Behavioral study after local injection of 6-hydroxydopamine into the nucleus accumbens in the rat

Brain Research, 344 (1985) 9-20 Elsevier

9

BRE 10976

Behavioral Study after Local Injection of 6-Hydroxydopamine into the Nucleus Accumbens in the Rat K. TAGHZOUTI, H. SIMON. A, LOUILOT, J. P. HERMAN and M. LE MOAL Laboratoire de Psychobiologie des Comportements Adaptatifs, 1. N. S. E. R.M. U259, Bordeaux (France) (Accepted December 1lth, 1984) Key words: dopamine - - nucleus accumbens - - limbic syndrome - - striatal sensorimotor syndrome - - Parkinson's disease

Anatomically, the nucleus accumbens (n.Acc.) has been considered as an interface between limbic and striatal scnsorimotor structures. In the light of this hypothesis we have investigated the behavioral effects of destruction of the dopamincrgic inncrvation ol the n.Acc, after local injection of 6-hydroxydopamine. The following behavioral deficits were observed: (1) hypoexploration in a 4-hole box and 2-compartment field, (2) failure to inhibit response strategies either with positive reinforcement in a straight alley test or negative reinforcement in a passive avoidance test. These disturbances comprise a syndrome of perseveration, reduced distraction by irrclcvant information, decreased behavioral switching and flexibility, and a paradoxical locomotor disinhibition in an emotional conlcxt. Very similar behavioral changes are found following lesions of limbic structures. In addition, these lesioned animals exhibit an enhanced latency to initiate motor responses. This deficit of behavioral initiation is classically observed in motor striatal disease. It is suggested that the n.Acc, is a key structure for the integration of limbic and striatal sensorimotor functions.

INTRODUCTION

cause of its d e v e l o p m e n t a l and anatomical relationships with the rest of the striatum"~,>,>,~s. Support

The nucleus accumbens (n.Acc.) has been the fo-

for considering the n.Acc, as both limbic and striatal

cus of an increasing n u m b e r of studies over the last

is also provided by the organization of the m aj o r af-

few years for at least three reasons. Firstly, it can be

ferents of the dorsal and ventral striatum, i.e. the D A

considered as both a striatal sensorimotor and a lim-

inputs. There is no clear-cut separation between do-

bic region 27, thereby conferring unique but still poor-

paminergic

ly understood integrative properties. Secondly, the n.Acc, receives a dense dopaminergic ( D A ) innerva-

pathways originating from the substantia nigra and from the ventral tegmental area (VTA)5,(',24,3% All

tion from the so-called A10-mesolimbic neurons

these anatomical considerations have led some au-

which are a m a j o r site of action of psychotropic

thors to suggest that the n.Acc, is an interface be-

drugs 19,22. Thirdly,

these functional and n e u r o c h e m -

tween limbic and sensorimotor structures, or in be-

ical characteristics suggest that various pathological states may involve this region3,~4.15,37.

havioral terms b et w een motivation and response output 2<29. H o w e v e r , this rather general hypothesis is of

The nucleus accumbens has long been considered

little value in explaining the functional role of n.Acc.

A9-nigrostriatal

and

A10-mesolimbic

to be a limbic structure. It sends projections to olfac-

and of its ascending D A afferents 3.12-3:. As pointed

tory, septal and hypothalamic regions 4-3~ and re-

out by Robbins and Everitt 3~, there is as yet little con-

ceives allocortical afferents from the hippocampus and the amygdala 3,1°,lT,ls,2s,30. H o w e v e r , the n.Acc.

vincing evidence from behavioral studies that the D A

might also be regarded as the ventral part of the stria-

processes in a similar way to the i n v o l v em en t of dorsal striatal D A neurons in m o t o r processes. It must

turn due to a similar architectonic organization be-

neurons of the n.Acc, are involved in motivational

Correspondence." H. Simon, Laboratoire de Psychobiologie des Comportements Adaptatifs, I.N,S.E,R.M. U259. Domainc dc Carreire. Rue Camille Saint-Saens, 33077 Bordeaux cedex, France. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

