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A water-maze discrimination learning deficit in the rat following lesion of the habenula
Physiology & Behavior, Vol. 49, pp. 819-822. o Pergamon Press plc, 1991. Printed in the U.S.A.
0031-9384/91 $3.00 + .00
BRIEF COMMUNICATION
A Water-Maze Discrimination Learning Deficit in the Rat Following Lesion of the Habenula EVERARD W. THORNTON 1 AND CLAIRE DAVIES
Department o f Psychology, University o f Liverpool, England R e c e i v e d 5 O c t o b e r 1989
THORNTON, E. W. AND C. DAVIES. A water-maze discrimination learning deficit in the rat following lesion of the habenula. PHYSIOL BEHAV 49(4) 819-822, 1991.--Although the habenula occupies a potentially important link between forebrain and midbrain, lesion of the complex produces little effect in most standard behavioral paradigms. More recently, it has been shown that such lesions may impair the ability to initiate or switch responses appropriate to environmental contingencies but only under demanding conditions. Although such deficits have been described as response failures, they could equally well be attributed to restricted attentional mechanisms. The present study was designed to further substantiate a role for the habenula in acquisition of adaptive behavior under demanding conditions and to examine the possible contribution of attentional failure. The initial response preference to 'escape' onto platforms situated in two chambers at the distal end of a water tank was established for groups of lesioned and sham-operated rats. Rats were subsequently trained in discrete trials to escape by choosing the side of the nonpreferred chamber. During training the choice of escape chamber was cued by distracting black or white visual stimuli displayed on the tank sides and above the entrances to the chamber. These were moved over trials so as to be nonpredictive of the appropriate escape position. Lesioned animals were significantly impaired in the acquisition of this positional discrimination. Analysis of response times suggested that both lesion and control animals were attending to the irrelevant visual cue. The results confirm a behavioral inflexibility following lesion of the habenula and suggest that this deficit cannot be explained simply in terms of a failure to attend to environmental cues. The lesion deficit also could not be attributed to a response perseveration. Habenula
Water maze
Attention
Discrimination learning
THE habenula has been described as an important relay linking forebrain to limbic midbrain structures, providing an alternative more dorsal route from the much studied medial forebrain bundle (11). Despite the well documented anatomical connections of the habenula and demonstrated physiological response changes following stimulation or lesion of the habenula (5, 12, 13), a functional role for the habenula in behavior has been less well documented and more difficult to specify. Indeed, until very recently, the most extensive study to define such a contribution concluded that there was little functional consequence of habenular damage (13), whilst a review on the habenula indicated the difficulties of integrating what positive effects have been reported (5). More recent findings demonstrate an inability to initiate or change response strategies appropriate to environmental contingencies under demanding conditions which may loosely be described as stressful. For example, although lesioned animals could learn a simple one-way active avoidance response, animals were
Rat
impaired in such learning when either shock levels were raised or when the motoric effort required for the specified avoidance response was increased (6). This supported an earlier study which showed that following a period of inescapable swimming in a confined space, lesioned rats were incapable of escaping when subsequently offered such an opportunity via a rope lowered to the water (8). Parallels between swim test responses and those of avoidance learning have been explicitly linked in learned helplessness models of depression and it is of relevance, therefore, that the failure to change a response strategy appropriate to environmental contingencies following habenular lesions appears most evident under more demanding or stressful conditions. Deficits in these paradigms, described in terms of an inflexibility of motor responses, are consistent with the interpretation that changes are effected through primary efferents to dopaminergic systems (6,10). Further indirect support for this notion is found in the demonstration of a change in corticofrontal dopamine turn-
1Requests for reprints should be addressed to Dr. E. W. Thornton, Department of Psychology, Eleanor Rathbone Building, University of Liverpool, P.O. Box 147, Liverpool L69 3BX, England.
