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Effects of interruption of limbic system pathways on different measures of activity
Physiology & Behavior, Vol. 17, pp. 65-72. Pergamon Press and Brain Research Publ., 1976. Printed in the U.S.A.
Effects of Interruption of Limbic System Pathways on Different Measures of Activity SALVATORE CAPOBIANCO z AND L E O N A R D W. HAMILTON 3
Department o f Psychology, Rutgers University, Busch Campus, New Brunswick, NJ 08903 (Received 16 April 1975) CAPOBIANCO, S. AND L. W. HAMILTON. Effects of interruption of limbic system pathways on difjerent measures of activity. PHYSIOL. BEHAV. 17(1) 65-72, 1976. - Activity levels were measured following selective knife-cuts of the fornix, medial forebrain bundle or diagonal band. Each of these manipulations increased running-wheel activity, but only fornix transection increased activity in the stabilimeter. The stabilimeter activity declined following diagonal band cuts, and did not change following medial forebrain bundle cuts. None of these manipulations changed open field or rearing activity. The different patterns of effects suggest different anatomical systems that control activity, and emphasize thc heterogeneity of activity indices. Activity
Fornix
Medialforebrain bundle
Diagonalband
RECENTLY some studies involving restricted disruption of neural activity in the forebrain have dissociated some of the disinhibitory effects attributed to structures such as the septum and hippocampus (e.g., [13,16]). Several researchers have suggested that the forebrain, particularly the limbic system, may perform an inhibitory function by modulating brainstem arousal mechanisms (cf. [17, 20, 22]). In view of the large number of studies which generally support this notion, surprisingly little work has been done to effectively isolate the neural systems involved in these phenomena. Lesions involving various portions of the limbic system and the subsequent measurement of changes in activity levels, have yielded a variety of responses depending upon the testing conditions, and the structure being investigated. For example, gross septal lesions, or lesions restricted to the medial septum, have been reported to result in a marked hypoactivity when the animals are tested in the running wheel [ 5,8 ]. Similarly, spontaneous or generalized baseline activity levels are reduced when rats with septal lesions are tested in small enclosures [8]. However, hyperactivity is reported if any change is introduced into the external or internal environment [ 3,9 ]. The hippocampus appears to be the limbic system site involved in modulating activity functions since surgical disruption frequently results in an increase in generalized activity levels in a variety of measuring devices [16]. Hyperactivity is often reported in the stabilimeter and open field [29], while locomotor changes utilizing the running wheel have shown either a decrease [2,16], or no change in activity levels following bilateral hippocampal lesions [29]. Other limbic system sites investigated for lesion-related
Limbic system
alterations in activity levels are the cingulum and amygdala. However, no changes from control baselines have been noted following lesions in either area [3,10]. One of the major problems in the investigation of anatomical functional relationships has been the inability to restrict damage to a specific chosen locus because conventional techniques for producing lesions often inadvertently destroy adjacent neural tissue. The importance of surgical specificity has been demonstrated by Lynch and associates [20,21] in their efforts to dissociate some of the anatomical and behavioral substrates of spontaneous activity. For example, Richter [271 originally demonstrated that bilateral removal of the frontal poles of the rat produced a large increase in wheel-running behavior. When tested in the stabilimeter these animals have been found to be more responsive to the activity potentiating effects of amphetamine and deprivation than controls [20]. Subsequently, Lynch [20] delimited separate neural pathways involved in wheel-running and stabilimeter activity. The system that influenced wheel running was found to extend from ventral frontal cortex into the medial forebrain bundle (MFB), while stabilimeter activity appeared to be under the control of fibers originating in the dorsal frontal cortex and passing through the inferior thalamic peduncle. It was suggested that these effects were produced by interactions with the reticular formation by interrupting a reticulo-cortico-reticulo loop which is then manifested by an increase in spontaneous activity [ 20]. Numerous studies have implicated the hippocampus and the reticular formation in the control of behavioral arousal and activity. For example, electrophysiological evidence suggests that reciprocal interactions may exist between
i This research was supported by grants to L. Hamilton from the U.S.P.H.S. (No. MH 2472) and an NIMH Predoctoral Fellowship awarded to S. Capobianco. 2 Now at Department of Psychology, University of Scranton, Scranton, Pennsylvania. 3 Requests/or reprints should be mailed to Leonard W. Hamilton, at above address. 65
66
(?APOBIAN( O AND HAMILTON
these 2 areas since stimulation of the reticular formation leads has been found to lead to synchronized hippocampal theta waves concomitant with desynchronized cortical EEG. Conversely, electrical stimulation of the hippocampus has been shown to produce inhibition of reticular activity [ 1,11 ]. Behaviorally, previous studies have indicated that hippocampal lesions result in increased activity in the stabilimeter [29] and open field [2]. Donovick [7t has demonstrated that lesions of the anteromedial septum both block hippocampal theta responses and result in greater intertrial activity. Further, it was found that lesions impinging upon the posterior septum (i.e., fornix-fimbria, and triangular nuclei) also increased activity. Recently, Capobianco and Hamilton [4] demonstrated a large increase in wheel-running behavior following discrete knife cuts of the fornix. These findings would seem to indicate a possible hippocampal mediation of arousal functions, as indexed by the running wheel and other measures of activity. Anatomical studies have demonstrated that a coarsefibered ascending pathway interconnects the limbic system and the brainstem via the MFB [ 12]. These ascending fibers were found to relay to the hippocampus in the lateral hypothalamus and bed nucleus of the diagonal band (DB). However, most fibers were found to synapse in the anteromedial nucleus of the septum. The anteromedial nucleus then projects these fibers from the septum to the hippocampus via the fornix bundle. Most hippocampat efferent fibers project through the fornix-fimbria system which becomes obvious at the level of the anterior pole of the hippocampus. From this point of convergence, the fibers arch downward and split into a rather diffuse precommissural component, which descends through the septum anterior to the level of the anterior commissure then into the preoptic area, and a more compact: postcommissural component which descends to the mammillary bodies [25]. Since this pathway implicates specific fiber tracts projecting between the limbic system and brainstem, knife cuts were placed at various points utilizing a modification [14] of the Scalafani and Grossman [28] microencephalotome to determine if interruption of this pathway would result in an alteration of behavioral excitability in a variety of testing situations. More specifically, transection was attempted in the anterior portion of the MFB, at the level of the anterior commissure, the DB ventrolateral to the septum, and the total fornix bundle just anterior to the hippocampus. EXPERIMENT 1 : WHEEL-RUNNING ACTIVITY METHOD
Animals The animals were 39 male albino rats of the SpragueDawley strain (CFE). The rats ranged in weight from 200 to 240 g, and were approximately 90 days old at the time of surgery. All animals were housed under ad lib food and water conditions, and were maintained in individual cages.
Surgical Procedures and Histology All surgery was performed under Equi-Thesin anesthesia (Jensen-Salsbury) by intraperitoneal injection of 0.25 cc/100 g body weight. Knife cuts of the total fornix bundle (FX) were made by positioning the knife such that when extended, the wire
formed an arc which projected 1.0 nmi below and 3.0 mm beyond the tip of the shaft. With the knife retracted, the shaft was angled 30 ° anterior lapproximately perpendicular to the fornix fibers) and lowered tO the following K6nig and Klippel [16] coordinates AP = 6.2, if = 1.5. [ = 1.5. Once the guide shaft had been towered to this point adjacent to the fornix, the knife was extended across ihe midline directly tinder the fornix b u n d l e and then raised 1.5 mm to effect the bilateral c'.~ through t:h( entire bundle. To effect transection of the diagonal bands IDB), the knife assembly was angled 15 ° laterally to the following coordinates: AP : 8.9, H = -1.0.. ~, .... ~ 0.3 ram. The extended knife formed an ar~: ~',"~,,, extended 2 . 0 r a m beyond and 1 . 0 m m below the tip ~;f the guide shaft. The assembly was then positioned such that the knife extended posteriorly from the guide shaft and passed through a t.5 mm vertical excursion to effect the cut. The medial forebrain bundle (MFB) was sectioned at the level of the anterior commissure, corresponding to coordinates: AP = 6.8, H = 3:0, [.... 21q. The knife extended 2.5 mm beyond and 1.0 mm ventral to the tip of the guide shaft. The carrier was adjusted Io an excursion 0f 2.5 ram, lowered to the proper coordinates where the knife extended medially, and the transection effected. The same procedures were again repeated contralaterally except that the tip of the knife pointed in a taieral direction. C o n t r o l animals were treated identically except lhat the knife was not lowered into the brain. Upon completion of behavioral iestmg the rats were sacrificed with an overdose of Equi-Thesin and perfused with Saline followed by a formol-saline solution. Ttie brains were removed and fixed in a 10% Formalin solution. Ever)' fourth 50-u thick frozen section was stained with cresyl violet and microscopically examined to determine the extent to damage.
