Effect of antibiotics on retention of visual discrimination training and on protein synthesis in the pigeon

Phystology& Behavior, Vol. 19, pp. 145--154. Pergamon Press and Brain Research Publ., 1977. Printed m the U.S.A.

Effect of Antibiotics on Retention of Visual Discrimination Training and on Protein Synthesis in the Pigeon L A U R E N C E J. S T E T T N E R

Department o f Psychology, Wayne State University, Detroit, MI AND R O B I N B A R R A C O A N D H E N R Y J. N O R M I L E

Department o f Physiology, ~Vayne State University Medical School, Detroit, MI ( R e c e i v e d 14 A u g u s t 1 9 7 6 ) STETTNER, L. J., R. BARRACO AND H J. NORMILE. Effect of antibiotics on retention of visual discrtmmatton trammg and on protem synthesis m the ptgeon. PHYSIOL. BEHAV. 19(1) 145-154, 1977. - Forty-three pigeons were trained for one day on a visual discrimination (horizontal vs. vertical stripes) and then immediately injected with either puromycm (PM), cyclohexlmlde (CXM), control saline, or combined PM and CXM solutmn. PM produced a marked amnesic effect, CXM a weaker effect. PM injected ammals (but not CXM's) also took sigmficantly more than 1 day longer than controls to reach criterion levels, indicating an effect on continued acquisition of the discriminatmn beyond the amnesm for Day 1. Combmatmn with CXM did not attenuate the PM effects. Protein mhlbitmn profiles showed maximum PM actlwty of 90% mhibltmn, whereas CXM's maximum was 98% and was distributed more widely throughout different brain regmns. In a second experiment, PM (N = 8), CXM (N = 8) or sahne (N = 8) were again rejected immedmtely after training, but the S+ was reversed on all postinjection training trials. In this case, PM ammals were superior m percentage of S+ pecks on Day 2 to controls and CXM's. Further, the continued acquisitmn deficit was absent m the PM group, indicating that this effect ~s highly specific to those behavioral clrcmts active at the time of mject~on. Antibiotics

Memory

Puromycm

Cycloheximlde

Protein synthesis

ily used r o d e n t s as s u b j e c t s a n d relatively simple t r a i n i n g p r o c e d u r e s (1.e., Y-maze e s c a p e / a v o i d a n c e involving posit i o n or b r i g h t n e s s d i s c r i m i n a t i o n ; one-trial passive avoida n c e ) u t t l i z m g s h o c k as an aversive stimulus. The m o s t extensive series of studies o n a n o n r o d e n t species, t h o s e b y A g r a n o f f a n d his c o - w o r k e r s w i t h goldfish, also used a s h o c k - a v o i d a n c e p a r a d i g m [ 1 , 2 ] . Studies in this area t e n d to a n a l y z e r e t e n t i o n results m a n all-or-none f a s h i o n 0 s t h e r e a m n e s i a or i s n ' t t h e r e ? ) a n d have n o t r e p o r t e d ( n o r t y p i c a l l y have b e e n designed to d e t e c t ) r e l e a r n i n g or n e w learning deficits p r o d u c e d by antlblotlc treatment F l e x n e r et al. [16] did n o t e t h a t s o m e o f t h e i r a m m a l s t o o k l o n g e r to relearn a Y-maze p o s i t i o n d i s c r i m i n a t i o n following p u r o m y c i n i n j e c t i o n , b u t t h e y did n o t p r e s e n t

F L E X N E R , F l e x n e r a n d Stellar [12] r e p o r t e d t h a t m t r a c erebral r e j e c t i o n s o f p u r o m y c m (PM) in mice f o l l o w i n g p o s i t i o n selective s h o c k - a v o i d a n c e t r a i n i n g in a Y-maze leads to loss of r e t e n t i o n of the Y-maze learning. T h e y related this b e h a v i o r a l result t o t h e i n h i b i t i o n of c e r e b r a l p r o t e i n s y n t h e s i s p r o d u c e d b y PM. S u b s e q u e n t investigat i o n s [3, 4, 13] e s t a b l i s h e d t h a t c y c l o h e x l m l d e (CXM) a n d a c e t o x y c y c l o h e x i m l d e ( A X M ) , w h i c h are even m o r e p o t e n t m h l b ~ t o r s of p r o t e i n s y n t h e s i s t h a n PM, also p r o d u c e d a m n e s i c e f f e c t s in mice. These results have led to a significant n u m b e r of studies ( r e c e n t l y reviewed b y G i b b s a n d Mark [20] a n d Barraco a n d S t e t t n e r [ 6 ] , w h i c h have u t l h z e d a n t i b i o t i c t r e a t m e n t s m o r d e r to p r o d u c e e x p e r i m e n t a l amnesia. These studies have p r i m a r -

~Ttus study Is based in part upon an M.A thesis by H. Normile, under the direction of R. Barraco. Support for this mvestagatlon was provided by Faculty Research Grants-In-Aid, Wayne State Umversity, to R. Barraco and L. Stettner. The authors wish to acknowledge the aid of Jeffrey Koscis, Diane Lemanek and Carolyn Clough in developing procedures used in this investigation. 145

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specific data n o r discuss the l m p h c a t t o n ol this obser~atlon In view o f the m a n y c o m p l e x m e s revolved m the I n t e r p r e t a t i o n o f a m n e s i c effects of c h e m i c a l t r e a t m e n t s , ~t seems i m p o r t a n t to assess these effects m an a p p e t i t i v e sxtuatmn a n d w i t h a t r a i n i n g p a r a d i g m t h a t can reveal relatively subtle f e a t u r e s of t h e i n t e r a c t i o n b e t w e e n behavioral and affmmical p a r a m e t e r s Danlels 19] and C o h e n and B a r o n d e s [8] have utilized a p p e t m v e t r a i n i n g procedures w i t h rats to successfully d e m o n s t r a t e a n t i b m n c - i n d u c e d r e t e n t i o n loss However, n e i t h e r p a r a d i g m (onetrial w a t e r - s p o u t a p p r o a c h and Y-maze learning for w a t e r r e i n f o r c e m e n t ) a p p e a r s to have the flex%fi~ty ot the one d e v e l o p e d b y M a y o r [ 2 2 , 2 3 ] w h o s t u d i e d the e f f e c t of several a n t l b l o t m s o n r e t e n t m n of a learned wsual discrimi n a t i o n u t l h z l n g an o p e r a n t k e y - p e c k f o o d - r e w a r d paradlgm in J a p a n e s e quaff He t r a i n e d animals for one session on a red-green color d i s c r i m i n a t i o n and f o u n d t h a t intracerebral r e j e c t i o n of PM l m m e d m t e l y following this session (Day 1) p r o d u c e d m e m o r y deficits d u n n g a s e c o n d session of r e t e s t l n g o n the same d l s c n m m a t m n t h r e e days l a t e r (Day 4) ( P u r o m y c m - a m m o n u c l e o s l d e , AXM, and sah n e h a d n o such effect ) M a y o r also n o t e d t h a t the PM effect m this i n s t a n c e was q m t e s e n s m v e to the degree of learning t h a t the a m m a l s were able to achmve A m m a l s t h a t were t r a i n e d long e n o u g h on Day 1 to reach a p o i n t of m a k i n g 90% of t h m r p e c k s to the r e w a r d e d s t i m u l u s still r e t a i n e d t h m r l e a r n i n g o n Day 4 despite the PM reJection The use of appetlt~vely r e i n f o r c e d v~sual disc n m m a t m n l e a r n i n g in c o n j u n c t i o n w i t h a n n b m n c treatm e n t in birds a p p e a r s to have several desirable f e a t u r e s A variety of stimuli can be used m s u c h t r a i n i n g to provide very specffm l e a r n i n g and to quite easily provide learning tasks of varying difficulties w i t h i n the same paradigm The t r a i n i n g can be so a r r a n g e d t h a t it takes o n l y a single session for t h e o c c u r r e n c e of sufficient t e a r n m g (m controis) t o p r o v i d e a b a s e h n e for a s s e s s m e n t of r e t e n t i o n loss, and at the same t i m e it takes several sessions for a l e a r n i n g c r i t e r i o n to be r e a c h e d p r o w d i n g a b a s e h n e for a s s e s s m e n t of any residual r e l e a r n i n g deficits F u r t h e r , the use of t h e f o o d - m o t w a t e d o p e r a n t k e y - p e c k r e s p o n s e allows for a sensitive, i n d e p e n d e n t m e a s u r e o f general p e r f o r m a n c e level (overall p e c k i n g r a t e ) t h a t can be used to assess s y s t e m i c as o p p o s e d t o specific effects of ant]bmnc treatment

