Influence of isolation and training on fighting in mice with olfactory bulb lesions

Phvstolog) & Behavu~r,Vol 31, pp 857-860 Pergamon Press Ltd, 1983 Printed in the U S A

BRIEF COMMUNICATION

Influence of Isolation and Training on Fighting in Mice with Olfactory Bulb Lesions JOHN

P DAVANZO,* MELODY SYDOW* DAVID R GARRIS?

AND

Departments o f Pharmacology* and Anatomy?, School o f Medtcme, East Carohna Umverstty Greenvdle, N C 27834 Recetved

19 M a y 1983

DAVANZO, J P , M SYDOW AND D R GARRIS Influence of tvolatton and oammg on fightmg tn mtce wtth olfactory bulb leston~ PHYSIOL BEHAV 31(6) 857-860, 1983 --Isolation-reduced aggressive mice receiving total bdateral bulbectomy faded to fight after chromc trammg Ammals receiving sub-total olfactory bulbectomy were capable of being trained to attack but the latency to attack was mcreased When mice were bulbectommed before being isolated they were incapable of attack regardless of training or completeness of the lesion These results indicated that ~solat~on mfluences aggresswe behavmr m bdaterally bulbectomlzed mice Isolation Induced aggressive behavior and olfactory bulbectomy Olfactory bulbectomy Training and fighting mice Total and subtotal olfactory lemons and fighting behavior m mice

IT is well recognized that male mice exhibit spontaneous c o m b a t i v e b e h a v i o r F a c t o r s which are k n o w n to affect social d o m i n a n c e in mice include the physiological state of the animal, weight differences, genetic differences m aggressiveness and conditioning [5] Individual mice may be conditioned to be made m o r e aggressive as a result of continued victories o v e r submissive mice [5] Further, ff mice are isolated for varying periods o f time the aggressive b e h a w o r is heightened with time [1] Thus, isolation-reduced fighting in male mice has b e c o m e a c o m m o n means of studying aggression Scott [8] d e v e l o p e d a t e c h m q u e for training isolated mice which resulted in the d e v e l o p m e n t of exceedingly aggressive ammals w h o s e latency for attack was reduced to a minimum In a d d m o n , fighters so d e v e l o p e d frequently did not show some o f the usual p r e h m m a r y mamfestatlons o f fighting such as tail rattling and h o m p d a t i o n but simply learned to attack o p p o n e n t s at once Ropartz [6] and others [3] d e m o n s t r a t e d that bilateral ablation of the olfactory bulbs of aggressive mice e h m m a t e d fighting b e h a v i o r We w e r e interested in learning w h e t h e r e x t e n s i v e training o f bulbectom~zed m~ce that were aggresswe before surgery would reverse the submissive b e h a v i o r H o w e v e r , it remains to be determined if the suppressive effects of b u l b e c t o m y on aggressive b e h a v i o r can be r e v e r s e d through training In a d d m o n , how w o u l d mice b e h a v e if they w e r e b u l b e c t o m l z e d before they w e r e isolated 9 The results o f these studies are the subject of this c o m m u m c a t l o n

METHOD

Ammals Male albino Dubhn (ICR) mice, 26-28 days old, e x c e p t where noted differently, were isolated for 21 days in selfcleaning cages 4 × 5 × 9 " , solid walled e x c e p t for front and b o t t o m screens The animals w e r e housed in an air conditloned room, used exclusively for these studies, and mamtamed at 76°F (___7°) with relative humidity uncontrolled Lights were controlled automatically to go on at 7 a m and off at 7 p m

Development of Aggresstve Behavtor The general aspects deahng with the d e v e l o p m e n t of agg r e s s w e b e h a v i o r were described previously ( D a V a n z o et al [1] Since the protocol for the present studms v a n e d somewhat from that of previous studies, the m e t h o d will be briefly r e d e s c n b e d , incorporating the changes U p o n completion of the 21 day isolation period, the mice, whde stdl m their isolation cages, were placed on a bench top for 3 minutes They were not r e m o v e d from the cages in which they were isolated During this 3 minute training period the animals were gently prodded on the lund quarters with long forceps and their tails w e r e pinched hard enough to e v o k e a squeak response, but not so hard as to cause injury On three occasions during the training period a non-isolated " n o r m a l "

