These authors contributed equally to this work.
Gene expression in accumbens GABA neurons from inbred rats with different drug-taking behavior
Article first published online: 1 AUG 2011
© 2011 The Authors. Genes, Brain and Behavior © 2011 Blackwell Publishing Ltd and International Behavioural and Neural Genetics Society
Genes, Brain and Behavior
Volume 10, Issue 7, pages 778–788, October 2011
How to Cite
Sharp, B. M., Chen, H., Gong, S., Wu, X., Liu, Z., Hiler, K., Taylor, W. L. and Matta, S. G. (2011), Gene expression in accumbens GABA neurons from inbred rats with different drug-taking behavior. Genes, Brain and Behavior, 10: 778–788. doi: 10.1111/j.1601-183X.2011.00716.x
- Issue published online: 9 OCT 2011
- Article first published online: 1 AUG 2011
- Accepted manuscript online: 11 JUL 2011 12:00AM EST
- Received 25 February 2011, revised 18 April 2011 and 23 May 2011, accepted for publication 1 July 2011
- Fisher 344 rats;
- laser-capture microdissection;
- Lewis rats;
- nucleus accumbens;
- ventral pallidum
Inbred Lewis and Fisher 344 rat strains differ greatly in drug self-administration; Lewis rats operantly self-administer drugs of abuse including nicotine, whereas Fisher self-administer poorly. As shown herein, operant food self-administration is similar. On the basis of their pivotal role in drug reward, we hypothesized that differences in basal gene expression in GABAergic neurons projecting from nucleus accumbens (NAcc) to ventral pallidum (VP) play a role in vulnerability to drug-taking behavior. The transcriptomes of NAcc shell-VP GABAergic neurons from these two strains were analyzed in adolescents, using a multidisciplinary approach that combined stereotaxic ionotophoretic brain microinjections, laser-capture microdissection (LCM) and microarray measurement of transcripts. Laser-capture microdissection enriched the gene transcripts detected in gamma-aminobutyric acid (GABA) neurons compared to the residual NAcc tissue: a ratio of neuron/residual >1 and false discovery rate (FDR) <5% yielded 6623 transcripts, whereas a ratio of >3 yielded 3514. Strain-dependent differences in gene expression within GABA neurons were identified; 322 vs. 60 transcripts showed 1.5-fold vs. 2-fold differences in expression (FDR < 5%). Classification by gene ontology showed that these 322 transcripts were widely distributed, without categorical enrichment. This is most consistent with a global change in GABA neuron function. Literature mining by Chilibot found 38 genes related to synaptic plasticity, signaling and gene transcription, all of which determine drug abuse; 33 genes have no known association with addiction or nicotine. In Lewis rats, upregulation of Mint-1, Cask, CamkII, Ncam1, Vsnl1, Hpcal1 and Car8 indicates that these transcripts likely contribute to altered signaling and synaptic function in NAcc GABA projection neurons to VP.
In rats, the inbred Lewis and Fisher 344 strains differ greatly in drug self-administration (SA) behavior; while Lewis rats operantly self-administer several drugs of abuse, including nicotine, Fisher rats self-administer poorly (Brower et al. 2002; Martin et al. 1999; Suzuki et al. 1988a,b). The nucleus accumbens shell (NAcc shell), located in ventral striatum, and its primary output target, the ventral pallidum (VP), are both critical to the integration and discrimination of information encoding the rewarding dimension of motivated behavior (Carlezon & Thomas 2009; Heimer & Wilson 1975; Shirayama & Chaki 2006; Smith et al. 2009). The vast majority of neurons in the NAcc are GABAergic medium spiny projection neurons (Meredith et al. 1993). The normal function of VP is essential for aspects of reward learning and to motivational ‘wanting’ and hedonic ‘liking’ (Smith et al. 2009). On the basis of their pivotal role in reward (Carlezon & Thomas 2009; Meredith et al. 1993), we hypothesized that differences in basal gene expression by these GABAergic neurons in the Lewis vs. Fisher 344 strains may contribute to drug-taking vulnerability, which for cigarette smoking is usually manifest during adolescence (Chassin et al. 1996; Dappen et al. 1996; SAMHSA 2001).