EXPERIMENT 1 Thin e x p e r i m e n t was designed p r i m a r i l y t o u t l h z e an appetltwely reinforced visual-discrimination paradigm m p]geons to p r o v i d e a detailed a s s e s s m e n t of the effects of a n t i b i o t i c s u p o n r e t e n t i o n of learned r e s p o n s e s A f t e r c o n s i d e r a b l e p i l o t w o r k , a p a r a d i g m utilizing b r i e f daffy t r a i n i n g sessions o f a l l n e - o n e n t a t m n d i s c r i m i n a t i o n (horiz o n t a l vs vertical stripes) was a d o p t e d a n d the effects o f l n t r a c e r e b r a l PM, c y c l o h e x i m l d e (CXM), a n d c o m b i n e d (PM and CXM) injection upon retentmn and continued learning o f this task were assessed. Specifically, pigeons were gwen o n e sessmn of successive d l s c n m m a t m n t r a i n i n g on h n e - o r i e n t a t l o n stimuli, a n d t h e n i n j e c t e d l m m e d m t e l y w i t h e i t h e r PM, CXM, a c o m b m e d m i x t u r e o f PM and CXM, or sahne s o l u t m n . T h e y were t e s t e d for r e t e n t m n o f this i m t i a l session 3 days later, a n d t h e n c o n t m u e d on t r a i n i n g each day t h e r e a f t e r u n t i l

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reaching c r i t e r i o n l']le {redtlllent w a s adtnHllS[Cit_d ~,llillt_dlately a f t e r t r a i n i n g because m thJs lmtlal cxpeHmen~ ,~, w a n t e d to m a x i m i z e the po~slbfllty ot the l r e a t m e n t havm~ an effect whale avoiding the c o m p h c a t l o n ol t r a i n i n g lhc a n i m a l mltlally while alread3, u n d e r lhe m t t u e m c ,.t a n t l b m t ] c rejections. C o m b i n e d CXM and PM mje~tlon~ were utilized t o assess the g e n e r a h t y to out paradigm (~I the findings o f F l e x n e r and F l e x n e r [131 and B a r o n d c s and C o h e n [3] t h a t c o m b i n i n g these a n t % l o t R s e h m m a t c d the amnesic effect t h a t would ordinarily be t o u n d ~ l t h PM w h e n it IS i n j e c t e d i n t o nnce 24 h r a l t e r a v o i d a n c e learning Finally, m an e f f o r t to pro~ lde some a n a t o m i c a l bpcclttclty as c o n t r a s t e d with the whole brain analyse~ wtnch p r e w o u s studies m this area have typlcall} e m p l o y e d t~c m e a s u r e d the regional i n h i b i t i o n ol p r o t e i n s~nthes~s p r o d u c e d by the various ant tblotlcs M 1' l H f) D

Ammals S e v e n t y - n i n e male pigeons of the G]ant White C a r n e a u x strain, 1 - 4 years old at the time of t r a i n i n g were used F o r t y - t h r e e were assigned to the various e x p e r i m e n t a l groups in the b e h a v i o r a l studies and 36 were u n h z e d to m e a s u r e m h i b m o n o f p r o t e i n s y n t h e s i s T h e y were h o u s e d l n d w l d u a l l y f r o m the time they e n t e r e d the l a b o r a t o r y u n t i l the e n d o f the e x p e r i m e n t F o l l o w i n g an m i t m l period ot a d a p t a t i o n a n d ad h b feeding (3 weeks), the pigeons wine fed a l i m i t e d a m o u n t o f food ( P u n n a pigeon c h o w ) so as to be m a i n t a i n e d at 8 5 ~ ol t h e i r ad h b b o d y wmght t h r o u g h o u t the course of the e x p e r i m e n t

Apparatus T r a i n i n g was c o n d u c t e d m a Lehigh Valley O p e r a n t c h a m b e r , w i t h a 25 × 37 ~ 58 cm high i n t e r i o r e q u i p p e d w i t h a fan a n d w i t h 3 t r a n s p a r e n t p e c k i n g keys of 2 54 cm d i a m e t e r m o u n t e d in a h o r i z o n t a l h n e on one wall 25 cm a b o v e the f l o o r of the c h a m b e r A s t i m u l u s p r o j e c t o r was m o u n t e d b e h i n d e a c h key A r e c t a n g u l a r a p e r t u r e of 5 × 6 4 c m was c e n t e r e d m the same wall and allowed access to grain t h r o u g h a f u n n e l - h k e food h o p p e r w h e n a solenoido p e r a t e d f o o d t r a y was activated. A 6 W i n c a n d e s c e n t b u l b m o u n t e d at t h e t o p o f the h o p p e r was lit w h e n e v e r the tray m a d e f o o d available A o n e - w a y o b s e r v a t i o n w i n d o w , 19 x 21.5 cm, was set i n t o one of the walls p e r p e n d i c u l a r to the r e s p o n s e key wall O p e r a t m n of the c h a m b e r was c o n t r o l l e d a u t o m a t i c a l l y and r e s p o n s e s r e c o r d e d t h r o u g h c o m b i n e d sohd-state and e l e c t r o - m e c h a n i c a l c i r c u i t r y

