Centrally and peripherally induced anosmia: Influences on maternal behavior in lactating female rats

Physiology and Behavior, Vol. 14, pp. 439-447. Brain Research Publications Inc., 1975. Printed in the U.S.A.

Centrally and Peripherally Induced Anosmia: Influences on Maternal Behavior in Lactating Female

Rats I

IRWIN BENUCK 2 AND F R A N K A. ROWE

Department o f Psychology, Illinois Institute o f Technology, Chicago, IL 60616

(Received 31 October 1974)

BENUCK, I. AND F. A. ROWE. Centrally and peripherally induced anosmia: influences on maternal behavior in lactating female rats. PHYSIOL. BEHAV. 14(4) 439-447, 1975. - Bilateral removal of the olfactory bulbs was found to produce deficits in several components of the maternal behavior of lactating, primiparous female rats. More dead pups were found in the cages of bilaterally bulbectomized females shortly after parturition than in the cages of unilaterally bulbectomized, sham operated, or unoperated females. Futhermore, bilaterally bulbe¢tomized females showed deficits in pup retrieval relative to females in the other surgical conditions, and pups reared by bilaterally bulbectomized females weighed less at weaning than pups reared by females in the other surgical conditions. In contrast to bilateral bulbecomy, zinc sulphate treatments had no influence on pup mortality at parturition. However, zinc sulphate treated females did exhibit deficits in pup retrieval relative to saline treated females. Maternal behavior

Olfactory bulbs

Zinc sulphate

MATERNAL behavior in female rats appears to be under the influence of a number of variables. Internally, a triad of hormones consisting of estrogen, progesterone, and prolactin facilitates the display of maternal behavior b y female rats [ 11 ]. In addition, limbic system structures appear to be important for the sequential coordination of the individual behavioral elements comprising maternal behavior [15,16]. Externally, a variety of environmental stimuli might be important determinants of the maternal responsiveness of female rats. In this context, there has been particular interest in the role played by olfactory stimuli. Most efforts to assess the importance of olfaction for maternal behavior have involved rendering female rats anosmic by destroying their olfactory bulbs. The results of these experiments indicate that the degree to which olfactory bulbectomy influences maternal behavior is largely determined b y the physiological state of the females (cycling vs. lactating), and whether or not the females have had experince with pups prior to surgery. Thus, bulbectomy-induced cannabalism of pups was reported to occur more often in cycling virgin females that were not exposed to pups preoperatively than in virgin females that had preoperative exposure to pups, or in lactating females [6,13 ]. In the case of lactating females, and cycling females that had preoperative maternal experi-

Anosmia

ence (either as virgins or as a consequence of an earlier pregnancy), bilateral bulbectomy was reported to have little or no adverse influence on components of maternal behavior such as pup retrieval or time spent on nests in a nursing posture [2, 6, 13, 17]. In fact, in some instances, the display of maternal behavior was facilitated by bulbectomy [6 ]. In most investigations of the influence of bulbectomy on maternal behavior in rats, behavioral observations were conducted for only a few days following the birth or presentation of pups. However, the maternal behavior of female rats normally undergoes changes as the pups mature. In general, these changes are manifested as a gradual decrease in the frequency with which female rats engage in most components of maternal behavior as their pups grow older [ 11 ]. These changes in the maternal responsiveness of female rats might partially reflect age-related changes in the stimulus properties of pups. If changes in the stimulus properties of maturing pups involve changes in olfactory stimuli, or alter the relative importance of existing olfactory stimuli, it is conceivable that bilateral olfactory bulb ablation might influence the responsiveness of female rats to pups of some ages, while having little or no influence on maternal responsiveness to pups of other ages. Therefore, Experiment 1 was performed to assess the influence of

~These experiments were part of a dissertation submitted to Illinois Institute of Technology by I. B. in partial fulfillment of the requirements for the Ph.D. in psychology. 2Present address: Northeast Georgia Genetic Counseling Program, 315 S. Enota Drive, Gainseville, GA 30501. 439

440

BENUCK AND ROWE

olfactory bulbectomy on maternal behavior in primiparous, lactating female rats that were allowed to rear their pups from birth to weaning. EXPERIMENT 1 METHOD

Animals Animals for this experiment were 28 female SpragueDawley rats. At the beginning of the experiment, all females were virgins and were between 1 0 0 - 1 1 0 days of age. Each female was housed individually in a 10 gal glass aquarium (45 cm X 25 cm X 30 cm high), the bottom of which was covered with San-I-Cel bedding. Food and water were available ad lib, and a reversed right-dark cycle was in effect in the colony room (12 hr light; lights on at midnight).