Differences in the gene expression profiles or transcriptomes between the NAcc shell-VP GABAergic neurons from these two strains were analyzed in adolescents [postnatal day 41 (PN41)]. We used a multidisciplinary approach, combining stereotaxic ionotophoretic brain microinjections, laser-capture microdissection (LCM) and microarray detection of transcript expression levels. Neuroanatomical specificity was obtained by ionotophoretic delivery of fluorogold (FG) into VP (Pieribone & Aston-Jones 1988) followed by capture of all FG+ neurons in sections from the NAcc shell by LCM. The efficacy of this approach was evaluated by measuring the ratio of gene expression for neurons compared to the remaining NAcc tissue after removal of the neurons. RNA extracted from both neurons and remaining NAcc was amplified and then analyzed by microarray. Differential gene expression by rat strain was validated for a subset of genes by real-time polymerase chain reaction (RT-PCR).
Animals and surgeries
Lewis rats were bred in the laboratory, using breeders purchased from Harlan Laboratories (Indianapolis, IN, USA). Adolescent male Lewis rats (PN35) were anesthetized with ketamine/xylazine (90 and 10 mg/kg, respectively, i.p.) and then placed in a stereotaxic frame. Fluorogold (2% in sterile saline, Fluorochrome, Englewood, CO, USA) was deposited into VP (Anterior–Posterior: −0.22, Medial–Lateral: ±2.2, Dorsal–Ventral: −8.0) bilaterally by ionotophoretic injection, using a Midgard high-voltage current source (Stoelting Co., Wood Dale, IL, USA). The current was set at +5 µA (7 seconds on, 7 seconds off, for 5 min). Micropipettes were left in place for 15 min before the scalp incision was sutured. Five days later, rats were killed, brains removed, immediately frozen, and stored at −70°C. All procedures were conducted in accordance with the NIH Guidelines Concerning the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of the University of Tennessee.
Chronic nicotine self-administration
Rats were acclimated to a reversed 12:12 h light:dark cycle. Standard rat chow and water were provided ad libitum. Nicotine (pH = 7.2; calculated as free base) was prepared weekly in 200 U heparinized saline to deliver 50 µl i.v. of 0.03 mg/kg body weight per injection. Self-administration was performed according to our published protocol (Valentine et al. 1997). Briefly, PN38 adolescent rats received jugular cannulae under xylazine-ketamine anesthesia and were individually housed in operant chambers (Coulbourn Instruments, Whitehall, PA, USA) inside a sound-attenuating chamber. Operant chambers were equipped with two levers positioned 4 cm above the floor, and a green cue light 1 cm above each lever was illuminated when nicotine was available. Lever presses were recorded and syringe pumps controlled by computers and interfaces, using L2T2 or Graphic State software (Ver 3.01, Coulbourn Instruments). Rats were given 3 days to recover from jugular surgery, during which time they received antibiotic daily and hourly aliquots (50 µl) of 200 U heparin. At PN41, rats received 0.03 mg/kg nicotine in heparinized saline i.v. with each lever press.
One lever was randomly designated as the active lever. Pressing this lever elicited a computer-driven i.v. injection of nicotine over 0.81 seconds on a fix ratio 1 (FR1) schedule. Each injection was followed by a 7-second period during which the green cue light was extinguished and lever presses were recorded, but nicotine was not injected. Pressing the inactive lever had no programmed consequence. Rats were given access to nicotine or saline SA 23 h/day and learned to self-administer nicotine without prior training, priming or food deprivation. The final hour of the lights-on cycle was reserved for housekeeping tasks. During this interval, environmental enclosure doors were opened and green cue lights turned off; levers were not retracted, and lever press activity was not recorded nor rewarded during this period. Self-administration day 1 (SA d 1) is the first day nicotine was made available and the rats were given 10 days unlimited free access to acquire nicotine SA.
Beginning on PN41, a separate cohort of adolescent male rats were trained on an FR1 schedule to self-administer food pellets (45 mg) during daily 2-h sessions in the same operant chambers used for nicotine SA. Animals were food deprived overnight prior to the first session. Thereafter, they received 16 g food per day.
