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Keywords:

  • gene expression;
  • microarray;
  • diazepam;
  • GABAA receptor;
  • plasticity

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. RNA isolation and cRNA target preparation for oligonucleotide arrays
  6. Data analysis
  7. Quantitative real-time PCR assay
  8. Results
  9. Diazepam-induced transcript changes in wild-type mice
  10. Diazepam-induced expression profile in α1(H101R) animals
  11. Confirmation of transcript changes by quantitative real-time PCR in wild-type and α1(H101R) mice
  12. Discussion
  13. Diazepam-induced transcript changes
  14. Change in calcium responsiveness
  15. Neurotrophic responses
  16. MAP kinase pathway
  17. Time-course
  18. Acknowledgements
  19. Supplementary Material
  20. References

Benzodiazepines are in wide clinical use for their sedative and tranquilizing actions, the former being mediated via α1-containing GABAA receptors. The signal transduction pathways elicited beyond the receptor are only poorly understood. Changes of transcript levels in cerebral cortex induced by acute diazepam administration were therefore compared by microarray analysis between wild-type and point mutated α1(H101R) mice, in which the α1 GABAA receptor subunit had been rendered insensitive to diazepam. In wild-type animals, diazepam reduced the expression levels of the α subunit of the calcium/calmodulin-dependent protein kinase II, as well as brain-derived neurotrophic factor, MAP kinase phosphatase, transcription factor GIF, c-fos and nerve growth factor induced gene-A. None of these transcripts was changed in the α1(H101R) mice after treatment with diazepam. Thus, the sedative action of diazepam is correlated with a selective down-regulation of transcripts involved in the regulation of neuronal plasticity and neurotrophic responses. Most transcript changes were transient except for the decrease of the CaMKIIα transcript which persisted even 40 h after the single dose of diazepam. This long-term alteration is likely to contribute to the resetting of the neuronal responsiveness, which may be involved in rebound phenomena and, under chronic treatment, in the development of tolerance and dependence.

Abbreviations used
BDNF

brain-derived neurotrophic factor

CaMKII

calcium/calmodulin-dependent kinase II

EST

expressed sequence tag

GIF

GDNF inducible transcription factor

MKP-1

MAP kinase phosphatase-1

NGFI-A

nerve growth factor induced gene-A

n.s.

not significant

Benzodiazepines exert their therapeutic effects in the treatment of generalized anxiety disorders, panic attacks, muscle spasms, sleep disturbances and seizure disorders by an enhancement of GABAA receptor-mediated chloride flux. However, it is largely unknown which downstream signaling events are triggered subsequent to the allosteric receptor modulation. Following chronic drug treatment, tolerance and withdrawal symptoms are manifestations of largely unknown neuronal adaptations. On the molecular level, chronic benzodiazepine treatment is known to result in a decrease of postsynaptic GABA sensitivity (Gallager et al. 1984), decrease of benzodiazepine binding (Wu et al. 1994a; Li et al. 2000) and in changes of GABAA receptor subunit transcripts, with the most consistent changes being the down-regulation of α1, α5 and γ2 subunit mRNAs (Heninger et al. 1990; Primus and Gallager 1992; Wu et al. 1994b; Holt et al. 1996; Impagnatiello et al. 1996; Longone et al. 1996). After an acute dose of diazepam, GABAA receptors in cortical and cerebellar membranes showed a decreased coupling between GABA and benzodiazepine binding sites (Holt et al. 1999), Fos protein expression varied depending on the brain subregion analyzed (Salminen et al. 1996) and striatal neuropeptide mRNA levels (dynorphin, tachykinin, enkephalin) were altered (Lucas et al. 1997). However, a systematic analysis of the downstream signaling events following the initial modulation of GABAA receptor would be required to identify neuronal adaptation induced by benzodiazepine treatment.

Using a microarray with 12 488 murine genes and expressed sequence tags (ESTs) an attempt was made to identify changes in the transcription profiles in the mouse brain following an acute dose of diazepam. This analysis was focused on the sedative action of diazepam and was therefore restricted to changes induced by the modulation of the α1 GABAA receptor which selectively mediates the sedative drug action (Rudolph et al. 1999; McKernan et al. 2000). This pharmacological focus was accomplished by comparing the diazepam-induced changes in gene expression between wild-type mice and mutant mice, in which the α1 GABAA receptor had been rendered insensitive to diazepam by the point-mutation α1(H101R). In these animals, even a high dose of diazepam (30 mg/kg) fails to induce sedation (Rudolph et al. 1999). Transcript changes that were present in the wild-type, but not in the α1(H101R) mice were therefore expected to provide insights into the neuronal signaling pathways that accompany the sedative action of acute benzodiazepine treatment. We chose to study the cerebral cortex since it expresses mainly the α1 GABAA receptor, although it also contains α2, α3 and α5 GABAA receptors. Thus, our study provides a molecular analysis of the downstream signaling events mediated selectively via α1 GABAA receptors. The transcript changes were expected to provide an indication for the drug-induced adaptive processes underlying rebound phenomena and, under chronic treatment, for the development of tolerance and dependence.

