NeuroscienceVol. 22, No. I, pp. 169-178, 1987 Printedin Great Britain
0306-4522/87 %3.00+ 0.00
Pergamon Journals Ltd 0 1987 IBRO
AUTOREGULATION OF DOPAMINE RELEASE AND METABOLISM BY INTRASTRIATAL NIGRAL GRAFTS AS REVEALED BY INTRACEREBRAL DIALYSIS R. E.
STRECKER,*
T. SHARP,t P. BRUNDIN, * T. ZETTERSTR&vl,t and A. BJ~RKLUND*
*Department of Histology, TDepartment of Pharmacology,
University Karolinska
of Lund, Institute,
u.
UNGERSTEDTt
Lund, Sweden Stockholm, Sweden
Abstract-The autoregulation of dopamine release and metabolism by intrastriatal grafts of mesencephalic dopamine neurons was examined in vioo using an intracerebral dialysis technique. Dopamine-rich cell suspension grafts were implanted into the head of the caudate putamen in rats with complete unilateral 6-hydroxydopamine lesion of the nigrostriatal dopamine pathway. Six months later behavioural tests indicated that the grafts had reversed the lesion-induced rotational behaviour. Extracellular levels of striatal dopamine and its metabolites 3,4-dihydroxyphenylacetic acid and homovanilhc acid were monitored bilaterally in the halothane-anaesthetized grafted rat, both under basal conditions, and also following low (0.05 mg/kg) and high (0.5 mg/kg) doses of the dopamine receptor agonist apomorphine. The perfusate from the grafted striatum showed levels of dopamine which were not statistically different from those of the intact contralateral striatum, indicating that the baseline release of dopamine from the graft was close to normal. Similarly, 3-4-dihydroxyphenylacetic acid and homovanillic acid levels were well recovered on the grafted side (67% and 52%, respectively, ofcontrol values). Consistent with previous observations, levels of the serotonin metabohte 5hydroxyindoleacetic acid measured in perfusate collected from the grafted side was elevated significantly above normal. Subsequent histological analysis revealed large grafts, rich in dopamine-containing neurons (mean + SEM number equalled 3138 f 630), giving rise to an approximately normal density of dopamine-containing fibres in the area of the host caudate putamen surrounding the probe. Treatment with 0.05 mg/kg (subcutaneous) apomorphine did not affect extracellular dopamine recovered from the grafted striatum, while extracellular DA decreased by a maximum of 30% on the intact side. However, a subsequent injection of 0.5 mg/kg apomorphine produced a large decrease of the dopamine recovered from both the grafted (maximum 40% decrease) and intact striata (maximum 80% decrease). Both the low and the high dose of apomorphine reduced extracellular dopamine metabolite levels, a response which was essentially similar for both the intact and grafted sides. Finally, the dopamine reuptake blocker nomifensine (IO-’ M) added to the perfusion medium produced similar large increases in dopamine in perfusates collected from both grafted and intact striata, while 3,4-dihydroxyphenylacetic acid and homovanillic acid did not change. The results indicate that the dopamine-receptor-mediated autoregulatory control and dopamine reuptake, which normally operate in the nigrostriatal dopamine pathway, are also functioning in the intrastriatal nigral grafts although dopamine release from grafted neurons is less sensitive to apomorphine treatment. It is proposed that dopamine autoregulation and negative feedback is an important physiological mechanism for tonic regulation of dopamine graft function, and that this may be sufficient for the reinstatement of motoric and sensorimotoric behaviour by intrastriatal nigral grafts.
Grafts of dopamine (DA)-rich fetal mesencephalic tissue to the DA-denervated striatum can compensate for a range of functional deficits induced by neurotoxic lesions of the ascending mesotelencephalic DA system *.20~30 (for recent reviews see Dunnett et u[.,‘~ Brundin and Bjorklund”). Rats with a unilateral 6-hydroxydopamine (6-OHDA) lesion of the nigrostriatal DA pathway develop contralateral sensory neglect, as well as postural and motor asymmetry, Address for partment kopsgatan
correspondence: Dr Robert Strecker, Deof Histology, University of Lund, Bis5, S-223 62 Lund, Sweden. Abbreuiarions: DA, dopamine; DOPAC, 3,4-dihydroxyphenylacetic acid; HVA, homovanillic acid; 5-HIAA, 5-hydroxyindoleacctic acid; 6-OHDA, 6-hydroxydopamine; VM, ventral mesencephalic.
which is characterized by spontaneous and druginduced rotation of the anima1.37~39~4’ Dopamine-rich grafts have been shown to produce dramatic improvement of all these behavioural deficits. Several studies have been directed towards understanding the mechanism by which graft-induced recovery in DA function is mediated. It is known that functional recovery is related to the number of DA neurons in the mesencephalic graft,” and the extent of DA fibre reinnervation,’ as well as its topographic distribution, within the host striatum.“~” Dopamine-containing neurons grafted to the dopaminergically denervated striatum re-establish synaptic connections with neuronal elements in the host striatum as observed at the ultrastructural leve1,2’,28 and there is electrophysiological evidence that grafted nigral neurons
