Effects of one-stage or serial transections of the lateral olfactory tracts on behavior and plasma testosterone levels in male hamsters

Brain Research, 109 (1976) 97-109

97

© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

EFFECTS OF ONE-STAGE OR SERIAL TRANSECTIONS OF THE LATERAL OLFACTORY TRACTS ON BEHAVIOR AND PLASMA TESTOSTERONE LEVELS IN MALE HAMSTERS

FOTEOS MACRIDES, A R T H U R C. FIRL, JR., STEPHEN P. SCHNEIDER, A N D R Z E J B A R T K E AND D O N A L D G. STEIN

Worcester Foundation for Experimental Biology, Shrewsbury, Mass. 01545 and Clark University, Worcester, Mass. 01610 (U.S.A.) (Accepted October 28th, 1975)

SUMMARY

Bilateral transections of the lateral olfactory tract (LOT) in male hamsters at the rostral border of the olfactory tubercle or anterior to the tubercle produced severe deficits in mounting, scent marking, food hoarding and nesting behaviors. There were no impairments of these behaviors following unilateral transections. Cutting of the LOT in two stages, and provision of additional experience in sexual and territorial behaviors during the interval between the first and second transection, did not alleviate the behavioral deficits. Impairments in mounting and scent marking were dissociated in some subjects, indicating individual differences in the hamsters' dependence on chemosensory cues for the regulation of these two types of social behavior. It is suggested that the apparent sparing of behavioral function which has been associated with gradual destruction of nervous tissue is due to the development of alternate mechanisms of response to environmental cues, and may be more difficult when brain damage is in structures which initially process these cues. Although sexual and territorial behaviors in the hamster are dependent on gonadal hormones, the observed impairments of these behaviors could not be explained as indirect consequences of lesion effects on the hypophysiogonadal axis. Circulating testosterone (T) levels in experimental subjects were not different from levels in control (sham-operated) subjects. An inverse correlation was observed betweenthecontrol subjects' circulating T levels and their incidence of scent marking.A similar correlation was observed between postoperative T levels and preoperative marking rates in experimental subjects, despite the fact that most subjects failed to scent mark after bilateral LOT transections. The latter observation strengthens the inference that the behavioral deficits were independent of changes in basal hormone levels.

98 INTRODUCTION

Within the last few years there have been a number of studies showing that destruction of bilaterally symmetrical CNS structures in two separate operations can reduce or eliminate many of the behavioral deficits which follow bilateral ablation inflicted in one stage, and that this reduction of deficits is facilitated if subjects are given additional behavioral experience during the interval between the first and second ablation16, is. Yet not all behaviors are spared following serial lesions. In male hamsters, for example, mating behavior is eliminated by one-stage lesions of the main and accessory olfactory bulbsla,ao, aS, and bilateral destruction of the olfactory bulbs in two stages does not preserve male copulatory behavior even if subjects are given interoperative sexual experience 4s. Olfactory and vomeronasal inputs to the main and accessory olfactory bulbs, respectively, have been shown to exert a cooperative influence on the maintenance of copulatory behavior in male hamsters 37. Sexual behavior ceased in the majority of subjects only when both vomeronasal and main olfactory inputs were eliminated. The main and accessory bulbs project centrally via the lateral olfactory tract (LOT) and synapse predominantly in temporal lobe structures such as the prepyriform cortex and amygdala 23,a~,45. Devor 10 has noted that lesions of the latter structures produce bizarre aberrations in the sexual behavior of male rats 2°, cats 21, monkeys 27 and humans 43, and he has attempted to dissociate their sexual functions in the hamster by cutting the LOT at progressively more caudal levels. He found that bilateral LOT transections eliminated mounting of females in all subjects, but that precopulatory courtship behavior was preserved with more caudal cuts which left bulbar inputs to the olfactory tubercle and adjacent parts of the prepyriform cortex intact. Many of Devor's transections would be expected to have spared collateral LOT projections to the anterior olfactory nuclei (AON). The AON are recognized primarily as pathways for contralateral interactions between the olfactory bulbs and for centrifugal influences to the bulbs from the prepyriform cortices 2~,aS,3s. However, there have been reports of centripetal projections from the AON to limbic and basal forebrain structures which also receive olfactory bulb influences by way of the LOT projections to the temporal 10be7,17,31. Devor's findings indicate that the alternate pathways via the AON normally cannot support copulatory behavior in the hamster. A possible interpretation of the failure to find sparing of sexual behavior in male hamsters after two-stage ablations of the olfactory bulbs is that no alternate pathways capable of assuming the necessary function during the interoperative interval remained after both bulbs were removed. That is, the central projections from the AON might have become capable of supporting mating behavior if only the LOT inputs to the temporal lobe had been severed in a serial fashion. Thus, one goal of the present study was to determine whether the elimination of olfactory and vomeronasal influences to the temporal lobe bilaterally in two stages, while sparing the olfactory bulbs and portions of the AON, can obviate the deficit in mounting behavior by male hamsters. Although studies of olfactory influences in hamsters have focused predominantly on sexual behavior, the olfactory bulbs also may be expected to play a role in the