Animals

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. RNA isolation and cRNA target preparation for oligonucleotide arrays
  6. Data analysis
  7. Quantitative real-time PCR assay
  8. Results
  9. Diazepam-induced transcript changes in wild-type mice
  10. Diazepam-induced expression profile in α1(H101R) animals
  11. Confirmation of transcript changes by quantitative real-time PCR in wild-type and α1(H101R) mice
  12. Discussion
  13. Diazepam-induced transcript changes
  14. Change in calcium responsiveness
  15. Neurotrophic responses
  16. MAP kinase pathway
  17. Time-course
  18. Acknowledgements
  19. Supplementary Material
  20. References

Wild-type and α1(H101R) male mice (Rudolph et al. 1999) were housed in groups of 10 under a 12-h light/dark cycle (light on from 06 : 30 h to 18 : 30 h), with food and water being provided ad libitum. Mice were kept in filter cages, health monitoring was performed every three months and mice were free of viral infections. The α1(H101R) mutation has been backcrossed to the 129/SvJ background for 13 generations. Homozygous mutant and wild-type offspring from heterozygote crosses were interbred for up to three generations to yield the experimental animals. Sixty minutes after dark onset, 8- to 10-week-old animals were injected either with diazepam (30 mg/kg, intraperitoneally) or vehicle (0.3% Tween80/saline, intraperitoneally). Diazepam was kindly provided by F. Hoffman-LaRoche (Basel, Switzerland). Drug-treated wild-type animals were killed at 6, 16 and 40 h after injection (four animals per each time point) and vehicle-treated wild-type animals (n = 4) at 16 h after injection. Thus, when comparing mice at 6 or 40 h post diazepam injection with mice 16 h post vehicle injection, differences in gene expression due to circadian timing and time-dependent effects of the vehicle may have to be taken into account. The cerebral cortices were rapidly removed and flash-frozen in liquid nitrogen, and each of the 16 cerebral cortex samples was analyzed by microarrays. Another group of wild-type as well as α1(H101R) mice were treated with diazepam (30 mg/kg, intraperitoneally, n = 4 per genotype) or vehicle (0.3% Tween80/saline, intraperitoneally, n = 4 per genotype) and killed 16 h after the treatment. The 16 h time-point was chosen in this experiment since it was considered to represent the time-point with the potentially most specific transcript changes in the initial time-course experiment. In a third experiment, α1(H101R) animals were treated with either diazepam (30 mg/kg, intraperitoneally) or vehicle, n = 4, and killed 16 h later. Thus, for the 16 h time-point, a total of 8 α1(H101R) cortex samples were available for microarray analysis. For real-time PCR analysis the same RNA preparations were used as for the microarray analysis.

RNA isolation and cRNA target preparation for oligonucleotide arrays

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. RNA isolation and cRNA target preparation for oligonucleotide arrays
  6. Data analysis
  7. Quantitative real-time PCR assay
  8. Results
  9. Diazepam-induced transcript changes in wild-type mice
  10. Diazepam-induced expression profile in α1(H101R) animals
  11. Confirmation of transcript changes by quantitative real-time PCR in wild-type and α1(H101R) mice
  12. Discussion
  13. Diazepam-induced transcript changes
  14. Change in calcium responsiveness
  15. Neurotrophic responses
  16. MAP kinase pathway
  17. Time-course
  18. Acknowledgements
  19. Supplementary Material
  20. References

Frozen tissue samples were homogenized in 1 mL RNAzol reagent (AMS Biotechnology, Bioggio-Lugano, Switzerland) and total RNA was isolated according to the manufacturer's instructions. Quality of the total RNA was monitored by gel electrophoresis and the OD260nm/OD280nm ratio. cRNA targets were prepared according to protocols provided by Affymetrix (Santa Clara, CA, USA; http://www.affymetrix.com). Briefly, 20 µg of total RNA was converted to double-stranded cDNA using SuperScript® Choice System (Invitrogen, Carlsbad, CA USA) in combination with a T7-dT24 primer (5′-GGCCAGTGAATTGTATACGACTCACTATAGGGAGGCGG-dT24-3′). cDNA samples were phenol–chloroform extracted using Phase-Lock Tubes (Eppendorf, Hamburg, Germany) and precipitated with 7.5 m NH4Ac and absolute ethanol. With this double-stranded cDNA serving as a template, biotinylated cRNA was synthesized with T7 MegaScript system (Ambion, Austin, TX, USA). Biotinylated, fragmented cRNA targets (15 µg of biotin-labeled cRNA) were hybridized to Affymetrix Murine Genome U74A_v2 arrays containing probes for 12 488 murine transcripts. Microarrays were hybridized, washed and stained according to the Affymetrix instructions (http://www.affymetrix.com). In total, 40 Murine Genome U74A_v2 arrays were hybridized with cRNA derived from individual cortices so that each array represented the analysis of a single animal. The values for 3′/5′ ratios for GAPDH and β-actin were in the 1.0–3.0 range. With respect to the spike controls, BioB was called present and BioC, BioD and CreX controls were called present with increasing intensities. Raw and normalized data are available from the authors upon request. All microarray data were scaled to an arbitrary target intensity (500) in order to permit comparisons between different arrays. Scaling factors of all arrays were within a threefold range. The average intensities and the percentage of transcripts called present were consistent for all the arrays in the experiment. The arrays had sensible background levels and Q scores as well as similar mean intensities. Visual checks were made for each array image for bubbles and scratches.

