Exp Brain Res (1987) 69:183-194

Experimental Brain Research �9 Springer-Verlag 1987

Dopamine neurons grafted unilaterally to the nucleus accumbens affect drug-induced circling and locomotion

P. Brundin, R.E. Strecker, E. Londos, and A. Bj6rklund

Department of Medical Cell Research, University of Lund, Biskopsgatan 5, S-22362 Lund, Sweden

Summary. The purpose of the present study was to investigate the amplifying function of the nucleus accumbens septi region (NAS) in 6-hydroxy- dopamine (6-OHDA)-induced rotational behaviour by implanting fetal dopamine (DA)-rich mesence- phalic cell suspensions unilaterally in the NAS of rats previously subjected to combined mesostriatal (MS) and NAS 6-OHDA lesions. First, all the rats received a unilateral 6-OHDA lesion of the ascending MS DA pathway, which produced an amphetamine-induced rotational asymmetry towards the lesioned side. In a second step, the rats received a local bilateral

6-OHDA lesion of the NAS which, as previously shown, caused a significant attenuation of the amphetamine-induced locomotor (1.5 mg/kg) and rotational (5 mg/kg) behaviour. Finally, some of these MS + NAS lesioned rats received a unilateral mesencephalic DA graft into the NAS ipsilateral to the original MS lesion. The unilateral DA-rich grafts in the NAS significantly elevated the amphetamine- induced locomotion and ipsilateral circling (opposite to the direction of rotation produced when a graft is placed in the ipsilateral caudate-putamen), suggest- ing that the NAS plays only an amplifier role in locomotor behaviour and not a directional role. In addition, these grafts significantly attenuated the supersensitive locomotor response observed in lesioned rats when given apomorphine (0.05 mg/kg). The findings emphasize the amplifying role of the NAS in locomotion and circling behaviour and they extend previous findings demonstrating the func- tional heterogeneity of the striatal complex as well as the regional specificity of the graft-derived functional effects. Moreover, the results argue against the notion that DA grafts can function through a diffu- sion of transmitter over large distances since, despite the large size of the grafts, the functional graft effects

Offprint requests to: P. Brundin (address see above)

were well localized to the reinnervated NAS and ventromedial striatal regions. We conclude, there- fore, that graft-induced amelioration of postural and locomotor deficits are affected through different parts of the striatal complex, and that multiple graft placements are required to produce more complete recovery of motoric behaviour in the DA-depleted brain.

Key words: Neural transplantation - Nucleus accum- bens - Dopamine - Circling behaviour - Locomotor activity - Amphetamine - Apomorphine

Introduction

A large majority of the dopamine (DA) neurons in the mammalian brain are found bilaterally in 2 partially overlapping, adjacent midbrain structures, the substantia nigra (A9 DA cell group after Dahl- str6m and Fuxe, 1964) and the ventral tegmental area (VTA; A10 DA cell group). The cell bodies and projections of these cell groups constitute, respec- tively, the mesostriatal (MS) DA system (A9) and the mesolimbocortical DA system (A10) (for reviews see Bj6rklund and Lindvall 1984, 1986).

The DA neurons of the MS pathway predomi- nantly innervate the caudate putamen (CP), particu- larly the dorsolateral portion, a region which has been implicated in the control of body posture (Bj6rklund and Lindvall 1986). Unilateral 6-hy- droxydopamine (6-OHDA) lesions of the MS system produce an asymmetry of striatal DA innervation and give rise to a complex behavioural syndrome characterized in part by both an asymmetric body posture, in which the head is turned towards the lesioned side, and by spontaneous circling, also

184

towards the lesioned side (Anddn et al. 1966). The spontaneous circling towards the lesioned side can be greatly enhanced by treatment with DA releasing drugs, such as amphetamine, which unilaterally increase functional DA activity on the intact side (Ungerstedt and Arbuthnott 1970). This so-called rotational model of DA function has been widely used, both in studies aimed at understanding the role of the nucleus accumbens septi region (NAS) in behaviour, and as an animal model of Parkinson's disease in neural grafting studies. For example, fetal mesencephalic DA neurons grafted to the dener- vated CP have been shown to reverse amphetamine- induced turning in this rotational model (Bj6rklund and Stenevi 1979; Perlow et al. 1979; Bj6rklund et al. 1980; Dunnett et al. 1981a, 1983; cf. Brundin and Bj6rklund 1987 for review).

A prominent target for the mesolimbocortical DA system is the NAS (Bj6rklund and Lindvall 1984, 1986; Swanson 1982), a structure thought to act as an interface between limbic and striatal systems, pos- sibly regulating motivational aspects of motor behaviour (Mogenson et al. 1980). Several studies indicate that DA receptors in the NAS mediate the increases in locomotor activity produced by the administration of direct or indirect DA receptor agonists. For example, both 6-OHDA lesions that denervate the NAS region (Kelly et al. 1975; Kelly and Iversen 1976; Fink and Smith 1980; Koob et al. 1981; Makanjuola and Ashcroft 1982; Carey 1983; Joyce et al. 1983; Kelly and Roberts 1983) and bilateral injections of DA receptor antagonists in the NAS (Jackson et al. 1975; Pijnenburg et al. 1975a) can block the locomotor activation caused by a peripheral injection of the DA releasing drug amphetamine. The blocking effect of 6-OHDA appears to specifically apply to motor stimulants acting via a DA mechanism (Joyce and Koob 1981). On the other hand, following 6-OHDA lesions that denervate the NAS, rats respond to low doses of directly-acting DA agonists, such as apomorphine, with a marked increase in locomotor behaviour, an effect thought to be mediated by supersensitive DA receptors (Kelly et al. 1975; Fink and Smith 1980; Koob et al. 1981; Carey 1983; Joyce et al. 1983; Kelly and Roberts 1983). Furthermore, the local injection of DA, or directly-acting DA agonists, into the NAS of rats produces a marked increase in locomotor activity in normal or reserpine treated rats (Pijnen- burg and van Rossum 1973; Jackson et al. 1975; Pijnenburg et al. 1975b; And6n and Johnels 1977; Costall et al. 1977; Makanjuola et al. 1980; Johnels 1982) which can be reversed by subsequent intra- NAS or systemic injection of haloperidol (Pijnenburg et al. 1975b; Makanjuola et al. 1980).

In agreement with these data, recent work has shown that the locomotor hyporesponsiveness to amphetamine, seen in rats with bilateral 6-OHDA lesions of the NAS or the VTA, can be reversed by bilateral implantation of fetal DA neurons into the NAS (Dunnett et al. 1984; Nadaud et al. 1984; Herman et al. 1985, 1986). Finally, Kelly and Moore (Kelly and Moore 1976; Moore and Kelly 1978) have clarified the role of the NAS versus the CP in amphetamine-induced circling and hypothesized that the DA released in the CP governs the direction of circling behaviour, whereas DA release in the NAS increases the speed of circling.