10 be borne in mind that the D A neurons are situated, in relation to the reticular formation, as a part of the neuronal system linking the limbic midbrain area and the forebrain limbic structures28. They are also sensitive to various external and internal inputs 23. The level of sensory input has been found to depend on variables such as context and novelty2. It is quite likely that they do not code for any specific information 35. The purpose of the present study was to investigate the behavioral consequences of a 6-hydroxydopamine (6-OHDA) lesions of the D A system at the level of the n.Acc. The working hypothesis was that the deficits would not be related to any specific function of D A neurons but would correspond to a general dysfunction of the n.Acc. In this study we show that such lesions induce a complex syndrome associated with reduction in the initiation of behavior, reduced distraction by irrelevant information, perseveration, decreased behavioral switching and flexibility and a paradoxical locomotor disinhibition in an emotional context.

acid. A 6 - O H D A solution of 8 k~g/2#l was infused bilaterally over 7 min via a 0.3 mm diameter stainless steel cannula connected by a polyethylene tubing to a 10 /zl microsyringe driven by a pump at constant speed. The cannulae were left in place for an additional 5 min after the end of the infusion. Control rats (C, n = 34) were treated in the same way except that they only received the vehicle solution. Behavioral tests started after a post-operative period of 2 weeks.

Behavioral procedure Four different groups of rats were used in four testing situations (i) measures of locomotor activity in a circular corridor; (ii) measures of exploration, initiation of responses to irrelevant information, behavioral flexibility in a 4-hole box and in a 2-compartment field; (iii) extinction of a learned paradigm for food reward in a straight alley test; and (iv) measures of locomotor disinhibition or perseveration in an aversive context using a step-through passive avoidance test. All the test sessions were carried out between 9.00-12.00 h and 16.00-19.00 h.

MATERIALS AND METHODS

Circular corridor: locomotor activity Animals and housing conditions Subjects were naive adult male rats of the Sprague-Dawley strain weighing 250-300 g. They were individually housed in a room maintained at a constant temperature (22 °C) and humidity and on a 12 h cycle of illumination (dark 20.00-8.00 h). The animals had free access to food and water.

6-OHDA injections into the nucleus accumbens Eighty-two rats were operated on under chloral hydrate anaesthesia (150 mg/kg, i.p.). The rats were pretreated 30 min before the 6 - O H D A injections with desmethylimipramine (DMI, Pertofran, Geigy, 25 mg/kg) in order to protect noradrenergic neurons. The rats were placed in a Kopf stereotaxic apparatus with tooth bar 5 mm above the interaural line. The skull was exposed and burr holes drilled above the appropriate targets. The experimental rats (n = 48) received bilateral 6 - O H D A injections at the level of n.Acc, according to the following coordinates: 0.8 mm anterior to the bregma, 1.7 mm lateral and 7.5 mm below the skull surface. The neurotoxin was dissolved at a concentration of 4/ag (free base) per 1 ~1 in a vehicle solution containing 0.2 mg/ml of ascorbic

Quantitative analysis of horizontal locomotor activity was measured in a circular corridor, 12 cm wide and 170 cm long equipped with 4 photocell beams situated 3 cm above the floor. Interruption of the beams was recorded automatically on an event recorder situated outside the testing room, Locomotor activity was measured for 1 h, 12 h of dark phase, and for the 24 h circadian cycle starting at 23.00 h. Sixteen rats were used, 8 rats with 6 - O H D A lesion and 8 controls.

Four-hole box: exploration The apparatus was a metallic square box (45 x 45 × 24 cm). There was a small corridor (8 x 6 cm) in each of the 4 corners, at the end of which a hole was drilled (3.5 cm diameter) situated 4 cm above the floor. This positioning of the holes was designed to eliminate a possible artefact related to an increase in locomotor activity and to only measure oriented responses. The box was closed with a transparent cover. At the beginning of the session the rat was placed in the middle of the arena and the parameters - - n u m ber, duration and time course of the nose pokes were recorded every 5 rain for 15 min on a computer. A 'spectrum analysis' of exploration (distribution of

11 visits as function of duration) was carried out by the computer. Twenty-six rats were used, fourteen 6-OHDA-lesioned rats and twelve controls.