169
R. E. STRECKERet al.
170
can be activated or inhibited by the host brain.’ Finally, there are electrophysioIogica13,3x~42 and biochemica119~3’-33 observations to indicate that the grafted DA neurons are spontaneously active. Using an intracerebral dialysis technique,25s40,” we recently examined the spontaneous in uiuo release of DA from nigral grafts implanted in the previously denervated rat striatum. 43 Small grafts, producing lO-20% DA reinnervation of the denervated striatum in the vicinity of the grafts, produced a significant recovery of extracellular DA, to between 20 and 40% of normal. In addition, amphetamine, a catecholamine-releasing agent, caused an increase in DA release from the graft which paralleled that observed from the intact normal striatum. However, it remains unknown whether DA released from the graft is tightly controlled or reflects a continuous nonregulated efflux. An important mechanism by which the activity of DA neurons is controlled in the intact brain is “self-regulation” via dopaminergic autoreceptors.13 This is exemplified by the ability of systemically or locally administered DA receptor agonists, such as apomorphine, to decrease both DA cell-body discharge activity and terminal DA release within the striatum in viva via activation of DA autoreceptors.‘2.22.36,47 In the present study we used intracerebral dialysis to determine whether DA released from graft DA neurons responds to subcutaneous doses of the DA agonist apomorphine in a similar manner to DA released from neurons in a normal brain. We chose to administer two doses of apomorphine; a low dose of 0.05 mg/kg, which is considered to decrease DA release via a selective action on the presynaptic DA autoreceptors, and a higher dose of 0.5 mg/kg which probably also activates the striatonigral postsynaptic negative feedback 10op.~’ A number of animals also received the DA uptake blocker, nomifensine, via local perfusion to examine whether the normal DA reuptake mechanism operates in grafted DA neurons. Moreover, as an extension of our earlier study we investigated whether the larger DA grafts used herein could further increase basal levels of DA release, and whether these DA measurements correlated with the degree of behavioural recovery. EXPERIMENTAL
PROCEDURES
Subjects, 6-hydroxydopamine lesion surgery and behavioural resting
Female Sprague-Dawley rats (ALAB, Stockholm, Sweden), weighing 18G200 g at the start of the experiment, were used. They were housed under 12 h light/12 h dark conditions with ad libifum access to food and water. For the initial unilateral striatal DA denervations, rats were anaesthetized with 50mg/kg i.p. methohexital (Brietal, Lilly), and GOHDA-HCl (Sigma) was stereotaxicallv iniected into the right ascending m&ostriatal DA fibre bundle-at the following coordinates: 4.4 mm caudal to bregma, I, 1. mm lateral from midline and 7.8 mm ventral to dura, with the tooth-bar set at -2.4 mm. Eight micrograms of free-base 6-OHDA, dissolved in 4 ~1 saline (containing 0.2 mg/ml ascorbic acid
as anti-oxidant), were injected over 4 min, with the injection cannula left in place for an additional 4 min. Eleven to 16 days after the 6 OHDA injection the rats were tested for rotational asymmetry in an automated rotomete?’ over a 90min period after the injection of metamphetamine (5 mg/kg, i.p.). Eight rats showing individual means of at least 6.8 full turns per minute, in the direction ipsilateral to the lesion, were selected for transplantation surgery. The rats were tested again for rotational asymmetry approximately 11weeks and 24 weeks after transplantation. Transplantation surgery Neural transplants were prepared from fetal ventral mesencephalic (VM) tissue, at two equivalent surgical sessions (3-5 weeks post lesion), according to the cell suspension method as described previously.9 At the first surgical session donor tissue was obtained from two litters (13 and 14 days gestational age) comprising a total of 20 fetuses. A VM cell suspension was prepared and the viability and concentration assessed.” Five rats were given three 2~1 graft injections (equivalent to a total of approx. 275,000 viable cells) into the denervated caudate putamen al the following coordinates (in mm) relative to bregma and the dural surface, and with the tooth-bar set at zero: (I) A = + 1.8, L = 2.5, V = 4.5; (2) A= +0.6, L=2.0, V=4.5; (3) A= +0.6, L=3.2, V = 4.5. A sixth rat received a 2 x 1.5 ~1 cell suspension injection (approx. 140,000 viable cells in total) at the following coordinates: A = + 0.9, L = 2.6, V = 5.1 and 4.2. At the second surgical session, 20 fetuses obtained from two litters (14 and 15 days gestational age) provided VM donor tissue. Cell suspension (3 x 2 ~1, equivalent to approx. 240,000 viable cells) was injected into the denervated caudate putamen in two additional rats, at the same three sites as above. All graft suspensions were injected at a rate of 1 pl/min, and the cannula was left in place for an additional 4min. Dialysis loop prepararion Dialysis loops were made from flexible Dow 50 cellulose tubing (Dow Co.) with an outer diameter of 0.30 mm and a molecular weight cut-off of about 5000. Both ends of the tubing were glued inside stainless steel cannulae (23 gauge), leaving a 4 mm length of dialysis membrane to be exposed to the brain. This tubing was then folded in half prior to implantation, making the dorsal-ventral distance covered by the dialysis probe equal to 2 mm, as illustrated schematically in Fig. I. For further details, see Sharp ef al.” Dialysis loop implantation and perfusate collection Throughout the dialysis experiment the rats were held in a stereotaxic frame under halothane anaeslhesia (1.3-I .5% halothane-air mixture) with body temperature being maintained at 37°C using a heating pad and an incandescent light. Six months after graft implantation the dialysis loop was stereotaxically placed in the middle of the head of the caudate putamen in a position equidistant from the three graft sites (about 0.6 mm from each graft site), and a second loop was placed in the contralateral intact striatum to serve as a normal control. With the tooth-bar set at zero the implantation coordinates (in mm) for the dialysis loop were as follows: A = + 1.2 from bregma, L = 2.6, D = 5.3 from dura. The dialysis loop was secured to the skull using several skull screws and dental cement. The loop inlet cannula was connected to a microinfusion system (Carnegie Medicin AB. Stockholm. Sweden) and continually perfused at 2 pl/min with Ringer solution. Striatal perfusates were collected during 30 min sampling periods in plastic Eppendorf microtubes, containing 10~1 1 M perchloric acid, which were placed on the outlet cannula. Figure I shows schematically the position of the dialysis loop in the striatum surrounded by the three graft implants. The first 30 min perfusate was discarded, as it typically contained excess amounts of DA released during probe implantation. At least three 30 min
Dopamine
+
release from nigral
to HPLC
171
grafts
and were processed I week later for catecholamine fluorescence histochemistry according to the ALFA method.*’ The remaining rat died following the dialysis experiment; its brain was cut in a cryostat and stained with
analysis
Cresyl Violet. Graft placements and probe location were identified under the microscope. The number of fluorescent DA cell bodies in the grafted striatum were counted in every third section, and this number was corrected according to the formula of Abercrombie.’ Finally, in a blind fashion, an observer rated the DA fibre density in the grafted striata within a distance of I mm from the probe tract, by expressing the perceived fibre density on the grafted side as a percentage of the intact contralateral striatum. RESULTS
Basal dopamine and metabolite levels in striatal dialysates
Fig. 1. Schematic drawing of the dialysis loop technique, showing the dialysis loop in the striatum surrounded by three grafts. An identical loop was positioned in the contralateral intact striatum of each rat.