In the present study we used the neural grafting technique to investigate the ability of the NAS to drive amphetamine-induced locomotor and turning behaviour by implanting fetal DA-rich mesence- phalic cells unilaterally in the NAS of rats (see Fig. 1 for experimental design). The design was as follows: All the rats first received a MS lesion which produced marked ipsilateral rotation. Next, the rats received a bilateral 6-OHDA injection into the NAS locally which, as shown by others, caused a significant reduction of the amphetamine-induced locomotor (Kelly et al. 1975; Kelly and Iversen 1976; Joyce and Koob 1981; Koob et al. 1981; Makanjuola and Ashcroft 1982; Joyce et al. 1983; Kelly and Roberts 1983) and rotational behaviour (Kelly and Moore 1976). Finally, some of the rats received a unilateral DA graft in the NAS ipsilateral to the original MS lesion. Note that according to previous graft studies a DA suspension graft in the ipsilateral CP of these animals would normalize the ipsilateral rotation (Dunnett et al. 1983; cf. Brundin and Bj6rklund 1987 for review). However, our hypothesis was as follows: if the NAS only plays an amplifying role in locomotor behaviour and not a directional role, then DA grafts in the ipsilateral NAS should actually produce an increase in ipsilateral rotation (opposite to the direc- tion resulting from a DA graft in the CP, as shown in Fig. 1A). Similarly, in open-field locomotion this unilateral graft was predicted to normalize the level of amphetamine-induced locomotor activity, which had been suppressed by previous MS and NAS lesions. Finally, apomorphine-induced locomotion was studied in order to examine graft-induced changes in DA receptor supersensitivity.

Material and methods

Experimental design and subjects

The time intervals for the different steps in the experiment are summarized in Fig. lB. Five normal control rats (female Sprague- Dawley rats, ALAB Stockholm, Sweden, 180-200 g at the start of

185

A NAS~

Amph. rotation

% f / z I t /

MS lesion

< [ ] =6-hydroxydopamine lesion d =dopamine rich neural graft MS = mesostriata[ NAS =nucleus accumbens septi CP =caudate putamen DA = dopamine AMPH = amphetamine

MOORE KEL Y, CP lesion NAS lesion

I PRESENT ~TUD NAS lesion DA graft in NAS

% ~ IEARLIER GRAFT STUDIESI

DA graft in CP

B - -1 -2 1 - 2 . - - 1.5 1 - - 1 ~g\ 1.5--0-1 ~, WEEKS

/ / / / , / .'-a, oe~ .o e .,--~-o e , - ~ d

Fig. 1. A The top diagram shows the present experimental design as well as the results of previous studies (cf. Moore and Kelly 1978; Brundin and Bj6rklund 1987). Note that a unilateral DA graft in the NAS is predicted to produce an increase in rotation towards the lesioned side, an effect opposite to that seen when similar grafts are placed in the ipsilateraI CP. B A summary of the sequence of surgery and behavioural tests with the time intervals indicated in weeks

the experiment) and 25 rats with extensive unilateral 6-OHDA- induced MS lesions were selected for the study. Five of the MS rats were left as MS lesion controls while the remaining 20 MS rats were given bilateral local injections of 6-OHDA into the NAS.

Fourteen of the 20 MS + NAS lesioned rats were selected for the rest of the study on the basis of showing diminished behavioural responses to amphetamine challenge (at least a 30% reduction in the rotation response compared to pre-NAS lesion level); they were divided into 2 groups matched with regard to

their behavioural performance. Seven of these MS + NAS lesioned rats received unilateral mesencephalic neural grafts in the NAS, ipsilateral to the MS lesion, while the remaining 7 MS + NAS lesioned rats were given sham injections of glucose-saline.

The rats were tested for amphetamine-induced rotation and locomotor activity, and apomorphine-induced locomotor activity, both before and after graft or sham injections. Finally, all 14 MS + NAS lesioned rats were analysed microscopically using a modified Falck-Hillarp catecholamine histofluorescence technique.

186

Unilateral 6-OHDA MS lesion

Forty rats were given a total of 13.5 ~g of 6-OHDA (Sigma) in two injections into the right ascending MS pathway under methohexi- tal barbiturate (Brietal) anaesthesia. Briefly, 2.5 ~tl of 6-OHDA (3 gg/~tl, free base, in 0.2 mg/kg ascorbate-saline) was injected at the following coordinates: -4.4 mm caudal to bregma; 1.2 mm lateral to midline; 7.8 mm ventral to dural surface; with the toothbar set at -2.4 mm below the interaural line. A second injection of 2 gl of 6-OHDA (same concentration as above) was performed at the following coordinates: -4.0 mm caudal to bregma; 0.8 mm lateral to midline; 8.0 mm ventral to dural surface; with the toothbar set at +3.4 mm above the interaural line. 6-OHDA was infused at a speed of 1 ~tl/min, and the cannula was left in place another 4 min before being withdrawn.

Bilateral 6-OHDA NAS lesion

Each injection consisted of 12 ~xg 6-OHDA (fi'ee base) in 3 btl ascorbate-saline. Stereotaxic coordinates: Toothbar: + 5.0 mm; AP: +3.2 ram; L: 1.7 ram; V: 7.0 and 6.5 mm (from bregma and dura respectively.). 1.5 ~tl was injected at each of the two sites over a period of about 4 min, and the cannula was left in place another 4 rain before it was withdrawn.

Grafting procedure

Ventral mesencephalic cells, obtained from 14-15 day old rat fetuses, were grafted unilaterally into the NAS, ipsilateral to the initial MS lesion, according to the cell suspension grafting tech- nique (see e.g. Brundin et al. 1985). Briefly, the ventral mesence- phalon was dissected, the tissue incubated for 20 min in trypsin at 37 ~ C, rinsed with sterile glucose-saline, before gentle mechanical dissociation using a fire-polished Pasteur pipette. Two • 1.5 [xl of cell suspension, equivalent to 36 x 10 4 viable cells, as determined with acridine orange and ethidium bromide as a vital stain (Brundin et al. 1985), was injected on the right side at coordinates identical to those used for the NAS lesion. Rats in the sham group received 2 x 1.5 ,ul of vehicle (glucose-saline) at the same coordinates.

Behavioural tests

Amphetamine-induced locomotor activity test. The rats were tested in automated locomotor activity boxes (50 x 50 cm) with 4 evenly spaced photocell beams perpendicular to each wall. Rats were habituated for 60 rain before monitoring of D-amphetamine- induced locomotor activity (1.5 mg/kg, i.p.) over 60 min. This test only monitored the rats' general locomotor activity and did not take into account any motor asymmetry exhibited by the rats.

Apomorphine-induced locomotor activity test. The rats were habituated for 40 rain before monitoring of apomorphine-induced locomotor activity (0.05 mg/kg, s.c. in the neck) over 40 min.

Amphetamine-induced rotation test. Rotational behaviour was followed for 90 min following a single injection of D-amphetamine (5.0 mg/kg, i.p.) in automated rotometer bowls (Ungerstedt and Arbuthnott 1970) which monitored half turns in either direction. A net rotation asymmetry score was calculated by subtracting the number of full turns performed in a direction contralateral to the MS lesion from the number of turns performed ipsilateral to the MS lesion.

