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Changes in striatal dopamine D2 receptors in relation to expression of and recovery from experimental parkinsonism
Brain Research 871 (2000) 281–287 www.elsevier.com / locate / bres
Research report
Changes in striatal dopamine D 2 receptors in relation to expression of and recovery from experimental parkinsonism T. Wade, D.S. Rothblat, J.S. Schneider* Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, 1020 Locust Street, 521 JAH, Philadelphia, PA 19107, USA Accepted 9 May 2000
Abstract Changes in dopamine D 2 receptor number in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated cats during various stages of experimental parkinsonism were examined. In the normal adult cat, D 2 receptors were expressed throughout the striatum. In symptomatic cats (assessed 7 days after the last MPTP administration), there was a slight elevation of D 2 receptors in all striatal regions. At 2 weeks after MPTP (animals still grossly symptomatic), D 2 receptor number was increased 60–75% above normal. At 3 weeks after MPTP (partial functional recovery), D 2 receptor number remained elevated at a level slightly less than that observed at 2 weeks. At 6 weeks after MPTP (full functional recovery), D 2 receptor levels were back to normal. Changes in D 2 receptor mRNA expression in the striatum essentially mirrored the changes in receptor number. Increases in D 2 receptor number and mRNA expression did not coincide with the onset of parkinsonian signs and peaked after the parkinsonism was established. Permanent reduction of parkinsonian signs corresponded to normalization of D 2 receptor number. 2000 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Degenerative disease: Parkinson’s Keywords: Dopamine; Receptor; Parkinsonism; Cat; Striatum; Recovery
1. Introduction Most symptoms of Parkinson’s Disease (PD) and experimentally induced parkinsonism occur as a result of loss of the nigrostriatal dopaminergic innervation. Postsynaptic dopamine (DA) receptors mediate the actions of DA and represent sites of action for antiparkinsonian drugs. Dopaminergic receptor stimulation that results in relief of parkinsonian motor signs has been consistently observed with drugs that interact with D 2 -like receptors [9,12]. Since D 2 receptors are present at high levels throughout the striatum [3] and since they represent potential sites of action for antiparkinsonian drugs, it is important to understand how D 2 receptors are regulated in response to loss of *Corresponding author. Tel.: 11-215-503-0370; fax: 11-215-9233808. E-mail address: [email protected] (J.S. Schneider)
DA and how changes in D 2 receptor number correspond with the expression of parkinsonian signs. In the present study, we examined changes in D 2 receptor number in animals that were severely parkinsonian and at different stages of spontaneous functional recovery in order to determine the extent to which changes in the expression of these receptors are related to expression of parkinsonian signs. The MPTP-treated cat was chosen for study because the initial parkinsonian syndrome has pathological features similar to those in PD [19,20,23] and because this model is characterized by a predictable recovery of function which has been previously characterized neurochemically [20]. Utilizing a radioligand selective for but with equivalent affinity for D 2 and D 3 receptors we were previously unable to detect changes in D 2 -like receptor number in either symptomatic or recovered MPTP-treated cats [5]. Therefore, we have now utilized a compound that does discriminate between these receptors
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and additionally examine D 2 receptor mRNA expression to assess changes related to parkinsonism and recovery in MPTP-treated animals.
2. Materials and methods
2.1. MPTP administration Adult cats were given daily intramuscular injections of MPTP–HCl (7.5 mg / kg) for 7 to 10 days until a severe parkinsonian syndrome developed [24]. Control animals received saline injections over a similar period of time. Following MPTP treatment, neurological / behavioral testing was performed twice weekly as described previously [20]. One group of cats (n56) still displaying prominent sensorimotor impairment, euthanized 7–10 days after the last MPTP injection. The other groups of MPTP-treated cats (n56 each) were assessed for degree of parkinsonism at 2, 3 and 6 weeks after the last MPTP injection and then euthanized. Control animals were euthanized at 10 days or 6 weeks after initial saline treatment. All cats were euthanized by lethal injection of sodium pentobarbital (150 mg / kg, i.v.). The brains were removed immediately after cessation of heartbeat and rinsed in ice-cold 0.1 M phosphate buffer. The brains were bisected along the midline and the hemispheres were frozen in powdered dry ice and stored at 2808C for subsequent autoradiographic studies. For these studies, brains were sectioned at 20 mm on a cryostat microtome, thaw-mounted onto gelatin-subbed slides, dried on a slide warmer, desiccated overnight at 48C and stored at 2808C until used.
