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Coordinated Expression of Dopamine Receptors in Neostriatal Medium Spiny Neurons
D. JamesSurmeier, Zhen Yan, and Wen-Jie-Song Department of Anatomy and Neurobiology University of Tennessee Memphis, Tennessee 38163
Coordinated Expression of Dopamine Receptors in Neostriatal Medium Spiny Neurons A clear picture of how dopamine (DA) receptor subtypes are distributed among the principal neurons of the neostriatum is of obvious importance to understanding how drugs like cocaine and amphetamine influence basal ganglia function. In recent years, this distribution has been the subject of debate (1, 2). For example, conventional anatomical and physiological approaches have yielded starkly different estimates of the extent to which D1- and D2-class DA receptors are colocalized. One plausible explanation for the discrepancy is that some DA receptors are present in physiologically significant numbers, but the mRNA for these receptors is not detectable with conventional techniques. To test this hypothesis, the expression of DA receptors in individual neostriatal neurons was examined using patch-clamp and single-cell reverse transcriptionpolymerase chain reaction (RT-PCR) techniques (3). As a first step in the study of cellular localization of DA receptor mRNAs, tissue expression was examined. Conventional RT-PCR analysis of dorsal neostriatal mRNA isolated from coronal slices revealed that all five receptor mRNAs were present, and the expression of D3, D4, and D l b mRNAs was not a consequence of de novo transcription. To determine the molecular identity of the receptors mediating the responses to the dopaminergic ligands in individual neurons, single-cell RT-PCR techniques were used. Neurons were initially divided into three groups on the basis of enkephalin (ENK) and substance P (SP) mRNA expression. The expression of these releasable peptides is strongly correlated with the axonal projection pattern of medium spiny neurons (1). An obvious problem in attempting to determine how the expression of several mRNAs is coordinated in the same cell is that low-abundance mRNAs may be inadvertently missed when the products from a single cell are broken up into different reaction vessels. To address this issue, PCR analysis of DAreceptor mRNAs used either a small fraction of the total cellular cDNA ( M O ) , an intermediate level (one-fourth), or the product of a preamplification stage using multiplex amplification of all five DA-receptor mRNAs (3). Medium spiny neurons having detectable levels of ENK but not SP mRNA (ENK+/SP- ) expressed high levels of D2-receptor mRNA (long splice variant). I020
Advances m Pharmacology. Volume 42 Copyright 0 1998 by Academic Press. All rights of reproduction in any form reserved. 1054-3589/98 $25.00
Expression of DA Receptors in Neostriatal Medium Spiny Neurons
102 I
With a 1/10 aliquot, D2 cDNA was detected in nearly 90% of this group. The other DA-receptor mRNAs were rarely detected with this small aliquot. As the fraction of the total cellular cDNA used in the PCR reactions increased, other DA-receptor mRNAs emerged. Doubling the template concentration for Dla, Dlb, D3, and D4 reactions led to a modest increase in the detection frequency of D l b mRNA (to 20% of the sample). The short isoform of the D2 mRNA was consistently seen in this condition. Using a two-stage multiplex procedure to maximize the detectability of low-abundance mRNAs increased the detection rate of D3 and D4 mRNAs primarily. An example of an ENK+/SP- cell in which D2, D3, and D4 mRNAs were seen is shown in Figure 1C. The data for ENK+/SP- neurons is summarized in Figure 1D. In this panel (and in subsequent summary diagrams), the extent to which particular mRNAs were found together is coded by the extent to which their lanes are shaded at similar points along the abscissa. The failure to detect D l b mRNA in as large a subject of neurons after multiplex amplification as after conventional PCR (OYOvs 20%) probably reflects sampling variation. Medium spiny neurons having detectable levels of SP but not ENK mRNA (ENK-/SP+) expressed high levels of Dla-receptor rnRNA. With 1/10 aliquots, D l a mRNA was detected in 80% of the sample. D3 mRNA was detected in half of this sample, but the other receptor mRNAs were either not seen or seen rarely. Doubling the aliquot size increased the D l a detection rate to 100% and did not change the detection rate for the D3 mRNA. Using the multiplex protocol, the detection frequencies for D l a and D3 mRNAs were similar to those seen with smaller aliquots. However, the detection frequency of D4 mRNA increased to 25% (as seen with one-fifth aliquots) (Figs. lA, B). In addition, D2 mRNA, which had not been seen with 1/10 or one-fifth aliquots, was seen in about 20% of the sample, suggesting that D2 mRNAs