7e.~llng Procedures Following surgery, the rats were placed in Wahmann activity wheels with attached living cages. Free access to the wheel was given during the entire testing period. Food and water were available ad lib. Both the colony room in which the anhnals were originally housed and the testing room were maintained on a 16 hr/8 hr light/dark cycle. One hr after the onset of the daily period of illumination, lhe following measures were taken: total activity, body weight, food and water intake. All animals were housed in the wheels for a period of 24 days. The last 12 clays of the session were used to assess differences between experimcnlal and control animals. R E S U k IS
The stained sections were examined microscopically to determine the locus and extent of damage produced by the various surgical procedures. Eight animals sustained bilateral total transection of the fornix bundle extending dorsally to the corpus callosum and ventrally to the third ventricle. All knife cuts sectioned the fOrnix completely at a point just anterior to the hippocampus. Five animals sustained cuts that passed through both limbs of the diagonal bands, typically extending dorso-ventrally from a point immediately ventral and lateral from the ventricles to the base of the brain: Typically, tlie anterior-posterior extent of these cuts extended anteriorly to portions of the
LIMBIC P A T H W A Y S A N D A C T I V I T Y
67 accumbens nuclei and posteriorly to the bed nucleus of the stria terminalis. In one case, the damage e x t e n d e d posteriorly b e y o n d the crossing of the anterior commissure. Seven animals sustained cuts that passed through the MFB fiber bundles bilaterally at levels extending from 1 to 2 mm anterior to the crossing of the anterior commissure, and ventrally to the base of the brain. The second phase of the e x p e r i m e n t required new surgical groups. The damage to these animals was virtually identical to the first phase, with final group sizes of 7, 6, 4, and 7, for the Groups FX, OC, DB, and MFB, respectively. The p h o t o m i c r o g r a p h s in Fig. 1 show representative stained sections through the midpoints of the various transections. The data for the running-wheel activity were collapsed across 3 day blocks for purposes of statistical analyses. As is usually the case in this type of procedure, the a m o u n t of running varied considerably with occasional animals showing little or no activity while others made over 14,000 revolutions during the 3 day block. Accordingly, a square root transformation was performed on the data before doing a 2-way analysis of variance. The results of this analysis revealed a significant Groups and Trials interaction (F(9,81) = 3.31, p < 0 . 0 0 2 , see Fig. 2). Subsequent 2-sample comparisons using the Fisher test of least significant differences (critical value = 23.1) indicate that the bases for this interaction lie in the increased activity of the MFB group and the steadily increasing level of activity of the DB group across days. Although the FX group was somewhat more active than controls, this difference did not reach statistical significance. Following surgery a mild aphagia and adipsia resulted in 2 of the animals which sustained knife cuts of the MFB. Consequently, this group was maintained on diluted sweetened condensed milk, supplem e n t a r y to their regular diet of Purina Lab Chow and water, for a period of 7 days. Since this procedure does c o n f o u n d food and water comparisons, the MFB group was excluded from data analysis on these measures, although they were included in tests for differences on body weights. No statistical differences ( F < 1) were found between food and water intake of control rats and rats with transections of the DB and FX. RUNNING
WHEEL
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FIG. 1. Photomicrographs of brain sections showing: (a)a parasaggital view of a MFB cut, (b) a parasaggital view of a FX cut, and (c) a coronal view of a DB cut.
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FIG. 2. Square root of wheel-running activity across 3 day blocks for controls (CON), rats with fornix cuts (FX), medial forebrain cuts (MFB) and diagonal band cuts (DB). The bracket (c.v.) indicates the critical value (t7 = 0.05) for Fisher's test of Least Significance Differences.
68
C A P O B I A N C O AND t t A M I L T O N
The analysis of variance for the percent of the preoperative b o d y weight revealed that the rats in the e x p e r i m e n t a l groups were marginally lighter than the control rats ( F ( 1 5 , 1 3 7 ) = 1.78, p < 0 . 0 5 , c.v. = 4.83). The mean b o d y weights on Day 12 expressed as a percent of preoperative weights, were 117, 117, 112, and 122 for groups FX, MFB, DB and CON, respectively.
E X P E R I M E N T 2: S T A B I L I M E T E R . OPEN F I E L D A N D REARING ACTIVITY Recent studies (e.g., 124]) ha~" d e m o n s t r a t e d thai various measures of activity may be differentially influenced by deprivation and a n u m b e r of o t h e r manipulations. In an effort to gain a better understanding of limbic system mechanisms of activity, the present e x p e r i m e n t assessed the effects of various knife cuts on 3 additional indices of l o c o m o t o r activity. METHOI
DISCUSSION
Transections of the MFB and DB were found to result in large increases in wheel running behavior. The present study did not utilize any procedures (e.g., deprivation, or amphetamine) o t h e r t h a n the knife cuts in the various tracts, to enhance the activity increases. However, it should be noted that wheel-running behavior has been d e m o n s t r a t e d to be sensitive to any change in the internal e n v i r o n m e n t [6]. This being the case, it may be argued that a change in h o m e o s t a t i c mechanisms could account for the increase m activity. Transections of the MFB did cause a mild aphasia and adipsia leading to lower b o d y weights, but these animals recovered to a point of virtually no difference from control (final means on Day 12; 122% and 111%, respectively, for Groups CON and MFB). Similarly, o t h e r investigators [16] have failed to find changes in food intake, water intake, or b o d y weights when knife cuts are placed in anterior portions o f the MFB. Also, it should be n o t e d that the increase in activity in the MFB group was c o m p a r a b l e with DB transections, and these animals did not differ significantly from control, in terms of food and water intake. Consequently, i n t e r o c e p t i v e changes do not seem to adequately account for the enhanced activity. The most plausible i n t e r p r e t a t i o n of these effects would seem to be a change in the arousal or excitability level of the animal. The magnitude of the increase in wheel activity was somewhat smaller, following fornix transection, than reported in a previous study [4]. The major procedural difference was that the previous study used female rats. The obvious suggestion is a h o r m o n a l l y based influence on activity, but the results, in the previous study, of vaginal smears indicated normal estrus cycles [4]. F o r n i x transections may also interact with the baseline level of activity in such a manner that differences f r o m control levels are greater when the activity levels are higher. Finally, it should be noted that the changes in running wheel activity bear no consistent relationship to the observed changes in reactivity to handling. Neither the rats with MFB transections nor the rats with FX transections showed changes in reactivity to handling at the time of testing (FX transections s o m e t i m e s result in a very transient hyperreactivity). Rats with transections o f the DB were mildly hyperreactive t h r o u g h o u t the course of the experiment. Unless there exists some very complicated interaction a m o n g the variables of brain damage, wheel running activity and reactivity to handling, it seems unlikely that the increased running wheel activity can be attributed to hyperreactivity. With respect to the differences in response to handling that were observed, our observations are consistent with Turner's [31] view that damage to the bed nucleus o f the stria terminalis is largely responsible for the increased reactivity that is observed following septal lesions: the DB transections in the present study rather consistently damaged this nucleus or fibers that would be closely associated with this nucleus.
Animals The animals were 32 male albino rats of the SpragueDawley strain (CFE). The rats ranged in weight from 220 to 250 g, and were a p p r o x i m a t e l y 100- 110 days old at the time of surgery. All animals were housed under ad lib food and water conditions, and were maintained in individual cages.
Surgical Procedures and HistoloKv The surgical procedures and histological procedures were identical to those described in E x p e r i m e n t I. The locus and extent of damage in the knife cut groups was microscopically e x a m i n e d and found to be similar to that sustained by animals in E x p e r i m e n t J.
Apparatus and Testing Procedures Stabilimeter. The stabilimeter cages, c o m p o s e d of wire mesh, measured 39 x 18 10 em high. They were m o u n t e d on a rack and placed m an insulated, soundattenuating, cabinet which was lightproof. Four microswitches were positioned under the floor and calibrated to the a p p r o x i m a t e weight of the animal, such that when m o v e m e n t s were made from one area of the cage to the o t h e r a c o n t a c t was closed, the activity was recorded on counting devices. Each testing session consisted of placing the animal m the stabilimeter for 20 min/day Ior a total of 4 days. Activity measures were taken at t0 rain intervals for tile within sessions comparisons. Open field. U p o n c o m p l e t i o n of lhe stabilimeter testing all animals were tested in the open field. This phase of testing consisted of placing the animal in a Ptexiglas box 60 60 x 37 cm high with a grid floor. The enclosure was divided into quadrants by placing photocells into the side of the box. Each time the animal crossed from one quadrant into the next. the beam from the photocell was m t e r r u p t e d , resulting in a circuit being closed and registering the total n u m b e r of crossings on counting devices. Within session measures were taken at 5 rain mtervals for a total of 15 rain. Daily sessions were continued for a total of 3 days. Rearing. A Plexiglas box measuring 63.5 cm in height with a 30 x 28 cm grid floor was placed in a soundproof. lightproof, ventilated enclosure. A bank of 8 photocells was arranged on the wall o f the c o m p a r t m e n t , and adjusted m the size of the animal such that when the animal rearea on its hindlegs, the head and shoulders would break the beams of the photocells resuRing in a circuit closure and the activity being recorded. Each animal was placed in the testing chamber for 10 min/day. The number and duration of rearing responses was recorded automatically each min for the daily session. All animals were tested for a total of 3 days.
