Dynamic regulation of striatal dopaminergic grafts during locomotor activity

BRAIN RESEARCH Brain Research 711) (1996) 45-55

ELSEVIER

Research report

Dynamic regulation of striatal dopaminergic grafts during locomotor activity S. Hattori

a,b Y.

Hashitani b, N. Matsui ", H. Nishino b.,

~ Department of Orthopedic Surgery, Nagoya City University Medical School, Mizuho-ku, Nagoya 467, Japan b Department of Physiology, Nagoya City Unit,ersity Medical School Mizuho-ku, Nagc>va46Z Japan Accepted 3 October 191)5

Abstract The present experiment was designed to estimate the neurochemical activity of dopaminergic grafts in hemiparkinsonian model rats during locomotion and to examine the functional importance of dynamic regulation of the grafted neurons in the host brain. Rats were trained to run on a straight treadmill at various speeds (300, 660, 1200, 1800 cm/min), and extracellular dopamine (DA) and its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), were measured by in vivo microdialysis during and after running. Grafted rats were divided into two groups depending on their running ability and data were compared with those of normal and lesioned controls. Although the tonic level of exlracellular DA in grafted rats recovered to 70% of control, levels of DOPAC and HVA remained 15-20% of controls. A small number of grafted rats showed full recovery in treadmill running tasks. In these animals, the percentage increase in DOPAC and HVA showed similar time courses and magnitudes as those in normal rats. Most grafted rats showed partial recovery in locomotor ability. The percentage increase in DOPAC and HVA in these animals remained at a lower level than that in normal rats, though the tonic levels of DA, DOPAC and HVA were not lower than those of fully recovered rats. Data suggest that grafted DAergic cells in functionally well recovered rats were dynamically regulated in the host brain in an actual behavior and that well-controlled release of DA might be involved in the recovery of complex motor behavior, such as high speed Iocomolion. Kevwords: Dopaminc; Locomotion; Treadmill; Striatum; Neural transplantation; 6-Hydroxydopamine; Dynamic regulation: In viw) microdialysis

1. Introduction Extensive studies on neural transplantation have been carried out in animal models of Parkinson's disease and the substantial functional effects of the transplants have been reported in several behavioral tests [ 5 , 1 4 16,24,39,44]. However, complex functional deficits such as aphasia, adipsia, exploring and hoarding behavior (without pretreatment with amphetamine), and skilled paw-reaching tasks have not been ameliorated in animal experiments [7,15,16,26], even if sufficient numbers of D A neurons survived in the host brain and extracellular levels of DA were increased to reverse stereotyped motor dysfunctions, such as s p o n t a n e o u s / d r u g - i n d u c e d motor asymmetry and sensory deficit [5,14,15,39]. Functional recovery following neural grafts may depend on multiple mechanisms such as d i f f u s e / s y n a p t i c release of neurotransmitter, trophic actions on the host, expression of substrate molecules and reconstruction of neurocircuitry

* Corresponding author. Fax: (81) (52) 842-3069. 00116-8993/96/$15.011

¢~, 1996 Elsevier Science B.V. All rights reserved

SSDI 0 0 / ) 6 - 8 9 9 3 t 9 5 ) 0 1 3 0 0 - 8

[4,29,50]. If grafted DAergic cells survive and can provide substantial innervation of the denervated striatum, released D A could exert various effects at a cellular level beyond reinnervated areas. The supersensitivity of DAD_, receptors, the increased level of preproenkephalin m R N A as well as the immunoreactivity of neuropeptide Y, and the presynaptic DA transporter activity might be normalized [3,13,19,33,41], and the released DA could restore inhibitory control over postsynaptic striatal cholinergic and G A B A e r g i c neurons [10,42]. Ultrastructural studies indicated that extensive efferent synaptic connections were formed from grafted to host neurons even in adult animals [20,30,38]. Furthermore, grafted DAergic neurons received afferent inputs both from local grafted neurons and from the host brain, however, host-to-graft connections were very sparse [8,36]. Electrophysiological studies have revealed that not only grafted neurons received afferent inputs from host neurons [2], but also that they responded to the host stimuli in a similar manner to normal host neurons [9,17,29,45,49]. Although, there are controversial data that denied functional afferent inputs in the grafts

46

S. Hattori et al. / Brain Research 710 (1996) 45-55

from the host brain [11,27,48], these observations suggested a possibility of partial restoration of reciprocal feedback regulation between grafted DAergic neurons and host brain [21,32]. Establishment of reciprocal innervation between the graft and host might be most important, because it makes it possible for the grafts to release DA phasically under the extrinsic regulation and to maintain DA levels within or close to the optimal range for each behavior. As shown in a previous report from our laboratory, extracellular levels of DA in intact rat striatum are maintained within a physiological range by the adjustment of DA turnover, in response to the intensity of treadmill exercise [25]. These results led us to hypothesize that recovery in regulated release of DA is essential for substantial functional improvement of complex motor behaviors in DA-depleted animals, although we need to consider that controlled DA release does not necessarily reflect restoration of reciprocal neurocircuitry [28,44]. The present experiment was designed to estimate the neurochemical activity of DAergic grafts during locomotion and to examine whether dynamic regulation of the grafted neurons in the host brain is essential for better behavioral recovery. Firstly, we assessed maximal locomotor ability of control, 6-OHDA-lesioned and fetal DAergic cell grafted rats quantitatively using a treadmill running test, as described in detail elsewhere [23-25]. Then, we investigated the time courses and magnitudes of alterations in DA turnover in the striatum during and after running at various speeds and therefore as a function of quantitatively measured exercise.

2. Materials and methods

2.1. Animals Young Std:Wistar/ST rats weighing 80-90 g at the start of the experiment (Chyubu Kagaku, Nagoya, Japan) were used and maintained on a 12-h light-dark cycle (lights on 07.00 h) in a temperature-controlled room at 25 _+ I°C. Animals were fed standard laboratory chow and had free access to water. Animal care was according to guidelines approved by the Nagoya City University Medical School.

2.2. 6-OHDA lesion Under sodium pentobarbital anesthesia (40 m g / k g , i.p., Pitman-Moore), 6-hydroxydopamine (6-OHDA, 8 /xg free base in 4 / z l of ascorbate-saline, Sigma) was injected into the left substantia nigra pars compacta (SNc, A, 2.0 mm from zero point; L, 1.6 mm; V, 7.2 mm from brain surface, according to the K6nig and Klippel stereotaxic atlas) to make chemical lesion in the unilateral nigrostriatal DAergic pathway (hemiparkinsonian models) [37].

2.3. Methamphetamine-induced rotations After 6-OHDA lesion, rats were tested for rotational behavior in a cylindrical container for 60 rain after methamphetamine (3 m g / k g , i.p.) injection. Rats that showed significant methamphetamine-induced ipsilateral rotations (more than 8 turns per min over 60 min) in two methamphetamine tests at 1 and 2 weeks after the lesion were grouped as 6-OHDA-lesioned rats. The test was repeated at 2, 4, 8, and 12 weeks after the grafting to evaluate the recovery.

