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Differentiation of basal ganglia dopaminergic involvement in behavior after hippocampectomy
Brain Research, 291 (1984)83-91 Elsevier
83
Differentiation of Basal Ganglia Dopaminergic Involvement in Behavior After Hippocampectomy JOHN H. HANNIGAN, Jr., JOE E. SPRINGER and ROBERT L. ISAACSON Department of Psychology and Center for Neurobehavioral Sciences, State University of New York at Binghamton, Binghamton, NY13901 (U.S.A.) (Accepted May 17th, 1983) Key words: hippocampal lesions - - nucleus accumbens - - caudate nucleus - - dopamine
Large bilateral aspiration lesions of the hippocampus in rats lead to a variety of changes in spontaneous behavior measured in an open field/hole board, relative to sham and neocortically lesioned controls. These changes include increased locomotion, and decreased grooming frequency and rearing bout duration. When animals were injected with the dopamine (DA) agonist 3,4-dihydroxyphenylamino-2-imidazoline (DPI: 0.5, 1.0 and 5.0 pg) into the nucleus accumbens one week after surgery, the behavior of hippocampally lesioned rats was restored to levels not different from control lesioned rats. Haloperidol injections (0.05, 0.1 and 0.5 pg) into the caudate nucleus were not able to do this. Further, DPI injected into the caudate nucleus one month after surgery was also able to attenuate some of the effects of hippocampal damage. On the other hand, haloperidol injections into the nucleus accumbens did not influence behavior. The results are interpreted in terms of hippocampal lesion-induced alteration of a balance in basal ganglia DA systems, indicated by modified response to pharmacological intervention and which mediate the behavioral effects of the lesion.
INTRODUCTION Direct effects of brain d a m a g e are a c c o m p a n i e d by a wide variety of s e c o n d a r y neural changes which can include altered local metabolism20, m e m b r a n e characteristics 1, sprouting and r e c e p t o r sensitivity 19 (see ref. 22). In general, all of these types of changes involve a n u m b e r of n e u r o t r a n s m i t t e r systems, and all could be of behavioral significance. Particular secondary influences on behavior can be distinguished from the p r i m a r y effects of a lesion in part on the basis of the attenuation of behavioral deficits by specific pharmacologic interventions. The behavioral effects of h i p p o c a m p e c t o m y in rats include e n h a n c e d locomotion and deficits in spontaneous alternation, avoidance conditioning, and discrimination tasks (see refs. 10, 12 and 18). W e r e p o r t here evidence of certain secondary influences on behavior involving nucleus accumbens dopaminergic system changes in animals with bilateral h i p p o c a m p a l damage. Because the h i p p o c a m p u s is richly connected to divergent areas, there are m a n y sites for secondary neural changes after lesions that could contribute to the behavioral effects of h i p p o c a m p a l lesions. O n e 0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B .V.
possible site is the nucleus accumbens which receives monosynaptic projections from the ventral subiculum and CA125, although these fibers m a y originate in the entorhinal cortex as well 15. These h i p p o c a m p a l projections are restricted largely to the medial aspects of the nucleus accumbens t6. Behaviorally potent secondary changes in the d e n e r v a t e d nucleus accumbens after h i p p o c a m p a l d a m a g e are suggested in part by the similarity of changes in locomotion, exploration, attention, and avoidance conditioning after h i p p o c a m p a l lesions and after various mesolimbic manipulations9,26-28. Potential secondary neural changes in the nucleus accumbens may involve d o p a m i n e ( D A ) systems' since, for example, the lesions m a d e by Tassin et al. 26 and the drug injections by W a c h t e l et al. 28, both involve this neurotransmitter. In fact, there are increased levels and decreased utilization of D A in the nucleus accumbens, but not in the caudate, after hippocampal lesions, and these changes vary with length of recovery p e r i o d 23. In addition, hippocampally lesioned rats d e m o n s t r a t e altered responsiveness to systemic D A manipulations. F o r example, they are less sensitive to a m p h e t a m i n e 29,3° and to neurolep-
84 tics 8. On the other hand, animals with hippocampal lesions are m o r e sensitive than controls to haloperidol inhibition of ACTH-induced excessive grooming11. Since the nucleus accumbens and the hippocampus appear to be complexly related, the nature of the mesolimbic dopamine system must be considered. Cools and Van Rossum4, 5 postulated the existence of two functionally and behaviorally distinct D A receptor populations in the basal ganglia. According to their model, an excitatory D A receptor (DAe subtype) predominates in the caudate nucleus, and an inhibitory receptor (DAi subtype) predominates in nucleus accumbens. This distinction has been supported by electrophysiological 2, pharmacological 7, behavioraP, and neurochemical evidence24. It has been argued that a relative balance in activity between the DAe-caudate and DAi-accumbens subsystems is critical for determining behaviorS. For example, the exhibition of ACTH-induced excessive grooming in rats can be attenuated by blocking or stimulating either system, but the behavior may be affected less when both systems are manipulated to maintain a balanced activity6. We have previously demonstrated that certain behavioral changes usually found in animals with hippocampal lesions can be attenuated by injections of the putatively specific DAi agonist, 3,4-dihydr0xyphenylamino-2-imidazoline (DPI) into the nucleus accumbens 21. Those results can be interpreted in terms of the Cools and Van Rossum model: destruction of the hippocampus disrupts the balance of the basal ganglia DAe/DAi systems and this contributes to the behavioral deficits found after such damage. Stimulation of the DAi subsystem in the nucleus accumbens by DPI may return the balance between the DAe and DAi subsystems and consequently alleviate some behavioral deficits. If this is the case, then blockade of the DAe system by haloperidol, the appropriate antagonist to D A in the caudate, should also attenuate the effects of hippocampal damage by similarly restoring a balance. This experiment was undertaken to test this hypothesis. In addition, the present study extends the environmental and pharmacological procedures of a previous study in this laboratory21 in order to determine if the conclusions of that study can be extended to other procedures. In the earlier study, independent groups of rats were
tested at one of 3 postoperative periods in a circular apparatus after intra-accumbens injections of 10/~g of DPI. In the present study animals were repeatedly tested in an open field-hole board apparatus after injections of lower doses of DPI into nucleus accumbens. Other animals were tested in the same fashion under a range of doses of haloperidol injected into the caudate. Three weeks after completion of the experiment, we tested the specificity of the drugs to the particular area injected, by injecting DPI into the caudate and haloperidol into the nucleus accumbens of these same animals. MATERIALS AND METHODS Fifty-three male, Long-Evans strain hooded rats (Blue Spruce Farms, Altamont, NY), with a mean weight of 310.3 _+ 4.3 g at the time of surgery, were used. Animals were individually housed in a 12/12 light-dark cycle with lights on at 07.00 h. Food and water were provided ad libitum except for the 18 h preceding surgery, when they were deprived of both. Animals were anesthetized with a solution of chloral hydrate (90 mg/kg), atropine sulfate (0.75 mg/kg), and sodium pentabarbitol (56 mg/kg) and received either neocortical or neocortical plus hippocampal lesions, using aspiration as described by Isaacson and Woodruff 13. Other animals receiving sham lesions were anesthetized, their scalps opened, bone drilled, and scalp sutured. Also, each rat was implanted unilaterally with a stainless steel cannula (0.022" O.D.) aimed at either the dorsal limit of the nucleus accumbens (A 8.9 mm, V 4.8 mm, L 1.2 mm; K6nig and Klippep4), or at the caudate nucleus (A 8.9 ram, V 3.8 mm, L 2.0 mm). The placement of cannulae was counterbalanced between lesion groups and between right and left sides. The cannulae were cemented into place with dental acrylic anchored to the skull by two stainless steel machine screws. Stainless steel wire stylets were placed inside the cannulae with the tip extending down to one-half of the distance of the beveled tip. After surgery, the animals were returned to their home cages in the vivarium and again provided freely with food and water. Starting on the sixth day after surgery, the rats were observed for 4 consecutive days in an open field. At testing, the animals were taken from the vi-
