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Regulation of dopamine receptor and neuropeptide expression in the basal ganglia of monkeys treated with MPTP
Experimental Neurology 189 (2004) 393 – 403 www.elsevier.com/locate/yexnr
Regulation of dopamine receptor and neuropeptide expression in the basal ganglia of monkeys treated with MPTP Ranjita Betarbet *, J. Timothy Greenamyre Department of Neurology and Center for Neurodegenerative Disease, Emory University, Atlanta, GA 30322, USA Received 22 October 2003; revised 21 May 2004; accepted 21 May 2004
Abstract In Parkinson’s disease (PD), striatal dopamine deficiency has been associated with complex changes in the functional and neurochemical anatomy of the basal ganglia. In this study, we simultaneously analyzed the regulation of D1 and D2 dopamine receptors and levels of the neuropeptides, substance P, and enkephalin (ENK) in various basal ganglia nuclei following 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine-induced dopaminergic denervation of striatum in nonhuman primates. Both unilateral and bilateral lesioned animals were used for this study. Striatal dopamine deficiency resulted in distinct alterations in D1, D2, substance P, and enkephalin levels and distribution: (1) Both D1 and D2 protein levels were significantly up-regulated in striatum. (2) There was an overall up-regulation of striatal substance P expression following dopamine denervation. (3) Substance P distribution was ‘reversed’ in dopamine depleted striatum: striosomes, which normally express higher levels of substance P, showed decreased expression, whereas substance P expression was upregulated in the matrix. (4) Substance P expression was up-regulated in the internal segment of the globus pallidus (GPi), but remained unchanged in substantia nigra (SN). (5) Enkephalin levels were increased in striatum and the external segment of the globus pallidus (GPe), but not in substantia nigra. All the changes were more pronounced in the bilateral lesioned monkeys, though the data represent a pooled statistical evaluation of unilateral and bilateral lesioned monkeys. Our studies indicate that D1 and D2 dopamine receptors and substance P and enkephalin undergo regulatory changes in response to nigrostriatal dopamine denervation. Simultaneous study of the alterations in these various components of the ‘direct’ and ‘indirect’ pathways in the same animals will enable better understanding of the pathophysiology of PD and its therapeutic targets. D 2004 Elsevier Inc. All rights reserved. Keywords: D1 dopamine receptor; D2 dopamine receptor; Substance P; Enkephalin; MPTP; Parkinson’s disease
Introduction Parkinson’s disease (PD) is a progressive neurodegenerative disease, the pathological hallmark of which is degeneration of the nigrostriatal dopaminergic pathway and subsequent dopamine deficiency in the striatum (Gerlach and Reiderer, 1993; Hornykiewicz and Kish, 1987; Wooten, 1997). The striatum is the major input nuclei of the basal ganglia (Divac et al., 1977; McGeorge and Faull, 1989). It receives glutamatergic input from the overlying cerebral cortex (Young et al., 1981) and thalamus (Beckstead, 1984; Parent et al., 1983; Royce and Mourey, 1985) and dopami* Corresponding author. Center for Neurodegenerative Diseases, Emory University, Suite 505, Whitehead Biomedical Research Building, 615 Michael Street, Atlanta, GA 30322. Fax: +1-404-727-3728. E-mail address: [email protected] (R. Betarbet). 0014-4886/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2004.05.041
nergic input from the substantia nigra pars compacta (Moore et al., 1971). The striatum is composed primarily of medium-sized spiny projection neurons (Gerfen and Wilson, 1996) that constitute approximately 95% of the striatal neuron population (Kemp and Powell, 1971). These projection neurons send GABAergic input to the basal ganglia output nuclei through two parallel ‘direct’ and ‘indirect’ pathways (Parent, 1986). Striatal neurons projecting directly to the output nuclei of the basal ganglia, the internal segment of globus pallidus (GPi; entopeduncular nucleus in rodents), and substantia nigra pars reticulata (SNr) express the D1 dopamine receptor and also co-localize the neuropeptide substance P (SP) (Gerfen et al., 1990; Yung et al., 1995). Neurons that indirectly project to the output nuclei through the external segment of the globus pallidus (GPe) and subthalamic nucleus (STN) express the D2 dopamine receptor and co-express the neuropeptide
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enkephalin (ENK) (Gerfen et al., 1990; Yung et al., 1995). Although recent studies have shown that both sets of striatal neurons express both D1 and D2 dopamine receptors, neurons of the direct pathway express significantly higher levels of D1 and very low levels of D2, whereas the neurons contributing to the indirect pathway express high levels of D2 (Aizman et al., 2000). Through the direct and indirect pathways, striatal neurons modulate the activity of the output nuclei of the basal ganglia. A family of at least five dopamine receptor genes, D1 – D5, has been identified in both rodents and humans (Niznik and Van Tol, 1992; Sibley and Monsma, 1992). Immunocytochemical studies have shown that the D1 and D2 receptors have similar regional distributions in the rat, monkey, and human brain, with highest densities in striatum and substantia nigra. Within each region, however, the precise distribution of each subtype is distinct and often complementary (Levey et al., 1993). D1 and D2 receptors are differentially enriched in the striosomes and matrix; however, in primates, the distinction between striosome and matrix is not always clear. Immunocytochemical identification of individual striatal cells expressing either protein selectively is difficult because of dense neuropil staining. As noted, striatal cells that express D1 co-localize SP. Normally, striosomes are more intensely stained for SP than matrix (Haber and Watson, 1985; Martin et al., 1991). Within the substantia nigra, D1 is enriched in the neuropil of substantia nigra pars reticulata (SNr) and D2 is enriched in perikarya and dendrites in the pars compacta. Following degeneration of the nigrostriatal pathway, numerous changes take place in basal ganglia circuitry. With loss of dopaminergic innervation and decreased activation of D1 and D2 receptors, there is disinhibition of the output nuclei via the direct pathway and increased excitatory input from the STN via the indirect pathway (Albin et al., 1989). This results in increased GABAergic inhibition of basal ganglia outflow targets, including ventrolateral thalamus and brainstem (Albin et al., 1989; Wichmann et al., 1994). Decreased thalamocortical feedback is believed to underlie many of the clinical manifestations of PD (Albin et al., 1989; Crossman, 1989; DeLong, 1990; Greenamyre, 1993; Klockgether and Turski, 1989). In the 6-hydroxydopamine (6-OHDA) model of PD, striatal neurons projecting to SNr show a reduction in the expression of mRNAs encoding the D1 dopamine receptor and SP. Conversely, striatal neurons projecting to globus pallidus show an elevation in the expression of mRNAs encoding the D2 dopamine receptor and ENK (Gerfen et al., 1990). In primates, however, D1 and D2 receptor binding studies, and studies of the distributions of SP and ENK in MPTP-treated primates and in PD patients have yielded conflicting and ambiguous results (Gnanalingham et al., 1993; Morissette et al., 1996; Piggott et al., 1999; Rioux et al., 1997). Furthermore, to date, there have been no reports of changes in D1 and D2 protein levels following striatal dopamine depletion.
In this study, we have analyzed the distributions of D1and D2 dopamine receptors and the distributions of neuropeptides SP and ENK in various basal ganglia nuclei after MPTP-induced striatal dopamine deficiency in nonhuman primates to better understand the pathophysiology and therapeutic targets of Parkinson’s disease.