2.4. Grafting of fetal nigral cells" Three weeks after 6-OHDA lesion, the ventral mesencephalon from fetal rats (E15-16 days) that contained DAergic neurons of SNc and ventral tegmental area was cut into small pieces and incubated in 0.05% trypsin (Sigma type II)-0.6% glucose-saline for 30 min at 37°C. After adding DNase and trypsin inhibiters, tissues were dissociated into cell suspensions by gentle pipetting (cell density, about 5 X l07 cells/ml; cell viability, more than 95%) [6,37]. An amount of 10 /xl of cell suspensions was grafted into two sites in the striatum ipsilateral to the lesions (5 /zl in each; A, _+0.5 mm from bregma; L, 2.8/3.2 mm; V, 5.0 mm from the surface) [36]. Rats that showed no ipsilateral methamphetamine-induced rotations at 3 months after the grafting were designated as grafted rats.

2.5. Treadmill running test Locomotor ability of control, 6-OHDA-lesioned and grafted rats was evaluated by a treadmill running test [23,24] 3 months after the lesion/graft. Rats were trained to run on a straight treadmill (15 cm wide, 40 cm long conveyor belt) for 20 min per day for 7 successive days at a speed of either 300 c m / m i n (n = 8 in each group), 660 c m / m i n (n = 8 in each group) or 1800 c m / m i n (n = 8 in control, n = 9 in lesioned and n = 13 in grafted rats). When rats could not keep up with the treadmill speed, they touched a grid located behind the belt that delivered a weak electric stimulation (ES). The number of ES that each rat received in the second 10 min was counted [23]. Daily training reduced the number of ES and an average number of ES that each rat received for the second 10 rain on the 5th to 7th day (termed 'treadmill running score (TRS)') seemed more accurately to reflect running ability on the treadmill [24]. Treadmill running ability is inversely proportional to the number of ES. Control, lesioned and grafted rats, that were tested at a speed of 300 c m / m i n , were re-evaluated their running ability at a speed of 1200 c m / m i n 1 week after the first test to complement the data of treadmill running. After completing the treadmill running test, control and grafted rats were trained to run at a speed of 1800 c m / m i n

S. Hattori et al. / Brain Research 710 (1996) 45-55

for another 7 days. However, lesioned rats were trained to run at a speed of 660 c m / m i n for another 7 days, because these rats could not follow higher speeds and received many ES shocks. During this period, grafted rats were divided into two groups depending on their running ability at a speed of 1800 c m / m i n for the following microdialysis study. Rats that could keep up with the treadmill as well as normal rats even at a speed of 1800 c m / m i n (TRS, _< 5) were classified as graft-1800 rats and utilized for microdialysis while running at 1800 c m / m i n . On the other hand, rats that received more ES at 1800 c m / m i n , but could run well at 1200 c m / m i n without receiving ES (checked just after finishing the second training), were termed graft-1200 rats and used for microdialysis study at slower speeds (randomly divided into 3 speed groups of either 300, 660 or 1200 cm/min).

2.6. Surgical procedures After completing treadmill training 4 months after the lesion/graft, rats were anesthetized with sodium pentobarbital (40 m g / k g , i.p., Pitman-Moore) and mounted in a stereotaxic apparatus (Takahashi, Tokyo, Japan). A guide cannula was implanted in the left striatum (coordination of the tip of the canula: A, 0.3 mm from bregma; L, 3.0 mm; V, 2.5 mm from the brain surface in control and 6OHDA-lesioned rats; and A, 0 mm from bregma; L, 3.0 mm; V, 2.0 mm from the brain surface in grafted rats, the center of the two grafting tracks; Fig. 1) and fixed to the skull with 3 anchoring screws and dental cement. A dummy probe was inserted into the guide canula till the intracerebral perfusion was done.

\

/ Ringer

/

/

solution

( 2 ~d/min )

!

,<

,~

i \~

~

/

. . . . .

.

. ,.

.,

.

~

/

A=0 ram. L=3.0 mm, D=60 mm (tip) Fig. 1. Schematic drawing showing the location of microdialysis probe in the grafted striatum (modified from G. Paxinos and C. Watson, The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1986).

47

2.7. In cit'o microdialysis Dialysis probes (membrane length, 4 mm; outer diameter, 0.15 ram; inner diameter, 0.02 mm: molecular cut-off, 5000; recovery rate, 20% at 37°C, Nikkiso, Japan), constructed as described by Nakahara [34], were implanted into the left striatum through the guide cannula 2 to 3 days after the implantation of the guide canula. They were continuously perfused with Ringer's solution (NaCI 147 raM, KC1 4 mM and CaCI~ 2.3 mM at pH 7.0) at a constant flow rate of 2 /xl/min. Following a 3-h stabilization period, perfusate samples were collected at 20-min intervals. After collecting 3 samples to determine the baseline level, each rat ran on the treadmill for 20 rain at a speed of either 300, 600, 1200 or 1800 c m / m i n (control rats, 300, 660 or 1800 c m / m i n ; lesion rats, 661) cm/min: graft-1200 rats, 300, 660 or 1200 c m / m i n ; graft-1800 rats, 1800 cm/min). One sample was collected during running and an additional 9 samples were collected after running (i.e. measurements of DA and metabolites were conducted during 180 rain after running). Dialysates were automatically injected into the high-performance liquid chromatography (HPLC) apparatus with electrochemical detection (ECD-100, EICOM, Japan) for analysis [24]. DA was separated on an Eicompack MA-5ODS (7 /,zm, 4.6 x 250 ram, EICOM). The working electrode was a WE-3G graphite electrode (detector potential against A g / A g C I reference electrode; +0.65 V, EICOM) and the mobile phase consisted of citric acid 70 mM, sodium acetate 100 raM, sodium 1-octanesulfonate 1.39 raM, EDTA 26.9 mM and methanol 15% at pH 4.0. The flow rate was set at 1.0 ml/min. Levels of DA, DOPAC and HVA in each brain perfusate were calculated as percentages of 3 stable baseline measurements before running, without correction fi~r recovery across the dialysis membrane [25].

2.8. lmmunocvtochemist~ of tyrosine hwtro.n,lase (TH) At the end of the experiment, all rats were deeply anesthetized with pentbarbital (50 m g / k g ) and perfused transcardially with modified Zamboni's solution (0.2% picric acid, 0.05% glutaraldehyde, 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.5) and the brains were frozen and cut into 50-/xm serial sections, lmmunocytochemistry for TH was conducted in 6-OHDA-lesioned and grafted rats to confirm the extent of the lesions and the survival of grafted cells. Each section was reacted with mouse anti-TH antibody (dilution, × 2000) and processed by the PAP method [22]. Tracks of the dialysis cannula were confirmed microscopically to be placed in the desired sites in the striatum from the histological sections.

2.9. Statistics Data are shown as means + S.E.M. Significant differences between groups in behavioral tests (metampheta-

48

S. Hattori et al. / Brain Research 710 (1996) 45-55

mine-induced rotations, the treadmill running test) and extracellular levels of DA, DOPAC and HVA under various conditions were analyzed using one-factor and twofactor repeated or factorial measures of analysis of variance (ANOVA), and a Games-Howell test was used as a post-hoc analysis for multiple comparisons (Super ANOVA, version 1.11). To determine the significantly increased sample bins compared to the prerunning basal values in the microdialysis measurements (within-group factor analysis of one-factor repeated measure), mean comparisons by contrast were done (Super ANOVA, version 1.11).