Procedure Beginning shortly after their arrival at the laboratory, the females' estrus cycles were monitored by observing vaginal smears taken daily at the same hour. Once it had been determined that a female was cycling normally, a proven male breeder was introduced during the period of proestrus or early estrus, and was left with the female for 24 hr. Females were then weighed on alternate days for 10 days. If a female did not gain at least 20 g within 10 days of being paired with a male, vaginal smears were repeated and the male was reintroduced during the next proestrus or early estrus stage. If a female failed to become pregnant after the second pairing with a male, she was discarded from the experiment. Ten to 12 days postcoitum, females underwent one of the following treatments: bilateral olfactory bulb ablation, unilateral olfactory bulb ablation, or sham surgery. There were 7 females in each surgical condition, and an additional 7 females served as unoperated controls. Bilateral bulbectomies were performed while females were anesthetized with ether. After securing a female's head in a stereotaxic apparatus, an incision was made in her scalp and the frontal bone was exposed. The bone overlying the olfactory bulbs was removed with a dental burr, the dura was silt, and the olfactory bulbs were aspirated. The resulting space was packed with Gelfoam, and the skin was closed with sutures. Unilaterally bulbectomized females (4 fight bulb and 3 left bulb) underwent t h e s a m e p r o c e d u r e , with the exception that one olfactory bulb was left intact. Sham operations consisted of removing the bone overlying the olfactory bulbs, and leaving the dura and olfactory bulbs intact. The following variables were used in evaluating maternal behavior in this experiment. Nest building. Both pre- and postpartum nest building activities were assessed. On the nineteenth and twentieth days after mating, females were presented with 15 g of paper towering. The paper was cut into strips (15.0 cm X 1.3 cm), and placed in a dispensing cylinder that was suspended 15 cm above the floor of each cage. Twentyfour hr after each presentation, the amount of paper remaining in the dispensers was weighed to the nearest gram. If on either day a female failed to empty the dispenser, the remaining paper was spread about the floor of her cage after it was weighed.

Judgements of nest quality were made on the 3 days immediately preceding parturition, and every day following parturition. On each of these days, two observers independently rated each nest. The rating assigned to a particular nest on a given day was the average of these two ratings. The following scale, adapted from Slotnick [15], was used in rating nest quality: (1) no nesting material pulled down from the dispenser; (2) nest material pulled down, but scattered over entire cage floor; (3) nest material concentrated in one area of the cage; (4) all nest material used in the construction of a good nest with compact walls and a hollow center; (5) all nest material used in the construction of an excellent nest with walls that were at least 2.5 cm high and reinforced with food and/or feces. Litter size and pup mortality. With in a few hours of parturition, the number of live and dead pups in each litter was determined. Subsequently, all litters were culled to 6 pups each. Nursing. Observations were made of the time that females spent on their nests in a nursing posture during 1 hr of each phase of the light-dark cycle. These observations were initiated within 24 hr of parturition, and were conducted at the same times each day until the pups were weaned. Pup retrieval. Tests of pup retrieval were initiated approximately 24 hr after parturition, and were conducted on alternate days until the pups were 13 days of age. Each test of retrieval consisted of gently nudging a female off her nest and forcing her to the opposite end of the cage, where she was restrained for 1 min by means of a wooden partition. During this minute, the pups were lifted from the nest with tongue forceps and scattered about the floor of the cage. Following removal of the restraining partition, the female was allowed 30 min to retrieve pups back into the nest. During this time, the number of pups retrieved by the female was noted, as was the latency to the retrieval of the first pup. Any pups not retrieved at the end of 30 rain were placed back into their nests by the observer. Weight of pups. Pups were weighed within 24 hr of their birth, and every fifth day thereafter until they were 20 days of age.

Histology At the conclusion of behavioral testing, unilaterally and bilaterally bulbectomized females were killed with an overdose of ether, and perfused intracardially with physiological saline followed by 10 percent Formalin. Their brains were removed from the skulls and examined under a dissecting microscope. Subsequently, the brains were embedded in paraffin, and the extent of neurological damage was assessed by examining 20 micra horizontal sections stained with cresyl violet. RESULTS

Bilateral olfactory bulb ablation differentially influenced the components of maternal behavior in lactating female rats.

Nest Building None of the surgical treatments influenced the amount of paper accumulated by female rats in the 2 days that it

A N O S M I A A N D M A T E R N A L B E H A V I O R IN R A T S

441

TABLE 1 MEAN (+ 1 S.E.) RATINGS OF NEST QUALITY IN EXPERIMENT 1 Postpartum Days 10-12

Surgical Group

Prepartum

1-3

4-6

7-9

13-15

16-18

Bilateral

1.1 2 0.12

2.7 2 0.26

3.0 2 0.25

2.4 -+ 0.37

2.5 2 0.33

1.9 -+ 0.22

1.0 -+ 0.04

Unilateral

1.2 +- 0.10

3.1 2 0.18

2.9 2 0.10

2.7 ± 0.21

2.5 2 0.25

2.1 -+ 0.18

1.3 2 0.13

Sham operated

1.3 2 0.12

3.0 2 0.19

3.0 -+ 0.13

2.9 2 0.20

2.5 -+ 0.11

2.2 2 0.24

1.1 2 0.06

Unoperated

1.3 2 0.09

3.2 +_ 0.10

2.9 2 0.12

2.5 2 0.16

1.9 2 0.12

1.1 2 0.10

1.0 2 0.00

TABLE 2 LITTER SIZE AND PUP MORTALITY

Surgical Group

Group Size

Mekn (2 1 S.E.) Litter Size Prior to Culling*

Mean (2 1 S.E.) Number of Dead Pups at Parturition

Mean Number of Pups Alive at Weaning t

Bilateral

7

9.7 2 1.8

2.1 +- 1.11:

6.0

Unilateral

7

10.7 2 1.4

0.4 2 0.8

6.0

Sham operated

7

12.0 2 1.6

0.3 2 0.5

6.0

Unoperated

7

11.4 2 2.2

0.3 2 0.5

6.0

*Based on number of live and dead pups found in cages shortly after parturition. tBased on culled litters of N = 6. :~Indicates significantly different from unilateral, sham operated, and unoperated females at p<0.05. was available prior to parturition. However, all females pulled more paper d o w n f r o m the dispensers on the first than on the second day o f access to paper, F(1,24) = 12.37, p<0.05. The quality o f nest c o n s t r u c t i o n was rated on each o f the three days i m m e d i a t e l y preceding parturition, and everyday following parturition until pups were 18 days old. Ratings for the 3 days prior to parturition were averaged to yield a single score. Similarly, p o s t p a r t u m nest scores were divided into blocks of 3 days. All females built b e t t e r nests in the first 9 days postp a r t u m than t h e y built prior to parturition. F u r t h e r m o r e , prior to and for the first 9 days after parturition, females in all surgical groups built nests of similar quality. However, nest quality began to decline shortly after parturition, the rate of decline being greatest in u n o p e r a t e d females. C o n s e q u e n t l y , from the t e n t h through the seventeenth days p o s t p a r t u m , u n o p e r a t e d females had nests o f p o o r e r quality than bilaterally b u l b e c t o m i z e d or sham-operated females (Surgical T r e a t m e n t × Days, F ( 3 , 1 4 4 ) = 211, p < 0 . 0 5 . By the eighteenth day p o s t p a r t u m , females in all surgical groups had ceased to maintain recognizable nests (Table 1).

Litter Size and Pup Mortality There were no differences a m o n g the surgical groups with regard to the n u m b e r of pups delivered by females (Table 2). However, when litter size was assessed a few hours after parturition, more dead pups were f o u n d in the cages o f bilaterally b u l b e c t o m i z e d females than in the cages

of females in the o t h e r surgical conditions, F(3,24) = 10.30, p<0.01. A f t e r litter size was determined, each litter was culled to 6 pups. There were no differences a m o n g the surgical groups with regard to the n u m b e r of these pups that survived to weaning.

Nursing N o n e of the surgical t r e a t m e n t s influenced the a m o u n t of time that females spent on their nests in a nursing posture in either phase of the l i g h t - d a r k cycle. However, all females spent m o r e t i m e on their nests during the light phase of the cycle than during the dark phase, F(1,48) = 85.85, p < 0 . 0 1 . As pups matured, all females spent a decreasing a m o u n t of time on their nests (Table 3).

Retrieval Bilateral olfactory bulb ablation resulted in a marked deficit in pup retrieval in tests c o n d u c t e d when pups were 24 hr old. In these tests, no bilaterally b u l b e c t o m i z e d females retrieved all of their pups in the allotted time, and t w o bilaterally b u l b e c t o m i z e d females failed to retireve any pups. In contrasts to bilaterally b u l b e c t o m i z e d females, 100 percent of the females in the o t h e r surgical conditions retrieved all o f their pups in the first test o f retrieval (Table 4). There were no differences among the surgical groups with regard to the n u m b e r of females that retrieved pups that were 3, 5, 7, 9, or 11 days of age. However, through-

442

BENUCK AND ROWE

TABLE 3 MEAN (5 1 S.E.) TIME (MIN) SPENT ON NESTS BY FEMALES DURING ONE HOUR OF EACH PHASE OF THE LIGHT-DARK CYCLE IN 3-DAY BLOCKS

Postpartum Days Surgical Group Bilateral

Unilateral

Sham operated

Unoperated

Phase of Lighting Cycle

1-3

4-6

7-9

10-12

13-15

16-18

Light

49.3 +_ 3.1

44.7 5 3.6

37.3 5 3.5

30.0 5 1.6

26.8 5 2.7

16.2 5 2.5

Dark

34.8 5 4.1

22.3 5 4.4

21.8 5 4.4

20.3 + 2.5

19.4 5 1.3

14.8 +- 1.4

Light

43.7 5 4.0

33.0 +- 5.4

39.4 5 4.3

32.5 5 2.8

20.2 ± 2.7

14.5 +- 2.2

Dark

29.0 5 3.8

15.4 5 2.8

21.3 +- 2.4

19.4 +- 2.2

15.9 5 1.6

11.6 + 2.5

Light

46.1 5 1.6

34.9 -+ 3.9

33.9 5 1.8

29.2 5 2.0

23.0 5 3.4

11.9 5 2.9

Dark

36.1 -+ 2.4

23.5 5 2.6

16.4 ± 2.3

21.8 5 1.7

16.9 +- 1.6

8.3 +- 0.9

Light

49.6 5 1.7

28.9 5 2.8

37.5 5 2.6

26.0 +- 1.2

19.2 5 1.1

12.9 ± 1.9

Dark

31.3 5 2.4

21.7 5 1.4

23.9 5 2.9

23.7 5 2.4

17.1 ± 1.8

9.4 ± 1.6

TABLE 4 RETRIEVAL BEHAVIOR IN FEMALE RATS Postpartum Day Surgical Group*

1

3

5

7

9

11

13

Percentage

BL

29

14

29

Retrieving No Pups

UL S N

0 0 0

0 0 14

0 0 0

58 0 0

58 0 28

58 0 14

86 0 57

14

14

28

72

BL

0

72

42

Variable

Percentage Retrieving All Pups

Number of Pups Retrieved (M +- 1 s.d.)