85 varium and transported in their home cages to a holding room in a nearby testing suite. Each animal was subsequently restrained by hand, the stylet removed from its cannula, and an inner injection cannula inserted. The inner cannula was designed to extend 0.5 mm below the beveled tip of the outer sleeve to allow direct injection into the respective brain region. Injections of 1.0 ~1 physiological saline were infused over 60 s. The inner cannula remained in place for an additional 30 s before it was removed and the stylet replaced. The animal was then returned to its home cage for 9-11 min whereupon it was placed into the open field apparatus located in an adjacent, quiet and dimly lit (red light) observation room. Observations were made for 10 min via a closed circuit video system by trained observers located in a third room. The open field was white Plexiglas, measured 71 x 71 x 46 cm high, and the floor was marked into 16 squares (a 4 x 4 matrix). A hole (3.2 cm diameter) was placed in the center of each square. The open field was cleaned between animals with a dilute Lysol solution. The measures employed over the 10 min session were the following: Peripheral locomotion. Movement of greater than 50% of the animal's head and body into one of the 12 squares bordering the 4 walls of the apparatus. Central locomotion. Movement of greater than 50% of the animal's head and body into one of the 4 central squares. Rearing. A lifting of both forepaws off the ground by a vertical extension of the body. Rearing against walls was included in this measure. Peripheral hole pokes. Placement of the animal's nose into one of the holes in the 12 squares bordering the apparatus' walls. Central hole pokes. Placement of the animal's nose into one of the 4 centrally located holes. Grooming. This measure included face washing, paw, body, and genital licking, scratching, and tail preening. In addition to measuring the frequency of occurrence of each behavior, the duration of individual hole poke, grooming, and rearing episodes was measured. All scored behavior was encoded as audio signals which were decoded for digital analysis by a microprocessor which approximated measures of continuous behavior using 0.1 s time sampling. This scoring procedure allowed the derivation of frequen-
cy of occurrence (Frequency), total duration per session (Duration), mean duration per episode, and occurrence over the 10 rain session for each behavior. This procedure was repeated for 4 successive days except that the animals within each lesion group received an injection of a specified drug and concentration. The animals with a cannula placed into the nucleus accumbens received 0.5, 1.0, and 5.0/~g of 3,4 dihydroxyphenylamino-2-imidazoline (DPI) on days 7, 8, and 9 post-lesion, respectively. Rats with a cannula in the caudate were injected with 0.05, 0.1, and 0.5 ~g of haloperidol. All drugs were dissolved in 0.9% saline and infused as described above. The doses were selected on the basis of work by Cools and Van RossumS. They reported that the dose of each drug yielding a maximal physiological effect was within the ranges we used. At the completion of the experiment, the animals remained in their home cages in the colony room until day 35 after surgery. At that time, the procedure followed on the first day of testing was repeated (saline injection). The next day (day 36), each rat was tested again in the open field, but this time rats with cannulae in the nucleus accumbens were injected with 0.5 ~g haloperidol, whereas those with a cannula in the caudate received 5.0 ktg of DPI. Four weeks later, all rats were placed under deep barbiturate anesthesia and perfused intracardially with saline followed by 10% formalin solution. Brains were removed, fixed in formalin, and subsequently sectioned at 80ktm, saving every fifth section through the extent of the lesion and every section through the cannula tracts. Sections were stained with thionin, and cannula location and lesion suitability verified. RESULTS
Histology Of the 53 rats used in the study, 13 were eliminated from analysis due to a physical blockade of the cannulae that occurred before completion of testing, an inability to locate the cannula placement histologically, an unintended cannula placement, or an improper lesion (e.g. some hippocampal damage in the cortical group, or thalamic or superior colliculus damage in the hippocampal group). Representative acceptable cannula tip placements and minimum and maximum
86
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Fig. 1. A: representative cannula tip placements in the nucleus accumbens and the caudate. B: the minimum (solid area) and maximum (striped) extent of cortical lesion C: idem for hippocampal lesions.
87
acceptable lesions are illustrated in Fig. 1. The final number of subjects in each group were: sham: accumbens, 8; caudate, 6; cortical: accumbens, 9; caudate, 6; hippocampah accumbens, 6; caudate, 5.
Behavior In a pre-experiment test of inter-observer reliability, 6 observers scored videotaped animals with the result that there was less than 4% variation among them over all behaviors. (1) Locomotion. There was no difference at any day for any group in thigmotaxia (the proportion of peripheral locomotion to peripheral plus central locomotion), so total locomotor scores were considered in all analyses. As expected, rats with hippocampal lesions were more active than sham or cortically lesioned animals on the first test session, 6 days after surgery (F(1, 34) = 20.70, P < 0.001). There was no difference between animals receiving control saline injections into nucleus accumbens and the caudate (F < 1.0). Over the subsequent 3 sessions with drug injections there was great variability between subjects within all groups, and the apparent mean dose response curves presented in Fig. 2 may not adequately reflect the nature of the drugs' effects in all lesion groups. There was no systematic manner in which increasing doses of either D P I injected into the nucleus accumbens or of haloperidol into tl~e caudate in any lesion group affected locomotion. When the 3 drug days are considered together, only one of 6 groups (cortical lesion with caudate cannula) significantly deviates from an expected null hypothesis that the highest locomotor score for each rat is equally likely to follow any dose injected ()~2 = 7.00, d.f. = 2; P < 0.05). This suggests that there is no dose-dependent relationship between the effectiveness of a drug in reducing enhanced locomotor activity when group means are considered. On the other hand, the large within-subject variability may conceal a drug effect. Of the 18 tests given after D P I to the 6 hippocampal rats, 55% had scores within the range of the sham and cortical rats receiving saline in nucleus accumbens, and 27% were below the median of the controls. Of the 15 haloperidol-into-caudate tests (5 hippocampal rats at 3 doses), 53% of scores overlapped the levels reached by control lesioned animals, but only 6.67% (1/15) were as low as the median of the
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Fig. 2. Mean total locomotion scores for rats measured in open field/hole board for 4 consecutive days starting day 6 after surgery. The key denotes lesion and where injections were given. On day 6 all rats received saline (1.0~1) into their respective injection sites. Caudate-injected animals received 0.05, 0.1, and 0.5/~g//A haoperidol on days 7, 8 and 9, respectively. Accumbens-injected animals received 0.5, 1.0 and 5.0 #g/gl of DPI on days 7, 8 and 9, respectively. control animals. This suggests that there was at least one dose of drug, especially in the DPI-accumbens group, which was effective in any given animal. Fig. 3 illustrates a comparison of the median total locomotor scores in saline-injected animals in each lesion group with the median of the lowest score (i.e. presumably at the most effective dose) for each rat across the 3 drug days. That is, for the comparison within each lesion group, the saline score was measured on day 6 and the drug score (haloperidol or DPI in caudate or nucleus accumbens, respectively) is the lowest of the three scores from days 7, 8 and 9. This would essentially bias the analysis to maximize the likelihood of demonstrating a drug effect despite individual differences. Sign tests for correlated samples show that the difference between the medians is significant only for the cortical and hippocampal lesion rats given D P I into the nucleus accumbens. Haloperidol injections into the caudate were not effective in significantly attenuating the increase in locomotor activity after the lesion. Thus, even when large individual differences in drug responsiveness are considered, only D P I injection into the nucleus ac-
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Over the drug administration regime of the next 3 days, all animals tended to rear less frequently (F(3, 102) = 4.00, P < 0.01). Animals receiving D P I into the nucleus accumbens had lower overall rearing frequencies than those injected with haloperidol into the caudate (F(1, 34) = 12.88; P < 0.001). Further, hippocampal lesioned rats maintained an overall lower number of rearing bouts than cortical lesioned rats (F(2, 34) = 6.06; P < 0.01). There were no significant interactions and no one group showed a significant change over days (largest F(3, 24) = 2.21; P <
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Fig. 3. Median total locomotion scores. A n i m a l s injected with saline (S) were scored on day 6. The m e d m n s for the animals injected with haloperidol (H) into caudate or D P I (D) into nucleus accumbens were derived from the lowest scores for each animal on days 7, 8 and 9. T h e * indicates significantly different from saline injected rats within same lesion group (sign test; P < 0.05).
