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A disconnection analysis of hippocampal function
Brain Research, 233 (1982) 241-253
241
Elsevier Biomedical Press
A DISCONNECTION ANALYSIS OF HIPPOCAMPAL FUNCTION
DAVID S. OLTON, JOHN A. WALKER* and WILLIAM A. WOLF Deparfment o f P~ychology, The John.s Hopkm~ Univer~tty, Bait:more, MD 21218 (U S.A /
(Accepted July 9th, 1981 Key words: hippocampus -- memory - - dtsconnectlon analysts - - working memory
hlppocdmpal
system
SUMMARY A dtsconne,.qion analys~s determmed the extent to which the fornix, hippocampus and entorhmal cortex are components of the same tunctional system m tasks that require workmg memory. Preoperattvely, rats were train~.d to perform accurately on a radial arm maze. Then various combinauons of umlateral and b,laterai le~ons were placed in the fornix and entorhinal cortex, either with or without a transection of the hippocampal commissures. When the lesions left intact at least one pathwa~ through the hippocantpu~ interconnecting the formx and entorhmal cortex, rats performed normally. Etther an uncrossed pathway (following a umlateraJ lesion o f the fornix, transection of the hippocampal commissures, and an ip~datcral E,ion of the entorhinal cortex) or a crossed pathway (following a unilateral Ic~lon of the formx and a contralateral lesion of the entorhinal cortex, leaving the hippocampai commisst, res intact) was sufficient, When the lesions produced a complete bilateral disconnection of the fornix and entorhinal cortex, rats performed poorly. The results indicate that the hippoeampal system provtdes a functional connection between the subcort~cal structures associated with the fornix and the neocortical structures a , s o o a t e d with the entorhinal cortex, and that without this connection normal processing of working memory can not occur.
INTRODUCTION Structures in the central nervous system function not as isolated umts, but as c o m p o n e n t s o f larger systems. One of the most effective ways o f d e t e r m i m n g the extent * Present address: Neurological Scnences Center. Good Samaritan itospJtal, 1015 NW 22nd Ave., Portland, OR 97210, U.S.A 0006-8993/82/0000-0000/$02.75 i ~Elsevier Biomedical Pr~~
242 of a functional system is through a disconnection analysisT,S, 13-15.20.zl.z¢.2s. In this analysis, unilateral lesions are combined with transections of interhemispheric commissures. Some combinations of lesions produce a complete, bilateral disconnection so that neural impulses are unable to flow through the system from one structure to another. Other combinations of lesions do not produce a disconnection, leaving at least one set of connections which is sufficient for normal neural transmission, if the structures in question participate in the same functional system, then all combinations of lesions that produce a disconnection should have a similar behavioral effect, and one that is the same as that found after bilateral lesions of these structures. If, on the other hand, the structures in question participate in different functional systems, then the disconnection should have little effect on behavior. The present experiment was designed to conduct a disconnection analysis of the hipgo~mpal system (septum, fornix, hippocampus, entorhinal cortex). For this analysis, three anatomical areas are of particular interest: (a) the fornix,which provides the extrinsic connections of the hippocampus with subcortical structures; (b) the perforant path and the entorhinal cortex, which provide the extrinsic connections of the hippocampus with neocortical structures; (c) the dorsal and ventral hippocampal commissures, which provide homotopic and heterotopic connections from the hippocampus on one side of the brain to the hippocampus on the other side of the brain (for reviews, se,: t-ors, i, ~, 5, 6, f0, 30, 45 and 47-51). Pr¢, !ous resea~ch has demoh~trated that bilateral lesions of the components of the hippocampal system produce an impairment in tasks that require working memory3.4.o.J~ 1~,31,34,:~5,3s-44..~3 ',fthese structures, which are closely related through their neuroanatomical connection~ ,articipate in the same functional system, then a disconnection analysis should produce a pattern of results as described above. The most critical dat "~ "ome from rats with a unilateral lesion of the fornix, unilateral lesion of the entorhinal cortex, and tran,~ection of the hippocampal commissures. Ipsilateral lesions of the fornix and entorhinal cortex do not produce a bilateral disconnection because a - ;ntact pathway between subcortical and neocortical structures through the htppocampus still exists in the side of the brain conttalateral to these lesions. Consequently, behavioral functions normally mediated by the hippocampus should remain intact, and performar,ce in tasks that require working memory should be normal. Co-:ralaterai lestons of the fornix and entorhinal cortex (in conjunction with transectmn of the hippocampai commissures), however, do produce a bilateral disconnection, in the side of the braia wtth the intact formx, neural impulses are able to travel between subcortical structt~res and the ipsilatcral hippocampus as usual. However, they are unable to reach neoeortical structures through hippocampai connectmns: the ipsilateral entorhinal cortex has been destroyed and, although the contralateral entorhinal cortex is intact, transection of the hippocampal commissures prevents communication through that pathway as well. A complementary pattern exists on the other side of the brain, the one w~th the intact entorhinal cortex. Here, neural impulses are able to travel between neocortieal structures and the ipsilateral hippocampus as usual. However, they are unable to reach sul:corticai structures
243
through hippocampal connections; the ipsilateral formx has been destroyed, and although the contralateral formx is intact, transection of the hippocampal commissures prevents communication through that pathway as well. Consequently, behavioral functions normally mediated by the hippocampus should be disrupted, and animals with these contralateral lesions and transection of the hippocarnpal commissures should behave the same as those with bilateral lesions of the hippecampus ~tself. An important point ~n t.he above analysis is the placement of the les~ons m the same 3 structures; only the ipsilateral-contralateral relationship of the lesions in the fornix and entorhinal cortex d~ffers in the two examples. Consequently, any differences in behavior following these two sets of lesions can be attributed to the difference in the topological relationship of the lesions, and the results interpreted m terms of the structures that have been d~sconneeted. The ability of rats to perform corre,.tly on a radial arm maze was used to assess hippocampal function. This test procedure requires working memory ~ 12.a3 3t, 112 52, /.t process that is severely disrupted following bilateral lesions in the hlppocampus or it~ extrinsic connections (see above). Nine d~fferent combinations of les~ons m the fornix. entorhinal cortex and hippocampal commlssures were used to evaluate the pred,ct,ons of the disconnection analysis (see above), and to answer other questions about the role of the hippocampal system m workmg memory (see Discussion). METHODS dpparatu.~ The test apparatus was a radial arm maze with 8 arms a~ described clsc~ hcrc ~.1 Each of the 8 arms extended from a central platform like a ~poke of a wheel The central platform was an octagon, 21 cm on each side. Each arm was 56 cm Iont~and 10 cm wide with a hole, 5 mm deep at the end away from the central platform Subiects Male albino rats derived from a Sprague-Dawley strain were raised m the John~ Hopkins Colony. When they were approximately 90 days old, they were placed m individual cages with water available at all times. Food deprivation reduced each rat's body weight to 85 % of its normal value. Body weights were maintained at this level, plus 5 g each week to allow for body growth, by giving the approprmte amount of Purina Lab Chow approximately ! h after each day's testing was completed Procedure Preoperative te.~tblg. All rats were shaped to go down the arms of the maze and eat the food in the hole at the end of each arm (see ref. 43). They then received one test session each day, 5 days a week. At the start of each test ~e~.sion, one 190 mg Noyes food pellet (P. J. Noyes Co.) was placed in the hole at the end ofeach arm. "[he rat was placed on the central platform and allowed to choose among the arms. A correct re.~ponse was going down an arm for the first time during a test session (in which case the rat obtained the food pellet at the end of it). An incorrect re~pon~e was going down