2.2. Quantitative receptor autoradiography Dopamine D 2 receptors were labeled with [ 3 H]spiperone, which exhibits higher affinity for D 2 than D 3 receptors [25]. Slide-mounted tissue sections were brought to 08C 30 min prior to incubation, and to room temperature just prior to incubation. Sections were pre-incubated in Tris buffer (50 mM Tris–HCl, pH 7.4, containing 120 mM NaCl, 5 mM KCl, and 1 mM MgCl 2 ) for 5 min at room temperature and then incubated for 60 min in 0.7 mM [ 3 H]-spiperone (97 Ci / mmol; Amersham, Inc.) in Tris buffer containing 1 mM ascorbic acid and 40 mM ketanserin. Non-specific binding was determined in the presence of 1 mM (1)-butaclamol. Slides were rinsed twice in ice-cold buffer for 20 s followed by a 10 s rinse in ddH 2 O, and blown dry under a stream of cool air.
2.3. D2 receptor in situ hybridization histochemistry Twenty micron thick sections through the striatum were processed for in situ hybridization histochemistry using an 35 S radiolabeled oligonucleotide probe for the common region (nucleotides 1057–1096) of the human DA D 2
receptor. The probe sequence was 59-GGG AGG TCC GGG TTT TGC CAT TGG GCA TGG TCT GCA TCT C-39. Although the sequence for the cat D 2 receptor is not known, a GenBank search revealed that the sequence used was completely conserved in rat, mouse and human. A search of the GenBank database further revealed that this sequence displayed no homology with other DA receptor subtypes (D 1 , D 3 , D 4 ). Tissue from animals representing all treatment groups were always hybridized together in the same run. Seven pmol of the oligonucleotide probe were incubated at 378C for 60 min with 5 ml [ 35 S]-deoxyadenosine 59-(alpha-thio)triphosphate ([ 35 S]-dATP) (.1000 Ci / mmol) (Amersham, Inc.), 10 ml TdT tailing buffer, 21.5 ml diethyl pyrocarbonate (DEPC)-treated water, 5 ml CoCl 2 and 1.5 ml TdT. The radiolabeled probe was then purified at room temperature and precipitated [18]. Tissue sections were fixed, subjected to pre-hybridization processing [18] and then hybridized overnight at 378C with 60 ml of hybridization cocktail (900 ml hybridization buffer, 50 ml salmon sperm DNA, 24 ml [ 35 S]-D2 probe (1310 6 counts per section) and 40 ml DTT). Posthybridization processing was as previously described [18]. The slides were emulsion coated in Kodak NTB2 emulsion diluted 1:1 with 300 mM ammonium acetate, exposed for 6–7 weeks at 48C, developed in Kodak D19 for 5 min at 168C, dried, counterstained with cresyl violet and coverslipped.
2.4. Quantification and analysis
2.4.1. Autoradiography Tissue sections and plastic tritium standards (American Radiolabeled Chemicals, Inc.) were loaded into X-ray cassettes and apposed to [ 3 H]-Hyperfilm (Amersham, Inc.) for 21 days. The film was developed in Kodak D19 developer and fixed. Autoradiographs were analyzed using a computer-driven analysis system (BRAIN version 3.0, Drexel University) which converts gray values to the amount of radioligand bound (fmol per milligram of protein). The brain areas analyzed included the head of the caudate nucleus (subdivided into dorsolateral, dorsomedial, ventrolateral and ventromedial regions), the putamen and the nucleus accumbens at approximately AP 16.5 according to the atlas of Berman and Jones [2]. A minimum of 4 sections for total and 2 sections for nonspecific binding of a radioligand were used. A mean value was calculated for each region per case. Analysis of variance (ANOVA) with regions compacted as repeated measures was performed to determine the group, region and group by region interaction effects. If a significant main effect for group existed, one factor ANOVA was used to determine the level of significance for each region. Statistical significance between groups was determined by ANOVA and NewmanKeuls post-hoc test.
T. Wade et al. / Brain Research 871 (2000) 281 – 287
2.4.2. In situ hybridization histochemistry Analysis of labeled cells was accomplished using NIH Image v. 1.6 and a grain counting macro [10]. The analysis technique utilized a series of filters which allowed cell bodies to be distinguished from overlying reduced silver grains. Using a 403 objective, cells in a particular area of the striatum (DL or VM CD) were first visualized using a green filter to accentuate the cresyl violet-stained cell bodies and a ‘cell’ image was captured. The same field was then visualized using a blue Wratten filter to accentuate the silver grains overlying the cells (‘grain’ image). A composite image of the cell and grain images was then created and filtered using a Laplace 535 transformation filter. The processed composite image was then compared to the original cell image so that the perimeter of cells with overlying silver grains could be traced and copied to the composite image. The area of the cell (mm 2 ) and the number of overlying silver grains were measured. Ten cells per section per area in two non-adjacent coronal sections were used per animal (4 animals in each of the 5 experimental conditions). Statistical significance between groups was tested by ANOVA followed by pair-wise comparisons using Newman-Keuls post-hoc test.