Data analysis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. RNA isolation and cRNA target preparation for oligonucleotide arrays
  6. Data analysis
  7. Quantitative real-time PCR assay
  8. Results
  9. Diazepam-induced transcript changes in wild-type mice
  10. Diazepam-induced expression profile in α1(H101R) animals
  11. Confirmation of transcript changes by quantitative real-time PCR in wild-type and α1(H101R) mice
  12. Discussion
  13. Diazepam-induced transcript changes
  14. Change in calcium responsiveness
  15. Neurotrophic responses
  16. MAP kinase pathway
  17. Time-course
  18. Acknowledgements
  19. Supplementary Material
  20. References

Absolute and comparison analysis.  Microarray data were analyzed with Affymetrix Microarray Suite (MAS) version 5.0 and Data Minig Tool version 3.0 according to the manufacturer's instructions. MAS 5.0 software uses one-sided Wilcoxon's signed rank test to calculate a detection p-value for each probe on the chip. Based on the p-value the software generates a present call (p < 0.04), marginal call (p = 0.04–0.059) or absent call (p≥ 0.06) for each probe. All probes included in subsequent analysis were required to have a detection p-value of less than 0.05, indicating that all transcripts were reliably detected by the microarrays. In the absolute analysis two criteria were applied to identify transcripts that were differentially expressed between drug- and vehicle-treated animals: (i) at least 1.3-fold change in the transcript expression level; and (ii) a Mann–Whitney p-value < 0.05. The threshold of 1.3 for detecting fold changes in neuronal tissue in the absolute analysis is in concordance with previous analyses of CNS tissue (Lewohl et al. 2000; Carter et al. 2001; Freeman et al. 2001; Hakak et al. 2001).

In the comparison analysis, each probe set on an experimental chip (i.e. a chip representing a diazepam-treated animal) is compared with its counterpart on the ‘baseline chip’ (i.e. a chip representing a vehicle-treated animal). Thus, for a group of eight diazepam- and eight vehicle-treated animals 64 comparisons are performed. Comparison analysis uses an algorithm that generates a qualitative difference call to indicate if a transcript in the experimental array is increased (I), marginally increased (MI), marginally decreased (MD), decreased (D) or equivalent to its baseline counterpart. The transcripts showing increase/marginal increase (I/MI) or decrease/marginal decrease (D/MD) calls were scored in such a way that a score of 100 denotes the same call for the transcript in all the 64 comparisons. In the comparison analysis a transcript was considered to be changed when the score was ≥ 50. The score cut-off level for comparison analysis significance was adapted from previous CNS expression profiling reports (Sandberg et al. 2000; Thibault et al. 2000; Middleton et al. 2002).

Quantitative real-time PCR assay

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. RNA isolation and cRNA target preparation for oligonucleotide arrays
  6. Data analysis
  7. Quantitative real-time PCR assay
  8. Results
  9. Diazepam-induced transcript changes in wild-type mice
  10. Diazepam-induced expression profile in α1(H101R) animals
  11. Confirmation of transcript changes by quantitative real-time PCR in wild-type and α1(H101R) mice
  12. Discussion
  13. Diazepam-induced transcript changes
  14. Change in calcium responsiveness
  15. Neurotrophic responses
  16. MAP kinase pathway
  17. Time-course
  18. Acknowledgements
  19. Supplementary Material
  20. References

The changes in transcript levels detected by microarray expression profiling were validated by TaqMan 5′ nuclease real-time PCR assay (Heid et al. 1996). Cerebral cortex samples from diazepam- and vehicle-treated wild-type and α1(H101R) mice (n = 8 for both genotypes) were subjected to real-time PCR assay for the amplicons of CaMKIIα, BDNF, MKP-1 and GIF. cDNA synthesis was performed using the InVitrogen SuperScript® cDNA synthesis kit (Invitrogen). In each reaction, 1200 ng total RNA from mouse cerebral cortex was reverse transcribed with oligo(dT)12−18 primer (0.5 µg/reaction). RT-reactions were performed as suggested by the manufacturer. Parallel reactions were run in the absence of SuperScript II® (– RT controls) to assess the degree of contaminating genomic DNA. Primers and TaqMan probes were designed using the Primer Express (Version 1, PE Applied Biosystems, FosterCity, CA, USA), a software provided with the 7700 Sequence Detection System. Amplicons (maximally 100 nucleotides in length) were designed to the 3′ region of the target sequences obtained from GenBank (http://www.ncbi.nlm.nih.gov/Entrez/). Forward and reverse primers were positioned as close as possible to each other without overlapping with the probe. Whenever possible, primers were targeted to intron/exon boundaries in order to prevent the signal originating from contaminating genomic DNA. Each probe was synthesized with the fluorescent reporter FAM (6-carboxy-fluorescein) in the 5′ end and the quencher TAMRA (6-carboxy-tetramethyl-rhodamine) in the 3′ end. Assays for each transcript were carried out in duplicates in 25 µL reaction volume on an ABI Prism 7700 Sequence Detection System (PE Applied Biosystems). Primer and probe sequences, as well as optimal primer concentrations are available upon request. The real-time PCR data were analyzed using the comparative CT (threshold cycle) method (ΔΔCT method, Applied Biosystems User Bulletin 2). The ΔΔCT method is applicable only when the efficiencies of the target and reference (cyclophilin) amplicons are approximately equal. To validate the amplification efficiency, the log of input template is plotted against the resulting CT; a slope of approximately zero (< 0.1) indicates that the amplification efficiencies are equal.