LIMBIC PATHWAYS AND ACTIVITY
69
RESULTS
Stabilimeter.
OPEN
The stabilimeter activity levels of the
various groups, s h o w n in t h e u p p e r p a n e l of Fig. 3, reveals a large increase in activity associated w i t h f o r n i x t r a n s e c t i o n , whereas rats sustaining t r a n s e c t i o n of t h e diagonal b a n d s h o w e d r e d u c e d activity ( F ( 3 , 1 9 ) = 18.5, p < 0 . 0 1 , c.v. = 162). All t r e a t m e n t groups s h o w e d declining activity across days ( F ( 3 , 5 7 ) = 3.03, p < 0 . 0 5 ) . T h e l o w e r p a n e l of Fig. 3 shows t h e within-session changes in activity. The A N O V A revealed a significant G r o u p s × I n t e r v a l i n t e r a c t i o n ( F ( 3 , 1 9 ) = 3.48, p < 0 . 0 5 , c.v. = 40.0). T w o - s a m p l e c o m p a r i s o n s a t t r i b u t e d this intera c t i o n to a higher initial level o f activity for t h e F X rats and a l o w e r overall level of activity for the DB rats.
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FIG. 4. Total activity in open field as a lhnction of days (upper panel). The lower panel shows activity changes during successive 5 rain periods within the session (collapsed across days). Legend is same as in Fig. 2.
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MINUTES FIG. 3. Stabilimeter activity as a function of days (upper panel) and during the first and second half of each session (lower panel) by collapsing across days. Legend is same as in Fig. 2.
Open field. T h e results o f t h e o p e n field test, s u m m a r i z e d in Fig. 4, show no c o n s i s t e n t d i f f e r e n c e s as a f u n c t i o n o f surgical t r e a t m e n t . T h e a p p a r e n t d i f f e r e n c e s in Day 1 o f the u p p e r p a n e l are a t r i b u t a b l e to e x t r e m e scores
r a t h e r t h a n a c o n s i s t e n t d i f f e r e n c e in central t e n d e n c i e s . A l t h o u g h t h e r e were no c o n s i s t e n t differences in overall activity across days ( F < I ) , all groups s h o w e d a significant decline in activity w i t h i n each daily session ( F ( 3 , 3 4 ) = 23.6, p < 0 . 0 0 1 ; see l o w e r panel of Fig. 4). Rearing. As in the case of o p e n field activity, the surgical t r e a t m e n t s had no c o n s i s t e n t effect on rearing responses. However, all g r o u p s s h o w e d a significant decline in the a m o u n t o f rearing b o t h across days ( F ( 2 , 3 7 ) = 16.3, p < 0 . 0 1 ; see u p p e r p a n e l o f Fig. 5), and w i t h i n daily sessions ( F ( 4 , 7 2 ) = 17.8, p < 0 . 0 1 ; see lower panel of Fig. 5). DISCUSSION The results o f these e x p e r i m e n t s suggest the existence of t w o s e p a r a t e and o p p o s i n g systems of c o n t r o l for the t y p e of activity m e a s u r e d by the s t a b i l i m e t e r device. T r a n s e c t i o n
70
CAPOBIAN(O REARING
fornix in the p r e s e n t study, These results, in c o m b i n a t i o n , suggest t h a t this b e h a v i o r may be m e d i a t e d by a septoh i p p o c a m p a l circuit. However, lhe s c p t u m may pJay a dual role in t h i s b e h a v i o r , since lesions of lhe ,~eptum reduce s t a b i l i m e t e r activity levels [ 1 0 i as did t r a n s e c t i o n o f ~he DB in l h c present study. Thus. the: overall p a t t e r n of activity changes i n d i c a t e t h a t d a m a g e i c~ ~he septot~ippocanlpal s y s t e m m a y increase this ~ype of l o c o m o t o r activity, whereas d a m a g e ~ !he ~ e p t o h y p o l h a ~ a m i c m i d b r a i n s y s t e m may r e d u c e such activi! y
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FIG. 5. Rearing activity as a function of days (upper panel), and as a function of intervals within the session flower panel). Legend is same as in Fig. 2. TABLE
I
EFFECTS OF INTERRUPTION OF LIMBIC SYSTEM PATHWAYS ON DIFFERENT ACTIVITY MEASURES Surgical Group
Running Wheel
Stabilimeter
Open Field
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*Abbreviations: FX = Fornix transection, DB = Diagonal band transection, MFB = Medial forebrain bundle transection. +Some evidence for transient increase. :~Some evidence for transient decrease.
o f the F X r e s u l t e d in large increases in activity, while t r a n s e c t i o n o f t h e DB r e d u c e d activity. T h e t r a n s e c t i o n o f M F B was i n e f f e c t i v e in altering activity levels. T h e results o f t h e s e k n i f e cut studies are generally c o n s i s t e n t w i t h t h o s e of studies involving lesions o f t h e s e p t u m a n d h i p p o c a m p u s . Lesions of t h e h i p p o e a m p u s increase s t a b i l i m e t e r activity [ 2 9 ] , as did t r a n s e c t i o n o f t h e
D i f f e r e n t measures of a c t i w t y , sucl~ ~s r u n n i n g wheel. s t a b i l i m e t e r , o p e n field and o t h e r :ypes of e x p l o r a t o r y b e h a v i o r have f r e q u e n t l y been discussed as shnply representing d i f f e r e n t methods ot measuring the same phenomenon l o c o m o t o r actwity. V u r t h e r m o r e . Hmre is an a h n o s t n a t u r a l a s s u m p t i o n that these behaviors are e q u a t e d with t h e t h e o r e t i c a l c o n c e p t , f b e h a v i o r a l arousal, t h e s u p p o s i t i o n being, thal the I o c o m ~ t o r activity reflects this c e n t r a l m o t i v a t i o n a l stale Evidence a c c u n m l a t e d during t h e past few years has challerlged the basic simplicity o f these n o t i o n s , and it is b e c o m i n g increasingly a p p a r e n t t h a t an u n d e r s t a n d i n g o f l o c o m o t o r activity and behavioral arousal will require a m u l t i f a c e t c d appr(mch P e r h a p s t h e first indication ol ihe c o m p l e x i t i e s to be e n c o u n t e r e d was t h e evidence_ p r e s e n t e d by H e b b 1 ! 5 l . t h a t t h e r e l a t i o n s h i p b e t w e e n central :lrousal stales and behavioral activity was not m o n o t o n i c but followed an i n v e r t e d U-shaped f u n c t i o n . ~_.. ~tc~ivtty was h i g h c s | ,'or i n t e r m e d i a t e levels o f arousal a n , dcctiaed witl" e i t h e r lower or higher levels of arousal. S u p e r i m p o s e d o n t h e r e l a l i o n s h i p s described by H e b b are a n u m b e r . f e x p e r i m e n t s ttlat fail to s h o w parallel changes in b e h a v i o r using different measures oi activity. Tapp [ 3 0 1 , for e x a m p l e , c o m p a r e d 5 different measures of activity a n d f o u n d virtually no i n t e r c o r r e l a t i o n s , the only m e a s u r e s l h a t c o r r e l a t e d highly were the r u n n i n g wheel activity and l i g h t - c o n t i n g e n t bar pressing. 1{ was suggested that this c o r r e l a t i o n m a y be a t t r i b u t a b l e to lhe fact | h a l b o t h of these types of activity invo!ve c o n s i d e r a b l e feedback from the e n v i r o n m e n L Bolh Tapp [30] and Collier c t al. [6] have r e p o r t e d that f o o d d e p r i v a t i o n can lead to increased aclivity in some situalJons~ Miezejeski c t a / . [24] have e x t e n d e d these findings by m a k i n g direct c o m p a r i s o n s of r u n n i n g wheel, s t a b i l i m e t e r and rearing activity,. Starvat i o n p r o d u c e d a d r a m a t i c increase in rnmning wheel activity, decreased s t a b i l i m e t e r activity and had little or no effect ~ n rearing. I n l e r e s t m g l y , the p r o c e d u r a l dermis tot lhc a s s e s s m e m o f d i f f e r e n t m e a s u r e s of activity have b e e n more c o n s i s t e n t t h a n t h e ensuing t h e o r e t i c a l views of ~hese behaviors. It i~ very difficult to o b t a i n stable measurc~ .,~t i u n n i n g whee] activity w h e n s h o r t test sessions are used It has been suggested (c.f., Collier [ 6 ] ) t h a i r u n n i n g wheeI activity is. in a sense, r e g u l a t o r y , and t h a t t h e final baselines of activity i n t e r a c t w i t h b o t h t h e n a t u r e a n d the q u a n t i t y o f the diel t h a t is c o n s u m e d . The stahilime~cr also r e p r e s e n t s m e a s u r e of l o c o m o t o r activity, bill t h e o p p o r t u m t y lor e n e r g y e x p e n d i t u r e is greatly curtailed Because ol the physical r e s t r i c t i o n s of this a p p a r a t u s , tesl so, stuns are typically s h o r t , and t h e i n t e r p r e t a t i o n s lean m o r e t o w a r d r e a c t i v i t y t h a n to m a i n t e n a n c e . Lat I t 9 ] a n d his associates have p e r f o r m e d a variety o f e x p e r H n e n t s involving the
LIMBIC PATHWAYS AND ACTIVITY
71