Prior to drug treatment, the baseline perfusate samples collected from the intact striatum collected over at least three 30 min periods, contained 0.07-0.55 pmol DA (mean f SEM = 0.22 f 0.05) 15.245.6 pmol DOPAC (35.0 k 3.6) 16.9-34.3 pmol HVA (23.0 + 2.4), and 5.2-18.1 pmol of the serotonin metabolite 5-HIAA (I 1.5 + 1.7; see Fig. 2). In 6-OHDA-lesioned rats, showing pregraft rotational behaviour identical to the rats used here, the content of DA and DA metabolites in the perfusates collected from the lesioned side was found to be substantially
Dopcmlne
baseline release samples were collected from each striatum before treatment with the low dose of apomorphine (0.05 mg/kg, s.c., prepared fresh in 0.9% saline), the effects of which were followed for up to 3 h. Then, the higher dose of apomorphine was administered (O.Smg/kg, s.c.) and monoamines were monitored for up to 3 additional hours. On the basis of previous experiments, a greater than 10% decrease of DA or DA metabolite for three or more of the six post-injection time points was considered a postive response to apomorphine. Using this criterion, two rats, which did not show a response to apomorphine on the intact side (believed to be the result of poor injections), were omitted from the analysis, leaving N = 6 for the low dose, and N = 5 for the high dose, of apomorphine. Following the apomorphine treatments nomifensine (10e5 M) was added to the perfusion medium for a 30 min sample in six of the rats. A final 30min sample was collected after nomifensine was removed from the perfusate.
Dopac
Intact
Tp
5-HIAA
Analysis of brain per&sates Immediately after collection, perfusates were assayed for DA, its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and the serotonin metabolite, S-hydroxyindoleacetic acid (5-HIAA) in a single run using high-performance liquid chromatography with electrochemical detection. Monoamines were separated on a Spherisorb SODS 5pm column (250 x 4 x 6mm), using a 0. I5 M sodium phosphate mobile phase (pH 3.8) containing 0.5 mM sodium octanyl sulphonic acid, 0. I mM ethylenediaminetetra-acetate and 12% methanol. Detection was by a carbon paste working electrode set at f0.65V. The perfusates were analysed within 20 min using a I .2 ml/min flow-rate (see Sharp et al.ls for further details of the assay). Histological analysis Seven of the eight rats survived
the dialysis
experiment
Intact
TP
Intact
Tp
Fig. 2. Basal levels of DA and the monoamine metabolites, DOPAC, HVA and S-HIAA in dialysates collected from the transplanted (TP) and intact contralateral striatum of the halothane-anaesthetized rat, 6 months after grafting. Each circle is the mean of three baseline 30 min samples obtained from individual animals before drug treatment. The horizontal bar represents the group mean. The hatching indicates levels obtained from non-grafted 6-OHDA lesioned striata (these latter data taken from Zetterstriim et a1.43).
R. E. STRECKER et al.
172
reduced, to between 0 and 8% of normal.43 As can be seen in Fig. 2, the mean baseline levels of DA, DOPAC and HVA in perfusates collected from the grafted striatum represented 85, 62 and SO%, respectively, of the mean levels detected in the contralateral intact striatum. The baseline perfusate samples collected from the 6-OHDA lesions plus grafted striatum contained 0.07XI.30 pmol DA (mean f SEM = 0. I8 f 0.03), 9.442.2 pmol DOPAC (24.3 _t 3.7), 5.4-22.9pmol HVA (11.9+2.1) and 16.l85.6 pmol 5-HIAA (46.2 k 9.4). The difference between intact and grafted baseline levels was not significant for either DA (P > 0.36, Wilcoxon signed rank tests, N = 8) or DOPAC (P > 0.24), although the HVA level was significantly lower on the grafted side (P < 0.01). In four of the eight rats the DA levels recovered from the grafted striatum were greater than the matched samples from the contralateral intact striatum. The mean level of 5-HIAA measured in the baseline perfusate samples was four times greater in the grafted striatum than it was in the intact side (P < 0.01, see Fig. 2), a finding that we have previously described. 43 In both the grafted and intact striata, the amount of DA in the perfusates was always approximately 100 times less than its metabolites DOPAC and HVA (Fig. 2). Effects
ef apomorphine and nomifensine
Dopamine in perfusates collected from the intact striatum decreased significantly following both the low (0.05 mg/kg, mean maximal decrease of 29%) and the high (0.5mg/kg, mean maximal decrease of 83%) dose of apomorphine, as has been previously reported46,47 (see Fig. 3). Although three out of the six grafted striata responded to the low dose of apomorphine with a decrease in DA (10% decrease or greater), as a group the grafted striata showed no significant decrease in extracellular DA after this treatment. The high dose of apomorphine consistently produced a reduction in DA released from the grafted striata, but this response (mean maximal decrease of 47%) was smaller than that observed on the contralateral intact side (t = 4.3, P < 0.05). Also, note that within 3 h of injection of the low dose of apomorphine, and immediately prior to the administration of the higher dose, the mean levels of DA, DOPAC and HVA on the intact side had returned to 75, 71 and 76%, respectively of their original baseline levels. Corresponding values for the grafted side were 121, 75 and 92%, respectively.