Materials and methods Animals All animal use was in accordance with NIH guidelines and was approved by the Emory University IACUC. In this study, brains of 10 adult, female rhesus monkeys (Macaca mulatta), ranging in age from 7 to 14 years, were examined. Of the 10 monkeys, 4 unlesioned monkeys were used as controls. Three monkeys received unilateral infusions of MPTP (0.4 mg/kg) into the internal carotid artery to produce a stable hemiparkinsonian syndrome. The remaining three monkeys were rendered bilaterally parkinsonian by intramuscular injections of MPTP (0.5 mg/kg every 2– 5 days) over a period of 2 –6 weeks. Two of the three monkeys with intramuscular MPTP injections were part of a separate study not reported here, wherein ibotenate lesion of the right STN was attempted unsuccessfully. No clear cell loss was apparent in the STN or in the surrounding structures in either monkey. The monkeys with unilateral lesions were euthanized about 2 years after receiving MPTP; those with bilateral parkinsonism were euthanized within 2 months of their last MPTP treatment. None of the monkeys received more than two or three treatments with a dopaminergic drug and none of the animals received any pharmacological treatment within 4 weeks of euthanasia. Table 1 gives the history of each monkey. After ketamine sedation, the monkeys were given an intravenous overdose of sodium pentobarbital. The animals were intracardially perfused with 3% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2. The brains were removed and cryoprotected in a 30% sucrose solution in 0.1 M phosphate buffer. Striatum, globus pallidus (both medial and lateral segments), and substantia nigra were analyzed. Double labeling with immunoautoradiography (IAR) and immunocytochemistry (ICC) Fifty-micrometer-thick brain sections were thoroughly washed in 0.1 M TBS (Tris-buffered saline) to remove the cryoprotectant and incubated in blocking serum consisting of 4% normal goat serum and 4% normal human serum with 0.4% Triton X-100 in TBS for 30 min. The sections were then incubated for 72 h in the same solution containing a combination of primary antibodies. The antibodies used for this study were against tyrosine hydroxylase (TH; 1:1000, mouse monoclonal antibody from Chemicon, CA, and 1:500, rabbit polyclonal antibody from Pelfreez, Rogers, AR); D1 and D2 dopamine receptors, (D1, 1:1000; D2,
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Table 1 History of each monkey used in this study Monkey
Treatment before MPTP
MPTP treatment
Sacrificed
Drugs
Bilateral 1
failed STN lesion with RF probe (no lesion) failed STN lesion with ibotenate
0.5 mg/kg 2 – 5 days for 6 weeks 0.5 mg/kg 2 – 5 days for 6 weeks 0.5 mg/kg 2 – 5 days for 6 weeks 0.4 mg/kg into internal carotid artery 0.4 mg/kg into internal carotid artery 0.4 mg/kg into internal carotid artery
55 days after last MPTP
no drugs
74 days after last MPTP
Sinemet 125 mg, po, for 1 week
70 days after last MPTP
Sinemet 125 mg, po, for 1 week; glutamate antagonist for first month
f2 years after last MPTP f2 years after last MPTP
glutamate antagonist for 18 months after MPTP no drugs
f2 years after last MPTP
no drugs
Bilateral 2
Bilateral 3
none
Unilateral 1
none
Unilateral 2
none
Unilateral 3
none
1:100; Levey et al., 1993) and neuropeptides, SP and methionine-enkephalin (SP and ENK; both used at 1:10,000, Diasorin, MN). The antibodies used in combination were from different hosts to avoid cross-reactivity. Following primary antibody incubation, sections were rinsed in 0.1 M TBS and incubated for 1 – 2 h in the blocking serum containing sheep anti-rat (for D1) or goat anti-rabbit (for D2, SP and ENK) antibody conjugated to 125 I and biotinylated goat anti-mouse or anti-rabbit (for TH). Immunostaining for TH and D1 (regular ICC to visualize the striosomes) was visualized using the avidin – biotin complex (ABC) method (ABC Elite Kit, Vector Laboratories, Burlingame, CA) and 3,3V-diaminobenzidine tetrachloride (DAB, Sigma, St. Louis, MO) was used to visualize the final product. For control sections, one or both the primary antibodies were omitted. Finally, the sections were rinsed in 0.1 M TBS, mounted on gelatin-coated slides and air-dried. The slides were placed in X-ray cassettes with calibrated 125 I standards (Amersham, Piscataway, NJ) and apposed to Hyperfilm3H (Amersham) for 2 –3 weeks. The films were developed in D-19, fixed in Kodak fixer, and dried. The slides were then dehydrated and coverslipped using Shandon mount (Pittsburgh, PA). The immunostained sections were examined using brightfield microscopy. Images were collected on a Leitz microscope linked to an MCID image analysis system (Imaging Research, St. Catharines, Ontario, Canada). For final output, images were composed using Adobe Photoshop. Quantitation For densitometric measurements of D1, D2, SP, and ENK-immunoreactivity (-ir), the autoradiograms were analyzed with a video-based MCID image analysis system (Imaging Research) by relating optical density to calibrated 125 I standards. Two sections were analyzed through the striatum, globus pallidus, and substantia nigra from each
of the 10 monkeys used for the study; a total of 80 sections were analyzed. The striatal sections selected for all analyses were from levels anterior to or at the level of the anterior commissure. The sections from control, unilateral, and bilateral lesioned brains were anatomically matched. For comparison purposes, data from the control or untreated side of the unilaterally lesioned monkeys were grouped together with the data from untreated control monkeys and the data from the lesioned side of unilaterally treated monkeys were grouped along with the bilaterally lesioned, MPTP-treated monkeys. Statistics Statistical analysis was performed using two-way ANOVA with Bonferroni’s post hoc correction to compare control values vs. values following MPTP treatment. A P value less than 0.05 was accepted as indicating a significant difference. Each data set for densitometric measurements is represented as a scatter plot with mean and standard error.
Results D1 dopamine receptor In normal monkeys D 1 was expressed uniformly throughout the striatum (Fig. 1). Striosomes and matrix were not distinctly demarcated, unlike what has been reported in humans (Levey et al., 1993). In the substantia nigra (SN), D1 was enriched in the pars reticulata region (Fig. 3). Following MPTP treatment, the striatum and SN were devoid of TH immunoreactivity (ir) (Figs. 1, 3, 5, 8, 9, 11, and 12) indicating degeneration of the nigrostriatal dopaminergic pathway. The distribution and density of D1 was a markedly increased following MPTP-induced striatal
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Fig. 3. TH- and D1-ir in the substantia nigra of a control monkey and a bilaterally MPTP-lesioned monkey. (Top panel) Macrograph shows TH-ir in the substantia nigra from a control monkey (A) and a lesioned monkey (B). Arrows indicate SNc. (Lower panel) The same nigral sections were double labeled for D1 by immunoautoradiography. The gray levels have been converted to a pseudocolor scale as in Fig. 1. Arrowheads indicate SN. Note the increase in D1-ir in the lesioned nigra (D) vs. the control (C). P, putamen.
Fig. 1. TH- and D1-ir in the striatum of a control monkey and a bilaterally MPTP-lesioned monkey. (Top panel) Macrograph shows TH-ir in the striatum from a control monkey (A) and a lesioned monkey (B). Note the lesioned striatum is devoid of TH-ir. (Lower panel) The same striatal sections were double labeled for D1 by immunoautoradiography. The gray levels have been converted to a pseudocolor scale wherein red depicts high immunoreactivity and blue depicts low immunoreactivity. Note the increase in D1-ir in the lesioned striatum (D) vs. the control (C).
dopamine depletion (Fig. 1); however, the changes were very pronounced in the bilateral MPTP-lesioned monkeys compared to unilateral lesioned ones. D1 up-regulation was more prominent in striosomes than in matrix such that intensely immunoreactive striosomes could be identified easily following striatal dopamine denervation (Fig. 2). Quantitative densitometric measurements of immunoautoradiographs indicated a significant increase in D1 expression following systemic MPTP treatment (Fig. 4). There was also a trend for up-regulation of D1 expression in the SN (Fig. 3) but this was not statistically significant (Fig. 4).
D2 dopamine receptor The distribution of D2-ir was similar to D1 in the striatum (Fig. 5). In the SN, D2 was enriched in the pars compacta region (not shown). Following MPTP, D2 expression was up-regulated in the striatum of both unilaterally and bilaterally lesioned monkeys (Figs. 5 and 6). The changes were more pronounced in the bilaterally lesioned animals. Densitometric measurements of autoradiographs showed that the increase in striatal D2 expression was statistically significant (Fig. 7). No change in D2 expression was detected in the SN (Fig. 7). Substance P SP has a unique distribution in the primate striatum. In normal monkeys, SP-ir was observed throughout the striatum; however, striosomes were more intensely stained than matrix (Fig. 8). GPi was stained intensely, while GPe expressed minimal levels of SP (Fig. 9). SP-ir was also detected in substantia nigra (not shown). Following striatal
Fig. 2. D1 distribution in the striatum from a control monkey (A) and a lesioned monkey (B). Note the increase in D1-ir in the lesioned striatum, especially in the striosomes (arrows). D1 is evenly distributed in the control striatum. In this experiment, D1 was visualized using DAB as a chromophore. Scale bar = 250 Am.