rearing repsonse and have concluded that it reflects both the general state of arousal of the organism, and the tendency for exploration. The operant level of this response drops very rapidly over time, so test sessions are typically very brief. Finally, the use of the open field has varied depending upon the interests of the investigator. The tendencies of the rat to exhibit thigmotaxis and to freeze in open areas are both exaggerated in this situation when the illumination levels are high. When tested in darkness or low-level illumination, the tendency to explore is much greater, and the rat will show the typical decline in activity that has been interpreted as the habituation of exploratory activity [22]. The procedural and theoretical differences among these measures accentuate the heterogeneity of activity indices and strongly suggest that the behaviors that are monitored in these situations may be under the control of different brain mechanisms. The present experiments have attempted to delineate some of these neural circuits by comparing the effects of the transection of three major limbic system pathways on four different measures of activity. Rather than attempting to make direct comparisons of the different indices under all conditions, the procedures were selected to represent the most commonly used parameters in each case. Thus, the rats were housed for a number of days in the running wheels, whereas, the other measures involved tests of short duration under low levels of illumination that result in relatively high levels of activity in normal rats. The results of these manipulations are summarized in Table 1. Of particular interest are the differing patterns of activity changes that follow the different knife-cuts. Knife cuts that produce increased activity in the running wheel can lead to an increase, a decrease, or no change in stabilimeter activity. In the present experiments, none of these manipulations significantly influenced activity as measured by open field activity or rearing. This lack of effect cannot be attributed to a simple lack of sensitivity, because lesions in the raphe nucleus greatly increase rearing but have little or no effect on running wheel activity (unpublished observations, Miezejeski and Hamilton). Furthermore, there is the possibility that the failure to observe large differences in activity in the open field and rearing tests may be attributable, in part, to sequence effects. Recently, it has been observed that rats with fornix transections show considerably higher levels of rearing responses when this is the initial test that occurs following the surgical disruption (MacDougall and Capobianco, unpublished observations). Consequently, there may be sufficient generality among the different test environments that the effects of novelty (in relation to the home cage) are reduced by previously exposing the same animals to a variety of testing situations. The results of the selective knife cuts are important regardless of the precise neural pathways that are disrupted; the fact that different patterns of changes are observed is, in and of itself, sufficient
evidence that the response measures are under separate neural control. The changes in activity levels can also be used as a tool to determine the anatomical pathways that may be involved in the control of different types of activity. The fact that all 3 manipulations increase wheel running activity suggests a long (probably polysynaptic) fiber system interconnecting the limbic midbrain with the septum and the hippocampus. The relatively smaller effects of the fornix transection would indicate lesser involvement by the hippocampus. The relationship of the fornix transections to the effects of septal lesions or hippocampal lesions are still somewhat difficult to explain. Both large septal lesions and smaller lesions that are restricted to the medial septum and nucleus of the diagonal band result in a decrease of wheel running activity [5,8], as do lesions of the hippocampus, at least in some cases (e.g., [2]). The fact that the transection of interconnections between the septum and more dorsal structures (FX cuts) or between the septum and more ventral structures (DB cuts) both lead to increased running wheel activity poses some interesting questions as to the nature of these neural substrates and their interactions. In the case of stabilimeter activity, the data clearly suggest 2 opposing systems of control. Damage to the septohippocampal system increase this type of activity, while damage inflicted upon the ventrolateral connections of the septum (via the diagonal band) decrease this activity. It should be noted that most of the interconnections between the forebrain structures and the limbic forebrain are via the medial forebrain bundle. The failure of medial forebrain transection to influence activity levels except in the running wheel may be attributable to the location of the cuts. The cuts were sufficiently anterior to allow many descending fibers of septal or hippocampal origin to pass uninterrupted behind the level of the cut. The nature of the differences among these tasks that could involve separate neural control remain somewhat ambiguous. It has been suggested that the running wheel involves a considerable amount of response feedback [30]. Stabilimeters may also involve varying degrees of feedback, owing to the fact that the floors of these cages are typically movable to allow recording, thus providing the animal with direct environmental cues. Other sources of external feedback, however, were reduced or eliminated by isolating the recording equipment from the open field and the rearing apparatus essentially eliminated any external feedback. Other differences are, of course, the degree of locomotor activity involved, the relative novelty of the environment, and the relative positions of the different responses in the organism's response heirarchy. The importance of these variables and their relationship to specific neural circuitry must await further investigation, but the present results suggest that the combination of different measures of activity with different types of neural disruption may be an effective means of establishing such relationships.
REFERENCES 1. Adey, W. R., J. P. Segundo and R. B. Livingston. Corticofugal influences on intrinsic brain-stem conduction in cat and monkey. J. Neurophysiology 20: 1-16, 1957. 2. Altman, J., R. L. Brunner and S. A. Bayer. The hippocampus and behavioral maturation. Behav. Biol. 8: 557-596, 1973. 3. Anderson, R. A. Appetitively motivated general activity in rats with limbic lesions. Physiol. Behav. 5: 755-761, 1970.
4. Capobianco, S. and L. W. Hamilton. Increased activity following fornix transection in the female rat. Physiol. Behap. 11: 407 410, 1973. 5. Clody, D. E. and P. L. Carlton. Behavior~ effects of lesions of the medial septum of rats. J. comp. physiol. Psychol. 67: 344-351, 1969.
72 6. Collier, G., E. Hirsch and A. I. Leshner. The metabolic cost of activity in activity-naive rats. PhysioL Behav. 8: 881-884, 1972. 7. Donovick, P. J. Effects of localized septal lesions on hippocampal EEG activity and behavior in rats. J. comp. physiol. Psychol. 66: 569-578, 1968. 8. Douglas, R. J. and A. C. Raphelson. Septal lesions and activity. J. comp. physioL PsychoL 62: 465-467, 1966. 9. Feigley, D. A. and L. W. Hamilton. Response to novel environment following septal lesions or cholinergic blockade in rats. J. comp. physiol. Psychol. 76: 496-504, 1971. 10. Gotsick, J. E. Factors effecting spontaneous activity in rats with limbic system lesions. Physiol. Behav. 4: 587-593, 1969. 11. Green, J. and A. Arduini. Hippocampat electrical activity in arousal. J. Neurophysiology 17: 533- 538, 1954. 12. Guillery, R. W. Degeneration in the hypothalamic connections of the albino rat. J. Anat. 91:91 -115, 1957. 13. Hamilton, L. W., R. A. McCleary and S. P. Grossman. Behavioral effects of cholinergic septal blockade in the cat. J comp. physiol. Psychol. 66: 563-568, 1968. 14. Hamilton, L. W., E. Worsham and S. Capobianco. A spring loaded carrier for transection of fornix and other large fiber bundles. Physiol. Behav. 10:157 -159, 1973. 15. Hebb, D. O. Drives and the C N. S. (Conceptual Nervous System). PsychoL Rev. 62: 243-254, 1955. 16. Jarrard, L. E. The hippocampus and motivation. PsychoL BulL 79: 1-11, 1973. 17. Kimble, D. E. Hippocampus and internal inhibition. PsychoL Bull 70: 285--293, 1968. 18. KBnig, J. S. R. and R. A. Klippel. The Rat Brain: A Stereotaxic Atlas. Baltimore: William and Wilkins, 1963. 19. Lat, J. The relationship of individual differences in regulation of food intake, growth and excitability of the central nervous system. Physiologia Bohemoslov. (suppl.) 5: 38-42, 1956. 20. Lynch, G. Separable forebrain systems controlling different manifestations of spontaneous activity. J. comp. physioL Ps:vchol. 70: 4 8 - 5 9 , 1970.