Brains were sectioned in a Leica cryostat at 10 µm, and sections were mounted onto uncharged glass slides and maintained at −20°C until dehydration. Slides were dehydrated by sequential immersion in the following: 100% methanol (3 min), 95% EtOH (2 min), 100% EtOH (1 min, twice) and xylene (5 min, twice), and then air dried (15 min). The Arcturus XT (Life Technologies, Carlsbad, CA, USA) was used to capture FG-labeled neurons. The infrared laser produced spots approximately 15 µm in diameter, which allowed consistent capture of neurons onto CapSure LCM caps (Life Technologies). Approximately 750 neurons were captured from each brain.
RNA trapped in the CapSure LCM caps was extracted using the PicoPure RNA isolation kit (Life Technologies); RNA was eluted with 13 µl nuclease-free H2O. RNA quality was analyzed using Bioanalyzer (Agilent Technologies, Foster City, CA, USA). The mean value of RNA Integrity Number (RIN) was 7.9 for the 1.84 ± 2.4 ng of total RNA obtained from each rat.
RNA amplification and Affymetrix microarray
Total RNA were amplified by NuGen Ovation Pico WTA system, followed by cDNA synthesis using the WT Ovation Exon module. Ovation Biotin kits were then used to fragment and label the cDNA (Ovation reagents: NuGen Inc., San Carlos, CA, USA). The labeled cDNA was then hybridized to the Rat Gene ST 1.0 array (Affymetrix, Santa Clara, CA, USA), according to the manufacturer's protocols for whole transcriptome arrays. Microarrays were then processed on a Fluidics Station 450 and scanned by the Affymetrix GeneChip 3000 7G Scanner. Expression Console software (version 1.1.2) was used to normalize data. All microarray samples were processed by the same technician consecutively, interleaving the two strains. No batch effect was observed. The Rat Gene ST 1.0 array probe sets are generated from the Brown Norway rat genome. Therefore, false-positive differences between Lewis and Fisher rat samples may occur because of the asymmetrical expression of single nucleotide polymorphisms (SNPs) by these two strains, differentially affecting cDNA hybridization to array probe sets.
Real-time quantitative PCR of eight genes (and two reference genes Ywhaz and Sdha) was performed on the LightCycler 480 (Basel, Switzerland) using probes from the Universal Probe Library. The original unamplified RNA from each sample was used to prepare cDNA. The critical threshold (Ct) of genes of interest was normalized to the geometric mean of the two reference genes.
Microarray data were analyzed using the R-statistical package (Team 2010). t-Tests were used to compare the expression level of each gene from the two strains of rats and then all genes were analyzed for false discovery rate (FDR). Gene ontology analysis was conducted using DAVID Bioinformatics Resources (Huang et al. 2009). Nicotine and food SA data were analyzed using repeated-measures analysis of variance (anova), with both lever and day treated as within-subject variables. Strain was treated as a between-subject variable.
The capacity to acquire operant nicotine vs. food SA was evaluated in adolescent Lewis compared to Fisher rats. For nicotine SA, rats were housed in operant chambers and were not shaped, primed or food deprived. Three days after jugular vein surgery, nicotine was made available for 23 h per day. Figure 1 (data analyzed by repeated-measures anova) indicates that Lewis rats rapidly acquired nicotine SA, manifest by a significant increase in active lever presses in the absence of any change in the inactive lever (effect of lever on number of presses, F1,7: 18.2, P < 0.001; effect of day on active lever presses, F9,63: 2.1, P < 0.05; effect of day on inactive lever presses, F9,63: 0.8, P > 0.05). In contrast, both active and inactive lever presses by Fisher rats failed to increase over time (F9,81: 1.5, P > 0.05). The number of lever presses was different between strains (F1,16: 111.8, P < 0.001) and the interaction between lever and strain was significant (F1,15: 17.5, P < 0.001). Thus, the number of active lever presses for nicotine was different between strains (F1,16: 66.6, P < 0.001), as was the number of inactive lever presses (F1,16: 53.5, P < 0.001). The higher number of inactive lever presses in Lewis rats is likely the result of stronger locomotor activation induced by a larger amount of nicotine intake. Both strains showed a specific increase in active lever presses reinforced by food (Fisher, F1,7: 47.1, P < 0.001; Lewis, F1,5: 35.1, P < 0.001). Neither active lever presses for food (F1,12: 0.7, P = 0.41) nor inactive lever presses (F1,12: 0.7, P = 0.41) differed by strain. The similar amount of inactive lever presses reflected similar level of arousal induced by food. Hence, both strains were capable of learning the operant task, but nicotine was only reinforcing in Lewis rats, under these conditions.
