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Spatial correlates of hippocampal unit activity are altered by lesions of the fornix and entorhinal cortex
Brain Research, 194 (1980) 311-323 © Elsevier/North-Holland Biomedical Press
311
S P A T I A L C O R R E L A T E S OF H I P P O C A M P A L U N I T A C T I V I T Y A R E A L T E R E D BY LESIONS OF T H E F O R N I X A N D E N T O R H I N A L C O R T E X
VIRGINIA M. MILLER* and PHILLIP J. BEST Department of Psychology, University of Virginia, Charlottesville, Va. 22901 (U.S.A.)
(Accepted January 24th, 1980) Key words: spatial cells - - spatial behavior - - hippocampus - - fornix lesions - - entorhinal lesions
SUMMARY Behavioral and electrophysiological evidence supports the role of the hippocampus in the processing of spatial information. In the present study, neuronal activity recorded from chronically implanted hippocampal microelectrodes was correlated with a rat's spatial orientation while traversing a radial maze for food reward. Place units were found in all fields of the dorsal hippocampus and dentate gyrus. Rotation of the maze relative to extramaze cues failed to disrupt the intact animal's spatial task performance or the spatial correlates of the unit activity. Lesions of the fornix or entorhinal cortex disrupted performance of the task. Unit activity correlated to the animal's spatial orientation was also disrupted by either lesion. There was no correlation between the disruption of the unit activity and location of the unit within hippocampal fields. Unit activity from lesioned animals showed correlation to the physical properties of the maze rather than to the orientation of the maze in space. These results further support the role of the hippocampus in the processing of spatial information.
INTRODUCTION Recent evidence indicates that the hippocampus is involved in the processing of spatial information. Behavioral support for this hypothesis is derived from experiments which demonstrate debilitated maze performance in animals with lesions of the hippocampus or its connectionsS,4,10,13. Further, electrophysiological evidence shows
* Present address for all correspondence: School of Life and Health Science, University of Delaware, Newark, Dela. 19711, U.S.A.
312 a high correlation between hippocampal neuronal activity and an animal's position within an environment6,8,11. Neurons showing spatial correlations typically display a significant change in firing rate when an animal is positioned in a defined place on a maze. This change in firing rate is independent of the animal's activity while located in that space. Such hippocampal units have been called place units 6. The fields of these place units are dependent upon the presence and location of such extra-maze cues as lights, cards or sound sources 7. An animal's behavior also is more dependent upon extra-maze cues than cues local to a maze. For example, an animal's spatial behavior is not altered by rotation of a maze within a fixed environment 9. To investigate the relationship between spatial task performance and place unit activity, the present study addresses several questions. Since maze rotations do not alter behavior, do they affect the response of place units? Secondly, do lesions of hippocampal connections, fornix or entorhinal cortex, which produce behavioral deficits in spatial task performance disrupt the place unit activity? Finally, since the flow of information through the hippocampus proceeds from the entorhinal cortex to the dentate gyrus and then to CA-3 and CA-I1, is the relative distribution of place units within the hippocampal fields and dentate gyrus affected differentially by lesion of hippocampal connections? An 8-arm radial maze was chosen as the test apparatus in this study. This maze offers several advantages as a testing device for spatial correlates of neuronal activity. The arms of the maze radiate symmetrically from the center platform thus representing an organized space with clearly differentiated subunits. Since the animal moves continuously between the arms of the maze, the statistical reliability of any change in neuronal firing rate can be tested. MATERIALS AND METHODS Male Sprague-Dawley rats (Flow Laboratories) of 350--450 g a d libitum weight were used in this study. Animals were housed individually at an ambient temperature of 22 :~ 2 °C; light/dark cycle of 12/12 h. The rats were deprived to 80 ~ of their ad libitum weight and trained following the procedure of Olton et ai. 8 to continuously traverse an elevated radial 8-arm maze for food reward. The maze was identical to that maze previously described 8 with the arms of the maze radiating symmetrically from a central platform like the spokes of a wheel. Located at the end of each arm was a depression into which the reward, 100 mg Noyes pellet, was placed. A water bottle was available only when the animal was confined to the center platform. The maze was located in a Faradic cage containing numerous fixed extra-maze cues, e.g. light, door, sound source, shelf. After a training period the animals were placed in one of the following surgical groups: lesion of the fornix (FL); bilateral lesion of the entorhinal cortex (EL); no lesion controls (C). All animals were implanted with chronic microelectrodes in the dorsal hippocampus. Surgical anesthesia was induced and maintained by 40 mg/kg sodium pento-
313 barbital given intraperitoneally. To reduce salivation, atropine sulfate (0.5 mg/kg) was administered subcutaneously. The surgery procedures described by Best et al. z were followed. Recording electrodes of 62 # m nichrome wire were positioned with a microdrive at a vertical placement where unit activity could be monitored on an oscilloscope. Placement coordinates for the electrodes with a level plane between bregma and lambda were: 2.0-4.5 m m posterior to bregma and 2.0-3.5 m m from midline. Electrodes were secured and the connector pins formed into a head block with dental acrylic. Seven recording electrodes and one ground electrode were placed in each animal. FL animals were lesioned and the microelectrodes were implanted on the same day. Coordinates for the fornix lesion were: 1.5 m m posterior to bregma, on the midline, and 4.0 m m ventral to the brain surface. The midsagittal sinus was teased to one side during lesioning to avoid rupture and bleeding. EL animals were lesioned and allowed to recover for 3-5 days before the electrodes were implanted. During this time the animals were placed on the maze daily for a period of 15 min. Coordinates for the entorhinal lesion were modified from those reported previously 4. At 1 m m anterior to lambda, lesions at vertical placements of 2, 4 or 6 m m below brain surface were made at 2.5, 3.5 or 4.0 m m lateral to midline. The