819
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THORNTON AND DAVIES
over during stress following lesion of the habenula (3). However, there is evidence to suggest that deficits in attentional mechanisms may also be evident following manipulation of dopaminergic systems or, indeed, lesion of several structures providing primary afferents to the habenula. Since the behavioral deficits evident under stress following lesion of the habenula require the initiation or change of response to an environmental cue, an alternative explanation couched in attentional rather than response terms should be considered. Unfortunately, there have been few studies examining a role for the habenula in attention in general or, more specifically, for attentional mechanisms under stress. Data from this laboratory have shown no effect of lesion of the habenula on orientation to novel stimuli or the distraction of ongoing operant responses by novel stimuli. Moreover, the acquisition of simple visual discriminations through either successive or simultaneous presentation of stimuli within both the Y-maze, a water maze or the conventional two-lever operant chamber are not influenced by lesion of the habenula (12). Such negative results have also been reported following lesion of limbic structures with afferents to the habenula. However, in contrast to other limbic lesions, lesions of the habenula also fail to influence reversal learning in such paradigms. The present study was carried out to elaborate the proposal that behavioral deficits may be observed following lesion of the habenula in alternative demanding conditions to those so far reported, and to determine more effectively whether any deficit is consistent with the failure to attend to salient visual cues. METHOD
Animals and Surgery Young male Wistar rats weighing 260 _ 8 g were individually housed at a temperature of 23-+2°(] in a 12-h light/dark cycle (light on 8:00 a . m . - - o f f 8:00 p.m.), given food and water ad lib, and assigned to lesion or sham operative procedures on a counterbalanced basis according to body weight. Animals were pretreated with atropine sulphate, 10 mg/kg, anaesthetised with sodium pentobarhital (50 mg/kg, IP) and subsequently positioned in a Kopf stereotaxic frame. Bilateral lesions were placed in the habenula according to coordinates derived from the atlas of Pellegrino and Cushman (4): A-P 3.8; L _+0.6; DV 0.7 above stereotaxic zero, by passage of radio frequency current from a Grass Model LM4 lesion maker through a stainless steel insulated needle with tip exposure of 0.4 mm. The current was gradually increased up to 10 mA over a period of l0 s until coagulation was attained. Sham-operated animals underwent similar procedures without delivery of current and with the electrode penetrating only through the cortex to terminate above the habenula. After removal of the electrode the wound was closed by suture and treated with an antibiotic powder. The normal procedure was to carry out one lesion and one sham operation on a single day. However, the process of attrition of animals from both groups (see the Results section) necessitated departure from this strategy for two sham-operated and two lesioned animals. For each animal, behavioral testing was carried out during the light phase of the cycle one week following surgery, after which the lesioned animals were sacrificed with pentobarbital, perfused, and their brains removed and stored in sucrose formaldehyde. The brains were subsequently frozen and sliced in 50 Ixm sections through the area of destruction. Sections were mounted on slides and examined to verify the extent of the lesion.
Behavioral Test Apparatus and Procedure The water-maze discrimination apparatus had similar parameters to that described in detail by Lee and Maier (2). A glass tank
(110 L × 30 W × 56 H cm) was filled to a depth of 30 cm water maintained at 34°C and made opaque by the addition of nontoxic white paint. The last 10 cm of the tank was divided into two chambers each of which was entranced via a black or white Plexiglas panel (13 W × 17 H cm), suspended 2 cm above the water surface. On each side wall was placed a larger black or white panel (30 W × 30 H cm), with the side for each shade conforming to the placement of the panel 'guarding' the end chambers. A starting platform, 9 cm square, with the level just above the surface of the water was placed at the end of the tank distant from the two chambers. During trials animals could climb onto an escape platform, also 9 cm square, with its surface 1.5 cm below the water and not visible, placed as appropriate into the end chambers. To minimize extra-maze cues, the apparatus was surrounded by a plain pastel sheet under a homogeneous ceiling. All behavioral testing was carried out by one experimenter who stood directly behind the end of the maze distal to the chambers throughout all trials. Each trial commenced by submersion of the start platform to the bottom of the tank with the rat required to swim to the other end of the tank, pass under the suspended stimulus card, and clamber onto the escape platform to terminate the trial. The time between submersion of the start platform and an escape with all four legs on the platform was recorded as the response time. Rats were allowed 45 s on the escape platform before being returned to the start platform facing the goal end of the apparatus and left for a period of 20 s prior to submersion of the platform and the start of the next trial. If the rat failed to reach the platform within a period of 90 s the trial was terminated, the rat removed and replaced on the start platform for 20 s prior to commencement of the next trial. In this instance, a response time of 90 s was recorded. The first 15 trials served to determine a position/shade preference. An escape platform was provided in both chambers and the preference defined as the more frequently chosen chamber. Commencing on the next trial, Trial 16, an escape platform was provided only on the nonpreferred side. If an incorrect choice was made the rat had to swim out of the inappropriate chamber into the compartment with the platform to complete the trial and in this instance an error was recorded. Animals were trained to a criterion of 8 trials in succession without an error (i.e., without recourse to the initial preferred side), or for a maximum of 40 discrimination trials. During these 40 trials the black and white stimuli were changed from one side of the tank to the other in a preprogrammed but randomised manner such that over the maximum number of trials there would be a balance between side and shade. The specific changes in position of the black/white shade cue was varied for each animal: For half the animals in each surgical treatment group, Group A, the switch of position on Trial 16 was associated with an incompatible change in the visual cue (e.g., a rat showing a preference for a left/black choice in the first 15 trials had to switch on Trial 16 to a right/white choice). For the remaining animals in each group, Group B, a shift of position but not visual cue was required on this trial (e.g., for a similar initial preference, it would be right/ black correct choice on Trial 16). For all animals, there was a change in the position of the visual cue between Trials 16 and 17. The reasoning behind these procedures was that if any animal in the initial, well-established preference had related this response to the visual cue, then rats in Group B would have an advantage over rats in Group A on Trial 16. For Group B, the visual stimulus and not position cue was predictive of escape at the time when the environment first changes for the animal. The more critical trial for the present study is the subsequent trial, Trial 17. For a rat in Group B any attention to the visual cue would be
DISCRIMINATION LEARNING FOLLOWING HABENULAR LESIONS
strengthened since the visual cue had been predictive of escape on the previous trial in which a change of positional response was required for the first time in order to escape. The visual cue should, therefore, even more strongly determine the response choice. In contrast, for rats in Group A the visual cue had been nonpredictive of escape and attention to that cue should diminish. Necessarily, over repeated trials in the acquisition of the posidon discrimination, this attention to the visual cue would be counterproductive and animals in Group B, who should be more 'switched-in' to the visual dimension, should be slower and make more errors in reaching the criterion for successful learning. The procedure required, therefore, a switch in position preference where attention to an external visual cue, which may initially have been appropriate in determining an adaptive response choice, would over trials interfere with the positional discrimination learning.