99 hamster's territorial behaviors such as scent marking, food hoarding and nesting 25,3z. A second aim of the present study was to compare the effects of one- and two-stage LOT transections on sexual v e r s u s territorial behaviors for individual subjects, and to determine whether such effects can be dissociated. Both mating and scent marking by male hamsters depend on gonadal hormones TM, and the degree of interest in a female related stimulus (vaginal odor) exhibited by individual males is positively correlated with their plasma testosterone (T) levels 32. A third goal in this study was to determine whether individual differences in scent marking are also correlated with differences in plasma T levels, and whether behavioral deficits after LOT cuts are associated with low T levels. MATERIALS AND METHODS

Adult, male, Syrian golden hamsters (Engle Laboratory Animals, Inc.) were housed individually in opaque fiberglass cages (46 cm × 28 cm × 17 cm; wire lids) containing corn cob bedding, Purina lab chow and cotton nesting material. A schedule of 14 h light- 10 h dark (red lighting) was maintained, and behavioral testing occurred during the dark period. Ovariectomized females were housed two per cage and were brought into estrus with subcutaneous injections of 50/~g estradiol benzoate in 0.05 ml sesame oil and 100/~g progesterone in 0.05 ml propylene glycol administered 48 and 4 h, respectively, prior to testing. Starting at least 3 weeks before surgery, the locations of the food hoard and nest maintained by individual subjects were recorded daily. On one day each week subjects had a receptive female placed in their cages for 30 min or until 15 separate mounts had occurred. On another day each week subjects were placed individually in a fiberglass cage containing a mixture of bedding from other males' cages, and the number of scent markings occurring during the 30-min residence in the strange cage were recorded. Subjects were assigned without preference to two-stage, one-stage and sham groups as they achieved 15 mounts on each of 3 successive exposures to a female. LOT transections were performed with the techniques developed by Devor 11. On the first day of surgery (day 0) the LOT was transected on one or both sides fol the two- and one-stage groups, respectively. The LOT was exposed by removing the overlying skull area at the anterior border of the orbit. The eye was carefully retracted and covered with cottonoid pads during LOT exposure so as to avoid damage to the optic nerve and Harderian gland. For the two-stage group, the intact LOT was exposed and transected on day 35. For the sham group, the LOT on both sides was exposed but not transected on day 35. Daily recording of food hoard and nest locations was continued until 4 weeks postsurgery for the one-stage and sham groups, and until 4 weeks after the second LOT transection for the two-stage group. Subjects had an estrous female placed in their home cages for 30 min or until 15 mounts on days 7, 21, 42 and 57. The incidence of scent marking during 30 min in a strange cage was recorded on days 8, 22, 43, and 58. In the week following completion of all behavioral testing, blood samples (0.3 ml) were collected by cardiac puncture under Metofane anesthesia a2. Samples

IN ONE (SOLID BAR)

Sham

H4S H8S HIOS H15S H16S

One-stage H I B H6B H7B HI2B HI4B

Two-stage H 2 R L H3LR H5RL H9RL HIILR HI3LR

Subject

15 15 15 15 15

ns

ns

15 15 15 15 15

15 15 15 15 15

ns

RS

15 15 15 15 15

15 15 15 15 15 15

15 15 15 15 15 15

15 15 15 15 15

ns

15 t5 15 15 15

RS

15 15 15 15 15 15 m

15 15 15 15 15

15

*

1 15 0 0 0

15 15 15 15 15 15 ns

15 15 15 15

ns

15 15 15 0 0

15 15 15 15 15 15 ns

Day 21

[1

~

~ ~

Day 35

15 15 15 15

15

*

0 t5 0 0 0

0 0 0 0 0 0 **

D~v 42

15 15 15 15

15

*

0 15 0 0 0

0 0 0 0 0 0 **

Day 57

3 2 1 0

2

ns

0 10 7 12 0

3 3 8 4 14 12 §

0 7 16 1

8

ns

0 13 5 |0 0

4 5 9 5 10 8 ns

2 3 3 0

2

ns

6 8 1 2 4

3 0 12 3 9 8 ns

!