were of relatively low abundance in this group. Neurons coexpressing detectable levels of SP and ENK (ENK+/SP+) consistently coexpressed D l a and D2 mRNAs. This group comprised about 20% of our total population of neurons. With 1/10 aliquots, D2 mRNA was the most commonly detected in this sample (78%). Dla-receptor amplicons were seen less frequently, and both amplicons were detected in about one-fifth of the sample. The other receptor mRNAs were either not detected or seen rarely with the small aliquot. Doubling the aliquot, did not change the D l a and D2 detection frequencies significantly. However, using the multiplex protocol, the detection rate for D l a mRNA increased to 90% (Fig. 1E). As a consequence, D l a and D2 mRNAs were detected in 70% of this group (Figs. lE, F). The detection frequency of D3, D4, and D l b mRNAs also increased with the size of the aliquot used, suggesting that they were expressed at relatively low levels in 20-30% of ENK+/SP+ neurons. In an attempt to determine whether these transcripts could give rise to functional protein, whole-cell recording was combined with conventional single-cell RT-PCR. Several neurons were found to express detectable levels of D2 and D1 b mRNA, but not D l a mRNA; these cells consistently had robust responses to D1-class agonists. As shown, in other cells, D3 or D4 mRNA was detected but not D2 mRNA; these cells consistently had responses to D2-class agonists. While these results do not prove that D3-, D4-, and Dlb-receptor
1022
D. James Surmeier et a/.
B
100% (n=16)
D
100% (n=9)
I
11%
D3
ENK SP D,,
D,
D3 D4 D,
D4
F
100% (n=lO)
FIGURE I Single-cell RT-PCR revealed that neostriatal neurons coexpress mRNAs for several dopamine receptors. (A) Photograph of a gel containing amplicons from an SP+ENKmedium spiny neuron in which a multiplex procedure employing three-fourths of the total cellular cDNA was used for detection of DA-receptor mRNAs. Note that D l a and D3 and D4 amplicons were detected. ( B ) Summary of coexpression detected with the multiplex procedure in 16 ENK-/SP+ neurons (C) Photograph of a gel containing amplicons from an SP-ENK+ medium spiny neuron in which a multiplex procedure was employed as before. ( D )Summary of coexpression detected with the multiplex procedure in nine ENK+/SP- neurons. ( E ) Photograph of a gel containing amplicons from an SP+iENK+ medium spiny neuron in which the multiplex procedure was employed. (F)Summary of coexpression detected with the multiplex procedure in 10ENK+/SP+ neurons. (Reprinted with permission from TheJoumalofNeuroscience 16, 6579-6591, 1996.)
Expression of DA Receptors in Neostriatal Medium Spiny Neurons
I023
mRNAs invariably give rise to functional protein, they do suggest that these mRNAs are capable of being translated and properly processed. The principal goal of this study was to understand why anatomical and functional studies of DA-receptor localization in the neostriatum have been so discordant (1,2). It was our working hypothesis that at the heart of the dispute was the assumption that only D l a and D2 receptors were functionally significant in the workings of the dorsal neostriatum. This inference allowed the equation of responsiveness to D1-class agonists with the presence of D l a receptors and responsiveness to D2-class agonists with the presence of D2 receptors. Our results argue that, in fact, this assumption is incorrect. Significant levels of D3-, D4-, and Dlb-receptor mRNAs are present in medium spiny neurons. The demonstration that D1- and D2-class receptors are coexpressed by a significant percentage of medium spiny neurons, particularly those within the striatonigral pathway, establishes a cellular and molecular foundation for the well-known physiological and biochemical responses of these neurons to D1and D2-class agonists. Our results also show that single-cell RT-PCR techniques yield a picture compatible with that derived from in situ hybridization if attention is restricted to only the most abundant receptor mRNAs. Because of their ability to evoke functionally significant physiological effects, the impact of the lower abundance DA receptors (D3, D4, D l b ) must be factored into models of drug action within the neostriatum.
References 1. Gerfen, C. R. (1992).The neostriatal mosaic: Multiple levels of compartmental organization. TINS 15, 133-139. 2. Surmeier, D. J., Reiner, A., Levine, M. S., and Ariano, M. A. (1993). Are neostriatal dopamine receptors co-localized? TINS 16, 299-305. 3. Sibley, D. R. (1995). Molecular biology of dopamine receptors. In Cellular and Molecular Mechanisms of the Neostriatum. (D. J. Surmeier, and M. A. Ariano, eds.), R. G. Landes, Austin, TX. 4. Surmeier, D. J., Song, W-J., and Yan, Z. (1997). Coordinated expression of dopamine receptors in neostriatal medium spiny neurons. J. Neurosci. 16, 6579-6591.