821
M e a n N u m b e r of T r i a l s to Criterion
B
RESULTS
Histology Histological verification of damage from frozen sections was performed by one experimenter without knowledge of behavioral outcomes and revealed consistent lesion size and destruction of both the medial and lateral habenula. Lesions showed no extension anteriorly into the stria medullaris or dorsomedial thalamus although there was some sparing of the anterior habenula. Misplacement of lesion for two animals necessitated two further operations with successful targetting of the lesion. The typical extent of the lesion was similar to that we have reported in previous studies using the same procedures and lesion parameters (6, 8, 9). Death of one sham-operated rat prior to testing also necessitated a substitute animal. One further sham-operated animal failed to swim satisfactorily past the suspended panel during the initial 15 trials and this animal was abandoned and a further operation performed.
B
A
A
Sham Operates
Mean Number of Errors
u m m m m m m m m m m m m m u m m m m m u m m m m m m m m m m
Lesion
B
A
A
i m
m
mm m m m m m m mm
I1" m
B
m "
Sham Operates
Lesion
Behavioral Analysis
FIG. 1. Presents the mean ( + SE) number of trials required to attain the criterion of 8 successive correct choices, together with the group mean ( + SE) number of errors made in attaining this criterion, for both habenular lesioned and sham-operated rats. Each of the two major treatment groups was subdivided on the basis of whether the correct switch of position choice on Trial 16, immediately after testing for the initial position preference, was also associated with a change of the visual cue. Thus, A = the correct switch of position response was also associated with a switch of shade and B = the correct switch of position response was associated with the same choice of shade as the initial preference.
The behavioral analyses were based on twelve lesion and twelve sham-operated controls. The left/black side was the preferred choice for four animals in each of the surgical groups during the initial 15 trials. The remaining rats showed preference for the right/white side. For half the animals in each of these groups the initial switch of position preference required on Trial 16 was also accompanied by a shift in the shade (from black to white or vice-versa). For remaining animals the required change of position on Trial 16 was compatible with the initial shade to which they had swum as a preference on the previous 15 trials. All animals showed a choice for their preferred side on a minimum of 10 of the initial 15 trials and at least two trials prior to position discrimination trials commencing on Trial 16. A two factor ANOVA of response times for Trials 13-15 revealed no effects of lesion group or trials (F values>l). The mean number of trials to criterion and the number of errors made prior to attainment of the criterion for each of the groups are presented in Fig. 1. Analyses of these data were undertaken by two-factor ANOVA with square root transformation applied for the nonhomogeneity of variance resulting from asymptotic scores for five lesioned animals. The analyses confirmed that lesioned rats made significantly more errors, F(1,20) = 10.3, p<0.01, and required significantly more trials, F(1,20)= 14, p<0.01, for acquisition of the discrimination. However, there were no significant, or near significant, interactions and effects related to the second factor of visual cue compatability for either dependent variable, although of the five lesioned animals failing to attain criterion over training, four were from Group B. All the sham
operates attained the criterion within the 40 trials. Evidence relating to the effects of the irrelevant visual cue on performance may be seen from the group mean response times for Trials 16 and 17 (Table 1). Statistical ANOVA of the data from Trial 16 confirm the longer response time for lesioned animals, F(1,20) = 4 . 8 , p<0.05, indicated in the previous analysis of trials to criterion. Although times on Trial 16 were longer for Group A as predicted, neither this second factor nor the interaction approached significance (F values>l). These data suggest that the initial preference over the first 15 trials for both lesion and control rats was not necessarily determined by the visual cue but may indicate a more direct response bias. A similar analysis for Trial 17 again showed the significant effect of the lesion, F = 8.8, p < 0 . 0 1 , but, in contrast to Trial 16, a significant effect of the visual cue factor [i.e., a Group A/Group B effect, F = 7.3, p<0.05] together with a significant interaction, F = 8 . 1 , p<0.01. Further analysis of the interaction using the Tukey HSD statistic confLrmed that the mean response time for lesion Group B was significantly different from each of the other three groups (p's<0.01), with no other comparison reaching significance. It would seem, therefore, that lesioned animals on Trial 17 were most impaired when, having escaped safely through the nonpreferred position in conjunction with the previously associated visual cue on Trial 16 (the first discrimination trial), they then on the next trial had to swim to the previously nonpreferred position with the preferred visual cue switched to the incorrect choice side.