244 an arm again during a test session (in which case the rat did not get food because he had eaten the pellet during his previous visit). Each test session continued until: (a) the rat had chosen all 8 arms, (b) 2 min had passed since the rat's previous response, or (c) a total o f l0 rain had passed since the start of the test session. Preope~'~five testing continued in this manner until the rat received at least 10 test sessions ar d reached a criterion of 7 correc4 responses in the first 8 choices for 5 consecutive te~t sessions. Surgery. Rats were given intraperitoneal injections of 0.02 mg methyl atropine (Sigma Chemical Company) and 2.2 ml/kg Chloropent (Fort Dodge Laboratories), and an intramuscular injection o f 60,000 units of Bicillin (Eli Lilly). They were placed in a stereotaxic instrument. The scalp was incised and retracted and holes drilled through the appropriate places in the skull. Entorhinal lesions were made by passing radio frequency current from a Grass LM-4A Lesion Maker through a stainless steel electrode insulated except for two mm on the posterior side of tip. This asymmetrical insulation directed the current posteriorly into entorhinal cortex, away from the hippocamp,Js and dentate gyruss2.4a. Selective lesions of the fornix and of the hippocampal commissures were made with a scalpel blade. For the fornical lesions, the blade was lowered in the coronal plane just posterior to the medial septal nucleus and moved laterally from the midline. For the commissurai lesions, it was lowered in the sagittal plane just posterior to the medial septal nucleus and moved posteriorly. Combined lesions of the fornix and hippocampal commissures were made either by combining both of the two knife cuts just described above, or by radiofrequency current, in this latter case, a stainless steel electrode, insulated except i'or 0.8 mm at the tip, was lowered into the fornix just anterior to the anterior pole of the hippocampus. Thus the lesions destroyed both the extrinsic fibers in the fornix, and the intrinsic fibers in the ventral hippocampal commissures *.~. Rats given control operations had various types of procedures. Some were designed to produce no brain damage, and consisted of only anesthesia and an incision in the scalp. Others were designed to spare t.he fornix, hippocampal commissures and entorhinal cortex, but produce damage in adjacent areas. Electrodes were lowered to the fornix or entorhinal cortex, but no current was passed. Scalpel blades were lowered to a position just dorsal to the fornix or the hippocampal commissurcs, and the appropriate cut made at this level. Postoperatire test~rig. After remaining in the home cage without testing for approxima'~?~, ~ne week following surgery, each rat was given at least 10 test sessions following the same procedure used preoperatively (except that no shaping was given). Some rats were then killed for histology. Some of the other rats, who had not received a complete disconnection, were tested until they reached a criteriof, of 7 correct responses in the first 8 choices and then given a second lesion. If this second lesion did not produce a complete disconnection, some of them were teste,' again until they reached criterion, given a third lesion, and tested once more. Those rats that had received a complete disconnection were given at least 10 test sessions; some received an additional 40 test sessions. Histology. Rats were deeply anesthe*ized with ether and then perfused with
245 0.09 ~o saline followed by 10 ~ formalin. The brains were removed from the skull and cut in two so that coronal sections could be taken at the level of the fornix and anterior hippocampus, and horizontal sections at the level of the posterior hippocampus and entorhinal cortex. Some rats with transections of fornix made by the scalpel blade had the entire brain cut in the horizontal plane. The brains were cut in a cryostat. In every 200/,m throughout the hippocampus and the area with the lesion, two sections were taken. One section, 20/,m thick, was stained with Luxol fast blue for myelinated fibers and cresyl violet for cell bodies. The other section, 50/,m thick, was stained for acetylcholinesterase (ACHE)~5.2e. These sections were mounted on a slide. The lesions were evaluated without knowledge of the rat's performance. RESULTS
Preoperative behavior Preoperatively, all rats learned to choose accurately. They reached the criterion of 7 correct responses in the first 8 choices for 5 consecutive test sessions in a mean of 9 test sessions.
Hi.~tology in all but 8 rats, the lesioas were as intended. The transection of the h~ppocampal commissures cut both the ventral and dorsal commissures. Dorsal structures, such as the corpus callosum and midline cortex, also received some damage. Damage occasionally extended more ventrally into the thalamus, but was always slight. An example of a representative lesion is presented in Fig. I (left top). The unilateral lesions of the fornix and ventral commissures made with radiofrequency current destroyed these fibers posterior to the septum and anter,or to the hippocampus. Slight damage was occasionally found in the dorsal part of the thalamus; other adjacent structures were spared. An example of a representative lesion is presented in Fig. 1 (left middle). The unilateral lesions of the fornix alone made with the scalpel blade were just posterior to the septum, and anterior to the ventral commissure. An example of a representative lesion is presented in Fig. 1 (left bottom). Both of these lesions had a profound effect on the density of AChE staining in the hippocampus. Normally, the hippocampus stains darkly with a regular laminated pattern (Fig. !, right top). After either type of fornix lesion, the hippocampus had little if any stain in it; whatever stain remained was usually in the most temporal portions 4~. An example of representative result following a fornix lesion is presented in Fig. I (right middle). The entorhinal lesions were large, destroying almost all of medial and lateral entorhinai cortex, along with some of the adjacent retrohippocampai areas. With the largest lesions, the most posterior edges of the hippocampus and dentate gyrus sustained some damage at their most posterior regions, but were otherwise intact. Following these lesions, an additional band of AChE stain was found in the outer twothirds of the apical dendrites of the dentate granule cells, reflecting the sprouting of the cholinergic fibers from the septum into the areas normally innervated by the afferents
b +° ,
~
~t,
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Fig I. Histological results• The figures on the left were all stained with Luxol fast blue and cresyl violet, the ones on the r,ght w e r e stained for ACHE. The top two figures on the left are in the coronal plane, all the others are in the horizontal plane. Left top" a transection of the hlppocampal comm,ssures completely tran~ected these fibers and produced damage to the overlying neocorte,~ and corpus callosum, but spared the thalamus• Left middle: a unilateral lesion of the formx and the ventral commissure destroyed v:rtually all of these fibers on the right side of the brain, while producing little damage to adjacent areas• Left bottom: a unilateral incision made with the scalpel blade transccted the fibers of the fornix as they reached the posterior portions of the septum, but spared the ventral commissure. Right top: a normal brain. Note the pattern of AChE staining in the hippocampus Right middle• a brain following an ipsdateral lesion of the formx. Note the very light stain for AChE in the hlppocampus as compared to the dense staining of the more medial structures Right bottom: a brain following a lesion of the entorhinal cortex. Note the extens,vc damage to retrohlppocampal area% and
247 from the entorhinal cortex zz,4n. An example of one o f the largest lesions Js presented m Fig. ! (right bottom). The 8 rats with inappropriate lesions were discarded because their lesions were either too large or too small. For example, one rat who was supposed to have a unilateral fornix les~on also had damage to the contralateral formx and no ACh E stare in the anterior portions of the h~ppocampus on that side. Another cat w~th a undateral formx lesion had damage to only the medial portions o f the fo, nix on the s~de o f the lesion, and AChE was absent only from the anterior portions o: the hlppocampus on that side. The data from all rats w~th inappropriate lesions were not mcluded m the behavioral analyses describe:l below.