Absolute analysis.  To monitor diazepam-induced changes in gene expression profile, wild-type mice were treated with either diazepam (30 mg/kg, intraperitoneally) or vehicle followed by detection of transcript levels by microarray analysis of the cerebral cortex at 6, 16, and 40 h after drug administration. The fold changes of the transcript levels were calculated as the average of the four individual hybridizations performed for each time point and treatment condition (absolute analysis method, see Methods). A fold change of at least 1.3 and a significance level of p < 0.05 were considered as threshold for a transcript change. At the 6 h time-point after diazepam injection 126 transcripts (1,0% of all the transcripts represented on the array) were differentially expressed in the cerebral cortex between drug- and vehicle-treated wild-type animals. At the 16 h time-point 52 transcripts (0.42% of all the transcripts on the array) met our criteria of significant change, and at the latest time-point analyzed (40 h), 99 transcripts (0.8% of all the transcripts on the array) were differentially regulated between the drug- and vehicle-treated wild-type animals.

When additional wild-type animals were analyzed at the 16 h time-point (n = 4 per treatment) and the microarray data from these animals were combined with the 16 h time-point data from the initial experiment (n = 8 per treatment in the combined dataset), 54 transcripts (0.43% of the transcripts on the array) met our criteria of being significantly changed with 34 transcripts being down-regulated and 20 transcripts being up-regulated (Table 1). The numerical fold changes identified in the present study were in keeping with previous microarray studies performed in the CNS (Lee et al. 2000; Lewohl et al. 2000; Mirnics et al. 2000; Hakak et al. 2001; Mayfield et al. 2002; Molteni et al. 2002).

Table 1.  54 significant transcript changes in cerebral cortex of wild-type mice ( n  = 8) 16 h after a single dose of diazepam (30 mg/kg) as determined by absolute analysis (Mann–Whitney test)
Fold changep-valueGenBank IDDescriptionBiological process/molecular function
−1.930.002AV345278EST 
−1.850.002V00727FBJ osteosarcoma oncogeneRegulation of cell cycle
−1.680.001X61940MKP1, Dusp1Dephosphorylation/protein phosphatase
−1.670.048AI845633EST 
−1.610.033AI643393EST 
−1.510.001AF038995Putative RNA helicase RCKNucleic acid binding
−1.490.024Y12474Centrin 3Cytokinesis/mitosis/Ca-binding
−1.440.001AF064088Transcription factor GIFRegulation of transcription/DNA binding
−1.430.005M59821Growth factor-inducible protein (pip92) 
−1.420.009M28845Early growth response 1Regulation of transcription/DNA binding
−1.420.046X87142CamKII alphaPhosphorylation/protein kinase
−1.410.001AF064088Transcription factor GIFRegulation of transcription/DNA binding
−1.410.043AF100694Pontin52ATP-binding
−1.390.002AI850463EST 
−1.390.012AW124735EST 
−1.390.017AI847837Caspase 8 associated protein 2ATP-binding
−1.380.018AB010297Actin-related protein 1 alpha-isoformCytoskeleton component
−1.380.036C76020EST 
−1.360.047AI836552EST 
−1.340.033AJ246002Spastin protein orthologue (Spast gene)ATP-binding, nucleotide binding
−1.330.002AA693125EST 
−1.330.022M64429Braf transforming geneZinc binding
−1.320.016X55573Brain derived neurotrophic factorGrowth factor
−1.320.027AI843178Cerebellar postnatal development protein 1Phosphatase inhibitor
−1.320.047M12660Complement component factor hComplement activation
−1.310.004L12030Stromal cell derived factor 1Immune response/growth factor/cytokine
−1.310.006AW046194EST 
−1.310.024AA684502EST 
−1.310.025AA738710EST 
−1.310.029AV278013EST 
−1.310.046AF049124Neuronal pentraxin 2Lectin
−1.30.012AI845584MKP-3, Dusp6Dephosphorylation/protein phosphatase
−1.30.021AF008574Potassium channel alpha subunit (Kv9.2)Potassium ion transport
−1.30.034L36829HIV enhancer binding protein 1Regulation of transcription
1.30.006X71978Fused toesMorphogenesis, pattern formation
1.30.046AI836143EST 
1.310.009AW124599EST 
1.320.003AI117157Sialyltransferase 1Protein glycosylation
1.340.027AI850638Thyrotroph embryonic factorRegulation of transcription
1.350.016M15832Procollagen type IVCell adhesion
1.360.032J02872Granzyme GProteolysis, peptidolysis
1.370.041AI853173RNA polymerase 1–3RNA polymerase
1.40.001AI844607EST 
1.410.041AW047017RAB14, member of RAS oncogene familyGTP binding
1.420.021X87257ELK1, member of ETS oncogene familyRegulation of transcription
1.480.009C77278EST 
1.480.018AB023619Methyltransferase related proteinMethyltransferase
1.480.043AW045808EST 
1.540.014AW228316EST 
1.550.021AW125442Protein kinase inhibitor, alphacAMP-dependent kinase inhibitor
1.580.009AI849035EST 
1.650.025AI839876EST 
1.660.029AW124271EST 
1.960.028AI835963Protein inhibitor of activated STAT 3DNA binding