Day 0

Pre-op

Day 7

Pre-op

Day 0

Number o f scent marks

Number o f mounts

6 5 5 4 0

2 6 3 7 2

*

2

1 *

0 0 0 0

11 2 5 7 ns

12 5 13 8 ns

1

5

4 0 0

2

1

Day 22 3

Day 8

~ ~

~

~

Day 35

5 4 5 7 2

*

2

0 0 0

0

0 0 0 0 **

I

0

Day 43

4 1 4 3 0

HS

3

0 0 0

0

0 0 0 0 **

0

0

Day 58

Statistical comparisons (Mann Whitney U-test) are between each experimental group versus the sham-operated (open bar) group on comparable test days. * = less than shams, P < 0.05; ** ~ less than shams, P -< 0.01: § = greater than shams, P < 0.05; ns = no significant difference, P > 0.05.

OR T W O ( H A T C H E D BARS) STAGES

INCIDENCE OF M O U N T I N G A N D SCENT M A R K I N G D U R I N G THE 3 PREOPERATIVE WEEKS A N D AFTER BILATERAL T R A N S E C T I O N OF THE L O T

TABLE I

101 were taken on two different days, separated by 3 days, during the second to sixth hours of dark. Blood was centrifuged and plasma was frozen. Plasma T levels were determined by radioimmunoassay g. The sensitivity of this assay is approximately 50 pg T. The mean of the two determinations for each subject was used as the baseline T level in correlations with scent marking. One subject (H3LR) died of unknown causes prior to blood collections. For histological verification of lesions, subjects were perfused intracardially with isotonic saline followed by 10 ~ formalin-saline after the second blood collection. Brains were inspected with a dissecting microscope and photographed prior to imbedding, and the locations and extents of transections were indicated on standard diagrams. Serial sections (frozen) were cut 30/~m thick and stained with cresyl violet. RESULTS

The effects of LOT transection on mounting and scent marking, and levels of significance, are indicated in Table I. Relative to the sham group, both the oneand two-stage groups showed a significant reduction of mounting and scent marking after the LOT had been cut on both sides. There was no significant reduction for the two-stage group after transection of one LOT, but there was a nearly complete absence of mounting and scent marking after the second transection. Prior to surgery all subjects maintained a food hoard and nest in separate regions of the cage and for individual subjects, their locations within the cage were consistent from one day to the next. To provide a quantitative measure of nesting (N) and food hoarding (F) behavior, the locations of food pellets and cotton nesting material were depicted daily on diagrams which represented each subject's cage divided into quadrants. The N and F scores were calculated separately. Each day, the subject received a score of 4 if the nest (hoard) was in the customary quadrant, and a score of 2 if it was in a different quadrant. One point was reduced from the score if some cotton (food pellets) also appeared in other quadrants. Zero scores were assigned if cotton or food pellets were distributed approximately evenly over two or more quadrants. The daily scores were averaged to provide weekly N and F scores. These are presented in Table II. The results parallel those for mounting and scent marking. There were no significant differences between the shams and the two-stage group after transection of one LOT, and both experimental groups exhibited significant reductions in N and F scores after bilateral transection (significance levels in Table II). The relative locations of LOT cuts for the experimental groups are depicted in Fig. 1. Transections were complete except for subject H6B (one-stage), which had the medial portion of the left LOT intact. This animal continued to mount estrous females vigorously during the postoperative period of testing, but ceased scent marking after the first postoperative week. Subject H6B changed the locations of its nest and food hoard during the evening following surgery and thereafter maintained them in their new locations. Subject H14B (one-stage) continued to scent mark during all postoperative tests despite complete bilateral transection of the LOT with concomi-

102 TABLE I1 EFFECTSOF LOT TRANSECTIONSON NESTING(N) AND FOODHOARDING(F) See text for scoring procedures. Pre-op scores are for the week immediately preceding surgery. Statistical comparisons and symbols are the same as in Table I. Subject

