Characterization of gaba release from intrastriatal striatal transplants: Dependence on host-derived afferents

Neuroscience Vol. 53, No. 2, pp. 403-415, 1993

0306-4522/93 $6.00 + 0.00 Pergamon Press Ltd © 1993 IBRO

Printed in Great Britain

CHARACTERIZATION OF GABA RELEASE FROM INTRASTRIATAL STRIATAL TRANSPLANTS: DEPENDENCE ON HOST-DERIVED AFFERENTS K. CAMPBELL,* P. KALt~N, K. WICTORIN, C. LUNDBERG, R. J. MANDEL and A. BJORKLUND Department of Medical Cell Research, University of Lund, S-223 62 Lund, Sweden A~traet--Extracellular levels of GABA, derived from cell suspension transplants of embryonic day 14-15 rat striatal primordia implanted into the previously excitotoxically lesioned straitum, were measured using intracerebral microdialysis in halothane-anaesthetized rats. GABA overflow was monitored using loop type dialysis probes implanted into grafted, age-matched ibotenic acid-lesioned and intact striata, under baseline conditions and after different pharmacological manipulations. Basal and evoked GABA release, which was reduced by 58 and 96%, repectively, in the excitotoxin-lesioned striatum, was restored by the striatal grafts to levels close to or above those observed in normal striata. The graft-derived release of GABA was most likely of neuronal origin, since the K+-evoked (100 mM) GABA overflow was reduced by almost 80% when Ca + ÷ was replaced by 20 mM Mg +÷ in the perfusion medium, and blockade of GABA uptake by nipecotic acid (0.5 mM), induced a greater than six-fold increase in GABA overflow. However, perfusion of the graft with 1/~M tetrodotoxin in combination with K ÷ (100 mM) resulted in little if any reduction in the K÷-evoked overflow. Histological analysis demonstrated a dense tyrosine hydroxylase-positive fibre network in the grafts, which was removed after a 6-hydroxydopamine lesion of the ipsilateral nigrostriatal pathway. The dopamine denervating lesion resulted in an increased K+-evoked GABA overflow both in the intact (+ 76%) and the grafted striata (+ 181%), suggesting that the tonic dopaminergic inhibitory control of GABA release, seen in the intact striatum, is also present in the grafted striata. The glutamate analogue, kainic acid (1 mM added to the perfusion fluid), evoked a 60-74% increase in GABA overflow both in intact striata (with or without dopaminergic denervation) and in the striatal grafts. This effect seemed to be dependent on an intact corticostriatal projection, since knife-cut transections of the frontal cortex at the level of the forceps minor, abolished the response in both the intact and grafted striata. These results demonstrate that grafts of fetal striatal tissue implanted into the excitotoxically lesioned straitum restore striatal GABA overflow in a neuron-dependent manner, close to or above that seen in the normal intact striatum. Furthermore, the graft-derived GABA release appears to be under normal regulatory control from the host dopaminergic and glutamatergic systems. Since the GABAergic striatal output system is critical for the expression of striatum-related behaviours, it is proposed that the graft-induced behavioural recovery in the striatal lesion model, at least in part, may depend on the restoration of striatal GABAergic neurotransmission.

Previous anatomical studies of fetal striatal grafts implanted into the excitotoxically lesioned striatum of adult rats, have demonstrated that such transplants develop into neuronal structures which partially resemble the normal intact striatum both morphologically and neurochemically. 23,3s,59 The striatum-like area of these grafts receive innervation from dopaminergic, serotonergic, cortical and thalamic host nuclei 23'38'57-6°,65,67 and in turn, send axons out to the globus pallidus and probably as far as the substantia nigra. 6'-63 Electron-microscopic studies have demonstrated that the host dopaminergic and cortical afferents preferentially form synaptic contacts with the dendritic spines of grafted striatal *To whom correspondence should be addressed. Abbreviations: DARPP-32, dopamine- and cyclic AMP-

regulated phosphoprotein with a molecular weight of 32,000; EDTA, ethylenediaminetetra-acetate; GAD, glutamate decarboxylase; HPLC, high-performance liquid chromatography; 6-OHDA, 6-hydroxydopamine; TB, tooth bar; TH, tyrosine hydroxylase; TTX, tetrodotoxin.

neurons, similar to that observed in the normal mature striatum, zL6°'65 Behavioural and electrophysiological studies of intrastriatal striatal grafts indicate that these grafts may be functionally integrated with the host brain circuitry. ~9'27'44'5°'66 Previous studies have shown that striatal grafts are rich in GABAergic neurons z6'49 and that these grafts reverse the lesion-induced reduction in glutamate decarboxylase ( G A D , the synthetic enzyme for G A B A ) levels, in the grafted striatum and ipsilateral globus pallidus. Additionally, in a push-pull perfusion study, Sirinathsinghji e t al. 51 have reported that striatal grafts can restore the lesion-induced deficits in extracellular G A B A overflow both in the ipsilateral globus pallidus and substantia nigra. On the basis of these observations it has been proposed that the intrastriatal striatal grafts may exert their functional effects at least in part, through the release of G A B A . In the normal intact striatum, the vast majority of the projection neurons are G A B A e r g i c 32 and send 403