Procedure Pretrammg When the p i g e o n had r e a c h e d 85% o f its ad h b weight, it was placed in the test c h a m b e r and allowed to eat f r o m t h e f o o d h o p p e r w h i c h was m a i n t a i n e d m the o p e r a t e d p o s i t i o n c o n t i n u o u s l y u n t i l the a n i m a l ate readily f r o m it. The f o o d tray was t h e n a c t i v a t e d i n t e r m i t t e n t l y so t h a t t h e pigeons were t r a i n e d to a p p r o a c h t h e f o o d h o p p e r a n d eat for a s h o r t p e r i o d of time w h e n e v e r the tray was o p e r a t e d , e a t i n g t i m e b e i n g gradually s h o r t e n e d u n t i l each p e r i o d o f access was l i m i t e d t o 4 sec. K e y - p e c k s h a p i n g was the n e x t stage, a n d was a c c o m p l i s h e d m a n u a l l y by operating t h e h o p p e r c o n t i n g e n t u p o n success]re a p p r o x l m a t]ons t o p e c k i n g at the c e n t e r k e y w h i c h was h t c o n t i n u ously b y a w h i t e circle o n the p r o j e c t o r m o u n t e d b e h i n d

E F F E C T OF ANTIBIOTICS ON RETENTION IN THE PIGEON the key. (The side keys were dark and inoperative throughout the entire course of the experiment.) Once the animal pecked the key with sufficient force to operate the micro-switch attached to it, each peck was reinforced by providing 4 sec of access to food. Pigeons were placed in the apparatus for 3 daily sessmns of continuous reinforcement which lasted until they made 80 pecks, during which time the center key was lit continuously except for 15 sec time-out periods which occurred at one-mxn intervals. Reinforcement was available only when the key was lit. In the next training phase a variable interval schedule (VI) which made food available only once every 25 sec on the average was programmed through use of a tape-loop, and the animals were run for 4 days of VI training to white light, 12 mxn per day. At this point, a persistent pecking response to the white light had been established. Grzd training. Extensive pilot work had indicated that the animals trained on white light often showed some initial mhlbitmn toward pecking the key when line stimuli were projected upon ~t during the first discrimination training session (see below) We believed it important to reduce this variablhty and ensure a prompt and consistent pecking response during this initial brief training period prior to Injection. Thus, a phase of training was introduced In which both the horizontal and vertical lines (each consisting of 3 black hnes, 2 mm wide and 2.5 cm long with a 4 mm white strip between them) were simultaneously projected thus producing a grid stimulus Pecks at the grid were reinforced on the VI schedule, with the same 1 min on, 15 sec off sequence that had been used in white light training. This procedure prowded exposure to the line stimuli (in compound form) prior to discrimination thus facilitating lmmedmte pecking during the initial discrimination session, but did not provide any differential reinforcement. VI grid training was continued for 5 days, 15 mIn per day. We considered the fact that exposure to the grid stimuli during this period could result in some differential response strength to the stimuli (despite the absence of any differential reinforcement) through selective attention mechanisms by which one aspect of the compound stimulus acquired greater stimulus control of pecking than others. However, since pilot work indicated no obvious tendency for this to occur and since the positive and negative stlmuh were counterbalanced within each group during dxscriminatmn training (thus prohibiting any systematic error from being introduced due to stimulus bias however generated), it was decided that the advantage of grid training outweighed possible disadvantages. Dzscrzmmatzon trainzng. Discrimination training was initiated on the day following the completion of grid training. Each discrimination session consisted of 6 presentations of the horizontal lines and six of the vertical hnes projected independently upon the response key for 1 min periods, with a 15 sec time out (during which the key was dark but the house light remained on) between stimulus presentations. During the presentation of the stimulus designated as positive (S+) pecks were reinforced on a VI 25 sec schedule; pecks at the other stimulus (S-) were never reinforced, nor were pecks during the time out period The stlmuh were presented in an irregular order, balanced to provide no more than 3 presentations in a row of one stimulus, and 6 presentations of each stimulus during each session. Half the animals in each condition were assigned the vertical stimulus as S+ and half the horizontal on an a

147

priori basis. The number of pecks during each 1 min stimulus presentation of each discrimination session were recorded on counters and tabulated by the experimenter as the animal was performing. The animals were allowed 2 full days of rest in their own cages following the initial day of discrimination training, and then were run for one discrimination session per day until they reached a criterion level of 90% of all pecks on a given day occurring dunng S+ periods. A lower hmit of 100 total pecks to S+ and S combined per session was estabhshed as a minimum for adequate performance. Any animals who failed to reach that total on the first day of discrimination training were dropped from the experiment. If the pigeon exceeded 100 pecks on the first session but failed to meet this level on any subsequent session, that single session was excluded from the analysis. (A total of 6 ammals were dropped from the e x p e n m e n t due to failure to respond on the first postlnjectlon day. Three were from the PM group, 2 were sahne controls, and 1 had received (CXM). Inlectzon procedure. Injections were made lntracerebrally, with the aid of a Kopf stereotaxxc apparatus, fitted with pigeon ear bars and beak holder. A 26 ga hypodermic needle was mounted on the arm of the stereotaxic apparatus and connected through cannula fittings to 0.036 in. diameter plastic tubing. The tubing in turn was fit over the end of a 1 ml syringe which was mounted in a microburette apparatus (Micrometric Model SB2) which allowed controlled delivery of solution graded In mlcroliters. Due to the thin and porous nature of the avian skull whach offered virtually no resistance to our needle, injections could be made directly through the scalp and into the forebrain by simply mounting the animal in the stereotaxlc apparatus and lowering the needle without drllhng through the skull and without the use of anesthesia. These rejections caused no apparent disturbance to our animals. All animals received injections into 4 sites on each side of the forebrain, with 10 ul of solution injected at each site. The sites of injection were all in the dorsal portion of the forebrain, in the regmn of the hyperstriatum ventral and the anterio-dorsal portion of the neostrxatum. (Fig. 1 indicates the injection sites ) Four solutions were used: PM (18 ug/~l), CXM (18 ug/ul), combined solution (18 t~g/ul PM - 18 ug/ul CXM), and control solutions of physiologic saline. The entire injection (all 8 sites) procedure took approximately five minutes to complete, after which the animal was immediately returned to its home cage. Analysts of mhibttton of protein synthests Animals were housed, fed, and rejected intracerebrally as described m previous sections. Puromycin dihydrochloride (Nutritional Biochemicals, Cleveland, OH) and cycloheximide (Sigma Chemical C o , St Louis, MO) solutions were prepared individually in 0.85% sahne with a final concentration of 18 ug/'ul The combined solution (PM-CXM) had a concentration of 18 ug/ul PM and 18 ug/ul CXM The solutions were tltrated to pH = 7 with 0.1 N NaOH. At various times following intracerebral injection of PM, CXM, P M - C X M combined solution, or saline animals were injected Intraperitoneally in the lower left quadrant of the breast with 12 5 uC of L-valine-l-C~ 4 (New England Nuclear, Boston, MA, specific activity 27.7 mC/mM) Ammals were sacrificed by decapitation 30 min following vallne rejection The brains were removed, briefly rinsed with saline, dissected into 5 regions and each region was weighed. The regions were. (1) right forebrain, (2) left forebraln, (3) cerebellum (4) optic tectum and (5) bralnstem-dlencephalon. The