3. Results MPTP administration caused a severe parkinsonian condition in all cats. Results of functional assessments
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performed at the conclusion of the study are shown in Fig. 1. Cats assessed 1 and 2 weeks after the last MPTP administration were similar in terms of extent of parkinsonian signs and are referred to as ‘symptomatic’. Three weeks after MPTP, cats showed partial functional recovery. At 6 weeks after MPTP, animals appeared grossly normal and are referred to as ‘recovered’.
3.1. D2 autoradiography In the normal adult cat, higher amounts of D 2 receptor binding were found in the dorsal caudate nucleus (CD) and lower binding was located ventrally and in the nucleus accumbens (NACC) but these differences were not statistically significant (Figs. 2 and 3). There were time dependent changes in the number of D 2 receptors as parkinsonian signs developed and then recovered. In symptomatic cats 1 week after MPTP, there was a slight (but not statistically significant) increase in D 2 receptor binding in all striatal regions (Figs. 2 and 3). At 2 weeks after MPTP (when animals were still grossly symptomatic), striatal D 2 receptor number was elevated an average of 71% (P, 0.01) in the dorsal CD and an average 62% (P,0.01) and 57% (P,0.01) in the ventral CD and NACC, respectively (Fig. 3). At 3 weeks after MPTP (partial functional recovery), D 2 receptor number was still elevated in the dorsal CD (average 43%) and ventral CD (average 32%) but these increases were significantly less than at 2 weeks (P,0.05). At 6 weeks after MPTP (gross functional
Fig. 1. Changes in behavioral ratings of cats across the different experimental conditions. Behaviors assessed were locomotion, posture, and responsiveness to tactile, visual and auditory stimuli. Note that parkinsonian symptoms were most severe at 1 and 2 weeks after MPTP exposure, were somewhat improved at 3 weeks, and for the most part were not present at 6 weeks. All measures at 1, 2 and 3 weeks were significantly different from normal (Kruskal-Wallis nonparametric analysis of variance with Dunn’s test post hoc pairwise comparisons, P,0.01). Measures taken at 6 weeks were not significantly different from normal.
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Fig. 2. Autoradiograms of total [ 3 H]-spiperone binding in striatum in normal animals (A, B) and animals at 1 week (C), 2 weeks (D), 3 weeks (E) and 6 weeks (F) after the last MPTP exposure. No consistent or significant changes were noted in [ 3 H]-spiperone binding at 1 week after MPTP. Elevation of D 2 receptor number was observed throughout the striatum but predominantly in the dorsal caudate nucleus at 2 weeks after MPTP and less so at 3 weeks after MPTP. At 6 weeks after MPTP, D 2 receptor binding was at normal levels.
recovery), D 2 receptor number was not significantly different from normal values in any striatal region (Figs. 2 and 3).
magnitude of the changes was much smaller (Figs. 4 and 5).
4. Discussion
3.2. D2 receptor in situ hybridization The pattern of changes in D 2 receptor mRNA in intrinsic neurons in the dorsal and ventral CD essentially mirrored the changes observed with [ 3 H] spiperone binding. In the DL CD, there was a small increase (11%) in D 2 mRNA expression per neuron at 1 week after MPTP which peaked (34% increase) at 2 weeks after MPTP. D 2 receptor mRNA expression was still elevated at 3 weeks after MPTP (21% increase) but was back to normal levels at 6 weeks after MPTP. Changes in D 2 mRNA expression in the VM CD mirrored this same temporal pattern but the
Consistent with previous reports [1,15], there were slight regional differences in D 2 receptor binding within the cat striatum with a somewhat ‘patchy’ appearance. D 2 binding was greatest in the sensorimotor striatum and present to a somewhat lesser extent in the limbic striatum [8]. In our previous analysis of changes in D 2 -like receptors utilizing [ 125 I] epidepride we were unable to identify changes with MPTP-induced loss of DA innervation to the cat striatum [5]. This may be because epidepride has approximately equivalent affinity for D 2 and D 3 receptors [17] and D 2 and D 3 receptors may be differently affected
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Fig. 3. Quantitative changes in [ 3 H]-spiperone binding to D 2 receptors in the striatum in normal and MPTP-lesioned cats. Specific binding of [ 3 H]-spiperone to dopamine D 2 receptors was significantly increased in all striatal subregions at 2 weeks following the last MPTP injection, still increased in the caudate nucleus at 3 weeks after MPTP, and not different from normal at 6 weeks after MPTP. *P,0.01 vs. normal; 1 P,0.05 vs. normal.