electrode was angled 9 ° away from midline for all entorhinal lesions. All lesions were produced by passing 1 m A current for 40 sec at each electrode placement. Following the implantation of the electrodes, a 3-day recovery period was allowed before the animals were tested on the maze. All electrodes were checked for activity when the animal was confined to the center platform. Once an active electrode was found, a recording was made while the animal traversed the maze in an initial position and following a 90 ° clockwise rotation of the maze from the initial position. Between tests the animal was confined to the center platform and allowed to drink. The animal was removed from the maze during the rotation and was replaced on the center platform after the maze was in the new position. All 8 arms of the maze were baited before release of the animal from the center platform. After the rat made 8 choices, removing the food from the end of those arms, the arms of the maze were rebaited randomly. That is, each traverse the rat made of an arm was not always rewarded. The unit signal was fed through a FET source follower attached to the animal's head block z. Amplification of the signal was through a Grass PI 5B AC preamplifier and AC solid state amplifier 8. Activity was monitored on an oscilloscope and recorded on the audio channel of a Sony AV-3600 video recorder simultaneously with a video recording of the animal's behavior. For analysis the audio portion of the tape was replayed through a discriminator circuit. This discriminated signal was input to a PDP-8E (Digital Equipment Corporation) computer where it was matched with the animal's arm selection. The firing rate of a unit was determined for the animal's visits to each arm. The mean firing rate for an entire trial taken during visits to all the arms was also calculated and designated the grand mean (Gm). In order for a unit to be classified as a place unit, the following criteria had to be met: (1) the mean firing rate on a given arm exceeded the
314 G m by three standard errors and (2) the number of visits to an arm during which the rate exceeded G m was significant by a sign test. A significance level o f P < 0.15 was used. For example, if the rate exceeded the G m in at least 6 of 7 or 6 of 8 traverses of an arm, then it was considered that one of the criterion for a place unit had been met. At the conclusion of the final testing period, the animals were anesthetized with sodium pentobarbital and were perfused via the heart with physiological saline followed by 10~L formalin. The brains were removed and 40/~m frozen coronal or horizontal sections were cut in order to establish the extent of the lesions as well as the location of the electrode tracts. Tissue was stained with cresyl violet. In 2 C animals, 3 FL animals, and 4 EL animals tissue was stained using an acetylcholine esterase (ACHE) technique to establish proliferation or diminution of acetylcholine terminals 5. The results of both staining techniques were used to establish the completeness of the lesions.
Fig. 1. Extent of fornix lesions shown in sections taken at regular intervals from serial coronal sections. Diagonals represent the most extensive lesion; cross-hatched area represents the least extensive lesion.
315 RESULTS The histological extents of both FL and EL lesions are shown in Figs. 1 and 2. In the FL group, all lesions included the fornix posterior to the anterior commissure. Lateral damage included portions of the fimbria. In cases where the lesion extended anterior to the anterior commissure, midline destruction was evident. AChE staining of this tissue indicated diminution of staining in the dentate gyrus stratum moleculare and the strata lacunosum and moleculare of the hippocampus proper. One animal in the study received a lesion extending only through the cingulate cortex to the dorsal edge of the corpus callosum and was included in the C group. The lesions in the EL group included lateral and medial entorhinal cortex as defined by StewardlL AChE staining indicated increased intensity of staining in the stratum moleculare of the dentate gyrus. In 3 of 11 animals, damage to the brain stem and colliculi was evident. The maze performance and nature of the unit activity of
Fig. 2. Extent of lesions to the entorhinal cortex shown in sections taken at regular intervals from serial horizontal sections. Diagonals represent the most extensive lesion; cross-hatched area represents the least extensive lesion.
316
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Fig. 3. Distribution of place units within the hippocampal fields in all lesion groups. Place units which showed directional persistence are represented by A; place units showing directional non-persistence by A ; Type IV and Type V units by 0. these animals did not differ from that of animals with no lesion of the medial structures. Behaviorally, lesioned animals displayed perseverative behavior as has been described by others a,l°. EL animals perseverated in distinct patterns repeating sequences of 2 or 3 choices several times in succession: e.g. 2, 4, 6, 2, 4, 6. FL animals tended to repeat the first two choices of a sequence followed by a third choice: e.g. 2, 4, 6, 2, 4, 8, 2, 4, 5. Distinct patterns varied within animals from trial to trial and from day to day. Units meeting the criteria for a place unit were found in all hippocampal fields in the C group. From 11 animals, 24 of the 24 units recorded were place units. Fewer place units (2 of 8) were found within the dentate gyrus than in the hippocampal fields in the C group. Place units were also found in animals from both lesion groups. However, the percentage of units which met the criteria for place units was reduced with the greatest deficit occurring in the EL group. Whereas all units from CA fields in the C group were place units, only 28 of 35 (80 ~ ) in FL and 13 of 30 (43 ~ ) in EL were place units. While it might be suggested that the number of place units could be correlated with the extent of the lesion, it should be noted that place units were found in animals with the most extensive lesions. Units from 15 animals were represented in the FL group and from I 1 animals in the EL group. No more than 5 units were recorded from any one animal in any of the groups. The relative distribution of place units within the hippocampus can be seen in Fig. 3. Units from all experimental groups were sorted according to the classification of Olton et al. s: Type I units with place fields restricted to one arm of the maze. Type 1I units with place fields restricted to two arms of the maze; the two arms need not necessarily be adjacent. Type IV or 'off' units showing significant decreases in firing rate restricted to one arm of the maze.