148

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FIG 1. Location of injection sites, indicated by drawing the needle tracks on tracings trom photomicro-graphs of the brim sections. 'lhe upper figure corresponds approximately to the A 10.25 section m the Karten and Hodos atlas [20] and the lower figure to A 9 50. Our birds were held m the stereotaxlc so that the anterior fixation pomt at the beak was more nearly parallel to the horizontal axis of the Instrument (30 °) than it was in the preparatlon of the Karten and Hodos atlas (45 °) This made rejections more convenient but correspondence with the atlas less precise. (HA Hyperstrlatum accessorum, HV Hyperstnatum ventrale, N: Neostrlatum. E Ectostrmtum) entire p r o c e d u r e o f sacrifice, dissection, and weighing t o o k a p p r o x i m a t e l y 10 rain I m m e d i a t e l y following weighing, each region was h o m o genized In 12 ml/g wet weight o f 2 5 M NaCI at 4"C w i t h a P o t t e r Elvehjam tissue h o m o g e n i z e r ( T a l b o y s Bodlne Mo-

tor) for five mln. P r o t e i n was e x t r a c t e d and isolated by the following m e t h o d . A 5 ml aliquot o f a q u e o u s brain h o m o genate was rapzdly papetted i n t o an equal volume ol 109; trlchloroacetxc acid ( T C A ) - 0 5% p h o s p h o t u n g s t l c acid (PTA). The samples were stirred for several minutes, stored

EFFECT OF ANTIBIOTICS ON RETENTION IN THE PIGEON for 30 mm at 4°C, and centrifuged at 4000 xg for 30 min (International Refrigerated Centrifuge). The supernatant was collected and the pellet was resuspended in 3 ml 0.3 N NaOH containing unlabelled L-valine (1% w/v) and heated at 80°C for 30 min for hydrolysis of labile nucleic acids and displacement of unincorporated labelled valine from nonspecific binding sites. The proteins were repreclpitated with 7 m l 10% T C A - 0 . 5 % PTA (4°C) and centrifuged for 30 min at 4000 g. The resultmg second supernate was combined with the supernate from the first centnfugation. The pellet was washed twice with 5% T C A - 0 . 2 5 % PTA. The llplds were extracted with a sequential series of solvents: the pellet was resuspended m 5 ml absolute ethanol buffered with 0.05 vols. 0.2 M KC1, stirred, and centrifuged. The supernate was decanted and discarded. The pellet was twice extracted with 10 ml chloroformmenthanol (2'1), heated at 50°C for 15 min, centrifuged, and the supernate was decanted and discarded. The pellet was then resuspended in absolute ether, centrifuged, and the supernate discarded The pellet was dried overnight in a vacuum desiccator. The dried protein pellet was dissolved in 3.0 ml in NaOH Triplicate 0.3 ml ahquots were used for FolinLowry assay and a 1 0 ml ahquot was placed in a polyethylene vial (Rochester Scientific)containing l0 ml insta-gel (Packard Instrument) and 0.1 glacial acetic acid for radioactive counting A 4.5 ml ahquot of the combined supernatant fraction containing unincorporated v a h n e 1-C' ' was placed in a polyethylene vial containing 10 ml lnsta-gel for radioactive counting. Samples were counted in a Packard Tri-Curb Liquid Scintillation Spectrometer (Model 3365). Quenching corrections were made from a plot of quenching vs channel ratios Efficiency was determined by internal standardization. As an index of inhibition of protein synthesis for each region and each animal, the ratio of dpm for the protein ahquots and supernatant (P/S ratio) were calculated according to Brink etal. [7]. Average percentage inhibition (%I) for the PM and CXM conditions was calculated from three separate samples. Further, the, P/S ratio and thus the %I were normahzed with respect to the saline controls Thus the percentage inhibition (%I) of protein synthesis was estimated for each brain region at various times following intracerebral rejection of antibiotic. The points analyzed for PM and CXM were 45 mm, l, 1.5, 2.5, 4 5, 8.5, 12.5 and 24 5 hr. For combined injections only the 2.5 and 4 5 hr time points were sampled, and data at each point was calculated as the average of two separate samples.

149

Experimental deszgn. All animals were given identical pretraining and then given 1 day of discrimination training as described above. Animals were randomly assigned to receive either CXM, PM, combined PM and CXM, or saline injections. They were removed from the training box and injected immediately following the initial discrimination training session. Animals were then maintained in their home cages without further treatment (except for weighing, feeding and general observation) for 2 additional full days following the injection treatment On the next day (Day 2 of the training, actually the 4th day of the experiment, counting the initial training session and injections as Day 1) discrimination training was resumed and the pigeons were run on discrimination training each day thereafter untd they reached criterion as described above. RESULTS

Behavioral In order to assess whether the ant:biotic treatments had any deleterious effects on performance per se, the total number of pecks made during the discrimination session just prior to injection and the first session run after injection were analyzed. As can be seen in Table 1, all the groups had a mean pecking rate which well exceeded the 100 response minimum criterion and there was no evidence of any decrease in overall pecking produced by antibiotic rejection. The only difference on Day 2 which even approaches significance is the comparison between the PM and the CXM group (t = 1.54, p<0.1), and even this is misleading, as the difference is clearly attributable to the fact that the random assignment of pigeons had resulted in a lower pecking rate of the PM animals on Day 1, prior to injection. (In fact, the PM ammals had a higher rate postinjectlon than prelnjection.) Analysis of variance of the scores for Day 1 revealed no significant differences in percentage of pecks to S+ between the groups prior to injection, F ( 3 , 3 9 ) = 1.22, p > 0 05, whereas on Day 2 there was a significant difference, F(3,39) = 3.62, p<0.05. Differences scores based on the change in percentage of pecks to the positive stimulus for each animal between Day 1 and Day 2 were computed as the primary measure reflecting retention of the Day 1 discrimination training. An analysis of variance of these scores revealed a highly significant effect of the experimental treatments, F(3,91) = 10.30, p<0.001 Individual comparisons showed that the control group's scores increased considerably and significantly more from Day 1 to

TABLE 1 NUMBER OF TOTAL PECKS ON TRAININGTRIAL (DAY 1) AND FIRST POSTINJECTIONTRIAL(DAY 2) Total Pecks Day 1

Day 2

Group

n

Mean

Standard Deviation

Saline CXM PM PM-CXM

13 10 10 10

389.6 499.9 313.8 437 0

153.8 136.7 188 8 128.2

Mean

Standard Deviation

433.2 444.88 332.7 398.7

213.9 120.9 174 2 155.8

150

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TABLE 2 P E R C E N T P E C K S TO S + ON DAY 1 A N D DAY 2, A N D C H A N G E IN P E R C E N T PROM DAY I-DAY 2 FOR P~VI'ERN DISCRIMINATION