by the MPTP-induced lesion [17]. In support of this is the observation that the binding of [ 3 H]-spiperone corresponded to labeling of D 2 mRNA in the striatum and both exhibited patterns of expression that differed significantly from that of the D 3 receptor ligand [ 125 I]trans 7-OHPIPAT in the cat striatum [22]. Thus, we believe that we have now selectively identified changes in numbers of D 2
receptors in cat striatum as a function of time from MPTP administration. In early symptomatic cats (1 week after MPTP), D 2 receptor number was only modestly increased but was significantly elevated throughout the striatum during the later symptomatic period (2 weeks after MPTP) and the early recovery period (3 weeks after MPTP). D 2 receptor
Fig. 4. Counts of reduced silver grains overlying striatal neurons expressing D 2 receptor mRNA in the dorsolateral (DL) and ventromedial (VM) areas of the head of the caudate nucleus across the various experimental conditions. The number of reduced silver grains per square micron was significantly increased in the DL caudate at 2 weeks and 3 weeks after MPTP administration and returned to normal levels at 6 weeks. **P,0.01; *P,0.05, vs. normal control.
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Fig. 5. Photomicrographs of D 2 receptor mRNA-expressing neurons in the dorsolateral portion of the head of the caudate nucleus in a normal cat (A) and cats at 1 week (B), 2 weeks (C), 3 weeks (D) and 6 weeks (E) after the last MPTP exposure. D 2 mRNA expression (arrowheads) increased and peaked at 2 weeks, was still slightly elevated at 3 weeks, and returned to normal at 6 weeks. Arrowheads point to D 2 -expressing neurons. Calibration530 microns.
levels were not significantly different from normal in fully recovered cats. In the DL CD, changes in D 2 receptor binding correlated with changes in D 2 receptor mRNA expression, although the changes in mRNA expression were of lesser magnitude. In the VM CD, a correlation between receptor binding and mRNA expression was less obvious due to only minor changes in mRNA expression across the conditions. This may suggest that D 2 receptors in DL and VM CD are differently regulated and that changes in D 2 receptor number in the VM CD in response to DA denervation may be more dependent on secondary post-transcriptional events than D 2 receptors in the DL CD. The observed changes in D 2 receptor binding and gene expression may reflect time-dependent changes in the DAergic innervation or levels of extracellular DA that occur during the recovery process. Striatal dopamine transporter (DAT) levels in the VM CD of the animals used in this study were previously found to be depleted by 85%, 85%, 84% and 81% at 1, 2, 3, and 6 wks., respectively, after MPTP. Dopamine transporter levels in the DL CD were depleted by 91%, 91%, 90% and 88% at 1, 2, 3, and 6 weeks, respectively, after MPTP [unpublished observations]. These data are consistent with a previous report of [ 3 H]-mazindol binding to striatal DA
uptake sites in normal, symptomatic and recovered MPTPtreated cats [5]. We have also shown that tissue and extracellular fluid (ECF) DA levels are maximally depleted in symptomatic parkinsonian cats (1 week after MPTP) and that ECF DA levels recover significantly in both ventral and dorsal striatal regions in recovered cats (6 weeks after MPTP), despite the continued extensive loss of the dorsal striatal DA innervation [16]. Extracellular fluid DA in the dorsal striatum of recovered cats primarily originates in the more highly recovered ventral striatal DA innervation and reaches the dorsal striatum via volume transmission [21]. At 1 week after MPTP, D 2 receptor number (and mRNA expression in the DL CD) is beginning to increase and reaches its peak increase at 2 weeks after MPTP when ECF DA levels are still likely significantly depleted. At 3 weeks post MPTP, ECF DA levels are likely beginning to recover, as evidenced by partial functional recovery. With a rise in ECF DA levels, D 2 receptors respond by beginning to down-regulate. At 6 weeks after MPTP the amount of ECF DA available throughout the striatum is sufficient to normalize D 2 receptor number. Several studies in different animal models of parkinsonism have shown that greater than 90% loss of DA results in elevation of D 2 receptor number which can be normalized by raising DA levels
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[4,6,11,13,14,23,26]. Our results in the cat are consistent with the idea that post-synaptic D 2 receptors in the striatum may be regulated at least in part by changes in ambient extracellular DA levels. It is still unclear as to how the observed changes in D 2 receptors may or may not be directly involved in the expression of or recovery from parkinsonism. Acute parkinsonism, partial recovery and full recovery may involve regulation of different DA receptor subtypes and their striatal efferent pathways and possible alterations in co-expression of DA receptors at least in a subset of striatal neurons. It is interesting that when cats are recovered from experimental parkinsonism, striatal D 2 receptor binding reverts back to normal levels but D 1 receptor binding is elevated to an even greater extent than when animals were symptomatic [5]. Perhaps functional recovery in this model is at least partially related to a persistent increase in D 1 receptor binding and a coexisting down-regulation of D 2 receptor binding.