Histology

Ten to eleven weeks post-grafting all the rats in the grafted and non-grafted MS + NAS lesioned groups were perfused for catecholamine histochemistry using the ALFA technique (Lordn et al. 1980). Briefly, the rats were perfused through the ascending aorta with Tyrode's buffer followed by ice-cold 2% formaldehyde solution containing a high concentration of A12SO4. The brains were rapidly dissected and the forebrain frozen in liquid propane cooled by liquid nitrogen. After 6 days of freeze-drying the specimens were reacted with formaldehyde vapour for 1 h at 80 ~ C. Finally, the specimens were embedded in paraffin and coronal sections cut through the rostral CP at 15 ~tm thickness. The extent of DA loss due to the 6-OHDA lesion was examined, and in grafted rats the graft derived DA innervation was moni- tored and surviving grafted DA neurons were counted on every third section with the final cell count being adjusted according to the formula of Abercrombie (1946). The statistical correlation between the number of DA neurons in the graft and behavioural changes in the grafted rats was then investigated.

Results

Effect o f unilateral 6 - O H D A M S lesion

F r o m t h e g r o u p of 40 ra ts w i t h 6 - O H D A les ions o f

t he M S D A sys t em, 25 ra ts w e r e s e l e c t e d to par t ic i -

p a t e in t he s tudy . T h e s e ra ts i n d i v i d u a l l y e x h i b i t e d a

m e a n a m p h e t a m i n e - i n d u c e d ips i l a t e ra l r o t a t i o n o f at

leas t 6.75 tu rns p e r ra in o v e r 90 rain w h i c h is

cons i s t en t w i t h at l eas t a 9 7 % d e p l e t i o n o f D A in t h e

C P on t h e l e s i o n e d s ide ( S c h m i d t e t al. 1982). T h e 25

rats w e r e d i v i d e d in to a M S l e s ion c o n t r o l g r o u p (n =

5, m e a n r o t a t i o n a s y m m e t r y p e r m i n o v e r 90 m i n =

9.2 + 0.8 S . E . M . ) and a g r o u p tha t s u b s e q u e n t l y

r e c e i v e d N A S les ions (n = 20, m e a n r o t a t i o n a sym-

m e t r y p e r ra in o v e r 90 m i n = 9 .2 + 0.5 S . E . M . ) .

W h e n t e s t e d f o r a m p h e t a m i n e - i n d u c e d loco-

m o t o r ac t iv i ty t h e M S l e s i o n e d c o n t r o l g r o u p (n = 5)

e x h i b i t e d a s ign i f i can t ly d e c r e a s e d m e a n l o c o m o t o r

r e s p o n s e c o m p a r e d to t h e n o r m a l c o n t r o l rats (n = 5)

(Fig. 4). T h e s a m e M S l e s i o n e d c o n t r o l g r o u p

s h o w e d , in a d d i t i o n , a s ign i f i can t ly i n c r e a s e d m e a n

l o c o m o t o r s c o r e in r e s p o n s e to a p o m o r p h i n e w h e n

c o m p a r e d to t h e rats in t h e n o r m a l c o n t r o l g r o u p

(Fig. 5) (p < 0.01, t (8) = 3 .50; S t u d e n t ' s t - tes t ) .

Effect o f bilateral 6 - O H D A N A S lesion

T h e b i l a t e ra l i n t r a - N A S i n j e c t i o n s o f 6 - O H D A re su l t ed in a s ign i f i can t r e d u c t i o n o f t h e

amphetamine- induced rotation r e s p o n s e fo r t h e c o m - p l e t e M S + N A S l e s i o n e d g r o u p (n = 20), f r o m 9.2

t u r n s / m i n to 5.3 t u r n s / m i n o v e r 90 m i n ( t (19) = 4 .97; p < 0.001 p a i r e d S t u d e n t ' s t - t e s t ) . F r o m this g r o u p ,

15-

AMPHETAMINE INDUCED ROTATION OF SELECTED MS RATS BEFORE AND AFTER BILATERAL NAS LESION

10-

Z - - _o,~ P-- o)

"-- 5 -

E

,N\'g

,x,\',?

unilateral DA rich graft to NAS

(n=7)

sham graft operation ~ n = 7 )

MS MS MS MS Division of NAS + lesioned rats into

NAS graft or sham graft (n=5) (n=14) (n=5) (n=l/,) groups

pre NAS lesion post NAS Lesion

Fig. 2. Net amphetamine-induced (5 mg/kg i.p.) rotation asym- metry of selected MS lesioned rats before and after bilateral NAS lesions. The group receiving bilateral 6-OHDA lesions of the NAS (hatched bars) showed a significant reduction in amphetamine- induced rotation when compared to either their pre-NAS lesion values (*indicates p < 0.001, t(13) = 10.82; paired Student's t- test), or to the MS lesioned group which did not receive a NAS lesion (solid bars) (+ indicates/) < 0.001, t(17) = 7.23; Student's t- test). There was no significant change in rotation between the two tests in the MS lesioned group, although there was a trend towards an increase, possibly related to amphetamine sensitization (p > 0.05, t(4) = 3.06; paired Student's t-test). As illustrated, the rats receiving NAS lesions were subsequently divided into two matched groups (n = 7 for each) used for either unilateral DA grafting to the NAS or sham graft surgery

the 14 rats that showed the greatest reduction in rotation were selected to participate in the rest of the study. These rats exhibited a significant mean reduc- tion in their rotational response to 36% of their individual pre-NAS-lesion rotation values (Fig. 2) (range 4%-67%, p < 0.001; paired Student's t-test).

Furthermore, the rats' locomotor activity response to amphetamine in the selected MS + NAS lesioned group (n = 14) was significantly reduced to only a mean of 14% of the mean value obtained in the normal control rats (Fig. 4) (range 2-57%, p < 0.001, t(17) = 14.27; Student's t-test). The selected MS + NAS lesioned group showed a significantly increased locomotor activity in response to a low dose of apomorphine, to a level of 597% of the mean for the normal control group (Fig. 5) (range 277-1104%, p < 0.001, t(17) --- 4.60; Student's t- test).

Z

t - - o Pc-

15-

E

10-

I/I

- - 5 - c Ig ID

E

AMPHETAMINE INDUCED ROTATION (5 mg/kg)

t ?* 187

t I PRE- GRAFT POST-GRAFT

J ~ - - - A MStesion only (n=5)

o .. . . . . O MS+NAS lesion (~=7) ~. MS+NAS lesion+unilateral graft (n=7)

Fig. 3. The effect of DA neurons grafted to the NAS on amphetamine-induced (5 mg/kg i.p.) rotation asymmetry. Mean amphetamine-induced rotation asymmetry ipsilateral to the mesostriatal lesion for the 3 experimental groups is shown at timepoints before and after grafting. The NAS lesion significantly decreased rotation (see also Fig. 2) illustrated here by the pre-graft timepoint where both the 2 groups with NAS lesions show significantly less rotation asymmetry than the MS lesion only group (ANOVA, F(2, 16) = 24.63 followed by Newman-Keuls' multiple comparisons; + indicates different from MS lesion group). The DA-rich transplants in the NAS, ipsilateral to the MS lesion, significantly restored the rotational behaviour that had been reduced by the bilateral NAS lesion (two-factor ANOVA, group x time interaction F(2, 16) = 16.65; p < 0.01). Nine weeks after grafting, the rotation in the grafted rats was significantly elevated compared to the MS + NAS lesioned rats (* indicates p < 0.05; ANOVA, F(2, 16) = 4.91, followed by Newman-Keuls' multiple comparisons) and it did not differ from the MS lesioned group (p > 0.05). The nngrafted MS + NAS lesioned group continued to show a significantly reduced rotational response compared to the MS group, demonstrating the long duration of the bilateral NAS lesions effect on amphetamine rotation (+ indicates p < 0.05; ANOVA followed by Newman-Keuls' multiple compari- sons as above)