Apomorphine also induced a decrease of DOPAC and HVA in the perfusates, an effect which was similar for both the grafted and intact striata (Fig. 3). At the high dose of apomorphine, the metabolites were actually slightly further depressed in the grafted striata compared to the intact striata, although this only reached significance for HVA (t = 4.07, P < 0.05). For DOPAC the mean maximal decrease following the low dose of apomorphine was 29% for intact striata, and 26% for grafted striata, while for HVA the mean maximal decrease was 24% for intact striata, and 32% for grafted striata. Following the high dose of apomorphine, the mean maximal decrease of DOPAC was 50% for intact striata and 61% for grafted striata, and for HVA 48% for intact and 64% for grafted striata. The apomorphine treatments had no consistent effects on 5-HIAA in the perfusates. Nomifensine, when added at a concentration of lo-‘M to the perfusion fluid, produced a large increase in the amount of DA in perfusate collected from both grafted and intact striata. Mean basal DA release was similar for both intact and grafted striata prior to nomifensine treatment (mean & SEM intact side = 0.12 + 0.03 pmole/30 min, mean + SEM grafted side = 0.13 + 0.05). During nomifensine treatment DA measured in 30min samples was 9.8 times greater than baseline on the intact side and 10.1 times greater on the grafted side. Since nomifensine was administered on top of a baseline that was suppressed by apomorphine, the percentage changes were somewhat larger than we have previously seen in normal brain (unpublished observations). Levels of the metabolite DOPAC did not change during nomifensine treatment in either the intact or grafted striata (DOPAC mean percentage of baseline f SEM for intact side = I I5 f 3%; grafted side = II3 + 6%). While the HVA levels during nomifensine did not change for the intact striata (I I9 k 1 I % of baseline), there was a significant increase in HVA in the grafted striata (I 59 + 22% of baseline; Wilcoxon signed rank test P < 0.05). Fluorescence
histochemistry
Histofluorescence analysis of the grafted striata indicated that all of the dialysis loops were positioned within the DA-reinnervated zone of the host caudate putamen, adjacent to the grafts (Fig. 4). In the intact striata the probe tracks ran through the central portion of the head of the caudate putamen and were
Fig. 3. Effects of two doses of apomorphine (0.05 mg/kg and 0.5 mg/kg, SC.) on the levels of DA (top), and its metabolites DOPAC (middle) and HVA (bottom) in perfusates from both 6-OHDA-lesioned plus grafted striata (dotted lines) and the contralateral normal intact striata (solid lines). Each 30 min sample provided 60~1 of perfusate (2 pI/min perfusion rate). Each data point represents the total amount of transmitter collected over the preceding 30 min period. The data points at zero hours represent the last baseline value prior to injection of the 0.05 mg/kg dose. Note that both doses were given to each animal, so that the 100% of baseline value for the high dose is defined as the last time-point in the left-hand panel, i.e. 3 h post injection of the low dose. Two animals that did not show a response to apomorphine on the intact side were omitted from this analysis. Hence, each data point represents the mean f SEM of six rats, although N = 4 for the last three data-points of the high dose (0.5 mgjkg).
173
Dopamine release from nigral grafts
DOPAMINE 150_
APO v.os mg/kg
* 0
-
I
6-OHD*.Grof,
4
Intact
,
,
,
0
side
APO 0.5 mg/kg
I
-150
side
,
,
,
2
I
, 3 Tvne
I
I
I
!
I
I
0 Ihours)
,
2
,
t
3
0
DOPAC 150_
APO 0.0s mg/kg
APO 0.5 mg/kg 4
i
o- -
0
-
,
+J
,
6-OHDA.Graft de I”lm,f s,& ( , , ,
0
2
I
, 3 Time
I
I
I
I
I
0 (hours)
I
I
2
I
t
0
I
0
3
HVA I so-
APO 0.05 mglkg 4
APO 0.5 mg/kg 4
u c lOO-i 2 z D 2 ;; r 50-
01,
u
0
,
Intact
,
I
51de
,
,
2
,
,
3 Time
I
0 Ihours)
Fig. 3.
I
1
I
I
I
2
,
1
3
174
R. E. STRECKER et al.
Fig. 4. Photomontage of surviving nigral cell suspension graft, injected into the denervated host caudate putamen. The highly fluorescent arras beginning at the border of the grafted tissue (G) and surrounding the diaiysis probe track (star) are caused by the dense @&-derived dopaminergi~ fibre reinnervation, which is absent in rats with &OHM ksions only (Zetterstriim er ~f.~~).Of the over 30 DA celi bodies visible in this photo~aph~ t&e are marked with arrows. Bar = 250 pm.
