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Selective olfactory system lesions in rats and changes in appetitive and aversive behavior
Physiology and Behavior, Vol. 10, lap. 731-'739. Brain Research Publications Inc., 1973. Printed in the U.S.A.
Selective Olfactory System Lesions in Rats and Changes in Appetitive and Aversive Behavior MICHAEL H. SIECK
D e p a r t m e n t o f Psychology, University o f California, Riverside, California 9 2 5 0 2
(Received 23 June 1972) SIECK, M. H. Selective olfactory system lesions in rats and changes in appetitive and aversive behavior. PHYSIOL. BEHAV. 10(4) 731-739, 1973.-Male rats with bilateral lesions in different parts of the olfactory system differed on a variety of behavioral measures. All lesioned animals were hyperreactive when handled and learned shuttle box avoidance faster than sham operates. However, activity in an open field, Y-maze, and startle chamber separated the groups. Animals with lesions confined to the olfactory bulb tip (LB) were least active whereas those sustaining peduncle destruction (DB) were most active. Bar pressing for continuous reinforcement also differentiated the groups. The LBs became inactive initially while DBs learned fastest but were more resistant to extinction. Only DBs failed to learn a passive avoidance task whereas, in one replication, only SHAMs failed to extinguish this task once learned. Differential lesions thus produced an activity or reactivity gradient that was low in LBs, moderate in MBs (bulb removed), and highest in DBs. The arousing properties of the measures used also interacted with lesions to produce a family of responses. The results are discussed in terms of possible olfactoryqimbic interactions and evidence for a role of the olfactory system in modulations of arousal or affect is considered. Olfaction
Limbic system
Emotionality
Behavior control mechanisms
ness after bulb lesions or olfactory nerve sections. Phillips et al. [12] later presented similar evidence in rats as measured by heart rate changes in a startle test. None of these authors found lesion correlated changes on any measure. Other workers have also noted changes in emotionality and activity after bulb lesions without lesion dependence [3,13]. Richman et al. [13] found strain differences after lesions: albino and hooded rats ambulated more in an open field than sham operates, but only albinos decreased exploration of an object-filled area. T h e y reported no change in spontaneous alternation behavior 8 - 1 0 days after operations although Douglas et al. [3] found a transient decrease 5 days after surgery. Richman et al. attributed possible differences in the two studies to differential emotionality changes. The fact that none of these authors found lesion dependent effects may be due to one or a combination of four factors: (a) most of them were not expecting them and therefore did not systematically vary lesion sites in sufficient numbers of animals; (b) the tasks used to measure lesion effects were not sensitive to possible differences; (c) the lesions were more or less confined to one area only, e.g., the olfactory bulb proper; and (d) possible species, strain, or sex differences were involved. Previous work from this laboratory has explored the first three of these issues
PREVIOUS research from this laboratory demonstrated that selective olfactory system lesions in rats produced different behavioral effects [ 17]. All lesioned animals were hyperactive in an open field and learned shuttle box avoidance faster than sham operates. However, only animals with both olfactory bulb and peduncle destruction failed to learn a step down passive avoidance task. A startle test also suggested that minimally lesioned animals were less reactive than any group although too few data were available to approach this conclusively. In addition, lesion correlated changes in open field defecation pointed to possible emotional differences between minimally and deeply lesioned animals. The results thus far published [16,17] reveal I~hat selective olfactory system lesions produce a family of responses dependent on lesion site, type of task involved, and level of arousal as defined by the apparent intensity of the environmental situation. Animals may appear hyporeactive in one situation and hyperreactive in another. Phillips [11 ] and Wenzel et al. [22] reported performance changes in tasks involving visual discrimination after bulb lesions in both rats [11] and pigeons [22]. They discussed their results in terms of both hypo- and hyperreactivity although a later study by Wenzel et al. [23] presented clear evidence of increased autonomic responsive-
1Research supported by NIH Grant MH 15585. The services of Barry Gordon and Jeffrey Turner are gratefully acknowledged. 731
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[16,17], and the present experiments are designed to confirm and extend those findings. EXPERIMENT 1 METHOD
Animals Animals were 30 naive male hooded rats from Simonsen Laboratories approximately three months old at the start of the experiments. They were individually caged in a temperature controlled room at 23°C on a 12 hr reversed cycle with the dark phase starting at 6:30 a.m. The animals were maintained on rat chow and water ad lib except during the operant conditioning phase. One group (N=5) received bilateral olfactory nerve sections and slight damage to the tips of the olfactory bulbs (LB); one group (N=5) received minimal to total bilateral bulbectomies (MB-B); one group (N=10) received deep bilateral bublectomies (DB) which removed the olfactory peduncle as well. A final group (N=10) received sham operations (SHAMs).
Surgical Procedure All animals were injected IP with atropine sulfate (0.4 mg/kg) followed in 15 min by sodium pentobarbital (50 mg/kg IP). The animals were placed in a stereotaxic apparatus, scalp incisions made, and the periosteum on the skull was reflected. The skull overlying the olfactory bulbs was drilled down to the dura in all animals and the dura was broken with a hook for all lesioned animals. All operations were carried out under direct visual guidance. For the LBs, lesions were effected by passing a specially designed curved cutting tool around the anterior tip and ventral surface of bulbs. Minimal damage to the outer layers generally occurred from this procedure. All bulbectomies were made by aspiration; tissue anterior to the frontal pole was removed in the MB-Bs while DBs were made by removing additional tissue underlying the frontal region (see Fig. 1A). Following surgery, sterile gelfoam was gently packed in the opened skull to effect hemostasis. Terramycin powder was sprinkled into the incision, and the skin flaps were sutured closed. The latter two steps were also part of the sham operations. All animals received bicillin (0.2 cc IM) and were returned to their home cages.
Histology The brains from five animals in each group were analyzed for regional concentrations of norepinephrine, serotonin, and dopamine [21]. These animals were killed by decapitation. The entire brain, including the olfactory bulbs, was removed and dropped into ice cold saline for a few seconds. Histological verification was done blind. Two experimenters who had no prior knowledge of the type of lesion the animals had sustained visually determined the extent of any lesion present and recorded it on a scale drawing of the rat brain from lateral, dorsal, and ventral views. The five DBs not used in the biochemical analysis were anesthetized with sodium pentobarbital, perfused intracardially with 0.9% saline followed by 10% formalin and decapitated. Most of the fascia and posterior skull were removed immediately and the remaining skull and brain remained for one week in 10% buffered formalin before completing skull removal. The amount of olfactory bulb
removal was measured as above and by comparison of cresyl violet stained sections with an anterior extension of the Pellegrino and Cushman Rat Atlas made in this laboratory [9,17]. Degeneration beyond any lesion was not followed. The LB lesions severed the olfactory nerves and typically damaged the most anterior tip of the olfactory bulbs. Other work has shown that such lesions destroy or damage the glomerular and external plexiform layers of the bulb tip and ventral surface near the tip (unpublished observations). The MB-B lesions removed all bulbar tissue in a range extending back between AP 15.1 and 13.0. Bulb removal in the DBs was complete and ranged back to AP 11.8 in the least and 10.4 in the greatest lesions. No frontal damage occurred in any group. The use of the present terminology standardizes results from this laboratory. Although variability in the AnteriorPosterior plane is high at the tip of the brain, approximate A - P measurements can define the four lesion groups now reported. However, really adequate measurement can only be made histologically. Combining these two approaches, the lesions are as follows: LB. Complete olfactory nerve section at bulb surface. Variable destruction of glomerular and external plexiform layers at bulb tip and ventral surface near the tip. Lesioned area at approximately AP 18.3-18.0. MB. Bulb ablation destroying all main bulbar layers back to AP 15.9-13.7. The range indicated typically leaves all or part of the accessory olfactory bulb intact. B. Complete bulbectomy destroying main and accessory bulbs and all of internal granular layer. Some damage to the ill-defined tip of the anterior olfactory nucleus usually occurs. AP 13.7-13.0. DB. Total bulb and olfactory peduncle removal. Lesion encompasses all or most of the anterior olfactory nucleus and occasionally reaches the olfactory tubercle. Ventral frontal pole damage occurs in a small percentage of the animals. AP 1 1.8-10.4. Figure IA indicates the extent of the lesions in the present study and Figure I B indicates the structures involved. The MB-B group of the present experiment encompassed the range of the MB and B categories.