Effects of unilateral intra-NAS grafts

Amphetamine-induced circling. The DA-rich grafts in the NAS significantly increased rotational behaviour over time in the rats with combined MS + NAS lesions (Fig. 3) (two-factor ANOVA, group x time interaction; p < 0.01). Nine weeks after transplanta- tion the rotation in the grafted rats was significantly elevated compared to the non-grafted MS + NAS lesioned rats and did not differ from the MS lesioned control group (ANOVA; p < 0.05, followed by

188

t -

E

0 3

- > 2

I - - - -

o 8 o

0 5 o , =

r - r~

E

250-

200-

150-

100-

50-

AMPHETAMINE INDUCED LOCOMOTOR ACTIVITY (1.5 mg/kg)

..........

I I

PRE-GRAFT POST-GRAFT

~ l r - - - A MS lesion only In=5) O ...... O MS+NAS lesion (n=7)

~. MS+NAS lesion+unilateral graft (n=7) I - . - . - I Normal rots in=5)

Fig. 4. Amphetamine-induced open field locomotor activity for the 3 experimental groups and normal controls at timepoints before and after grafting. Before grafting, the 3 lesioned groups all exhibited significantly decreased responses to amphetamine (+ indicates p < 0.05; AN OVA F(2, 20) = 62.84, p < 0.01; followed by Newman-Keuls' multiple comparisons). The DA-rich unilateral grafts in the NAS significantly restored amphetamine-induced locomotor activity over time (two-factor ANOVA, group x time interaction F(3, 20) = 20.13; p < 0.01), an effect which was similar to the grafts' effect on amphetamine rotation (Fig. 3). Nine weeks after grafting this amphetamine-induced locomotor activity was significantly elevated in grafted rats compared to the MS + NAS lesioned group and the MS lesioned group (* indicates p < 0.01), and did not differ from the normal control group (ANOVA F(3,

20 ) = 12.34, p < 0.01; followed by Newman-Keuls' multiple comparisons). However, in the same test both the MS + NAS lesioned group and the MS lesioned group still showed a signifi- cantly reduced amphetamine-induced locomotor activity com- pared to the normal controls (+ indicates p < 0.05). Note that the parallel increases in locomotor activity seen over time in the 3 control groups are believed to be due to amphetamine sensitiza- tion and the parallel nature of the increases indicates that the 3 groups were all affected equally

N e w m a n - K e u l s ' m u l t i p l e c o m p a r i s o n s ) . By con t r a s t , the MS + N A S l e s i o n e d g r o u p still s h o w e d a s igni f icant ly r e d u c e d r o t a t i o n a l r e s p o n s e c o m p a r e d to the MS l e s i o n e d g roup .

Amphetamine-induced locomotor activity. T h e D A - r ich grafts in the N A S s ign i f ican t ly r e s t o r e d the a m p h e t a m i n e - i n d u c e d l o c o m o t o r ac t iv i ty r e s p o n s e in the MS + N A S l e s i o n e d rats (Fig. 4) ( two fac tor A N O V A , g r o u p x t i m e i n t e r a c t i o n ; p < 0.01). N i n e

>

rw" 0 I-- 0

o

8 _ J

I00-

80- r-

In 03 C

o 60- b E

o ~ ~0- o

r -

0)

E 20-

APOMORPHINE INDUCED LOCOMOTOR ACTIVITY (0.05 mg/kg)

/ + /

/ / / / /

I PRE-GRAFT PO ST-GRAFT

A - ~ MS lesion only (n=5) O ...... o MS+NAS lesion (n=7)

-- M S + N A S lesion+unilateral graft (n=7) II-.--.-II Normal rots (n=5)

Fig. 5. Apomorphine-induced (0.05 mg/kg, s.c.) locomotor activity for the 3 experimental groups and normal controls at timepoints before and after grafting. At the pre-graft timepoint the 3 lesion groups all displayed a significantly elevated response to apomor- phine compared to the normal control group (+ indicates p < 0.05; ANOVA F(3, 20) = 8.29; p < 0.01, followed by Newman- Keuls' multiple comparisons). The DA-rich grafts in the NAS significantly reduced the supersensitive response to apomorphine over time (two-factor ANOVA, group x time interaction F(3, 20) = 7.68; p < 0.01). Nine weeks after transplantation the apomor- phine-induced locomotor activity in the grafted rats was signifi- cantly reduced compared to the MS + NAS and the MS lesioned groups (* indicates p < 0.01), and did not differ from the normal control group (p > 0.05) (ANOVA F(3, 20) = 5.91; p < 0.01, followed by Newman-Keuls' multiple comparisons). However, in the same test both the MS + NAS lesioned group and the MS lesioned group still showed significantly increased responses to apomorphine compared to normal controls (+ indicates p < 0.05)

weeks af ter t r a n s p l a n t a t i o n the a m p h e t a m i n e - i n d u c e d l o c o m o t o r ac t iv i ty in the g ra f t ed rats was s igni f icant ly e l e v a t e d c o m p a r e d to the MS + N A S and MS l e s i o n e d g roups , a n d it d id n o t differ f rom the n o r m a l con t ro l g r o u p ( A N O V A ; p < 0.01, fo l lowed by N e w m a n - K e u l s ' m u l t i p l e c o m p a r i s o n s ) . H o w e v e r , b o t h t he MS + N A S l e s i o n e d g r o u p a n d MS l e s i o n e d g roups still s h o w e d a s ign i f ican t ly

189

Fig. 6. Catecholamine histofluorescence photomicrograph showing a coronal section through the brain of a rat with a dopamine (DA)-rich neural transplant at the level of the nucleus accumbens septi region (NAS). This rat was first given a 6-OHDA lesion of the right mesostriatal (MS) pathway, and subsequently local injections of 6-OHDA were performed into the NAS bilaterally. Finally, the rat received the unilateral DA-rich transplant (t) into the right NAS. As a consequence of the MS lesion, the right hemisphere shows an absence of DA innervation in the dorsolateral striatum (dls). Conversely, the left hemisphere which did not receive this MS lesion has an intact DA innervation in the dorsolateral striatum, but lacks DA innervation in the NAS (star) and anteromedial striatum due to the local intra-NAS 6-OHDA injection. In the right hemisphere, where a similar 6-OHDA injection was given in the NAS region, the DA innervation has now been restored by the DA neurons in the cell suspension transplant. However, as the graft derived DA fibers do not reach the dorsolateral striatum, this region remains denervated. Cracks in the brain are artifacts due to the rapid freezing employed (Lordn et al. 1980) in order to obtain optimal catecholamine histofluorescence, ac = anterior commissure. The photograph was taken by Ragnar M~rtensson using his newly developed automated dark-field condensor scanner equipment (Mfirtensson and Bj6rklund 1984)

r educed a m p h e t a m i n e - i n d u c e d l o c o m o t o r act ivi ty c o m p a r e d to the n o r m a l cont ro ls .