317 TABLE I
Unit classification among groups Cell type Place unit
'off' IV
other V
1
H
Control regio-superior regio-inferior d e n t a t e gyrus
9 4 --
7 4 2
--3
--3
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15 4 1
8 1 --
2 1 5
4 -2
E n t o r h i n a l lesion regio-superior regio-inferior d e n t a t e gyrus
11 2 4
----
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1 4 1
Type V or non-place units. Type I units in this study fit the description of Olton et al.'s Type I and III units. The classification of all units can be seen in Table I. Since no differences in distribution between hippocampal fields were observed (Fig. 3), units were grouped as to the broad placement categories of regio-superior or -inferior. Not only did the percentage of place units differ among lesion groups, but the characteristics of the place units also differed. One such characteristic was the average firing rate or Gm over all the arms of the maze. The Gms of Type I units from all CA fields were compared since Type I units exhibited the most restricted place fields (Table II). The Gm of Type I place units from all hippocampal fields in all experimental groups was less than 1.0 imp./sec. In no case did individual Gms exceed 2.5 imp./sec. The C group demonstrated the lowest Gm, 0.39 _q:0.07 (mean 4- S.E.M.) imp./sec (n ----TABLE II
Firing rate characteristics o f Type I units Values are m e a n ± S.E.M., impulses/sec.
Grand mean Infield C h a n g e (robustness) No. units
Entorhinal lesion Control
Fornix lesion
0.79 ± 0.14" 1.25 ± 0.24 58 %* * 13
0.97 :i: 0.15" 1.79 4- 0.31" 90 %* * 19
* Significant f r o m c o n t r o l P < 0.05. ** Significant f r o m control P < 0.01.
0.39 ± 0.07 1.04 ± 0.13 201% 13
318 13), and was significantly less (P < 0.05) than the Gm of either the FL or EL groups, 0.97 :-: 0.15 (in = 19) and 0.79 ± 0.14 (n -: 13)imp./sec, respectively. The Gm between the FL and EL groups did not differ. The average firing rates for the place fields of Type I units also differed among groups (Table II). The place field rate for the FL group, 1.79 ± 0.31 imp./sec, was greater than those of theC, 1.04 5~ 0.13 imp./sec, or EL groups, 1.25 ± 0.24 imp./sec. Since the Gm for the FL group was greater than the C group, the elevated place field rate would not necessarily reflect increased stability of place units in the FL group. To obtain a measure of place unit stability or robustness, the percent change in place field activity was calculated for each group (the difference between the place field activity and Gm was divided by Gm and multiplied by 100). The largest increase in place field activity (Table II) was seen in the C group (201%). The place units from the FL group were more robust than the place units from the EL group, 9 0 ~ vs 58 i~'~; change in activity. A two tailed Mann-Whitney U-test was used to compare individual changes in place field firing rate of Type I units. Place unit robustness from the C group was significantly greater (P < 0.01) than that of place units in either the EL or FL groups (respective Z values of 4.54 and 3.72). The robustness of Type I units from EL and FL animals did not differ. Examples of firing patterns of Type I units from each lesion group are shown in Figs. 4, 5 and 6. A typical Type I unit from the regio-superior of a control animal is found in Fig. 4A. In this example, the unit rate increased when the animal traversed arm 1 of the maze. This change in firing rate was isolated and consistent in 7 of 9 traverses of
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320 T A B L E llI
Summary of place unit distribution and degree of directional persistence Control
Total Regio-superior Regio-inferior Dentate gyrus Animals
Fornix lesion
Entorhinal lesion
Total
Place
Persist
Total
Place
Persist
Total
Place
Persist
32 16 8 8 11
26 16 8 2
26 16 8 2
43 29 6 8 15
29 23 5 1
16 16 4 1
35 21 9 5 11
17 11 2 4
1 1 ----
arm 1 (P < 0.09). Place units from the regio-inferior also followed this pattern. The firing rate pattern of a regio-superior Type I unit from a F L group and dentate gyrus of EL group are seen in Figs. 5A and 6A, respectively. Although the activity was not as restricted to one arm of the maze in the FL animal as in the control (also reflected in elevated Gm values), a significant increase in firing rate was recorded in 10 of 11 traverses of arm 3. The overall unit activity from the EL animal (Fig. 6A) was more isolated to one arm of the maze than in the FL animal in Fig. 5A, but not as restricted as in the control (Fig. 4A). A significant increase in firing rate was seen in 8 of 10 traverses of arm 1. In addition to the number of place units and degree of robustness being reduced in the lesion groups, the degree of directional persistence demonstrated by those place units upon maze rotation was also reduced (Table III). Upon 90° rotation of the maze, all place units (26 of 26, P < 0.001) from the C group regardless of location within the hippocampus (Fig. 3) continued to show elevated firing rate in the same directional orientation as in the initial maze position. In no instance in the C group was the unit's place field related to the physical arm of the maze. That is, the place field of the units was not determined by maze cues. Only 1 of 17 place units in the EL group and only 16 of 29 place units in the FL group persisted to the directional orientation of the maze relative to extra-maze cues. An example of a Type I unit showing directional persistence is seen in Fig. 4. Following rotation, the unit in Fig. 4A displayed a significant increase in rate 8 of 9 times the animal traversed arm 3 (Fig. 4B). Arm 3 now occupied the directional orientation previously occupied by arm I. If the maze were returned to the initial position, the unit would again show an active place field on arm 1. For Type II units, this directional persistence was not always seen on both arms. However, directional persistence was always found on the arm showing the fastest rate. Units exhibiting directional persistence in the controls did so with no loss of robustness: 201 ~ with the maze in the initial position vs 181 ~ with the maze in the rotated position. There was also no difference in place unit robustness between units which were persistent and those which were non-persistent in the F L group: 117 ± 35 ~ for persistent vs 67 ± 13 ~ for nonpersistent units. The Gm for each of these subdivisions in the FL group did not differ from the Gm of the combined FL group (0.93