Two-compartment field test The same animals were used for this experiment and the exploration task described above. The apparatus was a wooden open field (100 x 100 x 50 cm), painted white and separated into two compartments by a barrier, in the middle of which a small door, which when open, allowed the rat to visit compartment B from the starting compartment A. Both compartments were identical with floors divided in 8 squares (25 x 25 cm) which enabled a quantitative measure of locomotion. The open field was placed in a darkened room (60 lux). The rats were submitted daily to a 10-min session for 9 days, at the same time of day. At the beginning of the test the rat was placed in the same start-square of the compartment A. On days 1, 2 and 3, the communicating door was closed. From the 4th until the 9th day the door was open and the rat could enter compartment B. The behavioral characteristics of the exploration in compartment B was measured during sessions 4, 5 and 6. For the daily sessions 7 and 8 a novel metallic object (3 x 3 x 7 cm) was placed and stuck in the center of the same square in a corner of compartment B. For the last session (Day 9) the object was removed. Depending on the sessions the following behavioral parameters were recorded: (a) start latency (time to leave startsquare); (b) latency to enter compartment B; (c) number of entries into compartment B; (d) locomotor activity in compartment A and B (square crossings); (e) number and duration of rearings in both compartments: (f) duration of the explorations of compartment B; (g) latency to touch the object in compartment B; (h) number and mean duration of the exploration of the square in which the novel object was placed.

Straight alley test The apparatus was a 3 m long runway in wood, painted black and divided into 3 compartments: a start box (30 x 15 x 30 cm), a central alley (240 x 15 x 30 cm), a goal box (30 x 15 x 30) and a door separating the start box from the alley. For one week before testing, the rats were placed on a restricted diet to maintain body weight at 85% of the normal

weight. In this experiment the animals had to run through the alley to obtain two food pellets as reinforcement. On each trial the rat was placed in the start box for 15 s, the door was then opened and the rats were allowed to reach the goal box where they were left for 20 s. The rats were put through 10 trials per day for 5 consecutive days. On the 6th day the food reinforcement was removed and 10 extinction trials were performed. The latency to leave the start box and the running time to reach the goal box were measured. Sixteen rats (8 lesioned and 8 controls) were used in this experiment.

Passive avoidance test The inhibitory avoidance test used was the stepthrough task 1. The apparatus was a square box made of wood (38 cm on one side) and painted black. The rat was placed under a light on a small ledge hanging over an empty space, communicating with the box. In order to avoid the aversive situation the animal learned to run into the hole and to enter the dark box. On the first day the rat was placed directly in the box for an exploration period of 2 min and then it was placed on the platform and allowed to enter the box where it was left for 20 s. On the second day the rat was put directly on the platform for another trial and left in the box for the 20 s. The procedure was the same on the third day except that the animal was given a scrambled foot shock (0.36 mA, 2 s) through the metallic bars of the floor on entering the box. After this training period, retention was tested and the latency to enter was measured 6 h, 24 h and 15 days later. Twenty-eight rats were used, 20 lesioned and 8 controls.

Neurochemical assay After the behavioral tests, the rats were killed by decapitation. The brain was rapidly removed and chilled on an ice-cold plate. Brain regions (n.Acc. prefrontal cortex, antero~median and postero-dorsal parts of striatum) were dissected and assayed for dopamine and noradrenaline according to the method of Fekete et al. 7.

Statistical analysis For data analysis, when appropriate, parametric statistics (Student's t-test, analysis of variance) or

12 non-parametric statistics

( M a n n - W h i t n e y U-test)

FOUR HOLE BOX" EXPLORATION 1 5MIN

were used.

