EXPERIMENTAL
NEUROLOGY
(1991)
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Development of Dl and D2 Dopamine Receptors and Associated Second Messenger Systems in Fetal Striatal Transplants GREGORY A. HELM,* MATTHEW W. ROBERTSoN,t GEORGE I. JALLO,* NATHAN SIMMONS,* AND JAMES P. BENNETT, JR.t$$ Departments
Stereotactic
of *Neurological liniversity
implantation
Surgery, tNeurology, *Behavioral Medicine of Virginia School of Medicine, Charlottesville,
of fetal brain regional an-
lage into adult host brain (“brain transplantation”) appears to be an increasingly viable strategy for therapy of neurodegenerative diseases. We have studied implantation of fetal striatum into adult striatum, previously lesioned by neurotoxic amino acid injection, as a model for transplantation therapy of Huntington’s disease. The beginning of behavioral recovery to apomorphine is not apparent until 6.5 months after implantation. By 4 months after implantation cerebral blood flow through the implants appears equal to that in the intact contralateral striatum. At this time, cerebral glucose utilization is reduced in the implants but increases following apomorphine treatment. The development of Dl and D2 dopamine (DA) receptors is markedly deficient in the striatal grafts at both 4 and 6.5 months after implantation. Very little D2 radioligand binding was observed in the grafts at either time point; Dl receptors appeared in a patchy fashion by 6.5 months at densities approaching normal striatum. In situ hybridization of D2 dopamine receptor mRNA demonstrated robust hybridization signal in normal striaturn and accumbens but no signal in 6.5-month-old striatal grafts. Adenylate cyclase (AC) activity, examined with high-affinity [3H]forskolin binding, also appeared in patches similar to Dl receptors at 6.5 months. In contrast, protein kinase C activity, labeled with [‘Hlphorbol ester, was very apparent in the grafts at both time points. Higher and generally homogenous densities of muscarinic cholinergic receptors, assessed with [‘H]QNB binding, develop in the grafts, but there appear to be few functioning choline&c terminals, as measured by [‘Hlhemicholinium binding. Our data demonstrate that very few Dl or D2 DA receptors develop in fetal striatal neurons removed from E15-16 embryos and subsequently implanted into adult ibotenic acid-lesioned striata. The lack of D2 receptor development appears to derive from lack of transcription of the D2 receptor gene. By 6.5 months, Dl receptor sites and AC activity both appear in discrete patches, consistent with the known in vitro association of Dl receptors with stimulation of AC activity. In this model, the majority of graft protein kinase C activity, to the extent that
and Psychiatry, and $Pharmacology, Virginia 22908
it is labeled with [3H]phorbol ester, is not associated with DA receptors. DA receptor development in fetal striatal implants may require coimplantation of DA neurons. 8 1991 Academic Press, Inc.
INTRODUCTION Over the past decade, multiple studies have confirmed the experimental utility of implanting fetal brain tissue into adult hosts as potential therapy for neurodegenerative diseasesin humans (14, 26). The general rationale behind brain transplantation strategies is to implant fetal anlage of adult neurons normally lost in the disease process. Clinical improvement should derive from normal biochemical andelectrophysiological maturation of implanted inactive neurons, which should establish anatomically appropriate afferent and efferent contacts with host neurons. Huntington’s disease (HD) is an autosomal dominantly inherited neurodegenerative disorder with typical onset of neuropsychiatric symptoms in early middle age. Brains from HD patients consistently have marked neuronal loss and atrophy in basal ganglia, involving particularly caudate, putamen, globus pallidus, and subthalamic nuclei. Such extensive loss of neurons can be produced in striata of experimental animals by stereotactic injection of excitotoxic amino acids, leading to a similar loss of neurotransmitter chemicals and neurotransmitter receptors (7, 8, 22). Implantation of fetal striatum into such lesioned striata leads to partial restoration of biochemical deficits and improvement in behavioral abnormalities (912,14,17,20,21,23). Such implanted fetal striatal neurons have been shown to develop adult striatal neuronal characteristics, based on morphological (5, 13, 19), immunohistochemical (5, 25) (Helm et al., submitted for publication), and functional neurochemical (11, 12) properties. They receive afferent innervation from several areas (24, 29, 33) but appear to establish limited efferent connection with the host brain (17, 24, 28). We have recently found with the section Golgi method that fetal striatal neurons implanted into ibo181 All
C opyright Q 1991 rights of reproduction
0014~&86/91$3.00 by Academic Press, Inc. in any form reserved.
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tenic acid-lesioned adult striata differentiate within 3 months into each of the seven types of adult intrinsic striatal neurons (19). Electron microscopic immunohistochemical studies of these implants have shown the presence of glutamic acid decarboxylase (GAD), choline acetyl transferase (ChAT), and substance P peptide within ultrastructurally normal neurons and nerve endings (Helm et al., submitted). Tyrosine hydroxylase (TH)-positive nerve endings, presumably from host afferent midbrain neurons, were seen making synaptic contacts with both GAD( +)- and GAD( -)-implanted neurons. These detailed morphological and immunohistochemical studies demonstrated that by 3 months after implantation the neurons have developed the anatomic and presynaptic neurochemical maturity of adult intrinsic striatal neurons and have begun both receiving host afferent dopaminergic innervation and establishing synaptic contacts with each other. It is presently unclear whether behavioral recovery after fetal striatal implantation in rodents with the animal model of HD requires development of functioning DA synapses within the graft, establishment of host afferent innervation and/or graft efferent innervation, or all of the above. In the present study we have characterized within our population of transplanted rats their behavioral recovery to apomorphine treatment, the appearance of markers of dopaminergic and cholinergic synaptic functions, associated second messenger systems, and regional cerebral 2-deoxyglucose uptake in response to DA receptor activation. METHODS The study population comprised 36 adult female Sprague-Dawley rats that received right striatal injections of ibotenic acid followed 2 weeks later by implantation of El%16 fetal striatal suspensions. Details of the lesioning and transplantation procedures and electron microscopic characterization of the transplants can be found elsewhere (19). Animals from the same transplantation group used to characterize the morphological and immunohistochemical properties of grafted neurons were used in the present study. All adult hosts exhibited brisk ipsilateral (toward the lesioned striatum) rotation in response to apomorphine 1 mg/kg ip or d-amphetamine 5 mg/kg ip. Net ipsilateral rotations were measured for 45 min after apomorphine treatment in an animal activity monitor (Columbus Instruments), beginning the day prior to implantation surgery and continuing for up to 26 weeks afterward. At 4 months after transplantation qualitative cerebral blood flow was demonstrated in three animals by iv injection of 25 &i [14C]iodoantipyrine and decapitation 2 min later. Brains were rapidly removed and frozen in Freon. Frozen sections (20 pm) were exposed to X-ray film for 1 week.