C A P O B t A N C O AND H A M I L T O N 21.
22.
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25. 26.
27.
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29.
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31.
Lynch, G. P. Ballantine and B. (ampbelL Potentiation ol behavioral arousal following eorlical lesions and subsequenl recovery. ExplNeurol. 23: 195-206, 1969. McCleary, R. A. Response modulating functions oi the limbic system initiation and suppression. In: Progress in Phys'iological Pvych. Vol. 1. edited by E. Stellar and L M. Sprague. New York: Academic Press, 1966. 209 -272. McReynolds, W. E., M. Weir and ~ t DeFries. ()pen field behavior in mice: Efl~ct of rest illumination. Ps'veh(m Sci 9: 277 -278, t967. Miezejeski, C. M., S. Lamon, G. Collier and L. W. Hamilton. Running wheel, stabilimeter, and rearing activity: Arousal or arousals? Presented at Eastern Psycholoeical Association. Ne~ York, 1975. Nauta, W. J. tl. An experimental s~ucty ol the fornix s~ stem m the rat../, comp. Neurol. 104: 247- 27[. Iq58. Paxinos, G. and D. Bindra Hypothalamic and midbrain neural pathways involved in eating, drinking, irritability, aggression, and copulation in rats. J. comp. phv~ioL Pwehol. 82: I 14_ 1973. Richter, ('~ P. and C. D. Hawkes. increased spontaneous activity and food intake produced by removal of the frontal poles of the brain. ~ NeuroL Psychiat. 2:231 -242. 1939. Sclafani, A. and S. P. Grossman. Hyperphagia produced by knife cuts, between medial and lateral hypotbalamus in the rat. Physiol. Behav. 4: 533-537. 1969. Strong, P. N. and W. J. Jackson. Effects of hippocampal lesions in rats on three measures of aetivity, r cornp, physiol. PsvchoI 70" 60 65, 1970. Tapp, J. T. Activity, reactivity, al~d the behavtor-directmg properties of stimuli. In: Reinfi)rcemenr and Behavior. edited by J. T. Tapp. New York: Academic Press. 1969. Turner, B. H. Neural structures involved in the rage syndrome ~f the rat. J. comp. physiol. P~ychot 71: 103-113. 1970.
Effects of Interruption of Limbic System Pathways on Different Measures of Activity SALVATORE CAPOBIANCO z AND L E O N A R D W. HAMILTON 3
Department o f Psychology, Rutgers University, Busch Campus, New Brunswick, NJ 08903 (Received 16 April 1975) CAPOBIANCO, S. AND L. W. HAMILTON. Effects of interruption of limbic system pathways on difjerent measures of activity. PHYSIOL. BEHAV. 17(1) 65-72, 1976. - Activity levels were measured following selective knife-cuts of the fornix, medial forebrain bundle or diagonal band. Each of these manipulations increased running-wheel activity, but only fornix transection increased activity in the stabilimeter. The stabilimeter activity declined following diagonal band cuts, and did not change following medial forebrain bundle cuts. None of these manipulations changed open field or rearing activity. The different patterns of effects suggest different anatomical systems that control activity, and emphasize thc heterogeneity of activity indices. Activity
Fornix
Medialforebrain bundle
Diagonalband
RECENTLY some studies involving restricted disruption of neural activity in the forebrain have dissociated some of the disinhibitory effects attributed to structures such as the septum and hippocampus (e.g., [13,16]). Several researchers have suggested that the forebrain, particularly the limbic system, may perform an inhibitory function by modulating brainstem arousal mechanisms (cf. [17, 20, 22]). In view of the large number of studies which generally support this notion, surprisingly little work has been done to effectively isolate the neural systems involved in these phenomena. Lesions involving various portions of the limbic system and the subsequent measurement of changes in activity levels, have yielded a variety of responses depending upon the testing conditions, and the structure being investigated. For example, gross septal lesions, or lesions restricted to the medial septum, have been reported to result in a marked hypoactivity when the animals are tested in the running wheel [ 5,8 ]. Similarly, spontaneous or generalized baseline activity levels are reduced when rats with septal lesions are tested in small enclosures [8]. However, hyperactivity is reported if any change is introduced into the external or internal environment [ 3,9 ]. The hippocampus appears to be the limbic system site involved in modulating activity functions since surgical disruption frequently results in an increase in generalized activity levels in a variety of measuring devices [16]. Hyperactivity is often reported in the stabilimeter and open field [29], while locomotor changes utilizing the running wheel have shown either a decrease [2,16], or no change in activity levels following bilateral hippocampal lesions [29]. Other limbic system sites investigated for lesion-related
Limbic system
alterations in activity levels are the cingulum and amygdala. However, no changes from control baselines have been noted following lesions in either area [3,10]. One of the major problems in the investigation of anatomical functional relationships has been the inability to restrict damage to a specific chosen locus because conventional techniques for producing lesions often inadvertently destroy adjacent neural tissue. The importance of surgical specificity has been demonstrated by Lynch and associates [20,21] in their efforts to dissociate some of the anatomical and behavioral substrates of spontaneous activity. For example, Richter [271 originally demonstrated that bilateral removal of the frontal poles of the rat produced a large increase in wheel-running behavior. When tested in the stabilimeter these animals have been found to be more responsive to the activity potentiating effects of amphetamine and deprivation than controls [20]. Subsequently, Lynch [20] delimited separate neural pathways involved in wheel-running and stabilimeter activity. The system that influenced wheel running was found to extend from ventral frontal cortex into the medial forebrain bundle (MFB), while stabilimeter activity appeared to be under the control of fibers originating in the dorsal frontal cortex and passing through the inferior thalamic peduncle. It was suggested that these effects were produced by interactions with the reticular formation by interrupting a reticulo-cortico-reticulo loop which is then manifested by an increase in spontaneous activity [ 20]. Numerous studies have implicated the hippocampus and the reticular formation in the control of behavioral arousal and activity. For example, electrophysiological evidence suggests that reciprocal interactions may exist between
i This research was supported by grants to L. Hamilton from the U.S.P.H.S. (No. MH 2472) and an NIMH Predoctoral Fellowship awarded to S. Capobianco. 2 Now at Department of Psychology, University of Scranton, Scranton, Pennsylvania. 3 Requests/or reprints should be mailed to Leonard W. Hamilton, at above address. 65
66
(?APOBIAN( O AND HAMILTON
these 2 areas since stimulation of the reticular formation leads has been found to lead to synchronized hippocampal theta waves concomitant with desynchronized cortical EEG. Conversely, electrical stimulation of the hippocampus has been shown to produce inhibition of reticular activity [ 1,11 ]. Behaviorally, previous studies have indicated that hippocampal lesions result in increased activity in the stabilimeter [29] and open field [2]. Donovick [7t has demonstrated that lesions of the anteromedial septum both block hippocampal theta responses and result in greater intertrial activity. Further, it was found that lesions impinging upon the posterior septum (i.e., fornix-fimbria, and triangular nuclei) also increased activity. Recently, Capobianco and Hamilton [4] demonstrated a large increase in wheel-running behavior following discrete knife cuts of the fornix. These findings would seem to indicate a possible hippocampal mediation of arousal functions, as indexed by the running wheel and other measures of activity. Anatomical studies have demonstrated that a coarsefibered ascending pathway interconnects the limbic system and the brainstem via the MFB [ 12]. These ascending fibers were found to relay to the hippocampus in the lateral hypothalamus and bed nucleus of the diagonal band (DB). However, most fibers were found to synapse in the anteromedial nucleus of the septum. The anteromedial nucleus then projects these fibers from the septum to the hippocampus via the fornix bundle. Most hippocampat efferent fibers project through the fornix-fimbria system which becomes obvious at the level of the anterior pole of the hippocampus. From this point of convergence, the fibers arch downward and split into a rather diffuse precommissural component, which descends through the septum anterior to the level of the anterior commissure then into the preoptic area, and a more compact: postcommissural component which descends to the mammillary bodies [25]. Since this pathway implicates specific fiber tracts projecting between the limbic system and brainstem, knife cuts were placed at various points utilizing a modification [14] of the Scalafani and Grossman [28] microencephalotome to determine if interruption of this pathway would result in an alteration of behavioral excitability in a variety of testing situations. More specifically, transection was attempted in the anterior portion of the MFB, at the level of the anterior commissure, the DB ventrolateral to the septum, and the total fornix bundle just anterior to the hippocampus. EXPERIMENT 1 : WHEEL-RUNNING ACTIVITY METHOD
Animals The animals were 39 male albino rats of the SpragueDawley strain (CFE). The rats ranged in weight from 200 to 240 g, and were approximately 90 days old at the time of surgery. All animals were housed under ad lib food and water conditions, and were maintained in individual cages.