cumbens attenuated the effects of hippocampectomy on locomotion. But this is not only because of the type of analysis since this same conclusion is drawn when (a) the dose of the median response across the 3 drug days is analyzed; and when (b) an analysis of variance about the means (rather than medians) of the lowest scored days is done. In this case, there is a significant drug x lesion interaction (F(2, 34) = 4.14, P < 0.05) consistent with the conclusion drawn above. One month later, the lesioned animals were still more active than controls (F(2, 34) = 6.69, P < 0.01; Fig. 4). When 5.0/~g of D P I was injected into the caudate, or 0.5 ~tg of haloperidol was injected into the nucleus accumbens the next day, there was a significant three-way interaction (F(2, 34) = 6.33; P < 0.01) which reflected the reduction of locomotor scores only in rats with hippocampal lesions that had received D P I injections into caudate (F(1, 4) = 12.12, P < 0.05). No other injections were effective. (2) Rearing. There was no difference between lesion groups in the frequency of rearing bouts in the open field after saline injections 6 days after surgery. There was, however, a reduction in the mean duration per rearing bout in hippocampal animals (F(2, 34) = 3.48, P < 0.05).
There was no effect of drug at any day in any lesion group on mean rearing duration. The reduced duration seen in hippocampal rats on day 6 is maintained throughout (F(2, 34) = 5.02; P < 0.05). One month later when the rats were again injected with saline, there were no differences between groups in rearing bout frequency, but hippocampal lesioned rats still reared for a significantly shorter time per bout (F(2, 34) = 3.57; P < 0.05). Neither D P I into caudate nor haloperidol into accumbens changed rearing frequency or duration on the next day. (3) Hole poking. All animals poked their heads into holes about the same number of times on day 6 DPI INTO CAUDATE [] z
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Fig. 4. Mean total locomotion scores for animals m e a s u r e d one m o n t h after surgery. O n day 35, all animals were injected with saline (S), and on day 36, rats received either haloperidol (H; 0.5/~g//~l) into nucleus accumbens or DPI (D; 5.0/~g/~tl) into caudate. The * indicates significantly different from saline injected group (F(1, 4) = 12.12, P < 0.05).
89 following saline injections. However, when haloperidol was injected into the caudate of cortically lesioned animals there was an immediate increase in the frequency of hole pokes observed, but primarily when the two highest doses were used (F(6, 102) = 2.51; P < 0.05). This 3-way interaction is accountable entirely by the cortically lesioned haloperidol group since it is the only one to change hole poke frequency significantly over days (F(3, 15) = 8.11; P < 0.01) although there was a significant overall increase in hole poke frequency over days as well (F(3, 102) = 4.08; P < 0.01). In addition, the haloperidolinjected groups showed overall higher frequencies of hole pokes than DPI-injected animals (F(1, 34) = 4.41; P < 0.05). One month later when saline was again injected into both areas, there appeared to be long-term effects of the haloperidol injections since hole poke frequency scores were still higher in animals that had been injected with haloperidol (F(1, 34) = 4.25; P < 0.05), with the largest difference apparently in the cortical and hippocampal lesioned groups. Administration of DPI the next day reduced, but did not eliminate, this effect. Mean hole poke durations per bout for hippocampally lesioned rats were shorter than for control lesioned rats on day 6 (F(2, 34) = 3.80; P < 0.05). The drug administration over the next 3 days did not systematically influence mean duration of hole pokes, and the lesion effect remained as on day 6 throughout (F(2, 34) = 3.49; P < 0.05). One month later hippocampal animals injected with saline still had shorter mean hole poke durations than controls (F(2, 34) = 4.57; P < 0.05). There was a significant drug x lesion interaction (F(2, 34) = 4.06; P < 0.05) which reflected an increase in the mean length of a hole poke for hippocampal animals receiving DPI into the accumbens (F(1, 4) = 8.66; P < 0.05). No other group significantly changed mean hole poke duration after drug injection on day 36. (4) Grooming. Six days after surgery there were no differences between groups in grooming bout frequency or in duration per bout following saline injections. With the subsequent drug injections, there was an overall increase in the frequency of grooming bouts in all groups (F(3, 102) = 7.44; P < 0.001). Fig. 5 illustrates the changes in grooming frequency over days. The apparent difference on day 8 in the
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Fig. 5. Mean grooming frequency scores. All animals received saline injection (1.0 ktl) on day 6 and increasing doses of DPI into nucleus accumbens (0.5, 1.0 and 5.0 ~tg/~l) or of haloperidol into caudate (0.05, 0.1 and 0.5/~g/#1) on days 7, 8 and 9, respectively. groups given DPI into nucleus accumbens is not significant, again attesting to large individual differences. Haloperidol injections into caudate systematically increased the number of bouts in shams (F(3, 15) = 3.87; P < 0.05), although hippocampally lesioned rats did not show this effect. On the other hand, there were no reliable changes in mean grooming bout duration over the 4 testing days. The saline injection given 35 days after surgery did not alter grooming frequency, but the mean duration per bout were longer in animals receiving caudate injections of saline than in rats with nucleus accumbens injections (F(1, 34) = 4.98; P < 0.05), and drug injections on day 36 do not change this difference. Grooming frequency was not altered by any injection although DPI into caudate does maintain the elongated grooming bouts seen after earlier haloperidol (days 8 and 9) or saline (day 35) injections into caudate (F(1, 34) = 5.74; P < 0.05). DISCUSSION In a previous study we demonstrated that the intraaccumbens injection of 10 ktg of 3,4-dihydroxyphenylamino-2-imidazoline (DPI) was effective in attenuating deficits in locomotion, exploration, and grooming found in rats with large bilateral destruction of the hippocampus 21. The present study supports those conclusions since DPI in a dose range of 1-5 ~g injected into the nucleus accumbens can also lower the enhanced locomotor scores of lesioned ani-