Apparatus Five different apparatuses were used to assess the lesion effects, The first was a white wood Y-maze consisting of three identical 3ftx9in.x9in. arms set at angles of t20 °. Each arm had a guillotine door 0 in. from the e n d that could be opened or dropped to control the ammals' movement. Several appetitive measures were made in BRS Foringer Skinner boxes programmed by solid state modules. Thirty-five mg Noyes pellets were used as reinforcement in these tasks. A stepdown passive avoidance apparatus, modified from a design previously used [16], consisted of a 3x3x3 ft dark Plexiglas box with a 4x4 in. platform raised 3 in. above the grid floor that could be electrified (150 V A.C. across 200 kP,, 0.68 ma) through a scrambler set to operate for 3 sec when an animal stepped down. The modification used m this experiment consisted of two amyl acetate saturated wicks at the top of the box and a pair of exhaust fans arranged to pull air over the wicks and grid floor and out the bottom of the box. Air was exhausted outside the building. This modification was made in order to minimize
SELECTIVE O L F A C T O R Y LESIONS
733
dropping a free swinging rod attached to one side of the box from a constant height to a platform on the bottom. Procedures
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FIG. 1. (A) Scale drawings of rat brain in mid-sagittal ialane showing lesion extent of the three surgical groups. Lightly shaded areas indicate least and darkly shaded areas greatest lesion extent. Locations of some major olfactory structures are indicated. For more complete anatomical discussion see Histology section and Fig. lB. (B) A transparent view of the anterior forebrain of the rat in midsagittal section. Outlines of major olfactory nuclei and tracts are drawn to scale. The lightly shaded area represents approximate origins and visible course of the lateral olfactory tract (LOT). The darkly shaded area is the approximate location of cortical LOT terminations and some secondary olfactory centers. Included in it are parts of the olfactory tubercle (TUO), prepiriform cortex, periamygdaloid and piriform cortices (PIR). AOB, accessory olfactory bulb; GLOM, glomerular layer; EPL, external plexfform layer; MIT, mitral layer; GRAN, granular layer; OAE, anterior olfactory nucleus pars externa; LOT, lateral olfactory tract; TUO, olfactory tubercle; PIR, piriform cortex; LPA, lateral paraolfactory area (nucleus accumbens, part of anterior olfactory nucleus); AC, anterior commissure; CC, corpus callosum; OAN, anterior olfactory nucleus (anterior end fuses with granular layer). Terminology adopted in part from PeUegrino and Cushman [9]. possible olfactory cues generated by the animals. In addition, the raised platform was wiped clean with a dilute water solution of amyl acetate after each animal was run. The active avoidance apparatus has been described previously [16] and consisted of a pair of dark Plexiglas enclosed boxes, 9 x 9 x 9 in. separated by a 3 x 3 in. closed alley. Raising two guillotine doors on either side of the alley started a buzzer and a clock followed 10 sec later by shock (80 V A.C. across 220 ks2, 0.36 ma). Shock was switched from one side to the other on alternate trials. The startle chamber previously described was also used [17]. It was a spring mounted 1 0 x l 0 x l 0 in. plastic box with a hinged front door and side ventilation holes. It was placed in a sealed chamber under controlled atmospheric and light conditions. A magnet attached to the under side of the box passed through a coil of wire to form a sensitive solenoid arrangement which transduced any movement into an electrical signal. This was amplified and recorded by a Beckman Type R Dynograph. Fixed amplification was used for all animals. Presession baseline calibrations of the instrument were made before each animal was tested by
A battery of six tests was run on each animal starting on postoperative Day 7. All testing was done in a quiet dimly red-lighted room. On postoperative Days 7 though 10, each animal was handled for 5 min per day. Difficulty in removal from the home cage, amount of escape behavior during handling, and vocalization were subjectively rated. On postoperative Days 11 through 16, animals were given two exploration trials daily in the Y-maze. One arm was designated the start box and all trials were started there. On Trial 1, the rat was placed in the start box for 20 sec and then the door was opened. Time taken to reach the center of the maze was recorded as was the first direction (right or left) taken from there. When an animal reached either goal box, the door was lowered and a 20-sec exploration period followed. The rat was then manually returned to the start box. Trial 2 was identical except that the animals were returned to their home cages afterward. The maze was cleaned with a dilute water solution of amyl acetate after each trial. Animals not reaching either goal box 3 min after starting a trial were picked up and returned to the start box or to their home cages depending upon the trial number. A bolus count was taken after each trial. On postoperative Day 17, the animals were divided into two groups consisting of approximately equal Ns from each surgical group. Members of one group were put on a food deprivation schedule that brought their weight down to 85% of ad lib feeding weight in five days. On the sixth day of deprivation they were started on a program of bar pressing for continuous reinforcement with three phases: ( 1) two days of magazine training in which 40 pellets were automatically delivered on a VI-45 sec schedule regardless of the rats' behavior; both bars were removed during this time; [2] two days of continuous reinforcement (CRF) training in which a response on either of the two bars delivered a pellet; sessions were automatically terminated after 60 pellets had been delivered and animals that were not pressing spontaneously were hand-shaped; [3] one day of extinction training on which 5 pellets were delivered on the CRF schedule and then reinforcement was discontinued. For CRF, latency of the first response each day and total time to obtain 60 pellets were recorded. For extinction, the number of responses and time to reach a criterion of no responses in 3 min were recorded. Animals failing to reach the extinction criterion after 60 min were returned to their home cages. After extinction, food and water ad lib were restored for six days and all animals were then run on the passive avoidance task. On the seventh day of ad lib food and water, three 5-min exploration periods 60 min apart were provided in the passive avoidance apparatus. On the next day, the rats were placed on the platform individually and were then shocked for 3 sec after they put three feet'on the grid. Time to step off the platform was measured. Three such trials were run each day until a criterion of 5 min without stepping down was met or 18 trials (6 days) had elapsed. Extinction training began on the day following criterion performance. All conditions were the same except that no shock was delivered. The number of trials to first step down within 5 min was recorded. Animals that did not reach the acquisition criterion after 6 days or failed to extinguish after 18
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734 trials t6 days) were started on the next task. Active avoidance training began the day following passive avoidance extinction or failure. It consisted of a two-way shuttle-box problem in which a buzzer started 10 sec prior to onset of shock to the grid floor. Guillotine doors opened to start each trial and also started a clock which stopped when the animal crossed to the opposite box. Crossing terminated the buzzer and the shock. Twenty trials per day were run until a criterion was reached of eight avoidances in any successive 10 trials or 180 trials (9 days) had elapsed. Nine days following active avoidance acquisition, the rats were tested for startle. Each session began with a 5-min exploration period of the enclosure before an 8JA-min testing period began with 6 trials per min. Activity levels were recorded throughout this time on the Dynograph. Startle responses were produced by a loud bell which rang for 2 sec in every lO-sec interval. Fifty trials per day were run for five consecutive days. The box was cleaned with water after each animal was returned to its home cage. As previously described [ 17] startle activity was quantified in each second by counting equal arbitrary units o f amplitude from the baseline. For tabulation purposes, amplitudes greater than 2.0 units per sec during the stimulus interval were assigned a count of 1, and activity greater than 0.5 unit per sec during the IT1 was assigned 1 count. Thus, during the 10-sec interval of one trial, a maximum of 2 for the stimulus period and 8 for the ITI was possible. Because of the great volume of data obtained, counts were omitted for Trials 11 through 15, 21 through 25,'31 through 35, and 4 l through 45. This produced a cumulative total for 30 trials per animal per day. Stimulus and ITI activity scores were treated separately This concluded the test battery. The second group of animals was run simultaneously on the same measures except that the avoidance tasks were given first followed by the CRF schedule. Eleven days elapsed from the end of CRF extinction to the first day of startle testing during which time food and water were available ad lib. All animals were left undisturbed in their cages until sacrifice 7 to 14 days after completion of all testing.
habits were noted in one animal from each group. However. time to choice point and number of failures to reach it in 180 sec clearly separated the groups. DBs and MB-Bs reached the choice point faster than SHAMs or LBs and were less likely to freeze in the start box. The LBs were somewhat faster than SHAMs and approached the speed of the most deeply lesioned groups by Day 5 (see Fig. 2). A ×= analysis using a running-time cut-off of less than 8 sec for positive criterion and 8 sec or over for negative criterion produced a x 2 of 67.40 (p<0.0001). The LB and SHAM scores were more likely to be over 8 sec than MB-Bs and DBs. The MB-Bs and DBs did not differ, but LBs were more likely than SHAMs to meet the positive criterion (×~- = 3.91, p< 0.05). Running times varied with session. Speed of SHAMs peaked on Day 3 and declined steadily after that. More failure to move and generally slower movement occurred on Days 5 and 6 presumably reflecting habituation (Fig. 2). Conversely, LBs gradually increased running speed and approximately equalled MB-Bs and DBs on Days 3, 5, and 6. The DBs reached peak speed by Day 2 and MB-Bs by Day 3. Neither group showed any sign of habituation by Day 6. One LB had consistently longer running times than the others and accounted for most of the high scores for this group. Figure 2 shows two LB curves-one including and one omitting this animal's scores.
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RESULTS No order effect was observed between Group 1 and Group 2 animals on any test. They were therefore pooled and treated together.
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Handling, Vocalization All lesioned animals tended to be hyperreactive when handled. They were also more difficult to remove from their home cages t h a n SHAMs. Many were aggressive and would attack the experimenter when approached. The condition persisted with only slight diminution throughout the experiment. The most aggressive animals were invariably members of the DB group. Vocalization followed the same pattern, viz., the most aggressive animals vocalized most. However, as previously found, wide variability existed in all groups. Some animals in every group were indistinguishable from SHAMs.
Y-Maze Performance All groups alternated above chance level ( 6 0 - 7 0 % ) and there were no differences among them. Slight position
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FiG. 2. Mean time to choice point in the Y-maze on successive days in Experiment l. All trials where the choice point was not reached before the 3 min cutoff were omitted. SHAM running time Iheavy line,~ shows Iiabituation trend not evident in lesioned groups. One LB was much slower than all others. Curves including (dotted line) and omitting (solid line) this animal's scores are shown (see textL
SELECTIVE OLFACTORY LESIONS Tabulation of the failures to reach the choice point in 180 see revealed that S = LB > MB-B > DB. Fifteen percent of all SHAM (18/120) and LB (9/60) trials ended in failure, 8.25% of MB-B (5/60) trials and only 1.67% of DB (2/120) trials were failures. No defecation differences were found (Six day means: SHAM = 1.8, LB = 1.7, MB-B = 1.I, DB = 1.2; Surgical F = 1.51, d f = 3/26, p < 0 . 2 5 ; Days F = 0.60, p > 0 . 7 5 ; Interaction F = 0.98, p<0.75).
Passive Avoidance One SHAM was accidentally omitted from this test. The data followed the same trend previously found [16,17]: 1 LB, 1 MB-B, 1 SHAM, and 4 DBs failed to reach the acquisition criterion. Since only DBs failed to reach the criterion in previous work and since scores for all groups were similar here, they were pooled and compared to the DB population. There was no significance in failures to criterion (p = 0.16,-Fisher exact test). Analysis of variance for trials to criterion computed on those animals reaching criterion revealed no differences in trials required (F = 0.670, d f = 3/18, p<0.50). As previously found, DBs that failed to reach acquisition criterion typically developed stereotyped jumping behavior as soon as they were put on the platform. In extinction, 0 LBs, 1 MB-B, 0 DBs, and 4 SHAMs failed to reach the criterion by the 6th day. Pooling the lesioned groups revealed this to be a significant effect (p = 0.04, Fisher exact test). SHAMs were less likely to step down than lesioned animals. Analysis of variance for trials to extinction criterion computed on those animals reaching that criterion again revealed no differences (F = 0.569, dr= 3/13, p<0.50).
Active Avoidance All lesioned animals reached acquisition criterion in approximately the same number of trails (LB = 24.80; MB-B = 26.00: DB = 29.50). SHAMs required approximately twice this number (41.03). However, because of the small Ns involved, the analysis of variance was not significant (F = 2.88, dr= 3/24, p<0.10). Since all lesioned animals had similar means, and since previous data indicated no differential lesion effect on this measure [16,17], the lesioned animals were pooled and compared to the SHAM operates as one group. SHAMs took significantly longer than all lesioned animals combined (F = 8.48, d f = 1/24, p<0.01). In addition, one SHAM and no lesioned animals failed to reach the acquisition criterion.