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Fig. 4. Densitometric measurements of D1-ir in the striatum (St) and substantia nigra (SN) from control (C) and MPTP-lesioned (M) monkeys. Two sections were analyzed at the level of the striatum and substantia nigra from each of the 10 monkeys used for the study. The control data presented here are from control monkeys and the control side of unilaterally lesioned monkeys, and the MPTP data are from bilaterally lesioned as well as the lesioned side of unilaterally lesioned monkeys. D1-ir for the MPTP-treated monkeys is presented as a percentage of the control D1-ir. Each data set is represented as a scatter plot with encircled symbols representing unilateral lesioned animals. Solid lines represent mean and broken lines represent F SE. *P < 0.05.
dopamine depletion, the pattern of SP expression was ‘‘reversed’’ such that the expression was down-regulated in striosomes and up-regulated in the matrix (Figs. 8 and 10) in both unilateral and bilateral models of MPTP. The changes were more distinct in the bilateral lesioned monkeys. As a result, in MPTP-treated animals, SP-ir was
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Fig. 6. D2-ir in the normal and dopamine depleted striatum. Immunoautoradiographs demonstrate D2-ir in the striatum of a control monkey (A) and a bilaterally lesioned monkey (B). The gray levels have been converted to a pseudocolor scale as in Fig. 1. Both images were processed simultaneously and identically. D2-ir is markedly increased in the MPTP-lesioned striatum.
more intense in matrix than in striosomes. In the GPi, the expression of SP was significantly up-regulated after MPTP (Figs. 9 and 10). In SN, SP expression showed a trend towards an increase but the change was not statistically significant (Fig. 10). Enkephalin Immunonreactivity for ENK was heterogeneous in the striatum, being most concentrated in the medial caudate nucleus and rostral putamen (Fig. 11). GPe was stained intensely but GPi had very low levels of ENK-ir (Fig. 12). Nigrostriatal dopaminergic degeneration resulted in increased levels of ENK-ir; in GPe there was a tendency
Fig. 5. TH-ir and D2-ir in the striatum. (A) Macrograph shows TH-ir in the striatum of a monkey with unilateral MPTP lesion. The striatum on the lesioned side is devoid of TH-ir. (B) The same striatal section has been double labeled for D2 by immunoautoradiography. The gray levels have been converted to a pseudocolor scale as in Fig. 1. Note the increase in D2ir in the striatum on the lesion side.
Fig. 7. Densitometric measurements of D2-ir in the striatum (St) and substantia nigra (SN) from control (C) and MPTP-lesioned (M) monkeys. The D2-ir for the MPTP-treated monkeys is presented as a percentage of the control D2-ir. Each data set is represented as a scatter plot with encircled symbols representing unilateral lesioned animals. Solid lines represent mean and broken lines represent F SE. *P < 0.05.
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D1 and D2 expression in striatum
Fig. 8. TH- and SP-ir in the striatum of a control and a bilaterally MPTPlesioned monkey. (Top panel) Macrograph shows TH-ir in the striatum from a control monkey (A) and a lesioned monkey (B). Note the lesioned striatum is devoid of TH-ir. (Lower panel) The same striatal sections were double labeled for SP by immunoautoradiography. The SP distribution is altered following striatal dopamine denervation. In the control striatum (C), the striosomes (dark patches) are intensely immunoreactive for SP. In the MPTP-lesioned striatum (D) SP-ir is down-regulated in the striosomes and up-regulated in the matrix.
towards an increase in ENK expression levels in the striatum (Figs. 11, 12 and 13). The changes were more evident in the bilateral lesioned monkeys. Densitometric measurements confirmed the changes seen in the ENK distribution in the striatum and the GPe (Fig. 13).
In this study, D1-ir was uniformly distributed in the normal striatum, with striosomes being barely distinct from the matrix, unlike what was observed in human tissue (Levey et al., 1993). Following MPTP treatment, up-regulation of both D1 and D2 receptors was observed in striatum. In addition, striosomes expressed increased levels of D1 compared to matrix and could be delineated easily after MPTP. This altered striatal compartmentation of D1 receptors has not been reported previously; however, a similar change in the striatal compartmentation of the GluR1 glutamate receptor subunit has been seen after dopamine depletion in nonhuman primates (Betarbet et al., 2000). Whether there is a functional interaction between D1 and GluR1-containing receptors in the denervated striatum remains to be explored. Consistent with our measurements of dopamine receptor protein levels, numerous studies have reported increased dopamine receptor binding in MPTP-treated monkeys and cats and in parkinsonian patients (Frohna et al., 1995; Gagnon et al., 1990; Gnanalingham et al., 1993; Piggott et al., 1999; Pope-Coleman et al., 2000; Raisman et al., 1985; Rinne et al., 1985; Rioux et al., 1997). Moreover, striatal D2 binding has been reported to be increased, in unilaterally lesioned monkeys, on the denervated side vs. the control side (Graham et al., 1990; Przedborski et al., 1991). It has been suggested that up-regulation of striatal D1 and D2 dopamine receptors is a compensatory response to
Discussion For the first time, we report changes in the protein levels of D1 and D2 dopamine receptors in striatum and substantia nigra following nigrostriatal dopaminergic denervation (both unilateral and bilateral) in MPTP-treated, nonhuman primates. In addition, in this model of PD, we have simultaneously studied alterations in SP and enkephalin levels and distribution in the same animals. In this study, we used both the unilateral and bilateral MPTP models. Just as in 6-OHDA-treated rats (Breese et al., 1987; Mileson et al., 1991), there are similarities and differences between unilaterally and bilaterally lesioned MPTP-treated monkeys (Betarbet et al., 2000, 2004). In this study, the pattern of receptor and neuropeptide responses to nigrostriatal dopaminergic denervation was similar (and always in the same direction) in the two models; therefore, data from the two models were pooled for statistical analysis. The changes in dopamine receptor levels and neuropeptide expression, however, were always more pronounced in the bilaterally lesioned monkeys.
Fig. 9. TH- and SP-ir in the globus pallidus of a control and a bilaterally MPTP-lesioned monkey. (Top panel) Macrograph shows TH-ir in brain sections, at the level of globus pallidus, from a control monkey (A) and a lesioned monkey (B). Note the lesioned striatum is devoid of TH-ir. (Lower panel) The same pallidal sections were double labeled for SP by immunoautoradiography. The gray levels have been converted to a pseudocolor scale as in Fig. 1. Note the increase in SP-ir in the internal segment of the globus pallidus from MPTP-lesioned (D) vs. control (C) monkeys.
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Fig. 10. Densitometric measurements of SP-ir in the striatum (St) and in the matrix (Stm) and striosomal (Sts) compartments of the striatum, internal segment of the GP (GPi), and substantia nigra (SN) from control (C) and MPTP-lesioned (M) monkeys. The SP-ir for the MPTP-treated monkeys is presented as a percentage of the control SP-ir. Each data set is represented as a scatter plot with encircled symbols representing unilateral lesioned animals. Solid lines represent mean and broken lines represent F SE. *P < 0.05.
nigrostriatal dopamine denervation. In MPTP-treated cats, which display a typical parkinsonian syndrome that recovers 4 –6 weeks after the last MPTP administration, receptor binding was used to examine changes in DA receptors in the basal ganglia of normal, symptomatic, and recovered cats (Frohna et al., 1995). In animals that recovered, there was increased D1 binding without evidence of dopaminergic sprouting (increased 3H-mazindol binding to dopamine transporters), suggesting that receptor up-regulation was a compensatory response to striatal dopamine loss that could lead to partial behavioral recovery. A second study involved the use of GM1 ganglioside in MPTP-treated monkeys
Fig. 11. TH- and enkephalin-ir in the striatum of a control and a bilaterally MPTP-lesioned monkey. (Top panel) Macrograph shows TH-ir in the striatum from a control monkey (A) and a lesioned monkey (B). Note the lesioned striatum is devoid of TH-ir. (Lower panel) The same striatal sections were double labeled for enkephalin by immunoautoradiography. The gray levels have been converted to a pseudocolor scale as in Fig. 1. Note the increase in enkephalin-ir in the lesioned striatum (D) vs. the control (C).
(Pope-Coleman et al., 2000). MPTP administration resulted in profound decreases in mazindol binding and increased D1 and D2 receptor binding in striatum. GM1 ganglioside treatment resulted in apparent dopaminergic sprouting (increased mazindol binding) and an associated decrease in D1 binding, suggesting that dopamine receptor expression is related to striatal dopamine levels. Similarly, D2 binding decreased with GM1 treatment but did not reach statistical significance. Thus, our results and the receptor binding studies indicate that one of the initial compensatory responses to striatal dopamine deficiency is an up-regulation of dopamine receptor expression. It must be noted, however, that some studies have reported no significant alterations in dopamine receptor
Fig. 12. TH- and enkephalin-ir in the globus pallidus of a control and a bilaterally MPTP-lesioned monkey. (Top panel) Macrograph shows TH-ir in brain sections, at the level of globus pallidus, from a control (A) and a lesioned (B) monkey. Note the lesioned striatum is devoid of TH-ir. (Lower panel) The same pallidal sections were double labeled for enkephalin by immunoautoradiography. The gray levels have been converted to a pseudocolor scale as in Fig. 1. Note the increase in enkephalin-ir in the external segment of the globus pallidus from MPTP-lesioned (D) vs. control (C) monkeys.
