Contralateral ‘neglect’ after unilateral dorsomedial prefrontal lesions in rats

53

Brain Research, 72 (1974) 53-63 !)) Elsevier Scientific Publishing Company, Amsterdam

CONTRALATERAL 'NEGLECT' AFTER P R E F R O N T A L LESIONS 1N RATS

Printed in The Netherlands

UNILATERAL

DORSOMEDIAL

ALAN COWEY AnD TOMAS BOT.EK

Department (if"Experinwntal P.£vchology, UniversiO, of Oxford, OA]/brd (Great Britain) (Accepted November 25th, 1973)

SUMMARY

Alter determining the side preferences of rats in a Y-maze either the dorsomedial or dorsolateral prefrontal cortex was removed unilaterally. The latter operation had no effect on choice behaviour whereas the former was followed by a significant reduction of turns to the side contralateral to the lesion. The results are discussed in relation to recent anatomical experiments which indicate that the dorsomedial prefrontal cortex in the rat may be homologous to the frontal eye-fields of monkeys, unilateral removal of which causes a contralateral neglect.

INTRODUCTION

The prefrontal cortical projection fields of the 3 principal divisions of the dorsomedial nucleus (MD) of the thalamus in monkeys are well known and quite separate, as are the behavioural effects that follow removal of each. In striking contrast, the homologous regions of prefrontal cortex and MD in rodents were obscure until Leonard 10,11 reported that 3 regions of M D could be discriminated in rats on the basis of their connexions with prefrontal cortex. She found that the most lateral, paralamellar portion of MD projected to the dorsal wall and 'shoulder' of medial prefrontal cortex, the internal segment projected to the cortex in the upper limb of the rhinal sulcus, and the anterior intervening region which surrounds the internal segment projected to the more ventral cortex on the medial wall of the prefrontal cortex. Leonard suggested that these 3 regions of MD, i.e. paralamellar, internal and intervening, might correspond to the pars multiformis, pars medialis and pars lateralis respectively in the rhesus monkey and that their prefrontal projection fields in dorsomedial cortex, sulcal cortex, and ventromedial cortex may be homologous to the frontal eye-fields (area 8), orbital region and dorsolateral region respectively in the frontal lobe of the rhesus monkey. The anatomical evidence that the dorsomedial

54

A. COWEY AND T. BOZEK

prefrontal cortex of the rat and the frontal eye-fields of monkeys may be homologous is particularly striking and was reviewed by Leonard 1~ who pointed out that (a) both cortical regions receive projections frcm the paralamellar segment of MD, (b) the latter receives fibres from the cerebellum, spinothalamic tract, superior colliculus, and pretectum in both rats and monkeys, (c) both cortical regions project to the superior colliculus, and (d) the parastriate cortex projects to the frontal eye-fields in monkeys and to medial frontal cortex in hamsters. Although the projection from parastriate cortex has not been studied in rats, it is known that medial frontal cortex in hamsters is related to the paratamellar segment of M D, as in rats. If Leonard's suggestions about homology are correct, it should be possible to reproduce in rodents some of the well-known effects of frontal ablation in monkeys: For example, unilateral removal of the frontal eye-fields in monkeys, corresponding to the projection field in the arcuate sulcus of MD pars multiformis, produces a clear contralateral neglect2,4,5,13. Although the gross symptoms disappear within a week or so, a much more enduring neglect is detectable if the animal's eye movements and visual fields are examined perimetrically 7 9. We therefore decided to remove the dorsomedial shoulder of the prefrontal cortex unilaterally in rats to determine whether the operation produced a contralaterat neglect in a Y-maze. METHODS

Subjects Fourteen male hooded Lister rats were used. They were approximately 6 months old and weighed from 350-400 g. During the experimental period they were housed singly and had unrestricted access to water. They were fed 15 g of diet 41B (Dixons) from 2 to 4 h after each testing session.