Apomorphine-induced locomotor activity. The D A - rich grafts in the N A S s ignif icant ly r e d u c e d hype r r e - spons iveness to a p o m o r p h i n e over t ime (Fig. 5) ( two- fac tor A N O V A , g roup x t ime in te rac t ion ; p < 0.01). Nine weeks af te r t r a n s p l a n t a t i o n the a p o m o r p h i n e - induced l o c o m o t o r act ivi ty in the g ra f ted rats was s ignif icant ly r e d u c e d c o m p a r e d to the MS + N A S les ioned and MS l e s ioned g roups , and d id no t differ f rom the n o r m a l con t ro l g roup ( A N O V A ; p < 0.01, fo l lowed by N e w m a n - K e u l s ' mu l t ip l e compar i sons ) . H o w e v e r , bo th the MS + N A S l e s ioned and the MS

les ioned group still showed a s ignif icant ly inc reased response to a p o m o r p h i n e when c o m p a r e d to the no rma l cont ro l s (p < 0.05).

Spontaneous locomotor activity. Befo re graf t ing, the spon taneous open f ield l o c o m o t o r act ivi ty of the two MS + N A S les ion groups (n = 7 each) and the MS les ioned g roup , as assessed in the 40 rain pre- a p o m o r p h i n e h a b i t u a t i o n p e r i o d ( m e a n pho toce l l b e a m cross ing/min + S. E. M. for the MS les ion only group = 29.7 + 5.1; 29.4 + 5.9 for MS + N A S + sham group; 20.3 + 3.1 for MS + N A S + graf t g roup) , was s ignif icant ly r e d u c e d c o m p a r e d to nor- mal cont ro l rats (59.6 + 7.2 for normals ; A N O V A ,

190

F(3, 20) = 9.40; p < 0.05, followed by Newman- Keuls' multiple comparisons). However , the lesioned groups did not differ f rom each other (p > 0.05).

Following grafting one might expect to see an increase in spontaneous locomotor activity in the rats with D A grafts in the NAS. However , all the lesioned groups displayed a recovery in spontaneous locomotion over t ime (2-factor A N O V A , time effect F(1, 20) = 14.94; p < 0.05) and the recovery did not differ between groups (group • t ime interaction F(3, 20) = 2.60; p > 0.05) (post-transplantation mean values + S .E .M. : MS _+ NAS lesion group = 44.9 + 6.3; MS + NAS lesion + unilateral graft group = 68.9 +_ 19.2; MS group = 49.2 +_ 3.5; normal control group = 63.2 _+ 6.2).

Histology

Denervation. On the side of the MS Iesion there was a total depletion of catecholamine histofluorescence in the dorsolateral CP. In the MS + NAS lesioned rats the NAS and the anteromedial striatum showed bilaterally a substantial loss of catecholamine fibers. Typically there was a total absence of histofluores- cence in the central NAS, dorsal olfactory tubercle and anteromedial striatum (Fig. 6), and very little trace of non-specific damage due to the local 6- O H D A injection.

Graft size and placement. All the grafts were mainly localized in the NAS in close proximity to the anterior commissure (Fig. 6). The DA-rich grafts typically gave rise to a virtually normal D A terminal network in most of the NAS and dorsal olfactory tubercle, reinnervating a zone of up to approximately 1.5 mm radius. Fluorescent cell bodies and graft derived reinnervation was also seen to a variable extent along the injection tract in the anteromedial striatum. Graft derived innervation was totally absent in the dorsolateral half of the CP. The number of D A cell bodies in the grafts ranged from 546 to 2826, with a mean of 1440 +_ 291 (+_ S .E .M. ) . A standard linear regression analysis revealed no sig- nificant linear correlation between the number of surviving graft D A neurons and changes in amphetamine-induced circling and locomotor activ- ity, or in apomorphine-induced locomotor activity.

Discussion

The present investigation confirms earlier studies that have shown that the selective destruction of catecholamine terminals by bilateral 6 - O H D A injec-

LEFT

cP INASI RIGHT

AMPLIFIER I

SIDEBIAS RIGHT i LEFT

ROTATIONAL RESPONSE

Fig. 7. Diagram illustrating the interaction between the caudate- putamen (CP) and nucleus accumbens septi region (NAS) in rotational behaviour (adapted from Moore and Kelly 1978). According to this model the side bias is governed from the CP, and the amplitude of the rotational behaviour by the NAS. If there is a relatively greater DA release in the CP on one side (e.g. if the contralateral side is denervated by 6-OHDA) the resulting motor behaviour will involve a rotational response in the contralateral direction. The magnitude or intensity of this rotational response, i.e., the number of body turns per unit time, can be increased by DA release in the NAS on either side of the brain (thej'amplifier" function). The results of the present study conform to this model as they show that increasing DA activity in a single 6-OHDA denervated NAS, by grafting DA rich fetal tissue unilaterally, does not affect the direction of the side bias in a rat with a 6-OHDA lesion of one CP. However, the same unilateral graft in the NAS amplifies the already existing rotational response and causes an increase in the speed of turning

tions directly into the NAS (Kelly et al. 1977) can dramatically reduce the open field locomotor activity induced by amphetamine t rea tment (Kelly et al. 1975; Kelly and Iversen 1976; Joyce and Koob 1981; Koob et al. 1981; Makanjuola and Ashcroft 1982; Joyce et al. 1983; Kelly and Rober ts 1983). In addition, we have found that a unilateral D A rich graft implanted in only one of the lesioned NAS is sufficient to reinstate and drive this amphetamine- induced locomotion. We also studied rotational behaviour since, in addition to the bilateral NAS lesion, these rats had a unilateral MS lesion which depleted D A throughout the ipsilateral striatal com- plex. In previous graft studies using this MS lesion rotational model, D A cell suspension grafts which had been placed in the central or dorsolateral striatum, ipsilateral to the MS lesion, produced a large decrease in amphetamine- induced rotation (Dunnett et al. 1983; He rman et al. 1985; Brundin et al. 1985, 1986). However , in the present study similar D A grafts placed unilaterally in the NAS ipsilateral to the MS lesion, gave rise to an increase in rotation,

191

i.e., the opposite effect of a DA graft placed further laterally in the same striatal complex.

These results support the hypothesis proposed by Moore and Kelly (1978) for the functional role of the NAS. According to this hypothesis (see Fig. 7), based on 6-OHDA lesion studies (Kelly 1975; Kelly and Moore 1976), the NAS is involved only in the amplitude of locomotor behaviour and not in the direction of locomotion, whereas the striatum is involved in the postural and directional aspects of locomotion (e.g. in rotational behaviour). Thus, as demonstrated in the present study, grafting of DA neurons into the NAS of a brain hemisphere previ- ously deprived of its DA innervation can paradoxi- cally also give rise to an increase in amphetamine- induced rotational asymmetry.