3. Results

3.1. Recovery of methamphetamine-induced rotations 6-OHDA-lesioned rats (n = 25) showed significant rotations ipsilateral to the lesion (14.1 _+ 1.0 turns/min and 18.0 _+ 1.2 turns/min) after the administration of methamphetamine 2 and 12 weeks after the lesion. In grafted rats (n = 29, 13.7 ___0.8 turns/rain before transplantation), ipsilateral rotation decreased to one-third of pregrafting values 2 weeks after grafting (4.2 _+ 1.0 turns/min) and disappeared 4 - 8 weeks after grafting ( - 1.7 +_ 0.5 and - 2.0 _+ 0.4 turns/rain, respectively). Twenty-three of these 29 grafted rats showed some contralateral rotation 8 - 1 2 weeks after the grafting ( - 2 . 2 + 0.5 at 12 weeks). Five rats showed no rotation and one turned ipsilaterally 1.5 turns/min.

Control rats could follow the treadmill easily at any speed. They received 5 - 1 0 ES during the second 10 min on the first day, but thereafter received only 0 - 4 ES, even at a speed of 1800 c m / m i n (n = 8 in each group, Fig. 2). Although 6-OHDA-lesioned rats could run on the treadmill at slow speed without receiving ES (300 and 660 c m / m i n , n = 8 in both groups, Fig. 2A, B), they could not keep up with the treadmill at higher speeds and received many ES, even after 7 days' training (40-50 E S / 1 0 min at a speed of 1200 c m / m i n (n = 8) and 80-100 E S / 1 0 min at a speed of 1800 c m / m i n (n = 9), respectively, Fig. 2C, D). On the other hand, grafted rats received only a few ES at a speed lower than 1200 c m / m i n (300, 660 and 1200 c m / m i n , n = 8 in each group) and 20-40 ES at a speed of 1800 c m / m i n (n = 13, Fig. 2). Two-factor analysis of variance indicated that the main group effects at speeds of 660, 1200 and 1800 c m / m i n were statistically significant (660 c m / m i n , F(2,21) = 6.41, P < 0.01; 1200 c m / m i n , F(2,21) = 8.65, P < 0.01; and 1800 c m / m i n , F(2,21) = 10.27, P < 0.001). Post-hoc tests showed significant differences between the control and lesioned rats and between the grafted and lesioned rats at 1200 c m / m i n and among 3 groups at 1800 c m / m i n (all P < 0.05). TRS at a speed of 660 c m / m i n did not differ among the 3 groups (F(2,21) = 1.0, P = 0.38) and the significance of the main group effect at this speed was due to the high score of the lesioned rats on the first 2 days. At a speed of 1200 c m / m i n , TRS of control (0.8 +_ 0.4) and grafted rats (1.4 ± 0.9) were smaller than that of lesioned rats (46.3 _+ 16.3),

300 cm/min

A

t~

20O

660 cm/min

200 . . . . . . . . . . . .

control lesion grafl

i

._ 150 E

~

3.2. Recovery of locomotor ability

150 . . . . . . . . . . .

10O

~

10O . . . . . . . . . . . . . . . . .

so

~

50 . . . . . . . . . . . . . . . .

dayl day2 day3 day4 day5 day6 day7

dayl day2 da~3 da)4 day5 da)6 da)7

1200 cm/min

C

200 .

D

1800 cm/min .

.

.

.

.

.

200 t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150 . . . . . . . . . . . . . . . . . . . . . . . . . . . .

=

150 ................................................

10O"

~

5@

~

.

?

_

_

~

50O

dayl day2 day3 day4 day5 day6 day7

0

dayl day2 day3 day4 day5 day6 da)7

Fig. 2. Effects of 6-OHDA lesion and DAergic cell grafts on treadmill running test at a speed of 300 c m / m i n (A), 660 c m / m i n (B), 1200 c m / m i n (C) and 1800 c m / m i n (D). Ordinate means ES counts that each rat received during the second 10 min in each day.

S. Hattori et al. /Brain Research 710 (1996) 45-55

Basal levels of DA, DOPAC, HVA 1200 T I(1110

// // //

80O ] II []

600" 400

control lesion

49

+ 19.6 fmol//~l, respectively, corresponding to 70, 12.9 and 17.2% of control levels. These values were significantly higher than those of 6-OHDA-lesioned rats (~P < 0.05 vs lesioned rats, followed by Games-Howell post-hoc analysis, Fig. 3). Basal levels of DA in grafted animals were slightly lower than controls; however, the difference was not significant.

graft

3.4. Dynamics of DA turnover during treadmill running in intact rats

-

2OO 0

IIA

D(-)PA("

ItVA

DA increased to 170 and 140% of prerunning basal values for 6 0 - 8 0 rain after running at moderate (660

x 1()()

Fig. 3. Basal levels of extracellular DA, D O P A C and H V A in the intact, 6-OHDA lesioned and grafted striata. Values of D A were represented in the 100-fold scalc. (* P <0.05, control vs lesion/graft; # P < 0 . 0 5 , lesion vs graft}

I)A

~

......., ....

It,~l

however, there were no differences between the control and grafted rats (F(2,21) = 7.68, P < 0.01). At a speed of 1800 c m / m i n , TRS of control rats (0.8 _+ 0.4) was smaller than grafted (97.4 _+ 28.7) and lesioned rats (26.7 _+ 7.7), but there was no difference between grafted and lesioned rats ( F ( 2 , 2 1 ) = 8.75, P < 0.01). No statistical difference in TRS between grafted and lesioned rats was due to large variation in the grafted group. Of the 13 grafted rats with treadmill training at a speed of 1800 c m / m i n , only 3 rats received a few ES (TRS, (1, 1.3, 3) as control rats (TRS, _< 5) and the other 10 rats received more ES (TRS, 5.7-87, mean = 34.3). In the microdialysis study, we separated grafted rats into two groups depending on the running ability at a speed of 1800 c m / m i n during the second training. Rats that could keep up with the treadmill as well as normal rats at a speed of 1800 c m / m i n (TRS, < 5) were classified as graft-1800 rats and utilized for microdialysis while running at 1800 c m / m i n (n = 6); rats that received more ES (TRS, > 5) were termed graft-1200 rats and used for microdialysis study at slower speeds (at a speed of either 300, 660 or 1200 c m / m i n in each rat, n = 6 in each speed group).