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axonal projections to the globus pallidus a n d substantia nigra (for review, see Ref. 24). Additionally, G A B A occurs in a p o p u l a t i o n of striatal interneurons, '2 a l t h o u g h they constitute only a small p r o p o r t i o n of the total G A B A e r g i c n e u r o n a l p o p u l a t i o n in the striatum. Earlier reports using the intracerebral microdialysis t e c h n i q u e : 5 have s h o w n t h a t the extracellular levels o f G A B A are only in part impulse-dependent (blocked by tetrodotoxin) a n d vesicular in n a t u r e (blocked by removal o f C a + +).,7,45 This implies t h a t a considerable p r o p o r t i o n of the G A B A recovered extracellularly m a y be derived from n o n - n e u r o n a l (metabolic a n d / o r glial) pools. However, in rats with neuron-depleting excitotoxic lesions we have recently s h o w n t h a t striatal G A B A overflow, u n d e r b o t h basal a n d K + - e v o k e d conditions, is reduced by 8 0 % to almost 100% w h e n the n e u r o n a l elements are d e s t r o y e d : This indicates t h a t intracerebral microdialysis can be used to m o n i t o r changes in n e u r o n a l G A B A release after striatal lesions a n d i m p l a n t a t i o n o f fetal striatal tissue. In the present series of experiments, we have used this a p p r o a c h to study G A B A release from transplants of fetal striatal tissue grafted to the excitotoxically lesioned striatum during baseline conditions a n d after pharmacological challenges. In order to determine the extent o f host afferent control over G A B A release from the grafted striatal neurons, we have employed m a n i p u l a t i o n s o f the d o p a m i n ergic or cortical afferents which are k n o w n to alter extracellular G A B A levels in the intact striatum. EXPERIMENTAL PROCEDURES

Lesion and transplantation surgery Thirty-six adult female Sprague-Dawley rats (c. 250 g body weight at the outset of the study, ALAB, Stockholm) were used. Lesion and transplantation surgery was performed under Equithesin anaesthesia (3.0ml/kg i.p.). Twenty-four animals received excitotoxic lesions with 14 pg of ibotenic acid (Sigma) administered unilaterally (on the animal's fight side) over three injection sites into the head of the striatum, as described previously)9 At seven to 10 days post-lesioning, 18 of the ibotenic acid-lesioned rats received intrastriatal transplants of cell suspensions prepared from fetal striatal primordia, dissected from embryonic day 14-15 rat embryos (crown-rumplength = 11-14.5 mm) as described earlier) 9 Approximately 1,000,000 cells were implanted into the lesioned striatum of each animal (4-5 #1 of cell suspension), divided over two needle penetrations at the coordinates: (1) A = 0.2; L = 3.0; V = 4 . 5 ; (2) A = 1 . 5 ; L = 2 . 5 ; V = 4 . 7 ; tooth bar (TB) = - 2 . 3 . At more than three months post-grafting, seven of the 18 grafted animals were subjected to 6-hydroxydopamine (6-OHDA, Sigma) lesions ipsilateral to the transplants while another seven intact age-matched animals also received 6-OHDA lesions. Two injections of 2.5 #1 of 6OHDA (3 pg/pl, free base, in 0.2 mg/kg ascorbate-saline) were made at the coordinates: (1) TB = -2.4; A = -4.4; L = l . 2 ; V = 7 . 8 ; (2) T B = + 3 . 4 ; A = - 4 . 0 ; L=0.8; V = 8.0. In another five of the 18 grafted animals, and in five of the intact controls, coronal knife-cuts were performed so as to undercut the frontal cortex ipsilateral to the transplant at the level of the forceps minor. The knife-cuts were made in the coronal plane using the following coordinates: TB=-3.2 (1) A = + 2 . 4 ; L = - I . 0 ; V=-5.4; (2)