Bar Pressing 1. Magazine training: All animals learned to eat from the hopper on the VI-45 sec schedule. No consistent differences were noted. 2. Acquisition: Three LBs and none of the MB-Bs, DBs, or SHAMs required hand shaping (Fisher exact test, p = 0.004). Further, one of the LBs failed hand shaping on both days and was not continued. These animals typically developed freezing behavior in one corner of the cage during the session. Mean scores are shown in Table I. Analysis of variance for time taken to first bar press revealed differences between groups (F = 6.328, dr= 3/23, p<0.O03). Day 1 scores did not differ from Day 2 scores (F = 1.059, df = 1/23, p > 0 . 5 0 ) and there was no interaction effect (F = 0.247, dr= 1/23, p>0.25). A Scheff~ analysis of mean differences showed that DBs pressed sooner than
735 other groups; a trend indicating DB < MB-B < LB < S was also observed (DB < LB, p<0.09, DB < SHAM, p<0.02; level has been set at 0.10 on this highly conservative measure to compensate for loss in power [25]). Detailed results appear in Table 1. Session time for 60 reinforcements did not differ among groups'(F -- 2.376, dr-- 3/23, p<0.09). However, a clear day effect occurred; Session I took significantly longer than Session 2 (F = 25.92, df = 1/23, p < 0 . 0 0 0 l ) . There was no interaction effect (F = 0.872, df = 3/23, p>0.50). The trend in the time required to obtain 60 reinforcements nearly paralleled that for seconds to first bar press, viz., DB = MB-B < S < LB. 3. Extinction. Number of responses to criterion showed significant surgical effects (F = 5.91, d f = 3/23, p< 0.004). The DBs made the most responses. A Scheff~ comparison of means showed that the MB-B < DB, p = 0.09, and S < DB,p = 0.006. The LBs and MB-Bs did not differ. Visual observation of the animals in extinction revealed that many DBs became very excited when reinforcement was cut off. They attacked the bars vigorously and often ran franctically around the chamber. No other animals did this. TABLE1 MEAN VALUES FOR CONTINUOUS REINFORCEMENT (CRF) ACQUISITION AND EXTINCTION. EXTINCTION DATA ARE MEAN NUMBERS OF BAR PRESSES AND MEAN TIME TO REACH A CRITERION OF NO RESPONSES IN 3 MIN
Sec to First Bar Press
Min for 60 Reinforcements
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Day 2
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Day 2
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69.75 24.50 27.80 94.89
73.50 38.50 25.85 45.22
29.00 13.80 14.60 17.56
14.00 8.00 8.30 8.11
Extinction Bar Presses Time 110.50 91.80 175.90 76.00
30.50 17.20 33.40 20.33
Startle A ctiviO, Because of equipment malfunction, Day 3 responses were incorrectly recorded and have been omitted from the analysis. Activity levels are given for Days I - 2 and 4 - 5 . The ITI activity and responses during the stimulus were treated separately. Analysis of variance for stimulus-induced activity revealed no significant group differences, (F = 2.22, d f = 3/19, p<0.14), no day effects (F = 1.757, dr= 3/67, p > 0 . 2 5 ) and no interaction between surgery and days (F = 1.701, df = 9/67, p<0.11). Analysis of Fig. 3 reveals that SHAMs, MB-Bs and DBs had similar scores which decreased from Day 1 to a relatively stable level for the remaining days. However, the LBs started at a much lower level and gradually increased over days until they equaled the other groups. A related trend was previously reported for less deeply lesioned animals [17] and is evident in the poor LB performance in the CRF and Y-maze tests in the present experiment. A post hoc analysis of variance for Day 1 demonstrated that LBs were less active than all other groups (F = 5.55, dr= 3/19, p<0.005). A Scheff~ analysis of mean differences showed that LBs < DBs (p<0.006), LBs < MB-Bs (p<0.05). and LBs < SHAMs (p<0.05). There were
736
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M E A N A C T I V I T Y S C O R E S IN T H E S T A R T L E C}tAMBER DURING AND BETWEEN STIMULUS PERIODS FOR DAY l A L O N E A N D F O R D A Y S 1. 2, 4 A N D 5 C O M B I N E D . M E A N N U M B E R O F B O L U S E S F O R A L L S E S S I O N S IS A L S O S H O W N
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46.85 81.47 85,03 61.09
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bred at this insitution from Simonsen Laboratories' hooded rats, They were approximately three months old at the start of the experiments and were individually caged in a temperature controlled room at 23°C on a 12 hr reversed cycle with the dark phase starting at 6:30 a.m. The animals were maintained on rat chow and water ad lib throughout the experiment. Surgical procedures duplicated those in Experiment 1. Three groups of 17 each consisted of LBs, DBs, and SHAMs.
Histology DAYS FIG. 3. Mean startle activity during the bell stimulus (lower graph) and ITI (upper graph) for Days 1-2 and 4-5. Each point represents the group average of 30 responses/day/animal or a total of 150 scores/pt for LB and MB-Bgroups and 300 scores/pt for SHAM and DB groups. Amplitudes are in arbitrary units (see text). no differences between SHAMs, MB-Bs, and DBs (See Table
2).
For ITI activity, overall surgical F = 3.06, d r = 3/19, p<0.05. An habituation effect over days was also observed (F = 4.90, d f = 3/67, p<0.004, see Fig. 3). Scheff6 analysis revealed that LBs were less active than DBs (p<0.05), and that Day 2 activity was lower than Day 5 activity (p<0.01). Day 1 scores again separated the groups markedly (Fig. 3). A post hoc analysis of variance for Day 1 was significant (F = 4.31, df = 3 / 19, p< 0.01 ). The Scheff6 tests revealed that LBs were less active than DBs (p<0.03) and SHAMs < DBs (p<0.09) (Table 2). There were no differences in bolus counts across days although all lesioned animals combined defecated more than SHAMs (F = 3.71, dr= 1/13, p < 0.05). EXPERIMENT 2 METHOD
In order to corroborate the results of Experiment l, several of the tests were rerun with larger Ns. In addition possible lesion induced activity differences were further explored in an open field setting.
Animals Animals were 51 naive male rats from an F, generation
Histological procedures duplicated those of Experiment 1 for the DBs not used in the bioamine assay, The lesions were essentially identical. The LBs were deafferented and sustained some damage to the bulb tips; DBs had complete bulb and peduncle removal to between AP 1 114 and 10.4. No frontal pole damage occurred.
Apparatus A white wood open field 4x4xl ft. high with i 6 l x l ft. squares marked on the floor was used to assess activity levels [18]. The Y-maze, step down passive avoidance and shuttle box avoidance apparatuses previously described were also used.
Procedure All testing was done in a quiet dimly red-lighted room. On postoperative Days 7 through 10 each animal was handled for 5 min/day. Difficulty in removal from the home cage, amount of escape behavior during handling and vocalization were subjectively rated. On postoperative Days 11 and 12, animals were given two 5 min exploration trials daily in the open field. Each rat was started in the same corner facing the center of the field and a count of 1 was made each time more than half of the rat's body crossed a line. The number of boluses dropped were counted after each trial and the maze was cleaned with a dilute water solution of amyl acetate before each trial began. On postoperative Days 13 through 18, animals were given two exploration trials daily in the Y-maze. The procedure duplicated that of Experiment I. On postoperative Day 19, the animals were started on the passive avoidance task which was followed immediately by the
SELECTIVE OLFACTORY LESIONS
737
shuttle box problem. Procedures described in Experiment 1 were followed. RESULTS Handling and Vocalization Members of the lesioned groups were the most difficult to handle and vocalized more. The DBs were harder to handle than LBs. However, some animals from each lesioned group were indistinguishable from SHAMs. Open Field Analysis of variance for number of lines crossed revealed differences between groups (F = 33.90, d f = 2/48, p<0.0001). There was no day (F = 1.49, d f = 1.48, p > 0 . 2 5 ) or interaction (F = 1.71, dr= 2/48, p > 0 . 2 5 ) effect. Scheff~ analysis of mean differences revealed that SHAMs were less active than both LBs (F - 3.80, p<0.03) and DBs (F = 32.79, p<0.O001) and that LBs were less active than DBs (F = 14.27, p<0.0001). Analysis of variance for boluses dropped showed a significant surgical effect (F = 5.86, d f = 2/48, p
N SHAM LB DB
(17) (17) (17)
Activity Day 1 Day 2
Boluses Day 1 Day 2
33.35 48.77 75.53
2.88 4.88 6.35
31.24 49.82 88.94
1.88 4.12 3.29
Y-Maze Performance All groups alternated above chance level ( 6 5 - 7 0 % ) and there were no differences among them. Analysis of variance for time to reach the choice point revealed significant surgical (F = 12.43, d f = 2/48, p<0.001 ), days (F = 3.57, d f = 5/240, p<0.O05), and interaction (F = 2.92, d f = 10/240, p
However, the DB failures occurred primarily on Days 1 and 2, the LB failures were evenly spread across days, and the SHAM failures were greater on the last 4 days. This reflects the differential habituation already discussed and is evident in comparison of Fig. 4A and 4B. Figure 4A shows the mean time to the choice point omitting the failures while 4B shows the mean time computed on all animals with 3 min scores given to all failures; 3.4% of the DB trials (7/204), 17.7% of the LB trials (36/204) and 31.1% of the SHAM trials (64/204) ended in failures. (Overall x a = 47.71, p < 0 . 0 0 0 1 ; S > LB, x ~ = 6.32, p < 0 . 0 2 5 ; LB > DB, x 2 = 17.77, p<0.001). B 120 .1OO
8,o O ~, 2 0
DAYS
FIG. 4. A. Mean time to the choice point in the Y-maze on successive days in Experiment 2. All trials in which the choice point was not actually reached were omitted. B. Same as above for all trials. Trials ending in failure to reach the choice point were given scores of 3 min each (see text). Analysis of variance for boluses dropped showed no surgical effect (mean boluses/day: SHAM = 0.86, LB = 1.31, and DB = 0.94; F = 1.84, dr= 2/48, p < 0 . 2 5 ) but did show a days effect (F = 7.74, d r = 5/240, p<0.0001). No interaction occurred (F = 1.27, d f = 10/240, p<0.25). Scheff6 analysis revealed that defecation rates were higher on the first three days of testing (Day 1 > Day 6, p < 0 . 0 0 2 ; Day 3 > Day 6, p<0.O001 ; Day 3 > Day 5, p<0.01). Passive Avoidance As previously found [17], more DBs failed to reach the acquisition criterion than any other group. Three SHAMs, 4 LBs, and 12 DBs failed (x ~ = 12.33, p<0.005). Analysis of variance for trials to criterion computed on those animals reaching criterion revealed no differences. (F = 0.48, d f = 2/29, p<0.50). In extinction 3 SHAMs, 1 LB, and no DB failed to reach that criterion. Analysis of variance for trials to extinction criterion was also insignificant (F = 0.69, d f = 2/25, p<0.50). Active Avoidance SHAMs took significantly more trials to reach criterion than lesioned animals. The LBs and DBs did not differ. (Means: SHAM = 45.72, LB'= 28.30, DB = 24.61 ; F = 6.23, d f = 2/47, p<0.O05). In addition, 2 SHAMs, 1 LB and O DBs failed to reach the criterion. DISCUSSION These experiments confirm and extend previous findings