These results emphasize the amplifying role of the NAS in locomotion and rotation and extend previous findings demonstrating the regional specific- ity of the functions of the striatal complex in rota- tional behaviour. A recent study by Sharp et al. (1987) has elegantly demonstrated this regional specificity by correlating in vivo changes in DA release with amphetamine-induced changes in behaviour. They found that increased locomotor activity was well-correlated with the time course and amount of DA released in the NAS in response to amphetamine, whereas it was not correlated with the DA released in the striatum. Striatal DA release, on the other hand, was closely correlated with the intensity of stereotypic head and forepaw move- ments. In a similar vein, Freed and Yamamoto (1985) found that the concentrations of DA and its metabolite 3,4-dihydroxyphenylacetic acid were increased in DA terminal regions in rats after run- ning on straight or circular treadmills, without drug treatment, The changes in DA metabolism in the NAS were more strongly linked to the speed of the movement, whereas changes in DA metabolism in the CP were more strongly related to the posture (Freed and Yamamoto 1985).

These studies are consistent with previous lesion studies (Kelly 1975; Kelly et al. 1975; Makanjuola and Ashcroft 1982; Carey 1983) as well as experi- ments involving direct intracerebral application of DA active drugs (Pijnenburg and van Rossum 1973; Jackson et al. 1975; Pijnenburg et al. 1975a; Anddn and Johnels 1977; Costall et al. 1977; Makanjuola et al. 1980; Johnels 1982), indicating that specific motor behaviours originate from distinct DA nerve terminal areas in the striatal complex. In agreement with this, grafts of DA neurons have previously been found to selectively affect different aspects of the behavioural syndrome that follows after an MS lesion depending

on the area of the CP innervated by the implant (Dunnett et al. 1981a, 1981b, 1983).

Previous grafting experiments have indicated that grafts of fetal mesencephalic DA neurons can rein- nervate the NAS (Bj6rklund et al. 1983) and when grafted bilaterally affect parameters of locomotor activity (Nadaud et al. 1984; Dunnett et al. 1984; Herman et al. 1985, 1986). In the present study the reinnervation of only one NAS was sufficient to completely restore amphetamine-induced locomo- tion. Even the smallest grafts, containing as few as 500 DA cell bodies, were able to restore amphetamine-induced locomotor activity and cir- cling. As it has been suggested that a relatively large proportion of the total of 18,000 DA neurons situ- ated in the VTA bilaterally project to the NAS (Swanson 1982), 500 grafted DA neurons probably represent only a small portion of the VTA DA neurons that normally bilaterally innervate the two NAS. Thus, as with DA grafts in the CP counteract- ing amphetamine-induced rotation (Brundin et al. 1985), it seems necessary to substitute for only a minor portion of the number of DA neurons nor- mally innervating the grafted region to obtain a marked locomotor effect under amphetamine stimu- lation in rats with grafts into the NAS. There was no linear correlation between the number of surviving grafted DA neurons and changes in behavioural parameters. However, when compared to a prior pilot study with smaller grafts (Poulsen et al. 1987), the effects on amphetamine-induced rotation in the present study were similar but more pronounced. Moreover, it is likely that the number of surviving grafted DA neurons in each rat in the present study saturated the NAS, and similar to grafts in the CP reversing amphetamine-induced turning, there is a ceiling effect beyond which additional DA neurons or graft-derived innervation will not further affect the behaviour (Bj6rklund et al. 1980; Brundin et al. 1987).

The effect of the unilateral DA graft in the NAS on spontaneous locomotion was more unclear. The spontaneous locomotion in the grafted group was normalized. However, due to a parallel partial recov- ery of locomotor behaviour in the MS + NAS lesioned group, there was no statistically significant difference between the recovery in spontaneous locomotion in the MS + NAS lesioned group and the grafted group.

Consistent with previous studies (Kelly et al. 1975; Fink and Smith 1980; Koob et al. 1981; Carey 1983; Joyce et al. 1983; Kelly and Roberts 1983), the rats with bilateral 6-OHDA lesions of the NAS showed marked increases in the locomotor response following a low dose of apomorphine. The unilateral

192

DA grafts in the NAS were also able to reduce this supersensitive locomotor response following apo- morphine, providing additional evidence that the grafts release DA also under spontaneous conditions. This was somewhat surprising since supersensitive areas are still expected to exist in the contralateral NAS and in the ipsilateral non-reinnervated striatum.

The results of the present study argue against the notion that intraparenchymal DA-containing grafts can function through a diffusion of transmitter over large distances, as could be suggested for grafts of adrenal medullary tissue and intraventricular DA neural grafts (Olson et al. 1985). Despite the rela- tively large size of the present grafts, and the graft- reinnervated region, the functional graft effects were well localized to the NAS and ventromedial striatal regions. Thus, had diffuse release of DA been responsible for the graft induced effects, one might expect this diffusion to reach and influence the ipsilateral dorsolateral striatum and cause a decrease in rotational asymmetry (opposite to that observed), The histofluorescence analysis showed that the region of fluorescent DA reinnervation around the graft did not extend more than approximately 1.5 mm around the actual graft borders (Fig. 6), Grafted DA neurons have previously been shown to form synapses with striatal neurons in the host brain (Freund et al. 1985, Mahalik et al. 1985). Possibly, the DA release is functionally efficient only at these synaptic sites and any "overflow" of DA may be rapidly metabolized, thus preventing extracellular diffusion over great distances. Indeed, using the in vivo voltammetry technique, recent data suggest that only DA released in the immediate vicinity (distances < 20 ~m) of the voltammetry probe can be moni- tored (Kuhr and Wightman 1986; Stamford et al. 1986). This idea is further supported by the voltam- metric data of Gonon and Buda (1985), which indicate that the DA concentration extrajunctionally is two orders of magnitude lower than within the synapses in a normal striatum. Moreover, experi- ments with local injections of DA into the NAS have shown that the amount of DA necessary to elicit a locomotor response is very high (bilateral injections of 1 ~g giving a significant, but not maximal, locomotor response; Pijnenberg and van Rossum 1973; Costall et al. 1977; Jackson et al. 1977). This amount exceeds the total normal hemisphere content of DA (approximately 0.6 ~tg; Schmidt et al. 1983), and is 20-30 fold greater than the total DA content of neural grafts of a similar type and size as used in the present study (Schmidt et al. 1983). This suggests that the DA synthesized in grafted DA neurons is much more efficient at affecting host DA receptors

than DA injected directly into the same region, thus supporting the view that the functional effects pro- duced by the mesencephalic grafts are mediated primarily through DA release at specific synaptic sites. The above discussion illustrates the usefulness of the neural grafting technique in exploring regional brain function. Either alone, or in combination with lesions, intracerebral neural grafting provides a tool to permanently alter local brain function and related behaviour, unlike local intracerebral infusions of neurotransmitters or drugs, whose effects are short- lived.

Recent data, showing that dissociated human fetal DA neurons can survive intracerebral grafting to rat striatum and give rise to functional effects (Brundin et al. 1986), has emphasized the possibility that this neural grafting technique may be applied clinically in patients with Parkinson's disease. The localized effect of cell suspension grafts demonstrates the importance of correct graft placement in order to obtain maximal functional recovery in 6-OHDA lesioned rats or, by inference, in Parkinsonian patients. Maximal recovery from the widespread DA depletion that occurs in Parkinson's disease (for review, see Scatton et al. 1984) would be best served by multiple implants of DA grafts throughout the caudate nucleus, putamen and NAS. Interestingly, recent work has quantitatively demonstrated that the relative severity of impairments in posture and locomotion varies among Parkinsonian patients and suggests that this variation may be attributable to individual variation in the degree and regional pat- tern of DA cell degeneration in the brain (Johnels et al. 1987). Although further knowledge about the functional role of the NAS in primates is necessary, based on the present results one may speculate that individual Parkinsonian patients with predominantly postural symptoms may be best served by grafts in the caudate nucleus and/or putamen, whereas cases that show impairments primarily in locomotor initia- tion and speed may particularly benefit from local grafts in the NAS.