Dopamine release from nigral grafts surrounded by a seemingly normal fluorescent DA terminal network. The grafts contained between 1700 and 5300 fluorescent DA cell bodies (mean +_ SEM = 3138 +_630, with N = 6; analysis of DA cell bodies was not possible in two rats). The degree of reinnervation around the probe on the grafted side (rated in a blind manner) varied between 80 and 140% of the terminal density seen on the normal intact side. The two smallest grafts in the present study, containing approx. 1700 DA neurons each, had 86 and 100% of normal DA levels in the baseline perfusates. The level of extracellular DA detected in these two rats was similar to that of the animals with the largest grafts (containing between 4700 and 5300 DA neurons), i.e. 82-136% of normal, but greater than that seen from the small-sized grafts (between 400 and 500 DA neurons) in our previous study, i.e. 3&50% of normal DA levels.43 Relationship between the biochemical and behavioural effects of grafts
Prior to transplantation all rats exhibited a high amphetamine-induced ipsilateral turning rate (mean + SEM = 11.9 f 1.2 full turns/min for 90 min), indicating a >97% DA depletion in the head of the caudate putamen. 32.33Eleven weeks after grafting the amphetamine-induced turning response was significantly reduced, from 11.9 + 1.2 to 1.4 + 2.1 turns/ min (t = 3.83, P < 0.01) indicating a graft-induced functional reinnervation of the previously denervated caudate putamen. ‘.I’ Rotational testing performed 24 weeks after transplantation produced a mean turning response of -0.6 + 0.9 turns/min, the minus sign indicating that as a group the grafted animals had reversed their net turning direction (t = 7.44, P < 0.001). The graft-induced reduction of the amphetamine-induced turning response ranged from 62% to 123%, with the mean +_SEM of 104 + 7.4%, again indicating that some of the grafted animals had exhibited more turns contralateral than ipsilateral to the operated side. Among behaviourally fully compensated or overcompensated rats, baseline DA release ranged from 32 to 136% of normal, baseline DOPAC levels from 25 to 117% and baseline HVA levels from 24 to 100% of normal. Within this range increased DA or DOPAC levels in the baseline perfusates did not correlate with increased reductions in amphetamineinduced turning. DISCUSSION
The present study used intracerebral dialysis to directly assess the level of DA release and metabolism in both normal and DA-neuron-grafted striata of rats in vivo, under basal conditions, and after administration of the DA agonist apomorphine and the DA reuptake inhibitor nomifensine. The results show that large grafts of fetal mesencephalic tissue, rich in DA-containing neurons, implanted into the DA-
175
denervated host striatum restore striatal DA release to normal levels in the area surrounding the grafts. In addition, the response of DA released from these grafted DA neurons to apomorphine and nomifensine suggests that grafted neurons exhibit a DA autoregulatory capacity. The rate of DA metabolism, as indicated by levels of DOPAC and HVA in the perfusates, was also close to the range of normal striatum and, indeed, the response of these metabolites to apomorphine was similar to that seen in the intact striatum. It is interesting to compare the present results with those obtained in our previous study4’ where the grafts were of considerably smaller size. In the latter case grafts containing between 300 and 550 surviving DA neurons restored DA levels in the perfusates to about 40% of normal, and DOPAC to about 15% of normal, whereas the graft-derived DA terminal density around the probes was estimated to between 10 and 20% of normal. In the present experiment grafts containing on the average seven times more surviving DA neurons restored perfusate levels of DA to about 85%, and DOPAC to about 80%, and the DA terminal density around the probes was estimated to between 80 and 140% of normal. These data suggest that in relation to the density of the graft-induced reinnervation the DA release rates were increased about twofold above normal in the small grafts that produced sparse reinnervation of the host striatum, but that DA release was close to normal in the large grafts where the sampled area had been completely reinnervated. This difference is consistent with previous observations on DA metabolism and synthesis rates in animals with intrastriatal nigral suspension grafts.24,32Small grafts, which restored up to 11% of normal DA levels in the head of the caudate putamen (corresponding to approximately 200-500 surviving DA neurons), had DA metabolism and synthesis rates that were two- to fourfold higher than normal, whereas large grafts, restoring up to about 50% of normal DA levels in the head of the caudate putamen (representing grafts with up to about 5000 DA neurons) had DA metabolism and synthesis rates close to the normal range.)* Taken together, these data indicate an inverse relationship between the density of DA reinnervation by the graft and the rate of DA release and synthesis in the reinnervated area. This suggests the presence of autoregulatory mechanisms within the graftreinnervated striatum, similar to those operating in the intrinsic nigrostriatal DA system after partial lesions.2*‘S*23 According to this autoregulatory principle small nigral grafts, which produce a sparse DA innervation of the host striatum, will have an increased transmitter synthesis and release, and with increasing size and reinnervation density the DA release rate will be gradually normalized. This mechanism may be important for functional recovery and adjustment of DA neurotransmission in the graftreinnervated striatum.
176
R. E.
STRECKER
et al.