90 mals to near-control levels, although there is no clear dose-response relationship. These supporting data come in a different testing situation and under a repeated injection regime, in contrast to the independent groups in the Reinstein et al. 21 study. However, whereas we expected that haloperidol injections into the caudate would also be effective, locomotor deficits were not attenuated by the dose used. This may mean that the balance between nucleus accumbens DAi receptor systems and caudate DAe systems is not critical to behavior in the way Cools and Van Rossum 5 propose. Alternatively, the hippocampal lesion may disrupt these systems in such a way that a balance between the D A e and DAi systems cannot be achieved bidirectionally. For example, a lesion could affect the sensitivity of the receptor subtypes to the drugs used in this study 19. Also, endogenous in vitro membrane phosphorylation is altered in both nucleus accumbens and caudate after hippocampal lesions 1. In fact, when DPI was injected into the caudate, it was effective in attenuating deficits in locomotion and mean hole poke duration in hippocampally lesoned rats. This is significant since the caudate is an area thought to have a low concentration of DAi receptors4, 5, and that has been shown to be insensitive to DPI in normal animals 6.24. However, Nauta 16 has shown that the D A projections from the ventral tegmental area innervate the mediolateral and medial anterodorsal caudate in the rat. This suggests that caudate D A systems may be indirectly (or secondarily) influenced by hippocampal lesions by way of a limbic-ventral tegmental connection16,17. It does not seem that the basal ganglia dopamine systems function in a 'balanced fashion' in mediating other behaviors affected by the lesions. As expected on the basis of previous results, rearing durations were shorter for the hippocampally lesioned animals, but this was not affected by any drug manipulation at any time. This is in contrast to earlier findings 21, although that study measured animals in a smaller apparatus and a higher dose of DPI was used. The changes in hole poke behavior are noteworthy. While there was no initial deficit in hole poke frequency due to either lesion, haloperidol induced a tremendous increase in hole poke frequency in rats receiving cortical lesions. The effect is of special interest because of its specificity and because it is a
long-term change. Also, as noted above, the restoration of normal hole poke durations in hippocampal animals by intra-caudate injections of DPI suggests again that caudate D A systems may be altered by the lesion. In the present study we failed to show any change in grooming after brain damage. However, the effects of hippocampal lesions on grooming in 'novelty' situations are not large, and longer observation periods may be required to detect the lesion effects. We did find that hippocampal animals do not increase the frequency of grooming bouts when injected with haloperidol into caudate as much as shams do one week after surgery. One month after surgery this increase persists and is not changed by DPI injections. We think that the absence of clear dose-response curves for locomotor and grooming effects suggests an enhanced variability in behavior induced by the lesion. If the lesion were removing a modulatory influence from the nucleus accumbens, the ventral tegmental area, or any other area, then the activity in those areas could fluctuate more in a given environmental situation or under a pharmacological challenge. In repeated treatment designs such as this, the contributions of repeated testing and increasing drug doses are confounded. Also, there is the possibility of carry-over effects which could make the dose relationships difficult to discern. Alternatively, the relatively lower doses of DPI we used could be near a threshold for behavioral effect. If so, this would explain the enhanced variability in performance. In conclusion, secondary dopaminergic changes in nucleus accumbens, perhaps involving DAi systems, seem to mediate an enhanced locomotor activity in hippocampally lesioned rats. In the present situation, rearing and hole poke deficits are not attenuated by DA manipulations, whereas grooming was not impaired by the lesion, although the increased grooming in control animals after the injection of haloperidol is not seen after hippocampal lesions. The predicted functional balance between basal ganglia DA systems did not seem be critical here since manipulations of D A e (caudate) and Dai (nucleus accumbens) in opposite directions did not yield similar effects. It may be that the lesion prevents a demonstration of the balanced systems as we hypothesized it. On the other hand, hippocampal lesions do
91 s e e m to alter b o t h D A e and D A i systems. First, t h e
inability of h a l o p e r i d o l to i n c r e a s e g r o o m i n g in hip-
l e s i o n e d animals are m o r e sensitive t h a n c o n t r o l s to
p o c a m p a l s . T h e s e results s u p p o r t t h e n o t i o n that the
D P I in nucleus a c c u m b e n s . S e c o n d , it a p p e a r s that
b e h a v i o r a l s e q u e l a e of h i p p o c a m p a l lesions are due
the lesion changes the specificity of p r e s u m e d cau-
in part to s e c o n d a r y c h a n g e s in m e s o l i m b i c d o p -
d a t e D A e system to the drugs used. This is s e e n in the
a m i n e r g i c systems.
REFERENCES
17 Nauta, W. J. H. and Domesick, V. B., Neural associations of the !imbic system. In A. Beckman (Ed.), Neural Substrates of Behavior, Spectrum Publications, New York, in press. 18 O'Keefe, J. and Nadel, L., The Hippocampus as a Cognitive Map, Oxford University Press, Oxford, 1978. 19 Reinstein, D. K., Behavioral and Biochemical Changes After Hippocampal Damage, Doctoral Dissertation, State University of New York at Binghamton, 1981. 20 Reinstein, D. K., Isaacson, R. L. and Dunn, A. J., Regional changes in 2-deoxyglucose uptake after neocortical and hippocampal destruction, Brain Research, 175 (1979) 392-397. 21 Reinstein, D. K., Hannigan, J. H. and Isaacson, R. L., Time course of certain behavioral changes after hippocampal damage and their alteration by dopaminergic intervention into nucleus accumbens, Pharmacol. Biochem. Behay., 17 (1982) 193-202. 22 Schoenfeld, T. and Hamilton, L., Secondary brain changes following lesions: a new paradigm for lesion experimentation, Physiol. Behav., 18 (1977) 951-967. 23 Springer, J. E. and Isaacson, R. L., Catecholamine alterations in basal ganglia after hippocampal lesions, Brain Research, 252 (1982) 185-188. 24 Springer, J. E., Hannigan, J. H. and Isaacson, R. L., Changes in dopamine and DOPAC following systemic administration of apomorphine and 3,4-dihydroxyphenylamino-2-imidazoline (DPI) in rats, Brain Research, 220 (1981) 226-230. 25 Swanson, L. and Cowan, M., An autoradiographic study of the organization of the efferent connections of the hippocampal formation in the rat, J. comp. Neurol., 172 (1977) 49-84. 26 Tassin, J. P., Stinus, L., Simon, H., Blanc, G., Thierry, A. M., Le Moal, M., Cardo, B. and Glowinski, J., Relationship between the locomotor hyperactivity induced by A10 lesions and the destruction of the frontocortical dopaminergic innervation in the rat, Brain Research, 141 (1978) 267-281. 27 Thierry, A. M., Tassin, J. P., Blanc, G. and Glowinski, J., Selective activation of the mesocortical DA system by stress, Nature (Lond.), 263 (1976) 242-244. 28 Wachtel, H., Ahlenius, S. and And6n, N.-E., Effects of locally applied dopamine to the nucleus accumbens on the motor activity of normal rats and following a-methyltyrosine or reserpme, Psychopharmacology, 63 (1979) 203-209. 29 Woodruff, M. L., Hippocampectomy attenuates the reduction of operant rate produced by D-amphetamine, Physiol. Psych., 9 (1981) 223-230. 30 Woodruff, M. L. and Isaacson, R. L., Attenuation of amphetamine-induced reduction in response rate in rats on FR operant schedule by hippocampal lesions, Behav. Biol., 20 (1977) 493-499.