Acknowledgements This research was supported by U.S. Public Health Service Grant NS23980.
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Research report
Changes in striatal dopamine D 2 receptors in relation to expression of and recovery from experimental parkinsonism T. Wade, D.S. Rothblat, J.S. Schneider* Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, 1020 Locust Street, 521 JAH, Philadelphia, PA 19107, USA Accepted 9 May 2000
Abstract Changes in dopamine D 2 receptor number in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated cats during various stages of experimental parkinsonism were examined. In the normal adult cat, D 2 receptors were expressed throughout the striatum. In symptomatic cats (assessed 7 days after the last MPTP administration), there was a slight elevation of D 2 receptors in all striatal regions. At 2 weeks after MPTP (animals still grossly symptomatic), D 2 receptor number was increased 60–75% above normal. At 3 weeks after MPTP (partial functional recovery), D 2 receptor number remained elevated at a level slightly less than that observed at 2 weeks. At 6 weeks after MPTP (full functional recovery), D 2 receptor levels were back to normal. Changes in D 2 receptor mRNA expression in the striatum essentially mirrored the changes in receptor number. Increases in D 2 receptor number and mRNA expression did not coincide with the onset of parkinsonian signs and peaked after the parkinsonism was established. Permanent reduction of parkinsonian signs corresponded to normalization of D 2 receptor number. 2000 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Degenerative disease: Parkinson’s Keywords: Dopamine; Receptor; Parkinsonism; Cat; Striatum; Recovery
1. Introduction Most symptoms of Parkinson’s Disease (PD) and experimentally induced parkinsonism occur as a result of loss of the nigrostriatal dopaminergic innervation. Postsynaptic dopamine (DA) receptors mediate the actions of DA and represent sites of action for antiparkinsonian drugs. Dopaminergic receptor stimulation that results in relief of parkinsonian motor signs has been consistently observed with drugs that interact with D 2 -like receptors [9,12]. Since D 2 receptors are present at high levels throughout the striatum [3] and since they represent potential sites of action for antiparkinsonian drugs, it is important to understand how D 2 receptors are regulated in response to loss of *Corresponding author. Tel.: 11-215-503-0370; fax: 11-215-9233808. E-mail address: [email protected] (J.S. Schneider)
DA and how changes in D 2 receptor number correspond with the expression of parkinsonian signs. In the present study, we examined changes in D 2 receptor number in animals that were severely parkinsonian and at different stages of spontaneous functional recovery in order to determine the extent to which changes in the expression of these receptors are related to expression of parkinsonian signs. The MPTP-treated cat was chosen for study because the initial parkinsonian syndrome has pathological features similar to those in PD [19,20,23] and because this model is characterized by a predictable recovery of function which has been previously characterized neurochemically [20]. Utilizing a radioligand selective for but with equivalent affinity for D 2 and D 3 receptors we were previously unable to detect changes in D 2 -like receptor number in either symptomatic or recovered MPTP-treated cats [5]. Therefore, we have now utilized a compound that does discriminate between these receptors
0006-8993 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )02484-7
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and additionally examine D 2 receptor mRNA expression to assess changes related to parkinsonism and recovery in MPTP-treated animals.
2. Materials and methods
2.1. MPTP administration Adult cats were given daily intramuscular injections of MPTP–HCl (7.5 mg / kg) for 7 to 10 days until a severe parkinsonian syndrome developed [24]. Control animals received saline injections over a similar period of time. Following MPTP treatment, neurological / behavioral testing was performed twice weekly as described previously [20]. One group of cats (n56) still displaying prominent sensorimotor impairment, euthanized 7–10 days after the last MPTP injection. The other groups of MPTP-treated cats (n56 each) were assessed for degree of parkinsonism at 2, 3 and 6 weeks after the last MPTP injection and then euthanized. Control animals were euthanized at 10 days or 6 weeks after initial saline treatment. All cats were euthanized by lethal injection of sodium pentobarbital (150 mg / kg, i.v.). The brains were removed immediately after cessation of heartbeat and rinsed in ice-cold 0.1 M phosphate buffer. The brains were bisected along the midline and the hemispheres were frozen in powdered dry ice and stored at 2808C for subsequent autoradiographic studies. For these studies, brains were sectioned at 20 mm on a cryostat microtome, thaw-mounted onto gelatin-subbed slides, dried on a slide warmer, desiccated overnight at 48C and stored at 2808C until used.