Apparatus This was a Y-maze. The internal dimensions of each arm were 50 cm long > 16 cm wide × 14 cm high. It was constructed of wood and painted matt grey. The two goal arms and the start arm were each covered with a removable wire-mesh ceiling. A small ceramic food-bowl filled to a depth of about 1 in. with wheat grains was placed at the far end of each goal arm. Clear perspex sliding doors could be inserted at the entrance of each goal arm to prevent access to either arm. These were only used during pretraining and in one experimental condition described below. A similar door formed the end wall of the start arm and was raised to place the animal in the maze for each trial. The maze stood on top of a table with the goal arms equidistant from the side walls of the room. It was illuminated diffusely by a 60-W bulb in a reflector mounted 60 cm above the choice point and pointing up at the white ceiling of the room,

Pretraining Each animal was allowed to explore the Y-maze for 20 min on 6 successive days. All found and ate the food in the bowls. They were then given 20 trials/day for 5 days

UNILATERAL'NEGLECT'IN RATS

55

to ensure that they ran promptly to the reward. On each trial the animal was placed in the start arm and removed 5 sec after finding and taking food in either goal arm. Retracing was prevented by lowering the door after the animal had entered a goal arm. Between trials the rat was placed in a black bowl behind the start arm and equidistant from the goal arms. It was allowed to remain in the bowl, where it usually finished eating the reward, for 15 sec. The next trial was then given. Side preferences during pretraining were not recorded but knowing them allowed us to ensure that animals with the same side preference were not tested consecutively as a group, which might have encouraged them to follow ineradicable odour trails.

Testing In the experiment proper the animals were given 20 trials/day, with an intertrial interval of 15 sec. After each trial the animal's choice, left or right, was recorded. Four experimental and two control animals were tested by the authors, who were aware of the type of lesion. The remainder were tested by an assistant who was unaware of the type of lesion or purpose of the experiment. The following stages were used. Stage 1. Immediately following pretraining the animals received a further 100 trials over 5 days to assess their side preferences. Stage 2. The following day each animal had the lids of one eye sutured to assess the effects of a unilateral visual deprivation on side preferences. It was hoped to compare the effects of a real unilateral sensory restriction with any effects o f subsequent unilateral lesions. The eyelids were sutured on the side ipsilateral to the preferred maze arm in half the animals, and on the contralateral side in the remainder. Starting the same day, they were given 20 trials/day for 5 days. Stage 3. Immediately after stage 2 the sutures were removed from the eyelids and the animals were given 100 trials with both eyes open again. These trials were used as the preoperative data in assessing lesion effects. Stage 4. Each animal was given a unilateral lesion in prefrontal cortex. With one exception the lesion was contralateral to the previously assessed preferred arm of the maze in stage 3. In the experimental group of 8 animals the dorsomedial shoulder of prefrontal cortex was removed. In 6 controls the adjacent dorsolateral prefrontal cortex, which does not receive a projection from MD, was removed. Testing began the day following surgery, and 20 trials/day were given, on days I, 2, 3, 4, 5, 7, 9 and 13. Stage .5. On postoperative day 14 all animals were given 20 forced trials to their non-preferred side to see if this would subsequently reduce or abolish any side preference caused by the operations. The effects of this procedure were examined by 60 free choices, on days 15-17. Stage 6. Oll days 18-19 the food was removed from the preferred arm of the maze to determine how rapidly the animals would learn to enter the other arm

Surgerl, (I) Eye closure. One eye was closed in stage 2 by briefly anaesthetisingthe animal with pure ether and inserting 3 nylon sutures through the eyelids. The animals were

56

A. COWEY AND t . BOZEK

not unconscious for more than 2-3 rain. At the end of stage 2 the sutures were removed using the same anaesthetic procedure. Most of the sutures were already loosening by this time. (2) Cortical lesions. The animal was anaesthetised with an intraperitoneal injection of Equithesin (Jensen Laboratories) 2.0 ml/kg, supplemented when necessary by pure ether. It was placed in a stereotaxic instrument, using non-rupture ear bars, and the top of the head was shaved. The skin was opened with a midline incision and the dura exposed by drilling a small hole in the skull with a hand-held burr to avoid thermal damage. The hole was enlarged with fine rongeurs and the dura cut over the lesion area. In the experimental group an attempt was made to remove all cortex of the upper half of the medial surface of the frontal lobe, from the genu of the corpus callosum posteriorly to the frontal pole anteriorly. This region corresponded to the prefrontal projection field of the far lateral, paralamellar, segment of MD. In the control group an attempt was made to remove an equivalent area of dorsolateral prefrontal cortex, which does not receive afferents from any part of MD x°,l]. All lesions were performed by aspiration using a 24-gauge sucker. When all bleeding had ceased the lesion was covered with a thin piece of sterile absorbable gelatin foam and the incision closed with nylon sutures.