1 Bfir, P. R., Gispen, W. H. and Isaacson, R. L., Behavioral and regional neurochemical sequelae of hippocampal destruction in the rat, Pharmacol. Biochem. Behav., 14 (1981) 304-312. 2 Bevan, P., Bradshaw, C., Pun, R., Slater, N. and Szabadi, L., The action of iontophoretically applied (3,4-dihydroxyphenylamino)-2-imidazoline (DPI) on single cortical cells, Brit. J. Pharmacol., 65 (1979) 701-706. 3 Cools, A. R., Two functionally and pharmacologically distinct dopamine receptors in the rat brain. In E. Costa and G. L. Gessa (Eds.), Advances in Biochemical Psychopharmacology, Vol. 16, Raven Press, New York, 1977. 4 Cools, A. R. and Van Rossum, J. M., Excitation-mediating and inhibition-mediating dopamine receptors: a new concept toward a better understanding of electrophysiological, biochemical, pharmacological, functional, and clinical data, Psychopharmacologia, 45 (1976) 243-254. 5 Cools, A. R. and Van Rossum, J. M., Minireview: multiple receptors for brain dopamine in behavior regulation: concept of dopamine-E and dopamine-I receptors, Life Sci., 27 (1980) 123%1353. 6 Cools, A. R., Wiegant, V. M. and Gispen, W. H., Distinct dopaminergic systems in ACTH-induced grooming, Europ. J. Pharmacol., 50 (1978) 265-268. 7 Costall, B. and Naylor, R. J., The behavioral effects of dopamine applied intracerebrally to areas of the mesolimbic system, Europ. J. Pharmacol., 32 (1975) 87-92. 8 Fish, B. S., Catecholamine Modulation of Behavior Following Bilateral Hippocampal Damage, Doctoral Dissertation, University of Florida, 1976. 9 Galey, D., Simon, H. and Le Moal, M., Behavioral effects of lesions in the A10 dopaminergic area of the rat, Brain Research, 124 (1977) 83-97. 10 Isaacson, R. L., The Limbic System, Plenum Press, New York, 1982. 11 Isaacson, R. L. and Colbern, D., Hippocampal lesions, haloperidol, and excessive grooming, Physiol. Psych., 9 (1981) 260-262. 12 Isaacson, R. L., and Pribram, K. H. (Eds.), The Hippocampus, Plenum Press, New York, 1975. 13 Isaacson, R. L. and Woodruff, M. L., Spontaneous alternation and passive avoidance behavior in rats after hippocampal lesions. In B. L. Hart (Ed.), Experimental Psychobiology, Freeman, San Francisco, 1975. 14 K6nig, J. and Klippel, R., The Rat Brain: A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem, Williams and Wilkins, Baltimore, 1963. 15 Krayniak, P. F., Meibach, R. C. and Siegel, A., A projection from the entorhinal cortex to the nucleus accumbens in the rat, Brain Research, 209 (1981) 427-431. 16 Nauta, W. J. H., The limbic innervation of the striatum. In Proceedings of the GiUes de la Tourette Syndrome Symposium, in press.
83
Differentiation of Basal Ganglia Dopaminergic Involvement in Behavior After Hippocampectomy JOHN H. HANNIGAN, Jr., JOE E. SPRINGER and ROBERT L. ISAACSON Department of Psychology and Center for Neurobehavioral Sciences, State University of New York at Binghamton, Binghamton, NY13901 (U.S.A.) (Accepted May 17th, 1983) Key words: hippocampal lesions - - nucleus accumbens - - caudate nucleus - - dopamine
Large bilateral aspiration lesions of the hippocampus in rats lead to a variety of changes in spontaneous behavior measured in an open field/hole board, relative to sham and neocortically lesioned controls. These changes include increased locomotion, and decreased grooming frequency and rearing bout duration. When animals were injected with the dopamine (DA) agonist 3,4-dihydroxyphenylamino-2-imidazoline (DPI: 0.5, 1.0 and 5.0 pg) into the nucleus accumbens one week after surgery, the behavior of hippocampally lesioned rats was restored to levels not different from control lesioned rats. Haloperidol injections (0.05, 0.1 and 0.5 pg) into the caudate nucleus were not able to do this. Further, DPI injected into the caudate nucleus one month after surgery was also able to attenuate some of the effects of hippocampal damage. On the other hand, haloperidol injections into the nucleus accumbens did not influence behavior. The results are interpreted in terms of hippocampal lesion-induced alteration of a balance in basal ganglia DA systems, indicated by modified response to pharmacological intervention and which mediate the behavioral effects of the lesion.
INTRODUCTION Direct effects of brain d a m a g e are a c c o m p a n i e d by a wide variety of s e c o n d a r y neural changes which can include altered local metabolism20, m e m b r a n e characteristics 1, sprouting and r e c e p t o r sensitivity 19 (see ref. 22). In general, all of these types of changes involve a n u m b e r of n e u r o t r a n s m i t t e r systems, and all could be of behavioral significance. Particular secondary influences on behavior can be distinguished from the p r i m a r y effects of a lesion in part on the basis of the attenuation of behavioral deficits by specific pharmacologic interventions. The behavioral effects of h i p p o c a m p e c t o m y in rats include e n h a n c e d locomotion and deficits in spontaneous alternation, avoidance conditioning, and discrimination tasks (see refs. 10, 12 and 18). W e r e p o r t here evidence of certain secondary influences on behavior involving nucleus accumbens dopaminergic system changes in animals with bilateral h i p p o c a m p a l damage. Because the h i p p o c a m p u s is richly connected to divergent areas, there are m a n y sites for secondary neural changes after lesions that could contribute to the behavioral effects of h i p p o c a m p a l lesions. O n e 0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B .V.