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2-[3H]Deoxyglucose (2-DG) accumulation was measured at 4 months in three groups of three animals each. Each animal received 500 &i of 2-t3H]DG iv followed by saline ip, d-amphetamine 5 mg/kg ip, or apomorphine 1 mg/kg ip and was decapitated 45 min later. Amphetamineand apomorphine-treated animals exhibited brisk ipsilateral rotation during the entire 45-min survival period. Brains were rapidly removed and frozen in Freon. Frozen sections (20 pm) were exposed to Amersham tritium-sensitive film for 3 weeks. Receptor autoradiography was performed on sections derived from the nine animals used in the 2-[3H]DG studies above at 4 months after transplantation and from four separate animals at 6.5 months after transplantation. Frozen sections (20 Km) were thaw-mounted onto poly-d-lysine-coated coverslips and stored at -70’ until assayed. They were brought to room temperature and washed in 25 n&f Tris acetate-buffered (pH 7.3) artificial cerebrospinal fluid (TA-CSF) to remove 2-[3H]DG and metabolites. Dl receptors were labeled by incubation in TA-CSF with [3H]SCH 23390 (1 nM) for 90 min at room temperature in the presence of 10 PM mianserin to block binding to S2 serotonin receptors. Nonspecific [3H]SCH 23390 binding was determined by adding 10 pM SKF 38393 to the medium. D2 receptors were labeled in TA-CSF with 1 nM [3H]spiperone in the presence of 10 pM mianserin, 4 nM [3H]-(-)-sulpiride, or 1 nM [3H]raclopride at room temperature for 90 min. (+)-Butaclamol (1 PM) determined nonspecific binding. [3H]quinuclidinyl benzilate (1 nM) in TA-CSF (QNB; nonspecific binding = 1 pM benztropine) and 10 nM [3H]hemicholinium in 0.15 M NaC1/50 mM glycylglycine, pH 7.8 (nonspecific binding = 10 pM hemicholinium), were used to label muscarinic choline@ receptors and high-affinity choline uptake sites, respectively. [3H]Forskolin (20 nM in the presence of 10 pM guanyldiphosphateimidophosphate, GppNHp; nonspecific binding = 10 pM forskolin) and [3H]phorbol 12,13dibutyrate (20 nM; nonspecific binding = 10 pM phorbol 12,13-dibutyrate), both in TA-CSF, were used to localize adenylate cyclase (15, 16, 32) and protein kinase C activities (31, 32), respectively. Following incubation with radioligand, sections were washed in three changes of ice-cold TA-CSF, dipped briefly into ice-cold water, blown dry, and apposed to Amersham tritium-sensitive film for periods of 3 weeks to 5 months. Quantitative autoradiography was performed on a DUMAS system with calibrated plastic tritium standards (Amersham). Typically lo-14 sections from each group were quantitated. Grain density in grafts was compared to that in dorsolateral normal (contralateral) striatum and converted to microcuries per gram of tissue. Regional 2-[3H]DG accumulation was normalized to corpus callosum values for each animal. For radioligand binding autoradiograms, pictures of to-
DA
0 I
DISTANCE CLOCKWISE
RECEPTOR
DEVELOPMENT
IN
TRAVELED ROTATIONS
0467
WEEKS AFTER
8
9
12
24
26
IMPLANTATION
FIG. 1. Recovery of motor responses to apomorphine after striatal grafting. Rats with right striatal ibotenic acid lesions received apomorphine 1 mg/kg ip immediately before (0 weeks) and at variable times after receiving fetal striatal grafts. Total distance traveled and clockwise (ipsilateral to lesioned side) turns were measured over the next 45 min. Data are means t SEM for 4-12 animals at each time point and are expressed as percentage of preimplantation values.