A = +2.4; L = -2.0; V = -5.3; (3) A = +2.4; L = -3.0; V = - 5 . 3 (for intact animals and TB = - 3 . 2 (1) A = +2.8; L = -1.0; V = -5.4; (2) A = +2.8; L = -2.0; V = -5.3; (3) A = +2.8; L = -3.0; V = - 5 . 3 (for grafted animals). The six ibotenic acid-lesioned animals which did not receive striatal grafts served as an age-matched lesion-only control group. These animals also contributed to a parallel study investigating the effects of striatal excitotoxic lesions on striatal GABA overflow. 6 Dialysis procedures Loop-type dialysis probes were constructed from flexible SCE cellulose tubing (CD Medical International Ltd) with an outer diameter of 0.3 mm and a molecular weight cut off of 10,000. 28 Four millimetres of dialysis membrane (2 mm in a dorsoventral aspect) were exposed to the brain tissue. Dialysis was performed at least three months after the ibotenic acid lesion and transplantation (four to six weeks after the 6-OHDA lesion, or six days after the frontal cortex transection). The probes were implanted under either chloral hydrate (350 mg/kg, i.p.) or halothane (1.5% halothane-air mixture) anaesthesia. The following coordinates were used: in the intact striatum, A = +0.7; L = __+2.6; V = - 4 . 5 ; T B = - 2 . 3 ; in the lesioned and transplanted striatum, A = +0.7; L = - 3 . 4 ; V = - 4 . 6 ; TB = --2.3; and in the ibotenic acid lesion-only striatum, A = +0.7; L = - 3.6; V = -4.6; TB = - 2 . 3 (in the lesioned and grafted striata the coordinates were modified in order to compensate for the tissue atrophy caused by the lesion). The dialysis probes were secured to the skull using screws and dental cement. In most cases, probes were implanted bilaterally into the normal intact side and experimental side in the transplanted and ibotenic acid lesioned animals. Starting on the day after probe implantation, dialysis experiments were conducted under halothane anaesthesia (1.2 1.5% halothane-air mixture) with the body temperature maintained at 37°C by incandescent light. Dialysis was performed over a two-day period. The inlet of the probe was connected to a CMA microinfusion system (Carnegie Medicin, Sweden) and perfused with Ringer solution (147 mM NaC1, 4 mM KC1, and 2.3 mM CaCI2, pH 6.0) at a rate of 2 pl/min. Nipecotic acid (0.5 mM), tetrodotoxin (TTX) (1 #M) and kainic acid (1 mM) were dissolved in Ringer solution and the pH adjusted to approximately 6.0. Both KC1 (100mM) and Ca++-free (20mM Mg ++) medium were prepared in compensated Ringer. The first 45 min of dialysate was discarded on each experimental day and samples collected every 15 min. Samples were immediately frozen in liquid nitrogen and stored at - 8 0 ° C for one to seven days before being assayed. Experiment L Probes were implanted bilaterally in animals which had received a unilateral ibotenic acid lesion and subsequent striatal graft (n = 6) or a unilateral striatal ibotenic acid lesion only (n = 6). The response to repeated K +-depolarization (100 mM, 15 min duration, separated by 60 min each) was tested subsequent to the first three baseline samples. The second stimulation was performed under Ca + +-free conditions (20 mM Mg ++ added), and the third with 1 p M TTX (Sigma) added to the perfusion fluid. The striata contralateral to the lesion or graft was used as the intact control (n = 7). On the following day, in the same animals, K+-depolarization was performed after the initial three baseline samples and 30 min thereafter nipecotic acid (0.5 mM, Sigma) was added to the perfusion fluid for an additional 30 min. (See Fig. 1). Experiment H. Four to six weeks after 6-OHDA lesions, the effect of K +-depolarization (I00 mM, 15 min) on GABA overflow was determined in seven lesioned and grafted rats and in seven intact (i.e. non-ibotenic acid lesioned) rats. In seven of the rats, dialysis probes were also implanted into the intact non-denervated contralateral striatum. On the second day of dialysis, the glutamate analogue, kainic acid (Sigma), was added at a concentration of 1 mM to the

405

Afferent regulation of GABA release in striatal grafts Exp. I: Pharmacological characteristics o f G A B A overflow

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Perfusion time (rain) Fig. 1. Schematic representation of the sequence of dialysis sampling and manipulations in experiments I-III. The placement of the dialysis probe is indicated for each experiment. See the Dialysis procedures section under Experimental Procedures for details. IBO, ibotenic acid; KA, kainic acid; Nip, nipecotic acid. perfusion fluid for 15min after the first three baseline samples were collected. (See Fig. 1). Experiment III. The effect of kainic acid on extracellular GABA levels was assessed in five intact and five lesioned and grafted animals which had received knife-cut transections of the frontal cortex six days prior to dialysis, in order to determine whether the kainic acid response was dependent on an intact corticostriatal pathway. In these rats, probes were implanted in the striatum ipsilateral to the knife-cut and kainic acid (1 mM) was added to the perfusion fluid for 15 min, as above. In this experiment, only two of the grafted animals were useful; two of the grafts showed poor survival and one had a misplaced probe. (See Fig. I).

GABA assay GABA was assayed using reverse phase high-performance liquid chromatography (HPLC) coupled to electrochemical detection, according to the method of Kehr and Ungerstedt. 3° For details and specificity of the GABA assay, see Refs 6 and 30. The detection limit for this assay was approximately 0.15 pmol/30#l of perfusate. Briefly, the dialysates were derivatized using O-phthaldialdehyde/tbutylthiol reagent prior to being injected onto a Nucleosil 3 C18 column (100 × 4mm) using a mobile phase of 0.15 M sodium acetate, 1 mM EDTA (pH 5.32) and 50% acetonitrile, passed at a rate of 0.8 ml/min. Compounds were detected by a glassy carbon working electrode set at +0.75 mV (with respect to a Ag/AgC1 reference electrode) using an LC4 amperometric controller (Bioanalytical Systems). Signals were processed through a Nelson computerized integrator (Perkin Elmer). GABA levels are reported as pmol/30pl of dialysate without correction for the recovery across the dialysis membrane. The recovery of extracellular GABA with the current 2-mm loop probes has previously been found to average about 13%. 6 Due to considerable inter- and intragroup variabilities the data were log-transformed before statistical analysis. Effects within groups were analysed using the Student's two-tailed paired t-test, and between

group effects were analysed by either the student's two-tailed unpaired t-test or a one-way analysis of variance (ANOVA) with the Newman-Keuls post hoc test. The null hypothesis was rejected with P < 0.05.