42

42

29

14

100

100

100

UL

100

100

100

100

S N

100 100

100 86

86 86

86

58

72

28

86

86

72

28

BL

2.4 5 2.0]-

4.8 +- 2.3

3.4 5 2.7~?

2.6 ± 3.3~

2.1 5 3.2t

2.4 5 3.0t

0.8 5 2.35

6.0 5 0.0 5.6 5 1.1 5.3 5 1.9

6.0 5 0.0

6.0 5 0.0

6.0 5 0.0

6.0 ± 0.0

5.6 ± 1.1 5.122.3

4.0 5 2.8 5.1+2.3

5.0 • 2.2 4.352.9

1.8 5 2.8 1.752.9

UL S N

6.0 5 0.0 6.0 +- 0.0 6.0 -+ 0.0

6.0 5 0.0 6.0 +- 0.0 5.1 5 2.3

*BL, UL, S, and N indicates bilateral, unilateral, sham operated, and unoperated females, respectively. J-Indicates significantly different from UL, S, and N females at the 0.05 level or better. $Indicates significantly different from UL females at the 0.05 level or better.

out this p e r i o d o f time, bilaterally b u l b e c t o m i z e d females retrieved f e w e r p u p s in t h e a l l o t t e d t i m e t h a n did females in t h e o t h e r surgical c o n d i t i o n s , F ( 3 , 2 4 ) = 7.95, p < 0 . 0 1 . N e u m a n - K e u l s analyses i n d i c a t e d t h a t bilaterally bulbect o m i z e d females retrieved significiantly ( p < 0 . 0 5 ) f e w e r p u p s t h a n unilaterally b u l b e c t o m i z e d females in all tests b u t t h o s e c o n d u c t e d w h e n p u p s were 3 days old. Similarly, bilaterally b u l b e c t o m i z e d females retrieved f e w e r p u p s t h a n

s h a m o p e r a t e d and u n o p e r a t e d females in all tests but t h o s e c o n d u c t e d w h e n p u p s were 3 and 13 days o f age. By t h e t i m e t h a t p u p s were 13 days o f age, m o s t females m a d e n o e f f o r t to retrieve t h e m b a c k i n t o t h e n e s t during t h e test period. However, 100 p e r c e n t o f t h e unilaterally b u l b e c t o m i z e d females c o n t i n u e d t o retrieve all o f their 13-day old p u p s in t h e a l l o t t e d time. No statistical analyses were p e r f o r m e d o n t h e latencies

ANOSMIA AND MATERNAL BEHAVIOR IN RATS

443 TABLE 5

MEAN (± 1 S.E.M.) BODY WEIGHT (G) OF PUPS IN EXPERIMENT 1" Postpartum Day Mother's Surgical Group

1

5

10

15

20

Bilateral

6.6 ± 0.1

13.5 -+ 0.9

22.0 ± 1.5"

31.9 -+ 1.5?

48.6 ± 2.6t

Unilateral

6.4 ± 0.2

14.3 _+0.4

26.8 ± 0.9

38.0 -+ 1.1

56.1 -+ 1.4

Sham operated

6.3 -+ 0.1

14.2 -+ 0.4

24.6 -+ 1.3

35.0 ± 1.25

51.2 -+ 1.75

Unoperated

6.8 ± 0.1

15.0 ± 0.5

27.4 ± 0.8

37.9 ± 0.8

56.0 ± 1.9

*Based on the data from 42 pups in each surgical condition. tlndicates significantly different from Unilateral, Sham operated, and Unoperated values at the 0.05 level or better. $Indicates significantly different from Unilateral and Unoperated values at the 0.05 level or better.

to retrieval of the first pup in each test. However, when pups were between the ages of 1 and 7 days, bilaterally bulbectomized females took approximately 2 to 5 times longer than the next slowest group to initiate pup retrieval.

Pup Weight There were no differences among the surgical groups with regard to the body weight of pups in the culled litters on the day of birth. Furthermore, at 5 days of age all pups weighed approximately the same. However, by the time that they were 10 days old, pups from bilaterally bulbectomized females weighed less than pups being reared by females in all other surgical groups, and this difference persisted through the age of 20 days (Surgical Group × Days, F(12,96) = 3.10, p<0.01). Furthermore, at 15 and 20 days of age, pups from sham operated females weighed less than pups from unilaterally bulbectomized and unoperated females (Table 5).

His to log)' Six of the 7 bilaterally bulbectomized females exhibited removal of at least the rostral two-thirds of both olfactory bulbs. In these 6 females, the accessory olfactory bulbs were either absent or extensively damaged, and in all but one case the lesion invaded the anterior olfactory nucleus on both sides. In one bilateral female, only the rostral half of both bulbs were removed. In no instance was there evidence of damage to the frontal, cingulate, or prepiriform cortices, or to the olfactory tubercles. In all unilaterally bulbectomized females, all of one main and accessory olfactory bulb were destroyed. In five of these females, the lesion invaded the anterior olfactory nucleus. Three females had slight damage to the superficial layers of the remaining bulb. EXPERIMENT 2 In Experiment 1, bilateral olfactory bulb ablation was found to produce deficits in several components of maternal behavior in primiparous, lactating female rats. While it is tempting to attribute these deficits to bulbectomy induced

anosmia, it has recently become evident that deficits seen in the social behaviors of several rodent species following bulbectomy cannot be attributed solely to anosmia (e.g. [ 5, 10, 12]). Recently, Fleming and Rosenblatt [7] found that, unlike bilateral bulbectomy, anosmia produced by applying zinc sulphate to the olfactory mucosa did not result in cannabalism of foster pups by virgin female rats. In fact, peripherally-induced anosmia shortened the latency to the display of maternal behavior by most virgin females. In view of the failure of peripherally-induced anosmia to mimic the effects of bilateral bulbectomy on maternal behavior in virgin female rats, this experiment was performed to investigate the influence of zinc sulphate-induced anosmia on maternal behavior in lactating, primiparous female rats. METHOD

Animals Animals for this experiment were 12 virgin female Sprague-Dawley rats. All females were 100-1 l 0 days old at the start of the experiment. Caging conditions, accessibility of food and water, and the light-dark cycle in effect in the colony room were the same as in Experiment 1.