Comparison analysis.  In principle, the results of an absolute data analysis can be distorted by individual values which strongly deviate from the mean due to potential interindividual differences among animals. Although this was not evident from inspection of the data sets, the combined wild-type data (n = 8 for each treatment group) were nevertheless analyzed with the comparison analysis algorithm using the Affymetrix Microarray Suite 5.0 and Data Mining Tool 3.0 software. This algorithm compares every expression value of each diazepam-treated animal (n = 8, 16 h time-point) with the expression values of each vehicle-treated animal (n = 8, 16 h time-point), thus performing 64 comparisons. In this analysis, transcripts were considered to be differentially expressed when at least 50% of the comparisons between drug- and vehicle-treated animals showed a consistent difference call. Of the 12 488 combined genes and ESTs represented on the chip, nine transcripts (0.07% of the transcripts on the array) were found to be significantly changed 16 h following diazepam treatment according to the 50% criterion (AB016424: mouse rbm3 mRNA, AI850638:EST, V00727:c-fos, AF064088:GIF, X61940:MKP-1, X55573:BDNF, M28845:NGFI-A, X16995:mouse N10 gene for a nuclear hormonal binding receptor, and X87142:CaMKIIα). Finally, when considering only expression changes that were found to meet the criteria in both absolute and comparison analyses, only six known genes, CaMKIIα, BDNF, MKP-1, GIF, c-fos and NGFI-A, were found to be changed following acute diazepam treatment of wild-type animals (Table 2). The decrease in the transcript levels of BDNF, MKP-1, GIF, c-fos and NGFI-A was transient peaking at the 16 h time-point, with a return to near baseline levels at the 40 h time-point. In contrast, CaMKIIα showed a rapid down-regulation which was fully apparent already 6 h after diazepam treatment and persisted for at least 40 h (Fig. 1).

Table 2.  Transcript changes in the cerebral cortex of wild-type ( n  = 8) and α1(H101R) mice ( n  = 8) 16 hours after an acute dose of diazepam (30 mg/kg i.p.)
Transcript changes in Wild-type miceTranscript changes in α1(H101R) mice
TranscriptGenBankFold changep -value TranscriptFold changep -value
  1. n.s., not significant (> 0.05, Mann–Whitney test).

CaMKIIαX87142− 1.40.046CaMKIIα1.1n.s.
BDNFX55573− 1.30.016BDNF1.1n.s.
MKP-1X61940− 1.70.001MKP-1− 1.1n.s.
GIFAF064088− 1.40.001GIF− 1.1n.s.
c-fosV00727− 1.90.002c-fos− 1.1n.s.
NGFI-AM28845− 1.40.009NGFI-A− 1.1n.s.
image

Figure 1. Time-course of transcript changes for CaMKIIα, BDNF, MKP-1, GIF, c-fos and NGFI-A in cerebral cortex of wild-type mice following acute treatment with diazepam (30 mg/kg, i.p.) or vehicle. Transcript levels were analysed using microarrays 6 h ( n  = 4, striped bars), 16 h ( n  = 8, grey bars) and 40 h ( n  = 4, black bars) after drug treatment. It is noteworthy that the transcript change of CaMKIIα persisted for at least 40 h.

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Diazepam-induced expression profile in α1(H101R) animals

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. RNA isolation and cRNA target preparation for oligonucleotide arrays
  6. Data analysis
  7. Quantitative real-time PCR assay
  8. Results
  9. Diazepam-induced transcript changes in wild-type mice
  10. Diazepam-induced expression profile in α1(H101R) animals
  11. Confirmation of transcript changes by quantitative real-time PCR in wild-type and α1(H101R) mice
  12. Discussion
  13. Diazepam-induced transcript changes
  14. Change in calcium responsiveness
  15. Neurotrophic responses
  16. MAP kinase pathway
  17. Time-course
  18. Acknowledgements
  19. Supplementary Material
  20. References

Point-mutated α1(H101R) animals fail to be sedated by diazepam (30 mg/kg, Rudolph et al. 1999). It was therefore tested whether the transcripts which were diazepam-sensitive in wild-type mice failed to be changed in the α1(H101R) mice. These animals served as genotypic control to determine the specificity of the changes induced by diazepam in wild-type animals. α1(H101R) animals were treated with either diazepam (30 mg/kg, intraperitoneally) or vehicle and sacrificed 16 h later for microarray analysis of the cerebral cortex samples. Following diazepam treatment, only three out of the 54 transcripts that were up- or down-regulated in wild-type mice according to the absolute analysis (see Table 1) were also changed in the α1(H101R) mice: cerebellar postnatal protein [fold change in α1(H101R) mice − 1.44, p = 0.043], HIV enhancer binding protein 1 [fold change in α1(H101R) mice − 1.44, p = 0.043] and ELK1 [fold change in α1(H101R) mice 1.63, p = 0.014]. Thus, the diazepam-induced regulation of these transcripts is independent of α1-containing GABAA receptors and thus likely mediated by α2-, α3- and α5-containing GABAA receptors. In contrast, the expression of the other 51 transcripts that were up- or down-regulated in wild-type mice according to the absolute analysis were not significantly changed in the α1(H101R) mice, indicating that the up- or down-regulation of these genes in the wild-type mice is strictly dependent on α1-containing GABAA receptors. Thus, the vast majority of diazepam-induced changes of gene expression in the cerebral cortex is mediated by this receptor subtype. The six transcripts which are changed by diazepam treatment in wild-type mice in the combined absolute and comparison analysis, CaMKIIα, BDNF, MKP-1, GIF, c-fos and NGFI-A were all unchanged in the α1(H101R) mice as determined by microarray analysis (Table 2).

