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. 1996 Oct 15;16(20):6579-91.
doi: 10.1523/JNEUROSCI.16-20-06579.1996.

Coordinated expression of dopamine receptors in neostriatal medium spiny neurons

D J Surmeier  1 , W J Song, Z Yan
Affiliations

Coordinated expression of dopamine receptors in neostriatal medium spiny neurons

D J Surmeier et al. J Neurosci. .

Abstract

In recent years, the distribution of dopamine receptor subtypes among the principal neurons of the neostriatum has been the subject of debate. Conventional anatomical and physiological approaches have yielded starkly different estimates of the extent to which D1 and D2 class dopamine receptors are colocalized. One plausible explanation for the discrepancy is that some dopamine receptors are present in physiologically significant numbers, but the mRNA for these receptors is not detectable with conventional techniques. To test this hypothesis, we examined the expression of DA receptors in individual neostriatal neurons by patch-clamp and RT-PCR techniques. Because of the strong correlation between peptide expression and projection site, medium spiny neurons were divided into three groups on the basis of expression of mRNA for enkephalin (ENK) and substance P (SP). Neurons expressing detectable levels of SP but not ENK had abundant mRNA for the D1a receptor. A subset of these cells (approximately 50%) coexpressed D3 or D4 receptor mRNA. Neurons expressing detectable levels of ENK but not SP had abundant mRNA for D2 receptor isoforms (short and long). A subset (10-25%) of these neurons coexpressed D1a or D1b mRNAs. Neurons coexpressing ENK and SP mRNAs consistently coexpressed D1a and D2 mRNAs in relatively high abundance. Functional analysis of neurons expressing lower abundance mRNAs revealed clear physiological consequences that could be attributed to these receptors. These results suggest that, although colocalization of D1a and D2 receptors is limited, functional D1 and D2 class receptors are colocalized in nearly one-half of all medium spiny projection neurons.