Procedure The manner in which females were paired with males and the criterion for a fertile mating were the same as in Experiment 1. Twenty days postcoitum, pregnant females had either a 5 percent solution of zinc sulphate or physiological saline applied to their olfactory mucosa. There were 6 females in each of these treatment conditions. The technique for applying zinc sulphate or saline to the olfactory mucosa has been described elsewhere [1]. Briefly, a female was anesthetized with ether, placed on her back, and her tongue held to one side with padded forceps. A 19 ga hypodermic needle, the tip of which had been blunted and bent to form a hook, was pushed back along the palate until it entered the esophagus. The needle was then retracted rostrally until the tip entered the nasopharynx. While the female's body was inclinded 45

444

BENUCK AND ROWE

degrees from horizontal (head down), 0.6 ml of zinc sulphate or saline were injected. Drops of either solution appearing at the external nares indicated a successful application of the solution. Following application of either solution, a female's head was kept lower than her body until she recovered from the anesthetic, and during this time her mouth and nares were aspirated to remove excess solution. To determine whether or not females were anosmic, a cotton-tipped applicator soaked in chloroform was presented in front of their noses. If zinc sulphate treated females did not try to avoid the applicator, they were considered to be anosmic. Tests for anosmia were performed daily for the duration of the experiment. The variables used to assess maternal behavior were the same as those described in Experiment 1. However, the transient nature of zinc sulphate induced anosmia necessitated some changes in the scheduling of behavioral observations. Beginning immediately after zinc sulphate or saline treatments, paper was presented to females on two consecutive days, and the amount of paper accumulated by females on each of these days was recorded. However, only one prepartum rating of nest quality was obtained on the day before parturition. Furthermore, in the present experiment tests of pup retrieval were conducted daily, rather than on alternate days as in Experiment 1. Finally, in the present experiment, maternal behavior was assessed through only the first 5 postpartum days. In order to compare maternal behavior in zinc sulphate and saline treated females with maternal behavior in untreated females, data from 6 of the 7 untreated females in Experiment I were used. In analysing data regarding preparturition nest quality, scores obtained by normal females in their first prepartum rating were used. Furthermore, since pup retrieval was assessed on alternate days in Experiment 1 and every day in the present experiment, the data from normal females were excluded from statistical analyses of retrieval data in the present experiment.

RESULTS

Nest Building Paper was presented to pregnant females immediately after administration of zinc sulphate or saline treatments. On the first day that it was available, zinc sulphate treated females accumulated less paper than saline treated or untreated females. However, on the second day that it was available, females in all treatment conditions pulled equal

amounts of paper down from the dispensers (Treatment × Days, F(2,15) = 4.38, p<0.05). None of the treatments influenced the quality of nests built by females either before or after parturition (Table 6). However, all postpartum nests were of better quality than prepartum nests, F(5,75) = 26.84, p<0.01.

Litter Size and Pup Mortality Zinc sulphate and saline-treated females gave birth to pups between 48 and 86 hr after treatment, with a mean of approximately 72 hr. None of the treatments influenced the number of pups delivered by females, and dead pups were infrequently observed in any of the litters (Table 7).

Nursing The amount of time that females spent on their nests in a nursing posture was recorded during 1 hr of each phase of the l i g h t - d a r k cycle on the first 5 days of lactation. There were no differences among the treatment groups with regard to the amount of time that females spent on their nests in either phase of the l i g h t - d a r k cycle (Table 8). However, all females spent more time on their nests during the light phase than during the dark phase of the cycle, F(1,20) = 84.24, p<0.01.

Retrieval Zinc sulphate and saline treated females were tested for pup retrieval on each of the first 5 postpartum days. There were no differences between the two treatment groups with regard to the number of females that retrieved either all or no pups in the five tests (Table 8). Similarly, females in both conditions had similar latencies to the initiation of pup retireval in all tests. However, zinc sulphate treated females retrieved fewer pups in all tests than did saline treated females, F(1,10) = 4.99, p<0.05. All zinc sulphate treated females used in the present study met our criterion of anosmia throughout the period during which behavioral observations were being made. Anosmic females not only made no attempt to avoid a chloroform-soaked applicator, they often attempted to chew the applicator when it was presented in front of their faces. In contrast to zinc sulphate treated females, saline treated females made a conspicuous effort to avoid the applicator. G EN ERA L DISCUSSION In the present study, bilateral olfactory bulb removal

TABLE 6 MEAN (+- 1 S.E.) RATING OF NEST QUALITY IN EXPERIMENT 2 Postpartum Day Treatment Group