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THORNTON AND DAVIES
TABLE 1 THE GROUP MEAN (_ SE) OF RESPONSE TIMES FOR LESIONED AND SHAM-OPERATED ANIMALSFOR TRIALS 16 AND 17 (i.e., THE FIRST TWO DISCRIMINATION TRIALS) Sham Operates A B Trial 16 Trial 17
17.5 -+ 4.8 7.0 -+ 3.5
13.3 _ 4.6 5.8 _+ 0.9
Lesion A
B
39.8 - 12.1 8.0 +_ 3.2
31.8 -- 15.6 54.0 _ 19.9
A: The switch of position response on Trial 16 was also associated with a switch of shade. B: The switch of position response on Trial 16 was associated with the same choice of shade as in the initial preference.
These analyses indicate that, somewhat paradoxically, lesioned rats were more influenced by the salient environmental visual cue than sham-operated controls. They attend more readily to extrinsic visual stimuli in the face of the changed environment on Trial 16 with this effect most readily seen as an interference to the required position change on the subsequent Trial 17 when their 'choice' is an approach to the now negative cue. DISCUSSION These data show an impairment of a rather complex discrimination acquisition following lesion of the habenular complex. This finding extends and is consistent with our previous suggestion that behavioral deficits following such lesions are apparent only under demanding conditions. These conditions require a switch from a prepotent response to optimise environmental contingencies (8). We have loosely labelled these conditions as 'stressful' and have argued that they invoke an activation of midbrain monoaminergic mechanisms in control animals. However, the physiological systems which are activated by the diverse test conditions we have utilised in demonstrating a contribution through the habenula have yet to be specified. Nevertheless, the present data provide further support for a functional contribution of the habenula. The deficit in discrimination acquisition in the presence of an irrelevant, distracting stimulus cue contrasts with the absence of effects of lesions of the habenula in more typical operant lever press, swim maze or Y-maze, discriminations with both simulta-
neous or successive presentation of relevant visual cues (12). The present study does not allow resolution as to whether it is the effects of the swim component, the irrelevant distracting visual cue, or their interaction which provides the conditions necessary to involve the habenular relay. However, the absence of any lesioninduced effects during Trials 13-15 indicates an absence of effect of the lesion on swimming alone and this is supported by our previous studies demonstrating no effect of lesions of the habenula on simple visual discrimination learning to black/white cues in the same test apparatus. Moreover, the data on performance during the initial preference testing support other studies showing an absence of effect of habenular lesions on motoric ability (1,7). The intrinsic requirement of the present test situation and an examination of both the pattern of response change and response latencies on Trials 16 and 17 also indicate that the impaired acquisition of the discrimination is not brought about by the simple perseveration of response that we have suggested might account for other data following lesion of the habenula (9). The present results are consistent with evidence suggesting that the habenula is involved in the ability of animals to switch response strategies under stress. Previous response deficits, such as the failure to utilise an escape route during a swim test, are interpretable in terms of a simple effect of reduced attention to 'external' cues. In the present study, an examination of the response times on Trials 16 and 17 convincingly demonstrates that lesion of the habenula does not result in a decreased attention to external cues when a response switch is demanded by the external contingencies. The significantly extended response time for lesioned animals in Group B on Trial 17 indicates that lesioned animals were attending to the visual cue on at least the two critical trials following the demand for a switch of response. Indeed, this increased attention to the irrelevant visual cue would contribute to the acquisition deficit. Neither a simple failure to attend nor the more paradoxical increase in attention to such stimulus dimension can explain the behavioral deficits reported in our previous studies (6, 8, 10). One possible alternative is that the present deficit is construed as a failure to switch from extrinsic to intrinsic stimulus control. A similar but inverse failure to switch from intrinsic to extrinsic stimulus control could be invoked to describe the escape deficit reported in our previous studies of swim tests (8,10). ACKNOWLEDGEMENT This work was supported by a Project Grant from the Medical Research Council of Great Britain.
REFERENCES 1. Lee, H, Y.; Huang, S. L. Role of the lateral habenula in the regulation of exploratory behavior and its relationship to stress in rats. Behay. Brain Res. 30:265-271; 1988. 2. Lee, K. L.; Maler, S. F. Inescapable shock and attention to internal versus external cues in a water discrimination escape task. J. Exp. Psychol. [Anita. Behav.] 14:302-310; 1988. 3. Lisoprawski, A.; Herve, D.; Blanc, G.; Glowinski, J.; Tassin, J. P. Selective activation of the mesocortico-frontal dopaminergic neurons induced by lesion of the habenula in rat. Brain Res. 183:229-234; 1980. 4. Pellegrino, L. J.; Cushman, A. S. A stereotaxic atlas of the rat brain. New York: Appleton-Century-Crofts; 1967. 5. Sutherland, R. J. The dorsal diencephalic conduction system: A review of the anatomy and function of the habenula complex. Neurosci. Biobehav, Rev. 6:1-13; 1982. 6. Thornton, E. W.; Bradbury, G. E. Effort and stress influence the effect of lesion of the habenula complex in one-way active avoidance learning. Physiol. Behav. 45:929-934; 1989. 7. Thornton, E. W.; Bradbury, G. E.; Davies, C. Increased immobility in an automated forced swimming test following lesion of the
8. 9.