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Figures

Fig. 1.
Fig. 1.
All five dopamine receptor mRNAs are expressed in the dorsal neostriatum. A, The region of dorsal striatum analyzed. Individual neurons were sampled from the same area.B, Photograph of an ethidium bromide-stained gel in which conventional PCR amplicons have been separated by electrophoresis. Note that D1a and D2 amplicons were the most abundant, but D3, D4, and D1b amplicons were clearly present. C, A photograph of an ethidium bromide-stained gel of amplicons derived as in B, but the tissue used as starting material was immersed in the RNA polymerase II inhibitor α-amanitin immediately after slicing. Note that there were no discernible changes in the pattern of expression.
Fig. 2.
Fig. 2.
Approximately one-half of all medium spiny neurons coexpress functional D1 and D2 class linked to modulation of voltage-dependent Ca2+ channels.A, Plot of peak current evoked by a voltage step to 0 mV from a holding potential of −80 mV. The application of the D2 class agonist quinpirole (5 μm) reversibly decreased peak current in this cell, as did application of the specific D1 class agonist 6-chloro-PB (2 μm). 5-HT had little or no effect on Ca2+ currents in this cell.Inset, A photomicrograph of an acutely isolated medium spiny neuron. B, Representative current traces from the records used to construct A, showing the effects of quinpirole. C, Representative current traces from the records used to construct A, showing the effects of 6-chloro-PB. D, Summary of recordings in which D1 and D2 class agonists were applied to the same cell and the effects on Ca2+ currents were monitored (n = 26). Modulations < 10% of the peak current were disregarded.
Fig. 3.
Fig. 3.
The probability of detecting a particular mRNA transcript decreases as the fraction of the total cellular cDNA used as a template decreases. A Poisson model (see Materials and Methods) was used to estimate the probability that a PCR reaction would fail to detect a transcript. Parametric curves generated by using 1/20th, 1/10th, 1/8th, 1/4, and 1/2 of the cellular cDNA are shown.
Fig. 4.
Fig. 4.
Neurons expressing detectable levels of enkephalin (ENK) but not substance P (SP) mRNA express D2 mRNA at high levels and other dopamine receptor mRNAs at lower levels. A, Photograph of an ethidium bromide-stained agarose gel in which dopamine receptor and peptide mRNA amplicons from a single medium spiny neuron have been separated by electrophoresis. One-tenth of the total cDNA was used for each PCR reaction. Note the presence of ENK and D2l amplicons.B, Summary of the coordinated DA receptor mRNA expression in 28 neurons expressing ENK, but not SP. Coexpression for any set of mRNAs can be deduced by the extent to which shaded bars in their lanes overlap. C, Photograph of a gel containing amplicons from another neuron in which one-fifth of the total cDNA was used for mRNAs not detected with one-tenth of the starting material (denoted by asterisks). Note the presence of both short and long isoforms of the D2 receptor mRNA. D, Summary of coexpression in eight neurons processed in the same manner. E, Photograph of a gel containing amplicons from a single medium spiny neuron in which a multiplex procedure with three-fourths of the total cellular cDNA was used for detection of DA receptor mRNAs. F, Summary of coexpression detected with the multiplex procedure in nine ENK+/SP− neurons.
Fig. 5.
Fig. 5.
Neurons expressing detectable levels of SP, but not ENK, mRNA express D1a mRNA at high levels and other dopamine receptor mRNAs at lower levels. A, Photograph of an ethidium bromide-stained agarose gel in which dopamine receptor and peptide mRNA amplicons from a single medium spiny neuron have been separated by electrophoresis. One-tenth of the total cDNA was used for each PCR reaction. Note the presence of SP and D1aamplicons. B, Summary of the coordinated DA receptor mRNA expression in 10 neurons expressing SP, but not ENK. Coexpression for any set of mRNAs can be deduced by the extent to whichshaded bars in their lanes overlap. C, Photograph of a gel containing amplicons from another neuron in which one-fifth of the total cDNA was used for mRNAs not detected with one-tenth of the starting material (denoted byasterisks). D, Summary of coexpression in four neurons processed in the same manner. E, Photograph of a gel containing amplicons from a single medium spiny neuron in which a multiplex procedure with three-fourths of the total cellular cDNA was used for detection of DA receptor mRNAs. Note that in these amplifications the D1a primer set yielding the longer amplicon was used. F, Summary of coexpression detected with the multiplex procedure in 16 ENK−/SP+neurons.
Fig. 6.
Fig. 6.
Neurons coexpressing detectable levels of ENK and SP mRNA coexpress D1a and D2 mRNAs.A, Photograph of an ethidium bromide-stained agarose gel in which dopamine receptor and peptide mRNA amplicons from a single medium spiny neuron have been separated by electrophoresis. One-tenth of the total cDNA was used for each PCR reaction. Note the presence of ENK, SP, and D2 amplicons. B, Summary of the coordinated DA receptor mRNA expression in nine neurons expressing both ENK and SP. C, Photograph of a gel containing amplicons from another neuron in which one-fifth of the total cDNA was used for mRNAs not detected with one-tenth of the starting material (denoted byasterisks). D, Summary of coexpression in four neurons processed in the same manner. E, Photograph of a gel containing amplicons from a single medium spiny neuron in which a multiplex procedure with three-fourths of the total cellular cDNA was used for detection of DA receptor mRNAs. F, Summary of coexpression detected with the multiplex procedure in 10 ENK+/SP+ neurons.
Fig. 7.
Fig. 7.
Low-abundance mRNAs give rise to functional protein. A, Photograph of an ethidium bromide-stained gel in which the amplicons from a single cell have been separated by electrophoresis. Note that this cell expressed detectable levels of D2 and D1b mRNA. B, Whole-cell recording from the same cell showing that the application of the D1 class agonist 6-chloro-PB (5 μm) reversibly reduced Ba2+ currents evoked by a voltage ramp.C, Photograph of an ethidium bromide-stained gel in which the amplicons from another cell have been separated by electrophoresis. Note that this cell expressed detectable levels of D4 but not D2 or D3 mRNA.D, Whole-cell recording from the same cell (fromC) showing that the application of the D2class agonist quinpirole (2 μm) reversibly reduced Ba2+ currents evoked by a voltage ramp.
Fig. 8.
Fig. 8.
D2 isoforms frequently were colocalized. A, Bar plot summarizing the coexpression of short and long transcripts in ENK+/SP−neurons. The extent of coexpression is coded by the extent to which the D2l and D2s lanes are shaded at the same point along the abscissa. B, Bar plot summarizing coexpression in ENK+/SP+ neurons.C, Summary of coexpression in ENK−/SP+ neurons.
Fig. 9.
Fig. 9.
D1 and D2 class receptor mRNAs were coexpressed most commonly in neurons expressing SP.A, Bar graph showing the percentage of ENK−/SP+ neurons expressing onlyD1 class (D1a, D1b), only D2class (D2, D3, D4), or at least one member of each class (Both). B, Bar graph showing the summary for ENK+/SP− neurons. C, Bar graph showing the summary for ENK+/SP+ neurons. All results were derived from multiplex PCR experiments.D, Bar graph showing pooled data in which ENK+/SP− and ENK−/SP+groups each were 40% of the total and the ENK+/SP+ group was 20% of the total. Note the similarity to Figure 2D.
Fig. 10.
Fig. 10.
A schematic summary of the pattern of dopamine receptor expression in medium spiny efferent populations (as inferred from peptide expression). Receptor abundance is coded by the size of the subtype label.
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