Prepartum

1

2

3

Zinc sulphate

1.3 -+ 0.21

2.7 -+ 0.21

2.7 -+ 0.21

2.8 -+ 0.17

3.2 +- 0.31

2.8 +- 0.17

Saline

1.5 -+ 0.22

3.0 + 0.00

3.0 +- 0.26

2.8 +- 0.17

3.2 -+ 0.17

3.2 +- 0.17

Normal

1.8 -+ 0.17

3.0 +- 0.00

3.5 +- 0.22

3.0 -+ 0.17

3.2 -+ 0.31

2.7 ± 0.21

ANOSMIA AND M A T E R N A L BEHAVIOR IN RATS

445 TABLE 7

LITTER SIZE AND PUP MORTALITY AFTER ZINC SULPHATE OR SALINE TREATMENT

Treatment

Group Size

Mean (± 1 S.D.) Litter Size Prior to Culling

Mean (± 1 S.D.) Number of Dead Pups at Parturition

Zinc sulphate

6

10.4 ± 1.4

0.3 ± 0.5

Saline

6

11.1 ± 1.4

0.0 ± 0.0

Untreated*

6

11.5 ± 1.8

0.2 -+ 0.4

*Selected from the 7 unoperated females in Experiment 1.

TABLE 8 NURSING AND RETRIEVAL BEHAVIOR IN ZINC SULPHATE AND SALINE TREATED FEMALES

1

2

Postpartum Day 3

4

5

Light Dark

53.6 ± 1.8 37.5 ± 3.2

46.3 ± 3.2 32.0 -+ 6.3

36.2 ± 4.1 31.7 ± 2.9

35.3 ± 2.2 23.5 ± 3.0

33.7 ± 3.5 27.5 ± 3.7

Light Dark

52.2 ± 1.9 40.0 ± 5.4

45.5 ± 3.9 23.0 ± 3.3

32.3 ± 3.0 30.5 ± 1.4

32.7 ± 3.9 29.8 -+ 2.6

25.7 +- 4.1 22.8 ± 3.3

0 0

17 0

33 0

50 100

83 100

83 100

67 100

4.2 ± 1.1 6.0 ± 0.0

5.2 ± 0.8 6.0 ± 0.0

5.2 ± 1.0 6.0 ± 0.0

4.0 ± 1.3 6.0 ± 0.0

Variable

Treatment

Mean (± 1 S.E.) time (min) spent in nursing posture in light and dark phases of lighting cycle

Zinc sulphate

Saline

Percentage of females retrieving no pups

Zinc sulphate Saline

17 0

17 0

Percentage of females retrieving all pups

Zinc sulphate Saline

83 100

Mean (± 1 S.D.) number of pups retrieved*

Zinc sulphate Saline

5.0 ± 1.0 6.0 ± 0.0

*Based on culled fitters of 6 pups each.

was found to produce deficits in several components of maternal behavior in primiparous, lactating female rats. Within a few hours of parturition, more dead pups were found in the cages of bilaterally bulbectomized females than in the cages of unilaterally bulbectomized, sham operated, or unoperated females. Furthermore, bilaterally bulbectomized females exhibited deficits in pup retrieval relative to females in the other surgical conditions, and pups reared by bilateral females weighed less throughout the last half of lactation than pups reared by females in the other surgical conditions. In contrast to bilateral bulbect o m y , peripherally-induced anosmia produced only a transient reduction in the amount of paper accumulated by pregnant females, and a reduction in the number of pups retrieved by female in the first 5 days postpartum. Our observation that bilateral bulbectomy produced deficits in components of maternal behavior that were not affected by zinc sulphate-induced anosmia is in accord with o t h e r observations indicating that behavioral changes observed after bulbectomy are not solely the consequence of the sensory deficit produced by bulbectomy (e.g. [5, 10, 12, 14]). Furthermore, when considered in light of recent

observations regarding the effects of bilateral bulbectomy and peripherally-induced anosmia on maternal behavior in virgin female rats [6,7], the present data indicate that these two procedures differentially influence maternal behavior in female rats, regardless of their physiological state (cycling vs. lactating). However, the effects of bulbectomy and peripherally-induced anosmia on maternal behavior do differ somewhat in cycling and lactating females. Fleming and Rosenblatt [7] found that, in most instances, virgin females rendered anosmic by zinc sulphate treatments exhibited shorter latencies to the display of maternal behavior than saline or air treated females. However, in the present study, zinc sulphate induced anosmia was never observed to facilitate maternal responsiveness in lactating females. Our failure to observe any facilitory influence o f zinc sulphate induced anosmia on maternal behavior was probably a consequence of the fact that the predisposition to respond maternally to pups was already maximal in our females as a consequence of the hormonal conditions associated with pregnancy and parturition. Bilaterally bulbectomized virgin female rats have been reported to exhibit a high incidence of cannabalism on