321 -4- 0.14 imp./sec, persistent; 0.92 4- 0.25 imp./sec, non-persistent; 0.97 4- 0.15 imp./sec, combined). There was no correlation between the directional persistence of a place unit and its location within the hippocampal fields. As can be seen in Fig. 3, the lesions effectively reduced the number of place units and degree of persistence of place units in all hippocampal fields. Increased firing rate correlated to the physical arm of the maze was observed in 5 cases in each of the FL and EL groups. An example of a Type I unit which persisted to the physical arm of the maze in the FL animal is shown in Fig. 5B. The increased activity observed initially on arm 3 (Fig. 5A) was again seen on arm 3 in 8 of 8 traverses of that arm when the maze was rotated. The remaining units from both the EL and FL groups either did not meet the criteria for a place unit upon maze rotation, or if the criteria were met, no correlation to the physical properties of the maze or spatial location of an arm was apparent. Also no correlation to the presence or absence of food reward could be made. Fig. 6B represents a typical example of a place unit from an EL animal that no longer met the criteria for a place unit once the maze was rotated. DISCUSSION Place units whose firing rate increased significantly when the animal traversed a particular arm of the maze are not differentially distributed within hippocampal CA fields in the rat. The dentate gyrus, however, which receives primary sensory input from the entorhinal cortex 1 displays a low percentage of place units in nonlesioned rats. Similar place unit distribution was noted by O'Keefe ~ where none of 8 dentate units showed spatial corrrelates but more than half showed activity correlated to sniffing or other behaviors. Contrary to these findings, Olton et al. 8 have found 5 of 5 units to have spatial correlates in the dentate gyrus. This discrepancy may be due to differences in recording techniques. Where the present study utilized fixed microelectrodes, the Olton group utilized a moveable microelectrode. They isolated units as the animal traversed the maze, thus perhaps biasing their selection of units studied. No distinctions were made in any of these studies of whether the units were located in the dentate granule cell layer or in the dentate hilus. The low number of place units in the dentate gyrus implies that input which establishes a place unit's field occurs not at the primary synapse of entorhinal cortex to dentate in the scheme described by Andersen et al. 1, but at a secondary step in information processing. Lesions of hippocampal connections did not differentially affect the distribution of place units found within the hippocampus but did significantly reduce the total number of place units in all hippocampal fields. The increase in the number of Type IV units found in the lesion groups suggests that facilitatory input to the units was disrupted by both lesions. That the place unit robustness was decreased by either lesion supports the suggestion that multiple inputs are necessary for the establishment of a unit's place field. The histological and behavioral data combine to verify the
322 completeness of the lesions. Therefore, it is unlikely that place units found in lesion groups were recorded from an isolated region of hippocampus where inputs were inadvertantly left intact. Of the units in the lesion groups not meeting the criteria for a place unit, none exhibited firing patterns related to food reward. Therefore, the classification of Ranck 11 of approach-consummate or approach-consummate-mismatch cells does not apply to units in the present study. The manipulation of maze rotation which did not disrupt the animal's task performance also failed to disrupt place unit integrity in nonlesioned animals. These data are consistent with the behavioral observation that following maze rotation animals avoided arms pointing in the directions of where food had been removed. They did not avoid previously traversed arms which now pointed in a new direction 9. That is, animals chose arms on the basis of direction and not on the basis of local intra-maze cues. Extra-maze cues have also been shown to be important in a 3-arm maze task. Only after 3 of 4 specific extra-maze cues had been removed was place unit activity and maze performance altered 7. The lesions which did disrupt task performance effectively altered not only the percentage of place units, robustness of place units but also the characteristic of directional persistence. The entorhinal lesion was the most effective in disrupting unit persistence. The loss of directional persistence could be the result of several factors. First, simple rotation of the maze could effectively alter place unit robustness. This was not the case, for in nonlesioned animals place units showed no loss of robustness once the maze was rotated. Secondly, lesioning hippocampal connections disrupts robustness such that persistence is reduced. Although the lesioned groups exhibited reduced place unit robustness, there were no differences in robustness between units which persisted and those which did not persist in the FL group. Therefore, a third alternative is needed. That is, nonpersistence results from the loss of information processing once the maze is rotated. This information processing involves utilization of a variety of cues some of which might be related to the physical properties of the maze. In some cases units from lesioned groups showed dependence on some intramaze cues. The exact nature of such cues remains undefined at this time. Intact animals have been shown not to rely on such intra-maze cues as scent trails or arm characteristics during behavioral tests 9. It may be that in the absence of normal information processing, the intra-maze cues become important in task performance. The decreased performance of blinded and anosmic rats over single sensory deprived rats on a radial maze task supports this suggestion 14. The ability of lesioned animals to relearn spatial tasks3,13 may also depend on the processing of alternative sets of cues. In summary, the integrity of place units in all hippocampal fields was undisturbed by manipulation of a maze which left the animal's behavior intact. Further, lesions of hippocampal connections which disrupt an animal's spatial task performance also alter place unit characteristics. These results further support the hypothesis that the hippocampus is involved in the processing of spatial information.
323 ACKNOWLEDGEMENTS T h e a u t h o r s wish to acknowledge the technical assistance of B. Crafton, J. Eisenberg a n d K. Glasheen. This research was f u n d e d by Sloan F o u n d a t i o n Fellowship to V . M . M . a n d N I M H G r a n t 16578 a n d N S F G r a n t SER76-18457 to P.J.B.