3.3. Basal levels of extracellular DA, DOPAC and HVA Basal levels of extracellular DA, DOPAC and HVA in control rats (n = 24) were 2.1 _+ 0.3, 1040.0 _+ 79.1 and 594.0 + 59.4 fmol/pA, respectively (Fig. 3). In 6-OHDAlesioned rats (n = 9), basal levels of DA, DOPAC and HVA were 0.4 _+ 0.1, 50.4 + 21.9 and 47.9 -t- 17.3 fmol//~l, and corresponded to 18.3, 4.8 and 8.1% of controls. These values were smaller than those of control rats (DA, F ( 2 , 4 8 ) = 6.45; DOPAC, F ( 2 , 4 8 ) = 93.8; and HVA, F(2,48) = 51.1; all P < 0.01; followed by GamesHowell post-hoe analysis, *P < 0.05 vs control rats, Fig. 3). In grafted rats overall (n = 24), basal levels of D A , D O P A C and H V A w e r e 1.6 + 0.2, 165.1 ___ 30.6 and 129.9

12o I(HF

ih • ,,11

t11

I1

0

lib

[ ~l

F,I)

,~11

J~{I

I~ll

INI}

)P \( '

t!H J IHD ]

1 r,tt

idl

IJ

~(1

a

~'~

~i~ i,~

ii

~ii

~,i,

,,:1 ii,

i~f, i~,~

Fig. 4. Time course of extracellular levels of DA (A), D O P A C (B) and H V A (C) in the intact, 6-OHDA-lesioned and grafted striata following 20 min running were expressed as percent change of thc mean of 3 samples prior to running, after normalization of each value (intact control rat: O, 300 c m / m i n ; [], 660 c m / m i n ; ,',, 1800 c m / m i n ; lesioned rat: × , 660 c m / m i n ; grafted rat: O , 300 c m / m i n ; B, 660 c m / m i n ; cross in square, 1200 c m / m i n ; A, 1800 c m / m i n ; all n - 6). Abscissas show time course (min). Each rat ran for 20 rain from time 0. Perfusate samples were collected at 20-rain intervals (40 /,d perfusate/20 rain). Significantly increased sample bins compared with prerunning basal level bins, determined by contrast, are shown in Table 1. Averaged S.E.M. of percentage increases in DA, D O P A C and HVA, and post-hoc analysis of all pairs following the global analysis with A N O V A are shown in Table 2 and Table 3, respectively.

S. Hattori et al. / Brain Research 710 (1990) 45-55

51)

cm/min) and high speed (1800 cm/min), respectively, but at a low speed (300 cm/min) no significant increase was observed (300 cm/min, F(12,60) = 1.73, P = 0.082; 660 cm/min, F(12,60) = 3.38, P < 0.001; and 1800 cm/min, F(12,60) = 2.90, P < 0.01; n = 6; Fig. 4A). Sample bins with significant difference from the prerunning basal level bins are shown in Table 1, and averaged standard error of means (S.E.M.) of percentage increases in DA, DOPAC and HVA are indicated in Table 2. Although no significant difference in the increases in DA between moderate and high speeds was observed by global analysis ( F ( 7 , 4 0 ) =

Table 1 Sample bins with significant difference from prerunning basal level bins in Fig. 4 Group and running speed(cm/min)

Sample number 0

l

2

*

*

a

Table 2 A v e r a g e d S.E.M. (standard error of the mean) of percent increases in extracellular DA, D O P A C and H V A during and after locomotion in Fig. 4 Group and running

DA

DOPAC

HVA

4.4 ± ~1.6

speed(cm/min) Control 300

3.0 _+(I.3

3.6 _+0.5

660

10.6_+2.1

4.2+0.7

5.3+_(I.7

1800

7.3+_ 1.(I

4.4+(/.5

5.4-+0.9

Lesion 660

5.1+0.6

5.1_+0.6

2.6±0.3

Graft 300

5.4_+0.8

2.1 ± 0 , 3

3.5 _~t).6

660 1200 1800

4.(I + 0.5 7.0 _+ 8.9 4.4-+0.8

2.1 +_0.3 2.6 + 0.3 4.7-+0.6

2.8 ± 0.4 2.2 + (/.2 7 . 9 ± 1.3

Values are Means + S.E.M. 3

4

5

6

7

8

9

(A) DA Control 300 660 1800 Lesion 660 Graft-1200 300 660 1200 Graft- 1800 1800

1.42, P = 0.23), there was a tendency for the levels of DA to rise and to return faster after moderate speed running than after high speed running. DOPAC increased to 120 and 135% of basal values at moderate and high speed, respectively. The increase in DOPAC at low speed was not significant (300 c m / m i n , F(12,60) = 1.41, P = 0.19; 660 c m / m i n , F(12,60)= 2.32, P < 0.05; and 1800 c m / m i n , F(12,60) = 17.68, P < 0.001; n = 6, Fig. 4B, Tables 1 and 2). HVA increased to 120 and 160% at moderate and high speed, respectively, and no significant increase was seen at low-speed running (300 c m / m i n , F(12,60)= 0.53, P = 0.88; 660 c m / m i n , F(12,60)= 2.94, P < 0.01; and 1800 c m / m i n , F(12,60) = 25.4, P < 0.001; n = 6, Fig. 4C, Tables 1 and 2). DOPAC reached its peak 40-60 rain after running, but HVA increased gradually over 3 h. The threshold speed of running for the increase of DA, DOPAC and HVA was evaluated to be between 300 and 660 cm/min.

n.s. b *

*

*

*

n.s. n.s. * n.s.

*

*

*

*

*

n.s. *

*

*

*

*

*

*

*

*

*

(B) DOPAC Control 300 660 18(/0

Lesion 660

n.s.

Graft-1200 300

*

*

660

*

*

*

1200 Graft-1800

n.s.

1800

*

*

*

*

*

*

* *

* *

* *

*

*

*

* * *

* * *

* *

*

*

*

*

*

*

3.5. Dynamics of DA turnol,er during treadmill running in 6-OHDA-lesioned rats

(C) HVA Control 300 660 1800 Lesion 660 Graft-t200 300 660 1200 Grafi-1800 1800

n.s. *

*

*

•

*

*

*

*

*

*

*

* P < 0.05, mean comparisons by contrast (vs mean of prerunning 3 samples). Perfusate samples were collected at 20-min intervals. One sample was collected during running (sample O) and an additional 9 samples were collected after running (samples 1 - 9 ) . b n.s., not significant.

Since 6-OHDA-lesioned rats could not run well on the treadmill at high speed (1200 or 1800 cm/min) and received many ES, microdialysis was done in 6 of 9 lesioned rats only at a speed of 660 cm/min. The remaining 3 rats showed rotation behavior ipsilateral to the lesion and could not run at all on the straight treadmill. Extracellular DA and DOPAC were not increased at this speed. Although HVA was increased up to 10%, it returned to the basal level 60 min after running (660 c m / m i n , DA F(12,60) = 1.04, P = 0.43; 660 c m / m i n , DOPAC F ( l ( ) , 6 0 ) = 1.48, P = 0.16; and 660 c m / m i n HVA, F(10,60) = 3.37, P < 0.001; n = 6, Fig. 4, Tables 1 and 2). Moreover, this increase was minuscule absolute amount, because the basal concentration of HVA in the lesioned rats was less than 10% of control value.