446 initial exposure to pups [6,13]. It has been hypothesized that pup cannabalism by bulbectomized virgin females is a consequence of the virgin female rats' tendency to respond aggressively to the novel stimulus situation created by the presentation of pups [6], compounded by bulbectomyinduced increases in irritable aggression [4]. In contrast to virgin females, lactating females seldom cannabalize their p u p s following bulbectomy. Our failure to observe cannabalism of pups in the present study is in accord with the suggestion that the hormonal state associated with pregnancy and lactation suppresses both natural and bulbectomy-induced tendencies of female rats to respond aggressively towards pups [ 6 ]. Although we never observed cannabalism of pups by bilaterally bulbectomized females, we did find more dead pups in the cages of bilaterally bulbectomized females shortly after parturition than in the cages of females in the other surgical conditions. It seems unlikely that these pups died as a consequence of having been attacked by their mothers, since in no instance did dead pups bear wounds that would be expected in this event. Furthermore, it did not appear that the greater incidence of pup mortality among bilaterally bulbectomized females was a consequnce of any deleterious influence that surgery might have had on the prenatal development of their pups. Dead pups found in the cages of these females appeared to be fully developed, a n d w e i g h e d approximately as much as their live littermates. In our opinion, the higher incidence of mortality among pups delivered by bilaterally bulbectomized females was a consequence of bulbectomy-induced deficits in the performance of behaviors at parturition that insure the subsequent survival of the pups. This conclusion was suggested by the observation that, in some instances, dead pups found in the cages of these females bore remnants of the fetal m e m b r a n e s . F u r t h e r m o r e , bilaterally bulbectomized females frequently failed to eat the placentae following delivery of their pups. The deficits in parturition behaviors evidence by bilaterally bulbectomized females did not appear to be a consequence of the sensory deficit produced by bulbectomy. The incidence of pup mortality in litters delivered by zinc sulphate treated females was no greater than in litters delivered by saline treated or untreated females, and zinc s u l p h a t e treated females never exhibited incomplete placentophagia. Bilaterally bulbectomized females retrieved fewer pups and tended to have longer latencies to the initiation of pup retireval than females in the other surgical conditions. These deficits might have been partially a consequence of bulbectomy-induced anosmia. That is, bilaterally bulbectomized females might have had difficulty in locating and/or recognizing pups that had been placed outside of their nests. However, the deficits in retrieval behavior exhibited by bulbectomized females in the present study appeared to be at least partially a consequence of the fact that bilateral bulbectomy disrupted the sterotyped sequencing of a c t s c o m p r i s i n g retrieval behavior. Bilateral bulbectomized females frequently exhibited disorganized retrieval behavior that was characterized by a female picking up a pup and wandering about the cage with it for an inordinately long time before depositing it in the nest, or abandoning it out of the nest. The disorganized pattern of retrieval behavior exhibited by these females did not appear to be a consequence of bulbectomy-induced anosmia, since

BENUCK AND ROWE similar disorganized retrieval behavior was never observed in zinc sulphate treated females. The inefficient retrieval behavior that was characteristic of our bilaterally bulbectomized females was similar to patterns of retrieval behavior that have been observed in female rats with lesions in the cingulate cortex [ 15,16] and septal area [ 14]. Although deficits in the retrieval behavior of bilaterally bulbectomized females did not appear to be solely a consequence of anosmia, a role for olfactory stimuli in the retrieval behavior of female rats is suggested by our observation that zinc sulphate treated as well as bilaterally bulbectomized females retrieved fewer pups than their respective controls. However, the present data provide no information regarding the nature of the rote played by olfactory stimuli in pup retrieval. Beginning midway through lactation, and continuing until weaning, pups being reared by bilaterally bulbectomized females weighed less than pups being reared by females in the other surgical conditions. The failure of pups reared by bilateral females to gain as much weight as pups reared by other females did not appear to be a consequence of deficits in the nursing behavior of bilaterally bulbectomized females. Throughout the experiment, bulbectomized females spent as much time on their nests in a nursing posture as females in the other surgical groups. We feel that there are at least two possible explainations for the lower body weight of pups reared by bilaterally b u l b e c t o m i z e d f e m a l e s . Recently, Herrenkohl and Rosenberg [8] observed that complete deafferentation of the medial basal hypothalamus in female rats suppressed lactation, and consequently suppressed weight gain in pups being reared by these females. It is conceivable that bulbectomy-induced disruption of olfactory input to the hypothalamus suppressed lactation in our female rats. Unfortunately, the effects of bulbectomy on lactation were not assessed in this experiment. In the present study, the difference in body weight between pups from bilaterally bulbectomized females and pups from females in the other surgical groups appeared at about the time that pups were beginning to leave the nest under their own power. In light of this fact, it is conceivable that the suppressed weight gain of pups from bilateral females might have been an indirect consequence of their mothers' deficit in pup retrieval. That is, in the interval in which they were 10 to 20 days of age, pups being reared by bilaterally bulbectomized females might have spent more time out of their nests, and consequently, less time nursing, than pups being reared by females in the other surgical groups. If the suppressed weight gain exhibited by pups reared by bilaterally bulbectomized females was, in fact, a consequence of their mothers' deficit in retrieval behavior, it is conceivable that pups reared by zinc sulphate treated females would have exhibited a similar suppression in weight gain after they became highly motile. Unfortunately, the transient nature of zinc sulphate-induced anosmia precluded observing pups being reared by anosmic females long enough to determine whether or not this is the case. Overall, the present data indicate that changes in the maternal behavior of bilaterally bulbectomized female rats are the result of both a neurophysiological and a sensory deficit. However, the sensory deficit produced by bulbectomy appears to be a minor contributing factor to postoperative deficits in maternal behavior. The conclusion