Immunohistochemistry At the completion of the dialysis experiments, grafted and ibotenic acid-lesioned animals were deeply anaesthetized with chloral hydrate and perfused transcardially with physiological saline followed by ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer (300 ml). Brains were immediately removed and stored in 20% sucrose in the same buffer overnight. Cornonal sections (30-40 #m) were cut on a freezing microtome. The sections were quenched of their endogenous peroxidase activity for I0 min in 3% H202 in 10% methanol and placed in 2% normal horse serum (for DARPP-32) or 2% normal swine serum [for tyrosine hydroxylase (TH)]. After a 1-h incubation in blocking serum, sections were incubated with either a mouse monoclonal antibody raised against DARPP-32 at a dilution of 1:20,000 (kindly provided by Dr P. Greengard) or with an anti-TH antiserum (Pel-freeze; dilution 1 : 500) produced in rabbit, overnight at room temperature. Sections were then rinsed and the primary antisera were detected by the avidin-biotin-peroxidase (ABC, Vectorlabs) method. Y,3'-Diaminobenzidine was used as the chromogen to detect the reaction product at a concentration of 0.05 mg/ml in 0.01% HzO2. The sections were mounted onto glass slides, osmium-intensified and coverslipped with DPX. In addition to DARPP-32- and TH-immunohistochemistry, one series of sections from each animal was also stained with Cresyl Violet. RESULTS

Lesion and graft morphology Ibotenic acid lesions o f the striatum resulted in a n almost complete loss of D A R P P - 3 2 - i m m u n o -

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labelled neurons and extensive gliosis throughout the head of the caudate-putamen. The loss of striatal neurons was accompanied by considerable atrophy of the head of the caudate-putamen and an enlargement of the adjacent lateral ventricle. All grafted animals, except two in Experiment III, possessed

surviving transplants. They were located within the gliotic neuron-depleted host striatum, surrounded by the densely packed myelinated fibre bundles of the internal capsule (Fig. 2). As reported earlier, 59 DARPP-32 immunoreactivity was non-homogeneous, occurring in discrete patches which covered

Fig. 2. Photomicrographs of DARPP-32 (A, C) and TH (B, D)-immunostained intrastriatal striatal grafts (outlined by arrowheads) implanted into the previously ibotenic acid-lesioned striatum, showing the location of the dialysisprobe tracts within the graft (indicated by asterisks). Note the close correspondence between the patches of DARPP-32 and TH immunoreactivities in adjacent sections of the graft shown in A and B. Dopamine depleting lesions using intracerebral 6-OHDA resulted in a loss of TH immunoreactivity (D) leaving DARPP-32 labelling unaffected (C). ac, anterior commissure; cc, corpus callosum; H, spared host striatum; Iv, lateral ventricle. Scale bar = 625 #m.

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Afferent regulation of GABA release in striatal grafts about 30-40% of the cross-sectional area through the transplant (Fig. 2A, C). Additionally, TH immunohistochemistry revealed a patch distribution of TH-positive terminals in the graft, corresponding well with the patches of DARPP-32 labelling on adjacent sections (Fig. 2B). Only grafted animals in which the dialysis probe had been placed within the boundaries of the striatal graft, were used in this study (Fig. 2); i5 of the 16 rats with surviving grafts fulfilled this criterion. Similarly, in all lesion-only animals included in the study the probe was located in the gliotic neuron-free area of the lesioned striatum. The dopamine-depleting lesions (induced by intracerebral 6-OHDA lesions on the side ipsilateral to the graft) resulted in a loss of dopaminergic innervation to the grafts as evidenced by the lack of TH-positive fibre staining within the graft and remaining host striatum (Fig. 2D).

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Fig. 4. Effect of membrane depolarization with K +, on GABA overflow in intact (n = 7), ibotenic acid-lesioned (n = 6), and lesioned and grafted striata (n = 6). (A) KC1 was added to the perfusion medium at 100mM over a 15-min sampling period. (B) K+-depolarization combined with Ca + +-free medium or TTX (1 yM) in intact, lesioned, and lesioned and grafted striata. Values give means _+ S.E.M.; **P < 0.01, ***P < 000.1 compared to the preceding baseline sample (A) or the initial K+-stimulated peak (B). Student's two-tailed paired t-test. the intact striatum (Fig. 3A). On the second day of dialysis, the basal levels of all three groups were not significantly different from the previous day, and the extracellular levels of GABA in the ibotenic acid-lesioned striata remained significantly reduced as compared to either the intact or the lesioned and grafted striata (Fig. 3B).

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Basal GABA overflow Basal GABA overflow in the intact striatum was 0.60 + 0.08 pmol/30 #1 of perfusate (mean + SEM of 14 animals, Fig. 3) on the first day of dialysis, which is comparable to the levels we have previously observed. 6 Excitotoxic ibotenic acid lesions of the striatum (performed more than three months prior to dialysis) produced a 58% reduction in GABA overflow (P < 0.01, Fig. 3A), down to a mean level of 0.25 + 0.08 pmol/30 pl (n = 6). In animals which had received transplants of fetal striatal tissue into the previously ibotenic acid-lesioned striatum (more than three months post-grafting), basal striatal GABA overflow was restored to levels similar to, or even slightly higher than those measured in the intact striatum. The recorded level, 0.84 + 0.14pmol/30 pl (n = 6), was significantly higher than that seen in the lesion-only animals (P < 0.01), but not different from

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Fig. 3. Basal GABA overflow in intact (n = 14, day 1; n = l l , day 2), ibotenate lesioned (n=6, day 1; n = 5 , day 2), and lesioned and grafted (n = 6, day 1; n = 5, day 2) striata on the first (A) and second day (B) of dialysis (i.e. the second and third days after probe implantation). Baseline levelswere determined from the average of the first three 15-minsamples on each day and are expressed as pmol/30/~1 of dialysate (without correction for recovery across the dialysis membrane). Values are means_S.E.M.; **P < 0.01, *P < 0.05 compared to intact or grafted striata, using one-way analysis of variance with the Newman Keuls post hoc test.