Change m Percent COJTeCt Respon,c Day 2-Da.~ i Standard Mean DevmUon

Percent Correct Response

Group

N

Day 1 Standard Mean Devmtmn

Saline CXM PM PM-CXM

13 10 10 10

48.2 51.8 54 9 50.6

96 72 102 55

Day 2 t h a n did t h e CXM (t = 2.02, p < 0 05), PM (t = 3 83, p < 0 0 0 1 ) g r o u p s or c o m b i n e d (t = 4 18, p < 0 . 0 0 1 ) groups. This i n d i c a t e s t h a t all t h e a n t i b i o t i c t r e a t m e n t s p r o d u c e d s o m e degree o f e x p e r i m e n t a l amnesia. As can be seen in T a b l e 2, t h e degree o f a m n e s i a was n o t as great in the CXM animals, w h o s e p e r c e n t a g e values were significantly greater t h a n t h o s e o f t h e PM ( t = 2 . 1 9 , p < 0 0 1 ) or c o m b i n e d g r o u p s (t = 0.37, p < 0 . 0 5 ) . C o m p a r i s o n of t h e scores for the a n t i b i o t i c anLmals as c o m p a r e d w i t h c o n t r o l s is an i n d e x as to the p r e s e n c e o f some degree of e x p e r i m e n t a l amnesia. In o r d e r to a n s w e r t h e q u e s t i o n of w h e t h e r t h e a m n e s i a is partial or total, we m u s t c o m p a r e p e r f o r m a n c e o f the a n t i b i o t i c g r o u p s w i t h t h a t of naive a n i m a l s o n t h e same task This c o m p a r i s o n can be m a d e s i m p l y b y l o o k i n g at t h e Day 2 - D a y 1 scores t h e m s e l v e s , if the e x p e r i m e n t a l a n i m a l s totally f o r g o t Day 1 training, t h e r e s h o u l d be as m a n y a n i m a l s decreasing t h e p e r c e n t a g e f r o m Day 1 to 2 as increasing it, a n d t h e m e a n s h o u l d n o t differ significantly f r o m 0. I n s p e c t i o n o f Fig. 2, d e p i c t i n g n o t o n l y t h e m e a n c h a n g e scores b y g r o u p s b u t t h e range a n d the score o f each i n d i v i d u a l in e a c h g r o u p , helps to clarify this q u e s t i o n T h e CXM g r o u p clearly h a s o n l y a partial a m n e s i a : t h e m e a n c h a n g e score of + 7 2, the fact t h a t 8 of 10 a n i m a l s in t h e g r o u p i m p r o v e d t h e i r scores, a n d t h e significant s u p e r i o r i t y i n d i c a t e t h a t CXM a n i m a l s did r e t a i n to some degree t h e effects o f t h e i r d i s c r i m i n a t i o n t r a i n i n g o n Day 1 j u s t p r i o r to i n j e c t i o n . The PM a n i m a l s also i n d i c a t e s o m e evidence o f r e t e n t i o n , albeit significantly l o w e r t h a n t h e CXM animals. I n s p e c t i o n of t h e i r scores i n d i c a t e s a highly c o n s i s t e n t p a t t e r n of a very small degree o f i m p r o v e m e n t Again, 8 of 10 a n i m a l s s h o w e d i m p r o v e m e n t , a l t h o u g h in n o case was t h e i m p r o v e m e n t greater t h a n 5% change, well b e l o w typical c o n t r o l p e r f o r m a n c e a n d m a n y of the CXM animals T h e c o m b i n e d PM a n d C X M animals, a l t h o u g h n o t significantly d i f f e r e n t f r o m the PM group, do n o t s h o w e v i d e n c e t h a t t h e y as a g r o u p r e t a i n e d t h e i r Day 1 t r a i n i n g Only 6 of the 10 a n i m a l s m this g r o u p s h o w e d i m p r o v e m e n t Table 3 summ a r i z e s this analysis, i n c l u d i n g the t-value for d e t e r m i n i n g w h e t h e r each g r o u p individually s h o w e d an increase signifi c a n t l y a b o v e 0. This increase was significant for the s a h n e ( p < 0 0 0 1 ) a n i m a l s and CXM a n i m a l s ( p < 0 0 0 5 ) b u t n o t significant for e i t h e r the PM or the c o m b i n e d groups. A n o t h e r facet of the a n t i b i o t i c effects can be revealed by c o m p a r i n g t h e m e a n days to c r i t e r i o n in e a c h g r o u p in the a p p r o p r i a t e m a n n e r Based simply o n the effect o f a m n e s i a (partial or t o t a l ) for Day 1 training, we w o u l d

Day 2 Mean

Standard Dewat~on

632 576 568 518

8.5 99 10.0 38

+15 q58 +19 +12

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-20 FIG. 2. Difference in the percentage of all pecks made to the positive stimulus between the first and second day of discrimination tralnmg in Experiment 1. Vertical lines display the entire range of the distribution for each group. Each point shows the score of an mdiwdual animal. e x p e c t a d i f f e r e n c e m days to c r i t e r i o n of at m a x i m u m 1 day ( t r a i n i n g session) if t h e r e were n o residual e f f e c t o f the a n t i b i o t i c t r e a t m e n t s on the animals d u r i n g s u b s e q u e n t t r a i n i n g A d i f f e r e n c e of m o r e t h a n o n e day w o u l d Indicate t h a t t h e r e were p r o l o n g e d effects o f the a n t i b i o t i c treatm e n t s w h i c h i n t e r f e r e d w i t h some aspect of the a c q u i s i t i o n of the d i s c r i m i n a t i o n task 3 days a f t e r i n j e c t i o n and t h e r e a f t e r Table 4 i n d i c a t e s t h a t this IS exactly w h a t did o c c u r in the PM a n d PM a n d CXM groups, b u t n o t in the CXM group. T h e d i f f e r e n c e b e t w e e n the CXM and c o n t r o l g r o u p s was less t h a n 1 full day ( c o n s i s t e n t with the partial a m n e s i a f o u n d in this group), and m o r e t h a n 3 days of t r a i n i n g in the o t h e r a n t i b i o t i c g r o u p s In o r d e r to assess w h e t h e r the days to c r i t e r i o n difference actually repres e n t e d a c o n t i n u e d a c q u i s i t i o n deficit, t h e h y p o t h e s i s tested in t h e c o m p a r i s o n s in Table 4 was t h a t the d i f f e r e n c e b e t w e e n t h e m e a n s is greater t h a n 1. This analysis shows a highly significant c o n t i n u e d a c q u i s i t i o n effect w h e n the PM a n d PM a n d CXM g r o u p s are c o m p a r e d to c o n t r o l s . (This h y p o t h e s i s is a u t o m a t i c a l l y rejected for the CXM g r o u p , w h e r e t h e o b t a i n e d m e a n difference is less t h a n 1.) Statistical c o m p a r i s o n of the days to c r i t e r i o n f o r the CXM g r o u p w i t h the o t h e r a n t i b i o t i c g r o u p s c o n f i r m s the fact

EFFECT OF ANTIBIOTICS ON R E T E N T I O N IN THE PIGEON

151

TABLE 3 AMNESIA ANALYSIS: PERCENTAGE INCREASE FROM DAY I-DAY 2 AND SIGNIFICANCELEVEL OF EACH GROUP'S INCREASEAS COMPAREDTO NO INCREASE Treatment Group PM CXM