Significant compensation of motor asymmetry, as the response of DOPAC and HVA compared well assessed by the amphetamine-induced turning re- with the control striata. Since the DA levels collected sponse, is seen with grafts containing as little as in the dialysis perfusates seem to reflect DA release, 120 DA neurons, restoring about 3-S% of the tissue and DOPAC and HVA predominantly indicate onDA levels in the head of the caudate putamen.7~“~32~33 going intraneuronal DA synthesis and metaboIn the dialysis experiments 50% recovery of extralism 26.44.45 this observation suggests that DA release cellular DA (as measured around the probe in the from the grafted DA neurons is less sensitive than central reinnervated zone) was observed with a minDA synthesis to autoreceptor-mediated feedback. imum of about 400 grafted DA neurons43 and 100% The reason for this is not clear. The studies of Bitran recovery of extracellular DA with a minimum of 1700 and Bustos6 and Zetterstriim et aI.& show that DA grafted DA neurons (this study). In grafts similar to receptor agonists can act differentially on DA release the ones used here, full compensation or overand synthesis, perhaps via different types of receptors compensation of the turning response has been asso- or via receptors located on different parts of the DA ciated with the restoration of between 3 and 50% of neurons (e.g. on terminals or cell bodies). normal tissue DA levels in the reinnervated head of The reduced effect of apomorphine on DA release the caudate putamen. 32 In the present and the pre- from the grafts could be due to subsensitivity of the vious study43 full compensation of the amphetamineDA autoreceptors regulating DA release, or perhaps, induced turning response was seen in animals with to the lack of an efficient postsynaptic negative baseline DA extracellular levels amounting to be- feedback system. Alternatively, it seems possible to tween 30 and 136% of normal. Within these ranges explain this effect on the basis of the mixed poputhere were no significant correlations between the lation of midbrain DA neuron types included in the effect on motor asymmetry and the magnitude of VM grafts. Part of the DA neurons in the ventral basal extracellular DA or whole tissue DA levels, tegmental area, specifically the ones projecting to the neocortex, appear to lack autoreceptors If these suggesting that above a certain level of graft-induced reinnervation the amount of DA released by am- mesocortical neurons participated in the reinnervation of the host striatum in the present study, it phetamine produces a maximal behavioural response. seems likely that the overall response to autoreceptor Thus, above a threshold level of graft reinnervation and DA release, the presynaptic hyperactivity and the activation would be reduced. However, the fact that the apomorphine-induced inhibition of intraneuronal postsynaptic receptor supersensitivity is gradually DA synthesis and metabolism (i.e. DOPAC and adjusted to maintain a relatively constant DA transHVA levels) was unimpaired may argue against this mission level. latter interpretation. The compensatory regulation of DA transmission seen in the intrinsic nigrostriatal DA pathway after There is electrophysiological evidence for the prespartial lesions has been proposed to be mediated both ence of DA autoreceptors on grafted nigral neurons by presynaptic autoreceptors (located either on the from a study on the effects of DA-receptor-active axonal terminals within the striatum, or on the cell drugs on neuronal firing rates in intraventricularly bodies or dendrites within the substantia nigra) and placed grafts.42 Similar to normal nigral DA neurons, by postsynaptic DA receptors located either on the discharge rate of grafted neurons decreased durstriatonigral feedback neurons or on local (e.g. ing application of apomorphine, and increased durcholinergic) interneurons within the striatum.23 In the ing application of the DA receptor blocker haloperidol. This is consistent with the observations of present study we tested the efficacy of DA-receptorHerman et aZ.24on the effects of haloperidol on DA mediated feedback mechanisms in the intrastriatal turnover in intrastriatal nigral grafts, as assessed by nigral grafts by giving systemic injections of apomeasurements of tissue DOPAC and DA levels. They morphine at two dose levels, a low dose (0.05 mg/kg) observed a marked two- to threefold increase in which is thought to act exclusively presynaptically, and a higher dose (0.5 mg/kg) which will activate also DOPAC and the DOPAC:DA ratio in the graftreinnervated striatum, although these effects were postsynaptic DA receptors.29.36,47Experiments in normal rats with kainic-acid-induced destruction of the somewhat smaller in magnitude to those seen in the striatonigral pathway’.“.” indicate, however, that the intact striatum. depression of both DA-neuron-firing in the substantia nigra and DA synthesis and metabolism induced CONCLUSIONS by apomorphine is unaffected by removal of the Taken together, the available data thus provide striatonigral feedback loop. Indeed, Seiger et aLI substantial evidence that the DA-receptor-mediated have shown that apomorphine is effective in reducing [3H]DA release in vitro from axons of nigral grafts autoregulatory control normally operating in the placed on the iris in the anterior eye chamber, i.e. in nigrostriatal system is also functioning in the intrastriatal nigral grafts. The finding that extracellular the absence of any neuronal target neurons.34 The present results show that the effect of apo- levels of DA increased in the graft following administration of nomifensine suggests that the normal DA morphine on extracellular levels of DA was smaller than normal in the graft-reinnervated striata, whereas reuptake mechanism also operates to control synaptic
Dopamine release from nigral grafts of DA. These autoregulatory mechanisms probably explain why tonic DA synthesis and release can be maintained at near-normal rates and do not “overshoot” in the grafted DA neurons despite their abnormal and ectopic location. As discussed above, the autoregulatory control is probably also important for the dynamic adjustment of DA release in relation to the degree and density of striatal reinnervation from the grafts. Indeed, DA-autoreceptor-mediated feedback control is likely to be an important physiological mechanism for the tonic regulation of DA graft function, which may be sufficient for the graftinduced effects on motor asymmetry, sensorimotor integration and akinesia, seen in rats with uni- or bilateral destruction of the nigrostriatal DA system (see Dunnett et al. I6 for review). There is some electrophysiological evidence for afferent host connections to intracortical mesencephalic grafts.3 Howlevels
177
ever, to what extent the function of nigral grafts can also be phasically regulated by the host brain, e.g. through direct afferent inputs to the grafted neurons or through local neuronal interactions, is so far poorly known. The intracerebral dialysis technique should, however, provide a valuable tool to provide more information on this important issue.
Acknowledgements-This work was supported by grants from the National Institutes of Health (NS-06701), the Swedish MRC (04X-3874,14X-0374 and 14X-3574) and the Karolinska Institute. T.S. was sponsored by an SERC postdoctoral fellowship, and R.E.S. is supported by an NIH postdoctoral fellowship (MH09229) and an IBRO/Swedish MRC postdoctoral fellowship. We thank Katarina Friberg, Yvette Jiinsson, Ragnar MBrtensson and Gertrude Stridsberg for skilled technical assistance, Arnold Vulto for helpful advice and Siv Carlson for her expert typing of the manuscript.
REFERENCES 1.