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Fig. 13. Densitometric measurements of enkephalin-ir in the striatum (St) and external segment of globus pallidus (GPe) from control (C) and MPTPlesioned (M) monkeys. The enkephalin-ir for the MPTP-treated monkeys is presented as a percentage of the control enkephalin-ir. Each data set is represented as a scatter plot with encircled symbols representing unilateral lesioned animals. Solid lines represent mean and broken lines represent F SE. *P < 0.05.
binding following striatal dopamine depletion in mice, monkeys, or PD patients (Alexander et al., 1991; Pierot et al., 1988; Pimoule et al., 1985; Tanji et al., 1999). In addition, a decrease in striatal D1 mRNA was reported in MPTP-treated monkeys (Morissette et al., 1996). The reasons for these discrepancies are unclear, but might be related to prior exposure to dopaminergic drugs before the receptor binding studies were done. D1 and D2 expression in the substantia nigra In normal monkeys, it has been reported that D1 dopamine receptor is enriched in the neuropil of the SN pars reticulata whereas the D2 receptor is localized to the perikarya and dendrites in the pars compacta (Levey et al., 1993). These distributions might lead to the predictions that in response to degeneration of the nigrostriatal pathway, D1 expression would increase, and D2 expression would decrease. However, we found that D1 and D2 expressions did not change significantly in the SN after MPTP treatment. In this regard, our results confirm previous findings in MPTPtreated mice, cats, and primates (Frohna et al., 1995; Gnanalingham et al., 1993; Przedborski et al., 1991; Tanji et al., 1999). Whether the lack of dopamine receptor changes in SN is real, or simply reflects a lack of sensitivity in the protocols used to date (i.e., receptor binding studies and immunoautoradiographic methods), is unclear. Substance P and enkephalin In rats, changes in overall striatal levels of SP and ENK following nigrostriatal dopaminergic denervation are well established. Levels of SP, expressed in neurons of the ‘direct’ pathway that project to the internal segment of the globus pallidus (entopeduncular nucleus) and substantia nigra, are decreased. In contrast, levels of ENK, expressed in striatal neurons of in the ‘indirect’ pathway that project
to the external segment of globus pallidus, are increased (Gerfen et al., 1991). However, reports on neuropeptide distribution in Parkinson’s disease patients and from MPTP-treated monkeys have been contradictory. For this reason, we studied the distributions of SP and ENK simultaneously, along with the distributions of D1 and D2 dopamine receptors in the same animals to better understand neurochemical changes that occur in striatum following dopamine denervation. In this study, increased expression of SP and ENK was observed in the primate striatum following MPTP-induced dopamine depletion. Similarly, levels of SP and ENK, as well as their mRNAs, have been reported to be increased in the caudate and putamen of MPTP-treated monkeys (Asselin et al., 1994; Augood et al., 1989; Bezard et al., 2001; Gudehithlu et al., 1991; Roeling et al., 1995; Schroeder and Schneider, 2000; Song and Haber, 2000; Taylor et al., 1991). Likewise, in postmortem striatal tissue from PD patients, levels of both SP and ENK were increased (Goto et al., 1990; Nisbet et al., 1995). Contrary to these results, there are studies that report either a decrease or no change in levels of SP and ENK in MPTP-treated monkeys (PerezOtano et al., 1992; Taquet et al., 1988; Tenovuo et al., 1984), as well as in PD patients (De Ceballos and LopezLozano, 1999; Fernandez et al., 1996; Levy et al., 1995; Waters et al., 1988; Zech and Bogerts, 1985). Such discrepancies may arise, in part, because neuropeptide expression may change differently depending on the extent of striatal DA depletion. For example, severe striatal dopamine loss (>80%) was correlated with a 3-fold increase in SP and ENK, while mild dopamine loss (<50%) correlated with a 80% decrease in SP and ENK levels (de Ceballos et al., 1993). Our MPTP-treated monkeys had severe dopamine denervation, as evident from TH immunocytochemistry, and we observed significant increases in both SP and ENK expression in striatum. In addition to an overall up-regulation in striatal SP levels in our study, there was a reversal of the normal pattern of SP distribution in striatum following dopamine denervation. SP levels, which are normally higher in striosomes (Martin et al., 1993), were reduced in striosomes and were up-regulated in the matrix. Similar changes in striatal SP expression were reported by Lavoie et al. (1991) in MPTP-treated monkeys. The significance of this change in striatal SP distribution is unclear. In our MPTP-treated monkeys, SP expression was elevated in GPi, and ENK expression was increased in GPe. Similar changes in pallidal SP and ENK expression have been reported in PD patients (De Ceballos and LopezLozano, 1999; Grafe et al., 1985). This up-regulation of neuropeptide expression in the GP, the projection area of the striatopallidal pathway, is consistent with the anatomy of the ‘direct’ and ‘indirect’ pathways (Gerfen, 1992). No significant changes were observed in SN neuropeptide expression in our study, similar to what was seen in a study of PD patients (Fernandez et al., 1996). In contrast, increased
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(Grafe et al., 1985) and decreased (Perez-Otano et al., 1992) nigral SP levels have been reported in PD patients and in MPTP-treated marmosets, respectively. The role of SP in the basal ganglia is not well understood. In rats, changes in peptide levels correspond to changes in the metabolic activity of the ‘direct’ and ‘indirect’ pathway, as measured by 2-deoxyglucose uptake (Kozlowski and Marshall, 1980; Wooten and Collins, 1983). In our study, and those of others, the apparent accumulation of presynaptic SP in GPi and ENK in GPe (increased immunoreactivity) may reflect reduced release or turnover of neuropeptide in striatopallidal projections after nigrostriatal degeneration, but this remains to be confirmed. Our studies in nonhuman primates suggest that D1 and D2 dopamine receptors might be involved in modulating the expression of neuropeptides. For example, in mice lacking the D1A receptor, there are reduced SP mRNA levels (Drago et al., 1994). Studies have also suggested that SP may have a stimulatory influence on dopaminergic neurons. SP administered intracerebroventricularly (Krasnova et al., 2000) or directly into substantia nigra (Reid et al., 1990) increased striatal dopamine concentrations. Furthermore, electrochemical studies have shown that SP, when applied directly to striatum, can induce striatal DA release (Tang et al., 1998). Together, these studies suggest that the increased SP expression observed in the present study may be a compensatory response to striatal DA deficiency. Similarly, increased expression of ENK might be a compensatory response to striatal dopamine deficiency. Enkephalins have been shown to modulate striatal dopamine release, though in a complex manner (Algeri et al., 1978; Diamond and Borison, 1978; Walczak et al., 1979). The presence of enkephalinergic interneurons in striatum and substantia nigra (Hokfelt et al., 1977) and the fact that opiates increase brain dopamine turnover (Costa et al., 1973) provide further support for this concept. It has also been proposed that increased preproenkephalin gene expression could represent a mechanism to partially offset the overactive GABAergic input from striatum to GPe in reserpinized rats (Maneuf et al., 1994) and MPTP-treated mice (Gudehithlu et al., 1991). Since this up-regulation occurred before the appearance of parkinsonian motor disabilities, the authors suggested that increased preproenkephalin gene expression is a compensatory response to striatal dopamine depletion (Bezard et al., 2001). Implications Hornykiewicz (1966) has proposed that the asymptomatic ‘preclinical’ phase of PD might be due to compensatory changes in the basal ganglia circuitry that permit residual dopaminergic neurons to subserve functions previously carried out by the entire nigrostriatal projection. Such compensatory mechanisms may include increased transmitter release from the remaining dopaminergic terminals (Agid
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et al., 1973), increased biosynthetic enzymes for dopamine synthesis (Zigmond et al., 1984), and increased dopamine receptor and neuropeptide levels in striatal neurons. However, with further progression of striatal dopamine loss, these compensatory mechanisms become insufficient to maintain adequate dopaminergic transmission, and clinical symptoms of the disease become apparent. Thus, a full appreciation of parkinsonian pathophysiology requires not only an understanding of the normal neurochemical anatomy of the basal ganglia, but knowledge of the changes that occur in PD. A more complete delineation of neurochemical anatomy in accurate animal models of PD will enable therapeutic targets to be approached more rationally.