Histology At the end of stage 6 in testing the animals were given ad libitum food and water and kept for a period of 6 weeks (6 animals) and 6 months (8 animals) to allow any retrograde degeneration to occur in the thalamus. They were then killed with an overdose of Nembutal and perfused through the heart with normal saline followed by 10 % formol-saline. The head was removed and placed in a stereotaxic instrument with the incisor bar at 2.4 mm below the inter-aural line to correspond with the stereotaxic atlas of Ktinig and Klippel 6. After removing the dorsal skull the brain was severed with a knife in the stereotaxic vertical plane at AP zero, and photographed from above after removal. In the 6 animals with a short survival time the brain was placed in sucroseformalin (30 % sucrose, 10% formalin) until infiltrated. Frozen sections were cut at 50/zm and every fourth section stained with thionin. The remaining 8 brains, for which the post-testing survival time was 6 months, were embedded in LVN and sectioned at 30 ~m. Every twentieth section was stained with cresyl violet, and additional intermediate sections through the lesion or the thalamus were subsequently stained where necessary and in the same way.

Histological reconstructions A dorsal view of each lesion was reconstructed by enlarging the coronal sections with a microprojector and marking the limits of the lesion at the appropriate AP position on the photograph of the dorsal surface, using proportional dividers. Since it was not possible to photograph the medial surface of the hemisphere without dividing the brain a reconstruction of the medial extent of the lesion for the experimental

UNILATERAL 'NEGLECT' IN RATS

57

group only was similarly prepared by marking the lesion on a standard diagram of the medial surface. The surface reconstructions are shown at the top of Fig. I. The dorsomedial lesions included the target area in every case but extended slightly too Jhr posteriorly on several instances and destroyed the genu of the corpus callosum. Their ventral limits were also unnecessarily low in several animals and encroached on the projection field of the anterior region of MD. The dorsolateral lesions of the control group were as intended except that in animals A and B the total extent of the lesion was smaller than in any of tile experimental animals, in this group too the lesion occasionally involved fibres of the corpus callosum, at their lateral extent. Cross sections from two representative animals are shown at the bottom of Fig. 1. Like Leonard ~0 we were unable to detect any retrograde degeneration in the thalamus in animals with dorso-

Fig. I. Top: reconstructions o f unilateral cortical lesions. Dorsolateral lesions are s h o w n f r o m above, dorsomedial lesions are s h o w n from above a n d from medial view. Below: outline d i a g r a m s of coronal sections t h r o u g h frontal lobes o f a representative animal from each group. Left hemisphere is s h o w n on left. For further details see text.

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A. COWEY AND T. BOZEK

medial prefrontal lesions. However, the 6 control animals all showed cell loss, pallor, and gliosis in the ipsilateral nucleus thalamus ventralis. RESULTS

Gross behaviour

F o r a few minutes daily in the first post-operative week each animal was placed inside a black bowl on a bench, and then on top o f the inverted bowl. In the first condition the animals tended to rear up and place their forepaws on the rim o f the bowl then to descend, turn, and repeat this action at another position. On the second condition they tended to place their forepaws on the edge o f the inverted bowl and to explore the perimeter by rotating a b o u t their hindquarters. The experimental and control g r o u p differed in that the former showed clear ipsiversive circling for the first few days after operation, whereas the latter explored in both directions. We made no attempt to measure this difference in this experiment. We did attempt to record the orientation to sounds (snapping fingers) on one side or the other o f the animal and to stroking the fur or vibrissae with a pencil. We observed no differences but the responses in general were unstable and difficult to score. Stages 1-3

The number of turns to the right made in 100 trials in each of stages 1, 2 and 3 are shown in Table I. I f a preference for the right is defined as any score greater than 50 then 11 out o f the 14 rats preferred the right in stage 1 (P - 0.05, 2-tailed). We have no explanation why the right arm was apparently more attractive in this stage, since the maze was placed centrally in the r o o m and both arms were cleaned regularly to delete o d o u r trails.