]

RESULTS

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n Acc

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Locomotor activity in the circular corridor As indicated in Table I the means for lesioned and control groups overlapped. There was no statistical difference in locomotor activity between lesioned and control groups over all the intervals studied.

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Exploration in four-hole box After the lesions, the exploratory behavior was reduced over the 15 min of the session (Fig, 1A and B) for both the n u m b e r of hole explorations (t = 6.8, df = 24, P < 0.001) and the total duration of explorations (t = 4.23, df = 24, P < 0.001). In addition, the

0 I

100-

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50-

latency to the first exploration was higher for the lesioned group (n.Acc. = 28 + 3 s, C = 7 + 2 s, t = 5.6, df = 24, P < 0.001). The time course of exploration for the control group was quite characteristic (Fig. 2B), i.e. a progressive reduction in the n u m b e r of explorations over the 15 min of the test with an increase in the duration of these explorations. For the lesioned group on the other hand, both the n u m b e r and the duration of the explorations r e m a i n e d stable over the 15 min of the test. Analysis of variance on the n u m b e r of hole visits indicated a group factor effect ( F = 9.78, df = 1,26, P < 0.005) and a time factor ef-

0-

Fig. 1. Exploratory behavior over 15 minutes in the 4-hole box. A: number (mean +_ S.E.M.) of hole visits. B: total duration (mean _+ S.E.M) of hole visits in seconds. *** P < 01001 Student's t-test.

shortest explorations ( 0 - 2 sec) decreased and the longest ( > 4 s) increased. This time course was not observed in the experimental group.

fect ( F = 9.29, df = 2,50, P < 0.001). The same difference was observed on the time course of duration of hole exploration, i.e. a group factor effect ( F = 5.64, df = 1, 26, P < 0.002), and time factor effect ( F = 4.32, df = 2, 50, P < 0.01). The differences are statistically significant at the beginning of the test for the n u m b e r of visits (t = 2.98, df = 24, P < 0.01) and at the end of the test for the duration (t = 2.82, df = 24, P < 0.01). The exploration spectrum analysis con-

FOUR HOLE BOX: E X P L O R A T I O N 15 MIN ~---4

_ 09 t09

.g

firmed these data (Fig. 3). For the control group the

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TABLE I

m

Groups (n) Control (8) n.Acc. (8)

1st hour 764 + 83 738 -4- 62

12 h dark phase 24 h circadian 187 1355 + 125 1686 +

2376 + 169 2459 + 221

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Effect of 6-OHDA lesions of n.Acc, on locomotor activity in a circular corridor

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Fig. 2. Time course of exploratory behavior in the 4-hole box (by 5 rain intervals). A: number of visitsl B:I duration Of visits: * P < 0.05, ** P < 0.01, Student's t-test.

13

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TIME ( M I N ) Fig. 3. Number of explorations in the 4-hole box (mean + S.E.M.), as a function of their duration. Representation by 5-rain periods: 0-2 short, 2-4 medium, > 4 s longer explorations in 4-hole box. ** P < 0.0l, Student's t-test.

T W O C O M P A R T M E N T FIELD : A C T I V I T Y IN C O M P A R T M E N T "A"

Two-compartment field test Exploration of the compartment A. D u r i n g the ses-

B~2Jn,Acc.

A LOCOMOTION

sions 1, 2 and 3 the rats w e r e not a l l o w e d to visit the

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B ( d o o r closed). B o t h g r o u p s w e r e

f o u n d to h a v e the s a m e l o c o m o t o r activity (Fig. 4 A ,

z >_2C o z Ld I,<~ .JIC I-r~ <~ I-U~ (3

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right). H o w e v e r , on the first day the start latency was

significantly l o n g e r for the e x p e r i m e n t a l g r o u p (t =

~3c

2.6, df = 24, P < 0.01). T h e n u m b e r and d u r a t i o n of rearings w e r e also significantly r e d u c e d o v e r the 3

sessions (Fig. 4B).