Pharmacological characteristics of GABA overflow (Experiment I) Depolarization with KC1 (100 mM), resulted in a 54-fold increase in GABA overflow in the intact striata (P < 0.001, 43.9___4.0 pmol/30 #1, Fig. 3). The K+-evoked GABA overflow was reduced by 96% in the lesion-only striata. Nevertheless, there was a small K+-evoked increase also in the lesiononly striatum which was significant when compared to the previous baseline sample (5.8-fold, P < 0.01, Fig. 4A). In the lesioned and grafted animals the K+-evoked GABA overflow was restored to

K. CAMPBELLet al.

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67.5 +5.0 pmol/30 pl (Fig. 4A), which was approximately 50% higher than that observed in the intact striatum. The magnitude of the K÷-evoked GABA overflow in the grafted striata on the second day of dialysis was 74.2 + 8.5pmol/30pl, similar to that observed on the first day. When Ca + ÷ was replaced by Mg ÷ ÷ in the perfusion medium little or no effect was observed on the basal GABA overflow in any of the groups. However, this manipulation did result in a considerable reduction of the K+-evoked levels. As shown in Fig. 4B, there was an 83% reduction (P < 0.001) in the K÷-evoked peak (compared to the preceding peak) in the intact striatum, whereas in the ibotenic acid-lesioned striatum the omission of Ca ÷ ÷ did not have any effect. In the grafted animals, the K+-evoked GABA overflow was reduced by 77% when Ca r÷ was omitted from the perfusion medium (P < 0.001). TTX added to the perfusion fluid at 1 p M concentration had no effects on the basal extracellular GABA levels in intact striata, but it reduced the KCl-induced peak by 40% (P < 0.001, Fig. 4B). In contrast, TTX did not have any effect on basal or evoked extracellular GABA overflow, neither in the lesioned controls nor in the grafted striata (Fig. 4B). The addition of the specific GABA uptake blocker, nipecotic acid (0.5mM), to the perfusion medium on the second day of manipulations induced an 11.5-fold increase (5.3 pmol/30 pl, P < 0.001, Fig. 5) in baseline GABA overflow over a 30-min perfusion period. This effect, which was abolished in the lesiononly animals, was restored in the grafted striata, showing a six-fold increase from basal GABA overflow during the 30-min perfusion with nipecotic acid (7.4 + 1.9 pmol/30/21, P < 0.01, Fig. 5).

Effect of 6-hydroxydopamine lesions of the host dopamine afferents (Experiment H) Previous studies using intracerebral microdialysis have reported that 6-OHDA lesions of the nigroEffect of GABA Uptake Blockade by Nipecotic Acid (Nip) 10"1 • Baseline

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Fig. 5. Effect of GABA uptake blockade in intact (n = 5), ibotenic acid-lesioned (n = 5), and lesioned and grafted striata (n = 5) during a 30-min perfusion with nipecotic acid (0.5 mM). GABA levels increased steadily over the perfusion period and peaked at 30 min. Values give means + S.E.M.; ***P < 0.001. **P < 0.01 compared to the previous baseline sample Student's two-tailed paired t-test.

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B Fig. 6. Effect of dopamine-denervating 6-OHDA lesions on basal and K+-depolarized GABA overflow. Basal (A) and K÷-depolarized (B) extracellular GABA levels in normal striata (n = 7), 6-OHDA denervated striata (n = 7), intact striata contralateral to the dopamine denervated striata (n = 7), non-denervated intrastriatal striatal grafts (n = 6) and 6-OHDA denervated striatal grafts (n = 7). Values represent means+S.E.M.; tP <0.05 compared to the contralateral striata using the Student's paired two-tailed t-test. *P < 0.05 compared to the normal striata or normal graft, using the Student's unpaired two-tailed t-test. striatal dopaminergic pathway result in increased basal and K÷-stimulated striatal GABA overflow in the striatum. 36'54 In the animals with a unilateral 6-OHDA lesion, basal overflow in the denervated striata was slightly higher than that recorded in either the non-denervated animals ( + 8 % ) or in the contralateral non-denervated striata ( + 14%); however, neither difference was statistically significant (Fig. 6A). The K÷-evoked GABA overflow was significantly higher in the dopamine deafferented striatum, either when compared to the non-denervated striatum contralateral to the lesion ( + 4 8 % , P < 0.05) or to the striata of animals with the dopamine inputs intact, on both sides ( + 7 6 % , P < 0.05, Fig. 6B). The magnitudes of these changes are similar to those previously reported. 36'54 As was the case in the intact striatum, basal GABA overflow collected from the striatal grafts was not

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Afferent regulation of GABA release in striatal grafts significantly different between non-denervated and 6-OHDA-denervated grafts (Fig. 6A). The dopaminedepleting lesions did, however, result in a potentiation of the K÷-stimulated G A B A overflow, from an average of 65.7 _ 5.0 pmol/30 #1 in non-denervated grafts, to 184.3 + 50.5 pmol/30 #1 in the 6-OHDA-denervated transplants, an increase of + 181% (P < 0.05, Fig. 6B).