10. 11. 12. 13.
habenula in rats: Absence of evidence for a contribution from motor impairment. Behav. Neurosci. 104:37--43; 1990. Thornton, E. W.; Evans, J. C. The role of the habenular nuclei in the selection of behavioural strategies. Physiol. Psychol. 10:361-367; 1982. Thornton, E. W.; Evans, J. A. C. The effects of lesions of the habenula nucleus on lever press behaviour during a tandem operant schedule with contrasting response requirements. Behav. Brain Res. 12:327-334; 1985. Thornton, E. W.; Evans, J. A. C.; Harris, C. Attenuated response to nomifensine in rats during a swim test following lesion of the habenula complex. Psychopharmacology (Berlin) 87:81-85; 1985. Wang, R. Y.; Aghajanian, G. K. Physiological evidence for habenula as a link between forebrain and midbrain raphe. Science 197:8991; 1977. Wickens, A. P. Neurochemical and behavioural functions of the habenula. Unpublished doctoral dissertation, University of Liverpool, Liverpool, England; 1988. Wirtshafter, R. D. Behavioral effects of habenular damage. Unpublished doctoral dissertation, University of lllinois, Chicago; 1982.
0031-9384/91 $3.00 + .00
BRIEF COMMUNICATION
A Water-Maze Discrimination Learning Deficit in the Rat Following Lesion of the Habenula EVERARD W. THORNTON 1 AND CLAIRE DAVIES
Department o f Psychology, University o f Liverpool, England R e c e i v e d 5 O c t o b e r 1989
THORNTON, E. W. AND C. DAVIES. A water-maze discrimination learning deficit in the rat following lesion of the habenula. PHYSIOL BEHAV 49(4) 819-822, 1991.--Although the habenula occupies a potentially important link between forebrain and midbrain, lesion of the complex produces little effect in most standard behavioral paradigms. More recently, it has been shown that such lesions may impair the ability to initiate or switch responses appropriate to environmental contingencies but only under demanding conditions. Although such deficits have been described as response failures, they could equally well be attributed to restricted attentional mechanisms. The present study was designed to further substantiate a role for the habenula in acquisition of adaptive behavior under demanding conditions and to examine the possible contribution of attentional failure. The initial response preference to 'escape' onto platforms situated in two chambers at the distal end of a water tank was established for groups of lesioned and sham-operated rats. Rats were subsequently trained in discrete trials to escape by choosing the side of the nonpreferred chamber. During training the choice of escape chamber was cued by distracting black or white visual stimuli displayed on the tank sides and above the entrances to the chamber. These were moved over trials so as to be nonpredictive of the appropriate escape position. Lesioned animals were significantly impaired in the acquisition of this positional discrimination. Analysis of response times suggested that both lesion and control animals were attending to the irrelevant visual cue. The results confirm a behavioral inflexibility following lesion of the habenula and suggest that this deficit cannot be explained simply in terms of a failure to attend to environmental cues. The lesion deficit also could not be attributed to a response perseveration. Habenula
Water maze
Attention
Discrimination learning
THE habenula has been described as an important relay linking forebrain to limbic midbrain structures, providing an alternative more dorsal route from the much studied medial forebrain bundle (11). Despite the well documented anatomical connections of the habenula and demonstrated physiological response changes following stimulation or lesion of the habenula (5, 12, 13), a functional role for the habenula in behavior has been less well documented and more difficult to specify. Indeed, until very recently, the most extensive study to define such a contribution concluded that there was little functional consequence of habenular damage (13), whilst a review on the habenula indicated the difficulties of integrating what positive effects have been reported (5). More recent findings demonstrate an inability to initiate or change response strategies appropriate to environmental contingencies under demanding conditions which may loosely be described as stressful. For example, although lesioned animals could learn a simple one-way active avoidance response, animals were
Rat
impaired in such learning when either shock levels were raised or when the motoric effort required for the specified avoidance response was increased (6). This supported an earlier study which showed that following a period of inescapable swimming in a confined space, lesioned rats were incapable of escaping when subsequently offered such an opportunity via a rope lowered to the water (8). Parallels between swim test responses and those of avoidance learning have been explicitly linked in learned helplessness models of depression and it is of relevance, therefore, that the failure to change a response strategy appropriate to environmental contingencies following habenular lesions appears most evident under more demanding or stressful conditions. Deficits in these paradigms, described in terms of an inflexibility of motor responses, are consistent with the interpretation that changes are effected through primary efferents to dopaminergic systems (6,10). Further indirect support for this notion is found in the demonstration of a change in corticofrontal dopamine turn-
1Requests for reprints should be addressed to Dr. E. W. Thornton, Department of Psychology, Eleanor Rathbone Building, University of Liverpool, P.O. Box 147, Liverpool L69 3BX, England.