Confirmation of transcript changes by quantitative real-time PCR in wild-type and α1(H101R) mice

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. RNA isolation and cRNA target preparation for oligonucleotide arrays
  6. Data analysis
  7. Quantitative real-time PCR assay
  8. Results
  9. Diazepam-induced transcript changes in wild-type mice
  10. Diazepam-induced expression profile in α1(H101R) animals
  11. Confirmation of transcript changes by quantitative real-time PCR in wild-type and α1(H101R) mice
  12. Discussion
  13. Diazepam-induced transcript changes
  14. Change in calcium responsiveness
  15. Neurotrophic responses
  16. MAP kinase pathway
  17. Time-course
  18. Acknowledgements
  19. Supplementary Material
  20. References

The validity of the diazepam-induced reduction of the transcripts of CaMKIIα, BDNF, MKP-1 and GIF, as determined by microarrays, was verified by analyzing wild-type and α1(H101R) mice at the 16 h time-point by quantitative real-time PCR (Fig. 2). Determination of the PCR efficiency between the four target amplicons and the internal control amplicon in a concentration range of 0.24–240 ng of total input RNA (1000-fold difference) revealed slope-values of less than 0.1 for all the three amplicons tested, allowing for ΔΔCT determinations of relative quantification of gene expression. Expression of target transcripts was normalized relative to cyclophilin. The results from real-time PCR confirmed the microarray data for all the candidate genes tested (Fig. 2) in that they were all down-regulated in wild-type mice to an extent which was comparable to the values detected by microarray analysis (Table 2). In addition, none of these transcripts was significantly altered in α1(H101R) mice. To address the question whether contaminating genomic DNA, potentially present in the total RNA preparations, could have influenced the target gene quantification, -RT controls (reverse transcription reactions performed in the absence of the Superscript II® enzyme) were subjected to real-time PCR assay. None of the samples gave a signal for any of the amplicons used.

image

Figure 2. Genotypic differences in transcript levels between wild-type and α1(H101R) mice. Using quantitative real-time PCR, transcripts for CaMKIIα, BDNF, MKP-1 and GIF were quantified in cerebral cortex from wild-type ( n  = 8) and α1(H101R) ( n  = 8) mice 16 h after diazepam treatment (30 mg/kg, i.p.). Target transcripts were normalized relative to cyclophilin expression and are expressed as mean transcription ratio for the eight independent RNA samples. SD of the measurements was less < 0.1 for all expression ratios except for MKP-1 in wild-type mice (SD 0.25) and GIF in α1(H101R) mice (SD 0.24).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. RNA isolation and cRNA target preparation for oligonucleotide arrays
  6. Data analysis
  7. Quantitative real-time PCR assay
  8. Results
  9. Diazepam-induced transcript changes in wild-type mice
  10. Diazepam-induced expression profile in α1(H101R) animals
  11. Confirmation of transcript changes by quantitative real-time PCR in wild-type and α1(H101R) mice
  12. Discussion
  13. Diazepam-induced transcript changes
  14. Change in calcium responsiveness
  15. Neurotrophic responses
  16. MAP kinase pathway
  17. Time-course
  18. Acknowledgements
  19. Supplementary Material
  20. References

A quantitative insight into the responses downstream of GABAA receptor modulation following diazepam treatment was obtained using high-density microarrays. A subset of genes was identified in wild-type mice that fulfill the criteria for being transcriptionally up-regulated or repressed by a single dose of diazepam. Almost all of these transcript changes are specifically related to responses mediated by α1-containing GABAA receptors. This conclusion is based on the finding that only three out of 54 transcripts up- or down-regulated by diazepam in wild-type mice according to the absolute analysis were unaffected by diazepam treatment in α1(H101R) mice. Thus, it appears that almost all diazepam-induced changes in gene expression in the cerebral cortex of wild-type mice are mediated by the GABAA receptor subtype which makes up approximately one-half of all GABAA receptors in the brain (McKernan and Whiting 1996). It is remarkable that the GABAA receptors containing the α2, α3 or α5 subunits which are targets for diazepam in the α1(H101R) mice and make up the other half of the GABAA receptor complement in the brain, are not sufficient to mediate these transcriptional actions. Thus, we have been able to identify α1-GABAA receptor subtype-specific downstream events. Since the sedative response to diazepam is also specifically mediated by α1-containing GABAA receptors (Rudolph et al. 1999), our results reveal an association between this major behavioral effect of diazepam and postreceptor events.

Although the focus of our study was on the identification of genes whose transcription is specifically regulated by α1-containing GABAA receptors, we also found that diazepam induces changes in gene expression in the cerebral cortex of α1(H101R) mice which are not seen in wild-type mice with 92 transcripts being down-regulated and 41 transcripts being up-regulated in the absolute analysis (see Table 3 Supplementary material in the online journal). These α1(H101R) mice-specific regulatory changes are thus clearly dependent on GABAA receptors containing the α2, α3 or α5 subunits. These α2, α3 or α5-specific regulations are not seen in wild-type mice presumably due to a functional interaction of α1 GABAA receptors and α2, α3, or α5 GABAA receptors. Diazepam actions which are present only in α1(H101R) mice but not in wild-type mice (as are these transcriptional changes) have previously been observed. In EEG studies, the diazepam-induced reduction in the number of brief awakenings, i.e. an increase in sleep continuity, is present in α1(H101R) mice but not in wild-type mice (Tobler et al. 2001). Similarly, in an unfamiliar (stressful) environment, diazepam induced a significant increase in locomotor activity in the α1(H101R) mice but not in wild-type mice (Crestani et al. 2000; McKernan et al. 2000).

