BACarray translational profiling combines genetic targeting of an EGFP-L10a ribosomal fusion protein to specific cell populations; simple, rapid, affinity purification of mRNA; and microarray technology to identify translated mRNAs in situ. Unlike traditional approaches, it combines coincident detection of all translated mRNAs with cell-type specificity; thus, one quantitative BACarray experiment can replace thousands of high-throughput qualitative in situ hybridization runs and, further, can be repeated easily for each experimental condition. Second, the information obtained is cell-type specific while abrogating the physiologic adaptations and mRNA degradation which can occur during the lengthy cell separation procedures used in other approaches that sever neuronal axons and dendrites and disrupt tissue-intrinsic signalling. Third, because polysomes are stabilized with cycloheximide in the extraction buffers and during the affinity purification steps, redistribution of the affinity tag within the mRNA pool during polysome isolation is prevented, obviating the problems inherent in using less stable mRNA binding proteins. Fourth, the use of established BACarray lines ensures that mRNA translation profiles can be reproducibly obtained and directly compared from the same cell population across experimental conditions. Crossing of these and other established BACarray lines to mouse models should overcome one of the major obstacles to the study of many neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease, which display both vulnerable and non-vulnerable cell populations. Fifth, the use of EGFP as the affinity tag greatly facilitates anatomic studies of candidate BACarray mouse lines, as well as electrophysiological studies of each cell type. Finally, the profiling of only translated mRNA pools will more accurately reflect actual protein levels in a cell than does conventional gene expression profiling, which relies on total mRNA pools.
The BACarray approach thus overcomes limitations of previous mRNA-tagging methods which have targeted other RNA-binding proteins, of which only Poly A–Binding Protein (PABP) has been used in vivo
for tissue-specific studies (Roy et al., 2002
; Kunitomo et al., 2005
), and which requires crosslinking of the tagged PABP to mRNA (and its inherent artifacts) due to its loose association with mRNA. The use of tagged ribosomal proteins to purify translating polysomes from yeast as well as plant cells has recently been reported (Inada et al., 2002
; Zanetti et al., 2005
), although in these cases cell-specific targeting to obtain cell-specific translational profiles was not attempted. From our own experience, we know that adapting this methodology to cell-specific targeting is not trivial, particularly for applications in the mammalian CNS, with its far more difficult conditions of low expression, limited material, and contamination by blood, myelin, and other biological factors. As a result of the optimization reported here, the BACarray methodology is now extremely robust to these conditions and broadly applicable (Doyle, Dougherty, et al., 2008; accompanying paper).
Furthermore, the BACarray approach was able to identify all previously known, well-studied striatonigral- and striatopallidal-enriched genes [with one exception: the striatonigral-enriched muscarinic receptor M4 (Chrm4
) (Ince et al., 1997
), for which probesets on the Affymetrix Mouse 430 2.0 GeneChips gave very little signal; however, real-time PCR analysis of Chrm4
expression demonstrated clear enrichment of Chrm4
mRNA in our striatonigral BACarray cell sample (Table S3
)]. This stands in distinction to a recent microarray study of FACS-sorted MSNs (Lobo et al., 2006
), in which well-known positive-control genes (e.g. Chrm4
, and Drd2
) were not identified as differentially expressed on microarrays and 16 of the 21 mRNAs reported in that study as striatopallidal-enriched in adult MSNs could not be confirmed in our analysis. Furthermore, we report here hundreds of distinguishing transcripts not identified in that study.
More importantly, our BACarray analysis of striatal MSN subtypes has identified novel physiological differences between striatonigral and striatopallidal cells, which may provide new therapeutic targets for various neurological diseases associated with pathophysiology in the striatum. As one example, we demonstrate that striatopallidal cells selectively express Gpr6, and that they correspondingly display a cell-type specific release of intracellular Ca2+ in response to sphingosine 1-phosphate. A second example of the distinct properties of these medically important cell types is provided by our studies of psychostimulant drug action, which give molecular and physiological evidence for upregulation of GABAA receptor subunits in striatonigral neurons following chronic cocaine administration.
The results presented here demonstrate that the BACarray methodology provides an enabling technology for studies of the biology of specific cell types in even the most heterogeneous cell populations, such as those that occur in the CNS. We have identified numerous distinguishing molecular characteristics among four distinct neuronal populations, including two closely related, critical cell types and have demonstrated that the BACarray methodology can be employed to analyze physiological adaptations of specific cell types in vivo. An accompanying study (Doyle, Dougherty, et al., 2008) has demonstrated the general applicability of the BACarray methodology by characterizing additional CNS cell types, each of which exhibits an enormously complex and cell-type specific molecular phenotype.