Postoperative behavtor Postoper~:twely, rats receiving control operations continued to perform well. making a mean o f 7.2 correct responses in the first 8 choices dunng the first 10 test sessions. These data demonstrate that the surgical procedure Jn general, and the damage to brain areas outside of the hsppocampal system, ¢2Jd not ~mpair ~holLe accuracy Rats with combinations of lesions that d~d not produce a bdateral disconnection also performed well postoperatively. The data from these grot:ps are ~ummarized m the top part o f Fig. 2. "l'he mean number of correct response~ m the first 8 tholces during the first 10 test sessions for all these groups ranged from 6 9 to 7.0. the ~or~t performance by any rat m any of these groups was a mean of (" 5 correct re~pon~cs m the first 8 choices. These data demonstrate that ~Jny cennectlon ~et~een the entol hmal cortex and the fornix through the hJppocampus was sufficJert to maintain normal performance in this task. Rats with combinations of lesions that did produce a bdatelal dssconnectlon performed worse than they had preoperatively. ~orse than the groups ~=thout ~t bilateral disconnection, and not significantly better than expected by chance. The datz; from these groups are sum:,mrized in the bottom part of Fig. 2 The mean number of correct responses in the first 8 choices during the first 10 test session~ ranged from 5 4 to 5.8; the best performance 5y any rat m any of these groups was 6.5 correct responses in the first 8 choices. Only one rat in the groups with bilateral disconnections chose as accurately as any rat in the groups w=thout bdateral disconnections. These data demonstrate that an intact connection between the entorhinal cortex and the fornix through the hippocampus is necessary to maintain normal performap, ce in this task. The sequentml order of the lesions had no obvious effect on choice accuracy For example, all rats in the U F - H C - C E group performed poorly after they had received all 3 lesions; this impairment occurred when the lesi~ms were given one at a time (w=th behavioral testing after each of them), and when "they were gs~en all m a single operation. Simdar results were found in all groups. All combinations of lesions that failed to produce a bilateral disconnection were followed by normal choice accurac~ (within the range of results reported for the first 6 groups in Fig 2). whde all combinations o f lesions that did produce a bilateral disconnection were followed b~
248 NUMBER OF CORRECT RESPONSES LESIONS N 5.O 5;5 6;0,, , 6 ~ 7.0 7:5
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Fig. 2. A summary of the experimenlal design and resulls. The left column $;ves the group name,
indicatingIhe placementof the lesionq. Lesion~were placed in either the fornix(F), ¢ntorhinalcortex (E~ or hippocampal commis,~ures (HC). The le~ions in the fornlJ; and antorhinal cortex were either unilaleral (U) or bilaleral (B), and the unilateral lesions were eilher ¢ontralaleral (C) or ipsilaleral (I). Thus group UF-HC-CE had a unilateral lesion of Ihe fornix, transection of Ihe hippocampal commissure% and a conlralateral lesion of the enlorhinal co-'~x. The r~.ond column presents a schematic diagram of a dorsal view of Ihe rat brain. The rounded structure in the middle al the lop repre~nls Ihe septum, the narrow area behind it is the fornix, the curved slruclure is the hippoeampus, and the area behind it is the entorhinal corlex. Lesions are indiczated by black areas. The third column presenls the number of rals in each group. The histograms present the mean (bar) and the range (superimposed line) of the mean number of correct responses in the first 8 choices during the first 10 lest sessionsfollowing the indicated lesion. Data from rats with control operations are in the top line (open bar). Dala from rats wilh at least one pathway interconnecting the fornix and enlorhinal cortex are presented in the next 5 I,nes (bars with lines slanted upwards to the right). Data from rats with a complete disconnection of fornix and entorhinal cortex are prcscnled in the next 4 lin¢~ (bar,, with lines slanted t,pwards to the left).
poor choice accuracy (within the range of results reported for the last 4 groups in Fig. 2). These data demonstrate that the behavioral effects of the various combinations of lesions did not depend on the order in which the rat received them. Each of the rats receiving more than one lesion contributed data to more than one group, prohibiting statistical analyses which require independent subjects. The clear nature of the results, however, does not require such analyses. First, the order in which the lesions were made had no effect on choice accuracy (see above). Second, all ~roups without disconnections (first 6 groups in Fig. 2) performed similarly; all groups with disconnections (last 4 groups in Fig. 2) performed similarly, and there were only two cases in which the scores of the disconnection and non-disconnection groups
249 overlapped. A binomial test, which does not require independent subjects, on these combined data indicated that the choice accuracy of rats with disconnections was significantly different than the choice accuracy of rats v'ithout disconnections (n ~ 82, X --- 2, P < 0.001). DISCUSSION
The present experiment used a disconnection analysis to examine the functional relationship of the hippocampus, the subcort~cal structures connected to ~t through the fornix, and the neocortical structures connected to it through the perforant path and the entorhinal cortex. All rats with an intact set of hippocampal connections between the subcortical structures associated with the formx and the neocort~cal structures associated with the entorhinal cortex performed well, whether this connection wa~ through one side of the brain (group UF-HC-IE), or from one side of the brain to the other through the hippocampai commissures (group UF-CE). In contrast, all rats with a complete bilateral disconnection performed poorly, this result was found following bilateral lesions m either or both the fornix and entorhmal cortex (groups BF, BE, BF-BE), and after a combination of contralateral unilateral lesions and transection of the hippocampal commissures (group UF-HC-CE). Thus at least m tasks that require working memory, the fornix, h~ppocampal formation and entorhmal cortex comprise a single fL,,ctional system which is critical for normal performance The role of thi~ system In working memory is relatively selective: damage to othc~ brain systems (amygdala, frontal cortex, postcrolatcral neocortex, caudate nucleus) did not produce similar behavioral impairments in tasks that reqmred working memory 4, and damage to the hippocampal system did not produce impa,rments i~1 tasks that didn't require working memory :~.:~:,,:~s,:~9,n4. The hippocampal con~missures are a major pathway interconnecting the hJpp,,campus of one hemisphere to the hippocampus of the other j,:'.4s~. In the prcscat experiment, transection of the hippocampal commissurcs alone (group HC) or contralateral lesions of the fornix and entorhinal cortex (groups UF-CE) did not impair performance. Transection of the commissures combined with contralateral lesions of the fornix and entorhinal cortex (group UF-HC-CE), however, produced an impairment of choice accuracy equivalent to that seen after bilateral destruction of either the fornix or the entorhinal cortex. This pattern of results demonbtrates that the hippocampal commissures act as functional connections between the forn,x and entorhinal cortex when they are the only pathway available through the hippocampus (group U F - C E as compared to group UF-HC-CE), but that they are not necessary for normal behavior when other, uncrossed pathways are present {group HC). Many studies have made lesions of the fornix and used the results of these experiments to draw conclusions about the behavioral functions of the hJppocampus. Such conclusions are appropriate, of course, only to the extent that lesion~ of the fornix and hippo,.ampus predate similar behavioral changes. The results of the present experiment demonstrate that at least with respect to working memory, the fornix and hippocampus are components of the same functional system, so that
250 conclusions about the behavioral functions of the hippoca~ ~ms can be made from the results of experiments demonstrating the behavioral effects produced by lesions in the fornix. Whether this assumption is justified for other types of behaviors, and for the entorhinal cortex, remains to be demonstratedts,s°. The hippocampus has a very low threshold for electrophysiological seizures, and many different events can produce abnormal electrophysiological activity in its3,2e. Thus the behavioral effects following a lesion of either the fornix or the entorhinal cortex might be due to the propagation of abnormal activity from the hippocampus through the remaining pathways to other brain structures. If such were the case, then combined bilateral lesions of the foruix and entorhinal cortex should eliminate these behavioral changes because this set of lesions effectively isolates the hippocampus from the rest of the brain and restricts any abnormal activity in the hippocampus to that structure alone. However, the performance of rats with bilateral lesions of both the fornix and entorhinal cortex was ~1obetter than that of rats with any other kind of disconnection. Thus the disconnecting lesions were effective because they interrupted normal electrophysiological activity in the hippncampus, rather than because they caused seizure activity in the hippocampus which was then propagated to other brain structures through the remaining intact pathways. in other experiments, unilateral lesions of the entorhinal cortex produced immediate behavioral changes which were followed by recovery of function during the next 10 days~4. The time course of this recovery was correlated with the growth of fibers from the intact entorhinal cortex through the dorsal hippocampal commissure to the contralateral dentate gyrus. Furthermore, transection of the dorsal hippocampal commissure reinstated the behavioral impairment, demonstrating that these commissural fibers were responsible for the observed recovery of function. In the present experiment, behavioral testing did not begin soon enough after surgery to determine if unilateral lesions of the entorhinal cortex (group UE) produced an immediate impairment of choice accuracy on the radial arm maze~ thus the present data are not releeant to this question. However, rats with a unilateral lesion of the entorhinal cortex and a transection of the hippocampal commissures (group UF-HCIE) performed well, demonstrating that in the present experiment, normal performance following a unilateral lesion of the entorhinal cortex was not dependent on commisural fibers. The reasons why the commissurai fibers were critical for normal performance in the Loesche and Steward"4 experiment, but not necessary for normal performance in the present experiment, are not clear, but may have to do with the types of behaviors examined and the amount of preoperative test experience. In sumn-,ary, the present experiment used a die,connection analysis to examine the functional relationships of the fornix, hippocam~us and entorhinal cortex in a task that required working memory. The results demonstrate that normal performance in this task requires that one of the interconncctions between the fornix and entorhinal cortex through the hippocampus be intact, but that any one of the alternative pathways, either crossed or uncrossed, is sufficient.