Acknowledgements. The technical assistance of Katarina Friberg, Yvette J6nsson, Ragnar Mfirtensson, Agneta Persson, and Ger- trude Stridsberg is gratefully acknowledged. This study was supported by grants from the Swedish MRC (04X-3874) and the Thorsten and Elsa Segerfalks Stiftelse.

References

Abercrombie M (1946) Estimation of nuclear population from microtome sections. Anat Rec 94:239-247

And6n N-E, Dahlstr6m A, Fuxe K, Larsson K (1966) Functional

193

role of the nigro-neostriatal dopamine neurons in the rat. Acta Pharmacol 24:263-274

And6n N-E, Johnels B (1977) Effect of local application of apomorphine to the corpus striatum and to the nucleus accumbens on the reserpine-induced rigidity in rats. Brain Res 133:386-389

Bj6rklund A, Stenevi U (1979) Reconstruction of the nigrostriatal pathway by intracerebral nigral transplants. Brain Res 177: 555-560

Bj6rklund A, Dunnett SB, Stenevi U, Lewis ME, Iversen SD (1980) Reinnervation of the denervated striatum by substantia nigra transplants: functional consequences as revealed by pharmacological and sensorimotor testing. Brain Res 199: 307-333

Bj6rklund A, Stenevi U, Schmidt RH, Dunnett SB, Gage FH (1983) Intracerebral grafting of neuronal cell suspensions. II. Survival and growth of nigral cell suspensions implanted in different brain sites. Acta Physiol Scand Suppl 522:9-18

Bj6rklund A, Lindvall O (1984) Dopamine-containing systems in the CNS. In: Bj6rklund A, H6kfelt T (eds) Handbook of chemical neuroanatomy, Vol 2. Classical transmitters in the CNS. Elsevier, Amsterdam, pp 55-122

Bj6rklund A, Lindvall O (1986) Catecholaminergic brain stem regulatory systems. In: Bloom FE (ed) Handbook of physiol- ogy. The nervous system, Vol 4. Intrinsic regulatory systems in the brain. Am Physiol Soc, Bethesda MD, pp 155-235

Brundin P, Isacson O, Bj6rklund A (1985) Monitoring of cell viability in suspension of embryonic CNS tissue and its use as a criterion for intracerebral graft survival. Brain Res 331: 251-259

Brundin P, Nilsson OG, Strecker RE, Lindvall O, ~stedt B, Bj6rklund A (1986) Behavioural effects of human fetal dopamine neurons grafted in a rat model of Parkinson's disease. Exp Brain Res 112:235-240

Brundin P, Barbin G, Strecker RE, Isacson O, Prochiantz A, Bj6rklund A (1987) Survival and function of dissociated dopamine neurons at different developmental stages or after being cultured in vitro. Dev Brain Res (in press)

Brundin P, Bj6rklund A (1987) Survival, growth and function of dopamine neurons grafted to the brain. In: Seil FJ, Herbet E, Carlson B (eds) Neural regeneration. Progress in brain research, Vol 71. Elsevier, Amsterdam, pp 293-308

Carey RJ (1983) Differential effects of limbic versus striatal dopamine loss on motoric function. Behav Brain Res 7: 283-296

Costall B, Naylor RJ, Cannon JG, Lee T (1977) Differentiation of the dopamine mechanisms mediating stereotyped behaviour and hyperactivity in the nucleus accumbens and caudate- putamen. J Pharm Pharmacol 29:337-342

Dahlstr6m A, Fuxe K (1964) Evidence for the existence of monoamine neurons in the central nervous system. I. Demon- stration of monoamines in the cell bodies of brain stem neurons. Acta Physiol Scand Suppl 232:1-55

Dunnett SB, Bj6rklund A, Stenevi U, Iversen SD (1981a) Behavioural recovery following transplantation of substantia nigra in rats subjected to 6-OHDA lesions of the nigrostriatal pathway. I. Unilateral lesions. Brain Res 215:14%161

Dunnett SB, Bj6rklund A, Stenevi U, Iversen SD (1981b) Grafts of embryonic substantia nigra reinnervating the ventrolateral striatum ameliorate sensorimotor impairments and akinesia in rats with 6-OHDA lesions of the nigrostriatal pathway. Brain Res 229:20%217

Dunnett SB, Bj6rklund A, Schmidt RH, Stenevi U, Iversen SD (1983) Intracerebral grafting of neuronal cell suspensions. IV. Behavioural recovery in rats with unilateral 6-OHDA lesions following implantation of nigral cell suspensions in different brain sites. Acta Physiol Scand Suppl 522:29-37

Dunnett SB, Bunch ST, Gage FH, Bj6rklund A (1984) Dopamine- rich transplants in rats with 6-OHDA lesions of the ventral tegmental area: effects on spontaneous and drug-induced locomotor activity. Behav Brain Res 13:71-82

Fink JS, Smith GP (1980) Relationships between selective dener- vation of dopamine terminal fields in the anterior forebrain and behavioral responses to amphetamine and apomorphine. Brain Res 201:107-127

Freed CR, Yamamoto BK (1985) Regional brain dopamine metabolism: a marker for the speed, direction, and posture of moving animals. Science 229:62-65

Freund TF, Bolam JP, Bj6rklund A, Stenevi U, Dunnett SB, Powell JF, Smith AD (1985) Efferent synaptic connections of grafted dopaminergic neurons reinnervating the host neo- striatum: a tyrosine hydroxylase immunocytochemical study. J Neurosci 5:603-616

Gonon FG, Buda MJ (1985) Regulation of dopamine release by impulse flow and by autoreceptors as studied by in vivo voltammetry in the rat striatum. Neuroscience 14:765-774

Herman J-P, Choulli K, Le Moal M (1985) Hyper-reactivity to amphetamine in rats with dopaminergic grafts. Exp Brain Res 60:521-526

Herman J-P, Choulli K, Geffard M, Nadaud D, Taghzouti K, Le Moal M (1986) Reinnervation of the nucleus accumbens and frontal cortex of the rat by dopaminergic grafts and effects on hoarding behavior. Brain Res 372:210-216

Jackson DM, And6n N-E, Dahlstr6m A (1975) A functional effect of dopamine in the nucleus accumbens and in some other dopamine-rich parts of the rat brain. Psychopharmacologia (Berl) 45:13%149

Johnels B (1982) Locomotor hypokinesia in the reserpine-treated rat: drug effects from the corpus striatum and nucleus accumbens. Pharmacol Biochem Behav 17:283-289

Johnels B, Ingvarsson P, Rydgren U, Thorselius M, Steg G (1987) Measuring motor function in Parkinson's disease. In: Benecke R, Conrad B, Marsden CD (eds) Motor disturbances II. Academic Press, London, pp 131-144