S. Hatfori et al. / Brain Research 710 (1 996) 45-55

3.6. Dynamic,s" of DA turnover during treadmill running in grafted rats Among grafted rats, only 6 rats could run well at a speed of 1800 c m / m i n without receiving ES (TRS, < 5) and they were used for microdialysis while running at 1800 c m / m i n (graft-1800 rats). The other grafted rats (n = 18) that could run well up to 1200 c m / m i n , but not at 1800 c m / m i n were used for microdialysis while running at slower speeds (at a speed of either 300, 660 or 1200 c m / m i n , n = 6 in each group, graft-1200 rats). In graft-1200 rats, DA was increased up to 115% by running at 660 c m / m i n , but the increase at 300 and 1200 c m / m i n did not reach significance. DOPAC was increased up to 110% at 300 and 660 c m / m i n , but the increase at 1200 c m / m i n was not significant. HVA was increased up to 110-115% at 300-1200 c m / m i n . Meanwhile, in graft1800 rats, DA, DOPAC and HVA were increased to 130, 120 and 135% at 1800 c m / m i n , respectively (660 c m / m i n DA, F(12,60) = 2.29, P < 0.05; 1800 c m / m i n DA, F(12,60) = 5.43, P < 0.001; 300 c m / m i n , DOPAC F(12,60) = 2.86, P < 0.01; 660 c m / m i n DOPAC, F(12,60) = 4.01, P < 0.001; 1800 c m / m i n DOPAC, F(12,60) = 4.80, P < 0.001; 300 c m / m i n HVA, F(12,60) = 2.42, P < I).05; 660 c m / m i n HVA, F(12,60) = 11.41, P < 0.001:1200 c m / m i n HVA, F(12,60) = 4.32, P < 0.001; 1800 c m / m i n HVA, F(12,60) = 4.28, P < 0.001: Fig. 4, Tables 1 and 2). The running-induced increases in graft-1200 rats were of shorter duration and more variable among test animals than those in control rats. In the global analysis of DA, DOPAC and HVA (treatment × speed ~
B. D O P A C control 1800 > graft 180~) = control 660 > graft 1200 = = = =

lesion 660 graft 300 graft 660 control 300

C. H V A control 1800 = graft 1800 > control 660 = graft 660 graft 6 6 0 = lesion 660 = graft 1200 = graft 300 = control 3 0 0 control 660


>

> lesion 660 = graft 1200 = graft 300 = control 3 0 0

51

(DOPAC, F ( 7 , 4 0 ) = 7.91; and HVA, F ( 7 , 4 0 ) = 9.12; all P values < 0.'001, Fig. 4). The results of post-hoc analysis of all pairs by the Games-Howell procedure were shown in Table 3. Briefly, there were significant differences in the increases of DOPAC and HVA among the 3 groups of control rats (control-1800 > control-660 > control-300), and between the 3 groups of graft-1200 rats and graft-1800 rats (graft-1800 > graft-1200 = graft-660 = graft-300). In graft-1200 rats, there was no definite increases in DOPAC and HVA in proportion to the increase of running speed (graft-1200 = graft-660 = graft-300). Moreover, DOPAC and HVA in graft-1200 was smaller than control-660, and graft-1200 and graft-660 did not differ from lesion-660.

3.7. Histology TH-positive cells in the left SNc disappeared completely in lesioned and grafted rats. Many TH-positive neurons (more than 400/rat) survived in the striatum of grafted rats (not shown). Tracks of dialysis probes were verified in the histological sections stained by TH or Cresyl violet,

4. Discussion

In the present study, using a treadmill running test, we demonstrated quantitatively motor and dopamine disturbances after 6-OHDA lesion and then measured these recoveries after neural transplantation. Lesioned rats received more ES with an increase in treadmill speed. Grafted rats with recovery in extracellular DA (70% of controls) could easily keep pace with treadmill speeds up to 12/)0 c m / m i n . Although we did not use sham controls such as cortical or spinal cord transplants, previous studies have shown that transplants of non-dopaminergic tissues do not lead to behavioral improvement [19,47]. We therefore attributed these behavioral effects to successful DAergic transplantations. However, the majority of grafted rats could not keep up with a higher treadmill speed of 1800 c m / m i n . Taken together with the result that extracellular DA levels of graft-1200 rats were not less than those of graft-1800 rats, these data suggest that restoration of extracellular DA levels up to 70% of normal controls is not sufficient for complete recovery of high speed running. In treadmill running, well-coordinated quadrupedal locomotor control of 4 limbs is needed. In addition, this test can detect subtle motor disturbances associated with moderate DA depletion with greater sensitivity by setting treadmill speed at higher levels. Although the treadmill running test is not a direct measurement of locomotion, it is a type of active avoidance test which can evaluate maximum running ability when used at various treadmill speeds. Earlier studies reported a change in sensory threshold following lesions of nigrostriatal DAergic system that might

52

S. Hattori et aL / B r a i n Research 710 (1996) 45-55

influence the results of the treadmill running test [14]. However, lesioned rats without grafts showed good running ability and received few ES at slow and moderate speeds (300 and 660 cm/min), similar to control rats. This suggests that almost all examined rats could perceive ES and learned to avoid ES during the early training periods (during 10 min habituation period before running and first 10 min running on the first day). Thus, the treadmill running test mainly reflects motor function rather than sensory threshold, although some reports have suggested that unilateral 6-OHDA lesions produced additional slight difficulties in using ipsilateral limbs [16]. In addition to incomplete recovery of treadmill running at high speeds observed in the present experiment, previous reports showed that DAergic grafts could not completely ameliorate complex functional deficits such as aphasia, adipsia, and skilled paw-reaching task [7,15,16,26]. Exploring and hoarding behaviors were not recovered without pretreatment with low dose of amphetamine [26]. Thus, questions remain why the functional effects are not general and restricted to certain behaviors and whether the differences in effects between functional tests are qualitative or quantitative [16]. There are multiple motor loops connecting motor inputs from the cortex, limbic area or thalamus to the subcortical motor output nuclei such as the globus pallidus and substantia nigra pars reticulata. These loops are topographically organized and modified by DAergic input throughout the striatum [1]. Thus, failure of recovery in some behaviors may be attributed to ectopic or inappropriate graft placement, resulting in incomplete or aberrant neural connections. In this experiment, we transplanted fetal DAergic cells into the mid portion of the striatum and this graft placement might explain part of the discrepancy between recovery in drug-induced behavior and treadmill running ability; several reports refer to differences in DA functions between medioventral and dorsolateral striatum. Along with the nucleus accumbens in the mesolimbic dopaminergic system, the medioventral striatum seems to mediate locomotor activity related to motivated behaviors [43], whereas the dorsolateral striatum has greater influence on stereotyped behaviors, such as drug-induced and postural asymmetry [18,40]. However, these segregations of function are not absolute and we reported running-dependent increase in DA turnover even in the mid striatum [25]. Ectopic grafts in the striatum cannot reconstruct nigronigral circuitry in the complex motor loops, although dendritic DA release within the substantia nigra may have an important role in functions of the basal ganglia [50]. These considerations have led to the recent development of a multiple microtransplantation technique, which may promote topographical integration in both homotypic (substantia nigra) and ectopic (striatum) sites with better functional results [35]. In addition to these topographical aspects, functional recovery after transplantation depends on various trophic,