Diazepam-induced transcript changes

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. RNA isolation and cRNA target preparation for oligonucleotide arrays
  6. Data analysis
  7. Quantitative real-time PCR assay
  8. Results
  9. Diazepam-induced transcript changes in wild-type mice
  10. Diazepam-induced expression profile in α1(H101R) animals
  11. Confirmation of transcript changes by quantitative real-time PCR in wild-type and α1(H101R) mice
  12. Discussion
  13. Diazepam-induced transcript changes
  14. Change in calcium responsiveness
  15. Neurotrophic responses
  16. MAP kinase pathway
  17. Time-course
  18. Acknowledgements
  19. Supplementary Material
  20. References

Genes were considered to have altered expression between vehicle- and diazepam-treated animals when meeting the following criteria: (i) at least 1.3-fold change in microarray expression level, and (ii) p < 0.05 in the Mann–Whitney test (absolute analysis). Some genes in addition displayed a consistent change in at least 50% of the files in the comparison analysis and thus fulfilled the criteria for the combined absolute and comparison analysis. Using this combined analysis, we were able to identify a subset of six transcripts (CaMKIIα, BDNF, MKP-1, GIF, c-fos and NGFI-A) which were significantly changed in the cerebral cortex of the wild-type mice following diazepam treatment. Since all of these transcripts remained unchanged in diazepam-treated α1(H101R) animals, the changes in wild-type animals are concluded to relate to diazepam-induced sedation, the major α1-mediated response in wild-type mice. Diazepam treatment resulted in a reduction of transcripts involved in regulating synaptic functions and plasticity (CaMKIIα, BDNF, GIF, c-fos and NGFI-A) and in an enhancement of the MAP-kinase pathway.

Change in calcium responsiveness

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. RNA isolation and cRNA target preparation for oligonucleotide arrays
  6. Data analysis
  7. Quantitative real-time PCR assay
  8. Results
  9. Diazepam-induced transcript changes in wild-type mice
  10. Diazepam-induced expression profile in α1(H101R) animals
  11. Confirmation of transcript changes by quantitative real-time PCR in wild-type and α1(H101R) mice
  12. Discussion
  13. Diazepam-induced transcript changes
  14. Change in calcium responsiveness
  15. Neurotrophic responses
  16. MAP kinase pathway
  17. Time-course
  18. Acknowledgements
  19. Supplementary Material
  20. References

Calcium/calmodulin-dependent kinase II (CaMKII) is a key element in Ca2+-dependent synaptic plasticity. The kinase consists of two main subunits, CaMKIIα and CaMKIIβ, the former being located in the cytosol while the latter acts as a targeting molecule localizing large numbers of CaMKIIα isoforms to synaptic and cytoskeletal sites of action (Shen et al. 1998). Diazepam induced a rapid and long lasting (> 40 h) down-regulation of the CaMKIIα while the CaMKIIβ mRNA levels were not affected by acute diazepam treatment. Diazepam may thus limit the neuronal responsiveness to changes in intracellular Ca2+ levels. Several studies have pointed to the presence of consensus sites for CaMKII phosphorylation of the GABAA receptor in γ2L, γ2S and β subunits (Machu et al. 1993; McDonald and Moss 1994), and phosphorylation of the α1 subunit has been demonstrated to lead to an increase in benzodiazepine binding (Churn et al. 2002). CaMKIIα has also been shown to positively modulate GABA-induced Cl currents (Wang et al. 1995). Furthermore, an early report proposed benzodiazepines (0.2–200 µm) to indirectly inhibit CaMKII activity (DeLorenzo et al. 1981). Thus, the down-regulation of the CaMKIIα transcript by diazepam may result in a decreased responsiveness of GABAA receptors.

Neurotrophic responses

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. RNA isolation and cRNA target preparation for oligonucleotide arrays
  6. Data analysis
  7. Quantitative real-time PCR assay
  8. Results
  9. Diazepam-induced transcript changes in wild-type mice
  10. Diazepam-induced expression profile in α1(H101R) animals
  11. Confirmation of transcript changes by quantitative real-time PCR in wild-type and α1(H101R) mice
  12. Discussion
  13. Diazepam-induced transcript changes
  14. Change in calcium responsiveness
  15. Neurotrophic responses
  16. MAP kinase pathway
  17. Time-course
  18. Acknowledgements
  19. Supplementary Material
  20. References

The mRNA level of BDNF was decreased following diazepam treatment of wild-type mice. BDNF is known to promote survival and differentiation of neurons and plays a key role in modulating activity-dependent neuronal plasticity (Black 1999). BDNF can also evoke excitatory actions even at low concentrations (Kafitz et al. 1999). Moreover, Brünig et al. (2001) demonstrated a fast down-regulation of GABAA receptor surface expression levels by BDNF. Thus, the down-regulation of the BDNF transcript after an acute dose of diazepam may reduce BDNF-induced excitatory responses and attenuate GABAA receptor turnover. Apart from BDNF, the transcription factor GIF was down-regulated after acute diazepam treatment. This zinc-finger protein is highly expressed in cerebral cortex, hippocampus and cerebellum. It contains putative MAP kinase target sites and is suggested to play a role in the negative transcriptional regulation of GDNF- and TGF-β- initiated cascades (Yajima et al. 1997). The diazepam-induced down-regulation of NGFI-A, a zinc-finger transcription factor (also known as Krox-24 or EGR-1) is likely to affect a variety of target genes regulating synaptic plasticity (Jones et al. 2001). The down-regulation of the c-fos transcript was in line with a previously reported diazepam-induced reduction of c-Fos protein (Salminen et al. 1996).