251 ACKNOWLEDGEMENTS
The research here was supported in part by Research Grant M H 24213 from the National Institutes of Health to D.S.O., and by a Biomedical Sciences Research Grant from the Biomedical Sciences Research Divi~ic,;. of the National Institutes of Health to the Johns Hopkins University. The author, ' hank C. Anderson, E. Breitenger, M. O'Reagan, T. Rumbarger, I. Wu, and S. Wu for testing rats; E. Philips and C. Tirado for histology; J. Becker, K. Caswell, G. Handelmann, J. Horel, L. Jarrard, M. Mishkin, S. Mitchell, N. Rawlins and O. Steward for comments on the manuscript: and J. Krach for typing. REFERENCES 1 Andersen, P , Organizauon of hippocampai neurons and their ir terconnectmns, in R. L. lsaacson and K. H. Prlbram (Eds.), The HIppocampu~, Vol. 1: StJ uctule and De..elopnte,t, Plenum Press, New York, 1975, pp. 155-175 2 Anderson, P., HolmqvJst, B. arid Voorhoeve, P E. Entorhinal activation of dentate granule cells, Aeta physto!, seand., 66 (1966) 448-460. 3 Becket, J. "i. and Olton, D. S., Cognitive mapping and hippocampal function Neloopsy~hologta. m press. 4 Bexker, J. T , Walker, J. A. and Olton, D. S., The neuroa a'omical bases of spatial memory. B~am Research, 203 (1980) 307-320. 5 Blackslad, T. W., Commissural connectmns of the hippocampal region m the rat with special reference to their mode of termination, J. camp. Neural, 105 (1956) 417-538 6 Chronlster, R. B. and White, L. E., Jr. Fibberarchitecture of the hippocampal formation: anatomy, projections, and structural significance. In R. L. lsaacson and K H. Prlbram. (Eds.}, The Hipp,campus, Val. I: Sirra'lute aml Development, Plenum Press, New York. 1975, pp. 9 39. 7 Gesehwind, N., Disconnection syndromes in animals and man. Part I, B~am 88 (1965~ 237 294. 8 Geschwind, N., Disconnection syndromes in animals and man. Part II, BJoie~, 88 (1965) 238 294 9 Handclman, G. E. and OIton, D. S., Spatial memory following damage Io hJppocampal CA3 pyramidal cells g ith kainic acid. impawment and recovery of functmn, Brain Reset, oh. 217 (1981) 41 -o58. 10 H.;orth-SJmonsen, A. and Jeune. B., Origin and termination of the happocampal perforant path in Ihe rat studied by silver impregnation, J. romp Neural., 144 (1972~ 215-232. ii Honig, W. K , Studies of working memory in the pigeon. In S. H. Hulse, H. Powler and W. K. Homg (Eds.), Cognitive Proce.sses in Animal Behavklr, Lawrence Erlbaum, HJllsdale. NJ, 1978, pp. 211-248. 12 Honig, W. K., Spatial aspects of working memory, Behav. Brain Srt., 2 (1979) 332-333. 13 Hotel, J. A. and Kcating, E. G., Partial Kluvcr-Bucy syndrome produced by cortical disconnection, Brain Re.searrh, 16 0969) 281-284. 14 Hotel, .L A. and Keating, E. G., Partial Kluver-Bucy syndrome in the monkey produced by disconnection, J camp. phyMo[. P~yehol., 79 (1972} 105-114 15 Hotel, J. A. and Misantone, L. G., Visual discrimination impaired by cutting temporal lobe connectmns, Science, 193 (1976) 336-338. 16 Jarrard, L. E., Anatomical and behavioral analysis of hippocampal cell fields, J. ~omp physwl P~ychoL, 90 (1976) 1035--1050. 17 Jarrard, L. E., Selective hippocampal lesions: d~fferential effects on performance by rats of a spatial lasS, with preoperauve versus postoperative training0 J. romp. physiol. P~ychol, 92 (! 978) I 119-1127. 18 Jarrard, L. E, Selective hlppocampal lesions and beh awor, Ph),~swl.Psychol, 8 (I 980) I ! 19- I 127. 19 Johnson, C. C., Olton, D S., Gage, F. H., i l l and Jenko, P. G.. Hippocampal connecuons and behavior: DRL-20 and spontaneous alternahon, J. cvmp. pto sml. P,yrhoL, 91 (1977) 508-522. 20 Jones, B. and Mishkin, M., Limbic lesions and the problem of stimulus-reinforcement associations, Exp. Neural., 36 (!972) 362-377. 21 Keatmg, E. G. and Hotel, J. A°, Somatosensory deficit produced by parietal-temporal disconnectmn m the monkey, Exp. NeuroL, 33 (1971) 547-565.