REFERENCES 1 Andersen, P., Bland, B. H. and Dudar, J. D., Organization of the hippocampal output, Exp. Brain Res., 17 (1973) 152-168. 2 Best, P. J., Knowles, W. D. and Phillips, I. M., Chronic brain unit recording for pharmacological applications, J. pharmacok Meth., 1 (1978) 161-170. 3 Jarrard, L. E., Selective hippocampal lesions: differential effects on performance by rats on a spatial task with preoperative versus postoperative training, J. comp.physiol. Psychol., 92 (1978) 1119-1127. 4 Loesche, J. and Steward, O., Behavioral correlates of denervation and reinnervation of the hippocampal formation of the rat: recovery of alternation performance following unilateral entorhinal cortex lesion, Brain Res. Bull., 2 (1977) 31-39. 5 Lynch, G., Matthews, D. A., Mosko, S., Parks, T. and Cotman, C. C., Induced AChE-rich layer in rat dentate gyrus following entorhinal lesions, Brain Research, 42 (1973) 311-318. 60'Keefe, J., Place units in the hippocampus of the freely moving rat, Exp. Neurol., 51 (1976) 78-109. 70'Keefe, J. and Conway, D. H., Hippocampal place units in the freely moving rat: Why they fire where they fire, Exp. Brain Res., 31 (1978) 573-590. 8 0 l t o n , D. S., Branch, M. and Best, P. J., Spatial correlates of hippocampal unit activity, Exp. Neurol., 58 (1978) 387-409. 9 0 l t o n , D. S. and Samuelson, R. J., Remembrance of places passed: spatial memory in rats, J. exp. Psychol., 2 (1976) 97-116. 10 Olton, D. S., Walker, J. A. and Gage, F. H., Hippocampal connections and spatial discrimination, Brain Research, 139 (1978) 295-308. 11 Ranck, J. B., Studies on single neurons in dorsal hippocampal formation and septum in unrestrained rats, Exp. Neurol., 41 (1973) 461-555. 12 Steward, O., Topographic organization of the projections from the entorhinal area to the hippocampal formation of the rat, J. comp. Neurol., 167 (1976) 285-314. 13 Winson, J., Loss of hippocampal theta rhythm results in spatial memory deficit in the rat, Science, 201 (1978) 160-163. 14 Zoladek, L. and Roberts, W. A., The sensory basis of spatial memory in the rat, Anita. Learn. Behav., 6 (1978) 77-81.
311
S P A T I A L C O R R E L A T E S OF H I P P O C A M P A L U N I T A C T I V I T Y A R E A L T E R E D BY LESIONS OF T H E F O R N I X A N D E N T O R H I N A L C O R T E X
VIRGINIA M. MILLER* and PHILLIP J. BEST Department of Psychology, University of Virginia, Charlottesville, Va. 22901 (U.S.A.)
(Accepted January 24th, 1980) Key words: spatial cells - - spatial behavior - - hippocampus - - fornix lesions - - entorhinal lesions
SUMMARY Behavioral and electrophysiological evidence supports the role of the hippocampus in the processing of spatial information. In the present study, neuronal activity recorded from chronically implanted hippocampal microelectrodes was correlated with a rat's spatial orientation while traversing a radial maze for food reward. Place units were found in all fields of the dorsal hippocampus and dentate gyrus. Rotation of the maze relative to extramaze cues failed to disrupt the intact animal's spatial task performance or the spatial correlates of the unit activity. Lesions of the fornix or entorhinal cortex disrupted performance of the task. Unit activity correlated to the animal's spatial orientation was also disrupted by either lesion. There was no correlation between the disruption of the unit activity and location of the unit within hippocampal fields. Unit activity from lesioned animals showed correlation to the physical properties of the maze rather than to the orientation of the maze in space. These results further support the role of the hippocampus in the processing of spatial information.
INTRODUCTION Recent evidence indicates that the hippocampus is involved in the processing of spatial information. Behavioral support for this hypothesis is derived from experiments which demonstrate debilitated maze performance in animals with lesions of the hippocampus or its connectionsS,4,10,13. Further, electrophysiological evidence shows
* Present address for all correspondence: School of Life and Health Science, University of Delaware, Newark, Dela. 19711, U.S.A.
312 a high correlation between hippocampal neuronal activity and an animal's position within an environment6,8,11. Neurons showing spatial correlations typically display a significant change in firing rate when an animal is positioned in a defined place on a maze. This change in firing rate is independent of the animal's activity while located in that space. Such hippocampal units have been called place units 6. The fields of these place units are dependent upon the presence and location of such extra-maze cues as lights, cards or sound sources 7. An animal's behavior also is more dependent upon extra-maze cues than cues local to a maze. For example, an animal's spatial behavior is not altered by rotation of a maze within a fixed environment 9. To investigate the relationship between spatial task performance and place unit activity, the present study addresses several questions. Since maze rotations do not alter behavior, do they affect the response of place units? Secondly, do lesions of hippocampal connections, fornix or entorhinal cortex, which produce behavioral deficits in spatial task performance disrupt the place unit activity? Finally, since the flow of information through the hippocampus proceeds from the entorhinal cortex to the dentate gyrus and then to CA-3 and CA-I1, is the relative distribution of place units within the hippocampal fields and dentate gyrus affected differentially by lesion of hippocampal connections? An 8-arm radial maze was chosen as the test apparatus in this study. This maze offers several advantages as a testing device for spatial correlates of neuronal activity. The arms of the maze radiate symmetrically from the center platform thus representing an organized space with clearly differentiated subunits. Since the animal moves continuously between the arms of the maze, the statistical reliability of any change in neuronal firing rate can be tested. MATERIALS AND METHODS Male Sprague-Dawley rats (Flow Laboratories) of 350--450 g a d libitum weight were used in this study. Animals were housed individually at an ambient temperature of 22 :~ 2 °C; light/dark cycle of 12/12 h. The rats were deprived to 80 ~ of their ad libitum weight and trained following the procedure of Olton et ai. 8 to continuously traverse an elevated radial 8-arm maze for food reward. The maze was identical to that maze previously described 8 with the arms of the maze radiating symmetrically from a central platform like the spokes of a wheel. Located at the end of each arm was a depression into which the reward, 100 mg Noyes pellet, was placed. A water bottle was available only when the animal was confined to the center platform. The maze was located in a Faradic cage containing numerous fixed extra-maze cues, e.g. light, door, sound source, shelf. After a training period the animals were placed in one of the following surgical groups: lesion of the fornix (FL); bilateral lesion of the entorhinal cortex (EL); no lesion controls (C). All animals were implanted with chronic microelectrodes in the dorsal hippocampus. Surgical anesthesia was induced and maintained by 40 mg/kg sodium pento-