Neurons projecting from NAcc shell to VP were identified by retrograde uptake of FG delivered by ionotophoresis. Figure 2 illustrates coronal sections of rat brain, identifying both NAcc shell and VP. Within VP, the central area of FG fluorescence for each of six rats is marked by an asterisk. A representative deposit of FG is shown in the inset. Within NAcc shell, the center of the distribution of retrograde-labeled FG+ neurons for each rat is shown by a green dot within the boxed area. The corresponding inset shows a representative fluorescence image of these FG+ neurons, located in ventral medial NAcc shell (20×). In this region of NAcc shell, virtually all neurons are FG+. All individual FG+ neurons were captured by LCM. Thus, this approach specifically captured a subpopulation of NAcc shell neurons that project from NAcc shell to VP, excluding neurons that project from NAcc shell to other brain regions.
Microarray analysis was used to detect the level of specific gene transcripts expressed by the transcriptome from laser-captured FG+ neurons compared to the tissue remaining in the area of NAcc shell after removal of these fluorescent neurons. A heat map of all genes was generated; Fig. 3a shows representative areas that show clustering of genes expressed at significantly greater or lesser levels in neurons vs. tissue remainder. At a ratio of neuron/remainder >1 and FDR < 5%, 6623 gene transcripts were identified in both Lewis and Fisher rats; at ratios >2.0 and >3.0, 5483 and 3514 genes, respectively, were enriched in the neuron populations from both inbred rat strains (Fig. 3b). Amongst the enriched transcripts, Table 1 shows those related to the neurotransmitter, GABA (e.g. GABA A receptor alpha 1, glutamate decarboxylase 1 and 2), and/or associated with regulation of GABA neuron-related functions (e.g. dopamine receptors D1A and D2; GABA vesicular transporter). These data indicate that LCM of individual FG+ neurons successfully enriched transcripts from GABA neurons projecting to VP. At three incremental levels of enrichment (i.e. neuron/remainder >1 to >3), the majority of these transcripts were common to both strains of rat. Nonetheless, LCM unavoidably introduces glial cells, in intimate contact with FG+ neurons, within each sample captured. For example, myelin basic protein and glial fibrillary acidic protein, standard markers for oligodendrocytes and astrocytes, respectively, were detected at neuron/remainder ratios of 2.7 and 1.5. This enrichment of glial markers indicates that targeting most of the neuronal soma for capture, by using 15-µm-diameter spot size, eliminates the ability to separate the neuronal vs. glial transcriptomes. This limitation of geometric capture by LCM of a high heterogeneous tissue was recently reported (Okaty et al. 2011).