tal ligand binding are presented, nonspecific bindingvalues were subtracted during quantitation. In situ hybridization utilized a complementary RNA (cRNA) probe synthesized from a 495-bp DNA insert
FETAL
STRIATAL
TRANSPLANTS
183
complementary to the third cytoplasmic loop of rat brain D2 dopamine receptors (3) (plasmid generously provided by Dr. 0. Civelli). The plasmid containing the insert was linearized with EcoRl, and [35S]UTP-cRNA was transcribed with T7 RNA polymerase (Riboprobe; Promega). The estimated probe specific activity was 8 X lo7 cpmlpg. Electrophoresis under denaturing conditions with formaldehyde in 1.2% agarose yielded a single peak of ca. 500 bases in size. In situ hybridization conditions, adapted from Alexander et al. (1) and Cox et aE.(6), utilized aldehyde-fixed, free-foating tissue sections, some of which were combined with immunohistochemistry and will be described in a separate publication. Rats were deeply anesthetized and perfused with freshly denatured, phosphate-buffered 4% paraformaldehyde. Brains were removed, postfixed for 24-48 h, and sunk in 30% sucrose. Frozen sections (30-40 pm) were incubated for 24 h at 45” in 25 ml hybridization buffer (50% formamideI0.3 M NaCl/lO mM Tris HCl, pH 8.0/l mM EDTA/0.2% bovine serum albumin/0.2% Ficoll/O.2% polyvinylpyrrolidone/yeast tRNA at 0.5 mg/ml/lO mM dithiothreitol) containing [35S]cRNA probe (6 pmol) in 24-well plastic chambers (Nason’s Machine, San Diego, CA). Sections were then washed three times for 30 min each in 4X SSC (1X SSC
FIG. 2. Cerebral blood flow and Z-deoxyglucose uptake patterns in striatal grafts. (A) At 4 months after receiving fetal striatal grafts, one group of rats received 25 rCi of [Wliodoantipyrene and were decapitated 2 min later. A representative autoradiographic image is shown of blood flow in the intact striatum (s) and the implant (i). (B-D) Separate rats with 4-month-old grafts received 500 &i of 2-[3H]deoxyglucose iv, followed by saline (B), d-amphetamine 5 mg/kg (C), or apomorphine 1 mg/kg (D).
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FIG. 3. Localization of Dl and D2 dopamine receptor sites in fetal striatal grafts at 4 and 6.5 months old. Representative images are shown from fetal striatal grafts 4 months old (A, C) and 6.5 months old (B, D). D2 receptor sites were labeled with or [3H]sulpiride (B). Dl receptor sites were labeled with [3H]SCH 23390 (C, D).
= 0.15 M NaCl, 0.015 M Na,citrate, pH 7) at room temperature, treated with RNase 20 kg/ml (Promega) for 30 min at 37”, washed for 30 min at 37” in RNase buffer (0.5 M NaCl/lO mM Tris HCl, pH 8), washed in 2~ SSC for 30 min at room temperature, and finally washed for 15 min at 40” in 0.1X SSC. Sections were dried on a slide warmer, dehydrated through graded alcohol with water replaced by 0.6 M NH,Ac, and apposed to Amersham film for 13 days. To determine the cRNA probe melting curve, sections were carried through the above hybridization, washing, and RNase procedures and washed for 15 min in 0.1X SSC at 40-80” in 5“ increments, and their 35S contents were assayed with liquid scintillation spectroscopy. .
RESULTS
Figure 1 presents the data for time course of recovery of asymmetric behavioral response to apomorphine challenge. Within 6 weeks of implantation surgery, motor responses to apomorphine were increased compared to preimplantation values and remained so until 24 weeks. By 26 weeks, motor responses were less than those observed at the time of surgery.
autoradiographic [3H]spiperone
(A)
Figure 2 shows that at 4 months after implantation surgery, cerebral blood flow within the striatal implants was the same as in the contralateral, intact striatum. Despite this apparently normal pattern of blood flow, 2-[3H]DG accumulation densities within the striatal grafts, reflecting absolute cerebral glucose utilization rates, were reduced 32 + 2% (P < 0.01, saline-treated group). Treatment with amphetamine did not affect 2-[3H]DG accumulation within the grafts. Apomorphine treatment increased 2-[3H]DG accumulation in the grafts by an average 16 + 4% (P < O.Ol), usually in an irregular pattern. Figure 3 shows Dl and D2 DA receptor localization in the grafts at 4 and 6.5 months after implantation. Very few, if any, D2 receptor sites were seen in the grafts at either time. These results were obtained with three different ligands commonly used to image D2 receptor sites. The same lack of D2 receptor development was seen in our most recent group of three B-month-old striatal implants, with [3H]raclopride as the ligand (results not shown). At 6.5 but not at 4 months, Dl receptor sites were consistently seen in discrete and dense patches. Dl receptor density in the transplant patches ranged from 71 to 100% of the mean Dl receptor density
DA
RECEPTOR
DEVELOPMENT
IN
FETAL
STRIATAL
FIG. 4. In situ hybridization autoradiogram of [?3]cRNA probe for D2 dopamine See Methods for details. The photograph is of a reconstructed image after subtraction intact striatum (s) and the marked lack of signal over transplant (t).
in contralateral normal striata. The mean patch Dl receptor density in the grafts was 84% of normal striatum and not significantly different. Figure 4 shows the pattern of localization of D2 receptor mRNA in a 6-month-old striatal transplant, as revealed by in situ hybridization with a cRNA probe. We determined that the melting temperature of the probetissue mRNA duplex in 0.1X SSC was 55°C (data not shown); therefore the final wash in 0.1X SSC was carried out at 40°C. High densities of hybridization signal are seen over normal striatum and accumbens; no significant signal is seen over the graft. Figure 5 shows the patterns of localization at 6.5 months after transplantation of adenylate cyclase, labeled with [3H]forskolin, and protein kinase C, labeled with [3H]phorbol ester. Patterns of [3H]forskolin labeling were also patchy and reminiscent of Dl receptor localization. On the other hand, specific [3H]phorbol ester binding was consistently and densely present throughout the grafts. Figure 6 reveals the patterns of development of preand postsynaptic markers for striatal acetylcholine function in the grafts. At both 4 and 6.5 months after
receptor of film
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messenger background.