252 22 Lewis, P. R. and Schute, C. C. D., The choliuergic limbic system. Projections to hippueampal form~on, medial cortex, nuclei of the ascending cholinergic reticular system, and the subfornicai organ and supra-optic crest, Bra/n, 90 (1967) 521-540. 23 Liberson, W. T. and Akert, IL, Hippocampal seizure states in guinea pig, Electroenceph. ch'n. 1Veurophysiol.~ 7 (1955) 211-222. 24 Loesche, J. and Steward, O., Behavioral correlates of dennervation and reinuervation of the hippocampal formation of the rat: recovery of alternation performance following unilateral entorhinal cortex lesions, Brain Res. Bull., 2 (1977) 31-39. 25 Lynch, G. and Carman, C. W., The hippocampus as a model for studying anatomical plasticity in the adult brain. In R. L. lsaac~n and K. H. Pribram (Eds.), The Hippocampus, gal. 1: Structure and Development. Plenum Press, New York, pp. 123 154. 26 Lynch, G., Mathews, D. A., Mosko, S., Parks, "IF.and Cotman, C. W., Induced acetylchohnesterase rich layer in rat dentate gyrus following entorhinal lesions, Brain Research, 42 (1972) 311-318. 27 Mishkin, M., Visual discrimination impairment after cutting cortical connections between the inferotemporal and striate areas in monkeys, Amer. Psychul., 13 (1958) 414--432. 28 Mishkin, M., Visual mechanisms beyond the striate cortex. In Frontiers of Physiological Psychulog); Academic Press, New York, 1962, pp. 93-119. 29 Nakajima, A., Hippocampal protein synthesis and spike discharges in relation to memory. In R. L. Isaacson and K. B. Pribram (Eds.), The Hippocampus, Vol. 1: Structure and Development, Plenum Press, New York, 1975, pp. 393-413. 30 O'Keefe, J. and Nadel, L., The Hippocampus as a Cognitive ,Map, Clarendon Press, Oxford, 1978. 31 alton, D. S., Discrimination reversal performance after hippocampal lesions: an enduring failure of reinforcement and non-reinforcement to direct behavior, Physiol. Behav., 9 (1972) 353-356. 32 alton, D. S., A technique for producing directionally specific brain lesions with radio frequency current, PhyMol. Behav., 14 (1975) 369-372. 33 alton, D. S., Characteristics of spatial memory. In S. H. Hul~, H. F. Fowler and W. K. Honig (Eds.), Cognitive Aspects of Animal Behavior, Lawrence Edbaum, Hillsdale, N J, 1978, pp. 341-373. 34 alton, D, S., The function of septo-hippocampal connections in spatially organized behavior. I , Ciba Foundalien Symposium ~8., Function~ of the Septo-Htppocampal System, Elsevier, New York, 1978, pp. 327-341. 35 alton, D. S., Spatial abilities of animals: Behavioral and neuroanatomical analysis, in M. Potebal (Ed.j, The Neural and Developmental Bases of Spatial Crientuliou, Academic Press, New York, in press. 36 alton, D. S., Mazes, maps, and memory, Amer. PmychoL, 34 (1979) 588-596. 37 alton, D. S., Spatially organized behaviors of animais: Behavioral and neurological studies. In M. Potegal (Ed.), The Neural and Det~lopmentul Ba.~esof Spatial Orientation, New York, Academic Press, Ifl press. 38 alton, D. S., Becket, J. T. and Ha,ldelmann, G. E., Hippueampus, space and memory, Behav. Brain Sci., 2 (1979) 313-322. 39 Ohon, D. S., Be':.ker, J. T. and Handelmann, G. E., A re-examination of the role of hippueampus in working memory, Behuv. Brain Sci., 2 (1979) 352-359. 40 Ol:on, D. S., Becket, J. 11".and Handelmann, G. E., Hippocampal function: working memory or cognitive mapping? PhysioL Psychol., 8 (1980) 239-246. 41 Oltop, D. S. and Feustle, W. A., Hippocampal function required for nonspatial working memory, E~p. #rum Res., 41 (1981) 380-389. 42 alton, D. S., and Papas, B. C., Spatial memory and hippocampal function, Neuropsychologio, 17 (1979) ~69-682. 43 alton, D. S., Walker, J. A , and Gage, F. H., I!I, Hippocampal connections and spataal discrimination, Brain Research, 139 (1978) 295-308. 44 alton, D. S. and Werz, M. A., Hippocampal functicn and behavior: Spatial discrimination and response inhibition, Physiol. Behav., 20 (1979) 597-605. 45 Powell, E. W. and Hines, G., Septohippocampal interface. In R. L. Isaaeson and K. H. Pribram Eds.), The Hippocumpus, VoL i: Structure and Development, Plenum Press, New York, 1975 pp. 41-59. 46 Rawlms, J. N. P., Feldon, J. and Gray, J. A., Septo-hippocampal theta rhythm, Exp. Brain Res., 37 (1979) 49-63. 47 Shepherd. G. H., The Synaptic Organization of the Brain, Oxford University Press. Oxford, 1979. 48 Steward, O., Topographic organization of the projections from the entorhinal area to the hippocampal format.:on of the rat, J. camp. Neural., 167 (1976) 285-314.
253 49 Steward, O., Loesche, J. and Horton, W. C.. Behavioral correlates of dennervation and remnervation of the hippocampal formation of the rat: open field activity and cue ut:lization following bdateral entorhinal cortex l~ions, Brain Res. Bull., 2 (1977) 41-48. 50 Steward, O. and Scoville, S. A., Cells of origin of entorhinal cortical afferents to the hippocampus and fascia dentata of the rat. J. comp. Neurol., 169 (1976) 347-370. 51 Swanson, L. W., The anatomical organization of septo-hippocampal projections. In Ciba Symposium No. 58, Functions of the Septo-Hippocampai System, Elsevier, Amsterdam, 1975, pp 25-q.3. 52 Walker, J. A. and Olton, D. S., Spatial memory deficit following fimbria-fornix lesions: Independent of time available for stimulus processing, Physiol. Behav., 23 (1979) 1!-15. 53 Walker, J. A. and OIton, D. S., Spatial memory deficit following fimbria-formx lesions: independent of time available for ~timulus processing, Physiol. Behav., 23 (1979) 1i-15 54 Walker, J. A. and Oilon, D. S., Fimbria-fornix lesions impair spatial working memory bm not cognitive n~apping, J. cmnp. Physiol. Psychol., in press. 55 Winson, J. and Abzug, C , Neuronal transmission through hlppocampal pathways dependent on behavior, J. Neurophysiol., 41 (1978) 716-732.
241
Elsevier Biomedical Press
A DISCONNECTION ANALYSIS OF HIPPOCAMPAL FUNCTION
DAVID S. OLTON, JOHN A. WALKER* and WILLIAM A. WOLF Deparfment o f P~ychology, The John.s Hopkm~ Univer~tty, Bait:more, MD 21218 (U S.A /
(Accepted July 9th, 1981 Key words: hippocampus -- memory - - dtsconnectlon analysts - - working memory
hlppocdmpal
system
SUMMARY A dtsconne,.qion analys~s determmed the extent to which the fornix, hippocampus and entorhmal cortex are components of the same tunctional system m tasks that require workmg memory. Preoperattvely, rats were train~.d to perform accurately on a radial arm maze. Then various combinauons of umlateral and b,laterai le~ons were placed in the fornix and entorhinal cortex, either with or without a transection of the hippocampal commissures. When the lesions left intact at least one pathwa~ through the hippocantpu~ interconnecting the formx and entorhmal cortex, rats performed normally. Etther an uncrossed pathway (following a umlateraJ lesion o f the fornix, transection of the hippocampal commissures, and an ip~datcral E,ion of the entorhinal cortex) or a crossed pathway (following a unilateral Ic~lon of the formx and a contralateral lesion of the entorhinal cortex, leaving the hippocampai commisst, res intact) was sufficient, When the lesions produced a complete bilateral disconnection of the fornix and entorhinal cortex, rats performed poorly. The results indicate that the hippoeampal system provtdes a functional connection between the subcort~cal structures associated with the fornix and the neocortical structures a , s o o a t e d with the entorhinal cortex, and that without this connection normal processing of working memory can not occur.