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Exploration of compartment B. F r o m the f o u r t h

session the d o o r was o p e n e d so that the rats could ex-

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32

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DAILY SESSIONS

plore c o m p a r t m e n t B ( n o v e l c o m p a r t m e n t ) f r o m the c o m p a r t m e n t A (familiar c o m p a r t m e n t ) . T h e latency

Fig. 4. General activity in compartment A of the 2-compartment field during the first three days of testing (10 min each day). Results are expressed in means + S.E.M. A: start latency to initiate locomotor activity (left panel), and number of squares crossed during the 10 min of test (right panel). B: number of rearings (left panel), and total duration of rearing in seconds (right panel). * P < 0.05, ** P < 0.01, *** P < 0.001, Student's t-test.

14

represented in Fig. 5A and B. The latency was signif-

n u m b e r of squares crossed was only significantly lower for the experimental group in session 4 (t = 3.5, df = 24, P < 0.01). The exploratory behavior of the

icantly higher for the lesioned group for session 4 (t = 3.01, df = 24, P < 0.05) and session 5 (t = 2.98, df =

duration of visits during the sessions 4, 5 and 6 (Fig.

24, P < 0.05) but was not significantly different for

5C). For the control group the time course of the

session 6. The locomotor activity measured by the

n u m b e r of explorations, high at the beginning and

to enter compartment B, and the n u m b e r of squares crossed in B (i.e. locomotor activity) for 10 rain are

TWO

COMPARTMENT FIELD • EXPLORATION AND ACTIVITY IN C O M P A R T M E N T "B"

Dn.Acc.

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novel e n v i r o n m e n t was evaluated by the n u m b e r and

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SESSIONS

Fig. 5. Exploration of the novel compartment (compartment B) of the 2-compartment field. Results are expressed in means _+_S.E.M. for days 4, 5 and 6 (10 rain each day). A: start latency in sec. to visit the novel compartment. B: number of squares crossed in compartment B. C: number of visits of compartment B. D: time spent in compartment B in seconds. * P < 0.05, ** P < 0.01. *** P < 0.001. Student's t-test.

15 declining with time (cf. Fig. 2), was not o b s e r v e d for the lesioned group whose e x p l o r a t o r y b e h a v i o r remained stable over time; the difference was only significant for session 4 (t = 2.51, df = 24, P < 0.05). Similar observations were m a d e for duration of the visits (Fig. 5D). Control animals begin with numerous visits of short duration and they gradually m a k e less visits each lasting longer. This adaptive behavioral strategy for exploration was not o b s e r v e d in lesioned rats (see also Fig. 2) and the differences between groups were significant for sessions 5 and 6 (day 5, t = 4.54, df = 24, P < 0.001, day 6, t = 3.8, df = 24, P < 0.001).

Exploration of a novel object and extinction (object omitted) A f t e r introducing an object in a corner square c o m p a r t m e n t B (sessions 7 and 8), the time spent this c o m p a r t m e n t increases in controls as well as rats with lesions c o m p a r a t i v e l y with the results

TWO

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COMPARTMENT FIELD • EXPLORATION E X T I N C T I O N IN C O M P A R T M E N T "B" ~

COMPARTMENT

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preceding day (session 6) without o b j e c t (Fig. 6). H o w e v e r , the time spent in the c o m p a r t m e n t (day 7, t = 3.02, df = 24, P < 0.01; day 8, t = 3.65, df = 24, P < 0.01) and also in the corner square (day 7, t = 2.99, df = 24, P < 0.001; day 8, t = 3.8, df = 24, P < 0.01) was shorter for lesioned rats (Fig. 6) comparatively to controls. H o w e v e r , the latency to the first contact with the object, the n u m b e r of contacts and the locom o t o r activity in c o m p a r t m e n t B was not significantly different b e t w e e n groups on days 7 and 8 (Table lI shows the data for day 7). On day 9, when the object was r e m o v e d , the exp l o r a t o r y behavior of control rats decreased. Comp a r e d to day 8 the time spent in the corner square when the object was present was significantly reduced (t = 7.05, df = 24, P < 0.001). The time spent in the square was: 250 ± 18 s on day 8 and 98 ± 9 s on day 9. Paradoxically, the lesioned rats did not reduce exploration either of the new c o m p a r t m e n t or in the square in which the object was placed (day 9, t = 5.9,

