not meet the stricter criterion for a loss of response.
for a significant avoidance response at this level. The criterion for avoidance r e s p o n s e w a s met on the last day of testing, and this rat was therefore not tested on level 5. The results for rats tested with 4-methyl-valeric acid are s h o w n in Table 3. Data for o n e control rat were discarded due to inconsistent licking TABLE 3 Results from Testing with 4-Methylvaleric Acid E x p o s u r e level Subjects Controls 10 Acetophenone exposed 11 12 4-Methylvaleric acid 13 14
Clear r e s p o n s e s
R e s p o n s e s lost
5
6
5 6
6 not tested
5 5
6 6a
Note. Level 5 c o r r e s p o n d s to a concentration of 4-methylvaleric acid of 1.1 x 10-7 M, level 6 to 4.2 x 10- 8 M. a This rat lost the clear r e s p o n s e at this level, but s h o w e d inconsistent r e s p o n s e that did not meet the stricter criterion for a loss o f response.
ODOR REACTIONS IN RATS
85
throughout the experiment. The remaining five rats responded at CS level 5 (1.1 x 10-7 M). Of these, three rats--one from each exposure condition--met the strict criterion for a loss of avoidance responding, while one rat (previously exposed to 4-methylvaleric acid) responded inconsistently for a long period. One rat (previously exposed to acetophenone) gave a significant response to level 6 on the last day of testing, after a long period of inconsistent response at this level.
Histology The sections of rat bulbs were examined with respect to the extent of degeneration of the mitral cell categorizing them as normal, with light cytoplasm and nucleus, degenerated with dark cytoplasm but pale nucleus, single-hatched in the drawings, and heavy degenerated cells where both cytoplasm and nucleus were dark. The patterns of degeneration of the mitral cells were drawn and some examples of these patterns are given in Fig. 1. Microscopic inspection confirmed grossly the previous findings as to the pattern and extent of selective degeneration. Within the bulbs of rats from groups II and III with long survival time there was a tendency for the patterns to be less precise than those found in rats from group I. In the rats with long survival time there were few mitral cells that could be characterized as heavily degenerated. The control rats from group I showed mitral cells with a slightly darker cytoplasm throughout the circumference of the bulb. In the control rats of groups II and III the degenerated mitral cells were found in the mediodorsal aspect of the bulb and also in patches of the lateral part of the bulb. The pattern of degenerated mitral cells in rats exposed to acetophenone and 4-methylvaleric acid was found to vary with the exposure time and the survival. Examples of sections from the three groups are shown in Fig. 1. In rats exposed to acetophenone a region of normal cells was regularly found in the dorsomedial part of the bulb. In rats exposed to 4-methylvaleric acid there were also variations in the pattern of selectivity of degenerated mitral cells. Generally the dorsal and lateroventral parts of the bulb showed normal cells while the more medial and ventral areas showed various degrees of degenerated cells. In some rats the granular cells showed conspicuous signs of degeneration. DISCUSSION Rats that were sacrificed for histological examination immediately after the exposure to acetophenone and 4-methylvaleric acid showed clear shrinkage and cytoplasmic darkening of mitral cells in distinct patterns. Rats that were left for behavioral experiments had a variable odor experience after leaving the exposure cages. In these anmals the selective degeneration of mitral cells was less precise with regard to both the
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DALLAND AND DOVING Control
Acetophenone
4- Me- valeric acid
Group~ I
Group H
! FIG. 1. Coronal sections of the middle part of the olfactory bulb showing the monolayer of mitral cell bodies. In the Group I1 control an outline is given of the olfactory bulb circumference to indicate the position of the mitral cell layer in the olfactory bulb. In this section the dorsal D, ventral V, medial M, and lateral L regions are indicated. All sections are oriented in the same way. Clear regions indicate the appearance of normal light-colored mitral cells, hatched areas indicate presence of mitral cell bodies with dark cytoplasm. Black regions mark areas where the mitral cells had a shrunken appearance, dark cytoplasm, and dark nucleus. c h a n g e s in m o r p h o l o g y o f t h e i n d i v i d u a l cells a n d t h e p a t t e r n o f s e l e c t i v e degeneration. T h e v a r i a t i o n s f o u n d in t h e m o r p h o l o g i c a l f e a t u r e s o f t h e m i t r a l cells most probably reflect the changing environment that rats have experie n c e d a f t e r e x p o s u r e . T h u s e v e n if s e l e c t i v e d e g e n e r a t i o n p e r s i s t s a f t e r considerable survival time after exposure, the patterns might lose their precision. These results confirm the observation by Laing and Panhuber
ODOR REACTIONS IN RATS
87