A N O S M I A A N D M A T E R N A L B E H A V I O R IN R A T S

that the neurophysiological consequences of bilateral bulbectomy c o n t r i b u t e m o r e to deficits in m a t e r n a l b e h a v i o r t h a n t h e s e n s o r y c o n s e q u e n c e s o f b u l b e c t o m y is n o t b a s e d solely o n c o m p a r i s o n s of t h e effects o f b i l a t e r a l b u b l e c t o m y a n d zinc s u l p h a t e t r e a t m e n t s o n m a t e r n a l b e h a v i o r . O b s e r v a t i o n s regarding t h e c o n s e q u e n c e s o f u n i l a t e r a l o l f a c t o r y b u l b a b l a t i o n are c o n s i s t e n t w i t h t h e idea t h a t changes in m a t e r n a l b e h a v i o r o b s e r v e d f o l l o w i n g b i l a t e r a l b u l b e c t o m y have a n e u r o p h y s i o l o g i c a l basis. F l e m i n g a n d R o s e n b l a t t [6] f o u n d t h a t u n i l a t e r a l b u l b e c -

447

t o m y resulted in c a n n a b a l i s m o f pups b y a small p e r c e n t a g e o f virgin female rats. F u r t h e r m o r e , in t h e p r e s e n t s t u d y , u n i l a t e r a l l y b u l b e c t o m i z e d females t e n d e d t o e x h i b i t m o r e efficient a n d m o r e p e r s i s t e n t retrieval o f p u p s t h a n s h a m o p e r a t e d or u n o p e r a t e d females. E v i d e n c e i n d i c a t i n g t h a t u n i l a t e r a l b u l b e c t o m y has little o r n o i n f l u e n c e o n o l f a c t o r y a c u i t y in rats [3] m a k e s it seem u n l i k e l y t h a t changes in t h e m a t e r n a l b e h a v i o r o f u n i l a t e r a l l y b u l b e c t o m i z e d females rats are a c o n s e q u e n c e of a sensory deficit.

REFERENCES 1. Alberts, J. R. and B. G. Galef. Acute anosmia in the rat. A behavioral test of a peripherally-induced olfactory deficit. Physiol. Behav. 6: 6 1 9 - 6 2 1 , 1971. 2. Beach, F. A. and J. Jaynes. Studies of maternal retrieving in rats. Ill. Sensory cues involved in the lactating female's response to her young. Behavior 10: 104-125, 1956. 3. Bennett, M. H. The role of the anterior limb of the anterior commissure in olfaction. Physiol. Behav. 3 : 5 0 7 - 5 1 5 , 1968. 4. Douglas, R., R. Isaacson and R. Moss. Olfactory lesions, emotionality, and activity. Physiol. Behav. 4: 3 7 9 - 3 8 1 , 1969. 5. Edwards, D. A., M. L. Thompson and K. G. Burge. Olfactory bulb removal vs. peripherally-induced anosmia: Differential effects on the aggressive behavior of male mice.Behav. Biol. 7: 8 2 3 - 8 2 8 , 1972. 6. Fleming, A. S. and J. S. Rosenblatt. Olfactory regulation of maternal behavior in rats: I. Effects of olfactory bulb removal in experienced and inexperienced lactating and cycling females. J. comp. physiol. Psychol. 8 6 : 2 2 1 - 2 3 2 , 1974. 7. Fleming, A. S. and J. S. Rosenblatt. Olfactory regulation of maternal behavior in rats: II. Effects of peripherally-induced anosmia and lesions of the lateral olfactory tract in pupinduced virgins. J. comp. physiol. Psychol. 86: 2 3 3 - 2 4 6 , 1974. 8. Herrenkohi, L. R. and P. A. Rosenberg. Effects of hypothalamic deafferentation late in gestation on lactation and nursing behavior in the rat. Hormones Behav. 5: 33--41, 1974.

9. Moltz, H., M. Lubin, M. Leon and M. Numan. Hormonal induction of maternal behvior in ovariectomized nulliparous rats. Physiol. Behav. 5: 1373-1377, 1971. 10. Powers, J. B. and S. Winans. Sexual behavior in peripherally anosmic male hamsters. Physiol. Behav. 10: 3 6 1 - 3 6 8 , 1973. 11. Rosenblatt, J. S. and D. S. Lehrman. Maternal behavior of the laboratory rat. In: Maternal Behavior in Mammals, edited by H. L. Rheingold. New York: Wiley, 1963. 12. Rowe, F. A. and W. E. Smith. Effects of peripherally induced anosmia on mating behavior of male mice. Psychonom. Sci. 27: 3 3 - 3 4 , 1972. 13. Schlein, P. A., M. X. Zarrow, H. A. Cohen, V. H. Denenberg and N. P. Johnson. The differential effect of anosmia on maternal behavior in the virgin and primiparous rat. J. Reprod. Fert. 30: 139-142, 1972. 14. Slotnick, B. M. Disturbances of maternal behavior in rats following lesions of the dorsal limbic cortex. Unpublished doctoral dissertation, University of Illinois, 1963. 15. Slotnick, B. M. Disturbances of maternal behavior in rats following lesions of the cinguate cortex. Behaviour 29: 2 0 4 - 2 3 6 , 1967. 16. Stamm, J. S. The function of the median cerebral cortex in maternal behavior of rats. J. comp. physiol. Psychol. 48: 3 4 7 - 3 5 6 , 1955. 17. Wiesner, B. P. and N. M. Sheard. Maternal Behaviour in the Rat. Lond: Oliver and Boyd, 1933.