738 [16,17] indicating that selective olfactory system lesions produce a family of behavior patterns involving at least lesion site, experimental setting, and the arousal state of the animal. Since all the data from this laboratory have been obtained from male hooded rats, the possibility that sex, strain, or species differences play roles not yet accounted for must be considered. It is evident that olfactory system lesions increase emotionality or reactivity in every species or strain thus far studied. Wenzel et al. found evidence for this after both nerve section and bulb ablation in pigeons [23] and others reported similar findings in both hooded [13] and albino rats [3,13]. However, some evidence suggests that female rats are more reactive after bulb ablations than males [3,11] and that male albinos are less reactive than male hoods after lesions [ 13 ]. Since no really systematic study of male-female differences has yet been published nor have variable lesion studies been made on any other strain or species, the generality of the present findings await further work. Nevertheless, some constant behavior changes emerge after selective lesion studies that correlate well with data from other laboratories. • All lesioned animals are more difficult to handle and remove from their home cages than SHAMs. Some lesion dependence occurs since DBs are the most difficult to work with. Also, all lesioned animals are more active than SHAMs in the open field although LBs ambulate less than other lesioned groups. These findings confirm other work demonstrating increased emotionality after bulb lesions and further suggest a lesion dependent emotionality continum. Emotionality in rats is often estimated by bolus counts and/or freezing behavior. Richman et al. [13] found increased defecation in the open field in their bulbectomized animals from a population that appears to encompass the MB and B categories of the present study. Sieck and Gordon found increased open field defecation in MBs but not DBs [17] although the present experiments did not confirm this. No bolus differences between lesioned groups were found in the Y-maze, open field, or startle tests of the present study, but all lesions increased defecation over SHAM levels in the startle test and open field. Thus, most of the defecation data support the hypothesis that any olfactory system lesion increases emotionality over SHAM levels. However, a lesion dependent defecation difference may still be present since correlations of 0.7719 (p<0.0001, Experiment 1) and 0.2730 (p<0.05. Experiment 2) were obtained between the number of boluses dropped and time to reach the choice point in the Y-maze. Animals running slowe'r or freezing more defecated most. Since LBs ran slower than other lesioned groups and had defecation means higher than all other lesioned animals, slight defecation differences may be present. Increased reactivity after any lesion may also be reflected in the lower shuttle box acquisition scores and more rapid passive avoidance extinction for all groups. However. some lesion dependence is again indicated since Bs and DBs were less likely to reach the passive avoidance acquisition criterion in this and previous work [ 17]. With the addition of the bulbar deafferented group (LB) in the present study, a continuum of central offactory system damage from minimal to essentially complete has been attained. An earlier report suggested that lesion dependent reactivity changes take two forms. In minimally bulbectomized animals, responsiveness is more internalized and is manifest as freezing and increased defecation; in more deeply bulbectomized subjects, responsiveness ap-
SIECK pears as increased motor activity [ 17 ]. This suggestion may now be extended. No consistent lesion dependent defecation differences occur but activity differences are clear. The LBs were less action oriented in several tasks. They were least active of all lesioned groups in the open field and on the first day in the startle apparatus they froze more than any other group. In the bar pressing task, three of the LBs and no other animals required hand shaping because they became inactive very quickly. They were also slower to reach the choice point and more likely to freeze in the Y-maze than either MB-Bs or DBs although they were more active than SHAMs - especially on the last test days. Likewise, their increased stimulus and ITI activity by Days 4 and 5 in the startle apparatus showed that their initially low responsiveness disappeared with experience. Together these results suggest that mildly aversive or changing situations increase the probability of inactivity in this group but that familiarity with the test situation decreases this to unmask motor hyperactivity. The MB-B group was a mixed lesion population in terms of the initial work [17]. This group was more active than the LBs in the startle apparatus, faster in the Y-maze, and quicker to bar press for continuous reinforcement. From other work [ 17] they were also as active as DBs in the open field. Although thus similar to the DBs in several tests, MB-Bs were unlike them in that fewer passive avoidance acquisition failures occurred and fewer bar presses and less agitated responsiveness appeared in CRF extinction. They also failed more in the Y-maze and reached their top Y-maze running speed one day later (Fig. 2). Thus the MB-B group was more action oriented than the LB group but somewhat less so than the DBs. The DBs were most responsive of all. Their initial startle and ITI activity was highest, they were fastest in the Y-maze and did not habituate over days. They pressed first in the bar pressing task and were more resistant to extinction. They failed to attain the passive avoidance criterion and were the most difficult to work with since many of them tended to attack the experimenter or attempt.to escape when handled. The lesion effects thus present a continuum of responsiveness that is partly situation dependent. There is an underlying hyperreactivity in all groups which appears clearly in strongly aversive situations and a variable degree of hyporeactivity in mildly arousing situations. This hyporeactive aspect is strongest in LBs and weakest in DBs. Possible CNS Mechanisms: S o m e Speculation
Lesion dependent changes in brain norepinephrine and serotonin were found in the animals of Experiment t [21]. Since these amines have been implicated in mechanisms of arousal and affect [4, 7, 20], it is tempting to link these with the behavioral results. For instance, a midbrain based serotoninergic system has been implicated in habituation to stimuli [14,15]. Reductions in the activity of this system correlate with decreased habituation. The greatest decrease in brainstem serotonin occurred in the DBs and their lack of habituation in the Y-maze, poor extinction performance in CRF, and genel~al hyperreactivity thus correlate. Furthermore, much increased brainstem norepinephrine in the DBs might also be associated with their increased reactivity. In the LBs, less striking changes in brainstem serotonin and norepinephrine perhaps correlate with their increased
SELECTIVE OLFACTORY LESIONS
739
freezing or internalized reactivity. There are many problems in such interpretations but the data point to a relationship between bioamine levels and olfactory system lesions and provide indirect evidence for olfactory system influences on arousal and affect [21 ]. From another approach, the increased behavioral effects observed as lesions were made deeper and the increased bioamine changes accompanying them suggest a mass effect after olfactory system destruction in the sense of Lashley's hypothesis on mass action. However, olfactory input was decreased in tlae LB group, the MB-B lesions ablated the olfactory bulbs, and the DB lesion added at least the anterior olfactory nucleus (AON). Since at least part of the AON projects to areas different from those receiving efferents from the bulbs proper [5, 8, 24], it is possible that differential modulation of more central structures occurs. There is also anatomical evidence demonstrating
that deep olfactory system lesions lead to greater degeneration in basal cerebrum than shallow qesions [5,24] and electrophysiological work suggesting that deeper lying olfactory structures are important in controlling the excitability of some anterior limbic areas [2,6]. Opposing this view are anatomical and electrophysiological data demonstrating relatively diffuse innervation of olfactory projection areas by bulbar elements [1, 5, 8, 24]. Until more evidence is available this question will remain unsettled. I n summary, behavioral, anatomical, and physiological evidence link selective olfactory system lesions with changes in presumed limbic and midbrain functions [18,19]. More broadly, a role of the olfactory system in the modulation of arousal and affect seems indicated. Titration of this syndrome in carefully controlled environments is currently clarifying this position.
REFERENCES I. Boudreau, J. and W. Freeman. Spectral analysis of electrical activity in the prepyriform cortex of the cat. Expl Neurol. 8: 423--439, 1963. 2. Callens, M. Peripheral and Central Regulatory Mechanisms o f the Excitability in the Olfactory Systent Brussels: Presses Acad6miques Europ6ennes, 1967. 3. Douglas, R. J., R. L. Isaacson and R. L. Moss. Olfactory lesions, emotionality, and activity. Physiol. Behav. 4: 379-381, 1969. 4. Fuxe, K., T. HiSkfelt and U. Ungerstedt. Morphological and functional aspects of central monoamine neurons. Int. Rev. Neurobiol. 13: 93-126, 1970. 5. Heimer, L. Synaptic distribution of centripetal and centrifugal nerve fibres in the olfactory system of the rat. An experimental anatomical study. J. Anat. Lend. 103: 413--432, 1968. 6. Highstone, H. H. Anterior Olfactory Nucleus and Forebrain Evoked Potentials. Unpublished Masters's Thesis, University of California at Berkeley, 1969. 7. Leaf, R. C. Pharmacology, Limbic Regulation, and Cortical Function. In: Dntgs and Cerebral Function, edited by W. L. Smith. Springfield: Thomas, 1970, pp. 201-213. 8. Lehman, A. H. M. and G. M. Mentink. The lateral olfactory tract, the anterior commissure and the cells of the olfactory bulb. Brain Res. 12: 396-413, 1969. 9. Pellegrino, L. J. and A. J. Cushman.A Stereotaxic Atlas o f the Rat Brain, edited by R. M. Elliott, G. Lindzey, and K. MacCorquodale, New York: Meredith Pub. Co., 1967. 10. Pfaff, D. and E. Gregory. Olfactory coding in olfactory bulb and medial forebrain bundle of normal and castrated male rats. J. Neurophysiol. 34: 208-216, 1971. 11. Phillips, D. S. The effects of olfactory bulb ablation on visual discrimination. Physiol. Behav. 4: 379-381, 1969. 12. Phillips, D. S. and G. K. Martin. Effects of olfactory bulb ablation on heart rate. Physiol. Behav. 7: 535-537, 1971. 13. Riehman, C. L., R. Gulkin, R. and K. Knoblock. Effects of bulbectomization, strain, and gentling on emotionality and exploratory behavior in rats. Physiol. Behav. 8: 447-452, 1972.