possible site is the nucleus accumbens which receives monosynaptic projections from the ventral subiculum and CA125, although these fibers m a y originate in the entorhinal cortex as well 15. These h i p p o c a m p a l projections are restricted largely to the medial aspects of the nucleus accumbens t6. Behaviorally potent secondary changes in the d e n e r v a t e d nucleus accumbens after h i p p o c a m p a l d a m a g e are suggested in part by the similarity of changes in locomotion, exploration, attention, and avoidance conditioning after h i p p o c a m p a l lesions and after various mesolimbic manipulations9,26-28. Potential secondary neural changes in the nucleus accumbens may involve d o p a m i n e ( D A ) systems' since, for example, the lesions m a d e by Tassin et al. 26 and the drug injections by W a c h t e l et al. 28, both involve this neurotransmitter. In fact, there are increased levels and decreased utilization of D A in the nucleus accumbens, but not in the caudate, after hippocampal lesions, and these changes vary with length of recovery p e r i o d 23. In addition, hippocampally lesioned rats d e m o n s t r a t e altered responsiveness to systemic D A manipulations. F o r example, they are less sensitive to a m p h e t a m i n e 29,3° and to neurolep-
84 tics 8. On the other hand, animals with hippocampal lesions are m o r e sensitive than controls to haloperidol inhibition of ACTH-induced excessive grooming11. Since the nucleus accumbens and the hippocampus appear to be complexly related, the nature of the mesolimbic dopamine system must be considered. Cools and Van Rossum4, 5 postulated the existence of two functionally and behaviorally distinct D A receptor populations in the basal ganglia. According to their model, an excitatory D A receptor (DAe subtype) predominates in the caudate nucleus, and an inhibitory receptor (DAi subtype) predominates in nucleus accumbens. This distinction has been supported by electrophysiological 2, pharmacological 7, behavioraP, and neurochemical evidence24. It has been argued that a relative balance in activity between the DAe-caudate and DAi-accumbens subsystems is critical for determining behaviorS. For example, the exhibition of ACTH-induced excessive grooming in rats can be attenuated by blocking or stimulating either system, but the behavior may be affected less when both systems are manipulated to maintain a balanced activity6. We have previously demonstrated that certain behavioral changes usually found in animals with hippocampal lesions can be attenuated by injections of the putatively specific DAi agonist, 3,4-dihydr0xyphenylamino-2-imidazoline (DPI) into the nucleus accumbens 21. Those results can be interpreted in terms of the Cools and Van Rossum model: destruction of the hippocampus disrupts the balance of the basal ganglia DAe/DAi systems and this contributes to the behavioral deficits found after such damage. Stimulation of the DAi subsystem in the nucleus accumbens by DPI may return the balance between the DAe and DAi subsystems and consequently alleviate some behavioral deficits. If this is the case, then blockade of the DAe system by haloperidol, the appropriate antagonist to D A in the caudate, should also attenuate the effects of hippocampal damage by similarly restoring a balance. This experiment was undertaken to test this hypothesis. In addition, the present study extends the environmental and pharmacological procedures of a previous study in this laboratory21 in order to determine if the conclusions of that study can be extended to other procedures. In the earlier study, independent groups of rats were
tested at one of 3 postoperative periods in a circular apparatus after intra-accumbens injections of 10/~g of DPI. In the present study animals were repeatedly tested in an open field-hole board apparatus after injections of lower doses of DPI into nucleus accumbens. Other animals were tested in the same fashion under a range of doses of haloperidol injected into the caudate. Three weeks after completion of the experiment, we tested the specificity of the drugs to the particular area injected, by injecting DPI into the caudate and haloperidol into the nucleus accumbens of these same animals. MATERIALS AND METHODS Fifty-three male, Long-Evans strain hooded rats (Blue Spruce Farms, Altamont, NY), with a mean weight of 310.3 _+ 4.3 g at the time of surgery, were used. Animals were individually housed in a 12/12 light-dark cycle with lights on at 07.00 h. Food and water were provided ad libitum except for the 18 h preceding surgery, when they were deprived of both. Animals were anesthetized with a solution of chloral hydrate (90 mg/kg), atropine sulfate (0.75 mg/kg), and sodium pentabarbitol (56 mg/kg) and received either neocortical or neocortical plus hippocampal lesions, using aspiration as described by Isaacson and Woodruff 13. Other animals receiving sham lesions were anesthetized, their scalps opened, bone drilled, and scalp sutured. Also, each rat was implanted unilaterally with a stainless steel cannula (0.022" O.D.) aimed at either the dorsal limit of the nucleus accumbens (A 8.9 mm, V 4.8 mm, L 1.2 mm; K6nig and Klippep4), or at the caudate nucleus (A 8.9 ram, V 3.8 mm, L 2.0 mm). The placement of cannulae was counterbalanced between lesion groups and between right and left sides. The cannulae were cemented into place with dental acrylic anchored to the skull by two stainless steel machine screws. Stainless steel wire stylets were placed inside the cannulae with the tip extending down to one-half of the distance of the beveled tip. After surgery, the animals were returned to their home cages in the vivarium and again provided freely with food and water. Starting on the sixth day after surgery, the rats were observed for 4 consecutive days in an open field. At testing, the animals were taken from the vi-
85 varium and transported in their home cages to a holding room in a nearby testing suite. Each animal was subsequently restrained by hand, the stylet removed from its cannula, and an inner injection cannula inserted. The inner cannula was designed to extend 0.5 mm below the beveled tip of the outer sleeve to allow direct injection into the respective brain region. Injections of 1.0 ~1 physiological saline were infused over 60 s. The inner cannula remained in place for an additional 30 s before it was removed and the stylet replaced. The animal was then returned to its home cage for 9-11 min whereupon it was placed into the open field apparatus located in an adjacent, quiet and dimly lit (red light) observation room. Observations were made for 10 min via a closed circuit video system by trained observers located in a third room. The open field was white Plexiglas, measured 71 x 71 x 46 cm high, and the floor was marked into 16 squares (a 4 x 4 matrix). A hole (3.2 cm diameter) was placed in the center of each square. The open field was cleaned between animals with a dilute Lysol solution. The measures employed over the 10 min session were the following: Peripheral locomotion. Movement of greater than 50% of the animal's head and body into one of the 12 squares bordering the 4 walls of the apparatus. Central locomotion. Movement of greater than 50% of the animal's head and body into one of the 4 central squares. Rearing. A lifting of both forepaws off the ground by a vertical extension of the body. Rearing against walls was included in this measure. Peripheral hole pokes. Placement of the animal's nose into one of the holes in the 12 squares bordering the apparatus' walls. Central hole pokes. Placement of the animal's nose into one of the 4 centrally located holes. Grooming. This measure included face washing, paw, body, and genital licking, scratching, and tail preening. In addition to measuring the frequency of occurrence of each behavior, the duration of individual hole poke, grooming, and rearing episodes was measured. All scored behavior was encoded as audio signals which were decoded for digital analysis by a microprocessor which approximated measures of continuous behavior using 0.1 s time sampling. This scoring procedure allowed the derivation of frequen-
cy of occurrence (Frequency), total duration per session (Duration), mean duration per episode, and occurrence over the 10 rain session for each behavior. This procedure was repeated for 4 successive days except that the animals within each lesion group received an injection of a specified drug and concentration. The animals with a cannula placed into the nucleus accumbens received 0.5, 1.0, and 5.0/~g of 3,4 dihydroxyphenylamino-2-imidazoline (DPI) on days 7, 8, and 9 post-lesion, respectively. Rats with a cannula in the caudate were injected with 0.05, 0.1, and 0.5 ~g of haloperidol. All drugs were dissolved in 0.9% saline and infused as described above. The doses were selected on the basis of work by Cools and Van RossumS. They reported that the dose of each drug yielding a maximal physiological effect was within the ranges we used. At the completion of the experiment, the animals remained in their home cages in the colony room until day 35 after surgery. At that time, the procedure followed on the first day of testing was repeated (saline injection). The next day (day 36), each rat was tested again in the open field, but this time rats with cannulae in the nucleus accumbens were injected with 0.5 ~g haloperidol, whereas those with a cannula in the caudate received 5.0 ktg of DPI. Four weeks later, all rats were placed under deep barbiturate anesthesia and perfused intracardially with saline followed by 10% formalin solution. Brains were removed, fixed in formalin, and subsequently sectioned at 80ktm, saving every fifth section through the extent of the lesion and every section through the cannula tracts. Sections were stained with thionin, and cannula location and lesion suitability verified. RESULTS
Histology Of the 53 rats used in the study, 13 were eliminated from analysis due to a physical blockade of the cannulae that occurred before completion of testing, an inability to locate the cannula placement histologically, an unintended cannula placement, or an improper lesion (e.g. some hippocampal damage in the cortical group, or thalamic or superior colliculus damage in the hippocampal group). Representative acceptable cannula tip placements and minimum and maximum
86
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Fig. 1. A: representative cannula tip placements in the nucleus accumbens and the caudate. B: the minimum (solid area) and maximum (striped) extent of cortical lesion C: idem for hippocampal lesions.