Coordinated Expression of Dopamine Receptors in Neostriatal Medium Spiny Neurons A clear picture of how dopamine (DA) receptor subtypes are distributed among the principal neurons of the neostriatum is of obvious importance to understanding how drugs like cocaine and amphetamine influence basal ganglia function. In recent years, this distribution has been the subject of debate (1, 2). For example, conventional anatomical and physiological approaches have yielded starkly different estimates of the extent to which D1- and D2-class DA receptors are colocalized. One plausible explanation for the discrepancy is that some DA receptors are present in physiologically significant numbers, but the mRNA for these receptors is not detectable with conventional techniques. To test this hypothesis, the expression of DA receptors in individual neostriatal neurons was examined using patch-clamp and single-cell reverse transcriptionpolymerase chain reaction (RT-PCR) techniques (3). As a first step in the study of cellular localization of DA receptor mRNAs, tissue expression was examined. Conventional RT-PCR analysis of dorsal neostriatal mRNA isolated from coronal slices revealed that all five receptor mRNAs were present, and the expression of D3, D4, and D l b mRNAs was not a consequence of de novo transcription. To determine the molecular identity of the receptors mediating the responses to the dopaminergic ligands in individual neurons, single-cell RT-PCR techniques were used. Neurons were initially divided into three groups on the basis of enkephalin (ENK) and substance P (SP) mRNA expression. The expression of these releasable peptides is strongly correlated with the axonal projection pattern of medium spiny neurons (1). An obvious problem in attempting to determine how the expression of several mRNAs is coordinated in the same cell is that low-abundance mRNAs may be inadvertently missed when the products from a single cell are broken up into different reaction vessels. To address this issue, PCR analysis of DAreceptor mRNAs used either a small fraction of the total cellular cDNA ( M O ) , an intermediate level (one-fourth), or the product of a preamplification stage using multiplex amplification of all five DA-receptor mRNAs (3). Medium spiny neurons having detectable levels of ENK but not SP mRNA (ENK+/SP- ) expressed high levels of D2-receptor mRNA (long splice variant). I020
Advances m Pharmacology. Volume 42 Copyright 0 1998 by Academic Press. All rights of reproduction in any form reserved. 1054-3589/98 $25.00
Expression of DA Receptors in Neostriatal Medium Spiny Neurons
102 I
With a 1/10 aliquot, D2 cDNA was detected in nearly 90% of this group. The other DA-receptor mRNAs were rarely detected with this small aliquot. As the fraction of the total cellular cDNA used in the PCR reactions increased, other DA-receptor mRNAs emerged. Doubling the template concentration for Dla, Dlb, D3, and D4 reactions led to a modest increase in the detection frequency of D l b mRNA (to 20% of the sample). The short isoform of the D2 mRNA was consistently seen in this condition. Using a two-stage multiplex procedure to maximize the detectability of low-abundance mRNAs increased the detection rate of D3 and D4 mRNAs primarily. An example of an ENK+/SP- cell in which D2, D3, and D4 mRNAs were seen is shown in Figure 1C. The data for ENK+/SP- neurons is summarized in Figure 1D. In this panel (and in subsequent summary diagrams), the extent to which particular mRNAs were found together is coded by the extent to which their lanes are shaded at similar points along the abscissa. The failure to detect D l b mRNA in as large a subject of neurons after multiplex amplification as after conventional PCR (OYOvs 20%) probably reflects sampling variation. Medium spiny neurons having detectable levels of SP but not ENK mRNA (ENK-/SP+) expressed high levels of Dla-receptor rnRNA. With 1/10 aliquots, D l a mRNA was detected in 80% of the sample. D3 mRNA was detected in half of this sample, but the other receptor mRNAs were either not seen or seen rarely. Doubling the aliquot size increased the D l a detection rate to 100% and did not change the detection rate for the D3 mRNA. Using the multiplex protocol, the detection frequencies for D l a and D3 mRNAs were similar to those seen with smaller aliquots. However, the detection frequency of D4 mRNA increased to 25% (as seen with one-fifth aliquots) (Figs. lA, B). In addition, D2 mRNA, which had not been seen with 1/10 or one-fifth aliquots, was seen in about 20% of the sample, suggesting that D2 mRNAs were of relatively low abundance in this group. Neurons coexpressing detectable levels of SP and ENK (ENK+/SP+) consistently coexpressed D l a and D2 mRNAs. This group comprised about 20% of our total population of neurons. With 1/10 aliquots, D2 mRNA was the most commonly detected in this sample (78%). Dla-receptor amplicons were seen less frequently, and both amplicons were detected in about one-fifth of the sample. The other receptor mRNAs were either not detected or seen rarely with the small aliquot. Doubling the aliquot, did not change the D l a and D2 detection frequencies significantly. However, using the multiplex protocol, the detection rate for D l a mRNA increased to 90% (Fig. 1E). As a consequence, D l a and D2 mRNAs were detected in 70% of this group (Figs. lE, F). The detection frequency of D3, D4, and D l b mRNAs also increased with the size of the aliquot used, suggesting that they were expressed at relatively low levels in 20-30% of ENK+/SP+ neurons. In an attempt to determine whether these transcripts could give rise to functional protein, whole-cell recording was combined with conventional single-cell RT-PCR. Several neurons were found to express detectable levels of D2 and D1 b mRNA, but not D l a mRNA; these cells consistently had robust responses to D1-class agonists. As shown, in other cells, D3 or D4 mRNA was detected but not D2 mRNA; these cells consistently had responses to D2-class agonists. While these results do not prove that D3-, D4-, and Dlb-receptor