Surgical Procedures and Histology All surgery was performed under Equi-Thesin anesthesia (Jensen-Salsbury) by intraperitoneal injection of 0.25 cc/100 g body weight. Knife cuts of the total fornix bundle (FX) were made by positioning the knife such that when extended, the wire
formed an arc which projected 1.0 nmi below and 3.0 mm beyond the tip of the shaft. With the knife retracted, the shaft was angled 30 ° anterior lapproximately perpendicular to the fornix fibers) and lowered tO the following K6nig and Klippel [16] coordinates AP = 6.2, if = 1.5. [ = 1.5. Once the guide shaft had been towered to this point adjacent to the fornix, the knife was extended across ihe midline directly tinder the fornix b u n d l e and then raised 1.5 mm to effect the bilateral c'.~ through t:h( entire bundle. To effect transection of the diagonal bands IDB), the knife assembly was angled 15 ° laterally to the following coordinates: AP : 8.9, H = -1.0.. ~, .... ~ 0.3 ram. The extended knife formed an ar~: ~',"~,,, extended 2 . 0 r a m beyond and 1 . 0 m m below the tip ~;f the guide shaft. The assembly was then positioned such that the knife extended posteriorly from the guide shaft and passed through a t.5 mm vertical excursion to effect the cut. The medial forebrain bundle (MFB) was sectioned at the level of the anterior commissure, corresponding to coordinates: AP = 6.8, H = 3:0, [.... 21q. The knife extended 2.5 mm beyond and 1.0 mm ventral to the tip of the guide shaft. The carrier was adjusted Io an excursion 0f 2.5 ram, lowered to the proper coordinates where the knife extended medially, and the transection effected. The same procedures were again repeated contralaterally except that the tip of the knife pointed in a taieral direction. C o n t r o l animals were treated identically except lhat the knife was not lowered into the brain. Upon completion of behavioral iestmg the rats were sacrificed with an overdose of Equi-Thesin and perfused with Saline followed by a formol-saline solution. Ttie brains were removed and fixed in a 10% Formalin solution. Ever)' fourth 50-u thick frozen section was stained with cresyl violet and microscopically examined to determine the extent to damage.
7e.~llng Procedures Following surgery, the rats were placed in Wahmann activity wheels with attached living cages. Free access to the wheel was given during the entire testing period. Food and water were available ad lib. Both the colony room in which the anhnals were originally housed and the testing room were maintained on a 16 hr/8 hr light/dark cycle. One hr after the onset of the daily period of illumination, lhe following measures were taken: total activity, body weight, food and water intake. All animals were housed in the wheels for a period of 24 days. The last 12 clays of the session were used to assess differences between experimcnlal and control animals. R E S U k IS
The stained sections were examined microscopically to determine the locus and extent of damage produced by the various surgical procedures. Eight animals sustained bilateral total transection of the fornix bundle extending dorsally to the corpus callosum and ventrally to the third ventricle. All knife cuts sectioned the fOrnix completely at a point just anterior to the hippocampus. Five animals sustained cuts that passed through both limbs of the diagonal bands, typically extending dorso-ventrally from a point immediately ventral and lateral from the ventricles to the base of the brain: Typically, tlie anterior-posterior extent of these cuts extended anteriorly to portions of the
LIMBIC P A T H W A Y S A N D A C T I V I T Y
67 accumbens nuclei and posteriorly to the bed nucleus of the stria terminalis. In one case, the damage e x t e n d e d posteriorly b e y o n d the crossing of the anterior commissure. Seven animals sustained cuts that passed through the MFB fiber bundles bilaterally at levels extending from 1 to 2 mm anterior to the crossing of the anterior commissure, and ventrally to the base of the brain. The second phase of the e x p e r i m e n t required new surgical groups. The damage to these animals was virtually identical to the first phase, with final group sizes of 7, 6, 4, and 7, for the Groups FX, OC, DB, and MFB, respectively. The p h o t o m i c r o g r a p h s in Fig. 1 show representative stained sections through the midpoints of the various transections. The data for the running-wheel activity were collapsed across 3 day blocks for purposes of statistical analyses. As is usually the case in this type of procedure, the a m o u n t of running varied considerably with occasional animals showing little or no activity while others made over 14,000 revolutions during the 3 day block. Accordingly, a square root transformation was performed on the data before doing a 2-way analysis of variance. The results of this analysis revealed a significant Groups and Trials interaction (F(9,81) = 3.31, p < 0 . 0 0 2 , see Fig. 2). Subsequent 2-sample comparisons using the Fisher test of least significant differences (critical value = 23.1) indicate that the bases for this interaction lie in the increased activity of the MFB group and the steadily increasing level of activity of the DB group across days. Although the FX group was somewhat more active than controls, this difference did not reach statistical significance. Following surgery a mild aphagia and adipsia resulted in 2 of the animals which sustained knife cuts of the MFB. Consequently, this group was maintained on diluted sweetened condensed milk, supplem e n t a r y to their regular diet of Purina Lab Chow and water, for a period of 7 days. Since this procedure does c o n f o u n d food and water comparisons, the MFB group was excluded from data analysis on these measures, although they were included in tests for differences on body weights. No statistical differences ( F < 1) were found between food and water intake of control rats and rats with transections of the DB and FX. RUNNING
WHEEL
8O
~"
z
FIG. 1. Photomicrographs of brain sections showing: (a)a parasaggital view of a MFB cut, (b) a parasaggital view of a FX cut, and (c) a coronal view of a DB cut.
o Iz) " o
60
n~
20
~,
~
MFB
C.V. 40
~
C
O
N
i
I
i
I
I
2
3
4
3-DAY
BLOCKS
FIG. 2. Square root of wheel-running activity across 3 day blocks for controls (CON), rats with fornix cuts (FX), medial forebrain cuts (MFB) and diagonal band cuts (DB). The bracket (c.v.) indicates the critical value (t7 = 0.05) for Fisher's test of Least Significance Differences.
68
C A P O B I A N C O AND t t A M I L T O N
The analysis of variance for the percent of the preoperative b o d y weight revealed that the rats in the e x p e r i m e n t a l groups were marginally lighter than the control rats ( F ( 1 5 , 1 3 7 ) = 1.78, p < 0 . 0 5 , c.v. = 4.83). The mean b o d y weights on Day 12 expressed as a percent of preoperative weights, were 117, 117, 112, and 122 for groups FX, MFB, DB and CON, respectively.
E X P E R I M E N T 2: S T A B I L I M E T E R . OPEN F I E L D A N D REARING ACTIVITY Recent studies (e.g., 124]) ha~" d e m o n s t r a t e d thai various measures of activity may be differentially influenced by deprivation and a n u m b e r of o t h e r manipulations. In an effort to gain a better understanding of limbic system mechanisms of activity, the present e x p e r i m e n t assessed the effects of various knife cuts on 3 additional indices of l o c o m o t o r activity. METHOI
DISCUSSION
Transections of the MFB and DB were found to result in large increases in wheel running behavior. The present study did not utilize any procedures (e.g., deprivation, or amphetamine) o t h e r t h a n the knife cuts in the various tracts, to enhance the activity increases. However, it should be noted that wheel-running behavior has been d e m o n s t r a t e d to be sensitive to any change in the internal e n v i r o n m e n t [6]. This being the case, it may be argued that a change in h o m e o s t a t i c mechanisms could account for the increase m activity. Transections of the MFB did cause a mild aphasia and adipsia leading to lower b o d y weights, but these animals recovered to a point of virtually no difference from control (final means on Day 12; 122% and 111%, respectively, for Groups CON and MFB). Similarly, o t h e r investigators [16] have failed to find changes in food intake, water intake, or b o d y weights when knife cuts are placed in anterior portions o f the MFB. Also, it should be n o t e d that the increase in activity in the MFB group was c o m p a r a b l e with DB transections, and these animals did not differ significantly from control, in terms of food and water intake. Consequently, i n t e r o c e p t i v e changes do not seem to adequately account for the enhanced activity. The most plausible i n t e r p r e t a t i o n of these effects would seem to be a change in the arousal or excitability level of the animal. The magnitude of the increase in wheel activity was somewhat smaller, following fornix transection, than reported in a previous study [4]. The major procedural difference was that the previous study used female rats. The obvious suggestion is a h o r m o n a l l y based influence on activity, but the results, in the previous study, of vaginal smears indicated normal estrus cycles [4]. F o r n i x transections may also interact with the baseline level of activity in such a manner that differences f r o m control levels are greater when the activity levels are higher. Finally, it should be noted that the changes in running wheel activity bear no consistent relationship to the observed changes in reactivity to handling. Neither the rats with MFB transections nor the rats with FX transections showed changes in reactivity to handling at the time of testing (FX transections s o m e t i m e s result in a very transient hyperreactivity). Rats with transections o f the DB were mildly hyperreactive t h r o u g h o u t the course of the experiment. Unless there exists some very complicated interaction a m o n g the variables of brain damage, wheel running activity and reactivity to handling, it seems unlikely that the increased running wheel activity can be attributed to hyperreactivity. With respect to the differences in response to handling that were observed, our observations are consistent with Turner's [31] view that damage to the bed nucleus o f the stria terminalis is largely responsible for the increased reactivity that is observed following septal lesions: the DB transections in the present study rather consistently damaged this nucleus or fibers that would be closely associated with this nucleus.