2.2. Quantitative receptor autoradiography Dopamine D 2 receptors were labeled with [ 3 H]spiperone, which exhibits higher affinity for D 2 than D 3 receptors [25]. Slide-mounted tissue sections were brought to 08C 30 min prior to incubation, and to room temperature just prior to incubation. Sections were pre-incubated in Tris buffer (50 mM Tris–HCl, pH 7.4, containing 120 mM NaCl, 5 mM KCl, and 1 mM MgCl 2 ) for 5 min at room temperature and then incubated for 60 min in 0.7 mM [ 3 H]-spiperone (97 Ci / mmol; Amersham, Inc.) in Tris buffer containing 1 mM ascorbic acid and 40 mM ketanserin. Non-specific binding was determined in the presence of 1 mM (1)-butaclamol. Slides were rinsed twice in ice-cold buffer for 20 s followed by a 10 s rinse in ddH 2 O, and blown dry under a stream of cool air.
2.3. D2 receptor in situ hybridization histochemistry Twenty micron thick sections through the striatum were processed for in situ hybridization histochemistry using an 35 S radiolabeled oligonucleotide probe for the common region (nucleotides 1057–1096) of the human DA D 2
receptor. The probe sequence was 59-GGG AGG TCC GGG TTT TGC CAT TGG GCA TGG TCT GCA TCT C-39. Although the sequence for the cat D 2 receptor is not known, a GenBank search revealed that the sequence used was completely conserved in rat, mouse and human. A search of the GenBank database further revealed that this sequence displayed no homology with other DA receptor subtypes (D 1 , D 3 , D 4 ). Tissue from animals representing all treatment groups were always hybridized together in the same run. Seven pmol of the oligonucleotide probe were incubated at 378C for 60 min with 5 ml [ 35 S]-deoxyadenosine 59-(alpha-thio)triphosphate ([ 35 S]-dATP) (.1000 Ci / mmol) (Amersham, Inc.), 10 ml TdT tailing buffer, 21.5 ml diethyl pyrocarbonate (DEPC)-treated water, 5 ml CoCl 2 and 1.5 ml TdT. The radiolabeled probe was then purified at room temperature and precipitated [18]. Tissue sections were fixed, subjected to pre-hybridization processing [18] and then hybridized overnight at 378C with 60 ml of hybridization cocktail (900 ml hybridization buffer, 50 ml salmon sperm DNA, 24 ml [ 35 S]-D2 probe (1310 6 counts per section) and 40 ml DTT). Posthybridization processing was as previously described [18]. The slides were emulsion coated in Kodak NTB2 emulsion diluted 1:1 with 300 mM ammonium acetate, exposed for 6–7 weeks at 48C, developed in Kodak D19 for 5 min at 168C, dried, counterstained with cresyl violet and coverslipped.
2.4. Quantification and analysis
2.4.1. Autoradiography Tissue sections and plastic tritium standards (American Radiolabeled Chemicals, Inc.) were loaded into X-ray cassettes and apposed to [ 3 H]-Hyperfilm (Amersham, Inc.) for 21 days. The film was developed in Kodak D19 developer and fixed. Autoradiographs were analyzed using a computer-driven analysis system (BRAIN version 3.0, Drexel University) which converts gray values to the amount of radioligand bound (fmol per milligram of protein). The brain areas analyzed included the head of the caudate nucleus (subdivided into dorsolateral, dorsomedial, ventrolateral and ventromedial regions), the putamen and the nucleus accumbens at approximately AP 16.5 according to the atlas of Berman and Jones [2]. A minimum of 4 sections for total and 2 sections for nonspecific binding of a radioligand were used. A mean value was calculated for each region per case. Analysis of variance (ANOVA) with regions compacted as repeated measures was performed to determine the group, region and group by region interaction effects. If a significant main effect for group existed, one factor ANOVA was used to determine the level of significance for each region. Statistical significance between groups was determined by ANOVA and NewmanKeuls post-hoc test.
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2.4.2. In situ hybridization histochemistry Analysis of labeled cells was accomplished using NIH Image v. 1.6 and a grain counting macro [10]. The analysis technique utilized a series of filters which allowed cell bodies to be distinguished from overlying reduced silver grains. Using a 403 objective, cells in a particular area of the striatum (DL or VM CD) were first visualized using a green filter to accentuate the cresyl violet-stained cell bodies and a ‘cell’ image was captured. The same field was then visualized using a blue Wratten filter to accentuate the silver grains overlying the cells (‘grain’ image). A composite image of the cell and grain images was then created and filtered using a Laplace 535 transformation filter. The processed composite image was then compared to the original cell image so that the perimeter of cells with overlying silver grains could be traced and copied to the composite image. The area of the cell (mm 2 ) and the number of overlying silver grains were measured. Ten cells per section per area in two non-adjacent coronal sections were used per animal (4 animals in each of the 5 experimental conditions). Statistical significance between groups was tested by ANOVA followed by pair-wise comparisons using Newman-Keuls post-hoc test.