OBJECT[+] 1

OBJECT[.-]

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9 DAILY

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7

8

9

SESSIONS

Fig. 6. Object exploration and extinction in the 2-compartment field. Object was placed in the compartment B for two days (day 7 and 8) of the experiment. The object was taken away on day 9 and exploratory responses were measured. Results are expressed as means +_ S.E.M. A: time spent (seconds) in compartment B with (object +), and without object (object -). B: time spent (seconds) in the square containing the object and after removal of the object. ** P < 0.01, *** P < 0.001, Student's t-test.

16 'FABLE II

Effect of 6-OHDA lesions of n.Acc, in the two-compartment field test. Locomotor activity and object exploration in compartment B (session 7) Groups (n)

Locomotor activity

Latency to the Mean number 1st contact of object of object (s) contacts

Control (12) n.Acc. (14)

47.5 + 2.6 45.1 _ 3.2

12 _.+ 3 15 + 2

11 _+ 1 13 _+_ 1

df = 24, P < 0.001). The lesioned rats seemed to perseverate in their previous pattern of behavior. The time spent in the square was not altered either when the object was present or not (165 + 7 s on day 8 and 167 + 8 s on day 9).

Straight alley test The results (Fig. 7) did not show any difference between the two groups either for the start latency from the start box (F = 1.55, df = 1, 13, n.s.) or for the running time to reach the goal box (F = 0.79, df = 1, 13, n.s.). Classical learning curves were obtained for both groups. All parameters decreased from session 1 to session 5. However, on day 6, during the extinction session the lesioned rats continued to run (extinguished less) more rapidly than the controls: start latencies (C: 19.1 + 1.5 s; n.Acc.: 7.3 + 1.3 s, t = 5.99,

Step-through passive avoidance test The median step-through latencies were similar in both groups for the day 1 and the day 2 pretraining sessions as well as for the day 3 acquisition session (foot-shock), (Table III). Significant differences appeared in retention tests 6 and 24 h and persisted 15 days after the shocks (Mann-Whitney U-test: 6 h, U = 22, P < 0.05; 24 h, U = 25, P < 0.01; 15 days, U = 24, P < 0.05).

Neurochemistry The injection of 6-OHDA into the n.Acc, resulted in a large reduction of endogenous dopamine concentrations in this structure (-93% in comparison with control group) and in prefrontal cortex (-60%) (Table IV). Dopamine concentration was also reduced in the antero-median striatum (-60%), and moderately in postero-lateral striatum (-30%). The pretreatment with DMI prior to 6 - O H D A injections led to partial protection of noradrenergic systems which were destroyed at 41% in the n.Acc., 30% in the prefrontal cortex and in the striatum. DISCUSSION

The biochemical data showed that the 6-OHDA injections into the ventral striatum-n.accumbens region almost completely depleted endogenous D A

STRAIGHT ALLEY RUNNING FOR FOOD REWARD ~-~

df = 14, P < 0.001; running time: C: 32 _+ 2.tL n.Acc.: 18_+ 3.1, t = 3.15, df = 14, P < 0.01).

n Acc

RUNNING

START LATENCY ACGUISLTION EXTINCTION

T A B L E III

TIME

30O

300

d 200

Effect of 6-OHDA lesions of n.Acc, on a passive avoidance task. Step-through latencies in s (medians), and semi-interquartile ranges Statistical significance with Mann-Whitney U-test.

Testing conditions 10o

Control (8)

n.Acc. (20)

100

, DALLY

o

ESSIONS

Fig. 7. Acquisition and extinction of the runway task. The acquisition was performed over 5 days, and the extinction phase (reinforcement omitted) took place the 6th day. Each rat was submitted to I0 trials per day. A: latency to leave the start box on the 5 days of test, and on the 6th day (extinction day). B: running time for reaching the goal box on the 5 days of test, and on 6th day (day of extinction).