INTRODUCTION Structures in the central nervous system function not as isolated umts, but as c o m p o n e n t s o f larger systems. One of the most effective ways o f d e t e r m i m n g the extent * Present address: Neurological Scnences Center. Good Samaritan itospJtal, 1015 NW 22nd Ave., Portland, OR 97210, U.S.A 0006-8993/82/0000-0000/$02.75 i ~Elsevier Biomedical Pr~~
242 of a functional system is through a disconnection analysisT,S, 13-15.20.zl.z¢.2s. In this analysis, unilateral lesions are combined with transections of interhemispheric commissures. Some combinations of lesions produce a complete, bilateral disconnection so that neural impulses are unable to flow through the system from one structure to another. Other combinations of lesions do not produce a disconnection, leaving at least one set of connections which is sufficient for normal neural transmission, if the structures in question participate in the same functional system, then all combinations of lesions that produce a disconnection should have a similar behavioral effect, and one that is the same as that found after bilateral lesions of these structures. If, on the other hand, the structures in question participate in different functional systems, then the disconnection should have little effect on behavior. The present experiment was designed to conduct a disconnection analysis of the hipgo~mpal system (septum, fornix, hippocampus, entorhinal cortex). For this analysis, three anatomical areas are of particular interest: (a) the fornix,which provides the extrinsic connections of the hippocampus with subcortical structures; (b) the perforant path and the entorhinal cortex, which provide the extrinsic connections of the hippocampus with neocortical structures; (c) the dorsal and ventral hippocampal commissures, which provide homotopic and heterotopic connections from the hippocampus on one side of the brain to the hippocampus on the other side of the brain (for reviews, se,: t-ors, i, ~, 5, 6, f0, 30, 45 and 47-51). Pr¢, !ous resea~ch has demoh~trated that bilateral lesions of the components of the hippocampal system produce an impairment in tasks that require working memory3.4.o.J~ 1~,31,34,:~5,3s-44..~3 ',fthese structures, which are closely related through their neuroanatomical connection~ ,articipate in the same functional system, then a disconnection analysis should produce a pattern of results as described above. The most critical dat "~ "ome from rats with a unilateral lesion of the fornix, unilateral lesion of the entorhinal cortex, and tran,~ection of the hippocampal commissures. Ipsilateral lesions of the fornix and entorhinal cortex do not produce a bilateral disconnection because a - ;ntact pathway between subcortical and neocortical structures through the htppocampus still exists in the side of the brain conttalateral to these lesions. Consequently, behavioral functions normally mediated by the hippocampus should remain intact, and performar,ce in tasks that require working memory should be normal. Co-:ralaterai lestons of the fornix and entorhinal cortex (in conjunction with transectmn of the hippocampai commissures), however, do produce a bilateral disconnection, in the side of the braia wtth the intact formx, neural impulses are able to travel between subcortical structt~res and the ipsilatcral hippocampus as usual. However, they are unable to reach neoeortical structures through hippocampai connectmns: the ipsilateral entorhinal cortex has been destroyed and, although the contralateral entorhinal cortex is intact, transection of the hippocampal commissures prevents communication through that pathway as well. A complementary pattern exists on the other side of the brain, the one w~th the intact entorhinal cortex. Here, neural impulses are able to travel between neocortieal structures and the ipsilateral hippocampus as usual. However, they are unable to reach sul:corticai structures
243
through hippocampal connections; the ipsilateral formx has been destroyed, and although the contralateral formx is intact, transection of the hippocampal commissures prevents communication through that pathway as well. Consequently, behavioral functions normally mediated by the hippocampus should be disrupted, and animals with these contralateral lesions and transection of the hippocarnpal commissures should behave the same as those with bilateral lesions of the hippecampus ~tself. An important point ~n t.he above analysis is the placement of the les~ons m the same 3 structures; only the ipsilateral-contralateral relationship of the lesions in the fornix and entorhinal cortex d~ffers in the two examples. Consequently, any differences in behavior following these two sets of lesions can be attributed to the difference in the topological relationship of the lesions, and the results interpreted m terms of the structures that have been d~sconneeted. The ability of rats to perform corre,.tly on a radial arm maze was used to assess hippocampal function. This test procedure requires working memory ~ 12.a3 3t, 112 52, /.t process that is severely disrupted following bilateral lesions in the hlppocampus or it~ extrinsic connections (see above). Nine d~fferent combinations of les~ons m the fornix. entorhinal cortex and hippocampal commlssures were used to evaluate the pred,ct,ons of the disconnection analysis (see above), and to answer other questions about the role of the hippocampal system m workmg memory (see Discussion). METHODS dpparatu.~ The test apparatus was a radial arm maze with 8 arms a~ described clsc~ hcrc ~.1 Each of the 8 arms extended from a central platform like a ~poke of a wheel The central platform was an octagon, 21 cm on each side. Each arm was 56 cm Iont~and 10 cm wide with a hole, 5 mm deep at the end away from the central platform Subiects Male albino rats derived from a Sprague-Dawley strain were raised m the John~ Hopkins Colony. When they were approximately 90 days old, they were placed m individual cages with water available at all times. Food deprivation reduced each rat's body weight to 85 % of its normal value. Body weights were maintained at this level, plus 5 g each week to allow for body growth, by giving the approprmte amount of Purina Lab Chow approximately ! h after each day's testing was completed Procedure Preoperative te.~tblg. All rats were shaped to go down the arms of the maze and eat the food in the hole at the end of each arm (see ref. 43). They then received one test session each day, 5 days a week. At the start of each test ~e~.sion, one 190 mg Noyes food pellet (P. J. Noyes Co.) was placed in the hole at the end ofeach arm. "[he rat was placed on the central platform and allowed to choose among the arms. A correct re.~ponse was going down an arm for the first time during a test session (in which case the rat obtained the food pellet at the end of it). An incorrect re~pon~e was going down
244 an arm again during a test session (in which case the rat did not get food because he had eaten the pellet during his previous visit). Each test session continued until: (a) the rat had chosen all 8 arms, (b) 2 min had passed since the rat's previous response, or (c) a total o f l0 rain had passed since the start of the test session. Preope~'~five testing continued in this manner until the rat received at least 10 test sessions ar d reached a criterion of 7 correc4 responses in the first 8 choices for 5 consecutive te~t sessions. Surgery. Rats were given intraperitoneal injections of 0.02 mg methyl atropine (Sigma Chemical Company) and 2.2 ml/kg Chloropent (Fort Dodge Laboratories), and an intramuscular injection o f 60,000 units of Bicillin (Eli Lilly). They were placed in a stereotaxic instrument. The scalp was incised and retracted and holes drilled through the appropriate places in the skull. Entorhinal lesions were made by passing radio frequency current from a Grass LM-4A Lesion Maker through a stainless steel electrode insulated except for two mm on the posterior side of tip. This asymmetrical insulation directed the current posteriorly into entorhinal cortex, away from the hippocamp,Js and dentate gyruss2.4a. Selective lesions of the fornix and of the hippocampal commissures were made with a scalpel blade. For the fornical lesions, the blade was lowered in the coronal plane just posterior to the medial septal nucleus and moved laterally from the midline. For the commissurai lesions, it was lowered in the sagittal plane just posterior to the medial septal nucleus and moved posteriorly. Combined lesions of the fornix and hippocampal commissures were made either by combining both of the two knife cuts just described above, or by radiofrequency current, in this latter case, a stainless steel electrode, insulated except i'or 0.8 mm at the tip, was lowered into the fornix just anterior to the anterior pole of the hippocampus. Thus the lesions destroyed both the extrinsic fibers in the fornix, and the intrinsic fibers in the ventral hippocampal commissures *.~. Rats given control operations had various types of procedures. Some were designed to produce no brain damage, and consisted of only anesthesia and an incision in the scalp. Others were designed to spare t.he fornix, hippocampal commissures and entorhinal cortex, but produce damage in adjacent areas. Electrodes were lowered to the fornix or entorhinal cortex, but no current was passed. Scalpel blades were lowered to a position just dorsal to the fornix or the hippocampal commissurcs, and the appropriate cut made at this level. Postoperatire test~rig. After remaining in the home cage without testing for approxima'~?~, ~ne week following surgery, each rat was given at least 10 test sessions following the same procedure used preoperatively (except that no shaping was given). Some rats were then killed for histology. Some of the other rats, who had not received a complete disconnection, were tested until they reached a criteriof, of 7 correct responses in the first 8 choices and then given a second lesion. If this second lesion did not produce a complete disconnection, some of them were teste,' again until they reached criterion, given a third lesion, and tested once more. Those rats that had received a complete disconnection were given at least 10 test sessions; some received an additional 40 test sessions. Histology. Rats were deeply anesthe*ized with ether and then perfused with
245 0.09 ~o saline followed by 10 ~ formalin. The brains were removed from the skull and cut in two so that coronal sections could be taken at the level of the fornix and anterior hippocampus, and horizontal sections at the level of the posterior hippocampus and entorhinal cortex. Some rats with transections of fornix made by the scalpel blade had the entire brain cut in the horizontal plane. The brains were cut in a cryostat. In every 200/,m throughout the hippocampus and the area with the lesion, two sections were taken. One section, 20/,m thick, was stained with Luxol fast blue for myelinated fibers and cresyl violet for cell bodies. The other section, 50/,m thick, was stained for acetylcholinesterase (ACHE)~5.2e. These sections were mounted on a slide. The lesions were evaluated without knowledge of the rat's performance. RESULTS
Preoperative behavior Preoperatively, all rats learned to choose accurately. They reached the criterion of 7 correct responses in the first 8 choices for 5 consecutive test sessions in a mean of 9 test sessions.