Pre-op

Week Week Week Week 1 2 3 4 N-F N-F N-F N-F

N-F

Two-stage

H2RL H3LR H5RL H9RL HI1LR H13LR

4-4 4-4 4-4 4-4 4-4 4-4

One-stage

HIB H6B H7B HI2B HI4B

Sham

H4S H8S H10S H15S HI6S

~ ~

4M 4M 4-4 4M 4-3 3-3

4-4 4-4 4-4 4-4 4-3 34

4-4 ~4 4-4 4-4 3-3 34

4M 4M ~4 4-4 3-3 3-4

4-4 4-4 4-4 4-4 4-4

2-0 2-2 2-2 4-3 4-2

2-0 2-2 2-2 31 4-3

2-0 2-2 2-3 2-0 21

10 2-2 I~ 3-0 0-1

4-4 4-4 4-4 4-4 4-4

3-4 4-4 3-4 4-4 4-4

3-4 4-4 3-4 4-4 4-4

3-3 44 3-4 4-4 4-4

3-4 4-4 3-4 4-4 4-4

Week Week Week Week 6 7 8 9 N-F N-F N-F N-F

~ ~

2-2 2-2 4-0 4-1 3-1 3-3

0-0 2-0 2-0 4-4 I 0 2-1

2- I 0-0 0-0 2-2 1-0 10

0-2 0-0 0-0 0-0 0-0 %0

tant cessation of m o u n t i n g and deterioration o f nesting and food h o a r d i n g behaviors. Three other subjects ( H I B , H 3 L R and H7B) with complete bilateral transection showed a persistence o f scent marking and/or vigorous mating during the first postoperative week, but eventually ceased to exhibit either behavior. D a m a g e to the anterior olfactory nuclei and/or olfactory tubercle, as judged from stained sections (see Fig. 2), was minimal for all subjects. There was substantial retrograde degeneration o f mitral and tufted ceils in the olfactory bulbs. However, normal neurons were present (approximately half as many as were seen in shamoperated controls). There were no systematic differences in extent or locus o f damage for subjects with one- or two-stage transections. G r a n d means and ranges for baseline T levels and for body weights at the time o f blood collections are presented in Table III. All subjects had detectable levels o f plasma T. The range o f baseline T levels for the experimental groups was somewhat larger than for shams, but variances were homogeneous (Fmax test, P > 0.05) and analysis o f variance revealed no significant differences (P > 0.05). There was a non-significant trend toward greater weight after bilateral transection o f the LOT. Subjects H 12B, H7B, H 11 LB and H 1B had the highest weights, respectively (218-169 g), and appeared obese. Their cages were notable in having become carpeted with partially nibbled food pellets.

103 TWO-STAGE

ONE- STAGE

&& Fig. 1. Relative locations of LOT cuts as viewed on the ventral surface and plotted on standard diagrams. Dark shading indicates the extents to which the white matter of the LOT was absent in the regions of the cuts. The order of cuts is indicated for the two-stage group. The completeness of

transections for subject H3LR was assessed only by inspection with a dissecting microscope. For the remaining subjects, the completeness of cuts and the extents of invasion into the adjacent gray matter of the AON and olfactory tubercles were judged from stained sections (see Fig. 2). Plasma T levels for individual subjects in the sham group were compared with their mean number of scent markings during the 7 tests. There was a nearly perfect inverse correlation (Pearson r = -0.94; df -----4; P < 0.01, two-tailed). Since scent marking was virtually eliminated after bilateral LOT transections, the postoperative T levels for individual subjects in each experimental group were compared with their mean number of scent markings during the 3 pre-operative tests. The one-stage group also showed an inverse correlation comparable to that for the sham group (r = -0.93; df = 4; P < 0.01), and the two-stage group showed a non-significant trend in the same direction (r = -0.56; df = 4; P > 0.05). DISCUSSION

Transections of the LOT near the anterior border of the olfactory tubercle

104

¸ ii !i

!IE ¸

ii~

~

i

i

~

ii~i

! !! !~

~

Fig. 2. Representative cresyl violet stained sections taken at 180-ttm intervals and showing the extent of invasion into the AON and olfactory tubercle for the right LOT transection in subject HI B. The LOT was completely destroyed at the level of section C and the knife cut was extended mediocaudally to the anterior border of the olfactory tubercle. Calibration in F is 1.0 ram.