Abercrombie M. (1946) Estimation of nuclear population from microtome sections. Anar. Rec. 94, 239-247. of the remaining dopaminergic neurons after partial destruction in the nigrostriatal dopaminergic system in the rat. Nature New Biol. 245, 15(r151. Arbuthnott G., Dunnett S. B. and MacLeod N. (1985) Electrophysiological recording from nigral transplants in the rat. Neurosci. L&t. 47, 205-210. Bannon M. J. and Roth R. H. (1983) Pharmacology of mesocortical dopamine neurons. Pharmac. Reu. 35, 53-68. Baring M. D., Walters J. R. and Eng N. (1980) Action of systemic apomorphine on dopamine cell firing after neostriatal kainic acid lesion. Brain Res. 181, 214-218. Bitran M. and Bustos G. (1982) On the mechanism of presynaptic auto-receptor-mediated inhibition of transmitter svnthesis in dooaminergic nerve terminals. Biochem. Pharmac. 31, 2851-2860. Bjorklund A., Dunnett S.B., Stenevi U., Lewis M. E. and Iversen S. D. (1980) Reinnervation of the denervated striatum by substantia nigra transplants: functional consequences as revealed by pharmacological and sensorimotor testing.
2. Agid Y., Javoy F. and Glowinski J. (1973) Hyperactivity 3. 4. 5.
6. 7.
Brain Res. 199, 307-333.
8. Bjiirklund A. and Stenevi U. (1979) Reconstruction of the nigrostriatal dopamine pathway by intracerebral nigral transplants. Brain Res. 177, 555-560. 9. Bjiirkhmd A., Stenevi U., Schmidt R. H., Dunnett S. B. and Gage F. H. (1983) Intracerebral grafting of neuronal cell suspensions. I. Introduction and general methods of preparation. Acta physiol. stand., Suppl. 522, I-10. IO. Brundin P. and Bjijrklund A. (1987) Survival, growth and function of dopaminergic neurons grafted to the brain. In Neural Regeneration (eds Seil F. J., Herbert E. and Carlson B. M.), Progress in Bruin Research, Vol. 71, pp. 293-308. 11. Brundin P., Isacson 0. and Bjiirklund A. (1985) Monitoring of cell viability in suspensions of embryonic CNS tissue and its use as a criterion for intracerebral graft survival. Brain Res. 331, 251-259. 12. Bunney B. S. and Aghajanian G. K. (1978) d-Amphetamine-induced depression of central dopamine neurons: evidence for mediation by both autoreceptors and a striato-nigral feedback pathway. Naunyn-Schmiedebergs Arch. Pharmak. 304, 255-26
I.
13. Carlsson A. (1975) Receptor-mediated control of dopamine metabolism. In Pre- and Postsynaptic Receptors (eds Usdin E., Bunney E. and Bunney W. E., Jr.), pp. 4966. Marcel Dekker, New York. 14. DiChiara G., Porceddu M. L., Fratta W. and Gessa G. L. (1977) Post-synaptic receptors are not essential for dopaminergic feedback regulation. Nature 267, 27&272. 15. Dravid A., Jaton A. L., Enz A. and Frei P. (1984) Spontaneous recovery from motor asymmetry in adult rats with 6-hydroxydopamine-induced partial lesions of the substantia nigra. Brain Res. 311, 361-365. 16. Dunnett S. B., Bjiirklund A., Gage F. H. and Stenevi U. (1985) Transplantation of mesencephalic dopamine neurons to the striatum of adult rats. In Neurul Graffing in the Mammalian CNS (eds Bjijrklund A. and Stenevi U.), pp. 451469. Elsevier, Amsterdam. 17. Dunnett S. B., Bjiirklund A., Schmidt R. H., Stenevi U. and Iversen S. D. (1983) lntracerebral 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. Stand., Suppl. 522, 2937. 18. Dunnett S. B., Bjiirklund A., Stenevi U. and Iversen S. D. (1981) 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, 209-217. 19. Forni C., Brundin P., Strecker R. E., El Ganouni S., Bjijrklund A. and Nieoullon A. (Unpublished) Continuous monitoring of neurotransmitter release by in uioo voltammetry during reinnervation of the denervated striatum by mesencephalic dopamine grafts. 20. Freed W. J., Perlow M. J., Karoum F., Seiger A., Olson L., Hoffer B. J. and Wyatt R. J. (1980) Restoration of dopaminergic function by grafting of fetal rat substantia nigra to the caudate nucleus: long-term behavioural, biochemical and histochemical studies. Ann. Neural. 8, 510-519.