313 barbital given intraperitoneally. To reduce salivation, atropine sulfate (0.5 mg/kg) was administered subcutaneously. The surgery procedures described by Best et al. z were followed. Recording electrodes of 62 # m nichrome wire were positioned with a microdrive at a vertical placement where unit activity could be monitored on an oscilloscope. Placement coordinates for the electrodes with a level plane between bregma and lambda were: 2.0-4.5 m m posterior to bregma and 2.0-3.5 m m from midline. Electrodes were secured and the connector pins formed into a head block with dental acrylic. Seven recording electrodes and one ground electrode were placed in each animal. FL animals were lesioned and the microelectrodes were implanted on the same day. Coordinates for the fornix lesion were: 1.5 m m posterior to bregma, on the midline, and 4.0 m m ventral to the brain surface. The midsagittal sinus was teased to one side during lesioning to avoid rupture and bleeding. EL animals were lesioned and allowed to recover for 3-5 days before the electrodes were implanted. During this time the animals were placed on the maze daily for a period of 15 min. Coordinates for the entorhinal lesion were modified from those reported previously 4. At 1 m m anterior to lambda, lesions at vertical placements of 2, 4 or 6 m m below brain surface were made at 2.5, 3.5 or 4.0 m m lateral to midline. The electrode was angled 9 ° away from midline for all entorhinal lesions. All lesions were produced by passing 1 m A current for 40 sec at each electrode placement. Following the implantation of the electrodes, a 3-day recovery period was allowed before the animals were tested on the maze. All electrodes were checked for activity when the animal was confined to the center platform. Once an active electrode was found, a recording was made while the animal traversed the maze in an initial position and following a 90 ° clockwise rotation of the maze from the initial position. Between tests the animal was confined to the center platform and allowed to drink. The animal was removed from the maze during the rotation and was replaced on the center platform after the maze was in the new position. All 8 arms of the maze were baited before release of the animal from the center platform. After the rat made 8 choices, removing the food from the end of those arms, the arms of the maze were rebaited randomly. That is, each traverse the rat made of an arm was not always rewarded. The unit signal was fed through a FET source follower attached to the animal's head block z. Amplification of the signal was through a Grass PI 5B AC preamplifier and AC solid state amplifier 8. Activity was monitored on an oscilloscope and recorded on the audio channel of a Sony AV-3600 video recorder simultaneously with a video recording of the animal's behavior. For analysis the audio portion of the tape was replayed through a discriminator circuit. This discriminated signal was input to a PDP-8E (Digital Equipment Corporation) computer where it was matched with the animal's arm selection. The firing rate of a unit was determined for the animal's visits to each arm. The mean firing rate for an entire trial taken during visits to all the arms was also calculated and designated the grand mean (Gm). In order for a unit to be classified as a place unit, the following criteria had to be met: (1) the mean firing rate on a given arm exceeded the
314 G m by three standard errors and (2) the number of visits to an arm during which the rate exceeded G m was significant by a sign test. A significance level o f P < 0.15 was used. For example, if the rate exceeded the G m in at least 6 of 7 or 6 of 8 traverses of an arm, then it was considered that one of the criterion for a place unit had been met. At the conclusion of the final testing period, the animals were anesthetized with sodium pentobarbital and were perfused via the heart with physiological saline followed by 10~L formalin. The brains were removed and 40/~m frozen coronal or horizontal sections were cut in order to establish the extent of the lesions as well as the location of the electrode tracts. Tissue was stained with cresyl violet. In 2 C animals, 3 FL animals, and 4 EL animals tissue was stained using an acetylcholine esterase (ACHE) technique to establish proliferation or diminution of acetylcholine terminals 5. The results of both staining techniques were used to establish the completeness of the lesions.
Fig. 1. Extent of fornix lesions shown in sections taken at regular intervals from serial coronal sections. Diagonals represent the most extensive lesion; cross-hatched area represents the least extensive lesion.
315 RESULTS The histological extents of both FL and EL lesions are shown in Figs. 1 and 2. In the FL group, all lesions included the fornix posterior to the anterior commissure. Lateral damage included portions of the fimbria. In cases where the lesion extended anterior to the anterior commissure, midline destruction was evident. AChE staining of this tissue indicated diminution of staining in the dentate gyrus stratum moleculare and the strata lacunosum and moleculare of the hippocampus proper. One animal in the study received a lesion extending only through the cingulate cortex to the dorsal edge of the corpus callosum and was included in the C group. The lesions in the EL group included lateral and medial entorhinal cortex as defined by StewardlL AChE staining indicated increased intensity of staining in the stratum moleculare of the dentate gyrus. In 3 of 11 animals, damage to the brain stem and colliculi was evident. The maze performance and nature of the unit activity of
Fig. 2. Extent of lesions to the entorhinal cortex shown in sections taken at regular intervals from serial horizontal sections. Diagonals represent the most extensive lesion; cross-hatched area represents the least extensive lesion.
316
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Fig. 3. Distribution of place units within the hippocampal fields in all lesion groups. Place units which showed directional persistence are represented by A; place units showing directional non-persistence by A ; Type IV and Type V units by 0. these animals did not differ from that of animals with no lesion of the medial structures. Behaviorally, lesioned animals displayed perseverative behavior as has been described by others a,l°. EL animals perseverated in distinct patterns repeating sequences of 2 or 3 choices several times in succession: e.g. 2, 4, 6, 2, 4, 6. FL animals tended to repeat the first two choices of a sequence followed by a third choice: e.g. 2, 4, 6, 2, 4, 8, 2, 4, 5. Distinct patterns varied within animals from trial to trial and from day to day. Units meeting the criteria for a place unit were found in all hippocampal fields in the C group. From 11 animals, 24 of the 24 units recorded were place units. Fewer place units (2 of 8) were found within the dentate gyrus than in the hippocampal fields in the C group. Place units were also found in animals from both lesion groups. However, the percentage of units which met the criteria for place units was reduced with the greatest deficit occurring in the EL group. Whereas all units from CA fields in the C group were place units, only 28 of 35 (80 ~ ) in FL and 13 of 30 (43 ~ ) in EL were place units. While it might be suggested that the number of place units could be correlated with the extent of the lesion, it should be noted that place units were found in animals with the most extensive lesions. Units from 15 animals were represented in the FL group and from I 1 animals in the EL group. No more than 5 units were recorded from any one animal in any of the groups. The relative distribution of place units within the hippocampus can be seen in Fig. 3. Units from all experimental groups were sorted according to the classification of Olton et al. s: Type I units with place fields restricted to one arm of the maze. Type 1I units with place fields restricted to two arms of the maze; the two arms need not necessarily be adjacent. Type IV or 'off' units showing significant decreases in firing rate restricted to one arm of the maze.