105 interrupt at least 3 neural pathways: (i) centripetal projections from the main olfactory bulb to extensive portions of the temporal lobe2a,36; (ii) centripetal projections from the accessory bulb to the corticomedial amygdala4~; and (iii) centrifugal projections to the olfactory bulbs from the nucleus of the horizontal limb of the diagonal band of Broca (HLDB) in the basal forebrain as. In hamsters, the present findings indicate that some or all of these pathways are of major importance not only for copulatory behavior, but also for the maintenance or elicitation of territorial behaviors. However, our data also can be taken to indicate that deficits in mounting and scent marking behaviors may be dissociated for individual subjects. Following bilateral damage to the LOT, one subject failed to mount females but continued to scent mark when placed in a strange cage. Another subject did mount females but ceased to scent mark. In neither case can the sparing of behavioral function be attributed to a beneficial effect of serial lesions, since both subjects were in the one-stage group. All animals in the two-stage group showed deficits in mounting, scent marking, food hoarding and nesting behaviors despite the occurrence of additional experience in these behaviors during the 5 week interval between the first and second transection. The deficits in mounting and territorial behaviors do not appear to be indirect consequences of lesion effects on the hypophysio-gonadal axis. For both experimental groups the range of plasma T levels completely overlapped that for the shams. The effects of LOT transections on feeding patterns were poorly reflected in the weight measurements. However, our informal observations of 4 experimental subjects (3 one-stage, one two-stage) in their home cages complement those of Murphy (personal communication) who has noted that hamsters with bilateral ablations of the olfactory bulbs tend to manipulate and mouth food pellets during much of their waking time. The finding of inverse correlations between individual subjects' plasma T levels and their incidence of scent marking is counterintuitive. Scent marking in hamsters is reduced following castration and returns to high levels with replacement therapy 44, so that a positive correlation might be expected. However, the inverse correlations for both the sham and one-stage groups were quite high and should not be too readily dismissed as spurious. Ak, o, T levels were determined 5 weeks sooner after surgery for the two-stage than the one-stage groups, and the weaker correlation for the twostage group may reflect residual effects of brain trauma. All subjects had mean T levels within the range of baseline values previously reported for sexually experienced hamstersSL Presumably, each subject's mean T level was adequate for long-term, trophic influences on the nervous system. The biological significance of variations within the normal range is not obvious. For example, the sensitivity of neural tissue to hormones need not be identical across subjects and relatively lower baseline levels could reflect a much higher sensitivity or ability to utilize the hormone 22,46. Also, intuitions based on castration-replacement studies can lead to erroneous behavioral predictions. In the hamster, exposure of sexually experienced males to vaginal odor produces acute surges in plasma T levelsaL Since aggressiveness in hamsters has been correlated with the presence v e r s u s absence of gonadal hormones44, we might expect vaginal odor to make male hamsters more aggressive. In fact, vaginal odor reduces

106 TABLE llI GRAND MEANS, STANDARD DEVIATIONS AND RANGES FOR POSTOPERATIVE PLASMA TESTOSTERONE LEVELS AND BODY WEIGHTS

Two-stage (N -- 5) One-stage (N = 5) Sham (N = 5)

Mean T ± S.D. (range T) in ng/ml

Mean Wt. ± S.D. (range Wt.) in g

1.65 & 1.80 (4.80-0.28) 2.44 ± 1.79 (4.51-0.42) 2.05 ± 0.71 (3.02-1.38)

141 ~ 22 (173-120) 168 ± 40 (218-127) 138 ± 9 (150-129)