Effect of intrastriatal application of kainic acid (Experiments H and III) Previous investigators have demonstrated that striatal perfusion with the glutamate analogue, kainic acid, results in increased release of the excitatory amino acids glutamate and aspartate. 5'68 Moreover, Young et al. 68 have demonstrated that the kainic acid-induced increases in glutamate and aspartate depend on an intact corticostriatal pathway. These findings are in line with previous suggestions that the neurotoxic effects of kainic acid function by increasing extracellular striatal glutamate from corticostriatal terminals. 2,3,42 In order to determine whether kainic acid-induced increases in striatal glutamate and aspartate can affect striatal G A B A release, 1 m M kainic acid was added to the perfusion medium for 15 min (Experiment II). This kainic acid infusion induced a transient 70% (P <0.01) increase in G A B A overflow, to 1.34 +_ 0.32 pmol/30/al, which returned to baseline levels in the subsequent sample (Fig. 7A); all six animals in this group responded to this challenge. This effect was completely abolished in animals which were subjected to a coronal knife transection undercutting the frontal cortex at the level of the forceps minor (Figs 7A, 8A). Unlike the frontal cortical lesions, the dopamine-depleting 6 - O H D A lesions had no effect on the kainic acid response (Fig. 7B). In the 6-OHDA-lesioned rats kainic acid infusion increased extracellular G A B A levels to 1.51 _ 0.35 pmol/30/~1, ( + 6 0 % from the baseline, i.e. an increase similar to that observed in the normal striatum). In the grafted animals, kainic acid infusion was performed on the second day of dialysis in animals with previous 6 - O H D A lesions. All six rats showed a transient, on average 74%, increase in G A B A overflow, to 1.53 + 0.33 pmol/30/al (P < 0.01; Fig. 9), closely resembling that observed in the intact striatum. In two grafted rats in Experiment III (where the grafts had survived and the probe was properly placed) the kainic acid-induced effect on the graft derived G A B A overflow was seen to be totally abolished by the cortical transection (dashed line in Fig. 9). The camera lucida drawing in Fig. 8B shows the position of the cortical knife cut and the location of the dialysis probe tract within the striatal graft, in one of these rats. DISCUSSION The present results show that intrastriatal striatal grafts restore excitotoxic lesion-induced deficits in

extracellular striatal G A B A to levels very similar to those observed in the intact striatum. The graftinduced restoration of striatal G A B A overflow appears, for the most part, to be neuronal in nature since the K+-depolarized G A B A levels were severely reduced when Ca ++ was replaced with Mg ++, and blockade of the G A B A uptake system produced considerable elevations in the grafts' G A B A overflow. These results also provide evidence that the host dopaminergic and cortical afferents, which are known to innervate the striatal grafts, exert regulatory influences over G A B A release from the grafted neurons. Effect of Kainic Acid (KA) on Non-decorticated and Frontal Decorticated Striata 2.0 1.8 1.6 --t

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Excitotoxic lesions are known to result in extensive destruction of neuronal elements within the striatum (for review, see Ref. 15), accompanied by a considerable reduction in tissue levels of the synthetic enzyme for G A B A , by as much as - 8 0 to - 9 0 % in the lesioned area) 3'26'41This matches well the reduction in basal G A B A overflow as measured by intrastriatal microdialysis, which is about -800/0. 6 It is unclear whether any of the residual G A B A is neuronal: K+-evoked G A B A overflow, as well as the increase in extracellular G A B A levels induced by the neuronal uptake blocker, nipecotic acid, are reduced from - 7 0 % to almost - 1 0 0 % in the neuron-depleted striatum. Over the first 20 weeks after the excitotoxic lesion, G A D activity has been shown to recover partially; to about 25% of normal when expressed in total striatal enzyme activity, and to about 40°,6 of normal when expressed in activity per wet weight. 26 This apparent recovery has been suggested to arise, at least in part, from the shrinkage and subsequent condensation of the remaining striatum over the long term. 26 Indeed, in the long-term excitotoxically lesioned striata studied here, G A B A release remained