Joyce EM, Koob G (1981) Amphetamine-, scopolamine- and caffeine-induced locomotor activity following 6-hydroxy- dopamine lesions of the mesolimbic dopamine system. Psychopharmacology 73:311-313

Joyce EM, Stinus L, Iversen SD (1983) Effects of injections of 6- OHDA into either nucleus accumbens septi or frontal cortex on spontaneous and drug-induced activity. Neuropharmacol- ogy 22:1141-1145

Kelly PH (1975) Unilateral 6-hydroxydopamine lesions of nigro- striatal or mesolimbic dopamine-containing terminals and the drug-induced rotation of rats. Brain Res 100:163-169

Kelly PH, Seviour PW, Iversen SD (1975) Amphetamine and apomorphine responses in the rat following 6-OHDA lesions of the nucleus accumbens septi and corpus striatum. Brain Res 94:507-522

Kelly PH, Iversen SD (1976) Selective 6-OHDA induced destruc- tion of mesolimbic dopamine neurons: abolition of psycho- stimulant induced locomotor activity in rats. Eur J Pharmacol 40:45-56

Kelly PH, Moore KE (1976) Mesolimbic dopaminergic neurones in the rotational model of nigrostriatal function. Nature 263: 695-696

Kelly PH, Joyce EM, Minneman KP, Phillipson OT (1977) Specificity of 6-hydroxydopamine-induced destruction of mesolimbic or nigrostriatal dopamine-containing terminals. Brain Res 122:382-387

Kelly PH, Roberts DCS (1983) Effects of amphetamine and apomorphine on locomotor activity after 6-OHDA and elec- trolytic lesions of the nucleus accumbens septi. Pharmacol Biochem Behav 19:137-143

194

Koob GF, Stinus L, Le Moal M (1981) Hyperactivity and hypoactivity produced by lesions to the mesolimbic dopamine system. Behav Brain Res 3:341-359

Kuhr WG, Wightman RM (1986) Real-time measurement of dopamine release in rat brain. Brain Res 381:168--171

Lordn I, Bj6rklund A, Falck B, Lindvall O (1980) The aluminium- formaldehyde (ALFA) method for improved visualization of catecholamines and indolamines. I. A detailed account of the methodology for central nervous tissue using paraffin, cryo- stat or vibratome sections. J Neurosci Meth 2:277-300

Makanjuola ROA, Dow RC, Ashcroft GW (1980) Behavioural responses to stereotactically controlled injections of mono- amine neurotransmitters into the accumbens and caudate- putamen nuclei. Psychopharmacology 71:227-235

Makanjuola ROA, Ashcroft GW (1982) Behavioural effects of electrolytic and 6-hydro• lesions of the accumbens and the caudate-putamen nuclei. Psychopharmacology 76: 333-340

M~rtensson R, Bj6rklund A (1984) Low-power photography in the fluorescence microscope using an automated dark-field condensor scanner. In: Bj6rklund A, H6kfelt T (eds) Hand- book of chemical neuroanatomy, Vol 2. Classical transmitters in the CNS Elsevier, Amsterdam, pp 380-386

Mogensen GJ, Jones DL, Yim CY (1980) From motivation to action: functional interface between the limbic system and the motor system. Prog Neurobiol 14:69-97

Moore KE, Kelly PH (1978) Biochemical pharmacology of mesolimbic and mesocortical dopaminergic neurons. In: Lip- ton MA, Di Mascio A, Killam KF (eds) Psychopharmacology: a generation of progress. Raven Press, New York, pp 221-234

Mahalik TJ, Finger TE, Str6mberg I, Olson L (1985) Substantia nigra transplants into denervated striatum of the rat: ultra- structure of graft and host interconnections. J Comp Neurol 240:60-70

Nadaud D, Herman JP, Simon H, Le Moal M (1984) Functional recovery following transplantation of ventral mesencephalic cells in rats subjected to 6-OHDA lesions of the mesolimbic dopaminergic neurons. Brain Res 304:137-141

Olson L, Backlund E-O, Freed W, Herrera-Marschitz M, Hoffer B, Seiger •, Str6mberg I (1985) Transplantation of mono- amine-producing cell systems in oculo and intracranially: experiments in search of a treatment for Parkinson's disease. Ann NY Acad Sci 457:105-126

Perlow MJ, Freed WJ, Hoffer BJ, Seiger A, Olson L, Wyatt RJ (1979) Brain grafts reduce motor abnormalities produced by destruction of nigrostriatal dopamine system. Science 204: 643-647

Pijnenburg AJJ, van Rossum JM (1973) Stimulation of locomotor activity following injection of dopamine into the nucleus accumbens. J Pharm Pharmacol 25:1003-1005

Pijnenburg AJJ, Honig WMM, van Rossum JM (1975a) Inhibition of d-amphetamine-induced locomotor activity by injection of haloperidol into the nucleus accumbens of the rat. Psychopharmacologia (Berl) 41:87-95

Pijnenburg AJJ, Honig WMM, van Rossum JM (1975b) Effects of antagonists upon locomotor stimulation induced by injection of dopamine and noradrenaline into the nucleus accumbens of nialamide-pretreated rats. Psychopharmacologia (Berl) 41: 175-180

Poulsen E, Brundin P, Strecker RE, Bj6rklund A (1987) Fetal dopamine neurons implanted unilaterally into the nucleus accumbens drive amphetamine-induced locomotion and cir- cling. Ann NY Acad Sci 495:774-776

Scatton B, Javoy-Agid F, Montfort JC, Agid Y (1984) Neurochemistry of monoaminergic neurons in Parkinson's disease. In: Usidin E, Dahlstr6m A, Engel J (eds) Catecholamines, Part C. Neuropharmacology and central nervous system - therapeutic aspects. Alan R Liss Inc, New York, pp 43-52

Schmidt RH, Ingvar M, Lindvall O, Stenevi U, Bj6rklund A (1982) Functional activity of substantia nigra grafts reinner- vating the striatum: neurotransmitter metabolism and [14C]2- deoxy-D-glucose autoradiography. J Neurochem 38:737-748

Schmidt RH, Bj6rklund A, Stenevi U, Dunnett SB, Gage FH (1983) Intracerebral grafting of neuronal cell suspensions. III. Activity of intrastriatal nigral suspension implants as assessed by measurements of dopamine synthesis and metabolism. Acta Physiol Scand Suppl 522:19-28

Sharp T, Zetterstr6m T, Ljungberg T, Ungerstedt U (1987) A direct comparison of amphetamine-induced behaviours and regional brain dopamine release in the rat using intracerebral dialysis. Brain Res 401:322-330

Stamford JA, Kruk ZL, Millar J (1986) Sub-second striatal dopamine release measured by in vivo voltammetry. Brain Res 381:351-355

Swanson LW (1982) The projections of the ventral tegmental area and adjacent regions: a fluorescent retrograde tracer and immunofluorescence study in the rat. Brain Res Bull 9: 321-353

Ungerstedt U, Arbuthnott GW (1970) Quantitative recording of rotational behavior in rats after 6-hydroxy-dopamine lesions of the nigrostriatal dopamine system. Brain Res 24:485-493

Received February 26, 1987 / Accepted July 31, 1987