Acknowledgments This work was supported by U.S. Public Health Service grant NS33779. We thank Dr. Todd Sherer for a critical reading of the manuscript.
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Regulation of dopamine receptor and neuropeptide expression in the basal ganglia of monkeys treated with MPTP Ranjita Betarbet *, J. Timothy Greenamyre Department of Neurology and Center for Neurodegenerative Disease, Emory University, Atlanta, GA 30322, USA Received 22 October 2003; revised 21 May 2004; accepted 21 May 2004
Abstract In Parkinson’s disease (PD), striatal dopamine deficiency has been associated with complex changes in the functional and neurochemical anatomy of the basal ganglia. In this study, we simultaneously analyzed the regulation of D1 and D2 dopamine receptors and levels of the neuropeptides, substance P, and enkephalin (ENK) in various basal ganglia nuclei following 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine-induced dopaminergic denervation of striatum in nonhuman primates. Both unilateral and bilateral lesioned animals were used for this study. Striatal dopamine deficiency resulted in distinct alterations in D1, D2, substance P, and enkephalin levels and distribution: (1) Both D1 and D2 protein levels were significantly up-regulated in striatum. (2) There was an overall up-regulation of striatal substance P expression following dopamine denervation. (3) Substance P distribution was ‘reversed’ in dopamine depleted striatum: striosomes, which normally express higher levels of substance P, showed decreased expression, whereas substance P expression was upregulated in the matrix. (4) Substance P expression was up-regulated in the internal segment of the globus pallidus (GPi), but remained unchanged in substantia nigra (SN). (5) Enkephalin levels were increased in striatum and the external segment of the globus pallidus (GPe), but not in substantia nigra. All the changes were more pronounced in the bilateral lesioned monkeys, though the data represent a pooled statistical evaluation of unilateral and bilateral lesioned monkeys. Our studies indicate that D1 and D2 dopamine receptors and substance P and enkephalin undergo regulatory changes in response to nigrostriatal dopamine denervation. Simultaneous study of the alterations in these various components of the ‘direct’ and ‘indirect’ pathways in the same animals will enable better understanding of the pathophysiology of PD and its therapeutic targets. D 2004 Elsevier Inc. All rights reserved. Keywords: D1 dopamine receptor; D2 dopamine receptor; Substance P; Enkephalin; MPTP; Parkinson’s disease
Introduction Parkinson’s disease (PD) is a progressive neurodegenerative disease, the pathological hallmark of which is degeneration of the nigrostriatal dopaminergic pathway and subsequent dopamine deficiency in the striatum (Gerlach and Reiderer, 1993; Hornykiewicz and Kish, 1987; Wooten, 1997). The striatum is the major input nuclei of the basal ganglia (Divac et al., 1977; McGeorge and Faull, 1989). It receives glutamatergic input from the overlying cerebral cortex (Young et al., 1981) and thalamus (Beckstead, 1984; Parent et al., 1983; Royce and Mourey, 1985) and dopami* Corresponding author. Center for Neurodegenerative Diseases, Emory University, Suite 505, Whitehead Biomedical Research Building, 615 Michael Street, Atlanta, GA 30322. Fax: +1-404-727-3728. E-mail address: [email protected] (R. Betarbet). 0014-4886/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2004.05.041
nergic input from the substantia nigra pars compacta (Moore et al., 1971). The striatum is composed primarily of medium-sized spiny projection neurons (Gerfen and Wilson, 1996) that constitute approximately 95% of the striatal neuron population (Kemp and Powell, 1971). These projection neurons send GABAergic input to the basal ganglia output nuclei through two parallel ‘direct’ and ‘indirect’ pathways (Parent, 1986). Striatal neurons projecting directly to the output nuclei of the basal ganglia, the internal segment of globus pallidus (GPi; entopeduncular nucleus in rodents), and substantia nigra pars reticulata (SNr) express the D1 dopamine receptor and also co-localize the neuropeptide substance P (SP) (Gerfen et al., 1990; Yung et al., 1995). Neurons that indirectly project to the output nuclei through the external segment of the globus pallidus (GPe) and subthalamic nucleus (STN) express the D2 dopamine receptor and co-express the neuropeptide
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enkephalin (ENK) (Gerfen et al., 1990; Yung et al., 1995). Although recent studies have shown that both sets of striatal neurons express both D1 and D2 dopamine receptors, neurons of the direct pathway express significantly higher levels of D1 and very low levels of D2, whereas the neurons contributing to the indirect pathway express high levels of D2 (Aizman et al., 2000). Through the direct and indirect pathways, striatal neurons modulate the activity of the output nuclei of the basal ganglia. A family of at least five dopamine receptor genes, D1 – D5, has been identified in both rodents and humans (Niznik and Van Tol, 1992; Sibley and Monsma, 1992). Immunocytochemical studies have shown that the D1 and D2 receptors have similar regional distributions in the rat, monkey, and human brain, with highest densities in striatum and substantia nigra. Within each region, however, the precise distribution of each subtype is distinct and often complementary (Levey et al., 1993). D1 and D2 receptors are differentially enriched in the striosomes and matrix; however, in primates, the distinction between striosome and matrix is not always clear. Immunocytochemical identification of individual striatal cells expressing either protein selectively is difficult because of dense neuropil staining. As noted, striatal cells that express D1 co-localize SP. Normally, striosomes are more intensely stained for SP than matrix (Haber and Watson, 1985; Martin et al., 1991). Within the substantia nigra, D1 is enriched in the neuropil of substantia nigra pars reticulata (SNr) and D2 is enriched in perikarya and dendrites in the pars compacta. Following degeneration of the nigrostriatal pathway, numerous changes take place in basal ganglia circuitry. With loss of dopaminergic innervation and decreased activation of D1 and D2 receptors, there is disinhibition of the output nuclei via the direct pathway and increased excitatory input from the STN via the indirect pathway (Albin et al., 1989). This results in increased GABAergic inhibition of basal ganglia outflow targets, including ventrolateral thalamus and brainstem (Albin et al., 1989; Wichmann et al., 1994). Decreased thalamocortical feedback is believed to underlie many of the clinical manifestations of PD (Albin et al., 1989; Crossman, 1989; DeLong, 1990; Greenamyre, 1993; Klockgether and Turski, 1989). In the 6-hydroxydopamine (6-OHDA) model of PD, striatal neurons projecting to SNr show a reduction in the expression of mRNAs encoding the D1 dopamine receptor and SP. Conversely, striatal neurons projecting to globus pallidus show an elevation in the expression of mRNAs encoding the D2 dopamine receptor and ENK (Gerfen et al., 1990). In primates, however, D1 and D2 receptor binding studies, and studies of the distributions of SP and ENK in MPTP-treated primates and in PD patients have yielded conflicting and ambiguous results (Gnanalingham et al., 1993; Morissette et al., 1996; Piggott et al., 1999; Rioux et al., 1997). Furthermore, to date, there have been no reports of changes in D1 and D2 protein levels following striatal dopamine depletion.
In this study, we have analyzed the distributions of D1and D2 dopamine receptors and the distributions of neuropeptides SP and ENK in various basal ganglia nuclei after MPTP-induced striatal dopamine deficiency in nonhuman primates to better understand the pathophysiology and therapeutic targets of Parkinson’s disease.