PM + CXM Mean Standard Deviatmn t

+1.2 3.3 1.02 9 <0.20 6/10

df p N showing increase

+1.9 4.5 1.27 9 <0.20 8/10

+5.8 64 2.71 9 <0.05 8/10

Saline +15 00 9.5 5.43 12 <0.001 13/13

TABLE 4 DAYS TO CRITERION FOR ACQUISITION OF A PATTERN DISCRIMINATIONAND STATISTICAL COMPARISON BETWEEN GROUPS

Group

N

Mean

Standard Deviation

Saline

13

3.8

1.7

Sa. vs CXM

CXM PM PM-CXM

10 I0 10

4.2 7.3 7.1

1.5 2.0 11

Sa. vs PM Sa. vs PM-CXM CXM vs PM CXM vs PM-CXM PM vs PM-CXM

Comparison

T-Value Difference
df

p*

21 21 18 18

0.001 0.001 0.005 0 001

*Significance levels are one-tailed. that the latter took significantly longer to reach criterion than the former.

Inhzbztion of Protein Synthesls In Fig. 3 are presented the profiles for percent inhibition of protein synthesis for each of 5 brain regions at various times following antibiotic injection. For PM (Fig. 3 (A)), maximal inhibition of protein synthesis does not occur until 2 5 hr after injection while for CXM (Fig. 3(B)), maximal inhibition occurs almost immediately, within 45 min after injection. Further, CXM exerts its maxmaal effect on protein synthesis for at least 4 hr in contrast to the briefer period of maximum mh:bltlon for PM. Although the injections occur only in the forebrain, CXM apparently diffuses rapidly to other brain regions, and exerts a maximal effect on protean synthesis m these regions within 45 min. On the other hand, PM takes much longer to produce a marked effect on protein synthesis in the cerebellum, brainstem, and optic tectum Moreover, PM during its maximum effect did not produce more than 90% inhibition of proetin synthes~s in the forebrain and never more than 80% inhibition in any of the other bram regions. Combined injections produced essentially the same values as did CXM. DISCUSSION The results of Experiment 1 confirm Mayor's report [2,3] that PM injected intracerebrally immediately after training has an amnesic effect on an appet:tlvely reinforced

visual discrimination In birds. It extends this finding specifically from color discrimination in quail to line orientation discrimination in pigeons. Our results also indicate that CXM has an amnesic effect in this situation, although a less potent one than PM. The partial amnesia produced by CXM immediately after injection differs from Mayor's finding of no CXM produced amnesia. However, in a sense our results are broadly consistent with the inconsistent findings concerning effects of injecting CXM after training, where it has sometimes led to amnesia [ 5, 18, 25 ] and sometimes not [3, 4, 5, 8, 9, 19]. Injection of CXM immediately after training is a marginal condition for the production of amnesia depending on species and nature of the task [6]. The combined PM and CXM injection had an amnesic effect that was at least as great as PM injected alone. There was no indication of any attenuation of the PM effect similar to that found when the combined solution is injected 24-hr after Y-maze shock-avoidance training in rats [3,13]. Since our paradigm utilized a different class of animals and an appetitive paradigm, there are many possible interpretations of our failure to fred attenuation with combined solutions, however, this pattern of results suggests that the mechanism of PM-lnduced amnesia may be quite different when injected at long delays of 24 hr or more than when injected immediately after training. The most surprising finding of Experiment 1 was the appearance of a continued acquisition deficit over and above the amnesic deficit in the PM and combined groups. This did not occur with CXM, further supporting the

152

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Lrlterlon ot correct responses over and dbO~c allm~I~J :,,: Day 1 t r a i n i n g This acquis]tlon deficit could fc, r e \ a m p k , be a d e h c l t in r e t e n t i o n such that the anlmat Ica)n~ n o r m a l l y d u r i n g each day ot t r a m m g , b u t Iorgets muLh ~)I this l e a r n i n g before the n e x t t r a l n l n g s e s s } o n Alternatlvel~. t h e r e c o u l d be some t o r m of deflcH m registering or sit)ling m f o r m a h o n d u r i n g the training sessions themselves J)elelm m a t l o n of any specific beha~loral locus ol the Lc,n l l n u e d a c q u l s l h o n deficit is n o t m e a n t to be l m p h e d m this p i p e ) This Is a s u b j e c t t o r f u r t h e r investigation The p r o t e i n - s y n t h e s i s - i n h i b i t ] o n p r o t d e o b t a i n e d loJ the b r i m of the pigeon is ver 5 snnflar (m b o t h tune-course and degree of m h l b ] t l o n p r o d u c e d ) to t h a t o b t a i n e d prev]oust~ In r o d e n t s {14] and m goldfish [7] CXM (or ~ X M ) p r o d u c e s m o r e rapid and m o r e p r o f o u n d e t t e c t s on p i o t e m s y n t h e s i s t h a n does PM m all three v e r t e b r a t e da.sses By s e c t i o n i n g the brain prior to analysis, we have f u r t h e r d e t e r m i n e d t h a t CXM p r o d u c e s effects m o r e rapldl~ and m o r e p r o f o u n d l y m all brain regions It is, of Lourse. possible t h a t PM m i g h t aLt m o r e rapldl~y and m o r e p r o f o u n d l y o n some c n t l c a l subset of n e u r o n a l structure~ close to the r e j e c t i o n site t h a n appears evident from an analysis w h i c h treats the f o r e b r a m as a whole

80 EXPERIMENT 2

~m ~ "6 "

50 40

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Hours after Cycloheximide Injection FIG. 3 Prec~nt mhibmon of protein synthesis for each of 5 brain regions at various tunes following antibiotic injection' puromycm (A); cycloheximide (B). Right forebram (o), left forebram (~), cerebellum (~), bramstem (A), and optic tectum ( ' ) Each point represents the average of 3 ammals h y p o t h e s i s [6] t h a t g l u t a r l m i d e s a n d p u r o m y c i n o p e r a t e t h r o u g h d i f f e r e n t m e t a b o l i c m e c h a n i s m s in t h e i r p r o d u c t i o n of b e h a v i o r a l effects F u r t h e r , t h e p r o l o n g e d e f f e c t of PM o n a c q u i s i t i o n of the d i s c r i m i n a t i o n task raases quest i o n s t h a t have n o t b e e n addressed b y a n y p r e v i o u s a u t h o r s T h e s e effects are o c c u r r i n g at least 4 and u p to 10 or m o r e days a f t e r i n j e c t i o n , l o n g a f t e r PM has ceased t o p r o d u c e i n h i b i t i o n o f p r o t e i n s y n t h e s i s or f o r m a t i o n o f p e p t i d y l puromycln fragments [10]. Mayor reported that Japanese quail t r e a t e d w i t h PM a f t e r o n e day o f t r a i n i n g t o o k an average of 13 1 days to r e a c h c r i t e r i o n o n a visual p a t t e r n d l s c r l m i n a t a o n , whereas saline c o n t r o l s t o o k 9.4 days This w o u l d a p p e a r to be a c o n t i n u e d a c q u i s i t i o n e f f e c t similar to t h e o n e r e p o r t e d h e r e , a l t h o u g h M a y o r did n o t r e p o r t it as such n o r d e t e r m i n e w h e t h e r this d i f f e r e n c e was sagnifi c a n t l y greater t h a n o n e day It s h o u l d be n o t e d t h a t c o n t i n u e d a c q u i s i t i o n is used in this c o n t e x t only to refer to the q u e s t i o n of w h e t h e r the s u b j e c t s are i m p a i r e d w i t h r e s p e c t to t h e degree of t r a i n i n g r e q u i r e d to r e a c h a 90%