RNA in a &month-old Note the high signal
striatal intensity
graft. over
implantation, there is generous and fairly homogenous development of muscarinic cholinergic receptors, labeled with the nonselective ligand [3H]QNB. Receptor site densities within the grafts approximate those seen in superficial layers (higher [3H]QNB density) of overlying cortex (83 and 90% of cortical values at 4 and 6.5 months, respectively) and are equal to those in intact striatum. In contrast, there is very little development at either time point of high-affinity choline uptake sites, labeled with [3H]hemicholinium and normally associated with cholinergic nerve terminals. At both 4 and 6.5 months after implantation, [3H]hemicholinium binding densities were 35-40% of those in the dorsolateral normal striatum. DISCUSSION
The present study confirms and extends results of previous studies that have shown behavioral recovery in rats with striatal intrinsic neuronal lesions following fetal striatal implants (9-12, 20, 21, 23), reduced density of D2 DA receptors in striatal grafts (10, 12), and more normal development of muscarinic cholinergic recep-
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FIG. 5. Localization of adenylate cyclase and protein kinase C in fetal striatal grafts. Shown are autoradiographic images old transplants incubated with [3H]forskolin to label adenylate cyclase (A) and [3H]phorbol 12,13dibutyrate to label protein labeling of adenylate cyclase but extensive and dense labeling of protein kinase C was observed in 4-month-old grafts (not
tom in striatal grafts (11). Our results show that by 4 months after fetal striatal transplantation (the earliest time point examined), there is no asymmetry of blood flow within the grafts. 2-[3H]DG studies after apomorphine treatment suggest the presence of limited numbers of functional DA receptors, but the results after amphetamine treatment do not support the presence of many functional DA synapses. Apomorphine-induced increases in basal ganglia 2-DG accumulation have been found on striatonigral endings in substantia nigra reticulata and have been shown to be mediated by super-
from 6.5-monthkinase C (B). No shown).
sensitive Dl dopamine receptors (30). It is possible that our 2-DG patterns after apomorphine in the 4-monthold grafts derive from activation of dopamine receptors not present in high enough concentrations to be visualized by autoradiography with tritiated ligands. GABA release in globus pallidus ipsilateral to striatal grafts 6-8 months old has been reported to be under control of DA synapses (14), suggesting the development of functional DA receptors in striatal grafts of that age. By 6.5 months after transplantation, partial behavioral recovery in our rats was first apparent, and Dl, but
DA
RECEPTOR
DEVELOPMENT
IN
FETAL
STRIATAL
TRANSPLANTS
FIG. 6. Development of presynaptic and postsynaptic markers of cholinergic synapses in striatal grafts. Presynaptic on cholinergic terminals were labeled with [3H]hemicholinium (A, B) and postsynaptic muscarinic receptors were labeled on sections from rats with 4-month-old (A, C) or 6.5.month-old (B, D) striatal grafts.
not D2, receptors were being expressed in discrete islands or patches. In situ hybridization autoradiography at 6 months showed that the absence of detectable D2 receptors, sought for with three different 3H ligands, appeared to derive from lack of transcription of the D2 receptor gene. It is unclear why our results are different from those of an earlier study that found limited recovery of D2 receptors in much smaller striatal implants (12). High-affinity [3H]forskolin binding in the presence of GppNHp, believed to be associated with adenylate cyclase catalytic activity (15, 16, 32), also was found in similar Dl receptor-like patch patterns at 6.5 months. Although we did not attempt colocalization of these patches with Dl receptors in the grafts, their similar autoradiographic patterns are consistent with the biochemical association between striatal Dl DA receptors and activation of adenylate cyclase (4). The marked reduction of DA receptor development in the grafts was not found for all receptors, as muscarinic cholinergic receptors developed much more extensively. Others have found similar development of cholinergic receptor sites (11). We observed, however, very little development of cholinergic terminals, as manifested by high affinity [3H]hemicholinium binding. Our electron
choline uptake sites with [aH]QNB (C, D)
microscopic immunohistochemical studies have shown that the grafts contain ChAT in ultrastructurally normal neurons and axon terminals, demonstrating that the neurochemical differentiation of the implanted cells into cholinergic neurons does take place in the fetal grafts. At the light microscopic level we observed very few ChAT-positive terminals in the grafts, consistent with the lack of significant [3H]hemicholinium binding. Electrophysiological studies suggest similarities between neuronal ion channels operated by forskolin and phorbol ester (18). To the extent that protein kinase C activity is reliably labeled with [3H]phorbol ester (31, 32), our results in chronic striatal implants suggest that the majority of protein kinase C activity develops independently of and is most likely not associated with DA receptors. The marked reduction at 6.5 months after grafting of DA receptor development on fetal striatal neurons, shown by our previous studies of the same group of animals to have developed into morphological and immunohistochemical maturity by 3 months after transplantation, suggests that dopaminergic afferent innervation of developing fetal striatal neurons may be necessary for DA receptor expression. This hypothesis is supported by similarities in our patterns of Dl DA receptor ex-
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pression to TH (+) patches (5) and islands of DA histofluorescence (24) seen in striatal grafts and biochemical studies of striatal DA receptor postnatal development (2). One test of this hypothesis could involve cotransplantation of fetal midbrain and striatal anlage into the same lesioned striatum. The factors responsible for behavioral recovery from excitotoxic lesions of striatal neurons remain unclear. If development of DA receptor function on grafted fetal striatal neurons is one necessary component of behavioral recovery, our results suggest that the Dl subtype of receptors is more important. It remains to be shown whether DA receptor expression in striatal grafts can be accelerated and, if so, whether more rapid behavioral recovery follows. ACKNOWLEDGMENTS This work has been supported by NIH-NINDS NS00978, NS26581, and NS07199. We thank Dr. Olivier Civelli for his gift of plasmid containing the D2 receptor cDNA, Ms. Joan Gilrain for technical help, and Ms. Rose Powell for editorial assistance.
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2.