inter-male aggression in hamsters26, 3~. Effects of gonadal hormones on tissues other than the brain also should be considered. Scent marking in hamsters is performed by rubbing the dorsal flank glands against environmental objects14, 25, and the size of these glands is testosterone-dependent 45. Subjects with lower basal secretory rates may have less well developed or less active glands, and the higher incidence of scent marking in these subjects may be compensatory. If the observed correlations between scent marking and hormone levels measured many weeks apart are valid, they suggest a far more stable basal secretory rate for T in the hamster than has been reported in other rodent species 3. They also strengthen the inference that behavioral effects of LOT transections were independent of effects on gonadal function. That is, the rank order of postoperative T levels for subjects in the one-stage group was almost exactly that predicted by their preoperative marking scores, yet all but one of these subjects stopped marking postoperatively. The present findings highlight the importance of determining hormone levels in individual subjects, and not relying solely on extrapolations from castration-replacement studies, in attempts to relate endocrine function and behavior. The present findings also may help to define the limits of recovery that are found to occur after sequential lesions in the CNS. Serial lesion procedures have been shown to minimize behavioral deficits following brain damage to both cortical and subcortical structures, and it has been hypothesized that gradual destruction provides an opportunity for remaining neural tissue which was not involved, or was minimally involved, in the behavioral function to assume a greater role 16,1s. In most cases the damage was to central structures which may be viewed as multisensory and/ or motor. A noteworthy exception to this generalization is a report in the mouse that ablation of the olfactory bulbs in two stages resulted in sparing of copulatory behavior which was eliminated following simultaneous bulb removal 41. However, the two-stage group in the latter study had less complete lesions than the one-stage group and may have retained some chemosensory function. In the present study, the one experimental subject which continued to mount postoperatively had an incomplete transection of one LOT. It appears that copulatory behavior can be maintained in male hamsters when only a few of the LOT fibers remain intact. Although complete ablations of the olfactory bulbs impair male sexual behavior in hamsters 15,~°,35, mice 40,41,47, rats 4,6,9,24,28 and rams 19,29,39 (but not in rabbits 8 or cats'), the hamster (and possibly the mouse) is relatively unique among laboratory mammals in being

107 critically dependent on chemosensory information for the regulation of its social behaviors. Peripheral olfactory and vomeronasal blockage eliminates self-initiated sexual and aggressive behaviors in male hamsters (anosmic subjects will fight back if physically attacked), and thus produces a syndrome which has been termed 'social agnosia '1z,83. If the present findings can be fully replicated following peripheral anosmia, this syndrome might be extended to include deficits in territorial behaviors in the subjects' home cages and in the cages of other conspecifics. The LOT transections may have produced a generally insurmountable sensory deficit for this species, although the dissociation of deficits in mounting and scent marking for two subjects suggests that individual hamsters may be differentially dependent on chemosensory cues for these behaviors. The behavioral sparing which commonly accompanies gradual neural destruction may be due, in part, to the development of alternate mechanisms of response to environmental cues, and may be more difficult or impossible when damage is to structures which initially process these cues. If this interpretation is correct, the centripetal projections from the A O N which may have survived LOT transections do not appear to represent a 'parallel' pathway to the basal forebrain potentially capable of providing the chemosensory cues which are processed via LOT projections to the temporal lobe. However, we should not exclude the possibility that the A O N play an important complementary role in the processing of chemosensory cues which regulate the hamster's social behavior. ACKNOWLEDGEMENTS

We thank Dr. Marshall Devor for demonstrating the surgical procedures used in this study. We also thank Dr. B. V. Caldwell for antiserum to testosterone, and Susan Dalterio for assistance with the radioimmunoassays. This investigation was supported by NINCDS Grant 1 RO1 NS12344, NSF Grant BMS75-07652, N I C H D Grant 5 RO1 HD06867, Research Career Development Awards MH70177 (to D.G.S.) and HD70369 (to A.B.), and funds made available through the generosity of Clark University. REFERENCES 1 ARONSON, L.R., AND COOPER,M.L., Olfactory deprivation and mating behavior in sexually experienced male cats, Behav. BioL, 11 (1974) 459--480. 2 BAOR, F. M., AND BARrrOS,A., Effect of ethyl alcohol on plasma testosterone level in mice, Steroids, 23 (1974) 921-928. 3 BARrg~, A., Sr~I~, R. E., MusTo, N., ANDCALDWELL,B. V., Fluctuations in plasma testosterone levels in adult male rats and mice, Endocrinology, 92 (1973) 1223-1228. 4 BEACH,F. A., An analysis of the stimuli adequate to elicit mating behavior in the sexually inexperienced male rat, J. comp. PsychoL, 33 (1942) 163-207. 5 BEACH,F. A., ANDPAUr~R, R. S., Effects of castration and subsequent androgen administration upon mating behavior in the male hamster, Endocrinology, 45 (1949) 211-221. 6 BeRMAt,rr, G., ANOTAYLOR,L., Interactive effects of experienceand olfactory bulb lesions in male rat copulation, PhysioL Behav., 4 (1969) 13-17. 7 BROADWELL,R., The Afferent andEfferent Connections of the Anterior Olfactory Nucleus (AON) in the Rabbit as studied with the Autoradiographic and Horseradish Peroxidase (HRP) Axon Tracing Methods, Paper presented to Society for Neuroscience, St. Louis, 1974.

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