TABLE I STAGES 1-3. TURNS MADE TO THE RIGHT

Values are of number of turns to the right in 100 trials when tested with both eyes open (stage I), then one eye closed (stage 2), and finally with both eyes open again (stage 3). Animals"

Eyes open Right eye closed Eyes open

Eyes open Left eye closed Eyes open

A

B

C

D

E

F

G

20 3 5

22 24 29

46 14 8

54 60 88

67 85 79

70 75 86

82 69 78

H

/

J

K

L

M

N

55 40 44

58 14 15

65 59 69

66 90 92

71 67 40

72 80 83

76 29 61

UNILATERAL'NEGLECT' IN RATS

59

The main result of stage 2 is that closing one eye had no consistent effect on side preference. Closing the right eye was followed by an intensified left preference in rats A and C, whereas closing the left eye was followed by a reversal of a right preference in rats l and N and an intensification in rat K. Furthermore, the preferences shown in stage 2 were retained in stage 3 with the exception of rat N, which was the only animal to show a reversal of preference from stages 1-2, and 2-3. We conclude that testing the animals monocularly had little effect on binocular side preferences.

Stage 4 The effects of the operations are shown in Figs. 2 and 3, and in detail in Table II. Fig. 2 shows the mean percentage of turns to the right in 5 rats with a left medial frontal lesion, and in 3 with a similar lesion on the right. In each experimental subgroup the mean side preference was clearly reversed in stage 4 following the operation and the effect was stable over 13 days. No such change occurred after the control lesion (Fig. 3). Since one animal (rat L) in the sub-group with a left medial lesion actually had a slight ipsilateral preference pre-operatively, a more sensitive way of examining the data is to ask whether the lesion reduced the number of contralatera[ choices irrespective of pre-operative side preference. As is shown in Table il, a reduction in contralateral choices occurred in every experimental animal in stage 4 (P 0.008, sign test, 2-tailed). No such reduction occurred in the control group (P ~> 0.65, sign test, 2-tailed). The experimental and control groups were compared directly by ranking their choices to the contralateral side. Pre-operatively they did not differ in stage 3 (nl 8, n2 ~ 6, U 19, P -- 0.57, 2-tailed). Post-operatively, in stage 4, the experimental group made significantly fewer turns to the side contralateral to the 100

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STAGES 3 - 6 . TURNS MADE TO THE RIGHT

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UNIL.A~I-ERAL

'NEGLECT'

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DAYS F'ig. 3. Effect of lateral frontal lesions on choice behaviour in Y-maze. Labelling as in Fig, 2. lesion (n, 8, n 2 - - 6, U l, P 0.002, 2-tailed). When the differences between stage 3 and stage 4 scores for each animal were ranked the same result was obtained (nl : 8, n._, 6. U -- 1, P -~ 0.002, 2-tailed). The medial frontal lesion therefore significantly reduced the proportion of turns to the contralateral side. Nevertheless, it should be noted that the reduction in one control animal (rat G) was slightly greater than in one experimental (rat D) yet there was no evidence that medial cortex had been inadvertently damaged in the former and spared in the latter.

Stage 5 In this stage an attempt had been made to diminish any side preference by blocking that alley for 20 trials then giving a further 60 free choice trials over 3 days. Table II shows that this had no effect. The side preferences of both groups remained untiltered.

Sla,ge 6 Here the reward was permanently removed from the preferred alley and retained on the non-preferred side. Both groups promptly learned not to enter the previously preferred alley and by the second session all but two animals (controls G and J) entered the rewarded alley on at least 85 jo{,o f trials. The experimental and control groups were clearly not different from each other. I)ISCUSSION

The present experiment demonstrated that removal of the cortex on the upper half of tile medial surface o f the frontal lobe in rats produced a significant reduction