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NEUROLOGY
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Development of Dl and D2 Dopamine Receptors and Associated Second Messenger Systems in Fetal Striatal Transplants GREGORY A. HELM,* MATTHEW W. ROBERTSoN,t GEORGE I. JALLO,* NATHAN SIMMONS,* AND JAMES P. BENNETT, JR.t$$ Departments
Stereotactic
of *Neurological liniversity
implantation
Surgery, tNeurology, *Behavioral Medicine of Virginia School of Medicine, Charlottesville,
of fetal brain regional an-
lage into adult host brain (“brain transplantation”) appears to be an increasingly viable strategy for therapy of neurodegenerative diseases. We have studied implantation of fetal striatum into adult striatum, previously lesioned by neurotoxic amino acid injection, as a model for transplantation therapy of Huntington’s disease. The beginning of behavioral recovery to apomorphine is not apparent until 6.5 months after implantation. By 4 months after implantation cerebral blood flow through the implants appears equal to that in the intact contralateral striatum. At this time, cerebral glucose utilization is reduced in the implants but increases following apomorphine treatment. The development of Dl and D2 dopamine (DA) receptors is markedly deficient in the striatal grafts at both 4 and 6.5 months after implantation. Very little D2 radioligand binding was observed in the grafts at either time point; Dl receptors appeared in a patchy fashion by 6.5 months at densities approaching normal striatum. In situ hybridization of D2 dopamine receptor mRNA demonstrated robust hybridization signal in normal striaturn and accumbens but no signal in 6.5-month-old striatal grafts. Adenylate cyclase (AC) activity, examined with high-affinity [3H]forskolin binding, also appeared in patches similar to Dl receptors at 6.5 months. In contrast, protein kinase C activity, labeled with [‘Hlphorbol ester, was very apparent in the grafts at both time points. Higher and generally homogenous densities of muscarinic cholinergic receptors, assessed with [‘H]QNB binding, develop in the grafts, but there appear to be few functioning choline&c terminals, as measured by [‘Hlhemicholinium binding. Our data demonstrate that very few Dl or D2 DA receptors develop in fetal striatal neurons removed from E15-16 embryos and subsequently implanted into adult ibotenic acid-lesioned striata. The lack of D2 receptor development appears to derive from lack of transcription of the D2 receptor gene. By 6.5 months, Dl receptor sites and AC activity both appear in discrete patches, consistent with the known in vitro association of Dl receptors with stimulation of AC activity. In this model, the majority of graft protein kinase C activity, to the extent that
and Psychiatry, and $Pharmacology, Virginia 22908
it is labeled with [3H]phorbol ester, is not associated with DA receptors. DA receptor development in fetal striatal implants may require coimplantation of DA neurons. 8 1991 Academic Press, Inc.
INTRODUCTION Over the past decade, multiple studies have confirmed the experimental utility of implanting fetal brain tissue into adult hosts as potential therapy for neurodegenerative diseasesin humans (14, 26). The general rationale behind brain transplantation strategies is to implant fetal anlage of adult neurons normally lost in the disease process. Clinical improvement should derive from normal biochemical andelectrophysiological maturation of implanted inactive neurons, which should establish anatomically appropriate afferent and efferent contacts with host neurons. Huntington’s disease (HD) is an autosomal dominantly inherited neurodegenerative disorder with typical onset of neuropsychiatric symptoms in early middle age. Brains from HD patients consistently have marked neuronal loss and atrophy in basal ganglia, involving particularly caudate, putamen, globus pallidus, and subthalamic nuclei. Such extensive loss of neurons can be produced in striata of experimental animals by stereotactic injection of excitotoxic amino acids, leading to a similar loss of neurotransmitter chemicals and neurotransmitter receptors (7, 8, 22). Implantation of fetal striatum into such lesioned striata leads to partial restoration of biochemical deficits and improvement in behavioral abnormalities (912,14,17,20,21,23). Such implanted fetal striatal neurons have been shown to develop adult striatal neuronal characteristics, based on morphological (5, 13, 19), immunohistochemical (5, 25) (Helm et al., submitted for publication), and functional neurochemical (11, 12) properties. They receive afferent innervation from several areas (24, 29, 33) but appear to establish limited efferent connection with the host brain (17, 24, 28). We have recently found with the section Golgi method that fetal striatal neurons implanted into ibo181 All
C opyright Q 1991 rights of reproduction
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tenic acid-lesioned adult striata differentiate within 3 months into each of the seven types of adult intrinsic striatal neurons (19). Electron microscopic immunohistochemical studies of these implants have shown the presence of glutamic acid decarboxylase (GAD), choline acetyl transferase (ChAT), and substance P peptide within ultrastructurally normal neurons and nerve endings (Helm et al., submitted). Tyrosine hydroxylase (TH)-positive nerve endings, presumably from host afferent midbrain neurons, were seen making synaptic contacts with both GAD( +)- and GAD( -)-implanted neurons. These detailed morphological and immunohistochemical studies demonstrated that by 3 months after implantation the neurons have developed the anatomic and presynaptic neurochemical maturity of adult intrinsic striatal neurons and have begun both receiving host afferent dopaminergic innervation and establishing synaptic contacts with each other. It is presently unclear whether behavioral recovery after fetal striatal implantation in rodents with the animal model of HD requires development of functioning DA synapses within the graft, establishment of host afferent innervation and/or graft efferent innervation, or all of the above. In the present study we have characterized within our population of transplanted rats their behavioral recovery to apomorphine treatment, the appearance of markers of dopaminergic and cholinergic synaptic functions, associated second messenger systems, and regional cerebral 2-deoxyglucose uptake in response to DA receptor activation. METHODS The study population comprised 36 adult female Sprague-Dawley rats that received right striatal injections of ibotenic acid followed 2 weeks later by implantation of El%16 fetal striatal suspensions. Details of the lesioning and transplantation procedures and electron microscopic characterization of the transplants can be found elsewhere (19). Animals from the same transplantation group used to characterize the morphological and immunohistochemical properties of grafted neurons were used in the present study. All adult hosts exhibited brisk ipsilateral (toward the lesioned striatum) rotation in response to apomorphine 1 mg/kg ip or d-amphetamine 5 mg/kg ip. Net ipsilateral rotations were measured for 45 min after apomorphine treatment in an animal activity monitor (Columbus Instruments), beginning the day prior to implantation surgery and continuing for up to 26 weeks afterward. At 4 months after transplantation qualitative cerebral blood flow was demonstrated in three animals by iv injection of 25 &i [14C]iodoantipyrine and decapitation 2 min later. Brains were rapidly removed and frozen in Freon. Frozen sections (20 pm) were exposed to X-ray film for 1 week.