Materials and methods Animals All animal use was in accordance with NIH guidelines and was approved by the Emory University IACUC. In this study, brains of 10 adult, female rhesus monkeys (Macaca mulatta), ranging in age from 7 to 14 years, were examined. Of the 10 monkeys, 4 unlesioned monkeys were used as controls. Three monkeys received unilateral infusions of MPTP (0.4 mg/kg) into the internal carotid artery to produce a stable hemiparkinsonian syndrome. The remaining three monkeys were rendered bilaterally parkinsonian by intramuscular injections of MPTP (0.5 mg/kg every 2– 5 days) over a period of 2 –6 weeks. Two of the three monkeys with intramuscular MPTP injections were part of a separate study not reported here, wherein ibotenate lesion of the right STN was attempted unsuccessfully. No clear cell loss was apparent in the STN or in the surrounding structures in either monkey. The monkeys with unilateral lesions were euthanized about 2 years after receiving MPTP; those with bilateral parkinsonism were euthanized within 2 months of their last MPTP treatment. None of the monkeys received more than two or three treatments with a dopaminergic drug and none of the animals received any pharmacological treatment within 4 weeks of euthanasia. Table 1 gives the history of each monkey. After ketamine sedation, the monkeys were given an intravenous overdose of sodium pentobarbital. The animals were intracardially perfused with 3% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2. The brains were removed and cryoprotected in a 30% sucrose solution in 0.1 M phosphate buffer. Striatum, globus pallidus (both medial and lateral segments), and substantia nigra were analyzed. Double labeling with immunoautoradiography (IAR) and immunocytochemistry (ICC) Fifty-micrometer-thick brain sections were thoroughly washed in 0.1 M TBS (Tris-buffered saline) to remove the cryoprotectant and incubated in blocking serum consisting of 4% normal goat serum and 4% normal human serum with 0.4% Triton X-100 in TBS for 30 min. The sections were then incubated for 72 h in the same solution containing a combination of primary antibodies. The antibodies used for this study were against tyrosine hydroxylase (TH; 1:1000, mouse monoclonal antibody from Chemicon, CA, and 1:500, rabbit polyclonal antibody from Pelfreez, Rogers, AR); D1 and D2 dopamine receptors, (D1, 1:1000; D2,
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Table 1 History of each monkey used in this study Monkey
Treatment before MPTP
MPTP treatment
Sacrificed
Drugs
Bilateral 1
failed STN lesion with RF probe (no lesion) failed STN lesion with ibotenate
0.5 mg/kg 2 – 5 days for 6 weeks 0.5 mg/kg 2 – 5 days for 6 weeks 0.5 mg/kg 2 – 5 days for 6 weeks 0.4 mg/kg into internal carotid artery 0.4 mg/kg into internal carotid artery 0.4 mg/kg into internal carotid artery
55 days after last MPTP
no drugs
74 days after last MPTP
Sinemet 125 mg, po, for 1 week
70 days after last MPTP
Sinemet 125 mg, po, for 1 week; glutamate antagonist for first month
f2 years after last MPTP f2 years after last MPTP
glutamate antagonist for 18 months after MPTP no drugs
f2 years after last MPTP
no drugs
Bilateral 2
Bilateral 3
none
Unilateral 1
none
Unilateral 2
none
Unilateral 3
none
1:100; Levey et al., 1993) and neuropeptides, SP and methionine-enkephalin (SP and ENK; both used at 1:10,000, Diasorin, MN). The antibodies used in combination were from different hosts to avoid cross-reactivity. Following primary antibody incubation, sections were rinsed in 0.1 M TBS and incubated for 1 – 2 h in the blocking serum containing sheep anti-rat (for D1) or goat anti-rabbit (for D2, SP and ENK) antibody conjugated to 125 I and biotinylated goat anti-mouse or anti-rabbit (for TH). Immunostaining for TH and D1 (regular ICC to visualize the striosomes) was visualized using the avidin – biotin complex (ABC) method (ABC Elite Kit, Vector Laboratories, Burlingame, CA) and 3,3V-diaminobenzidine tetrachloride (DAB, Sigma, St. Louis, MO) was used to visualize the final product. For control sections, one or both the primary antibodies were omitted. Finally, the sections were rinsed in 0.1 M TBS, mounted on gelatin-coated slides and air-dried. The slides were placed in X-ray cassettes with calibrated 125 I standards (Amersham, Piscataway, NJ) and apposed to Hyperfilm3H (Amersham) for 2 –3 weeks. The films were developed in D-19, fixed in Kodak fixer, and dried. The slides were then dehydrated and coverslipped using Shandon mount (Pittsburgh, PA). The immunostained sections were examined using brightfield microscopy. Images were collected on a Leitz microscope linked to an MCID image analysis system (Imaging Research, St. Catharines, Ontario, Canada). For final output, images were composed using Adobe Photoshop. Quantitation For densitometric measurements of D1, D2, SP, and ENK-immunoreactivity (-ir), the autoradiograms were analyzed with a video-based MCID image analysis system (Imaging Research) by relating optical density to calibrated 125 I standards. Two sections were analyzed through the striatum, globus pallidus, and substantia nigra from each
of the 10 monkeys used for the study; a total of 80 sections were analyzed. The striatal sections selected for all analyses were from levels anterior to or at the level of the anterior commissure. The sections from control, unilateral, and bilateral lesioned brains were anatomically matched. For comparison purposes, data from the control or untreated side of the unilaterally lesioned monkeys were grouped together with the data from untreated control monkeys and the data from the lesioned side of unilaterally treated monkeys were grouped along with the bilaterally lesioned, MPTP-treated monkeys. Statistics Statistical analysis was performed using two-way ANOVA with Bonferroni’s post hoc correction to compare control values vs. values following MPTP treatment. A P value less than 0.05 was accepted as indicating a significant difference. Each data set for densitometric measurements is represented as a scatter plot with mean and standard error.
Results D1 dopamine receptor In normal monkeys D 1 was expressed uniformly throughout the striatum (Fig. 1). Striosomes and matrix were not distinctly demarcated, unlike what has been reported in humans (Levey et al., 1993). In the substantia nigra (SN), D1 was enriched in the pars reticulata region (Fig. 3). Following MPTP treatment, the striatum and SN were devoid of TH immunoreactivity (ir) (Figs. 1, 3, 5, 8, 9, 11, and 12) indicating degeneration of the nigrostriatal dopaminergic pathway. The distribution and density of D1 was a markedly increased following MPTP-induced striatal
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Fig. 3. TH- and D1-ir in the substantia nigra of a control monkey and a bilaterally MPTP-lesioned monkey. (Top panel) Macrograph shows TH-ir in the substantia nigra from a control monkey (A) and a lesioned monkey (B). Arrows indicate SNc. (Lower panel) The same nigral sections were double labeled for D1 by immunoautoradiography. The gray levels have been converted to a pseudocolor scale as in Fig. 1. Arrowheads indicate SN. Note the increase in D1-ir in the lesioned nigra (D) vs. the control (C). P, putamen.
Fig. 1. TH- and D1-ir in the striatum of a control monkey and a bilaterally MPTP-lesioned monkey. (Top panel) Macrograph shows TH-ir in the striatum from a control monkey (A) and a lesioned monkey (B). Note the lesioned striatum is devoid of TH-ir. (Lower panel) The same striatal sections were double labeled for D1 by immunoautoradiography. The gray levels have been converted to a pseudocolor scale wherein red depicts high immunoreactivity and blue depicts low immunoreactivity. Note the increase in D1-ir in the lesioned striatum (D) vs. the control (C).
dopamine depletion (Fig. 1); however, the changes were very pronounced in the bilateral MPTP-lesioned monkeys compared to unilateral lesioned ones. D1 up-regulation was more prominent in striosomes than in matrix such that intensely immunoreactive striosomes could be identified easily following striatal dopamine denervation (Fig. 2). Quantitative densitometric measurements of immunoautoradiographs indicated a significant increase in D1 expression following systemic MPTP treatment (Fig. 4). There was also a trend for up-regulation of D1 expression in the SN (Fig. 3) but this was not statistically significant (Fig. 4).
D2 dopamine receptor The distribution of D2-ir was similar to D1 in the striatum (Fig. 5). In the SN, D2 was enriched in the pars compacta region (not shown). Following MPTP, D2 expression was up-regulated in the striatum of both unilaterally and bilaterally lesioned monkeys (Figs. 5 and 6). The changes were more pronounced in the bilaterally lesioned animals. Densitometric measurements of autoradiographs showed that the increase in striatal D2 expression was statistically significant (Fig. 7). No change in D2 expression was detected in the SN (Fig. 7). Substance P SP has a unique distribution in the primate striatum. In normal monkeys, SP-ir was observed throughout the striatum; however, striosomes were more intensely stained than matrix (Fig. 8). GPi was stained intensely, while GPe expressed minimal levels of SP (Fig. 9). SP-ir was also detected in substantia nigra (not shown). Following striatal
Fig. 2. D1 distribution in the striatum from a control monkey (A) and a lesioned monkey (B). Note the increase in D1-ir in the lesioned striatum, especially in the striosomes (arrows). D1 is evenly distributed in the control striatum. In this experiment, D1 was visualized using DAB as a chromophore. Scale bar = 250 Am.