317 TABLE I
Unit classification among groups Cell type Place unit
'off' IV
other V
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Type V or non-place units. Type I units in this study fit the description of Olton et al.'s Type I and III units. The classification of all units can be seen in Table I. Since no differences in distribution between hippocampal fields were observed (Fig. 3), units were grouped as to the broad placement categories of regio-superior or -inferior. Not only did the percentage of place units differ among lesion groups, but the characteristics of the place units also differed. One such characteristic was the average firing rate or Gm over all the arms of the maze. The Gms of Type I units from all CA fields were compared since Type I units exhibited the most restricted place fields (Table II). The Gm of Type I place units from all hippocampal fields in all experimental groups was less than 1.0 imp./sec. In no case did individual Gms exceed 2.5 imp./sec. The C group demonstrated the lowest Gm, 0.39 _q:0.07 (mean 4- S.E.M.) imp./sec (n ----TABLE II
Firing rate characteristics o f Type I units Values are m e a n ± S.E.M., impulses/sec.
Grand mean Infield C h a n g e (robustness) No. units
Entorhinal lesion Control
Fornix lesion
0.79 ± 0.14" 1.25 ± 0.24 58 %* * 13
0.97 :i: 0.15" 1.79 4- 0.31" 90 %* * 19
* Significant f r o m c o n t r o l P < 0.05. ** Significant f r o m control P < 0.01.
0.39 ± 0.07 1.04 ± 0.13 201% 13
318 13), and was significantly less (P < 0.05) than the Gm of either the FL or EL groups, 0.97 :-: 0.15 (in = 19) and 0.79 ± 0.14 (n -: 13)imp./sec, respectively. The Gm between the FL and EL groups did not differ. The average firing rates for the place fields of Type I units also differed among groups (Table II). The place field rate for the FL group, 1.79 ± 0.31 imp./sec, was greater than those of theC, 1.04 5~ 0.13 imp./sec, or EL groups, 1.25 ± 0.24 imp./sec. Since the Gm for the FL group was greater than the C group, the elevated place field rate would not necessarily reflect increased stability of place units in the FL group. To obtain a measure of place unit stability or robustness, the percent change in place field activity was calculated for each group (the difference between the place field activity and Gm was divided by Gm and multiplied by 100). The largest increase in place field activity (Table II) was seen in the C group (201%). The place units from the FL group were more robust than the place units from the EL group, 9 0 ~ vs 58 i~'~; change in activity. A two tailed Mann-Whitney U-test was used to compare individual changes in place field firing rate of Type I units. Place unit robustness from the C group was significantly greater (P < 0.01) than that of place units in either the EL or FL groups (respective Z values of 4.54 and 3.72). The robustness of Type I units from EL and FL animals did not differ. Examples of firing patterns of Type I units from each lesion group are shown in Figs. 4, 5 and 6. A typical Type I unit from the regio-superior of a control animal is found in Fig. 4A. In this example, the unit rate increased when the animal traversed arm 1 of the maze. This change in firing rate was isolated and consistent in 7 of 9 traverses of
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Fig. 6. Sample activity from a unit in the dentate gyrus of an EL animal. The numbers represent the arms of the maze. A" maze in initial position; place field on arm 1. B: maze in rotated position; unit no longer meets criteria for a place unit.
320 T A B L E llI
Summary of place unit distribution and degree of directional persistence Control
Total Regio-superior Regio-inferior Dentate gyrus Animals
Fornix lesion
Entorhinal lesion
Total
Place
Persist
Total
Place
Persist
Total
Place
Persist
32 16 8 8 11
26 16 8 2
26 16 8 2
43 29 6 8 15
29 23 5 1
16 16 4 1
35 21 9 5 11
17 11 2 4
1 1 ----
arm 1 (P < 0.09). Place units from the regio-inferior also followed this pattern. The firing rate pattern of a regio-superior Type I unit from a F L group and dentate gyrus of EL group are seen in Figs. 5A and 6A, respectively. Although the activity was not as restricted to one arm of the maze in the FL animal as in the control (also reflected in elevated Gm values), a significant increase in firing rate was recorded in 10 of 11 traverses of arm 3. The overall unit activity from the EL animal (Fig. 6A) was more isolated to one arm of the maze than in the FL animal in Fig. 5A, but not as restricted as in the control (Fig. 4A). A significant increase in firing rate was seen in 8 of 10 traverses of arm 1. In addition to the number of place units and degree of robustness being reduced in the lesion groups, the degree of directional persistence demonstrated by those place units upon maze rotation was also reduced (Table III). Upon 90° rotation of the maze, all place units (26 of 26, P < 0.001) from the C group regardless of location within the hippocampus (Fig. 3) continued to show elevated firing rate in the same directional orientation as in the initial maze position. In no instance in the C group was the unit's place field related to the physical arm of the maze. That is, the place field of the units was not determined by maze cues. Only 1 of 17 place units in the EL group and only 16 of 29 place units in the FL group persisted to the directional orientation of the maze relative to extra-maze cues. An example of a Type I unit showing directional persistence is seen in Fig. 4. Following rotation, the unit in Fig. 4A displayed a significant increase in rate 8 of 9 times the animal traversed arm 3 (Fig. 4B). Arm 3 now occupied the directional orientation previously occupied by arm I. If the maze were returned to the initial position, the unit would again show an active place field on arm 1. For Type II units, this directional persistence was not always seen on both arms. However, directional persistence was always found on the arm showing the fastest rate. Units exhibiting directional persistence in the controls did so with no loss of robustness: 201 ~ with the maze in the initial position vs 181 ~ with the maze in the rotated position. There was also no difference in place unit robustness between units which were persistent and those which were non-persistent in the F L group: 117 ± 35 ~ for persistent vs 67 ± 13 ~ for nonpersistent units. The Gm for each of these subdivisions in the FL group did not differ from the Gm of the combined FL group (0.93