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2-[3H]Deoxyglucose (2-DG) accumulation was measured at 4 months in three groups of three animals each. Each animal received 500 &i of 2-t3H]DG iv followed by saline ip, d-amphetamine 5 mg/kg ip, or apomorphine 1 mg/kg ip and was decapitated 45 min later. Amphetamineand apomorphine-treated animals exhibited brisk ipsilateral rotation during the entire 45-min survival period. Brains were rapidly removed and frozen in Freon. Frozen sections (20 pm) were exposed to Amersham tritium-sensitive film for 3 weeks. Receptor autoradiography was performed on sections derived from the nine animals used in the 2-[3H]DG studies above at 4 months after transplantation and from four separate animals at 6.5 months after transplantation. Frozen sections (20 Km) were thaw-mounted onto poly-d-lysine-coated coverslips and stored at -70’ until assayed. They were brought to room temperature and washed in 25 n&f Tris acetate-buffered (pH 7.3) artificial cerebrospinal fluid (TA-CSF) to remove 2-[3H]DG and metabolites. Dl receptors were labeled by incubation in TA-CSF with [3H]SCH 23390 (1 nM) for 90 min at room temperature in the presence of 10 PM mianserin to block binding to S2 serotonin receptors. Nonspecific [3H]SCH 23390 binding was determined by adding 10 pM SKF 38393 to the medium. D2 receptors were labeled in TA-CSF with 1 nM [3H]spiperone in the presence of 10 pM mianserin, 4 nM [3H]-(-)-sulpiride, or 1 nM [3H]raclopride at room temperature for 90 min. (+)-Butaclamol (1 PM) determined nonspecific binding. [3H]quinuclidinyl benzilate (1 nM) in TA-CSF (QNB; nonspecific binding = 1 pM benztropine) and 10 nM [3H]hemicholinium in 0.15 M NaC1/50 mM glycylglycine, pH 7.8 (nonspecific binding = 10 pM hemicholinium), were used to label muscarinic choline@ receptors and high-affinity choline uptake sites, respectively. [3H]Forskolin (20 nM in the presence of 10 pM guanyldiphosphateimidophosphate, GppNHp; nonspecific binding = 10 pM forskolin) and [3H]phorbol 12,13dibutyrate (20 nM; nonspecific binding = 10 pM phorbol 12,13-dibutyrate), both in TA-CSF, were used to localize adenylate cyclase (15, 16, 32) and protein kinase C activities (31, 32), respectively. Following incubation with radioligand, sections were washed in three changes of ice-cold TA-CSF, dipped briefly into ice-cold water, blown dry, and apposed to Amersham tritium-sensitive film for periods of 3 weeks to 5 months. Quantitative autoradiography was performed on a DUMAS system with calibrated plastic tritium standards (Amersham). Typically lo-14 sections from each group were quantitated. Grain density in grafts was compared to that in dorsolateral normal (contralateral) striatum and converted to microcuries per gram of tissue. Regional 2-[3H]DG accumulation was normalized to corpus callosum values for each animal. For radioligand binding autoradiograms, pictures of to-
DA
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9
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FIG. 1. Recovery of motor responses to apomorphine after striatal grafting. Rats with right striatal ibotenic acid lesions received apomorphine 1 mg/kg ip immediately before (0 weeks) and at variable times after receiving fetal striatal grafts. Total distance traveled and clockwise (ipsilateral to lesioned side) turns were measured over the next 45 min. Data are means t SEM for 4-12 animals at each time point and are expressed as percentage of preimplantation values.
tal ligand binding are presented, nonspecific bindingvalues were subtracted during quantitation. In situ hybridization utilized a complementary RNA (cRNA) probe synthesized from a 495-bp DNA insert
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complementary to the third cytoplasmic loop of rat brain D2 dopamine receptors (3) (plasmid generously provided by Dr. 0. Civelli). The plasmid containing the insert was linearized with EcoRl, and [35S]UTP-cRNA was transcribed with T7 RNA polymerase (Riboprobe; Promega). The estimated probe specific activity was 8 X lo7 cpmlpg. Electrophoresis under denaturing conditions with formaldehyde in 1.2% agarose yielded a single peak of ca. 500 bases in size. In situ hybridization conditions, adapted from Alexander et al. (1) and Cox et aE.(6), utilized aldehyde-fixed, free-foating tissue sections, some of which were combined with immunohistochemistry and will be described in a separate publication. Rats were deeply anesthetized and perfused with freshly denatured, phosphate-buffered 4% paraformaldehyde. Brains were removed, postfixed for 24-48 h, and sunk in 30% sucrose. Frozen sections (30-40 pm) were incubated for 24 h at 45” in 25 ml hybridization buffer (50% formamideI0.3 M NaCl/lO mM Tris HCl, pH 8.0/l mM EDTA/0.2% bovine serum albumin/0.2% Ficoll/O.2% polyvinylpyrrolidone/yeast tRNA at 0.5 mg/ml/lO mM dithiothreitol) containing [35S]cRNA probe (6 pmol) in 24-well plastic chambers (Nason’s Machine, San Diego, CA). Sections were then washed three times for 30 min each in 4X SSC (1X SSC
FIG. 2. Cerebral blood flow and Z-deoxyglucose uptake patterns in striatal grafts. (A) At 4 months after receiving fetal striatal grafts, one group of rats received 25 rCi of [Wliodoantipyrene and were decapitated 2 min later. A representative autoradiographic image is shown of blood flow in the intact striatum (s) and the implant (i). (B-D) Separate rats with 4-month-old grafts received 500 &i of 2-[3H]deoxyglucose iv, followed by saline (B), d-amphetamine 5 mg/kg (C), or apomorphine 1 mg/kg (D).