neurochemical and synaptic interactions between host brain and grafts. Therefore, we should consider the extent of maturation and reorganization of these mechanisms after grafting as explanations for the outcome in each functional measurement [16]. Although drug-induced rotation is considered a useful parameter to estimate the net effects of asymmetry of DA release and receptor expression [31], it does not reflect cortically regulated DA release. The observation that electrophysiological properties of many grafted neurons in animals with complete reversal of rotational behaviors after transplantation remained immature indicates that recovery of rotational behavior may not require full maturation of functional neural connections between the grafted and host neurons [17]. On the other hand, treadmill running at high speeds requires integrated limb use as well as skilled paw-reaching; these types of behavior might need higher levels of integration of the graft into the host neural circuitry. The comparison of the dynamics of DA turnover in the striatum of control, lesioned and grafted animals provides insight into the relationship between functional recovery and the normalization of the tonic/phasic control of DA turnover. The study of the alterations in striatal DA turnover during treadmill running in control rats showed that there was a threshold speed between 300 and 660 c m / m i n at which increased DA turnover was observed. Above the threshold speed, DA turnover increased and extracellular levels of DA, DOPAC and HVA were elevated. This data is consistent with the result of previous study [18] that tissue levels of DA and DOPAC in the intact striatum increased after treadmill running at the speed of 72[I cm/min. Extracellular DA, which reflects the net balance of release and uptake of DA at terminals, increased for 60-80 min. However, the degree of increase of DA did not depend on the running speed. As discussed previously, this might be due to alterations of DA transporter activity during locomotion [25], which could be modulated by synaptic activity itself. Thus, extracellular DA levels do not necessarily increase because of an increase in DA turnover. Extracellular DOPAC and HVA, which mainly reflect DA metabolism of the de novo synthesis pool of DA [52], increased for over 3 h at moderate or high speed and the degree of increase depended on the running speed. These data suggest that DA turnover in the rat striatum increased during and even after treadmill running in proportion to the speed [18,25]. Thus, we could not demonstrate directly that more DA release occurred as rats ran at higher speed, but it was likely that striatal DA turnover was temporarily regulated by the exercise and increased quantitatively related to the intensity of exercise. Since both TH and MAO activities changed during/after running in a direction that would elevate extracellular levels of DA, DOPAC and HVA, both synthesis and metabolism of DA might be intimately involved in this regulation [25]. In graft-1200/graft-1800 rats that showed complete

S. Hattori et al. / Brain Research 710 (1996) 45-55

recovery of rotational asymmetry and could keep up with high treadmill speeds (1200 or 1800 cm/min), extracellular levels of DA recovered to 70% of normal (not significant vs normal control rats), while those of DOPAC and HVA remained only 15-20% of normals. The discrepancy in recovery of extracellular levels of DA and D O P A C / H V A suggests low density of DAergic terminals consistent with the survival of small to moderate numbers of DAergic neurons a n d / o r immaturity of DA uptake and metabolism at nerve terminals. An earlier report indicated a reverse correlation between the terminal density and turnover rate of grafted DAergic neurons [46]. In small grafts that contained 300-550 DA neurons, both the DA terminal density and extracellular levels of D O P A C / H V A remained 10-20% of normals, whereas DA release rate increased 2 times higher than normal and extracellular levels of DA recovered to 40% of normal [51]. Recovery rate of extracellular DA was compatible with the product of % terminal density and increasing rate of DA release (20% × 2). Thus, in a small graft, a high turnover rate may compensate for the lack of surviving DAergic neurons and maintain extracellular levels of DA close to normal. The result that recovery rates of extracellular levels of D O P A C / H V A remained less than one-third that of DA also indicates that the majority of newly synthesized DA in the grafted DAergic terminals is released preferentially into the extracellular space and that a smaller amount of de novo synthesized DA than normal is directly metabolized without release. Another possible explanation for lower levels of D O P A C / H V A is immaturity of DA metabolism in the presynaptic terminals; extracellular D O P A C / H V A levels did not recover completely (67 and 52% of normals) even in the large grafts where both DAergic terminal density and DA release rate were close to normal [46]. As numbers of surviving DA neurons increase, both terminal density and extracellular levels of DA increased over threshold and presynaptic hyperactivity and postsynaptic receptor supersensitivity could be normalized. In a large graft that contained over 1700 DAergic neurons, the DA receptor-mediated autoregulatory system and DA transporter activity were functioning, the DA release rate recovered nearly to normal, and extracellular tonic levels of DA were maintained within a nearly normal range [46]. Taken together, grafts in the present experiment are considered small to moderate, and the net effects of low terminal density, high turnover rate of DA release and immaturity of DA transporter activity/metabolism at nerve terminals might be responsible for relatively high levels of extracellular DA and low levels of DOPAC and HVA. Although extracellular basal levels of DA, DOPAC and HVA of graft-1200 rats were not less than those of graft1800 rats, the alterations of DA turnover after running were significantly different in magnitude and time course between the two groups. In graft-1800 rats, running-induced % increases of extracellular DA, DOPAC and HVA were similar to control rats both in magnitude and time

53

course (DOPAC was slightly smaller than control). Therefore, grafted DAergic neurons in rats with full recovery of locomotor ability can respond to physiological stimuli, such as locomotion, and release, and metabolize DA in similar proportions as the DAergic neurons in the intact nigrostriatal system, even though extracellular basal levels of D O P A C / H V A remained 15-20% of normals and an increase of basic DA release rate is postulated. Methamphetamine caused a proportional increase in DA release (1350%) compared to intact striatum (1500%), without decrease in DOPAC, even in the small grafts [51]. Since methamphetamine is known to release DA from the cytoplasmic pool of newly synthesized DA [12], these data suggest that grafted DAergic neurons have enough capacity to synthesize DA in response to the increased demand for further DA release or following actual DA release. As in the intact striatum, an increase in extracellular DA did not relate to running speeds. However, the question remains why extracellular levels of DOPAC and HVA in the striatum of graft-1800 rats were increased by running proportionally to those in intact striatum in spite of lower basal levels. One possible explanation is that grafted DAergic neurons might receive dynamic control from the host brain, so that synthesis, release and metabolism of DA could be controlled close to the manner of intact DAergic neurons, although there are controversies regarding the underlying mechanisms responsible for the phasic control of grafted neurons [4,44,50]. Thus, an increase in DA synthesis by running may result in a proportional increase in DOPAC and HVA, even in the small to moderate grafts. On the other hand, in graft-1200 rats that could not follow the treadmill at a speed of 1800 c m / m i n , runninginduced % increases of DA turnover were less than those in control animals (graft-1200 < control-1660) and there was no correlation between increase in DA turnover and running speeds (graft-1200 = graft-660 = graft-300). Thus, in less functionally recovered rats, grafted DAergic neurons could not increase DA turnover enough during actual behaviors, suggesting that they might be less controlled by host brain. Although we could not examine the degree and density of efferent and afferent reinnervation between the graft and host and underlying mechanisms are unknown, our results raise the possibility that the phasic regulation of graft function is important for better recovery in complex motor functions. In conclusion, treadmill running ability appears to be a very good indicator of DA turnover in the striatum, and can be coupled with microdialysis to quantitatively evaluate the maturation of host-graft interactions and their involvement in functional recovery. The results demonstrate that DA turnover in both the intact and grafted striatum of the functionally well recovered rats is increased by physical exercise, and that it depends on the intensity of the exercise. This suggests that grafted neurons can release and metabolize DA in the host striatum tonically and phasically in response to the actual behavior.

54

S. Hattori et al. / Brain Research 710 (1996) 45-55

Acknowledgements W e t h a n k Dr. Q . M . Li, M i s s M. K u m a z a k i , M i s s T. S a k u r a i for t e c h n i c a l support, Dr. H. H a t a n a k a for p r o v i d ing us w i t h a n t i - T H a n t i b o d y , a n d Dr. A. T e s s l e r for e d i t i n g the m a n u s c r i p t . T h i s r e s e a r c h w a s e n t r u s t e d to the N a g o y a City U n i v e r s i t y M e d i c a l S c h o o l b y the S c i e n c e a n d T e c h n o l o g y A g e n c y o f J a p a n , u s i n g the Special C o o r d i n a t i o n F u n d s for p r o m o t i n g S c i e n c e a n d T e c h n o l o g y . T h i s r e s e a r c h w a s s u p p o r t e d in part b y the M i n i s t r y o f E d u c a t i o n a n d Culture, G r a n t - i n - A i d for Scientific Research 03454133 and Developmental Scientific Research 04557005.