Afferent regulation of GABA release in striatal grafts severely depressed both after K÷-induced depolarization ( - 9 6 % ) and during uptake blockade ( - 9 0 % ) , indicating that there is very little spontaneous recovery of neuronal GABA release in the lesioned striatum over time. Previous biochemical and immunohistochemical studies have demonstrated that intrastriatal striatal grafts are rich in GABA-containing neurons. 26,49Consistent with this, striatal grafts have been shown to replenish the lesion-induced reduction of G A D activity26 as well as lesion-induced changes in G A B A overflow in the globus pallidus and substantia nigra, as determined by the push-pull perfusion technique) l In line with the previous results, the findings of the present study show that neuron-depleting excitotoxic lesion-induced deficits in basal striatal extracellular GABA, which amounts to about - 8 0 % in shortterm ibotenic acid lesions (seven to 10 days postlesioning6), are completely reversed by striatal grafts. Furthermore, evoked GABA overflow, which is reduced by more than 96% in the neuron-depleted striatum, was restored by the grafts to levels that were similar to or slightly higher than those obtained in the normal intact striatum. These data indicate that the graft-derived extracellular G A B A levels are largely of neuronal origin. In the normal striatum, it appears that the response of striatal G A B A overflow to high K ÷ levels may be composed of three components: 6 firstly, a Ca+÷-dependent TTX-sensitive portion (which may represent impulse-dependent vesicular release); secondly, a Ca ÷ ÷-dependent TTX-insensitive portion (which may correspond to dendritic release); and finally a small component that is both Ca ÷÷independent and TTX-insensitive. There is evidence from in vitro studies that a significant component of striatal G A B A release may occur through Ca ÷÷independent mechanisms. L47 In fact, in halothaneanaesthetized rats, the G A B A overflow that was seen after blockade of G A B A uptake by nipecotic acid was Ca+ +-dependent and TTX-sensitive only to about 20-30%. This suggests that under baseline conditions only a small fraction of the G A B A overflow in the normal striatum may reflect impulsedependent vesicular G A B A release.6 Indeed, previous electrophysiological data have shown that most striatal neurons (the majority of which are GABAergic32), are electrically silent in animals at rest. 14'64 Similar to the normal striatum, basal G A B A overflow in the grafts was not responsive to Ca+÷-free conditions or TTX perfusion. Unlike the basal extracellular G A B A levels, the K÷-evoked G A B A overflow in striatal grafts displayed a similar Ca ÷ +dependence as that in the normal intact striatum. This suggests that most of the G A B A released from the grafted striatal neurons in response to elevated levels of K ÷ is vesicular in nature. In addition, nipecotic acid, a specific G A B A uptake blocker devoid of any effect in the ibotenic acid-lesioned (neuron-depleted) striatum, increased graft-derived

411

G A B A overflow to levels which were close to those seen in the intact striatum. Interestingly, the K +evoked G A B A overflow in the grafts did not respond to perfusion with the sodium channel blocker, TTX. This finding may suggest that a significant proportion of the K+-evoked G A B A release within the grafts is dendritic in origin.

Dopaminergic regulation of GABA release in the striatal grafts In the normal striatum, dopamine afferents are known to terminate specifically on the dendritic spines of medium-sized GABAergic neurons. 21'34 In this respect, the nigrostriatal dopamine system is ideally situated to modulate the activity of striatal GABAergic neurons. Indeed, recent reports have shown that disruptions of striatal dopamine transmission resulted in altered basal and K+-stimulated G A B A overflow. 36'54 Recent light-microscopic investigations have shown that dopamine afferents specifically innervate the striatum-like regions of the grafts, as distinguished by acetylcholinesterase histochemistry, DARPP-32 or calbindin immunoreactivity. 23'38"59In addition, Clarke et al)' have previously demonstrated, using electron-microscopic techniques, that dopamine terminals invading grafts of fetal striatal tissue form synapses with grafted neurons, a number of which, could be identified as densely spiny medium-sized neurons. In the present study, 6-OHDA lesions of the nigrostriatal dopamine projection in normal intact animals resulted in a marked increase in striatal G A B A overflow in response to K+-depolarization, similar to previous reports. 36'54 Unlike previous studies, however, we did not see any significant effect on baseline G A B A overflow. This difference may be due to the fact that the dialysis procedures in the present experiment commenced on the day after the probes were implanted, while in the previous studies they began immediately following probe implantation. The effect of dopamine denervation on challenged G A B A overflow is consistent with the view that striatal dopamine afferents contribute an inhibitory component to the K+-evoked G A B A overflow from striatal target neurons. Indeed, dopamine D2 receptor stimulation in combination with depolarizing levels of KC1 has been shown to result in a significantly reduced K÷-evoked G A B A overflow. 53 In the grafted animals, 6-OHDA lesions of the host nigrostriatal dopamine afferents increased the K ÷-stimulated G A B A overflow by 181% above that seen in grafts where the dopamine afferents were left intact. These results provide evidence that host dopamine afferents to striatal grafts modulate extracellular G A B A levels and therefore are functional in nature. We have recently observed that the host dopamine afferents to striatal grafts have a regulatory influence on neuropeptide gene expression within grafted striatal neurons, inducing changes in the

412

K. CAMPBELLet al.

expression of the mRNAs encoding for the neuropeptides enkephalin and substance P that correspond well with those seen after dopamine denervation in the intact striatum. 7 Additionally, studies in grafted animals have demonstrated that pharmacological manipulation of the host dopamine system can induce the expression of the immediate early gene c-fos in transplanted striatal neurons in a manner similar to that observed in the intact s t r i a t u m . 16'39'4° Growing data thus support the notion that the hostderived dopamine afferents to striatal grafts exert a prominent functional regulation over grafted striatal neurons. Cortical regulation o f G A B A release f r o m the striatal grafts