A l t h o u g h the analysis oI t o t a l p e c k i n g responses t e n d s to p r e c l u d e a gross side effects i n t e r p r e t a t i o n ot E x p e r i m e n t 1, t h e r e is still t h e possibility t h a t some m o r e subtle form ot i n t e r f e r e n c e (e g , s e n s o r y - p e r c e p t u a l ) was p r o d u c e d b y the a n t i b i o t i c s , r a t h e r t h a n aninesia per se F u r t h e r , the c o n t i n u e d a c q u i s i t i o n d e h c t t in a m m a l s receiwng purom y c i n ( e i t h e r alone or with CXM) raises q u e s n o n s a b o u t w h e t h e r t h e low scores for these a m m a l s were ieally due t,~ amnesia, or w h e t h e r t h e y started at the same level as the c o n t r o l s at the o u t s e t of Day 2, b u t slmply learned at a m u c h slower rate t h e r e a f t e r Th~s a l t e r n a t i v e i n t e r p r e t a t i o n ~s p a r t i c u l a r l y plausible m the PM g r o u p , where g of t 0 animals s h o w e d an i m p r o v e m e n t on Day 2 as c o m p a i e d to Day 1 Does this really indicate p a r n a l a m n e s m for Day I t r a i n i n g or is ~t simply m a r k e d i n h i b i t i o n of c o n t i n u e d a c q u i s i t i o n o n Day 29 In o r d e r to test this directly, m E x p e r i m e n t 2 the r e i n f o r c e m e n t c o n t i n g e n c i e s were reversed o n Day 2 T h u s an amnesic effect would p r o d u c e s u p e r i o r p e r f o r m a n c e by the a n t i b i o t i c - r e J e c t e d a m m a l s ( w h o w o u l d have f o r g o t t e n the c o n f l i c t i n g e x p e r i e n c e ot Day 1) whereas a l e a r n i n g deficit w i t h o u t amnesia would still leave the a n t i b i o t i c a m m a l s p e r f o r n i m g at a level i n f e r i o r to c o n t r o l s on Day 2 METHOD

Ammals T w e n t y - t w o male G i a n t White C a r n e a u x Pigeons, 6 m o n t h s - 2 years old at the n i n e of t h e e x p e r i m e n t , were used

Apparatus and Procedure A p p a r a t u s , p r e t r a i n i n g , d i s c r i m i n a t i o n training, i n j e c t i o n p r o c e d u r e , a n d p o s t l n j e c t l o n interval b e f o r e t h e r e s u m p t i o n of t r a i n i n g were identical t o E x p e r i m e n t 1

Experiment Design After

Day 1 of d i s c r i m i n a t i o n , the animals were ran-

EFFECT OF ANTIBIOTICS ON RETENTION IN THE PIGEON domly assigned to either Saline, CXM, or PM treatment groups (No combined injections were admimstered.) On Day 2, and thereafter, all antmals were reinforced only for pecking at the stimulus which was unremforced during Day 1. Animals were run until they reached a criterion of 90% correct pecks to the stimulus established as S+ on Day 2 RESU LTS AND DISCUSSION

Figure 4 shows individual Day 2-Day 1 scores for all groups as well as displaying the mean and range. Table 5 shows the results of Experiment 2 In terms of Day 1 and Day 2 scores, mean change in percentage scores from Day 1 to Day 2, and days to criterion. Analysis of variance of Day 1 scores revealed no significant differences (F< 1), whereas on Day 2 the groups did differ significantly, F(2,19) = 7.46, p<0.01 Analysis of the difference scores also revealed an overall significant difference between the groups F ( 2 , 1 9 ) = 3.96, p<0.05 It IS clear that the Day 1 training interfered with Day 2 learning for the control animals. Their mean score decreased from Day 1 to Day 2. No such effect is found in the PM animals, whose performance is significantly superior to the controls. The PM animals actually showed an Increase In mean percentage scores, but it was not a significant increase ( t = 2 04, p < 0 1) As shown by Fig 4, four of the antmals had higher Day 2 scores and 3 had lower ones The CXM animals, like the controls, exhibited considerable negative transfer from Day 1 to Day 2 (showing a decrease in correct percentage), being only slightly and nonsignificantly (t = 0.030, p< 1 0) superior to the controls, and inferior though not significantly so (t = 1 8 1 , p < 0 1) to the PM group. The days to criterion analysis reveals the same trends in terms of between group comparisons. Saline animals took the most sessions to reach criterion and were significantly poorer than the PM animals ( t = 3 9 7 , p < 0 0 1 ) . CXM animals were only very slightly and insignificantly better than the controls and were on this measure significantly poorer than the PM injected birds. Of considerable significance is the fact that comparison of the days to criterion for the PM animals in Experiment 2 with those In Experiment 1 shows that the former actually took significantly fewer days to reach criterion (t = 2.97, p<0.005). The demonstration of superior performance in the PM animals when retention of the training experience just prior to injection produces negative effects on subsequent discrimlnatIon performance clearly supports an amnesic interpretation of the deficits in Day 2 performance of the PM group found in Experiment 1. (This confirms a similar

153

30 20

÷O3

Puromycin CyclohexJmlde

I0

o .~.

Saline 0 .....

"6 -IO

c

--I Mean

8~ 2 -20 -30 -40

FIG. 4. Difference in the percentage of all pecks made to the positive stimulus between the f'trst and second day of discrimination training in Experiment 2. The positive stimulus was reversed between the first and second day. Vertical lines display the entire range of the distribution for both groups, each point shows the score of an individual antmal.

finding by Mayor [23] who used reversal of a color discrimination in Japanese quail.) The continued acquisition deficit shown by these ammals could not account for the results of Experiments 1 and 2 These experiments together confirm the existence of both an amnesic and a continued acquisition effect of PM. The fact that there was also negative transfer in the CXM groups sheds further hght on the partial amnesia reported in this group in Expertment 1 This indicates unequivocally that there was, in fact, considerable retention of the Day 1 training expenence in this group, and further differentiates (at least quantitatively if not quahtatlvely) the CXM and PM effects. The superiority of the performance level of the PM animals on days to criterion m Experiment 2 as compared to Experiment 1 provides something of an enigma. This finding means that these subjects actually reached criterion on a reversal of their Day 1 training more rapidly than animals who were trained to peck at the same stimulus as had been reinforced on Day,1 Amnesia alone can not account for this; it could at most lead to equal performance in the two groups. The answer appears to be not in the amnesic effects of PM (which appear to occur in both the