62

A. COWEY AND T. BOZEK

of the number of turns to the contralateral side in a Y-maze. This behaviour resembles the neglect of food or light flashes presented contralaterally in monkeys after unilateral removal of the frontal eye-fields (area 8) and the result supports Leonard's suggestion10,1 t that these two regions are homologous. Furthermore, we observed i psiversive circling in the experimental rats for the first few days after operation and this phenomenon is also seen in monkeys after unilateral removal of area 8. One clear difference between the effects of medial frontal lesions in rats and frontal eye-field lesions in monkeys is that no retrograde degeneration was found in the paralamellar portion of M D in rats. Leonard l°,H also noted this but did describe a dense dust-like deposit in silver stained sections, which may represent retrograde degenerative changes in presynaptic dendrites. Two important questions are: how similar in function are the medial frontal cortex of rats and the frontal eye-fields of monkeys, and what is the nature of the contralateral neglect following removal of these areas'? In monkeys the gross symptoms of neglect disappear in a week or two, although careful perimetric testing shows that it is present for a very much longer period 7. Electrical stimulation of the frontal eye-fields in monkeys provokes contralateral saccadic eye movements whose direction and extent depend on the locus of stimulation 12. Cells in this region are also excited during and after voluntary saccadic eye movements and head movements t. Whether these phenomena occur in rats remains to be investigated. However, two things are clear. First, the avoidance or neglect of the contralateral side in rats was rapidly overcome when the ipsilateral reward was removed. The neglect therefore reflects a preference for the ipsilateral side and not an inability to turn contralaterally. Second, Collin and Cowey (in preparation) have found that the contralateral neglect in rats following dorsomedial frontal lesions occurs even if all pre- and post-operative testing takes place in total darkness. The neglect is therefore not restricted to vision and may not even involve vision. Welch and StutevillO 3 found evidence of a polymodal neglect in their monkeys with frontal eye-field lesions so it is still possible that the defect in rats and monkeys may be similar. There is one earlier finding that appears to conflict with ours. Clark and Goldberg 3 studied choice behaviour in rats in a Y-maze before and after excising the lateral three-quarters of the frontal lobe on one side. The animals showed a striking contralateral neglect when tested either I or 14 days after operation. Now the tissue they did n o t remove corresponded closely to our experimental lesion and their result apparently conflicts with ours. However, it is very doubtful whether the remaining medial one-quarter of the frontal lobe was functionally intact following such a radical removal of the lateral region. Certainly, their schematic diagram indicates that the fibres from M D to medial prefrontal cortex must have been damaged or even severed. Since they presented no histological results it is impossible to confirm this. ACKNOWLEDGEMENTS

This research was supported by M R C Grant G 971/397/B. We thank Miss M. McAnulty for assistance with the histology, Mrs. S. Perry for

UNILATERAL 'NEGLECT' IN RATS

63

p r e p a r i n g t h e figures, M i s s A. S k u l l f o r h e r h e l p in t e s t i n g t h e a n i m a l s , a n d D r . R. P a s s i n g h a m a n d Dr. P. D e a n f o r t h e i r c o m m e n t s o n t h e m a n u s c r i p t .

REFERENCES 1 Blzzl, E., AND SCHILLER, P. H., Single unit activity in the frontal eye fields of unanesthetized monkeys during head and eye movements, Exp. Brain Res., 10 (1970) 151-158. 2 BRUCH~:R, J. M., The frontal eye field of the monkey, htt. J. Neurol. (Montevideo), 5 (1966) 262 28 I. 3 CLARK, G., AND GOLDBERG, S. E., Visual disturbances after unilateral frontal lesions in the rat, J. comp. physiol. P~Tchol., 44 (1951) 487 491. 4 KE~NARO, M. A., Alterations in response to visual stimuli following lesions of frontal lobe in monkeys, Arch. NeuroL Psychiat. (Chic.), 41 (1939) 1153-1165. 5 KENNARD, M. A., AND ECTORS, L., Forced circling in monkeys following lesions of the frontal lobes, J. Neurophysiol., I (1938) 45-54. 6 K(JNIG, J. F. R., AND KLIPPEL, R. A., The Rat Brahz: A Stereotaxic Atlas o f the Forebrain and Lower Parts (~[ the Brain Stein, Williams and Wilkins, Baltimore, Md., 1963. 7 LATTO, R., ANO COWEr, A., Visual field defects after frontal eye-field lesions in monkeys, Brain Re.search, 30 (1971) 1-24. 8 LATTO, R., AND COWEr, A., Fixation changes after frontal eye-field lesions in monkeys, Brahz Research, 30 (1971) 25 36. 9 LATTO, R., AND COWEY, A., Frontal eye-field lesions in inonkeys, Bib/. ophthaL (Basel), 82 (1972) 159-168. 10 LEONARD, C. M., The prefrontal cortex of the rat. 1. Cortical projections of the mediodorsal nucleus. I1. Efferent connections, Brain Research, 12 (1969) 321-343. I 1 L~ONARD, C. M., The connections of the dorsomedial nuclei, Brain Behav. Eva/., 6 (I 972) 524 -541. 12 ROBINSO~q,D. A., AND FUCHS, A. F., Eye movements evoked by stimulation of frontal eye fields, J. Neurophysiol., 32 (1969) 637-648. 13 WELCH, K., AND STUTEWLLE, P., Experimental production of unilateral neglect in monkeys, Brain, 81 (1958) 341-347.