(1978) who found that the degeneration in animals exposed to acetophenone or cyclohexanone for 4 months followed by 5 months of exposure to normal rat and laboratory odors was less extensive and less severe than that directly following 2 months of exposure. These observations point to the importance of keeping the experimental animals in exposure conditions during the behavioral studies. The results of the present study did not seem to be influenced by these variations in the pattern of selective degeneration. Behavioral results showed that all rats had similar reactions to the different odors. The presence of obvious morphological changes in the bulb did not strongly affect the olfactory behavior in our test situation. The rats were still able to respond to the odor that they had been exposed to and also reacted to other odors. In our test situation we could not detect any differences in the behavior of the rats which could be related to their previous odor experience that had led to morphological changes in the relay cells of the bulb. There was, however, some individual variation between the rats in the exact level at which the avoidance response disappeared. This is to be expected considering that discrete steps of stimulation were used and that the testing procedure depended on motivation levels and learning and generalization abilities as well as on sensory capacity. However, the variation may also reflect true individual differences in lower threshold. In each odor condition, the majority of rats lost the avoidance response at the same CS level. It seems likely that for these rats the threshold was between 3.6 and 7.2 x 10-~ M for acetophenone and between 1.1 and 4.2 x 10-8 M for 4-methylvaleric acid. However, in each condition, one rat finally made a clear response at the lower of these levels, after a long period of inconsistent responding. In addition, in each condition one of the rats that lost the clear avoidance response responded inconsistently for a long period without meeting the strict criterion for an absence of reaction to that level. Inconsistent response is a typical reaction to stimulation near threshold level. For these rats, therefore, the conclusion may be that the lower threshold was very close to the last presented CS level. The aim of the present experiment was not to define the absolute threshold for the two odor substances used, but to investigate possible behavioral differences between odor-exposed and control rats. The concentrations at which the rats ceased to show avoidance reactions are not necessarily identical to the thresholds for these odors tested by other methods. The fact that the rats in the present experiment were water deprived and were drinking at the time of CS presentation might have caused them to stop responding at a stimulation level that would have given a response under different circumstances. In conclusion, no effect on the ability to use an olfactory CS as a cue to avoid shock was found to result from early exposure to the same odor, or
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DALLAND AND D~VING
to an odor that gives a different degenerative pattern. Loss of response occurred at approximately the same odor level for all groups. The term "selective degeneration" was adopted by Dcving and Pinching (1973) because the morphological changes in the mitral cells following odor exposure were similar to those found upon severing the olfactory nerve (Pinching & Powell, 1971a). It was stressed that the term selective degeneration did not necessarily imply cell death. Electrophysiological experiments made on odor-exposed rats (Oakley, Dcving, & Pinching, unpublished observation) show that mitral cells in the degenerated areas reacted to odors. The behavioral results in the present experiment are another indication that the morphological changes do not represent a loss of function in the olfactory pathways. REFERENCES Dcving, K. B., & Pinching, A. J. (1973). Selective degeneration of neurones in the olfactory bulb following prolonged odour exposure. Brain Research, 52, 115-129. Dcving, K. B., & Schieldrop, B. (1975). An apparatus based on turbulent mixing for delivery of odorous stimuli. Chemical Senses and Flavor, 1, 371-374. Laing, D. G., & Panhuber, H. (1978). Neuronal and behavioral changes in rats following continuous exposure to an odour. Journal of Comparative Physiology, 124, 259-265. Oakley, B., Dcving, K. B., & Pinching, A. J. (1980). Physiological effects of chronic odour exposure on rat olfactory bulb neurones. In preparation. Pinching, A. J., & Dcving, K. B. (1974). Selective degeneration in the rat olfactory bulb following exposure to different odours. Brain Research, 82, 195-204. Pinching, A. J., & Powell, T. P. S. (1971). Ultrastructural features of transneuronal cell degeneration in the olfactory system. Journal of Cell Science, 8, 253-287. (a) Pinching, A. J., & Powell, T. P. S. (1971). The neuron types of the glomenalar layer of the olfactory bulb. Journal of Cell Science, 9, 305-345. (b) Richardson, K. C., Jarett, L., & Finke, E. H. (1960). Embedding in epoxy resins for ultrathin sectioning in electron microscopy. Stain Technology, 35, 313-323. Siegel, S. (1956). Nonparametric Statistics for Behavioural Sciences. New York: McGraw-Hill.