819
820
THORNTON AND DAVIES
over during stress following lesion of the habenula (3). However, there is evidence to suggest that deficits in attentional mechanisms may also be evident following manipulation of dopaminergic systems or, indeed, lesion of several structures providing primary afferents to the habenula. Since the behavioral deficits evident under stress following lesion of the habenula require the initiation or change of response to an environmental cue, an alternative explanation couched in attentional rather than response terms should be considered. Unfortunately, there have been few studies examining a role for the habenula in attention in general or, more specifically, for attentional mechanisms under stress. Data from this laboratory have shown no effect of lesion of the habenula on orientation to novel stimuli or the distraction of ongoing operant responses by novel stimuli. Moreover, the acquisition of simple visual discriminations through either successive or simultaneous presentation of stimuli within both the Y-maze, a water maze or the conventional two-lever operant chamber are not influenced by lesion of the habenula (12). Such negative results have also been reported following lesion of limbic structures with afferents to the habenula. However, in contrast to other limbic lesions, lesions of the habenula also fail to influence reversal learning in such paradigms. The present study was carried out to elaborate the proposal that behavioral deficits may be observed following lesion of the habenula in alternative demanding conditions to those so far reported, and to determine more effectively whether any deficit is consistent with the failure to attend to salient visual cues. METHOD
Animals and Surgery Young male Wistar rats weighing 260 _ 8 g were individually housed at a temperature of 23-+2°(] in a 12-h light/dark cycle (light on 8:00 a . m . - - o f f 8:00 p.m.), given food and water ad lib, and assigned to lesion or sham operative procedures on a counterbalanced basis according to body weight. Animals were pretreated with atropine sulphate, 10 mg/kg, anaesthetised with sodium pentobarhital (50 mg/kg, IP) and subsequently positioned in a Kopf stereotaxic frame. Bilateral lesions were placed in the habenula according to coordinates derived from the atlas of Pellegrino and Cushman (4): A-P 3.8; L _+0.6; DV 0.7 above stereotaxic zero, by passage of radio frequency current from a Grass Model LM4 lesion maker through a stainless steel insulated needle with tip exposure of 0.4 mm. The current was gradually increased up to 10 mA over a period of l0 s until coagulation was attained. Sham-operated animals underwent similar procedures without delivery of current and with the electrode penetrating only through the cortex to terminate above the habenula. After removal of the electrode the wound was closed by suture and treated with an antibiotic powder. The normal procedure was to carry out one lesion and one sham operation on a single day. However, the process of attrition of animals from both groups (see the Results section) necessitated departure from this strategy for two sham-operated and two lesioned animals. For each animal, behavioral testing was carried out during the light phase of the cycle one week following surgery, after which the lesioned animals were sacrificed with pentobarbital, perfused, and their brains removed and stored in sucrose formaldehyde. The brains were subsequently frozen and sliced in 50 Ixm sections through the area of destruction. Sections were mounted on slides and examined to verify the extent of the lesion.
Behavioral Test Apparatus and Procedure The water-maze discrimination apparatus had similar parameters to that described in detail by Lee and Maier (2). A glass tank
(110 L × 30 W × 56 H cm) was filled to a depth of 30 cm water maintained at 34°C and made opaque by the addition of nontoxic white paint. The last 10 cm of the tank was divided into two chambers each of which was entranced via a black or white Plexiglas panel (13 W × 17 H cm), suspended 2 cm above the water surface. On each side wall was placed a larger black or white panel (30 W × 30 H cm), with the side for each shade conforming to the placement of the panel 'guarding' the end chambers. A starting platform, 9 cm square, with the level just above the surface of the water was placed at the end of the tank distant from the two chambers. During trials animals could climb onto an escape platform, also 9 cm square, with its surface 1.5 cm below the water and not visible, placed as appropriate into the end chambers. To minimize extra-maze cues, the apparatus was surrounded by a plain pastel sheet under a homogeneous ceiling. All behavioral testing was carried out by one experimenter who stood directly behind the end of the maze distal to the chambers throughout all trials. Each trial commenced by submersion of the start platform to the bottom of the tank with the rat required to swim to the other end of the tank, pass under the suspended stimulus card, and clamber onto the escape platform to terminate the trial. The time between submersion of the start platform and an escape with all four legs on the platform was recorded as the response time. Rats were allowed 45 s on the escape platform before being returned to the start platform facing the goal end of the apparatus and left for a period of 20 s prior to submersion of the platform and the start of the next trial. If the rat failed to reach the platform within a period of 90 s the trial was terminated, the rat removed and replaced on the start platform for 20 s prior to commencement of the next trial. In this instance, a response time of 90 s was recorded. The first 15 trials served to determine a position/shade preference. An escape platform was provided in both chambers and the preference defined as the more frequently chosen chamber. Commencing on the next trial, Trial 16, an escape platform was provided only on the nonpreferred side. If an incorrect choice was made the rat had to swim out of the inappropriate chamber into the compartment with the platform to complete the trial and in this instance an error was recorded. Animals were trained to a criterion of 8 trials in succession without an error (i.e., without recourse to the initial preferred side), or for a maximum of 40 discrimination trials. During these 40 trials the black and white stimuli were changed from one side of the tank to the other in a preprogrammed but randomised manner such that over the maximum number of trials there would be a balance between side and shade. The specific changes in position of the black/white shade cue was varied for each animal: For half the animals in each surgical treatment group, Group A, the switch of position on Trial 16 was associated with an incompatible change in the visual cue (e.g., a rat showing a preference for a left/black choice in the first 15 trials had to switch on Trial 16 to a right/white choice). For the remaining animals in each group, Group B, a shift of position but not visual cue was required on this trial (e.g., for a similar initial preference, it would be right/ black correct choice on Trial 16). For all animals, there was a change in the position of the visual cue between Trials 16 and 17. The reasoning behind these procedures was that if any animal in the initial, well-established preference had related this response to the visual cue, then rats in Group B would have an advantage over rats in Group A on Trial 16. For Group B, the visual stimulus and not position cue was predictive of escape at the time when the environment first changes for the animal. The more critical trial for the present study is the subsequent trial, Trial 17. For a rat in Group B any attention to the visual cue would be