MAP kinase pathway

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. RNA isolation and cRNA target preparation for oligonucleotide arrays
  6. Data analysis
  7. Quantitative real-time PCR assay
  8. Results
  9. Diazepam-induced transcript changes in wild-type mice
  10. Diazepam-induced expression profile in α1(H101R) animals
  11. Confirmation of transcript changes by quantitative real-time PCR in wild-type and α1(H101R) mice
  12. Discussion
  13. Diazepam-induced transcript changes
  14. Change in calcium responsiveness
  15. Neurotrophic responses
  16. MAP kinase pathway
  17. Time-course
  18. Acknowledgements
  19. Supplementary Material
  20. References

MAP kinase phosphatase-1 (MKP-1, also termed Dusp-1, 3CH134 and PTPN16) a dual-specific protein phosphatase (Sun et al. 1993) is considered to be a key inhibitory element in controlling the MAP kinase-activated pathways and to selectively inactivate ERK, JNK and p38 kinases (Brondello et al. 1997). MKP-1 was significantly down-regulated in the cerebral cortex of wild-type but not in α1(H101R) mice pointing to an enhancement of the MAP kinase pathway following acute diazepam treatment. Interestingly, also MKP3 (Dusp-6), another member of dual-specific protein phosphatases was significantly down-regulated in the wild-type mice (Table 1) according to the absolute analysis (16 h time-point, n = 8).

Time-course

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. RNA isolation and cRNA target preparation for oligonucleotide arrays
  6. Data analysis
  7. Quantitative real-time PCR assay
  8. Results
  9. Diazepam-induced transcript changes in wild-type mice
  10. Diazepam-induced expression profile in α1(H101R) animals
  11. Confirmation of transcript changes by quantitative real-time PCR in wild-type and α1(H101R) mice
  12. Discussion
  13. Diazepam-induced transcript changes
  14. Change in calcium responsiveness
  15. Neurotrophic responses
  16. MAP kinase pathway
  17. Time-course
  18. Acknowledgements
  19. Supplementary Material
  20. References

The transcript changes of BDNF, MKP-1, GIF, c-fos and NGFI-A, observed after an acute dose of diazepam, were transient with a return to normal levels 40 h after drug administration. In contrast, the CaMKIIα transcript was rapidly reduced (6 h time-point) and persisted on this level even at 40 h after diazepam treatment (Fig. 1). Since in this analysis all three time points after diazepam treatment were compared to the 16 h time point after vehicle treatment, it cannot be excluded that circadian changes in gene expression of time-dependent vehicle-induced changes in gene expression might also have contributed to these findings. The long-term alteration of CaMKIIα may be the initial signal in the resetting of the neuronal responsiveness, which may underlie rebound phenomena after acute treatment (Kales et al. 1978) and may contribute to the development of tolerance and dependence under chronic treatment. It is noteworthy that molecular and cellular adaptations to chronic antidepressant treatment include changes of the same signaling pathways albeit in opposite directions. Antidepressant treatment leads to an up-regulation of BDNF as well as to an activation of CaMKII (Shirayama et al. 2002). Thus, the diazepam-dependent signaling pathways identified in the present study are, at least in part, known to be able to contribute to drug-induced adaptive plasticity.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. RNA isolation and cRNA target preparation for oligonucleotide arrays
  6. Data analysis
  7. Quantitative real-time PCR assay
  8. Results
  9. Diazepam-induced transcript changes in wild-type mice
  10. Diazepam-induced expression profile in α1(H101R) animals
  11. Confirmation of transcript changes by quantitative real-time PCR in wild-type and α1(H101R) mice
  12. Discussion
  13. Diazepam-induced transcript changes
  14. Change in calcium responsiveness
  15. Neurotrophic responses
  16. MAP kinase pathway
  17. Time-course
  18. Acknowledgements
  19. Supplementary Material
  20. References

We thank Hanns Möhler for his help in initiating this study, invaluable discussions and critical reading of the manuscript. We also thank Sabine Küttel for excellent technical assistance. This work was supported by a Swiss Federal Institute of Technology (ETH Zürich) grant TH-16, 01–1 and the NCCR (National Center of Competence in Research), Neural Plasticity and Repair of the Swiss National Science Foundation.

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  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. RNA isolation and cRNA target preparation for oligonucleotide arrays
  6. Data analysis
  7. Quantitative real-time PCR assay
  8. Results
  9. Diazepam-induced transcript changes in wild-type mice
  10. Diazepam-induced expression profile in α1(H101R) animals
  11. Confirmation of transcript changes by quantitative real-time PCR in wild-type and α1(H101R) mice
  12. Discussion
  13. Diazepam-induced transcript changes
  14. Change in calcium responsiveness
  15. Neurotrophic responses
  16. MAP kinase pathway
  17. Time-course
  18. Acknowledgements
  19. Supplementary Material
  20. References
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