|Symbol||Gene name||Neuron/remainder (mean of both strains)||Neuron fluorescence intensity (mean of both strains)|
|Akap9||A kinase (PRKA) anchor protein (yotiao) 9||5.18||7.28|
|Dbi||Diazepam binding inhibitor (GABA receptor modulator acyl-Coenzyme A binding protein)||8.25||8.43|
|Drd1a||Dopamine receptor D1A||9.83||9.20|
|Drd2||Dopamine receptor D2||2.74||6.81|
|Gabarap||GABA(A) receptor-associated protein||10.55||9.41|
|Gabarapl1||GABA(A) receptor-associated protein like 1||9.18||8.73|
|Gabarapl2||GABA(A) receptor-associated protein like 2||6.01||8.03|
|Gabarapl2||GABA(A) receptor-associated protein like 2||5.32||7.78|
|Gabra1||Gamma-aminobutyric acid (GABA) A receptor alpha 1||3.42||7.48|
|Gabra2||Gamma-aminobutyric acid (GABA) A receptor alpha 2||3.90||6.69|
|Gabra4||Gamma-aminobutyric acid (GABA) A receptor alpha 4||10.74||8.73|
|Gabra5||Gamma-aminobutyric acid (GABA) A receptor alpha 5||1.72||5.97|
|Gabrb1||Gamma-aminobutyric acid (GABA) A receptor beta 1||2.92||6.24|
|Gabrb2||Gamma-aminobutyric acid (GABA) A receptor beta 2||6.04||7.45|
|Gabrb3||Gamma-aminobutyric acid (GABA) A receptor beta 3||6.90||8.87|
|Gabrg1||Gamma-aminobutyric acid (GABA) A receptor gamma 1||3.32||6.68|
|Gabrg2||Gamma-aminobutyric acid (GABA) A receptor gamma 2||5.37||7.92|
|Gabrg3||Gamma-aminobutyric acid (GABA) A receptor gamma 3||4.11||7.24|
|Gabbr1||Gamma-aminobutyric acid (GABA) B receptor 1||3.93||7.42|
|Gabbr2||Gamma-aminobutyric acid (GABA) B receptor 2||1.64||5.63|
|Gad1||Glutamate decarboxylase 1||2.23||6.69|
|Gad2||Glutamate decarboxylase 2||6.44||9.25|
|Slc32a1||Solute carrier family 32 (GABA vesicular transporter) member 1||6.41||8.38|
|Slc6a1||Solute carrier family 6 (neurotransmitter transporter GABA) member 1||3.21||6.86|
|Slc6a11||Solute carrier family 6 (neurotransmitter transporter GABA) member 11||2.90||6.59|
|Snap25||Synaptosomal-associated protein 25||14.77||11.79|
|Stxbp1||Syntaxin binding protein 1||9.05||9.15|
Genes that were differentially expressed in Lewis vs. Fisher NAcc shell GABA neurons projecting to VP were identified. One caveat about array analysis across strains concerns the potential for false positives because of asymmetrical expression of SNPs by the two strains, differentially affecting hybridization to the array probes. Table 2 shows the total number of gene transcripts as a function of the expression ratio in Lewis compared to Fisher rats (i.e. >1.5-fold and >2-fold); total transcript number declined with the application of increasingly stringent criteria to achieve statistical significance. A total of 322 genes were differentially expressed at a Lewis vs. Fisher ratio of >1.5-fold (P < 0.05 and FDR < 5%; see Table S1). Of these, 174 gene transcripts could be mapped to biological processes, as shown in Fig. 4. Transcripts mapped across a wide range of biological processes and molecular functions, showing no specific enrichment by either biological or molecular ontology.
|Criteria||Number of genes|
|Lewis vs. Fisher > 1.5-fold||1210|
|t-Test P < 0.05||608|
|Lewis vs. Fisher> twofold||267|
|t-Test P < 0.05||90|
The differential gene expression detected by microarray analysis was validated by PCR using the same RNA samples. Eight gene transcripts were selected for validation. By microarray analysis, it was found that the expression ratio of these transcripts in Lewis vs. Fisher rats varied by 1.5- to 2-fold (see Fig. 5). In addition, six of the eight transcripts were expressed at higher levels in Lewis rats, whereas two (i.e. A disintegrin and metalloproteinase with thrombospondin motifs 3 and Neuromedin B) transcripts were expressed at higher levels in Fisher rats. Significant strain-dependent differences were detected by PCR analysis of all eight transcripts.
Gene interactions between the 322 differentially expressed transcripts were sought using Chilibot, an established data-mining software, to analyze relationships documented in all the abstracts compiled in PubMed (Chen & Sharp 2004). We identified genes likely to determine drug abuse behavior based on their known relationships to cell signaling, synaptic plasticity, transcription, addiction and nicotine. Table 3 shows 22 genes that mapped to synaptic plasticity, 24 to signaling, 18 to transcription, 10 to addiction and 4 to nicotine. Twenty-one of 22 genes mapping to synaptic plasticity were also related to cell signaling. However, the strength of these gene relationships to signaling vs. synaptic plasticity varied in that gene × signaling interactions were based on a median of 14.5 unique publications, whereas gene × synaptic plasticity interactions were derived from a median of 3.5 publications. Only 4 of 18 genes mapped to transcription regulation were common to signaling and synaptic plasticity. Strong interactions between gene × transcription regulation were evident based on a median of 15.5 publications. Lastly, 7 of 10 genes mapped to addiction and all 4 genes mapped to nicotine were common to signaling and synaptic plasticity. Overall, 33 of the 41 genes in Table 3 have not previously been associated with addiction or nicotine. CASK (calcium/calmodulin-dependent serine protein kinase) exemplifies a gene strongly associated with all three biological processes, but not with addiction or nicotine.