3. Results MPTP administration caused a severe parkinsonian condition in all cats. Results of functional assessments
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performed at the conclusion of the study are shown in Fig. 1. Cats assessed 1 and 2 weeks after the last MPTP administration were similar in terms of extent of parkinsonian signs and are referred to as ‘symptomatic’. Three weeks after MPTP, cats showed partial functional recovery. At 6 weeks after MPTP, animals appeared grossly normal and are referred to as ‘recovered’.
3.1. D2 autoradiography In the normal adult cat, higher amounts of D 2 receptor binding were found in the dorsal caudate nucleus (CD) and lower binding was located ventrally and in the nucleus accumbens (NACC) but these differences were not statistically significant (Figs. 2 and 3). There were time dependent changes in the number of D 2 receptors as parkinsonian signs developed and then recovered. In symptomatic cats 1 week after MPTP, there was a slight (but not statistically significant) increase in D 2 receptor binding in all striatal regions (Figs. 2 and 3). At 2 weeks after MPTP (when animals were still grossly symptomatic), striatal D 2 receptor number was elevated an average of 71% (P, 0.01) in the dorsal CD and an average 62% (P,0.01) and 57% (P,0.01) in the ventral CD and NACC, respectively (Fig. 3). At 3 weeks after MPTP (partial functional recovery), D 2 receptor number was still elevated in the dorsal CD (average 43%) and ventral CD (average 32%) but these increases were significantly less than at 2 weeks (P,0.05). At 6 weeks after MPTP (gross functional
Fig. 1. Changes in behavioral ratings of cats across the different experimental conditions. Behaviors assessed were locomotion, posture, and responsiveness to tactile, visual and auditory stimuli. Note that parkinsonian symptoms were most severe at 1 and 2 weeks after MPTP exposure, were somewhat improved at 3 weeks, and for the most part were not present at 6 weeks. All measures at 1, 2 and 3 weeks were significantly different from normal (Kruskal-Wallis nonparametric analysis of variance with Dunn’s test post hoc pairwise comparisons, P,0.01). Measures taken at 6 weeks were not significantly different from normal.
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Fig. 2. Autoradiograms of total [ 3 H]-spiperone binding in striatum in normal animals (A, B) and animals at 1 week (C), 2 weeks (D), 3 weeks (E) and 6 weeks (F) after the last MPTP exposure. No consistent or significant changes were noted in [ 3 H]-spiperone binding at 1 week after MPTP. Elevation of D 2 receptor number was observed throughout the striatum but predominantly in the dorsal caudate nucleus at 2 weeks after MPTP and less so at 3 weeks after MPTP. At 6 weeks after MPTP, D 2 receptor binding was at normal levels.
recovery), D 2 receptor number was not significantly different from normal values in any striatal region (Figs. 2 and 3).
magnitude of the changes was much smaller (Figs. 4 and 5).
4. Discussion
3.2. D2 receptor in situ hybridization The pattern of changes in D 2 receptor mRNA in intrinsic neurons in the dorsal and ventral CD essentially mirrored the changes observed with [ 3 H] spiperone binding. In the DL CD, there was a small increase (11%) in D 2 mRNA expression per neuron at 1 week after MPTP which peaked (34% increase) at 2 weeks after MPTP. D 2 receptor mRNA expression was still elevated at 3 weeks after MPTP (21% increase) but was back to normal levels at 6 weeks after MPTP. Changes in D 2 mRNA expression in the VM CD mirrored this same temporal pattern but the
Consistent with previous reports [1,15], there were slight regional differences in D 2 receptor binding within the cat striatum with a somewhat ‘patchy’ appearance. D 2 binding was greatest in the sensorimotor striatum and present to a somewhat lesser extent in the limbic striatum [8]. In our previous analysis of changes in D 2 -like receptors utilizing [ 125 I] epidepride we were unable to identify changes with MPTP-induced loss of DA innervation to the cat striatum [5]. This may be because epidepride has approximately equivalent affinity for D 2 and D 3 receptors [17] and D 2 and D 3 receptors may be differently affected
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Fig. 3. Quantitative changes in [ 3 H]-spiperone binding to D 2 receptors in the striatum in normal and MPTP-lesioned cats. Specific binding of [ 3 H]-spiperone to dopamine D 2 receptors was significantly increased in all striatal subregions at 2 weeks following the last MPTP injection, still increased in the caudate nucleus at 3 weeks after MPTP, and not different from normal at 6 weeks after MPTP. *P,0.01 vs. normal; 1 P,0.05 vs. normal.