Animals The animals were 32 male albino rats of the SpragueDawley strain (CFE). The rats ranged in weight from 220 to 250 g, and were a p p r o x i m a t e l y 100- 110 days old at the time of surgery. All animals were housed under ad lib food and water conditions, and were maintained in individual cages.
Surgical Procedures and HistoloKv The surgical procedures and histological procedures were identical to those described in E x p e r i m e n t I. The locus and extent of damage in the knife cut groups was microscopically e x a m i n e d and found to be similar to that sustained by animals in E x p e r i m e n t J.
Apparatus and Testing Procedures Stabilimeter. The stabilimeter cages, c o m p o s e d of wire mesh, measured 39 x 18 10 em high. They were m o u n t e d on a rack and placed m an insulated, soundattenuating, cabinet which was lightproof. Four microswitches were positioned under the floor and calibrated to the a p p r o x i m a t e weight of the animal, such that when m o v e m e n t s were made from one area of the cage to the o t h e r a c o n t a c t was closed, the activity was recorded on counting devices. Each testing session consisted of placing the animal m the stabilimeter for 20 min/day Ior a total of 4 days. Activity measures were taken at t0 rain intervals for tile within sessions comparisons. Open field. U p o n c o m p l e t i o n of lhe stabilimeter testing all animals were tested in the open field. This phase of testing consisted of placing the animal in a Ptexiglas box 60 60 x 37 cm high with a grid floor. The enclosure was divided into quadrants by placing photocells into the side of the box. Each time the animal crossed from one quadrant into the next. the beam from the photocell was m t e r r u p t e d , resulting in a circuit being closed and registering the total n u m b e r of crossings on counting devices. Within session measures were taken at 5 rain mtervals for a total of 15 rain. Daily sessions were continued for a total of 3 days. Rearing. A Plexiglas box measuring 63.5 cm in height with a 30 x 28 cm grid floor was placed in a soundproof. lightproof, ventilated enclosure. A bank of 8 photocells was arranged on the wall o f the c o m p a r t m e n t , and adjusted m the size of the animal such that when the animal rearea on its hindlegs, the head and shoulders would break the beams of the photocells resuRing in a circuit closure and the activity being recorded. Each animal was placed in the testing chamber for 10 min/day. The number and duration of rearing responses was recorded automatically each min for the daily session. All animals were tested for a total of 3 days.
LIMBIC PATHWAYS AND ACTIVITY
69
RESULTS
Stabilimeter.
OPEN
The stabilimeter activity levels of the
various groups, s h o w n in t h e u p p e r p a n e l of Fig. 3, reveals a large increase in activity associated w i t h f o r n i x t r a n s e c t i o n , whereas rats sustaining t r a n s e c t i o n of t h e diagonal b a n d s h o w e d r e d u c e d activity ( F ( 3 , 1 9 ) = 18.5, p < 0 . 0 1 , c.v. = 162). All t r e a t m e n t groups s h o w e d declining activity across days ( F ( 3 , 5 7 ) = 3.03, p < 0 . 0 5 ) . T h e l o w e r p a n e l of Fig. 3 shows t h e within-session changes in activity. The A N O V A revealed a significant G r o u p s × I n t e r v a l i n t e r a c t i o n ( F ( 3 , 1 9 ) = 3.48, p < 0 . 0 5 , c.v. = 40.0). T w o - s a m p l e c o m p a r i s o n s a t t r i b u t e d this intera c t i o n to a higher initial level o f activity for t h e F X rats and a l o w e r overall level of activity for the DB rats.
FIELD
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FIG. 4. Total activity in open field as a lhnction of days (upper panel). The lower panel shows activity changes during successive 5 rain periods within the session (collapsed across days). Legend is same as in Fig. 2.
\ \
~ -
i
5
MINUTES
CONO-.._
I~I 9 o
z
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MINUTES FIG. 3. Stabilimeter activity as a function of days (upper panel) and during the first and second half of each session (lower panel) by collapsing across days. Legend is same as in Fig. 2.
Open field. T h e results o f t h e o p e n field test, s u m m a r i z e d in Fig. 4, show no c o n s i s t e n t d i f f e r e n c e s as a f u n c t i o n o f surgical t r e a t m e n t . T h e a p p a r e n t d i f f e r e n c e s in Day 1 o f the u p p e r p a n e l are a t r i b u t a b l e to e x t r e m e scores
r a t h e r t h a n a c o n s i s t e n t d i f f e r e n c e in central t e n d e n c i e s . A l t h o u g h t h e r e were no c o n s i s t e n t differences in overall activity across days ( F < I ) , all groups s h o w e d a significant decline in activity w i t h i n each daily session ( F ( 3 , 3 4 ) = 23.6, p < 0 . 0 0 1 ; see l o w e r panel of Fig. 4). Rearing. As in the case of o p e n field activity, the surgical t r e a t m e n t s had no c o n s i s t e n t effect on rearing responses. However, all g r o u p s s h o w e d a significant decline in the a m o u n t o f rearing b o t h across days ( F ( 2 , 3 7 ) = 16.3, p < 0 . 0 1 ; see u p p e r p a n e l o f Fig. 5), and w i t h i n daily sessions ( F ( 4 , 7 2 ) = 17.8, p < 0 . 0 1 ; see lower panel of Fig. 5). DISCUSSION The results o f these e x p e r i m e n t s suggest the existence of t w o s e p a r a t e and o p p o s i n g systems of c o n t r o l for the t y p e of activity m e a s u r e d by the s t a b i l i m e t e r device. T r a n s e c t i o n
70
CAPOBIAN(O REARING
fornix in the p r e s e n t study, These results, in c o m b i n a t i o n , suggest t h a t this b e h a v i o r may be m e d i a t e d by a septoh i p p o c a m p a l circuit. However, lhe s c p t u m may pJay a dual role in t h i s b e h a v i o r , since lesions of lhe ,~eptum reduce s t a b i l i m e t e r activity levels [ 1 0 i as did t r a n s e c t i o n o f ~he DB in l h c present study. Thus. the: overall p a t t e r n of activity changes i n d i c a t e t h a t d a m a g e i c~ ~he septot~ippocanlpal s y s t e m m a y increase this ~ype of l o c o m o t o r activity, whereas d a m a g e ~ !he ~ e p t o h y p o l h a ~ a m i c m i d b r a i n s y s t e m may r e d u c e such activi! y
TOTAL I00
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GENERAL DISCUSSION
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FIG. 5. Rearing activity as a function of days (upper panel), and as a function of intervals within the session flower panel). Legend is same as in Fig. 2. TABLE
I
EFFECTS OF INTERRUPTION OF LIMBIC SYSTEM PATHWAYS ON DIFFERENT ACTIVITY MEASURES Surgical Group
Running Wheel
Stabilimeter
Open Field
FX*
1"
~, ~,
l
DB
1' ~"
~,
:~
MFB
1' 1"
--
Rearing
--
*Abbreviations: FX = Fornix transection, DB = Diagonal band transection, MFB = Medial forebrain bundle transection. +Some evidence for transient increase. :~Some evidence for transient decrease.