Pretraining Day 1 Day 2 Acquisition Day 3 Retention + 6h + 24 h + 15 days * P < 0.05. ** P < 0.01.

12.5 (7-17) 7 (5-20)

14 (11-25) 7 (6-20)

5.5 (4-9)

6 (4-20)

140 (45-300) 173 (60-300) 95 (30-300)

75 (10-300)* 62 (5-3(K1)~* 28 (4-300)*

17 TABLE IV Biochemical analysis of dopamine and noradrenaline contents in n.A cc. and other regions after 6-OHDA lesions of the nucleus aceumbens

Statistical significance with Student's t-test. Groups (n)

Dopamine (ng/g tissue) n.Accumbens

Anteromedian striatum

Posterolateral striatum

Prefrontal cortex

Control(12) n.Acc.(14) Depletion (%)

9570 ± 720 670 ± 140 -93%***

10450 ± 370 4170 ± 690 -60%***

10950 ± 800 7460 ± 650 -30%*

89.2 ± 49 36.1 ± 17 -60%

Groups (n)

Noradrenaline (ng/g tissue)

Control (12) n.Acc. (14) Depletion (%)

n.A ccumbens

Anteromedian striatum

Posterolateral striatum

Prefrontal cortex

434.9 _+ 28 259.5 _+ 28 -41%**

94.3 + 9 67.9 ± 13.0 -28%*

76.1 _+ 8.3 51.7 _+ 9.0 -32%*

425.9 +_ 20.4 298.0 ± 43.0 -30%*

* P < 0.05. ** P < 0.01. *** P < 0.001.

( - 9 3 % ) . A more limited, but significant reduction in D A was also found in other A10 p r o j e c t i o n areas, such as the a n t e r o - m e d i a n striatum and prefrontal cortex. U n f o r t u n a t e l y it seems impossible with the 6 - O H D A lesion m e t h o d to wholly d e p l e t e D A levels in the n.accumbens, and avoid lesion of D A fiber innervating prefrontal cortex and striatum. It is therefore possible, given the anatomical relationships of these structures, that some aspects of the behavioral syndrome observed may be due to disorders in the prefrontal cortex. H o w e v e r , data from this laboratory 34 and elsewhere21. 25 have shown that specific lesions of the a n t e r o - m e d i a l prefrontal cortex lead to a different pattern of deficits. It is generally accepted that lesion of the supra-rhinal region rather than that of antero-medial part of the prefrontal cortex produces disturbances reminiscent of the 'limbic syndrome'. Bilateral lesions of the dopaminergic terminals within the ventral striatum-n,accumbens induce a complex behavioral s y n d r o m e with deficits in exploration which do not a p p e a r to be due to changes in spontaneous l o c o m o t o r activity. P a r a m e t e r s such as mean duration of a single exploration or percentage of alternating choices are not affected by overall locomotor activity. Results from the 4-hole box and the t w o - c o m p a r t m e n t field showed, not only that the number and duration of explorations were reduced,

but that the time course and overall pattern of these p a r a m e t e r s (exploration spectrum) were affected by the lesions. This deficit in e x p l o r a t o r y behavior was d e m o n s t r a t e d in a forced exploration task (4-hole box) as well as in a choice exploration task (two-comp a r t m e n t field). M o r e o v e r , this deficit was also observed when the novelty was either an open space or a discrete object. As has been pointed out by Kohler and Srebro 20, only comparison of several tests of exploratory behavior and m e a s u r e m e n t of different p a r a m e t e r s enable the various forms of exploratory behavior to be distinguished. W e also found that amount of exposure to the test situation r e p r e s e n t e d a significant variable. Using the terminology of Berlyne 2 we investigated the 'inquisitive' exploration of a new environment (first minutes of 4-hole box exploration, first sessions of the t w o - c o m p a r t m e n t field), and an 'inspective' exploration caused by the physical presence of a discrete novel object, or as elicited in preference testing (e.g. during days 4, 5 and 6 in the t w o - c o m p a r t m e n t field). These behaviors represent a complex interplay of curiosity (approach) and emotionality (fear-avoidance)2.20, 33. In this study we used situations where the rat could either explore a novel object in a familiar environment (directed e x p l o r a t o r y behavior), or situations of forced exploration where the levels of emotionality and fear are p r e s u m e d to be different. It has been