DISCRIMINATION LEARNING FOLLOWING HABENULAR LESIONS
strengthened since the visual cue had been predictive of escape on the previous trial in which a change of positional response was required for the first time in order to escape. The visual cue should, therefore, even more strongly determine the response choice. In contrast, for rats in Group A the visual cue had been nonpredictive of escape and attention to that cue should diminish. Necessarily, over repeated trials in the acquisition of the posidon discrimination, this attention to the visual cue would be counterproductive and animals in Group B, who should be more 'switched-in' to the visual dimension, should be slower and make more errors in reaching the criterion for successful learning. The procedure required, therefore, a switch in position preference where attention to an external visual cue, which may initially have been appropriate in determining an adaptive response choice, would over trials interfere with the positional discrimination learning.
821
M e a n N u m b e r of T r i a l s to Criterion
B
RESULTS
Histology Histological verification of damage from frozen sections was performed by one experimenter without knowledge of behavioral outcomes and revealed consistent lesion size and destruction of both the medial and lateral habenula. Lesions showed no extension anteriorly into the stria medullaris or dorsomedial thalamus although there was some sparing of the anterior habenula. Misplacement of lesion for two animals necessitated two further operations with successful targetting of the lesion. The typical extent of the lesion was similar to that we have reported in previous studies using the same procedures and lesion parameters (6, 8, 9). Death of one sham-operated rat prior to testing also necessitated a substitute animal. One further sham-operated animal failed to swim satisfactorily past the suspended panel during the initial 15 trials and this animal was abandoned and a further operation performed.
B
A
A
Sham Operates
Mean Number of Errors
u m m m m m m m m m m m m m u m m m m m u m m m m m m m m m m
Lesion
B
A
A
i m
m
mm m m m m m m mm
I1" m
B
m "
Sham Operates
Lesion
Behavioral Analysis
FIG. 1. Presents the mean ( + SE) number of trials required to attain the criterion of 8 successive correct choices, together with the group mean ( + SE) number of errors made in attaining this criterion, for both habenular lesioned and sham-operated rats. Each of the two major treatment groups was subdivided on the basis of whether the correct switch of position choice on Trial 16, immediately after testing for the initial position preference, was also associated with a change of the visual cue. Thus, A = the correct switch of position response was also associated with a switch of shade and B = the correct switch of position response was associated with the same choice of shade as the initial preference.
The behavioral analyses were based on twelve lesion and twelve sham-operated controls. The left/black side was the preferred choice for four animals in each of the surgical groups during the initial 15 trials. The remaining rats showed preference for the right/white side. For half the animals in each of these groups the initial switch of position preference required on Trial 16 was also accompanied by a shift in the shade (from black to white or vice-versa). For remaining animals the required change of position on Trial 16 was compatible with the initial shade to which they had swum as a preference on the previous 15 trials. All animals showed a choice for their preferred side on a minimum of 10 of the initial 15 trials and at least two trials prior to position discrimination trials commencing on Trial 16. A two factor ANOVA of response times for Trials 13-15 revealed no effects of lesion group or trials (F values>l). The mean number of trials to criterion and the number of errors made prior to attainment of the criterion for each of the groups are presented in Fig. 1. Analyses of these data were undertaken by two-factor ANOVA with square root transformation applied for the nonhomogeneity of variance resulting from asymptotic scores for five lesioned animals. The analyses confirmed that lesioned rats made significantly more errors, F(1,20) = 10.3, p<0.01, and required significantly more trials, F(1,20)= 14, p<0.01, for acquisition of the discrimination. However, there were no significant, or near significant, interactions and effects related to the second factor of visual cue compatability for either dependent variable, although of the five lesioned animals failing to attain criterion over training, four were from Group B. All the sham
operates attained the criterion within the 40 trials. Evidence relating to the effects of the irrelevant visual cue on performance may be seen from the group mean response times for Trials 16 and 17 (Table 1). Statistical ANOVA of the data from Trial 16 confirm the longer response time for lesioned animals, F(1,20) = 4 . 8 , p<0.05, indicated in the previous analysis of trials to criterion. Although times on Trial 16 were longer for Group A as predicted, neither this second factor nor the interaction approached significance (F values>l). These data suggest that the initial preference over the first 15 trials for both lesion and control rats was not necessarily determined by the visual cue but may indicate a more direct response bias. A similar analysis for Trial 17 again showed the significant effect of the lesion, F = 8.8, p < 0 . 0 1 , but, in contrast to Trial 16, a significant effect of the visual cue factor [i.e., a Group A/Group B effect, F = 7.3, p<0.05] together with a significant interaction, F = 8 . 1 , p<0.01. Further analysis of the interaction using the Tukey HSD statistic confLrmed that the mean response time for lesion Group B was significantly different from each of the other three groups (p's<0.01), with no other comparison reaching significance. It would seem, therefore, that lesioned animals on Trial 17 were most impaired when, having escaped safely through the nonpreferred position in conjunction with the previously associated visual cue on Trial 16 (the first discrimination trial), they then on the next trial had to swim to the previously nonpreferred position with the preferred visual cue switched to the incorrect choice side.