87
acceptable lesions are illustrated in Fig. 1. The final number of subjects in each group were: sham: accumbens, 8; caudate, 6; cortical: accumbens, 9; caudate, 6; hippocampah accumbens, 6; caudate, 5.
Behavior In a pre-experiment test of inter-observer reliability, 6 observers scored videotaped animals with the result that there was less than 4% variation among them over all behaviors. (1) Locomotion. There was no difference at any day for any group in thigmotaxia (the proportion of peripheral locomotion to peripheral plus central locomotion), so total locomotor scores were considered in all analyses. As expected, rats with hippocampal lesions were more active than sham or cortically lesioned animals on the first test session, 6 days after surgery (F(1, 34) = 20.70, P < 0.001). There was no difference between animals receiving control saline injections into nucleus accumbens and the caudate (F < 1.0). Over the subsequent 3 sessions with drug injections there was great variability between subjects within all groups, and the apparent mean dose response curves presented in Fig. 2 may not adequately reflect the nature of the drugs' effects in all lesion groups. There was no systematic manner in which increasing doses of either D P I injected into the nucleus accumbens or of haloperidol into tl~e caudate in any lesion group affected locomotion. When the 3 drug days are considered together, only one of 6 groups (cortical lesion with caudate cannula) significantly deviates from an expected null hypothesis that the highest locomotor score for each rat is equally likely to follow any dose injected ()~2 = 7.00, d.f. = 2; P < 0.05). This suggests that there is no dose-dependent relationship between the effectiveness of a drug in reducing enhanced locomotor activity when group means are considered. On the other hand, the large within-subject variability may conceal a drug effect. Of the 18 tests given after D P I to the 6 hippocampal rats, 55% had scores within the range of the sham and cortical rats receiving saline in nucleus accumbens, and 27% were below the median of the controls. Of the 15 haloperidol-into-caudate tests (5 hippocampal rats at 3 doses), 53% of scores overlapped the levels reached by control lesioned animals, but only 6.67% (1/15) were as low as the median of the
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Fig. 2. Mean total locomotion scores for rats measured in open field/hole board for 4 consecutive days starting day 6 after surgery. The key denotes lesion and where injections were given. On day 6 all rats received saline (1.0~1) into their respective injection sites. Caudate-injected animals received 0.05, 0.1, and 0.5/~g//A haoperidol on days 7, 8 and 9, respectively. Accumbens-injected animals received 0.5, 1.0 and 5.0 #g/gl of DPI on days 7, 8 and 9, respectively. control animals. This suggests that there was at least one dose of drug, especially in the DPI-accumbens group, which was effective in any given animal. Fig. 3 illustrates a comparison of the median total locomotor scores in saline-injected animals in each lesion group with the median of the lowest score (i.e. presumably at the most effective dose) for each rat across the 3 drug days. That is, for the comparison within each lesion group, the saline score was measured on day 6 and the drug score (haloperidol or DPI in caudate or nucleus accumbens, respectively) is the lowest of the three scores from days 7, 8 and 9. This would essentially bias the analysis to maximize the likelihood of demonstrating a drug effect despite individual differences. Sign tests for correlated samples show that the difference between the medians is significant only for the cortical and hippocampal lesion rats given D P I into the nucleus accumbens. Haloperidol injections into the caudate were not effective in significantly attenuating the increase in locomotor activity after the lesion. Thus, even when large individual differences in drug responsiveness are considered, only D P I injection into the nucleus ac-
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Over the drug administration regime of the next 3 days, all animals tended to rear less frequently (F(3, 102) = 4.00, P < 0.01). Animals receiving D P I into the nucleus accumbens had lower overall rearing frequencies than those injected with haloperidol into the caudate (F(1, 34) = 12.88; P < 0.001). Further, hippocampal lesioned rats maintained an overall lower number of rearing bouts than cortical lesioned rats (F(2, 34) = 6.06; P < 0.01). There were no significant interactions and no one group showed a significant change over days (largest F(3, 24) = 2.21; P <
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Fig. 3. Median total locomotion scores. A n i m a l s injected with saline (S) were scored on day 6. The m e d m n s for the animals injected with haloperidol (H) into caudate or D P I (D) into nucleus accumbens were derived from the lowest scores for each animal on days 7, 8 and 9. T h e * indicates significantly different from saline injected rats within same lesion group (sign test; P < 0.05).
cumbens attenuated the effects of hippocampectomy on locomotion. But this is not only because of the type of analysis since this same conclusion is drawn when (a) the dose of the median response across the 3 drug days is analyzed; and when (b) an analysis of variance about the means (rather than medians) of the lowest scored days is done. In this case, there is a significant drug x lesion interaction (F(2, 34) = 4.14, P < 0.05) consistent with the conclusion drawn above. One month later, the lesioned animals were still more active than controls (F(2, 34) = 6.69, P < 0.01; Fig. 4). When 5.0/~g of D P I was injected into the caudate, or 0.5 ~tg of haloperidol was injected into the nucleus accumbens the next day, there was a significant three-way interaction (F(2, 34) = 6.33; P < 0.01) which reflected the reduction of locomotor scores only in rats with hippocampal lesions that had received D P I injections into caudate (F(1, 4) = 12.12, P < 0.05). No other injections were effective. (2) Rearing. There was no difference between lesion groups in the frequency of rearing bouts in the open field after saline injections 6 days after surgery. There was, however, a reduction in the mean duration per rearing bout in hippocampal animals (F(2, 34) = 3.48, P < 0.05).
There was no effect of drug at any day in any lesion group on mean rearing duration. The reduced duration seen in hippocampal rats on day 6 is maintained throughout (F(2, 34) = 5.02; P < 0.05). One month later when the rats were again injected with saline, there were no differences between groups in rearing bout frequency, but hippocampal lesioned rats still reared for a significantly shorter time per bout (F(2, 34) = 3.57; P < 0.05). Neither D P I into caudate nor haloperidol into accumbens changed rearing frequency or duration on the next day. (3) Hole poking. All animals poked their heads into holes about the same number of times on day 6 DPI INTO CAUDATE [] z
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Fig. 4. Mean total locomotion scores for animals m e a s u r e d one m o n t h after surgery. O n day 35, all animals were injected with saline (S), and on day 36, rats received either haloperidol (H; 0.5/~g//~l) into nucleus accumbens or DPI (D; 5.0/~g/~tl) into caudate. The * indicates significantly different from saline injected group (F(1, 4) = 12.12, P < 0.05).