References [1] Alexander, G.E., Delong, M.R. and Strick, P.L., Parallel organization of functionally segregated circuits linking basal ganglia and cortex, Annu. Rev. Neurosci., 9 (1986) 357-381. [2] Arbuthnott, G., Dunnett, S.B. and MacLeod, N., Electrophysiological properties of single units in dopamine-rich mesencephalic transplants in rat brain, Neurosci. Lett., 57 (1985) 205-210. [3] Bal, A., Savasta, M., Chritin, M., Mennicken, F., Abrous, D.N., Le Moal, M., Feuerstein, C. and Herman, J.P., Transplantation of fetal nigral cells reverse the increase of preproenkephalin mRNA levels in the rat striatum caused by 6-OHDA lesion of the dopaminergic nigrostriatal pathway: a quantitative in situ hybridization study, Mol. Brain Res., 18 11993) 221-227. [4] Bj6rklund, A., Lindvall, O., Isacson, O., Brundin, P., Wictorin, K., Strecker, R.E., Clarke, DJ. and Dunnett, S.B., Mechanisms of action of intracerebral neural implants: studies on nigral and striatal grafts to the lesioned striatum, Trends Neurosci., 10 (1987) 509-516. [5] Bj6rklund, A. and Stenevi, U., Reconstruction of the nigrostriatal dopamine pathway by intracerebral nigral transplants, Brain Res., 177 (19791 555-560. [6] Bj6rklund, A., Stenevi, U., Schmidt, R.H., Dunnett, S.B. and Gage, F.H., lntracerebral grafting of neuronal cell suspensions. I. Introduction and general methods of preparation, Acta Physiol. Scand., Suppl., 522 11983) 1-7. [7] Bj6rklund, A., Stenevi, U., Schmidt, R.H., Dunnett, S.B. and Gage, F.H., Intracerebral grafting of neuronal cell suspensions. II. Survival and growth of nigral cell suspensions implanted in different brain sites, Acta Physiol. Scand., Suppl., 522 (1983) 9-18. [8] Bolam, J.P., Freund, T.F., Bj6rklund, A., Dunnett, S.B. and Smith, A.D., Synaptic input and local output of dopaminergic neurons in grafts that functionally reinnervate the host neostriatum, Exp. Brain Res., 68 (1987) 131-146. [9] Buzsaki, G., Gage, F.H., Kellenyi, K. and Bj6rklund, A., Behavioral dependence of the electrical activity of intracerebrally transplanted fetal hippocampus, Brain Res., 400 (1987) 321-333. [10] Carder, R.K., Jackson, D., Morris, H., Lund, R.D. and Zigmond, MJ., Dopamine released from mesencephalic transplants restores modulation of striatal acetylcoline release after neonatal 6-hydroxydopamine: an in vitro analysis, Exp. Neurol., 105 (1989) 251-259. [11] Cenci, M.A., Nilsson, O.G., Kal~n, P. and Bj6rklund, A., Characterization of in vivo noradrenaline release from superior cervical ganglia or fetal locus coeruleus transplanted to the subcortically deafferented hippocampus in the rat, Exp. Neurol., 122 (1993) 73-87. [12] Chiueh, C.C. and Moore, K.E., ~Amphetamine-induced release of "newly synthesized' and stored dopamine from the caudate nucleus in vivo, J. Pharmacol. Exp. Ther., 192 (1975) 642-653.

[13] Dawson, T.M., Dawson, V.L., Gage, F.H., Fisher, L.J., ttunt, M. and Wamsley, J.K., Functional recovery of supersensitive dopamine receptors after intrastriatal grafts of fetal substantia nigra, Exp Neurol., 111 (1991) 282-292. [14] Dunnett, S.B., Bj6rklund, A., Stenevi, U. and Iversen, S.D., Grafts of embryonic substantia nigra reinnervating the ventrolateral striaturn ameliorate sensorimotor impairments and akinesia in rats with 6-OHDA lesions of the nigrostriatal pathway, Brain Res., 229 (1981) 209-217. [15] Dunnett, S.B., Bj6rklund, A., Stenevi, U. and lversen, S.D., Behavioural recovery following transplantation of substantia nigra in rats subjected to 6-OHDA lesions of the nigrostriatal pathway, Brain Res., 229 (1981) 457-471/. [16] Dunnett, S.B., Wishaw, I.Q., Rogers, D.C. and Jones, G.H., Dopamine rich grafts ameliorate whole body motor asymmetry and sensory neglect but not independent limb use in rats with 6-hydroxydopamine lesions, Brain Res., 415 (1987) 63-87. [17] Fisher, 1,3., Young, S.J., Tepper, J.M., Groves, P.M. and Gage, F.H., Electrophysiological characteristics of cells within mesencephalon suspension grafts, Neuroscience, 40 (1991) 109-122. [18] Freed, C.R. and Yamamoto, B.K., Regional brain dopamine metabolism: a marker for the speed, direction and posture of moving animals, Science, 229 (1985) 62-65. [19] Freed, W.J., Ko, G.N., Niehoff, D.L., Kuhar, M.J., Hoffcr, BJ., Olson, L., Cannon-Spoor, H.E., Morihisa, J.M. and Wyatt, R.J., Normalization of spiroperiodol binding in the denervated rat striaturn by homologous grafts of substantia nigra, Science, 222 (19831 937-939. [20] Freund, T.F, Bolam, J.P., Bj6rklund, A., Stenevi, U., Dunnett, S.B., Powell, J.F. and Smith, A.D., Efferent synaptic connections of grafted dopaminergic neurons reinnervating the host neostriatum: a tyrosine hydroxylase immunocytochemical study, J. Neurosci., 5 (1985) 6113-616. [21] Groves, P.M., A theory of the functional organization of thc neostriatum and the neostriatal control of voluntary movement, Brain Res. Rev., 5 (19831 109-132. [22] Hatanaka, H. and Arimatsu, Y., Monoclonal antibodies to tyrosine hydroxylase from rat pheochromocytoma PC12 with special reference to nerve growth factor-mediated increase of the immunoprccipitable enzymes, Neurosci. Res., I (1984)253-263. [23] Hattori, S., Li, Q.M., Matsui, N. and Nishino, H., Treadmill running test for evaluating locomotor activity after 6-OHDA lesions and DAergic cell grafts in the rats, Brain Res. Bull., 31 (1993) 433-435. [24] Hattori, S., Li, Q.M., Matsui, N. and Nishino, H., Treadmill running combined with microdialysis can evaluate motor deficit and improvement following dopaminergic grafts in 6-OHDA lesioned rats, Restor. Neurol. Neurosei., 6 (1993) 65-72. [25] Hattori, S., Naoi, M. and Nishino, H., Striatal dopamine turnover during treadmill running in the rat: relation to the speed of running, Brain Res. Bull., 35 (1994) 41-49. [26] Herman, J.P., Choulli, K., Geffard, M., Nadaud, D., Taghzouti, K. and Lc Moal, M., Reinnervation of the nucleus accumbens and frontal cortex of the rat by dopaminergic grafts and effects on hoarding behavior, Brain Res., 372 (19861 21(I-216. [27] Herman, J.P., Rivet, J.M,, Abrous, N. and Lc Moal, M., lntracerebral dopaminergic transplants are not activated by electrical foolshock stress activating in situ mesocorticolimbic neurons, Neurosci. Lett., 911 (1988) 83-88. [28] Keller, R.W., Jr., Synder-Keller, A.M, Lund, R.D. and Zigmond, M.J., Stress-induced release of dopamine from mesencephalic transplants in striatum monitored by microdialysis, Soc. Neurosci. Abstr., 14 (19881 734. [29] Lee, S.M. and Ebner, F.F., Response characteristics of neocortical graft neurons to host somatosensory input, Prog. Brain Res., 82 (1990) 301-308. [30] Mahalik, T.J., Finger, T.E., Stromberg, I. and Olson, L., Substantia nigra transplants into denervated striatum of the rats: ultrastructure