Electron-microscopic studies have shown that fibres originating from cell bodies in the cerebral cortex form synaptic contacts with the dendritic spines of medium-sized neurons in the normal striatum. ~8'52 A recent study by Fu and Beckstead, 22 has demonstrated that electrical stimulation of the frontal cortex induces the expression of c-fos in striatal output neurons (most of which are medium-sized and GABAergic32). In combination with previous electrophysiological data, 33 this suggests that cortical inputs to the striatum directly regulate the activity of their medium-sized postsynaptic target neurons. Earlier anatomical studies of striatal grafts have shown that host cortical fibres innervate the striatumlike regions of the grafts and terminate rather specifically on the dendritic spines of grafted striatal neurons. 58'6°'61'65 Moreover, Rutherford et al. 5° and Xu et al. 66 have provided electrophysiological evidence for the restoration of a functional corticostriatal pathway in intrastriatal striatal transplants of the type studied here. Since the first discovery that the glutamate analogue, kainic acid, produces a severe and nonreversible destruction of neurons in the striatum, leaving the fibres of passage u n a f f e c t e d , 13'41 a great deal of interest has been concentrated on studying both the physiological and excitotoxic activity of this molecule. Previous investigations have suggested that the excitotoxic effect of kainic acid depends on an intact corticostriatal pathway. 2'3'42 Recent studies using intracerebral microdialysis for the excitatory amino acids, glutamate and aspartate, have shown that kainic acid dramatically increases the levels of these amino acid transmitters. 5'6s The mechanism by which kainic acid increases extracellular glutamate/aspartate is somewhat controversial since Butcher et al. 5 observed no effect after decortication, while Young et al. 68 found that the kainic acid-induced increase in these amino acid transmitters was considerably reduced following a decorticating lesion. The results of the present study show that kainic acid (1 mM) perfusion of the striatum induces significant increases in extracellular GABA overflow,

which is consistent with a previous in vitro study. 68 Although studies have shown that kainic acid elevates dopamine levels in the striatum, s'9'29 it appears that the increased dopamine overflow has only a small effect, if any, on kainic acid-induced GABA levels, since the response of G A B A release to kainic acid in striata receiving prior 6-OHDA lesions was effectively the same as in the dopamine intact striatum. In the present study, knife transections undercutting the frontal cortex abolished the kainic acid-induced effect on striatal G A B A release, suggesting that the integrity of cortical afferents is indeed essential for the response. It is clear that the cortical lesions employed here do not provide a complete cortical denervation of the entire striatum. However, the dialysis probes were centered in the rostral most part of the striatum, which is an area known to receive dense innervation from the frontal cortex. 43 The lack of a direct postsynaptic effect of kainic acid on GABA release from striatal neurons is notable, since in vitro studies have demonstrated that kainic acid can induce GABA release from cultured s t r i a t a l n e u r o n s . 46'4s'56 We could, however, not detect this effect in vivo. In accordance with the existence of a substantial anatomical input from the cortex to grafted striatal neurons, 5s,6°,61'65kainic acid applied locally in the graft through the dialysis probe induced a significant increase in graft-derived G A B A overflow. As was the case in the normal striatum, the kainic acidinduced increase in graft-derived GABA release was dependent on intact cortical afferents to the graft, since this effect was abolished by the frontal cortical knife cut. These findings suggest that excitatory amino acids, released from host cortical afferents are able to modulate GABA release from the implanted striatal neurons. Consistent with previous electrophysiological studies, 5°'66these data indicate that host cortical inputs to the graft functionally regulate the activity of grafted striatal neurons. We are currently attempting to further characterize this host cortical influence over the graft-derived G A B A release in experiments with electrical stimulation of the frontal cortex. CONCLUSIONS

The present results show that intrastriatal striatal grafts are capable of reversing the excitotoxic lesioninduced deficits in basal and evoked striatal G A B A overflow in a neuron-dependent manner. The results obtained by manipulation of the nigrostriatal and corticostriatal afferents to the grafts indicate that the graft-derived G A B A release is under regulatory control from the host dopaminergic and glutamatergic systems, similar to that occuring in the intact striatum. There is considerable evidence that the GABAergic striatal output neurons are critical for the expression of striatum-related behaviours. ~° Moreover, excitotoxic lesions of the striatum have been

Afferent regulation of GABA release in striatal grafts s h o w n to induce alterations in nerve cell activity 25'3L37 in the p r i m a r y striatal target regions, consistent with the removal o f inhibitory (GABAergic) striatal outp u t neurons. Lesions o f this inhibitory striatal outp u t system are also k n o w n to result in p r o f o u n d m o t o r a n d cognitive b e h a v i o u r a l deficits, which are ameliorated by intrastriatal striatal grafts o f the type studied here. 4,2° O n the basis o f the present results we p r o p o s e t h a t these graft-induced behavioural effects,

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at least in part, depend o n the restoration of striatal G A B A e r g i c neurotransmission. thank Alicja Flasch, Birgit Haraldson, Ulla Jarl, Anna-Karin Old6n, Anne-Marie Olsson and Gertrude Stridsberg for their excellent technical assistance. This work was supported by grants from the Swedish MRC (04X-3874, 12X-09882), the National Institutes of Health (NS 06701) and the G6ran Gustafsson Foundation. K.C. is a MRC of Canada student. Acknowledgements--We

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