822
THORNTON AND DAVIES
TABLE 1 THE GROUP MEAN (_ SE) OF RESPONSE TIMES FOR LESIONED AND SHAM-OPERATED ANIMALSFOR TRIALS 16 AND 17 (i.e., THE FIRST TWO DISCRIMINATION TRIALS) Sham Operates A B Trial 16 Trial 17
17.5 -+ 4.8 7.0 -+ 3.5
13.3 _ 4.6 5.8 _+ 0.9
Lesion A
B
39.8 - 12.1 8.0 +_ 3.2
31.8 -- 15.6 54.0 _ 19.9
A: The switch of position response on Trial 16 was also associated with a switch of shade. B: The switch of position response on Trial 16 was associated with the same choice of shade as in the initial preference.
These analyses indicate that, somewhat paradoxically, lesioned rats were more influenced by the salient environmental visual cue than sham-operated controls. They attend more readily to extrinsic visual stimuli in the face of the changed environment on Trial 16 with this effect most readily seen as an interference to the required position change on the subsequent Trial 17 when their 'choice' is an approach to the now negative cue. DISCUSSION These data show an impairment of a rather complex discrimination acquisition following lesion of the habenular complex. This finding extends and is consistent with our previous suggestion that behavioral deficits following such lesions are apparent only under demanding conditions. These conditions require a switch from a prepotent response to optimise environmental contingencies (8). We have loosely labelled these conditions as 'stressful' and have argued that they invoke an activation of midbrain monoaminergic mechanisms in control animals. However, the physiological systems which are activated by the diverse test conditions we have utilised in demonstrating a contribution through the habenula have yet to be specified. Nevertheless, the present data provide further support for a functional contribution of the habenula. The deficit in discrimination acquisition in the presence of an irrelevant, distracting stimulus cue contrasts with the absence of effects of lesions of the habenula in more typical operant lever press, swim maze or Y-maze, discriminations with both simulta-
neous or successive presentation of relevant visual cues (12). The present study does not allow resolution as to whether it is the effects of the swim component, the irrelevant distracting visual cue, or their interaction which provides the conditions necessary to involve the habenular relay. However, the absence of any lesioninduced effects during Trials 13-15 indicates an absence of effect of the lesion on swimming alone and this is supported by our previous studies demonstrating no effect of lesions of the habenula on simple visual discrimination learning to black/white cues in the same test apparatus. Moreover, the data on performance during the initial preference testing support other studies showing an absence of effect of habenular lesions on motoric ability (1,7). The intrinsic requirement of the present test situation and an examination of both the pattern of response change and response latencies on Trials 16 and 17 also indicate that the impaired acquisition of the discrimination is not brought about by the simple perseveration of response that we have suggested might account for other data following lesion of the habenula (9). The present results are consistent with evidence suggesting that the habenula is involved in the ability of animals to switch response strategies under stress. Previous response deficits, such as the failure to utilise an escape route during a swim test, are interpretable in terms of a simple effect of reduced attention to 'external' cues. In the present study, an examination of the response times on Trials 16 and 17 convincingly demonstrates that lesion of the habenula does not result in a decreased attention to external cues when a response switch is demanded by the external contingencies. The significantly extended response time for lesioned animals in Group B on Trial 17 indicates that lesioned animals were attending to the visual cue on at least the two critical trials following the demand for a switch of response. Indeed, this increased attention to the irrelevant visual cue would contribute to the acquisition deficit. Neither a simple failure to attend nor the more paradoxical increase in attention to such stimulus dimension can explain the behavioral deficits reported in our previous studies (6, 8, 10). One possible alternative is that the present deficit is construed as a failure to switch from extrinsic to intrinsic stimulus control. A similar but inverse failure to switch from intrinsic to extrinsic stimulus control could be invoked to describe the escape deficit reported in our previous studies of swim tests (8,10). ACKNOWLEDGEMENT This work was supported by a Project Grant from the Medical Research Council of Great Britain.
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