by the MPTP-induced lesion [17]. In support of this is the observation that the binding of [ 3 H]-spiperone corresponded to labeling of D 2 mRNA in the striatum and both exhibited patterns of expression that differed significantly from that of the D 3 receptor ligand [ 125 I]trans 7-OHPIPAT in the cat striatum [22]. Thus, we believe that we have now selectively identified changes in numbers of D 2
receptors in cat striatum as a function of time from MPTP administration. In early symptomatic cats (1 week after MPTP), D 2 receptor number was only modestly increased but was significantly elevated throughout the striatum during the later symptomatic period (2 weeks after MPTP) and the early recovery period (3 weeks after MPTP). D 2 receptor
Fig. 4. Counts of reduced silver grains overlying striatal neurons expressing D 2 receptor mRNA in the dorsolateral (DL) and ventromedial (VM) areas of the head of the caudate nucleus across the various experimental conditions. The number of reduced silver grains per square micron was significantly increased in the DL caudate at 2 weeks and 3 weeks after MPTP administration and returned to normal levels at 6 weeks. **P,0.01; *P,0.05, vs. normal control.
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Fig. 5. Photomicrographs of D 2 receptor mRNA-expressing neurons in the dorsolateral portion of the head of the caudate nucleus in a normal cat (A) and cats at 1 week (B), 2 weeks (C), 3 weeks (D) and 6 weeks (E) after the last MPTP exposure. D 2 mRNA expression (arrowheads) increased and peaked at 2 weeks, was still slightly elevated at 3 weeks, and returned to normal at 6 weeks. Arrowheads point to D 2 -expressing neurons. Calibration530 microns.
levels were not significantly different from normal in fully recovered cats. In the DL CD, changes in D 2 receptor binding correlated with changes in D 2 receptor mRNA expression, although the changes in mRNA expression were of lesser magnitude. In the VM CD, a correlation between receptor binding and mRNA expression was less obvious due to only minor changes in mRNA expression across the conditions. This may suggest that D 2 receptors in DL and VM CD are differently regulated and that changes in D 2 receptor number in the VM CD in response to DA denervation may be more dependent on secondary post-transcriptional events than D 2 receptors in the DL CD. The observed changes in D 2 receptor binding and gene expression may reflect time-dependent changes in the DAergic innervation or levels of extracellular DA that occur during the recovery process. Striatal dopamine transporter (DAT) levels in the VM CD of the animals used in this study were previously found to be depleted by 85%, 85%, 84% and 81% at 1, 2, 3, and 6 wks., respectively, after MPTP. Dopamine transporter levels in the DL CD were depleted by 91%, 91%, 90% and 88% at 1, 2, 3, and 6 weeks, respectively, after MPTP [unpublished observations]. These data are consistent with a previous report of [ 3 H]-mazindol binding to striatal DA
uptake sites in normal, symptomatic and recovered MPTPtreated cats [5]. We have also shown that tissue and extracellular fluid (ECF) DA levels are maximally depleted in symptomatic parkinsonian cats (1 week after MPTP) and that ECF DA levels recover significantly in both ventral and dorsal striatal regions in recovered cats (6 weeks after MPTP), despite the continued extensive loss of the dorsal striatal DA innervation [16]. Extracellular fluid DA in the dorsal striatum of recovered cats primarily originates in the more highly recovered ventral striatal DA innervation and reaches the dorsal striatum via volume transmission [21]. At 1 week after MPTP, D 2 receptor number (and mRNA expression in the DL CD) is beginning to increase and reaches its peak increase at 2 weeks after MPTP when ECF DA levels are still likely significantly depleted. At 3 weeks post MPTP, ECF DA levels are likely beginning to recover, as evidenced by partial functional recovery. With a rise in ECF DA levels, D 2 receptors respond by beginning to down-regulate. At 6 weeks after MPTP the amount of ECF DA available throughout the striatum is sufficient to normalize D 2 receptor number. Several studies in different animal models of parkinsonism have shown that greater than 90% loss of DA results in elevation of D 2 receptor number which can be normalized by raising DA levels
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[4,6,11,13,14,23,26]. Our results in the cat are consistent with the idea that post-synaptic D 2 receptors in the striatum may be regulated at least in part by changes in ambient extracellular DA levels. It is still unclear as to how the observed changes in D 2 receptors may or may not be directly involved in the expression of or recovery from parkinsonism. Acute parkinsonism, partial recovery and full recovery may involve regulation of different DA receptor subtypes and their striatal efferent pathways and possible alterations in co-expression of DA receptors at least in a subset of striatal neurons. It is interesting that when cats are recovered from experimental parkinsonism, striatal D 2 receptor binding reverts back to normal levels but D 1 receptor binding is elevated to an even greater extent than when animals were symptomatic [5]. Perhaps functional recovery in this model is at least partially related to a persistent increase in D 1 receptor binding and a coexisting down-regulation of D 2 receptor binding.
Acknowledgements This research was supported by U.S. Public Health Service Grant NS23980.
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