321 -4- 0.14 imp./sec, persistent; 0.92 4- 0.25 imp./sec, non-persistent; 0.97 4- 0.15 imp./sec, combined). There was no correlation between the directional persistence of a place unit and its location within the hippocampal fields. As can be seen in Fig. 3, the lesions effectively reduced the number of place units and degree of persistence of place units in all hippocampal fields. Increased firing rate correlated to the physical arm of the maze was observed in 5 cases in each of the FL and EL groups. An example of a Type I unit which persisted to the physical arm of the maze in the FL animal is shown in Fig. 5B. The increased activity observed initially on arm 3 (Fig. 5A) was again seen on arm 3 in 8 of 8 traverses of that arm when the maze was rotated. The remaining units from both the EL and FL groups either did not meet the criteria for a place unit upon maze rotation, or if the criteria were met, no correlation to the physical properties of the maze or spatial location of an arm was apparent. Also no correlation to the presence or absence of food reward could be made. Fig. 6B represents a typical example of a place unit from an EL animal that no longer met the criteria for a place unit once the maze was rotated. DISCUSSION Place units whose firing rate increased significantly when the animal traversed a particular arm of the maze are not differentially distributed within hippocampal CA fields in the rat. The dentate gyrus, however, which receives primary sensory input from the entorhinal cortex 1 displays a low percentage of place units in nonlesioned rats. Similar place unit distribution was noted by O'Keefe ~ where none of 8 dentate units showed spatial corrrelates but more than half showed activity correlated to sniffing or other behaviors. Contrary to these findings, Olton et al. 8 have found 5 of 5 units to have spatial correlates in the dentate gyrus. This discrepancy may be due to differences in recording techniques. Where the present study utilized fixed microelectrodes, the Olton group utilized a moveable microelectrode. They isolated units as the animal traversed the maze, thus perhaps biasing their selection of units studied. No distinctions were made in any of these studies of whether the units were located in the dentate granule cell layer or in the dentate hilus. The low number of place units in the dentate gyrus implies that input which establishes a place unit's field occurs not at the primary synapse of entorhinal cortex to dentate in the scheme described by Andersen et al. 1, but at a secondary step in information processing. Lesions of hippocampal connections did not differentially affect the distribution of place units found within the hippocampus but did significantly reduce the total number of place units in all hippocampal fields. The increase in the number of Type IV units found in the lesion groups suggests that facilitatory input to the units was disrupted by both lesions. That the place unit robustness was decreased by either lesion supports the suggestion that multiple inputs are necessary for the establishment of a unit's place field. The histological and behavioral data combine to verify the
322 completeness of the lesions. Therefore, it is unlikely that place units found in lesion groups were recorded from an isolated region of hippocampus where inputs were inadvertantly left intact. Of the units in the lesion groups not meeting the criteria for a place unit, none exhibited firing patterns related to food reward. Therefore, the classification of Ranck 11 of approach-consummate or approach-consummate-mismatch cells does not apply to units in the present study. The manipulation of maze rotation which did not disrupt the animal's task performance also failed to disrupt place unit integrity in nonlesioned animals. These data are consistent with the behavioral observation that following maze rotation animals avoided arms pointing in the directions of where food had been removed. They did not avoid previously traversed arms which now pointed in a new direction 9. That is, animals chose arms on the basis of direction and not on the basis of local intra-maze cues. Extra-maze cues have also been shown to be important in a 3-arm maze task. Only after 3 of 4 specific extra-maze cues had been removed was place unit activity and maze performance altered 7. The lesions which did disrupt task performance effectively altered not only the percentage of place units, robustness of place units but also the characteristic of directional persistence. The entorhinal lesion was the most effective in disrupting unit persistence. The loss of directional persistence could be the result of several factors. First, simple rotation of the maze could effectively alter place unit robustness. This was not the case, for in nonlesioned animals place units showed no loss of robustness once the maze was rotated. Secondly, lesioning hippocampal connections disrupts robustness such that persistence is reduced. Although the lesioned groups exhibited reduced place unit robustness, there were no differences in robustness between units which persisted and those which did not persist in the FL group. Therefore, a third alternative is needed. That is, nonpersistence results from the loss of information processing once the maze is rotated. This information processing involves utilization of a variety of cues some of which might be related to the physical properties of the maze. In some cases units from lesioned groups showed dependence on some intramaze cues. The exact nature of such cues remains undefined at this time. Intact animals have been shown not to rely on such intra-maze cues as scent trails or arm characteristics during behavioral tests 9. It may be that in the absence of normal information processing, the intra-maze cues become important in task performance. The decreased performance of blinded and anosmic rats over single sensory deprived rats on a radial maze task supports this suggestion 14. The ability of lesioned animals to relearn spatial tasks3,13 may also depend on the processing of alternative sets of cues. In summary, the integrity of place units in all hippocampal fields was undisturbed by manipulation of a maze which left the animal's behavior intact. Further, lesions of hippocampal connections which disrupt an animal's spatial task performance also alter place unit characteristics. These results further support the hypothesis that the hippocampus is involved in the processing of spatial information.
323 ACKNOWLEDGEMENTS T h e a u t h o r s wish to acknowledge the technical assistance of B. Crafton, J. Eisenberg a n d K. Glasheen. This research was f u n d e d by Sloan F o u n d a t i o n Fellowship to V . M . M . a n d N I M H G r a n t 16578 a n d N S F G r a n t SER76-18457 to P.J.B.
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