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FIG. 3. Localization of Dl and D2 dopamine receptor sites in fetal striatal grafts at 4 and 6.5 months old. Representative images are shown from fetal striatal grafts 4 months old (A, C) and 6.5 months old (B, D). D2 receptor sites were labeled with or [3H]sulpiride (B). Dl receptor sites were labeled with [3H]SCH 23390 (C, D).
= 0.15 M NaCl, 0.015 M Na,citrate, pH 7) at room temperature, treated with RNase 20 kg/ml (Promega) for 30 min at 37”, washed for 30 min at 37” in RNase buffer (0.5 M NaCl/lO mM Tris HCl, pH 8), washed in 2~ SSC for 30 min at room temperature, and finally washed for 15 min at 40” in 0.1X SSC. Sections were dried on a slide warmer, dehydrated through graded alcohol with water replaced by 0.6 M NH,Ac, and apposed to Amersham film for 13 days. To determine the cRNA probe melting curve, sections were carried through the above hybridization, washing, and RNase procedures and washed for 15 min in 0.1X SSC at 40-80” in 5“ increments, and their 35S contents were assayed with liquid scintillation spectroscopy. .
RESULTS
Figure 1 presents the data for time course of recovery of asymmetric behavioral response to apomorphine challenge. Within 6 weeks of implantation surgery, motor responses to apomorphine were increased compared to preimplantation values and remained so until 24 weeks. By 26 weeks, motor responses were less than those observed at the time of surgery.
autoradiographic [3H]spiperone
(A)
Figure 2 shows that at 4 months after implantation surgery, cerebral blood flow within the striatal implants was the same as in the contralateral, intact striatum. Despite this apparently normal pattern of blood flow, 2-[3H]DG accumulation densities within the striatal grafts, reflecting absolute cerebral glucose utilization rates, were reduced 32 + 2% (P < 0.01, saline-treated group). Treatment with amphetamine did not affect 2-[3H]DG accumulation within the grafts. Apomorphine treatment increased 2-[3H]DG accumulation in the grafts by an average 16 + 4% (P < O.Ol), usually in an irregular pattern. Figure 3 shows Dl and D2 DA receptor localization in the grafts at 4 and 6.5 months after implantation. Very few, if any, D2 receptor sites were seen in the grafts at either time. These results were obtained with three different ligands commonly used to image D2 receptor sites. The same lack of D2 receptor development was seen in our most recent group of three B-month-old striatal implants, with [3H]raclopride as the ligand (results not shown). At 6.5 but not at 4 months, Dl receptor sites were consistently seen in discrete and dense patches. Dl receptor density in the transplant patches ranged from 71 to 100% of the mean Dl receptor density
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FIG. 4. In situ hybridization autoradiogram of [?3]cRNA probe for D2 dopamine See Methods for details. The photograph is of a reconstructed image after subtraction intact striatum (s) and the marked lack of signal over transplant (t).
in contralateral normal striata. The mean patch Dl receptor density in the grafts was 84% of normal striatum and not significantly different. Figure 4 shows the pattern of localization of D2 receptor mRNA in a 6-month-old striatal transplant, as revealed by in situ hybridization with a cRNA probe. We determined that the melting temperature of the probetissue mRNA duplex in 0.1X SSC was 55°C (data not shown); therefore the final wash in 0.1X SSC was carried out at 40°C. High densities of hybridization signal are seen over normal striatum and accumbens; no significant signal is seen over the graft. Figure 5 shows the patterns of localization at 6.5 months after transplantation of adenylate cyclase, labeled with [3H]forskolin, and protein kinase C, labeled with [3H]phorbol ester. Patterns of [3H]forskolin labeling were also patchy and reminiscent of Dl receptor localization. On the other hand, specific [3H]phorbol ester binding was consistently and densely present throughout the grafts. Figure 6 reveals the patterns of development of preand postsynaptic markers for striatal acetylcholine function in the grafts. At both 4 and 6.5 months after
receptor of film
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messenger background.
RNA in a &month-old Note the high signal
striatal intensity
graft. over
implantation, there is generous and fairly homogenous development of muscarinic cholinergic receptors, labeled with the nonselective ligand [3H]QNB. Receptor site densities within the grafts approximate those seen in superficial layers (higher [3H]QNB density) of overlying cortex (83 and 90% of cortical values at 4 and 6.5 months, respectively) and are equal to those in intact striatum. In contrast, there is very little development at either time point of high-affinity choline uptake sites, labeled with [3H]hemicholinium and normally associated with cholinergic nerve terminals. At both 4 and 6.5 months after implantation, [3H]hemicholinium binding densities were 35-40% of those in the dorsolateral normal striatum. DISCUSSION
The present study confirms and extends results of previous studies that have shown behavioral recovery in rats with striatal intrinsic neuronal lesions following fetal striatal implants (9-12, 20, 21, 23), reduced density of D2 DA receptors in striatal grafts (10, 12), and more normal development of muscarinic cholinergic recep-
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FIG. 5. Localization of adenylate cyclase and protein kinase C in fetal striatal grafts. Shown are autoradiographic images old transplants incubated with [3H]forskolin to label adenylate cyclase (A) and [3H]phorbol 12,13dibutyrate to label protein labeling of adenylate cyclase but extensive and dense labeling of protein kinase C was observed in 4-month-old grafts (not
tom in striatal grafts (11). Our results show that by 4 months after fetal striatal transplantation (the earliest time point examined), there is no asymmetry of blood flow within the grafts. 2-[3H]DG studies after apomorphine treatment suggest the presence of limited numbers of functional DA receptors, but the results after amphetamine treatment do not support the presence of many functional DA synapses. Apomorphine-induced increases in basal ganglia 2-DG accumulation have been found on striatonigral endings in substantia nigra reticulata and have been shown to be mediated by super-
from 6.5-monthkinase C (B). No shown).