Copyright © 1983 Pergamon Press Ltd--0031-9384/83/120857-04503 00

858

DAVANZO, SYDOW AND GARRIS

FIG 1 Olfactory bulb lesions (A Bx) were confined to the regions rostral to the prepynform cortex (pc) A normal (N) brain is exhibited for companson Olfactory tract neurons m the prepyrlform cortex (C) were observed to be undergoing degeneration m lesioned (D) animals Neurons m the pc of normal (D) and Bx (E) ammals are demonstrated (×20), w~th degenerated neurons exhibiting an intense staining of the perlkanon

m o u s e o f smaller size a n d r e a r e d m aggregation, w a s picked up b y the tad a n d b u m p e d into the ~solated m o u s e A t the e n d o f the t r a m l n g p e r i o d the n o r m a l s t i m u l u s m o u s e w a s introd u c e d into the ~solated m o u s e ' s h o m e cage a n d the l a t e n c y o f attack recorded with a stopwatch At the end of 3 minutes (180 sec), tf n o a t t a c k o c c u r r e d , the s u m u l u s a m m a l w a s r e m o v e d f r o m the h o m e cage a n d the a m m a l was s c o r e d as a non-fighter

5urgtcal Procedule and Tleatrnent oJ Tissue M~ce w e r e a n e s t h e U z e d with I P a d m t m s t r a t ~ o n o f s o d m m

p e n t o b a r b l t a l (35-50 m g - k g ) and the skin was o p e n e d f r o m L a m b d a to the t~p o f the n o s e B r e g m a , the c o r o n a l s u t u r e s a n d frontal suture w e r e identified a n d a dental drill was used to r e m o v e a 2 m m strip o f b o n e o v e r l y i n g the o l f a c t o r y b u l b s at a b o u t m t d - p o m t B o t h b u l b s w e r e s e v e r e d f r o m t h e b r a i n e i t h e r by c a u t e r y or s u c t i o n A f t e r p a c k i n g the s p a c e with gel f o a m , a topical antibiotic (Furacln) was a p p h e d T h e skin was c l o s e d with metal clips a n d s p r a y e d w i t h aerosol merthiolate Skin c h p s were r e m o v e d u n d e r e t h e r a n e s t h e s i a 7-10 days post-operatively A t t h e c o m p l e t i o n o f the b e h a v i o r a l studies, the a m m a l s w e r e again a n e s t h e t i z e d (50 m g - k g s o d m m p e n t o b a r b l t a l ,

OLFACTORY BULBECTOMY AND AGGRESSION

859

TABLE 1 TOTAL AND SUBTOTAL BULBECTOMY AND FIGHTING BEHAVIOR Number of Animals

Sham Operated 12

Subtotal Bulbectomy 11

Total Bulbectomy 11

2 2 - 031 1 7 - 0 20

50_+ 098 121 7 _+ 20 6*

1 3--- 045 179 6 ± 0 36*

2 7 --- 0 41

124 0 ± 18 6*

177 1 _+ 2 9*

100 100

100 75

100 9

100

84

9

Average Attack Latency (sec) Pre-operaUve(5d) 1st training session (10d 2nd training session (20d) Percent Fighting Pre-operative 1st training session

2nd training session

Percent Bulb Remalmng Range Average

100 100

10-50 31 ± 2 6

2

0-5 ± 10

*All values are represented as group means ( - S E M ), *indicates p < 0 001 vs controls TABLE 2 BILATERAL BULBECTOMY BEFORE ISOLATION

Number of Ammals Average Attack Latency (sec) 1st trammg session (7d) 2nd training sessmn (5d) Percent Fighting 1st training sesston 2nd training session Percent Bulb Remaining Range Average

Subtotal Bulbectomlzed

Nonbulbectomlzed

11

11

180 + 0* 180 -+ 0~

114 -+ 26 71 ± 27

0 0

73 64

5-80 28 ± 7 5

100 100

*Indicates p < 0 02 vs non bulbectomlzed, tlndlcates p < 0 001 vs non bulbectomized

IP), perfused via the ascending aorta with E a r l e ' s balanced salt solution containing s o d m m mtrate (0 1 mg/100 ml) and fixed by perfuslon with 2 5% glutaraldehyde m 0 1 M phosphate buffer The brain was r e m o v e d from the skull, submerged m the glutaraldehyde fixative for 1 hr and transferred to 70% alcohol A v~sual estLmate of the intact, proximal olfactory bulb was m a d e and photographs m a d e R e p r e s e n t a t i v e brains were collected for histological analysis of the olfactory bulb lesmns The forebram was isolated and prepared for hght mtcroscop~c examination using c o n v e n t i o n a l t e c h m q u e s N~ssel stamed, serial sections w e r e analyzed and the p r e s e n c e o f degenerated neurons m the olfactory tubercle and p r e p y n f o r m c o r t e x w e r e used as determining criteria for the c o m p l e t e disruption o f both the medml and lateral olfactory tracts (Fig 1)