S. Hattori et al. / B r a i n Research 710 11996) 45-55

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

of graft and host interconncctions, J. Comp. Neurol., 240 (1985) 60-71t. Marshall, ,I.F and Ungerstedt, U., Supersensitivity to apomorphine following destruction of the ascending dopamine neurons: quantification using the rotational model, Ear. J. Pharmacol., 41 (1977) 361-367. Melon(, R., (ihilds, J., Gerogan, F., Yurkofsky, S. and Gale, K., Effect of haloperidol on transplants of fetal substantia nigra: evidence for leedback regulation of dopamine turnover in the graft and projections, t'rog. Brain Res., 78 (1988)457-461. Moukhles. H.. Nieoullon, A. and Daszuta, A., Early and widespread normalization of dopamine-neuropeptide Y interactions in the rat striatum after transplantation of fetal mesencephalon cells, Neuroscienoe, 47 (1992) 781-792. Nakahara, D. Ozaki, N. and Nagatsu, T., A removable brain microdialysis probe unit for in vivo monitoring of neurochemical activity, Biogcnic Amines, 6 (1989) 559-564. Nikkhah, G., Cunningham, M.G., Bentlage, C. and Bj/Srklund, A., Behavioral recovery induced by intranigral fetal dopamine micrografts in the rat parkinson model, J. Neurosci., 14 (1994) 3449-3461. Nishino, tt.. lfashitani, T., Kumazaki, M., Sato, H., Furuyama, F., Isobe, Y.. ~A'atari, N., Kanai, M. and Shiosaka, S., Long-term survival ot grafted cells, dopamine synthesis/release, synaptic connections and functional recovery after transplantation of fetal nigral cells in rats with unilateral 6-OHDA lesions in the nigrostriatal dopamine pathway, Brain Res., 534 (1990) 83-93. Nishino, tt., Ono, T., Shibata, R., Kawamata, S., Watanabe, H., Shiosaka, S., l'ohyama, M. and Karadi, Z., Adrenal medullary cells transmute into dopaminergic neurons in dopamine-depleted rat caudate and ameliorate motor disturbances, Brain Res., 445 (1988) 325 337. Nishino, t1., Ono, T., Takahashi, J., Kimura, M., Shiosaka, S., Yamasaki, H.. Hatanaka, H. and Tohyama, M., The formation of new neuronal circuit between transplanted nigral dopamine neurons and non-immunoreactive axon terminals in the host rat caudate nucleus, Neurosci. Lett., 64 11986) 13-16. Perlow, M.J,, Freed, W.J., Hofter, B.J., Olson, L. and Wyatt, R.J., Brain grafts reduce motor abnormalities produced by destruction of nigrostriatal dopamine system, Science, 204 11979) 643-647. Pijnenburg, A.J.J., Honig, W.M.M. and Van Rossum, J.M., Antagonism of apomorphine and amphetamine-induced stereotyped behaviour by injection of low doses of haloperidol into the caudate nucleus and nucleus accumbens, P,~vchopharmacologia, 45 (1975) 65-71

55

[41] Savasta, M., Mennicken, F., Chritin, M., Abrous, D.N., Feuerstein, C. and Lc Moal, M., Instrastriatal dopamine-rich implants reverse the changes in dopamine D2 receptor densities caused by 6-hydroxydopamine lesion of the nigrostriatal pathway in the rats: an autoradiographic study, Neuroscience, 46 (11~92) 729-738. [42] Segovia, J., Melon(, R. and Gale, K., Effect of dopaminergic denervation and transplant-derived reinncrvation on a marker of striatal GABAergic function, Brain Res.. 493 ( 1989J 185-18tL [43] Sharp, T., Zctterstr6m, T., Ljungberg, T. and Ungerstcdt, U., A direct comparison of amphetamine-induced behaviours and regional brain dopamine release in the rat using intraccrebral dialysis. Brain Res., 401 11987) 322-330. [44] Snyder-Keller, A.M., Carder, R.K and Lund, R.D., Development of dopamine innervation and turning behavior in dopamine-dcpleted infant rats receiving unilateral nigral transplants, Neuroscience, 30 (1989) 779 794. [45] Sotelo, C. and Alvarado-Mallart, R.M., The reconstruction of ccrcbellar circuits, Trends Neurosci., 14 (1991) 350 355. [46] Strecker, R.E., Sharp, T., Brundin, P., Zctterstr6m, T., Ungcrstedt, U. and Bj6rklund, A., Autoregulation of dopamine release and metabolism by intrastriatal nigral grafts as revealed by intraccrebral dialysis, Neuroscience, 22 (1987) 169-178. [47] Str6mberg, l., Herrera-Marshitz, M., Ungerstedt, U., Ebendal, T. and Olson, L., Chronic implants of chromaffin tissue into the dopamincdenervated striatum, Effects of NGF on gral~t survival, fiber growth and rotational behavior, Exp. Brain Res., 60 (1985)335-349. [48] Trulson, M.E., Hosseini, A. and Trulson, T.J., Serotonin neuron transplants: electrophysiological unit activity of intrahippocampal raphe grafts in freely moving cats, Brain Res. Bull., 17 (19861 461-468. [49] Wilson, C.J., Xu, Z.C., Emson, P.C. and Feler, C., Anatomical and physiological properties of the cortical and thalamic innervations of neostriatal tissue grafts, Prog. Brain Res., 82 (1990) 417--426. [50] Yurek, D.M. and Sladek, J~R., Dopaminc cell replacement: Park(nson's disease, Annu. Rec. Neuros'ci., 13 (19911) 415 44t). [51] Zetterstr6m, T., Brundin, P., Gage, F.H., Sharp, "l., Isacson, O., Dunnett, S.B., Ungerstedt, U. and Bj6rklund, A., In vivo measurement of spontaneous release and metabolism of dopamine from intrastriatal nigral grafts using intracercbral dialysis, Brain Res., 362 (1986) 344-349. [52] Zetterstr(Sm, T., Sharp, T., Collin, A.K. and Ungerstedt, U., In viw~ measurement of extracellular dopamine and DOPAC in rat striatum alter various dopamine-releasing drugs; implications fi~r the origin of extracellular DOPAC, Eur. J. Pharmacol., 106 (11/88) 27-37.