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Fig. 4. Densitometric measurements of D1-ir in the striatum (St) and substantia nigra (SN) from control (C) and MPTP-lesioned (M) monkeys. Two sections were analyzed at the level of the striatum and substantia nigra from each of the 10 monkeys used for the study. The control data presented here are from control monkeys and the control side of unilaterally lesioned monkeys, and the MPTP data are from bilaterally lesioned as well as the lesioned side of unilaterally lesioned monkeys. D1-ir for the MPTP-treated monkeys is presented as a percentage of the control D1-ir. Each data set is represented as a scatter plot with encircled symbols representing unilateral lesioned animals. Solid lines represent mean and broken lines represent F SE. *P < 0.05.
dopamine depletion, the pattern of SP expression was ‘‘reversed’’ such that the expression was down-regulated in striosomes and up-regulated in the matrix (Figs. 8 and 10) in both unilateral and bilateral models of MPTP. The changes were more distinct in the bilateral lesioned monkeys. As a result, in MPTP-treated animals, SP-ir was
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Fig. 6. D2-ir in the normal and dopamine depleted striatum. Immunoautoradiographs demonstrate D2-ir in the striatum of a control monkey (A) and a bilaterally lesioned monkey (B). The gray levels have been converted to a pseudocolor scale as in Fig. 1. Both images were processed simultaneously and identically. D2-ir is markedly increased in the MPTP-lesioned striatum.
more intense in matrix than in striosomes. In the GPi, the expression of SP was significantly up-regulated after MPTP (Figs. 9 and 10). In SN, SP expression showed a trend towards an increase but the change was not statistically significant (Fig. 10). Enkephalin Immunonreactivity for ENK was heterogeneous in the striatum, being most concentrated in the medial caudate nucleus and rostral putamen (Fig. 11). GPe was stained intensely but GPi had very low levels of ENK-ir (Fig. 12). Nigrostriatal dopaminergic degeneration resulted in increased levels of ENK-ir; in GPe there was a tendency
Fig. 5. TH-ir and D2-ir in the striatum. (A) Macrograph shows TH-ir in the striatum of a monkey with unilateral MPTP lesion. The striatum on the lesioned side is devoid of TH-ir. (B) The same striatal section has been double labeled for D2 by immunoautoradiography. The gray levels have been converted to a pseudocolor scale as in Fig. 1. Note the increase in D2ir in the striatum on the lesion side.
Fig. 7. Densitometric measurements of D2-ir in the striatum (St) and substantia nigra (SN) from control (C) and MPTP-lesioned (M) monkeys. The D2-ir for the MPTP-treated monkeys is presented as a percentage of the control D2-ir. Each data set is represented as a scatter plot with encircled symbols representing unilateral lesioned animals. Solid lines represent mean and broken lines represent F SE. *P < 0.05.
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D1 and D2 expression in striatum
Fig. 8. TH- and SP-ir in the striatum of a control and a bilaterally MPTPlesioned monkey. (Top panel) Macrograph shows TH-ir in the striatum from a control monkey (A) and a lesioned monkey (B). Note the lesioned striatum is devoid of TH-ir. (Lower panel) The same striatal sections were double labeled for SP by immunoautoradiography. The SP distribution is altered following striatal dopamine denervation. In the control striatum (C), the striosomes (dark patches) are intensely immunoreactive for SP. In the MPTP-lesioned striatum (D) SP-ir is down-regulated in the striosomes and up-regulated in the matrix.
towards an increase in ENK expression levels in the striatum (Figs. 11, 12 and 13). The changes were more evident in the bilateral lesioned monkeys. Densitometric measurements confirmed the changes seen in the ENK distribution in the striatum and the GPe (Fig. 13).
In this study, D1-ir was uniformly distributed in the normal striatum, with striosomes being barely distinct from the matrix, unlike what was observed in human tissue (Levey et al., 1993). Following MPTP treatment, up-regulation of both D1 and D2 receptors was observed in striatum. In addition, striosomes expressed increased levels of D1 compared to matrix and could be delineated easily after MPTP. This altered striatal compartmentation of D1 receptors has not been reported previously; however, a similar change in the striatal compartmentation of the GluR1 glutamate receptor subunit has been seen after dopamine depletion in nonhuman primates (Betarbet et al., 2000). Whether there is a functional interaction between D1 and GluR1-containing receptors in the denervated striatum remains to be explored. Consistent with our measurements of dopamine receptor protein levels, numerous studies have reported increased dopamine receptor binding in MPTP-treated monkeys and cats and in parkinsonian patients (Frohna et al., 1995; Gagnon et al., 1990; Gnanalingham et al., 1993; Piggott et al., 1999; Pope-Coleman et al., 2000; Raisman et al., 1985; Rinne et al., 1985; Rioux et al., 1997). Moreover, striatal D2 binding has been reported to be increased, in unilaterally lesioned monkeys, on the denervated side vs. the control side (Graham et al., 1990; Przedborski et al., 1991). It has been suggested that up-regulation of striatal D1 and D2 dopamine receptors is a compensatory response to
Discussion For the first time, we report changes in the protein levels of D1 and D2 dopamine receptors in striatum and substantia nigra following nigrostriatal dopaminergic denervation (both unilateral and bilateral) in MPTP-treated, nonhuman primates. In addition, in this model of PD, we have simultaneously studied alterations in SP and enkephalin levels and distribution in the same animals. In this study, we used both the unilateral and bilateral MPTP models. Just as in 6-OHDA-treated rats (Breese et al., 1987; Mileson et al., 1991), there are similarities and differences between unilaterally and bilaterally lesioned MPTP-treated monkeys (Betarbet et al., 2000, 2004). In this study, the pattern of receptor and neuropeptide responses to nigrostriatal dopaminergic denervation was similar (and always in the same direction) in the two models; therefore, data from the two models were pooled for statistical analysis. The changes in dopamine receptor levels and neuropeptide expression, however, were always more pronounced in the bilaterally lesioned monkeys.
Fig. 9. TH- and SP-ir in the globus pallidus of a control and a bilaterally MPTP-lesioned monkey. (Top panel) Macrograph shows TH-ir in brain sections, at the level of globus pallidus, from a control monkey (A) and a lesioned monkey (B). Note the lesioned striatum is devoid of TH-ir. (Lower panel) The same pallidal sections were double labeled for SP by immunoautoradiography. The gray levels have been converted to a pseudocolor scale as in Fig. 1. Note the increase in SP-ir in the internal segment of the globus pallidus from MPTP-lesioned (D) vs. control (C) monkeys.
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Fig. 10. Densitometric measurements of SP-ir in the striatum (St) and in the matrix (Stm) and striosomal (Sts) compartments of the striatum, internal segment of the GP (GPi), and substantia nigra (SN) from control (C) and MPTP-lesioned (M) monkeys. The SP-ir for the MPTP-treated monkeys is presented as a percentage of the control SP-ir. Each data set is represented as a scatter plot with encircled symbols representing unilateral lesioned animals. Solid lines represent mean and broken lines represent F SE. *P < 0.05.
nigrostriatal dopamine denervation. In MPTP-treated cats, which display a typical parkinsonian syndrome that recovers 4 –6 weeks after the last MPTP administration, receptor binding was used to examine changes in DA receptors in the basal ganglia of normal, symptomatic, and recovered cats (Frohna et al., 1995). In animals that recovered, there was increased D1 binding without evidence of dopaminergic sprouting (increased 3H-mazindol binding to dopamine transporters), suggesting that receptor up-regulation was a compensatory response to striatal dopamine loss that could lead to partial behavioral recovery. A second study involved the use of GM1 ganglioside in MPTP-treated monkeys
Fig. 11. TH- and enkephalin-ir in the striatum of a control and a bilaterally MPTP-lesioned monkey. (Top panel) Macrograph shows TH-ir in the striatum from a control monkey (A) and a lesioned monkey (B). Note the lesioned striatum is devoid of TH-ir. (Lower panel) The same striatal sections were double labeled for enkephalin by immunoautoradiography. The gray levels have been converted to a pseudocolor scale as in Fig. 1. Note the increase in enkephalin-ir in the lesioned striatum (D) vs. the control (C).
(Pope-Coleman et al., 2000). MPTP administration resulted in profound decreases in mazindol binding and increased D1 and D2 receptor binding in striatum. GM1 ganglioside treatment resulted in apparent dopaminergic sprouting (increased mazindol binding) and an associated decrease in D1 binding, suggesting that dopamine receptor expression is related to striatal dopamine levels. Similarly, D2 binding decreased with GM1 treatment but did not reach statistical significance. Thus, our results and the receptor binding studies indicate that one of the initial compensatory responses to striatal dopamine deficiency is an up-regulation of dopamine receptor expression. It must be noted, however, that some studies have reported no significant alterations in dopamine receptor
Fig. 12. TH- and enkephalin-ir in the globus pallidus of a control and a bilaterally MPTP-lesioned monkey. (Top panel) Macrograph shows TH-ir in brain sections, at the level of globus pallidus, from a control (A) and a lesioned (B) monkey. Note the lesioned striatum is devoid of TH-ir. (Lower panel) The same pallidal sections were double labeled for enkephalin by immunoautoradiography. The gray levels have been converted to a pseudocolor scale as in Fig. 1. Note the increase in enkephalin-ir in the external segment of the globus pallidus from MPTP-lesioned (D) vs. control (C) monkeys.