Hi.~tology in all but 8 rats, the lesioas were as intended. The transection of the h~ppocampal commissures cut both the ventral and dorsal commissures. Dorsal structures, such as the corpus callosum and midline cortex, also received some damage. Damage occasionally extended more ventrally into the thalamus, but was always slight. An example of a representative lesion is presented in Fig. I (left top). The unilateral lesions of the fornix and ventral commissures made with radiofrequency current destroyed these fibers posterior to the septum and anter,or to the hippocampus. Slight damage was occasionally found in the dorsal part of the thalamus; other adjacent structures were spared. An example of a representative lesion is presented in Fig. 1 (left middle). The unilateral lesions of the fornix alone made with the scalpel blade were just posterior to the septum, and anterior to the ventral commissure. An example of a representative lesion is presented in Fig. 1 (left bottom). Both of these lesions had a profound effect on the density of AChE staining in the hippocampus. Normally, the hippocampus stains darkly with a regular laminated pattern (Fig. !, right top). After either type of fornix lesion, the hippocampus had little if any stain in it; whatever stain remained was usually in the most temporal portions 4~. An example of representative result following a fornix lesion is presented in Fig. I (right middle). The entorhinal lesions were large, destroying almost all of medial and lateral entorhinai cortex, along with some of the adjacent retrohippocampai areas. With the largest lesions, the most posterior edges of the hippocampus and dentate gyrus sustained some damage at their most posterior regions, but were otherwise intact. Following these lesions, an additional band of AChE stain was found in the outer twothirds of the apical dendrites of the dentate granule cells, reflecting the sprouting of the cholinergic fibers from the septum into the areas normally innervated by the afferents
b +° ,
~
~t,
,+o. -
•
Fig I. Histological results• The figures on the left were all stained with Luxol fast blue and cresyl violet, the ones on the r,ght w e r e stained for ACHE. The top two figures on the left are in the coronal plane, all the others are in the horizontal plane. Left top" a transection of the hlppocampal comm,ssures completely tran~ected these fibers and produced damage to the overlying neocorte,~ and corpus callosum, but spared the thalamus• Left middle: a unilateral lesion of the formx and the ventral commissure destroyed v:rtually all of these fibers on the right side of the brain, while producing little damage to adjacent areas• Left bottom: a unilateral incision made with the scalpel blade transccted the fibers of the fornix as they reached the posterior portions of the septum, but spared the ventral commissure. Right top: a normal brain. Note the pattern of AChE staining in the hippocampus Right middle• a brain following an ipsdateral lesion of the formx. Note the very light stain for AChE in the hlppocampus as compared to the dense staining of the more medial structures Right bottom: a brain following a lesion of the entorhinal cortex. Note the extens,vc damage to retrohlppocampal area% and
247 from the entorhinal cortex zz,4n. An example of one o f the largest lesions Js presented m Fig. ! (right bottom). The 8 rats with inappropriate lesions were discarded because their lesions were either too large or too small. For example, one rat who was supposed to have a unilateral fornix les~on also had damage to the contralateral formx and no ACh E stare in the anterior portions of the h~ppocampus on that side. Another cat w~th a undateral formx lesion had damage to only the medial portions o f the fo, nix on the s~de o f the lesion, and AChE was absent only from the anterior portions o: the hlppocampus on that side. The data from all rats w~th inappropriate lesions were not mcluded m the behavioral analyses describe:l below.
Postoperative behavtor Postoper~:twely, rats receiving control operations continued to perform well. making a mean o f 7.2 correct responses in the first 8 choices dunng the first 10 test sessions. These data demonstrate that the surgical procedure Jn general, and the damage to brain areas outside of the hsppocampal system, ¢2Jd not ~mpair ~holLe accuracy Rats with combinations of lesions that d~d not produce a bdateral disconnection also performed well postoperatively. The data from these grot:ps are ~ummarized m the top part o f Fig. 2. "l'he mean number of correct response~ m the first 8 tholces during the first 10 test sessions for all these groups ranged from 6 9 to 7.0. the ~or~t performance by any rat m any of these groups was a mean of (" 5 correct re~pon~cs m the first 8 choices. These data demonstrate that ~Jny cennectlon ~et~een the entol hmal cortex and the fornix through the hJppocampus was sufficJert to maintain normal performance in this task. Rats with combinations of lesions that did produce a bdatelal dssconnectlon performed worse than they had preoperatively. ~orse than the groups ~=thout ~t bilateral disconnection, and not significantly better than expected by chance. The datz; from these groups are sum:,mrized in the bottom part of Fig. 2 The mean number of correct responses in the first 8 choices during the first 10 test session~ ranged from 5 4 to 5.8; the best performance 5y any rat m any of these groups was 6.5 correct responses in the first 8 choices. Only one rat in the groups with bilateral disconnections chose as accurately as any rat in the groups w=thout bdateral disconnections. These data demonstrate that an intact connection between the entorhinal cortex and the fornix through the hippocampus is necessary to maintain normal performap, ce in this task. The sequentml order of the lesions had no obvious effect on choice accuracy For example, all rats in the U F - H C - C E group performed poorly after they had received all 3 lesions; this impairment occurred when the lesi~ms were given one at a time (w=th behavioral testing after each of them), and when "they were gs~en all m a single operation. Simdar results were found in all groups. All combinations of lesions that failed to produce a bilateral disconnection were followed by normal choice accurac~ (within the range of results reported for the first 6 groups in Fig 2). whde all combinations o f lesions that did produce a bilateral disconnection were followed b~
248 NUMBER OF CORRECT RESPONSES LESIONS N 5.O 5;5 6;0,, , 6 ~ 7.0 7:5
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"l
UF
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,o
! UF
' rlllllA
.
