Microarray analysis identifies cerebellar genes sensitive to chronic ethanol treatment in PKCgamma mice.
Neuroadaptive changes that occur in the development of ethanol tolerance may be the result of alterations in gene expression. We have shown that PKCgamma wild-type mice develop tolerance to the sedative-hypnotic effects of ethanol after chronic ethanol treatment; whereas, mutant mice do not, making these genotypes a suitable model for identifying changes in gene expression related to tolerance development. Using a two-stage process, several genes were initially identified using microarray analyses of cerebellar tissue from ethanol-treated PKCgamma mutant and wild-type mice. Subsequent confirmation of a subset of these genes using quantitative real time reverse transcriptase polymerase chain reactions (qRT-PCR) was done to verify gene expression changes. A total of 109 genes from different functional classifications were identified in these groups on the microarrays. Eight genes were selected for verification as follows: three, Twik-1, Plp, and Adk2, were chosen as genes related to tolerance; another three, Hsp70.2, Bdnf, and Th, were chosen as genes related to resistance to tolerance; and two genes, JunB and Nur77, were selected as candidate genes sensitive to chronic ethanol. The results from the verification experiments indicated that Twik-1, which codes for a potassium channel, was associated with tolerance and appeared to be dependent on the presence of PKCgamma. No genes were confirmed to be related to resistance to tolerance; however, expression of two of these, Hsp70.2 and Th, were found to be sensitive to chronic ethanol and were added to the transcription factors, JunB and Nur77, confirmed by qRT-PCR, as a subset of genes that respond to chronic ethanol.
Bowers
Barbara J
BJ
Institute for Behavioral Genetics, University of Colorado, 447 UCB, Boulder, CO 80309, USA. bbowers@colorado.edu
Radcliffe
Richard A
RA
Smith
Amy M
AM
Miyamoto-Ditmon
Jill
J
Wehner
Jeanne M
JM
D000437:Alcoholism / Q000235:genetics*; D000818:Animals; D002492:Central Nervous System Depressants / Q000494:pharmacology*; D002531:Cerebellum / Q000187:drug effects* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004361:Drug Tolerance / Q000235:genetics; D000431:Ethanol / Q000494:pharmacology*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D018991:Myelin Proteolipid Protein / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; D020411:Oligonucleotide Array Sequence Analysis; D024683:Potassium Channels, Tandem Pore Domain / Q000378:metabolism; D011493:Protein Kinase C / Q000235:genetics*; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D011987:Receptors, Steroid / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism
D002492:Central Nervous System Depressants; D004268:DNA-Binding Proteins; D018840:HSP70 Heat-Shock Proteins; C490434:Hspa2 protein, mouse; C486372:Kcnk1 protein, mouse; D018991:Myelin Proteolipid Protein; D009419:Nerve Tissue Proteins; C539818:Nr4a1 protein, mouse; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; C047516:Plp1 protein, mouse; D024683:Potassium Channels, Tandem Pore Domain; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D014157:Transcription Factors; D000431:Ethanol; D014446:Tyrosine 3-Monooxygenase; C087857:protein kinase C gamma; D011493:Protein Kinase C