14. Sheard, M. H., J. B. Appel and D. X. Freedman. The effect of central nervous system lesions on brain monoamines and behavior. J. psychiat. Res. 5: 237-242, 1967. 15. Sheard, M. H. and G. K. Aghajanian. Stimulation of midbrain raph~ neurons: Behavioral effects of serotonin release. Life Sci. 7: 19-25, 1968. 16. Sieck, M. H. The role of the olfactory system in avoidance learning and activity changes. Physiol. Behav. 8: 705-710, 1972. 17. Sieck, M. H. and B. L. Gordon. Selective olfactory bulb lesions: Reactivity changes and avoidance learning in rats. Physiol. Behav. 9: 545-552, 1972. 18. Smythies, J. R. Brain Mechanisms in Behavior, New York: Academic Press, 1970. 19. Thomas, G. T., G. Hostetter and D. J. Barker. Behavioral functions of the limbic system. In: Progress in Physiological Psychology, Vol. 2, edited by E. Stellar and J. M. Sprague. New York: Academic Press, 1968, pp. 230-311. 20. Ungerstedt, U. Adipsia and aphagia after 6-hydroxydopamine induced degeneration of the nigro-striatal dopamine system. Acta physiol, scand., suppl. 367: 95-122, 1971. 21. Walker, J. B., M. H. Sieck and J. P. Walker. The effects of selective olfactory bulb lesions on regional concentrations of biogenic amines in rat brain and some possible correlations with behavioral change. Pharmac. Biochem. Behav., in press. 22. Wenzel, B. M. and A. Salzman. Olfactory bulb ablation or nerve section and pigeons' behavior in non-olfactory learning. ExplNeuroi. 22: 62-69, 1968. 23. Wenzel, B. M., P. F. Albritton, A. Salzman and T. E. Oberjat. Behavioral changes in pigeons after olfactory nerve section or bulb ablation. In: Olfaction and Taste 111, edited by C. Pfaffmann, New York: Rockefeller Press, 1969, pp. 278-287. 24. White, L. E., Jr. Olfactory bulb projections of the rat. Anat. Rec. 152: 465-480, 1965. 25. Winer, B. J. Statistical Principles in Experimental Design. New York: McGraw-Hill, 1962.
Selective Olfactory System Lesions in Rats and Changes in Appetitive and Aversive Behavior MICHAEL H. SIECK
D e p a r t m e n t o f Psychology, University o f California, Riverside, California 9 2 5 0 2
(Received 23 June 1972) SIECK, M. H. Selective olfactory system lesions in rats and changes in appetitive and aversive behavior. PHYSIOL. BEHAV. 10(4) 731-739, 1973.-Male rats with bilateral lesions in different parts of the olfactory system differed on a variety of behavioral measures. All lesioned animals were hyperreactive when handled and learned shuttle box avoidance faster than sham operates. However, activity in an open field, Y-maze, and startle chamber separated the groups. Animals with lesions confined to the olfactory bulb tip (LB) were least active whereas those sustaining peduncle destruction (DB) were most active. Bar pressing for continuous reinforcement also differentiated the groups. The LBs became inactive initially while DBs learned fastest but were more resistant to extinction. Only DBs failed to learn a passive avoidance task whereas, in one replication, only SHAMs failed to extinguish this task once learned. Differential lesions thus produced an activity or reactivity gradient that was low in LBs, moderate in MBs (bulb removed), and highest in DBs. The arousing properties of the measures used also interacted with lesions to produce a family of responses. The results are discussed in terms of possible olfactoryqimbic interactions and evidence for a role of the olfactory system in modulations of arousal or affect is considered. Olfaction
Limbic system
Emotionality
Behavior control mechanisms
ness after bulb lesions or olfactory nerve sections. Phillips et al. [12] later presented similar evidence in rats as measured by heart rate changes in a startle test. None of these authors found lesion correlated changes on any measure. Other workers have also noted changes in emotionality and activity after bulb lesions without lesion dependence [3,13]. Richman et al. [13] found strain differences after lesions: albino and hooded rats ambulated more in an open field than sham operates, but only albinos decreased exploration of an object-filled area. T h e y reported no change in spontaneous alternation behavior 8 - 1 0 days after operations although Douglas et al. [3] found a transient decrease 5 days after surgery. Richman et al. attributed possible differences in the two studies to differential emotionality changes. The fact that none of these authors found lesion dependent effects may be due to one or a combination of four factors: (a) most of them were not expecting them and therefore did not systematically vary lesion sites in sufficient numbers of animals; (b) the tasks used to measure lesion effects were not sensitive to possible differences; (c) the lesions were more or less confined to one area only, e.g., the olfactory bulb proper; and (d) possible species, strain, or sex differences were involved. Previous work from this laboratory has explored the first three of these issues
PREVIOUS research from this laboratory demonstrated that selective olfactory system lesions in rats produced different behavioral effects [ 17]. All lesioned animals were hyperactive in an open field and learned shuttle box avoidance faster than sham operates. However, only animals with both olfactory bulb and peduncle destruction failed to learn a step down passive avoidance task. A startle test also suggested that minimally lesioned animals were less reactive than any group although too few data were available to approach this conclusively. In addition, lesion correlated changes in open field defecation pointed to possible emotional differences between minimally and deeply lesioned animals. The results thus far published [16,17] reveal I~hat selective olfactory system lesions produce a family of responses dependent on lesion site, type of task involved, and level of arousal as defined by the apparent intensity of the environmental situation. Animals may appear hyporeactive in one situation and hyperreactive in another. Phillips [11 ] and Wenzel et al. [22] reported performance changes in tasks involving visual discrimination after bulb lesions in both rats [11] and pigeons [22]. They discussed their results in terms of both hypo- and hyperreactivity although a later study by Wenzel et al. [23] presented clear evidence of increased autonomic responsive-
1Research supported by NIH Grant MH 15585. The services of Barry Gordon and Jeffrey Turner are gratefully acknowledged. 731
732
$1ECK
[16,17], and the present experiments are designed to confirm and extend those findings. EXPERIMENT 1 METHOD
Animals Animals were 30 naive male hooded rats from Simonsen Laboratories approximately three months old at the start of the experiments. They were individually caged in a temperature controlled room at 23°C on a 12 hr reversed cycle with the dark phase starting at 6:30 a.m. The animals were maintained on rat chow and water ad lib except during the operant conditioning phase. One group (N=5) received bilateral olfactory nerve sections and slight damage to the tips of the olfactory bulbs (LB); one group (N=5) received minimal to total bilateral bulbectomies (MB-B); one group (N=10) received deep bilateral bublectomies (DB) which removed the olfactory peduncle as well. A final group (N=10) received sham operations (SHAMs).
Surgical Procedure All animals were injected IP with atropine sulfate (0.4 mg/kg) followed in 15 min by sodium pentobarbital (50 mg/kg IP). The animals were placed in a stereotaxic apparatus, scalp incisions made, and the periosteum on the skull was reflected. The skull overlying the olfactory bulbs was drilled down to the dura in all animals and the dura was broken with a hook for all lesioned animals. All operations were carried out under direct visual guidance. For the LBs, lesions were effected by passing a specially designed curved cutting tool around the anterior tip and ventral surface of bulbs. Minimal damage to the outer layers generally occurred from this procedure. All bulbectomies were made by aspiration; tissue anterior to the frontal pole was removed in the MB-Bs while DBs were made by removing additional tissue underlying the frontal region (see Fig. 1A). Following surgery, sterile gelfoam was gently packed in the opened skull to effect hemostasis. Terramycin powder was sprinkled into the incision, and the skin flaps were sutured closed. The latter two steps were also part of the sham operations. All animals received bicillin (0.2 cc IM) and were returned to their home cages.
Histology The brains from five animals in each group were analyzed for regional concentrations of norepinephrine, serotonin, and dopamine [21]. These animals were killed by decapitation. The entire brain, including the olfactory bulbs, was removed and dropped into ice cold saline for a few seconds. Histological verification was done blind. Two experimenters who had no prior knowledge of the type of lesion the animals had sustained visually determined the extent of any lesion present and recorded it on a scale drawing of the rat brain from lateral, dorsal, and ventral views. The five DBs not used in the biochemical analysis were anesthetized with sodium pentobarbital, perfused intracardially with 0.9% saline followed by 10% formalin and decapitated. Most of the fascia and posterior skull were removed immediately and the remaining skull and brain remained for one week in 10% buffered formalin before completing skull removal. The amount of olfactory bulb
removal was measured as above and by comparison of cresyl violet stained sections with an anterior extension of the Pellegrino and Cushman Rat Atlas made in this laboratory [9,17]. Degeneration beyond any lesion was not followed. The LB lesions severed the olfactory nerves and typically damaged the most anterior tip of the olfactory bulbs. Other work has shown that such lesions destroy or damage the glomerular and external plexiform layers of the bulb tip and ventral surface near the tip (unpublished observations). The MB-B lesions removed all bulbar tissue in a range extending back between AP 15.1 and 13.0. Bulb removal in the DBs was complete and ranged back to AP 11.8 in the least and 10.4 in the greatest lesions. No frontal damage occurred in any group. The use of the present terminology standardizes results from this laboratory. Although variability in the AnteriorPosterior plane is high at the tip of the brain, approximate A - P measurements can define the four lesion groups now reported. However, really adequate measurement can only be made histologically. Combining these two approaches, the lesions are as follows: LB. Complete olfactory nerve section at bulb surface. Variable destruction of glomerular and external plexiform layers at bulb tip and ventral surface near the tip. Lesioned area at approximately AP 18.3-18.0. MB. Bulb ablation destroying all main bulbar layers back to AP 15.9-13.7. The range indicated typically leaves all or part of the accessory olfactory bulb intact. B. Complete bulbectomy destroying main and accessory bulbs and all of internal granular layer. Some damage to the ill-defined tip of the anterior olfactory nucleus usually occurs. AP 13.7-13.0. DB. Total bulb and olfactory peduncle removal. Lesion encompasses all or most of the anterior olfactory nucleus and occasionally reaches the olfactory tubercle. Ventral frontal pole damage occurs in a small percentage of the animals. AP 1 1.8-10.4. Figure IA indicates the extent of the lesions in the present study and Figure I B indicates the structures involved. The MB-B group of the present experiment encompassed the range of the MB and B categories.