sensitive Dl dopamine receptors (30). It is possible that our 2-DG patterns after apomorphine in the 4-monthold grafts derive from activation of dopamine receptors not present in high enough concentrations to be visualized by autoradiography with tritiated ligands. GABA release in globus pallidus ipsilateral to striatal grafts 6-8 months old has been reported to be under control of DA synapses (14), suggesting the development of functional DA receptors in striatal grafts of that age. By 6.5 months after transplantation, partial behavioral recovery in our rats was first apparent, and Dl, but
DA
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FIG. 6. Development of presynaptic and postsynaptic markers of cholinergic synapses in striatal grafts. Presynaptic on cholinergic terminals were labeled with [3H]hemicholinium (A, B) and postsynaptic muscarinic receptors were labeled on sections from rats with 4-month-old (A, C) or 6.5.month-old (B, D) striatal grafts.
not D2, receptors were being expressed in discrete islands or patches. In situ hybridization autoradiography at 6 months showed that the absence of detectable D2 receptors, sought for with three different 3H ligands, appeared to derive from lack of transcription of the D2 receptor gene. It is unclear why our results are different from those of an earlier study that found limited recovery of D2 receptors in much smaller striatal implants (12). High-affinity [3H]forskolin binding in the presence of GppNHp, believed to be associated with adenylate cyclase catalytic activity (15, 16, 32), also was found in similar Dl receptor-like patch patterns at 6.5 months. Although we did not attempt colocalization of these patches with Dl receptors in the grafts, their similar autoradiographic patterns are consistent with the biochemical association between striatal Dl DA receptors and activation of adenylate cyclase (4). The marked reduction of DA receptor development in the grafts was not found for all receptors, as muscarinic cholinergic receptors developed much more extensively. Others have found similar development of cholinergic receptor sites (11). We observed, however, very little development of cholinergic terminals, as manifested by high affinity [3H]hemicholinium binding. Our electron
choline uptake sites with [aH]QNB (C, D)
microscopic immunohistochemical studies have shown that the grafts contain ChAT in ultrastructurally normal neurons and axon terminals, demonstrating that the neurochemical differentiation of the implanted cells into cholinergic neurons does take place in the fetal grafts. At the light microscopic level we observed very few ChAT-positive terminals in the grafts, consistent with the lack of significant [3H]hemicholinium binding. Electrophysiological studies suggest similarities between neuronal ion channels operated by forskolin and phorbol ester (18). To the extent that protein kinase C activity is reliably labeled with [3H]phorbol ester (31, 32), our results in chronic striatal implants suggest that the majority of protein kinase C activity develops independently of and is most likely not associated with DA receptors. The marked reduction at 6.5 months after grafting of DA receptor development on fetal striatal neurons, shown by our previous studies of the same group of animals to have developed into morphological and immunohistochemical maturity by 3 months after transplantation, suggests that dopaminergic afferent innervation of developing fetal striatal neurons may be necessary for DA receptor expression. This hypothesis is supported by similarities in our patterns of Dl DA receptor ex-
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pression to TH (+) patches (5) and islands of DA histofluorescence (24) seen in striatal grafts and biochemical studies of striatal DA receptor postnatal development (2). One test of this hypothesis could involve cotransplantation of fetal midbrain and striatal anlage into the same lesioned striatum. The factors responsible for behavioral recovery from excitotoxic lesions of striatal neurons remain unclear. If development of DA receptor function on grafted fetal striatal neurons is one necessary component of behavioral recovery, our results suggest that the Dl subtype of receptors is more important. It remains to be shown whether DA receptor expression in striatal grafts can be accelerated and, if so, whether more rapid behavioral recovery follows. ACKNOWLEDGMENTS This work has been supported by NIH-NINDS NS00978, NS26581, and NS07199. We thank Dr. Olivier Civelli for his gift of plasmid containing the D2 receptor cDNA, Ms. Joan Gilrain for technical help, and Ms. Rose Powell for editorial assistance.
ET AL. covery
2.
ALEXANDER, M. J., M. A. MILLER, D. M. DORSA, B. P. BULLOCK, R. H. MELLONI, JR., P. R. DOBNER, AND S. E. LEEMAN. 1989. Distribution of neurotensin/neuromedin N mRNA in rat forebrain: Unexpected abundance in hippocampus and subiculum. Proc. N&l. Acad. Sci. USA 86: 5202-5206. BROADDUS, W. C., AND J. P. BENNETT, JR. 1990. Postnatal development of striatal dopamine function. II. Effects of 6-hydroxydopamine treatments on Dl and D2 receptors, adenylate cyclase activity and markers for presynaptic dopamine function. Deu. Brain Res. 62: 273-277.
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BUNZOW, J. R., H. H. M. VAN TOL, D. K. GRANDY, P. ALBERT, J. SALON, M. CHRISTIE, C. A. MACHIDA, K. NEVE, AND 0. CIVELLI. 1988. Cloning and expression of a rat D, dopamine receptor cDNA. Nature (London) 336: 783-787.
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