1022
D. James Surmeier et a/.
B
100% (n=16)
D
100% (n=9)
I
11%
D3
ENK SP D,,
D,
D3 D4 D,
D4
F
100% (n=lO)
FIGURE I Single-cell RT-PCR revealed that neostriatal neurons coexpress mRNAs for several dopamine receptors. (A) Photograph of a gel containing amplicons from an SP+ENKmedium spiny neuron in which a multiplex procedure employing three-fourths of the total cellular cDNA was used for detection of DA-receptor mRNAs. Note that D l a and D3 and D4 amplicons were detected. ( B ) Summary of coexpression detected with the multiplex procedure in 16 ENK-/SP+ neurons (C) Photograph of a gel containing amplicons from an SP-ENK+ medium spiny neuron in which a multiplex procedure was employed as before. ( D )Summary of coexpression detected with the multiplex procedure in nine ENK+/SP- neurons. ( E ) Photograph of a gel containing amplicons from an SP+iENK+ medium spiny neuron in which the multiplex procedure was employed. (F)Summary of coexpression detected with the multiplex procedure in 10ENK+/SP+ neurons. (Reprinted with permission from TheJoumalofNeuroscience 16, 6579-6591, 1996.)
Expression of DA Receptors in Neostriatal Medium Spiny Neurons
I023
mRNAs invariably give rise to functional protein, they do suggest that these mRNAs are capable of being translated and properly processed. The principal goal of this study was to understand why anatomical and functional studies of DA-receptor localization in the neostriatum have been so discordant (1,2). It was our working hypothesis that at the heart of the dispute was the assumption that only D l a and D2 receptors were functionally significant in the workings of the dorsal neostriatum. This inference allowed the equation of responsiveness to D1-class agonists with the presence of D l a receptors and responsiveness to D2-class agonists with the presence of D2 receptors. Our results argue that, in fact, this assumption is incorrect. Significant levels of D3-, D4-, and Dlb-receptor mRNAs are present in medium spiny neurons. The demonstration that D1- and D2-class receptors are coexpressed by a significant percentage of medium spiny neurons, particularly those within the striatonigral pathway, establishes a cellular and molecular foundation for the well-known physiological and biochemical responses of these neurons to D1and D2-class agonists. Our results also show that single-cell RT-PCR techniques yield a picture compatible with that derived from in situ hybridization if attention is restricted to only the most abundant receptor mRNAs. Because of their ability to evoke functionally significant physiological effects, the impact of the lower abundance DA receptors (D3, D4, D l b ) must be factored into models of drug action within the neostriatum.
References 1. Gerfen, C. R. (1992).The neostriatal mosaic: Multiple levels of compartmental organization. TINS 15, 133-139. 2. Surmeier, D. J., Reiner, A., Levine, M. S., and Ariano, M. A. (1993). Are neostriatal dopamine receptors co-localized? TINS 16, 299-305. 3. Sibley, D. R. (1995). Molecular biology of dopamine receptors. In Cellular and Molecular Mechanisms of the Neostriatum. (D. J. Surmeier, and M. A. Ariano, eds.), R. G. Landes, Austin, TX. 4. Surmeier, D. J., Song, W-J., and Yan, Z. (1997). Coordinated expression of dopamine receptors in neostriatal medium spiny neurons. J. Neurosci. 16, 6579-6591.