178
R. E. STRECKER et al
21. Freund T. F., Bolam J. P., Bjtirkhmd A., Stenevi U., Dunnett S. B., Powell J. F. and Smith A. D. (1985) Efferent synaptic connections of grafted dopaminergic neurons reinnervating the host neostriatum: a tyrosine hydroxylase immunocytochemical study. J. Neurosci. 5, 603616. 22. Gonon F. and Buda M. (1985) Regulation of dopamine release by impulse flow and by autoreceptors as studied by in oiuo voltammetry in the rat striatum. Neuroscience 14, 765-774. 23. Hefti F., Melamed E. and Wurtman R. J. (1980) Partial lesions of the dopaminergic nigrostriatal system in rat brain: biochemical characterization. Brain Res. 195, 123-137. 24. Herman J. P., Choulli K. and Le Meal M. (1985) Activation of striatal dopaminergic grafts by haloperidol. Brain Res. Bull. 15, 543-546. 25. Imperato A. and DiChiara G. (1984) Trans-striatal dialysis coupled to reverse phase high performance liquid chromatography with electrochemical detection: a new method for the study of the in tjivo release of endogenous dopamine and metabolites. J. Neurosci. 4, 966977. 26. Imperato I. and DiChiara G. (1985) Dopamine release and metabolism in awake rats after systemic neuroleptics as studied by transtriatal dialysis. J. Neurosci. 5, 297-306. 27. Lortn I., Bjlirklund A., Falck B. and Lindvall 0. (1980) The aluminum-formaldehyde (ALFA) method for improved visualization of catecholamines and indoleamines. I. A detailed account of the methodology for central nervous tissue using paraffin, cryostat or Vibratome sections. J. neurosci. Meth. 2, 277-300. 28. Mahalik T. J., Finger T. E., Strijmberg I. and Olson L. (1985) Substantia nigra transplants into denervated striatum of the rat: ultrastructure of graft and host interconnections. J. camp. Neurol. 240, 6&70. 29. Meltzer H. Y. (1982) Dopamine autoreceptor stimulation: clinical significance. Pharmac. Biochem. Eehuc. 17, Suppl. I, I-10. 30. Perlow M. J., Freed W. J., Hoffer B. J., Seiger A., Olson L. and Wyatt R. J. (1979) Brain grafts reduce motor abnormalities produced by destruction of nigrostriatal dopamine system. Science 204, 643-647. 31. Rose G., Gerhardt G., Strijmberg I., Olson L. and Hoffer B. (1985) Monoamine release from dopamine-depleted rat caudate nucleus reinnervated by substantia nigra transplants: an in oivo electrochemical study. Bruin Res. 341, 92-100. 32. Schmidt R. H., Bjiirklund A., Stenevi U., Dunnett S. B. and Gage F. H. (1983) Intracerebral grafting of neuronal cell suspension. III. Activity of intrastriatal nigral suspension implants as assessed by measurements of dopamine synthesis and metabolism. Acta physiol. stand., Suppl. 522, 23-32. 33. Schmidt R. H., Ingvar M., Lindvall O., Stenevi U. and Bjiirklund A. (1982) Functional activity of substantia nigra grafts reinnervating the striatum: neurotransmitter metabolism and (‘4C)-2-deoxy-D-glucose autoradiography. J. Neurochem. 38, 737-748. 34. Seiger A., Olson L., Famebo L.-O. (1976) Brain tissue transplanted to the anterior chamber of the eye: 4. Drug-modulated transmitter release in central monoamine nerve terminals lacking normal postsynaptic receptors. Cell Tiss. Res. 165, 157-170. T. and Ungerstedt U. (1986) An in viva study of dopamine release and metabolism in rat brain 35. Sharp T., Zetterstriim regions using intracerebral dialysis. J. Neurochem. 47, 113-122. 36. Skirboll L. R., Grace A. A. and Bunney B. S. (1979) Dopamine auto- and postsynaptic receptors: electrophysiological evidence for differential sensitivity to dopamine agonists. Science 206, 8(1-82. 37. Stricker E. M. and Zigmond M. J. (1976) Recovery of function following damage to central catecholamine-containing neurons: a neurochemical model for the lateral hypothalamic syndrome. In Progress in Psychobiology and Physiological Psychology (eds Sprague J. M. and Epstein A. N.), pp. 121-189. Academic Press, New York. of dopamine-denervated striatum by substantia 38. StrGmberg I., Johnson S., Hoffer B. and Olson L. (1985) Reinnervation nigra transplants: immunohistochemical and electrophysiological correlates. Neuroscience 14, 98 I-990. 39. Ungerstedt U. (1974) Brain dopamine neurons and behavior. In The Neurosciences, Third Study Program (eds Schmitt F. 0. and Worden F. G.), pp. 695-703. MIT Press, Cambridge, Massachusetts. U. (1984) Measurement of neurotransmitter release by intracranial dialysis. In Measurement sf‘ Neuro40. Ungerstedt transmitrer release in vivo (ed. Marsden C. A.), pp. 81-105. Wiley, New York. G. W. (1970) Quantitative recording of rotational behaviour in rats after 41. Ungerstedt U. and Arbuthnott 6-hydroxydopamine lesions of the nigrostriatal system. Brain Res. 24, 485493. 42. Wuerthele S. M., Freed W. J., Olson L., Morihisa J., Spoor L., Wyatt R. J. and Hoffer B. J. (1981) Effect ofdopamine agonists and antagonists on the electrical activity of substantia nigra neurons transplanted into the lateral ventricle of the rat. Expl Brain Res. 44, ILIO. T., Brundin P., Gage F. H., Sharp T., Isacson O., Dunnett S. B., Ungerstedt U. and Bjiirklund A. (1986) 43. Zetterstrdm In uiuo measurement of spontaneous release and metabolism of dopamine from intrastriatal nigral grafts using intracerebral dialysis. Brain Res. 362, 344-350. T., Sharp T., Marsden C. A. and Ungerstedt U. (1983) In uiuo measurement of dopamine and its 44. Zetterstriim metabolites by intracerebral dialysis: changes after d-amphetamine. J. Neurochem. 41, 1769-l 773. 45. Zetterstriim T., Sharp T. and Ungerstedt U. (1984) Effect of neuroleptic drugs on striatal dopamine release and metabolism in the awake rat studied by intracerebral dialysis. Eur. J. Pharmac. 106, 27-37. T., Sharp T. and Ungerstedt U. (1986) Effect of dopamine D-l and D-2 receptor selective drugs on 46. Zetterstriim dopamine release and metabolism in rat striatum in Go. Naunyn-Schmiedebergs Arch. Pharmak. 334, 117-124. 47. Zetterstriim T. and Ungerstedt U. (1984) Effects of apomorphine on the in uiuo release of dopamine and its metabolites. studied by brain dialysis. Eur. J. Pharmuc. 97, 29-36. (Accepted 8 Seprember
1986)