Testing Procedure F r o m a group o f mice treated as described a b o v e 11 w e r e r a n d o m l y selected that attacked a sttmulus ammal Wltlun 1-10 sec on 5 c o n s e c u U v e days U p o n completion o f training the ammals w e r e parUally b u l b e c t o m t z e d The goal here was to leave intact approxunately 50% of the bulbar t~ssue A f t e r a 3 day r e c o v e r y p e n o d the animals w e r e retramed for 10 c o n s e c u U v e days, rested one w e e k and retrained for 20 consecutlve days A second group o f 11 animals was treated essentially as described e x c e p t that 100% o f the olfactory bulb was r e m o v e d A third group o f 11 animals was sham operated in which skull holes w e r e drilled but the olfactory bulbs w e r e not m a m p u l a t e d In a separate experiment, the o b j e c t w e was to ascertmn h o w b u l b e c t o m y before isolation

860

DAVANZO, SYDOW AND GARRIS

influenced the d e v e l o p e m e n t o f aggressive b e h a v i o r O n e g r o u p of e l e v e n animals, 40-50 d a y s old, weighing 22-35 g w a s b u l b e c t o m l z e d and isolated l m m e d m t e l y after surgery Adult a n i m a l s w e r e utd~zed here b e c a u s e p r e v i o u s experie n c e r e v e a l e d that 26--28 day bulbectom~zed m~ce d~d not s u r v w e the Isolation p r o c e s s A s e p a r a t e g r o u p o f 11 a n i m a l s s e r v e d as c o n t r o l s T h e y w e r e not s h a m o p e r a t e d since preh m l n a r y e x p e r i m e n t s i n d i c a t e d t h a t p r o c e d u r e d~d not influe n c e fighting b e h a v i o r In th~s e x p e r i m e n t , v a r y i n g a m o u n t s o f o l f a c t o r y tissue, ranging from 5-80%, as verified at autopsy, were left intact RESULTS T h e results of the e x p e r i m e n t s dealing w~th total a n d subtotal b u l b e c t o m y are i n c l u d e d in T a b l e 1 In the g r o u p o f a m m a l s w~th total b u l b e c t o m y f e w e r t h a n 20% o f the a n i m a l s w e r e e v e r o b s e r v e d to a t t a c k d u n n g e i t h e r o f the training periods T h e a t t a c k latency time for the two mice t h a t did a t t a c k w a s greatly p r o l o n g e d w h e n c o m p a r e d to m t a c t controls In c o m p a r i s o n , a n i m a l s w h i c h w e r e f o u n d to be subtotally b u l b e c t o m l z e d d e m o n s t r a t e d a h i g h e r p e r c e n t a g e o f fightmg b e h a v i o r , but a similar latency time to a t t a c k , as c o m p a r e d w t t h mtce w~thout o l f a c t o r y b u l b s All a m m a l s that w e r e b u l b e c t o m t z e d b e f o r e they w e r e isolated failed to d e v e l o p aggressive b e h a v i o r r e g a r d l e s s o f the a m o u n t o f bulb a r ttssue w h t c h w a s left intact (Table 2) T h e latency p e r m d and p e r c e n t a g e of fighters t h a t d e v e l o p e d from the nonb u l b e c t o m l z e d a m m a l s in this e x p e r i m e n t w a s u n e x p e c t e d l y long, p e r h a p s due to the o l d e r age o f the a n i m a l s w h e n lsolau o n was initiated DISCUSSION R e m o v a l of b o t h o l f a c t o r y b u l b s in m~ce results m a n u m b e r o f s o c m l - b e h a v l o r a l deficits o n e o f w h i c h ~s the e h m l n a t l o n o f aggressive b e h a v i o r m male m~ce A s pointed out b y R o w e a n d E d w a r d s [7], the t e r m a g g r e s s i o n has b e e n u s e d to d e s c r i b e a v a r i e t y of b e h a v i o r s ranging f r o m ratermale fighting a m o n g m~ce to s t r u g g h n g a m o n g food d e p r i v e d m~ce m a s l t u a t m n w h e r e they were m c o m p e t l t m n for food T h e s e s a m e i n v e s t i g a t o r s s h o w e d t h a t i n t a c t olfactory b u l b s are not an a b s o l u t e r e q m r e m e n t for fighting b e h a v i o r to take place m a s l t u a t m n w h e r e the a m m a l s were c o m p e t i n g for food In t h e i r studies bulbectom~es r a n g e d in c o m p l e t e n e s s from total r e m o v a l o f b o t h olfactory b u l b s and p e d u n c l e s to