TABLE 5 PERCENT CORRECT RESPONSE AND DAYS TO CRITERION (DTC) FOR HABIT REVERSAL OF A SUCCESSIVE PATTERNDISCRIMINATIONFOR CONTROL AND EXPERIMENTALGROUPS

Group

N

Percent Correct Response Day 1 Day 2 Standard Standard Mean Dev,atlon Mean Deviation Day 2-Day 1

Saline CXM PM

8 7 7

49.7 51.2 50.0

83 10 4 8.2

41.1 42.2 54.2

7.1 6.2 8.0

-8 6 -8.2 +4.2

DTC Mean

Standard Deviation

8.4 8.0 5.1

1.4 0.82 0 69

154

SI'[ F ] N I R, BARR,\(~(I

reversed and n o n r e v e r s e d g r o u p s ) b u t in the c o n t i n u e d a c q u i s l t m n deficit p r e s e n t in E x p e r i m e n t 1 This appears a b s e n t f o r the PM animals in E x p e r i m e n t 2 The m e a n o f 5 1 days to criterion for these subjects is only slightly greater t h a n a d i f f e r e n c e o f 1 day longer to reach criterion t h a n the c o n t r o l s o f E x p e r i m e n t 1 One day d i f f e r e n c e is premsely w h a t would be e x p e c t e d ff animals m Experim e n t 2 had t o t a l a m n e s i a for t h e i r Day 1 training but were equally p r o f i c i e n t at a c q u i s i t i o n starting f r o m scratch on Day 2 (Testing the h y p o t h e s i s t h a t the d i f f e r e n c e b e t w e e n days to c r i t e r m n for PM animals in E x p e r i m e n t 2 and c o n t r o l s m E x p e r i m e n t 1 is greater t h a n + 1 s u p p o r t s this by yielding a value o f t = 0 39, p < 0 4 0 , tar s h o r t o f significance ) It t h u s appears t h a t PM p r o d u c e d amnesia for the Day 1 training in b o t h e x p e r i m e n t s , but p r o d u c e d an a d d i t i o n a l deficit on f u r t h e r acquisition only in Experim e n t 1 w h e n the s u b s e q u e n t training involved r e i n f o r c e m e n t for p e c k i n g at the same stimulus as in the first session This Indicates t h a t the c o n t i n u e d a c q u l s m o n deficit prod u c e d by PM is n o t general b u t is very highly specific right d o w n to the selection o f the r e i n f o r c e d s t i m u l u s This e f f e c t can be c o n c e p t u a h z e d by assuming that PM impairs the f u r t h e r d e v e l o p m e n t o f n e u r o c h e m l c a l circuits that are active at the t i m e o f Its Injection but has no e f f e c t on the s u b s e q u e n t laying d o w n o f n e w circuits which r e p r e s e n t different response choices

\'~l) \ ( ~ R M I [ I

S U M M A R Y A N D ( ' O N C L U S I O N ~, The results of k x p e r l m e n t s 1 and 2 cleat1} mdlcatt_ th,lt PM and CXM have amnesic e l f e c t s in a lralmng s~tuat~on w h e r e general p e r f o r m a n c e d e c r e m e n t (m terms t,l oxerall p e c k i n g rate) can be assessed m d e p e n d e n t l 3 and ~ a s l o u n d to be absent The p a t t e r n o f results o b t a m e d provides l urttier evlde,l~C c o n s i s t e n t w i t h the h y p o t h e s i s that a l t h o u g h PM arid ('XM b o t h p r o d u c e amnesia their e f f e c t s & f r e t substantially {61 F u r t h e r , the nature of the d i f f e r e n c e s is nol L.orisistenl with the differential e f f e c t s p r o d u c e d by these s u b s t a n c e s on p r o t e i n synthesis per se CXM p r o d u c e d a higher level t~l Inhibition o f p r o t e i n synthesis, m o r e p r o f o u n d dffluslon t h r o u g h o u t the brain, and a longer d u r a t m n oI peak i n h i b i t i o n However, It p r o d u c e d considerably les~ ol an amnesic e f f e c t than PM in b o t h e x p e r n n e n l s , and had no effect on c o n t i n u e d acquisition Thus we can lnler t h a t the amnesic and c o n t i n u e d a c q m s l t I o n effects ot PM must be associated with some relatwely unique effects ot th~s s u b s t a n c e n o t reflected directly in general level oI p r o t e i n s y n t h e s i s inhibition It appears that the m o s t promising avenue o f investigation to pursue in this regard would follow l r o m the work o f F l e x n e r [11,171 which indicates that the key to u n d e r s t a n d i n g the behavioral e l t e c t s ol PM involves Its relationship to p e p t i d e activity m the nervous system

REFERENCES 1. Agranoff, B W Memory and protein synthesis. Sctent A m 216: 115-122, 1967 2 Agranoff, B W, R E. Davis and J J. Brink. Memory fixation in goldfish. Proc natn Acad. Sct U S A . 54: 788-793, 1965 3 Barondes, S H. and H D Cohen. Comparative effects of cycloheximide and puromycin on cerebral protein synthesis and consolidation of memory In mice Brain Res 4: 4 4 - 5 1 , 1967 4. Barondes, S. H and H. D. Cohen. Delayed and sustained effect of acetoxy-cycloheximIde on memory in mice. Proc natn Acad. Scl. U.S.A 58: 157-164, 1967 5 Barondes, S. H. and H. D Cohen Memory impairment after subcutaneous rejection of acetoxycyclohexlmlde Sctence 160: 556-557, 1968 6 Barraco, R. A. and L. J Stettner. Antibiotics and memory Psychol Bull 83: 242-302, 1976 7 Brink, J J., R E. Davis and B. W. Agranoff. Effects of puromycln, acetoxycycloheximide, and actinomycm-D on protein synthesis In goldfish. J. Neurochem. 13: 889-896, 1966 8 Cohen, H D and S. H Barondes Cycloheximlde Impairs memory of an appetitive task. Communs Behav Biol 1: 337-340, 1968. 9. DanIets, D. Effects of acetoxycycloheximide on appetitive learning and memory Q Jl exp Psychol. 24: 102-114, 1972 10. Flexner, J. B and L B. Flexner. Restoration of expression of memory lost after treatment with puromycln. Proc natn Acad Sct U.SA. 57: 1651-1654, 1967 11. Flexner, J. B and L. B. Flexner. Pituitary peptides and the suppression of memory by puromycin. Proc natn A c i d Scl U S A 68:2519 2521, 1971 12. Flexner, J B , L. B. Flexner and E Stellar. Memory In mice as affected by mtracerebral puromycm. Science 141: 57-59, 1963 13. Flexner, L. B. and J B. Flexner. Effect of acetoxycyclohexlmide and of an acetoxycycloheximide-puromycmmixture on cerebral protein synthesis and memory in mice. Proc natn Acad Sct U S A 55: 369-374, 1966

14

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