Apparatus Five different apparatuses were used to assess the lesion effects, The first was a white wood Y-maze consisting of three identical 3ftx9in.x9in. arms set at angles of t20 °. Each arm had a guillotine door 0 in. from the e n d that could be opened or dropped to control the ammals' movement. Several appetitive measures were made in BRS Foringer Skinner boxes programmed by solid state modules. Thirty-five mg Noyes pellets were used as reinforcement in these tasks. A stepdown passive avoidance apparatus, modified from a design previously used [16], consisted of a 3x3x3 ft dark Plexiglas box with a 4x4 in. platform raised 3 in. above the grid floor that could be electrified (150 V A.C. across 200 kP,, 0.68 ma) through a scrambler set to operate for 3 sec when an animal stepped down. The modification used m this experiment consisted of two amyl acetate saturated wicks at the top of the box and a pair of exhaust fans arranged to pull air over the wicks and grid floor and out the bottom of the box. Air was exhausted outside the building. This modification was made in order to minimize
SELECTIVE O L F A C T O R Y LESIONS
733
dropping a free swinging rod attached to one side of the box from a constant height to a platform on the bottom. Procedures
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FIG. 1. (A) Scale drawings of rat brain in mid-sagittal ialane showing lesion extent of the three surgical groups. Lightly shaded areas indicate least and darkly shaded areas greatest lesion extent. Locations of some major olfactory structures are indicated. For more complete anatomical discussion see Histology section and Fig. lB. (B) A transparent view of the anterior forebrain of the rat in midsagittal section. Outlines of major olfactory nuclei and tracts are drawn to scale. The lightly shaded area represents approximate origins and visible course of the lateral olfactory tract (LOT). The darkly shaded area is the approximate location of cortical LOT terminations and some secondary olfactory centers. Included in it are parts of the olfactory tubercle (TUO), prepiriform cortex, periamygdaloid and piriform cortices (PIR). AOB, accessory olfactory bulb; GLOM, glomerular layer; EPL, external plexfform layer; MIT, mitral layer; GRAN, granular layer; OAE, anterior olfactory nucleus pars externa; LOT, lateral olfactory tract; TUO, olfactory tubercle; PIR, piriform cortex; LPA, lateral paraolfactory area (nucleus accumbens, part of anterior olfactory nucleus); AC, anterior commissure; CC, corpus callosum; OAN, anterior olfactory nucleus (anterior end fuses with granular layer). Terminology adopted in part from PeUegrino and Cushman [9]. possible olfactory cues generated by the animals. In addition, the raised platform was wiped clean with a dilute water solution of amyl acetate after each animal was run. The active avoidance apparatus has been described previously [16] and consisted of a pair of dark Plexiglas enclosed boxes, 9 x 9 x 9 in. separated by a 3 x 3 in. closed alley. Raising two guillotine doors on either side of the alley started a buzzer and a clock followed 10 sec later by shock (80 V A.C. across 220 ks2, 0.36 ma). Shock was switched from one side to the other on alternate trials. The startle chamber previously described was also used [17]. It was a spring mounted 1 0 x l 0 x l 0 in. plastic box with a hinged front door and side ventilation holes. It was placed in a sealed chamber under controlled atmospheric and light conditions. A magnet attached to the under side of the box passed through a coil of wire to form a sensitive solenoid arrangement which transduced any movement into an electrical signal. This was amplified and recorded by a Beckman Type R Dynograph. Fixed amplification was used for all animals. Presession baseline calibrations of the instrument were made before each animal was tested by
A battery of six tests was run on each animal starting on postoperative Day 7. All testing was done in a quiet dimly red-lighted room. On postoperative Days 7 though 10, each animal was handled for 5 min per day. Difficulty in removal from the home cage, amount of escape behavior during handling, and vocalization were subjectively rated. On postoperative Days 11 through 16, animals were given two exploration trials daily in the Y-maze. One arm was designated the start box and all trials were started there. On Trial 1, the rat was placed in the start box for 20 sec and then the door was opened. Time taken to reach the center of the maze was recorded as was the first direction (right or left) taken from there. When an animal reached either goal box, the door was lowered and a 20-sec exploration period followed. The rat was then manually returned to the start box. Trial 2 was identical except that the animals were returned to their home cages afterward. The maze was cleaned with a dilute water solution of amyl acetate after each trial. Animals not reaching either goal box 3 min after starting a trial were picked up and returned to the start box or to their home cages depending upon the trial number. A bolus count was taken after each trial. On postoperative Day 17, the animals were divided into two groups consisting of approximately equal Ns from each surgical group. Members of one group were put on a food deprivation schedule that brought their weight down to 85% of ad lib feeding weight in five days. On the sixth day of deprivation they were started on a program of bar pressing for continuous reinforcement with three phases: ( 1) two days of magazine training in which 40 pellets were automatically delivered on a VI-45 sec schedule regardless of the rats' behavior; both bars were removed during this time; [2] two days of continuous reinforcement (CRF) training in which a response on either of the two bars delivered a pellet; sessions were automatically terminated after 60 pellets had been delivered and animals that were not pressing spontaneously were hand-shaped; [3] one day of extinction training on which 5 pellets were delivered on the CRF schedule and then reinforcement was discontinued. For CRF, latency of the first response each day and total time to obtain 60 pellets were recorded. For extinction, the number of responses and time to reach a criterion of no responses in 3 min were recorded. Animals failing to reach the extinction criterion after 60 min were returned to their home cages. After extinction, food and water ad lib were restored for six days and all animals were then run on the passive avoidance task. On the seventh day of ad lib food and water, three 5-min exploration periods 60 min apart were provided in the passive avoidance apparatus. On the next day, the rats were placed on the platform individually and were then shocked for 3 sec after they put three feet'on the grid. Time to step off the platform was measured. Three such trials were run each day until a criterion of 5 min without stepping down was met or 18 trials (6 days) had elapsed. Extinction training began on the day following criterion performance. All conditions were the same except that no shock was delivered. The number of trials to first step down within 5 min was recorded. Animals that did not reach the acquisition criterion after 6 days or failed to extinguish after 18
S I E(" K
734 trials t6 days) were started on the next task. Active avoidance training began the day following passive avoidance extinction or failure. It consisted of a two-way shuttle-box problem in which a buzzer started 10 sec prior to onset of shock to the grid floor. Guillotine doors opened to start each trial and also started a clock which stopped when the animal crossed to the opposite box. Crossing terminated the buzzer and the shock. Twenty trials per day were run until a criterion was reached of eight avoidances in any successive 10 trials or 180 trials (9 days) had elapsed. Nine days following active avoidance acquisition, the rats were tested for startle. Each session began with a 5-min exploration period of the enclosure before an 8JA-min testing period began with 6 trials per min. Activity levels were recorded throughout this time on the Dynograph. Startle responses were produced by a loud bell which rang for 2 sec in every lO-sec interval. Fifty trials per day were run for five consecutive days. The box was cleaned with water after each animal was returned to its home cage. As previously described [ 17] startle activity was quantified in each second by counting equal arbitrary units o f amplitude from the baseline. For tabulation purposes, amplitudes greater than 2.0 units per sec during the stimulus interval were assigned a count of 1, and activity greater than 0.5 unit per sec during the IT1 was assigned 1 count. Thus, during the 10-sec interval of one trial, a maximum of 2 for the stimulus period and 8 for the ITI was possible. Because of the great volume of data obtained, counts were omitted for Trials 11 through 15, 21 through 25,'31 through 35, and 4 l through 45. This produced a cumulative total for 30 trials per animal per day. Stimulus and ITI activity scores were treated separately This concluded the test battery. The second group of animals was run simultaneously on the same measures except that the avoidance tasks were given first followed by the CRF schedule. Eleven days elapsed from the end of CRF extinction to the first day of startle testing during which time food and water were available ad lib. All animals were left undisturbed in their cages until sacrifice 7 to 14 days after completion of all testing.
habits were noted in one animal from each group. However. time to choice point and number of failures to reach it in 180 sec clearly separated the groups. DBs and MB-Bs reached the choice point faster than SHAMs or LBs and were less likely to freeze in the start box. The LBs were somewhat faster than SHAMs and approached the speed of the most deeply lesioned groups by Day 5 (see Fig. 2). A ×= analysis using a running-time cut-off of less than 8 sec for positive criterion and 8 sec or over for negative criterion produced a x 2 of 67.40 (p<0.0001). The LB and SHAM scores were more likely to be over 8 sec than MB-Bs and DBs. The MB-Bs and DBs did not differ, but LBs were more likely than SHAMs to meet the positive criterion (×~- = 3.91, p< 0.05). Running times varied with session. Speed of SHAMs peaked on Day 3 and declined steadily after that. More failure to move and generally slower movement occurred on Days 5 and 6 presumably reflecting habituation (Fig. 2). Conversely, LBs gradually increased running speed and approximately equalled MB-Bs and DBs on Days 3, 5, and 6. The DBs reached peak speed by Day 2 and MB-Bs by Day 3. Neither group showed any sign of habituation by Day 6. One LB had consistently longer running times than the others and accounted for most of the high scores for this group. Figure 2 shows two LB curves-one including and one omitting this animal's scores.
A SHAM
\~
LB
~
MB-B
,,, LB ;
~t
3O
RESULTS No order effect was observed between Group 1 and Group 2 animals on any test. They were therefore pooled and treated together.
i //
k
/ / / P SHAM
•,
7
DB
o O 2O
Handling, Vocalization All lesioned animals tended to be hyperreactive when handled. They were also more difficult to remove from their home cages t h a n SHAMs. Many were aggressive and would attack the experimenter when approached. The condition persisted with only slight diminution throughout the experiment. The most aggressive animals were invariably members of the DB group. Vocalization followed the same pattern, viz., the most aggressive animals vocalized most. However, as previously found, wide variability existed in all groups. Some animals in every group were indistinguishable from SHAMs.
Y-Maze Performance All groups alternated above chance level ( 6 0 - 7 0 % ) and there were no differences among them. Slight position
0
i
i
i
~
i
i
t
2
3
4
5
6
DAYS
FiG. 2. Mean time to choice point in the Y-maze on successive days in Experiment l. All trials where the choice point was not reached before the 3 min cutoff were omitted. SHAM running time Iheavy line,~ shows Iiabituation trend not evident in lesioned groups. One LB was much slower than all others. Curves including (dotted line) and omitting (solid line) this animal's scores are shown (see textL
SELECTIVE OLFACTORY LESIONS Tabulation of the failures to reach the choice point in 180 see revealed that S = LB > MB-B > DB. Fifteen percent of all SHAM (18/120) and LB (9/60) trials ended in failure, 8.25% of MB-B (5/60) trials and only 1.67% of DB (2/120) trials were failures. No defecation differences were found (Six day means: SHAM = 1.8, LB = 1.7, MB-B = 1.I, DB = 1.2; Surgical F = 1.51, d f = 3/26, p < 0 . 2 5 ; Days F = 0.60, p > 0 . 7 5 ; Interaction F = 0.98, p<0.75).