UF
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,,
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Fig. 2. A summary of the experimenlal design and resulls. The left column $;ves the group name,
indicatingIhe placementof the lesionq. Lesion~were placed in either the fornix(F), ¢ntorhinalcortex (E~ or hippocampal commis,~ures (HC). The le~ions in the fornlJ; and antorhinal cortex were either unilaleral (U) or bilaleral (B), and the unilateral lesions were eilher ¢ontralaleral (C) or ipsilaleral (I). Thus group UF-HC-CE had a unilateral lesion of Ihe fornix, transection of Ihe hippocampal commissure% and a conlralateral lesion of the enlorhinal co-'~x. The r~.ond column presents a schematic diagram of a dorsal view of Ihe rat brain. The rounded structure in the middle al the lop repre~nls Ihe septum, the narrow area behind it is the fornix, the curved slruclure is the hippoeampus, and the area behind it is the entorhinal corlex. Lesions are indiczated by black areas. The third column presenls the number of rals in each group. The histograms present the mean (bar) and the range (superimposed line) of the mean number of correct responses in the first 8 choices during the first 10 lest sessionsfollowing the indicated lesion. Data from rats with control operations are in the top line (open bar). Dala from rats wilh at least one pathway interconnecting the fornix and enlorhinal cortex are presented in the next 5 I,nes (bars with lines slanted upwards to the right). Data from rats with a complete disconnection of fornix and entorhinal cortex are prcscnled in the next 4 lin¢~ (bar,, with lines slanted t,pwards to the left).
poor choice accuracy (within the range of results reported for the last 4 groups in Fig. 2). These data demonstrate that the behavioral effects of the various combinations of lesions did not depend on the order in which the rat received them. Each of the rats receiving more than one lesion contributed data to more than one group, prohibiting statistical analyses which require independent subjects. The clear nature of the results, however, does not require such analyses. First, the order in which the lesions were made had no effect on choice accuracy (see above). Second, all ~roups without disconnections (first 6 groups in Fig. 2) performed similarly; all groups with disconnections (last 4 groups in Fig. 2) performed similarly, and there were only two cases in which the scores of the disconnection and non-disconnection groups
249 overlapped. A binomial test, which does not require independent subjects, on these combined data indicated that the choice accuracy of rats with disconnections was significantly different than the choice accuracy of rats v'ithout disconnections (n ~ 82, X --- 2, P < 0.001). DISCUSSION
The present experiment used a disconnection analysis to examine the functional relationship of the hippocampus, the subcort~cal structures connected to ~t through the fornix, and the neocortical structures connected to it through the perforant path and the entorhinal cortex. All rats with an intact set of hippocampal connections between the subcortical structures associated with the formx and the neocort~cal structures associated with the entorhinal cortex performed well, whether this connection wa~ through one side of the brain (group UF-HC-IE), or from one side of the brain to the other through the hippocampai commissures (group UF-CE). In contrast, all rats with a complete bilateral disconnection performed poorly, this result was found following bilateral lesions m either or both the fornix and entorhmal cortex (groups BF, BE, BF-BE), and after a combination of contralateral unilateral lesions and transection of the hippocampal commissures (group UF-HC-CE). Thus at least m tasks that require working memory, the fornix, h~ppocampal formation and entorhmal cortex comprise a single fL,,ctional system which is critical for normal performance The role of thi~ system In working memory is relatively selective: damage to othc~ brain systems (amygdala, frontal cortex, postcrolatcral neocortex, caudate nucleus) did not produce similar behavioral impairments in tasks that reqmred working memory 4, and damage to the hippocampal system did not produce impa,rments i~1 tasks that didn't require working memory :~.:~:,,:~s,:~9,n4. The hippocampal con~missures are a major pathway interconnecting the hJpp,,campus of one hemisphere to the hippocampus of the other j,:'.4s~. In the prcscat experiment, transection of the hippocampal commissurcs alone (group HC) or contralateral lesions of the fornix and entorhinal cortex (groups UF-CE) did not impair performance. Transection of the commissures combined with contralateral lesions of the fornix and entorhinal cortex (group UF-HC-CE), however, produced an impairment of choice accuracy equivalent to that seen after bilateral destruction of either the fornix or the entorhinal cortex. This pattern of results demonbtrates that the hippocampal commissures act as functional connections between the forn,x and entorhinal cortex when they are the only pathway available through the hippocampus (group U F - C E as compared to group UF-HC-CE), but that they are not necessary for normal behavior when other, uncrossed pathways are present {group HC). Many studies have made lesions of the fornix and used the results of these experiments to draw conclusions about the behavioral functions of the hJppocampus. Such conclusions are appropriate, of course, only to the extent that lesion~ of the fornix and hippo,.ampus predate similar behavioral changes. The results of the present experiment demonstrate that at least with respect to working memory, the fornix and hippocampus are components of the same functional system, so that
250 conclusions about the behavioral functions of the hippoca~ ~ms can be made from the results of experiments demonstrating the behavioral effects produced by lesions in the fornix. Whether this assumption is justified for other types of behaviors, and for the entorhinal cortex, remains to be demonstratedts,s°. The hippocampus has a very low threshold for electrophysiological seizures, and many different events can produce abnormal electrophysiological activity in its3,2e. Thus the behavioral effects following a lesion of either the fornix or the entorhinal cortex might be due to the propagation of abnormal activity from the hippocampus through the remaining pathways to other brain structures. If such were the case, then combined bilateral lesions of the foruix and entorhinal cortex should eliminate these behavioral changes because this set of lesions effectively isolates the hippocampus from the rest of the brain and restricts any abnormal activity in the hippocampus to that structure alone. However, the performance of rats with bilateral lesions of both the fornix and entorhinal cortex was ~1obetter than that of rats with any other kind of disconnection. Thus the disconnecting lesions were effective because they interrupted normal electrophysiological activity in the hippncampus, rather than because they caused seizure activity in the hippocampus which was then propagated to other brain structures through the remaining intact pathways. in other experiments, unilateral lesions of the entorhinal cortex produced immediate behavioral changes which were followed by recovery of function during the next 10 days~4. The time course of this recovery was correlated with the growth of fibers from the intact entorhinal cortex through the dorsal hippocampal commissure to the contralateral dentate gyrus. Furthermore, transection of the dorsal hippocampal commissure reinstated the behavioral impairment, demonstrating that these commissural fibers were responsible for the observed recovery of function. In the present experiment, behavioral testing did not begin soon enough after surgery to determine if unilateral lesions of the entorhinal cortex (group UE) produced an immediate impairment of choice accuracy on the radial arm maze~ thus the present data are not releeant to this question. However, rats with a unilateral lesion of the entorhinal cortex and a transection of the hippocampal commissures (group UF-HCIE) performed well, demonstrating that in the present experiment, normal performance following a unilateral lesion of the entorhinal cortex was not dependent on commisural fibers. The reasons why the commissurai fibers were critical for normal performance in the Loesche and Steward"4 experiment, but not necessary for normal performance in the present experiment, are not clear, but may have to do with the types of behaviors examined and the amount of preoperative test experience. In sumn-,ary, the present experiment used a die,connection analysis to examine the functional relationships of the fornix, hippocam~us and entorhinal cortex in a task that required working memory. The results demonstrate that normal performance in this task requires that one of the interconncctions between the fornix and entorhinal cortex through the hippocampus be intact, but that any one of the alternative pathways, either crossed or uncrossed, is sufficient.
251 ACKNOWLEDGEMENTS
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