lX suggested that different central nervous system lesions can discriminate between the approach and avoidance components 20. After 6-OHDA lesions of the n.accumbens, both exploratory behaviors (inspective and forced inquisitive) were markedly altered. For the exploration of a novel object, it was only the duration of exploration that was affected (Fig. 7B). There were no differences between control and experimental groups in locomotor activity, latency to first contact with the object and mean number of contacts with the object (Table II). This indicated that the deficit is not a result of non-specific motor or emotional disturbances, or of failure to detect novelty. Latency to respond has also been shown to be an important parameter in exploratory behavior. Our results showed that the lesioned rats had an increased latency to initiate a response in a novel situation. This was observed in the 4-hole box test, on the first day in the two-compartment arena, and after the door between compartment A and B had been opened. This deficit is observed mainly in exploration of a new space rather than a new object (Table II). It should be pointed out that this disturbance also depends on the motivational state of the animal. Motivational and learning deficits were not observed in the experimental group. They behaved like controls in the runway test and showed normal acquisition in the T-maze test 39. There was also no difference between the groups in latency to run, when food deprived. The other main characteristic of the n.accumbens syndrome is the absence of behavioral flexibility. The animals have difficulty in suppressing an ongoing behavior and switching to another. This was shown in the extinction trials of the runway test, and in exploration of the corner square after removal of the object. In the 4-hole box (number and duration of visits) and the two-compartment field (time and number of periods spent in compartment B on days 4, 5 and 6) the behavior was not just a simple reduction in exploration, but rather a perseveration of a previous behavior. These results were indicative of both temporal and spatial perseveration. Although deficits in memory cannot be excluded as an explanation of the

results in the passive avoidance task, we reel that they can more readily be explained in terms of an exaggerated response while in a state of high emotional arousal. Our results are in partial agreement with data from other laboratories. We did not find a significant reduction in locomotor activity13.~2 after n.accumbens lesions. However, it should be borne in mind that this parameter is dependent on duration of the measurement period32, the nature of the test, and maybe on effects of the lesions external to the n.accumbens itself. It has been shown, for example, that after large 6-OHDA lesions in the hypothalamus, denervating large catecholamine terminal fields in the forebrain with less destruction in the A9 and 10 cell groups, that rats showed less rearing and were less active in an open fieldS.9. These animals also displayed abnormal exploratory behavior, and investigated novel objects to a lesser extent. There were no signs of 'hyperemotionality' or sensorimotor disturbances. On the basis of these and the present results it would appear that this syndrome is more complex than previously

thoughtS.9,22,32. In view of the behavioral results reported in this study we have been led to wonder whether the motor-striatal and limbic components of the n.accumhens have a functional as well as anatomical basis, and whether it might be possible to dissociate the two. The behavioral disturbances resulting from lesions to these two systems have been amply described, although the exact functions of each are still unclear. It does appear that deficits in behavioral initiation may be due to sensorimotor disturbances 40 while deficits in exploration and lack of behavioral flexibility result from limbic dysfunction 11.~,. It is therefore possible that the ensemble of the behavioral disturbances that we observed is due to a dysfunction in limbic/sensorimotor coordination carried out by the n.accumbens. The D A neurons in this structure appear to be involved in the modulation of both types of functions and none of the deficits described above can be fitted into a specific dopaminergic role 35. Additionally, these results may help throw light on the non-motor disturbances seen in Parkinson's disease.

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