Passive Avoidance One SHAM was accidentally omitted from this test. The data followed the same trend previously found [16,17]: 1 LB, 1 MB-B, 1 SHAM, and 4 DBs failed to reach the acquisition criterion. Since only DBs failed to reach the criterion in previous work and since scores for all groups were similar here, they were pooled and compared to the DB population. There was no significance in failures to criterion (p = 0.16,-Fisher exact test). Analysis of variance for trials to criterion computed on those animals reaching criterion revealed no differences in trials required (F = 0.670, d f = 3/18, p<0.50). As previously found, DBs that failed to reach acquisition criterion typically developed stereotyped jumping behavior as soon as they were put on the platform. In extinction, 0 LBs, 1 MB-B, 0 DBs, and 4 SHAMs failed to reach the criterion by the 6th day. Pooling the lesioned groups revealed this to be a significant effect (p = 0.04, Fisher exact test). SHAMs were less likely to step down than lesioned animals. Analysis of variance for trials to extinction criterion computed on those animals reaching that criterion again revealed no differences (F = 0.569, dr= 3/13, p<0.50).
Active Avoidance All lesioned animals reached acquisition criterion in approximately the same number of trails (LB = 24.80; MB-B = 26.00: DB = 29.50). SHAMs required approximately twice this number (41.03). However, because of the small Ns involved, the analysis of variance was not significant (F = 2.88, dr= 3/24, p<0.10). Since all lesioned animals had similar means, and since previous data indicated no differential lesion effect on this measure [16,17], the lesioned animals were pooled and compared to the SHAM operates as one group. SHAMs took significantly longer than all lesioned animals combined (F = 8.48, d f = 1/24, p<0.01). In addition, one SHAM and no lesioned animals failed to reach the acquisition criterion.
Bar Pressing 1. Magazine training: All animals learned to eat from the hopper on the VI-45 sec schedule. No consistent differences were noted. 2. Acquisition: Three LBs and none of the MB-Bs, DBs, or SHAMs required hand shaping (Fisher exact test, p = 0.004). Further, one of the LBs failed hand shaping on both days and was not continued. These animals typically developed freezing behavior in one corner of the cage during the session. Mean scores are shown in Table I. Analysis of variance for time taken to first bar press revealed differences between groups (F = 6.328, dr= 3/23, p<0.O03). Day 1 scores did not differ from Day 2 scores (F = 1.059, df = 1/23, p > 0 . 5 0 ) and there was no interaction effect (F = 0.247, dr= 1/23, p>0.25). A Scheff~ analysis of mean differences showed that DBs pressed sooner than
735 other groups; a trend indicating DB < MB-B < LB < S was also observed (DB < LB, p<0.09, DB < SHAM, p<0.02; level has been set at 0.10 on this highly conservative measure to compensate for loss in power [25]). Detailed results appear in Table 1. Session time for 60 reinforcements did not differ among groups'(F -- 2.376, dr-- 3/23, p<0.09). However, a clear day effect occurred; Session I took significantly longer than Session 2 (F = 25.92, df = 1/23, p < 0 . 0 0 0 l ) . There was no interaction effect (F = 0.872, df = 3/23, p>0.50). The trend in the time required to obtain 60 reinforcements nearly paralleled that for seconds to first bar press, viz., DB = MB-B < S < LB. 3. Extinction. Number of responses to criterion showed significant surgical effects (F = 5.91, d f = 3/23, p< 0.004). The DBs made the most responses. A Scheff~ comparison of means showed that the MB-B < DB, p = 0.09, and S < DB,p = 0.006. The LBs and MB-Bs did not differ. Visual observation of the animals in extinction revealed that many DBs became very excited when reinforcement was cut off. They attacked the bars vigorously and often ran franctically around the chamber. No other animals did this. TABLE1 MEAN VALUES FOR CONTINUOUS REINFORCEMENT (CRF) ACQUISITION AND EXTINCTION. EXTINCTION DATA ARE MEAN NUMBERS OF BAR PRESSES AND MEAN TIME TO REACH A CRITERION OF NO RESPONSES IN 3 MIN
Sec to First Bar Press
Min for 60 Reinforcements
Group
N
Day 1
Day 2
Day 1
Day 2
LB MB-B DB SHAM
4 5 10 10
69.75 24.50 27.80 94.89
73.50 38.50 25.85 45.22
29.00 13.80 14.60 17.56
14.00 8.00 8.30 8.11
Extinction Bar Presses Time 110.50 91.80 175.90 76.00
30.50 17.20 33.40 20.33
Startle A ctiviO, Because of equipment malfunction, Day 3 responses were incorrectly recorded and have been omitted from the analysis. Activity levels are given for Days I - 2 and 4 - 5 . The ITI activity and responses during the stimulus were treated separately. Analysis of variance for stimulus-induced activity revealed no significant group differences, (F = 2.22, d f = 3/19, p<0.14), no day effects (F = 1.757, dr= 3/67, p > 0 . 2 5 ) and no interaction between surgery and days (F = 1.701, df = 9/67, p<0.11). Analysis of Fig. 3 reveals that SHAMs, MB-Bs and DBs had similar scores which decreased from Day 1 to a relatively stable level for the remaining days. However, the LBs started at a much lower level and gradually increased over days until they equaled the other groups. A related trend was previously reported for less deeply lesioned animals [17] and is evident in the poor LB performance in the CRF and Y-maze tests in the present experiment. A post hoc analysis of variance for Day 1 demonstrated that LBs were less active than all other groups (F = 5.55, dr= 3/19, p<0.005). A Scheff~ analysis of mean differences showed that LBs < DBs (p<0.006), LBs < MB-Bs (p<0.05). and LBs < SHAMs (p<0.05). There were
736
SIECK TABLE ~
I001 ~
oB
80!
SHAM L8
MB-B
MS-B
M E A N A C T I V I T Y S C O R E S IN T H E S T A R T L E C}tAMBER DURING AND BETWEEN STIMULUS PERIODS FOR DAY l A L O N E A N D F O R D A Y S 1. 2, 4 A N D 5 C O M B I N E D . M E A N N U M B E R O F B O L U S E S F O R A L L S E S S I O N S IS A L S O S H O W N
Stimulus
<
LB
0i
ITI
Boluses
Group
N
4 Days
Day t
4 Days
Day' l
LB MB-B DB SHAM
5 5 10 10
18.10 28.63 27.42 26.97
12.20 31.60 34.70 30.t3
46.85 81.47 85,03 61.09
14.80 75.00 101.50 39.38
5.00 5.44 5.28 3.88
DB MB'-B
~3C
SHAM
MB~ SHAM
DB Z <
LB
D O 15 LB
bred at this insitution from Simonsen Laboratories' hooded rats, They were approximately three months old at the start of the experiments and were individually caged in a temperature controlled room at 23°C on a 12 hr reversed cycle with the dark phase starting at 6:30 a.m. The animals were maintained on rat chow and water ad lib throughout the experiment. Surgical procedures duplicated those in Experiment 1. Three groups of 17 each consisted of LBs, DBs, and SHAMs.
Histology DAYS FIG. 3. Mean startle activity during the bell stimulus (lower graph) and ITI (upper graph) for Days 1-2 and 4-5. Each point represents the group average of 30 responses/day/animal or a total of 150 scores/pt for LB and MB-Bgroups and 300 scores/pt for SHAM and DB groups. Amplitudes are in arbitrary units (see text). no differences between SHAMs, MB-Bs, and DBs (See Table
2).
For ITI activity, overall surgical F = 3.06, d r = 3/19, p<0.05. An habituation effect over days was also observed (F = 4.90, d f = 3/67, p<0.004, see Fig. 3). Scheff6 analysis revealed that LBs were less active than DBs (p<0.05), and that Day 2 activity was lower than Day 5 activity (p<0.01). Day 1 scores again separated the groups markedly (Fig. 3). A post hoc analysis of variance for Day 1 was significant (F = 4.31, df = 3 / 19, p< 0.01 ). The Scheff6 tests revealed that LBs were less active than DBs (p<0.03) and SHAMs < DBs (p<0.09) (Table 2). There were no differences in bolus counts across days although all lesioned animals combined defecated more than SHAMs (F = 3.71, dr= 1/13, p < 0.05). EXPERIMENT 2 METHOD
In order to corroborate the results of Experiment l, several of the tests were rerun with larger Ns. In addition possible lesion induced activity differences were further explored in an open field setting.
Animals Animals were 51 naive male rats from an F, generation
Histological procedures duplicated those of Experiment 1 for the DBs not used in the bioamine assay, The lesions were essentially identical. The LBs were deafferented and sustained some damage to the bulb tips; DBs had complete bulb and peduncle removal to between AP 1 114 and 10.4. No frontal pole damage occurred.
Apparatus A white wood open field 4x4xl ft. high with i 6 l x l ft. squares marked on the floor was used to assess activity levels [18]. The Y-maze, step down passive avoidance and shuttle box avoidance apparatuses previously described were also used.