89 following saline injections. However, when haloperidol was injected into the caudate of cortically lesioned animals there was an immediate increase in the frequency of hole pokes observed, but primarily when the two highest doses were used (F(6, 102) = 2.51; P < 0.05). This 3-way interaction is accountable entirely by the cortically lesioned haloperidol group since it is the only one to change hole poke frequency significantly over days (F(3, 15) = 8.11; P < 0.01) although there was a significant overall increase in hole poke frequency over days as well (F(3, 102) = 4.08; P < 0.01). In addition, the haloperidolinjected groups showed overall higher frequencies of hole pokes than DPI-injected animals (F(1, 34) = 4.41; P < 0.05). One month later when saline was again injected into both areas, there appeared to be long-term effects of the haloperidol injections since hole poke frequency scores were still higher in animals that had been injected with haloperidol (F(1, 34) = 4.25; P < 0.05), with the largest difference apparently in the cortical and hippocampal lesioned groups. Administration of DPI the next day reduced, but did not eliminate, this effect. Mean hole poke durations per bout for hippocampally lesioned rats were shorter than for control lesioned rats on day 6 (F(2, 34) = 3.80; P < 0.05). The drug administration over the next 3 days did not systematically influence mean duration of hole pokes, and the lesion effect remained as on day 6 throughout (F(2, 34) = 3.49; P < 0.05). One month later hippocampal animals injected with saline still had shorter mean hole poke durations than controls (F(2, 34) = 4.57; P < 0.05). There was a significant drug x lesion interaction (F(2, 34) = 4.06; P < 0.05) which reflected an increase in the mean length of a hole poke for hippocampal animals receiving DPI into the accumbens (F(1, 4) = 8.66; P < 0.05). No other group significantly changed mean hole poke duration after drug injection on day 36. (4) Grooming. Six days after surgery there were no differences between groups in grooming bout frequency or in duration per bout following saline injections. With the subsequent drug injections, there was an overall increase in the frequency of grooming bouts in all groups (F(3, 102) = 7.44; P < 0.001). Fig. 5 illustrates the changes in grooming frequency over days. The apparent difference on day 8 in the
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DAYS POST- LESION
Fig. 5. Mean grooming frequency scores. All animals received saline injection (1.0 ktl) on day 6 and increasing doses of DPI into nucleus accumbens (0.5, 1.0 and 5.0 ~tg/~l) or of haloperidol into caudate (0.05, 0.1 and 0.5/~g/#1) on days 7, 8 and 9, respectively. groups given DPI into nucleus accumbens is not significant, again attesting to large individual differences. Haloperidol injections into caudate systematically increased the number of bouts in shams (F(3, 15) = 3.87; P < 0.05), although hippocampally lesioned rats did not show this effect. On the other hand, there were no reliable changes in mean grooming bout duration over the 4 testing days. The saline injection given 35 days after surgery did not alter grooming frequency, but the mean duration per bout were longer in animals receiving caudate injections of saline than in rats with nucleus accumbens injections (F(1, 34) = 4.98; P < 0.05), and drug injections on day 36 do not change this difference. Grooming frequency was not altered by any injection although DPI into caudate does maintain the elongated grooming bouts seen after earlier haloperidol (days 8 and 9) or saline (day 35) injections into caudate (F(1, 34) = 5.74; P < 0.05). DISCUSSION In a previous study we demonstrated that the intraaccumbens injection of 10 ktg of 3,4-dihydroxyphenylamino-2-imidazoline (DPI) was effective in attenuating deficits in locomotion, exploration, and grooming found in rats with large bilateral destruction of the hippocampus 21. The present study supports those conclusions since DPI in a dose range of 1-5 ~g injected into the nucleus accumbens can also lower the enhanced locomotor scores of lesioned ani-
90 mals to near-control levels, although there is no clear dose-response relationship. These supporting data come in a different testing situation and under a repeated injection regime, in contrast to the independent groups in the Reinstein et al. 21 study. However, whereas we expected that haloperidol injections into the caudate would also be effective, locomotor deficits were not attenuated by the dose used. This may mean that the balance between nucleus accumbens DAi receptor systems and caudate DAe systems is not critical to behavior in the way Cools and Van Rossum 5 propose. Alternatively, the hippocampal lesion may disrupt these systems in such a way that a balance between the D A e and DAi systems cannot be achieved bidirectionally. For example, a lesion could affect the sensitivity of the receptor subtypes to the drugs used in this study 19. Also, endogenous in vitro membrane phosphorylation is altered in both nucleus accumbens and caudate after hippocampal lesions 1. In fact, when DPI was injected into the caudate, it was effective in attenuating deficits in locomotion and mean hole poke duration in hippocampally lesoned rats. This is significant since the caudate is an area thought to have a low concentration of DAi receptors4, 5, and that has been shown to be insensitive to DPI in normal animals 6.24. However, Nauta 16 has shown that the D A projections from the ventral tegmental area innervate the mediolateral and medial anterodorsal caudate in the rat. This suggests that caudate D A systems may be indirectly (or secondarily) influenced by hippocampal lesions by way of a limbic-ventral tegmental connection16,17. It does not seem that the basal ganglia dopamine systems function in a 'balanced fashion' in mediating other behaviors affected by the lesions. As expected on the basis of previous results, rearing durations were shorter for the hippocampally lesioned animals, but this was not affected by any drug manipulation at any time. This is in contrast to earlier findings 21, although that study measured animals in a smaller apparatus and a higher dose of DPI was used. The changes in hole poke behavior are noteworthy. While there was no initial deficit in hole poke frequency due to either lesion, haloperidol induced a tremendous increase in hole poke frequency in rats receiving cortical lesions. The effect is of special interest because of its specificity and because it is a
long-term change. Also, as noted above, the restoration of normal hole poke durations in hippocampal animals by intra-caudate injections of DPI suggests again that caudate D A systems may be altered by the lesion. In the present study we failed to show any change in grooming after brain damage. However, the effects of hippocampal lesions on grooming in 'novelty' situations are not large, and longer observation periods may be required to detect the lesion effects. We did find that hippocampal animals do not increase the frequency of grooming bouts when injected with haloperidol into caudate as much as shams do one week after surgery. One month after surgery this increase persists and is not changed by DPI injections. We think that the absence of clear dose-response curves for locomotor and grooming effects suggests an enhanced variability in behavior induced by the lesion. If the lesion were removing a modulatory influence from the nucleus accumbens, the ventral tegmental area, or any other area, then the activity in those areas could fluctuate more in a given environmental situation or under a pharmacological challenge. In repeated treatment designs such as this, the contributions of repeated testing and increasing drug doses are confounded. Also, there is the possibility of carry-over effects which could make the dose relationships difficult to discern. Alternatively, the relatively lower doses of DPI we used could be near a threshold for behavioral effect. If so, this would explain the enhanced variability in performance. In conclusion, secondary dopaminergic changes in nucleus accumbens, perhaps involving DAi systems, seem to mediate an enhanced locomotor activity in hippocampally lesioned rats. In the present situation, rearing and hole poke deficits are not attenuated by DA manipulations, whereas grooming was not impaired by the lesion, although the increased grooming in control animals after the injection of haloperidol is not seen after hippocampal lesions. The predicted functional balance between basal ganglia DA systems did not seem be critical here since manipulations of D A e (caudate) and Dai (nucleus accumbens) in opposite directions did not yield similar effects. It may be that the lesion prevents a demonstration of the balanced systems as we hypothesized it. On the other hand, hippocampal lesions do
91 s e e m to alter b o t h D A e and D A i systems. First, t h e
inability of h a l o p e r i d o l to i n c r e a s e g r o o m i n g in hip-
l e s i o n e d animals are m o r e sensitive t h a n c o n t r o l s to
p o c a m p a l s . T h e s e results s u p p o r t t h e n o t i o n that the
D P I in nucleus a c c u m b e n s . S e c o n d , it a p p e a r s that
b e h a v i o r a l s e q u e l a e of h i p p o c a m p a l lesions are due
the lesion changes the specificity of p r e s u m e d cau-
in part to s e c o n d a r y c h a n g e s in m e s o l i m b i c d o p -
d a t e D A e system to the drugs used. This is s e e n in the
a m i n e r g i c systems.
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