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Fig. 13. Densitometric measurements of enkephalin-ir in the striatum (St) and external segment of globus pallidus (GPe) from control (C) and MPTPlesioned (M) monkeys. The enkephalin-ir for the MPTP-treated monkeys is presented as a percentage of the control enkephalin-ir. Each data set is represented as a scatter plot with encircled symbols representing unilateral lesioned animals. Solid lines represent mean and broken lines represent F SE. *P < 0.05.
binding following striatal dopamine depletion in mice, monkeys, or PD patients (Alexander et al., 1991; Pierot et al., 1988; Pimoule et al., 1985; Tanji et al., 1999). In addition, a decrease in striatal D1 mRNA was reported in MPTP-treated monkeys (Morissette et al., 1996). The reasons for these discrepancies are unclear, but might be related to prior exposure to dopaminergic drugs before the receptor binding studies were done. D1 and D2 expression in the substantia nigra In normal monkeys, it has been reported that D1 dopamine receptor is enriched in the neuropil of the SN pars reticulata whereas the D2 receptor is localized to the perikarya and dendrites in the pars compacta (Levey et al., 1993). These distributions might lead to the predictions that in response to degeneration of the nigrostriatal pathway, D1 expression would increase, and D2 expression would decrease. However, we found that D1 and D2 expressions did not change significantly in the SN after MPTP treatment. In this regard, our results confirm previous findings in MPTPtreated mice, cats, and primates (Frohna et al., 1995; Gnanalingham et al., 1993; Przedborski et al., 1991; Tanji et al., 1999). Whether the lack of dopamine receptor changes in SN is real, or simply reflects a lack of sensitivity in the protocols used to date (i.e., receptor binding studies and immunoautoradiographic methods), is unclear. Substance P and enkephalin In rats, changes in overall striatal levels of SP and ENK following nigrostriatal dopaminergic denervation are well established. Levels of SP, expressed in neurons of the ‘direct’ pathway that project to the internal segment of the globus pallidus (entopeduncular nucleus) and substantia nigra, are decreased. In contrast, levels of ENK, expressed in striatal neurons of in the ‘indirect’ pathway that project
to the external segment of globus pallidus, are increased (Gerfen et al., 1991). However, reports on neuropeptide distribution in Parkinson’s disease patients and from MPTP-treated monkeys have been contradictory. For this reason, we studied the distributions of SP and ENK simultaneously, along with the distributions of D1 and D2 dopamine receptors in the same animals to better understand neurochemical changes that occur in striatum following dopamine denervation. In this study, increased expression of SP and ENK was observed in the primate striatum following MPTP-induced dopamine depletion. Similarly, levels of SP and ENK, as well as their mRNAs, have been reported to be increased in the caudate and putamen of MPTP-treated monkeys (Asselin et al., 1994; Augood et al., 1989; Bezard et al., 2001; Gudehithlu et al., 1991; Roeling et al., 1995; Schroeder and Schneider, 2000; Song and Haber, 2000; Taylor et al., 1991). Likewise, in postmortem striatal tissue from PD patients, levels of both SP and ENK were increased (Goto et al., 1990; Nisbet et al., 1995). Contrary to these results, there are studies that report either a decrease or no change in levels of SP and ENK in MPTP-treated monkeys (PerezOtano et al., 1992; Taquet et al., 1988; Tenovuo et al., 1984), as well as in PD patients (De Ceballos and LopezLozano, 1999; Fernandez et al., 1996; Levy et al., 1995; Waters et al., 1988; Zech and Bogerts, 1985). Such discrepancies may arise, in part, because neuropeptide expression may change differently depending on the extent of striatal DA depletion. For example, severe striatal dopamine loss (>80%) was correlated with a 3-fold increase in SP and ENK, while mild dopamine loss (<50%) correlated with a 80% decrease in SP and ENK levels (de Ceballos et al., 1993). Our MPTP-treated monkeys had severe dopamine denervation, as evident from TH immunocytochemistry, and we observed significant increases in both SP and ENK expression in striatum. In addition to an overall up-regulation in striatal SP levels in our study, there was a reversal of the normal pattern of SP distribution in striatum following dopamine denervation. SP levels, which are normally higher in striosomes (Martin et al., 1993), were reduced in striosomes and were up-regulated in the matrix. Similar changes in striatal SP expression were reported by Lavoie et al. (1991) in MPTP-treated monkeys. The significance of this change in striatal SP distribution is unclear. In our MPTP-treated monkeys, SP expression was elevated in GPi, and ENK expression was increased in GPe. Similar changes in pallidal SP and ENK expression have been reported in PD patients (De Ceballos and LopezLozano, 1999; Grafe et al., 1985). This up-regulation of neuropeptide expression in the GP, the projection area of the striatopallidal pathway, is consistent with the anatomy of the ‘direct’ and ‘indirect’ pathways (Gerfen, 1992). No significant changes were observed in SN neuropeptide expression in our study, similar to what was seen in a study of PD patients (Fernandez et al., 1996). In contrast, increased
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(Grafe et al., 1985) and decreased (Perez-Otano et al., 1992) nigral SP levels have been reported in PD patients and in MPTP-treated marmosets, respectively. The role of SP in the basal ganglia is not well understood. In rats, changes in peptide levels correspond to changes in the metabolic activity of the ‘direct’ and ‘indirect’ pathway, as measured by 2-deoxyglucose uptake (Kozlowski and Marshall, 1980; Wooten and Collins, 1983). In our study, and those of others, the apparent accumulation of presynaptic SP in GPi and ENK in GPe (increased immunoreactivity) may reflect reduced release or turnover of neuropeptide in striatopallidal projections after nigrostriatal degeneration, but this remains to be confirmed. Our studies in nonhuman primates suggest that D1 and D2 dopamine receptors might be involved in modulating the expression of neuropeptides. For example, in mice lacking the D1A receptor, there are reduced SP mRNA levels (Drago et al., 1994). Studies have also suggested that SP may have a stimulatory influence on dopaminergic neurons. SP administered intracerebroventricularly (Krasnova et al., 2000) or directly into substantia nigra (Reid et al., 1990) increased striatal dopamine concentrations. Furthermore, electrochemical studies have shown that SP, when applied directly to striatum, can induce striatal DA release (Tang et al., 1998). Together, these studies suggest that the increased SP expression observed in the present study may be a compensatory response to striatal DA deficiency. Similarly, increased expression of ENK might be a compensatory response to striatal dopamine deficiency. Enkephalins have been shown to modulate striatal dopamine release, though in a complex manner (Algeri et al., 1978; Diamond and Borison, 1978; Walczak et al., 1979). The presence of enkephalinergic interneurons in striatum and substantia nigra (Hokfelt et al., 1977) and the fact that opiates increase brain dopamine turnover (Costa et al., 1973) provide further support for this concept. It has also been proposed that increased preproenkephalin gene expression could represent a mechanism to partially offset the overactive GABAergic input from striatum to GPe in reserpinized rats (Maneuf et al., 1994) and MPTP-treated mice (Gudehithlu et al., 1991). Since this up-regulation occurred before the appearance of parkinsonian motor disabilities, the authors suggested that increased preproenkephalin gene expression is a compensatory response to striatal dopamine depletion (Bezard et al., 2001). Implications Hornykiewicz (1966) has proposed that the asymptomatic ‘preclinical’ phase of PD might be due to compensatory changes in the basal ganglia circuitry that permit residual dopaminergic neurons to subserve functions previously carried out by the entire nigrostriatal projection. Such compensatory mechanisms may include increased transmitter release from the remaining dopaminergic terminals (Agid
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et al., 1973), increased biosynthetic enzymes for dopamine synthesis (Zigmond et al., 1984), and increased dopamine receptor and neuropeptide levels in striatal neurons. However, with further progression of striatal dopamine loss, these compensatory mechanisms become insufficient to maintain adequate dopaminergic transmission, and clinical symptoms of the disease become apparent. Thus, a full appreciation of parkinsonian pathophysiology requires not only an understanding of the normal neurochemical anatomy of the basal ganglia, but knowledge of the changes that occur in PD. A more complete delineation of neurochemical anatomy in accurate animal models of PD will enable therapeutic targets to be approached more rationally.
Acknowledgments This work was supported by U.S. Public Health Service grant NS33779. We thank Dr. Todd Sherer for a critical reading of the manuscript.
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