r e m o v a l of only the rostral third o f b o t h bulbs T h u s , e v e n partml d a m a g e to t h e olfactory b u l b s m a y i n d u c e nearm a x i m a l b e h a v i o r a l (aggressive) c h a n g e s m mice T h e p a r h c u l a r p a r a d i g m t h a t was s e l e c t e d for the p r e s e n t studies dealt with an e n d p o i n t t h a t is, at least m part. a l e a r n e d r e s p o n s e (~ e , aggression) S e l e c t i o n o f a t t a c k beh a v m r as the end p o i n t was u s e d b e c a u s e d u n n g the training p r o c e s s m a n y o f the m t e r m e d m t e and p r e h m m a r y b e h a v i o r a l m a n i f e s t a t i o n s fall to take place or o c c u r m a variable mann e r T h e role o f lsolatzon m the d e v e l o p m e n t o f fighting beh a w o r ~s not well u n d e r s t o o d Isolation is not an e s s e n t m l r e q m r e m e n t for fighting b e h a v t o r to o c c u r [2] but is o b w o u s l y h e i g h t e n s the b e h a v i o r T h e results o f the p r e s e n t studies d e a h n g w~th b u l b e c t o m y b e f o r e ~solat~on are o f interest m this regard since t h e y suggest t h a t isolation a c c e n t u a t e s the d e v e l o p m e n t o f fighting b e h a w o r if the a n i m a l s are b u l b e c t o m l z e d before t h e y are isolated T h e s e findings are m a g r e e m e n t w~th p r e w o u s studies [1] indicating that a relat i o n s h i p b e t w e e n learned aggressive b e h a v i o r a n d ~solat~on exists m m~ce W h a t ~s o f f u r t h e r interest ~s that ~t does not s e e m to m a k e a d~fference h o w m u c h olfactory t~ssue ~s left intact m a m m a l s b u l b e c t o m l z e d prior to ~solatzon with regard to e x p r e s s e d aggression T h e situation with regard to the a m m a l s that were m a d e a g g r e s s w e b e f o r e they w e r e bulbectom~zed ~s quite the oppostte E d w a r d s and Burge ( u n p u b h s h e d o b s e r v a t i o n s o t e d m [4]) o b s e r v e d that at least 90% o f the olfactory bulbs must be r e m o v e d to r e n d e r an a m m a l r e h a b l y anosm~c Th~s s e e m s to be m a g r e e m e n t w~th o u r w o r k d e a h n g with fighting beh a r b o r in w h i c h e x p r e s s e d aggression was m o r e p r o n o u n c e d m s u b t o t a l bulbectom~zed m~ce t h a n m the total bulbectom~zed g r o u p (Table I) In c o n t r a s t , m the a m m a l s partmlly bulbectom~zed prior to ~solat~on learned aggressive b e h a v i o r was totally e h m l n a t e d (Table 2) T h u s , the effects of~solat~on are a p p a r e n t l y as ~mportant m the d e v e l o p m e n t o f l e a r n e d , aggressxve b e h a v i o r as ~s the e x t e n t of olfactory b u l b rem o v a l T h e specific C N S s~tes w h i c h are a s s o c i a t e d w~th ~solat~on-mduced, aggressive b e h a w o r m bulbectom~zed m~ce are p r e s e n t l y u n d e r i n v e s t i g a t i o n AC KNOWLEDGEMENTS The authors wish to acknowledge Mrs Llnda Pope for assistance with the preparation of this manuscript, Dr Larry Means for his critical review of the text and Dr M Evelyn McNed for assistance with the surgical procedures

REFERENCES 1 DaVanzo, J P , M Daugherty, R Ruckart and L Kang Pharmacological and biochemical studies m isolation-reduced fighting mice Pswhopharmatologta 9 210-219. 1966 2 DaVanzo, J P Observations related to drug-reduced alterations m aggresstve behawor In Aggre~:ve Behavior edited by S Garatum and E B Slgg New York Wdey lntersclence 1969 pp 263-272 3 Denenberg, V H , E Gauhn-Kremer, R Gandelman and M X Zarrow The development of standard stimulus animals for mouse (Mus mus~ ulus) aggressmn testing by means of olfactory bulbectomy Ammal Behav 21 590-598, 1973 4 Edwards, D A Non-sensory involvement of the olfactory bulbs m the medmtlon of socml behawors Behav Btol It 287-302 1974

5 Ginsburg, G and W C Allee Some effects of ~.ondttlonmg on socml dominance and subordination in inbred stratus of m~ce Phvsu~l Zool 15 485-506, 1942 6 Ropartz, P The relation between olfactory stimulation and aggressive behavior in mice Amrn Beha~ 16 97-100 1968 7 Rowe, F A and D A Edwards Olfactory bulb removal Influences on the aggressive behaviors of male mice Phwtol Behav 7 889-892, 1971 8 Scott J P Incomplete adjustment caused by frustration of untrained fighting mice J Comp Phwtol P ~ ~hol 39 379-390, 1946