Procedure All testing was done in a quiet dimly red-lighted room. On postoperative Days 7 through 10 each animal was handled for 5 min/day. Difficulty in removal from the home cage, amount of escape behavior during handling and vocalization were subjectively rated. On postoperative Days 11 and 12, animals were given two 5 min exploration trials daily in the open field. Each rat was started in the same corner facing the center of the field and a count of 1 was made each time more than half of the rat's body crossed a line. The number of boluses dropped were counted after each trial and the maze was cleaned with a dilute water solution of amyl acetate before each trial began. On postoperative Days 13 through 18, animals were given two exploration trials daily in the Y-maze. The procedure duplicated that of Experiment I. On postoperative Day 19, the animals were started on the passive avoidance task which was followed immediately by the
SELECTIVE OLFACTORY LESIONS
737
shuttle box problem. Procedures described in Experiment 1 were followed. RESULTS Handling and Vocalization Members of the lesioned groups were the most difficult to handle and vocalized more. The DBs were harder to handle than LBs. However, some animals from each lesioned group were indistinguishable from SHAMs. Open Field Analysis of variance for number of lines crossed revealed differences between groups (F = 33.90, d f = 2/48, p<0.0001). There was no day (F = 1.49, d f = 1.48, p > 0 . 2 5 ) or interaction (F = 1.71, dr= 2/48, p > 0 . 2 5 ) effect. Scheff~ analysis of mean differences revealed that SHAMs were less active than both LBs (F - 3.80, p<0.03) and DBs (F = 32.79, p<0.O001) and that LBs were less active than DBs (F = 14.27, p<0.0001). Analysis of variance for boluses dropped showed a significant surgical effect (F = 5.86, d f = 2/48, p
N SHAM LB DB
(17) (17) (17)
Activity Day 1 Day 2
Boluses Day 1 Day 2
33.35 48.77 75.53
2.88 4.88 6.35
31.24 49.82 88.94
1.88 4.12 3.29
Y-Maze Performance All groups alternated above chance level ( 6 5 - 7 0 % ) and there were no differences among them. Analysis of variance for time to reach the choice point revealed significant surgical (F = 12.43, d f = 2/48, p<0.001 ), days (F = 3.57, d f = 5/240, p<0.O05), and interaction (F = 2.92, d f = 10/240, p
However, the DB failures occurred primarily on Days 1 and 2, the LB failures were evenly spread across days, and the SHAM failures were greater on the last 4 days. This reflects the differential habituation already discussed and is evident in comparison of Fig. 4A and 4B. Figure 4A shows the mean time to the choice point omitting the failures while 4B shows the mean time computed on all animals with 3 min scores given to all failures; 3.4% of the DB trials (7/204), 17.7% of the LB trials (36/204) and 31.1% of the SHAM trials (64/204) ended in failures. (Overall x a = 47.71, p < 0 . 0 0 0 1 ; S > LB, x ~ = 6.32, p < 0 . 0 2 5 ; LB > DB, x 2 = 17.77, p<0.001). B 120 .1OO
8,o O ~, 2 0
DAYS
FIG. 4. A. Mean time to the choice point in the Y-maze on successive days in Experiment 2. All trials in which the choice point was not actually reached were omitted. B. Same as above for all trials. Trials ending in failure to reach the choice point were given scores of 3 min each (see text). Analysis of variance for boluses dropped showed no surgical effect (mean boluses/day: SHAM = 0.86, LB = 1.31, and DB = 0.94; F = 1.84, dr= 2/48, p < 0 . 2 5 ) but did show a days effect (F = 7.74, d r = 5/240, p<0.0001). No interaction occurred (F = 1.27, d f = 10/240, p<0.25). Scheff6 analysis revealed that defecation rates were higher on the first three days of testing (Day 1 > Day 6, p < 0 . 0 0 2 ; Day 3 > Day 6, p<0.O001 ; Day 3 > Day 5, p<0.01). Passive Avoidance As previously found [17], more DBs failed to reach the acquisition criterion than any other group. Three SHAMs, 4 LBs, and 12 DBs failed (x ~ = 12.33, p<0.005). Analysis of variance for trials to criterion computed on those animals reaching criterion revealed no differences. (F = 0.48, d f = 2/29, p<0.50). In extinction 3 SHAMs, 1 LB, and no DB failed to reach that criterion. Analysis of variance for trials to extinction criterion was also insignificant (F = 0.69, d f = 2/25, p<0.50). Active Avoidance SHAMs took significantly more trials to reach criterion than lesioned animals. The LBs and DBs did not differ. (Means: SHAM = 45.72, LB'= 28.30, DB = 24.61 ; F = 6.23, d f = 2/47, p<0.O05). In addition, 2 SHAMs, 1 LB and O DBs failed to reach the criterion. DISCUSSION These experiments confirm and extend previous findings
738 [16,17] indicating that selective olfactory system lesions produce a family of behavior patterns involving at least lesion site, experimental setting, and the arousal state of the animal. Since all the data from this laboratory have been obtained from male hooded rats, the possibility that sex, strain, or species differences play roles not yet accounted for must be considered. It is evident that olfactory system lesions increase emotionality or reactivity in every species or strain thus far studied. Wenzel et al. found evidence for this after both nerve section and bulb ablation in pigeons [23] and others reported similar findings in both hooded [13] and albino rats [3,13]. However, some evidence suggests that female rats are more reactive after bulb ablations than males [3,11] and that male albinos are less reactive than male hoods after lesions [ 13 ]. Since no really systematic study of male-female differences has yet been published nor have variable lesion studies been made on any other strain or species, the generality of the present findings await further work. Nevertheless, some constant behavior changes emerge after selective lesion studies that correlate well with data from other laboratories. • All lesioned animals are more difficult to handle and remove from their home cages than SHAMs. Some lesion dependence occurs since DBs are the most difficult to work with. Also, all lesioned animals are more active than SHAMs in the open field although LBs ambulate less than other lesioned groups. These findings confirm other work demonstrating increased emotionality after bulb lesions and further suggest a lesion dependent emotionality continum. Emotionality in rats is often estimated by bolus counts and/or freezing behavior. Richman et al. [13] found increased defecation in the open field in their bulbectomized animals from a population that appears to encompass the MB and B categories of the present study. Sieck and Gordon found increased open field defecation in MBs but not DBs [17] although the present experiments did not confirm this. No bolus differences between lesioned groups were found in the Y-maze, open field, or startle tests of the present study, but all lesions increased defecation over SHAM levels in the startle test and open field. Thus, most of the defecation data support the hypothesis that any olfactory system lesion increases emotionality over SHAM levels. However, a lesion dependent defecation difference may still be present since correlations of 0.7719 (p<0.0001, Experiment 1) and 0.2730 (p<0.05. Experiment 2) were obtained between the number of boluses dropped and time to reach the choice point in the Y-maze. Animals running slowe'r or freezing more defecated most. Since LBs ran slower than other lesioned groups and had defecation means higher than all other lesioned animals, slight defecation differences may be present. Increased reactivity after any lesion may also be reflected in the lower shuttle box acquisition scores and more rapid passive avoidance extinction for all groups. However. some lesion dependence is again indicated since Bs and DBs were less likely to reach the passive avoidance acquisition criterion in this and previous work [ 17]. With the addition of the bulbar deafferented group (LB) in the present study, a continuum of central offactory system damage from minimal to essentially complete has been attained. An earlier report suggested that lesion dependent reactivity changes take two forms. In minimally bulbectomized animals, responsiveness is more internalized and is manifest as freezing and increased defecation; in more deeply bulbectomized subjects, responsiveness ap-
SIECK pears as increased motor activity [ 17 ]. This suggestion may now be extended. No consistent lesion dependent defecation differences occur but activity differences are clear. The LBs were less action oriented in several tasks. They were least active of all lesioned groups in the open field and on the first day in the startle apparatus they froze more than any other group. In the bar pressing task, three of the LBs and no other animals required hand shaping because they became inactive very quickly. They were also slower to reach the choice point and more likely to freeze in the Y-maze than either MB-Bs or DBs although they were more active than SHAMs - especially on the last test days. Likewise, their increased stimulus and ITI activity by Days 4 and 5 in the startle apparatus showed that their initially low responsiveness disappeared with experience. Together these results suggest that mildly aversive or changing situations increase the probability of inactivity in this group but that familiarity with the test situation decreases this to unmask motor hyperactivity. The MB-B group was a mixed lesion population in terms of the initial work [17]. This group was more active than the LBs in the startle apparatus, faster in the Y-maze, and quicker to bar press for continuous reinforcement. From other work [ 17] they were also as active as DBs in the open field. Although thus similar to the DBs in several tests, MB-Bs were unlike them in that fewer passive avoidance acquisition failures occurred and fewer bar presses and less agitated responsiveness appeared in CRF extinction. They also failed more in the Y-maze and reached their top Y-maze running speed one day later (Fig. 2). Thus the MB-B group was more action oriented than the LB group but somewhat less so than the DBs. The DBs were most responsive of all. Their initial startle and ITI activity was highest, they were fastest in the Y-maze and did not habituate over days. They pressed first in the bar pressing task and were more resistant to extinction. They failed to attain the passive avoidance criterion and were the most difficult to work with since many of them tended to attack the experimenter or attempt.to escape when handled. The lesion effects thus present a continuum of responsiveness that is partly situation dependent. There is an underlying hyperreactivity in all groups which appears clearly in strongly aversive situations and a variable degree of hyporeactivity in mildly arousing situations. This hyporeactive aspect is strongest in LBs and weakest in DBs. Possible CNS Mechanisms: S o m e Speculation
Lesion dependent changes in brain norepinephrine and serotonin were found in the animals of Experiment t [21]. Since these amines have been implicated in mechanisms of arousal and affect [4, 7, 20], it is tempting to link these with the behavioral results. For instance, a midbrain based serotoninergic system has been implicated in habituation to stimuli [14,15]. Reductions in the activity of this system correlate with decreased habituation. The greatest decrease in brainstem serotonin occurred in the DBs and their lack of habituation in the Y-maze, poor extinction performance in CRF, and genel~al hyperreactivity thus correlate. Furthermore, much increased brainstem norepinephrine in the DBs might also be associated with their increased reactivity. In the LBs, less striking changes in brainstem serotonin and norepinephrine perhaps correlate with their increased
SELECTIVE OLFACTORY LESIONS
739
freezing or internalized reactivity. There are many problems in such interpretations but the data point to a relationship between bioamine levels and olfactory system lesions and provide indirect evidence for olfactory system influences on arousal and affect [21 ]. From another approach, the increased behavioral effects observed as lesions were made deeper and the increased bioamine changes accompanying them suggest a mass effect after olfactory system destruction in the sense of Lashley's hypothesis on mass action. However, olfactory input was decreased in tlae LB group, the MB-B lesions ablated the olfactory bulbs, and the DB lesion added at least the anterior olfactory nucleus (AON). Since at least part of the AON projects to areas different from those receiving efferents from the bulbs proper [5, 8, 24], it is possible that differential modulation of more central structures occurs. There is also anatomical evidence demonstrating
that deep olfactory system lesions lead to greater degeneration in basal cerebrum than shallow qesions [5,24] and electrophysiological work suggesting that deeper lying olfactory structures are important in controlling the excitability of some anterior limbic areas [2,6]. Opposing this view are anatomical and electrophysiological data demonstrating relatively diffuse innervation of olfactory projection areas by bulbar elements [1, 5, 8, 24]. Until more evidence is available this question will remain unsettled. I n summary, behavioral, anatomical, and physiological evidence link selective olfactory system lesions with changes in presumed limbic and midbrain functions [18,19]. More broadly, a role of the olfactory system in the modulation of arousal and affect seems indicated. Titration of this syndrome in carefully controlled environments is currently clarifying this position.
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