o f the F X r e s u l t e d in large increases in activity, while t r a n s e c t i o n o f t h e DB r e d u c e d activity. T h e t r a n s e c t i o n o f M F B was i n e f f e c t i v e in altering activity levels. T h e results o f t h e s e k n i f e cut studies are generally c o n s i s t e n t w i t h t h o s e of studies involving lesions o f t h e s e p t u m a n d h i p p o c a m p u s . Lesions of t h e h i p p o e a m p u s increase s t a b i l i m e t e r activity [ 2 9 ] , as did t r a n s e c t i o n o f t h e
D i f f e r e n t measures of a c t i w t y , sucl~ ~s r u n n i n g wheel. s t a b i l i m e t e r , o p e n field and o t h e r :ypes of e x p l o r a t o r y b e h a v i o r have f r e q u e n t l y been discussed as shnply representing d i f f e r e n t methods ot measuring the same phenomenon l o c o m o t o r actwity. V u r t h e r m o r e . Hmre is an a h n o s t n a t u r a l a s s u m p t i o n that these behaviors are e q u a t e d with t h e t h e o r e t i c a l c o n c e p t , f b e h a v i o r a l arousal, t h e s u p p o s i t i o n being, thal the I o c o m ~ t o r activity reflects this c e n t r a l m o t i v a t i o n a l stale Evidence a c c u n m l a t e d during t h e past few years has challerlged the basic simplicity o f these n o t i o n s , and it is b e c o m i n g increasingly a p p a r e n t t h a t an u n d e r s t a n d i n g o f l o c o m o t o r activity and behavioral arousal will require a m u l t i f a c e t c d appr(mch P e r h a p s t h e first indication ol ihe c o m p l e x i t i e s to be e n c o u n t e r e d was t h e evidence_ p r e s e n t e d by H e b b 1 ! 5 l . t h a t t h e r e l a t i o n s h i p b e t w e e n central :lrousal stales and behavioral activity was not m o n o t o n i c but followed an i n v e r t e d U-shaped f u n c t i o n . ~_.. ~tc~ivtty was h i g h c s | ,'or i n t e r m e d i a t e levels o f arousal a n , dcctiaed witl" e i t h e r lower or higher levels of arousal. S u p e r i m p o s e d o n t h e r e l a l i o n s h i p s described by H e b b are a n u m b e r . f e x p e r i m e n t s ttlat fail to s h o w parallel changes in b e h a v i o r using different measures oi activity. Tapp [ 3 0 1 , for e x a m p l e , c o m p a r e d 5 different measures of activity a n d f o u n d virtually no i n t e r c o r r e l a t i o n s , the only m e a s u r e s l h a t c o r r e l a t e d highly were the r u n n i n g wheel activity and l i g h t - c o n t i n g e n t bar pressing. 1{ was suggested that this c o r r e l a t i o n m a y be a t t r i b u t a b l e to lhe fact | h a l b o t h of these types of activity invo!ve c o n s i d e r a b l e feedback from the e n v i r o n m e n L Bolh Tapp [30] and Collier c t al. [6] have r e p o r t e d that f o o d d e p r i v a t i o n can lead to increased aclivity in some situalJons~ Miezejeski c t a / . [24] have e x t e n d e d these findings by m a k i n g direct c o m p a r i s o n s of r u n n i n g wheel, s t a b i l i m e t e r and rearing activity,. Starvat i o n p r o d u c e d a d r a m a t i c increase in rnmning wheel activity, decreased s t a b i l i m e t e r activity and had little or no effect ~ n rearing. I n l e r e s t m g l y , the p r o c e d u r a l dermis tot lhc a s s e s s m e m o f d i f f e r e n t m e a s u r e s of activity have b e e n more c o n s i s t e n t t h a n t h e ensuing t h e o r e t i c a l views of ~hese behaviors. It i~ very difficult to o b t a i n stable measurc~ .,~t i u n n i n g whee] activity w h e n s h o r t test sessions are used It has been suggested (c.f., Collier [ 6 ] ) t h a i r u n n i n g wheeI activity is. in a sense, r e g u l a t o r y , and t h a t t h e final baselines of activity i n t e r a c t w i t h b o t h t h e n a t u r e a n d the q u a n t i t y o f the diel t h a t is c o n s u m e d . The stahilime~cr also r e p r e s e n t s m e a s u r e of l o c o m o t o r activity, bill t h e o p p o r t u m t y lor e n e r g y e x p e n d i t u r e is greatly curtailed Because ol the physical r e s t r i c t i o n s of this a p p a r a t u s , tesl so, stuns are typically s h o r t , and t h e i n t e r p r e t a t i o n s lean m o r e t o w a r d r e a c t i v i t y t h a n to m a i n t e n a n c e . Lat I t 9 ] a n d his associates have p e r f o r m e d a variety o f e x p e r H n e n t s involving the
LIMBIC PATHWAYS AND ACTIVITY
71
rearing repsonse and have concluded that it reflects both the general state of arousal of the organism, and the tendency for exploration. The operant level of this response drops very rapidly over time, so test sessions are typically very brief. Finally, the use of the open field has varied depending upon the interests of the investigator. The tendencies of the rat to exhibit thigmotaxis and to freeze in open areas are both exaggerated in this situation when the illumination levels are high. When tested in darkness or low-level illumination, the tendency to explore is much greater, and the rat will show the typical decline in activity that has been interpreted as the habituation of exploratory activity [22]. The procedural and theoretical differences among these measures accentuate the heterogeneity of activity indices and strongly suggest that the behaviors that are monitored in these situations may be under the control of different brain mechanisms. The present experiments have attempted to delineate some of these neural circuits by comparing the effects of the transection of three major limbic system pathways on four different measures of activity. Rather than attempting to make direct comparisons of the different indices under all conditions, the procedures were selected to represent the most commonly used parameters in each case. Thus, the rats were housed for a number of days in the running wheels, whereas, the other measures involved tests of short duration under low levels of illumination that result in relatively high levels of activity in normal rats. The results of these manipulations are summarized in Table 1. Of particular interest are the differing patterns of activity changes that follow the different knife-cuts. Knife cuts that produce increased activity in the running wheel can lead to an increase, a decrease, or no change in stabilimeter activity. In the present experiments, none of these manipulations significantly influenced activity as measured by open field activity or rearing. This lack of effect cannot be attributed to a simple lack of sensitivity, because lesions in the raphe nucleus greatly increase rearing but have little or no effect on running wheel activity (unpublished observations, Miezejeski and Hamilton). Furthermore, there is the possibility that the failure to observe large differences in activity in the open field and rearing tests may be attributable, in part, to sequence effects. Recently, it has been observed that rats with fornix transections show considerably higher levels of rearing responses when this is the initial test that occurs following the surgical disruption (MacDougall and Capobianco, unpublished observations). Consequently, there may be sufficient generality among the different test environments that the effects of novelty (in relation to the home cage) are reduced by previously exposing the same animals to a variety of testing situations. The results of the selective knife cuts are important regardless of the precise neural pathways that are disrupted; the fact that different patterns of changes are observed is, in and of itself, sufficient
evidence that the response measures are under separate neural control. The changes in activity levels can also be used as a tool to determine the anatomical pathways that may be involved in the control of different types of activity. The fact that all 3 manipulations increase wheel running activity suggests a long (probably polysynaptic) fiber system interconnecting the limbic midbrain with the septum and the hippocampus. The relatively smaller effects of the fornix transection would indicate lesser involvement by the hippocampus. The relationship of the fornix transections to the effects of septal lesions or hippocampal lesions are still somewhat difficult to explain. Both large septal lesions and smaller lesions that are restricted to the medial septum and nucleus of the diagonal band result in a decrease of wheel running activity [5,8], as do lesions of the hippocampus, at least in some cases (e.g., [2]). The fact that the transection of interconnections between the septum and more dorsal structures (FX cuts) or between the septum and more ventral structures (DB cuts) both lead to increased running wheel activity poses some interesting questions as to the nature of these neural substrates and their interactions. In the case of stabilimeter activity, the data clearly suggest 2 opposing systems of control. Damage to the septohippocampal system increase this type of activity, while damage inflicted upon the ventrolateral connections of the septum (via the diagonal band) decrease this activity. It should be noted that most of the interconnections between the forebrain structures and the limbic forebrain are via the medial forebrain bundle. The failure of medial forebrain transection to influence activity levels except in the running wheel may be attributable to the location of the cuts. The cuts were sufficiently anterior to allow many descending fibers of septal or hippocampal origin to pass uninterrupted behind the level of the cut. The nature of the differences among these tasks that could involve separate neural control remain somewhat ambiguous. It has been suggested that the running wheel involves a considerable amount of response feedback [30]. Stabilimeters may also involve varying degrees of feedback, owing to the fact that the floors of these cages are typically movable to allow recording, thus providing the animal with direct environmental cues. Other sources of external feedback, however, were reduced or eliminated by isolating the recording equipment from the open field and the rearing apparatus essentially eliminated any external feedback. Other differences are, of course, the degree of locomotor activity involved, the relative novelty of the environment, and the relative positions of the different responses in the organism's response heirarchy. The importance of these variables and their relationship to specific neural circuitry must await further investigation, but the present results suggest that the combination of different measures of activity with different types of neural disruption may be an effective means of establishing such relationships.
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