|Gene symbol||Signaling||Synaptic plasticity||Transcription regulation||Addiction||Nicotine||Gene name|
|ADRBK1||345||14||5||Adrenergic, beta, receptor kinase 1|
|AHI1||6||2||Abelson helper integration site 1|
|APBA1||15||30||15||1||Amyloid beta (A4) precursor protein-binding, family A, member 1|
|CAMK2D||8||2||136||41||15||Calcium/calmodulin-dependent protein kinase II delta|
|CAR8*||3||2||Carbonic anhydrase 8|
|CASK||30||51||16||Calcium/calmodulin-dependent serine protein kinase|
|DAAM1||25||2||Dishevelled associated activator of morphogenesis 1|
|ETFA||1||3||1||Electron-transfer-flavoprotein, alpha polypeptide|
|GRIA4||6||Glutamate receptor, ionotrophic, AMPA 4|
|GRB10||78||Growth factor receptor-bound protein 10|
|GABRG1*||1||Gamma-aminobutyric acid (GABA) A receptor, gamma 1|
|HBP1||48||HMG-box transcription factor 1|
|HMBOX1||4||Homeobox containing 1|
|ILF3||35||Interleukin enhancer binding factor 3|
|MTSS1||14||Metastasis suppressor 1|
|NCAM1||277||675||11||5||Neural cell adhesion molecule 1|
|NFATC3||76||Nuclear factor of activated T-cells, calcineurin-dependent 3|
|NMI||15||N-myc (and STAT) interactor|
|POLE3||5||Polymerase (DNA directed), epsilon 3 (p17 subunit)|
|PPP1R14A*||26||Protein phosphatase 1, regulatory (inhibitor) subunit 14A|
|PSMD7||1||Psmd7 proteasome 26S subunit, non-ATPase, 7|
|PTAFR||68||10||Platelet-activating factor receptor|
|RAD21||44||RAD21 homolog (Schizosaccharomyces pombe)|
|RICTOR||90||2||1||RPTOR independent companion of MTOR, complex 2|
|RUFY2||6||4||2||RUN and FYVE domain containing 2|
|SSH2||2||2||Slingshot homolog 2|
|SNAPC2||9||Small nuclear RNA activating complex, polypeptide 2|
|SPARC||99||21||Secreted protein, acidic, cysteine-rich (osteonectin)|
|STXBP3||1||Syntaxin binding protein 3|
|TERF1||111||Telomeric repeat binding factor (NIMA-interacting) 1|
|VLDL-R||17||4||Very low density lipoprotein receptor|
|ZCCHC12||2||4||Zinc finger, CCHC domain containing 12|
|ZFP410||6||Zinc finger protein 410|
|Number of genes||24||22||18||10||4|
|Median PubMed hits||14.5||3.5||15.5||1.5||3.5|
These studies show significant rat strain-dependent differences in the transcriptomes from neuroanatomically defined sets of enriched GABA neurons that regulate the reinforcing dimension of motivated behavior. Differences in gene expression may impact the motivational function of these GABA neurons, because Lewis vs. Fisher rats differ greatly in their propensity to operantly self-administer nicotine and other drugs (Brower et al. 2002; Martin et al. 1999; Suzuki et al. 1988a,b). In contrast, acquisition of operant food SA was similar between these strains. Therefore, differential gene expression is potentially related to drug reinforcement, rather than operant learning per se.
We combined neuroanatomical identification of specific neurons labeled with FG and laser capture of individual neurons. Fluorogold was transported from VP by GABA neurons in ventral medial NAcc shell. Despite RNA yield of only 1.8 ng/rat, requiring amplification before microarray, this protocol enriched >6600 genes, including GABA-related transcripts. Strain-dependent effects on gene expression were identified; 322 vs. 60 transcripts showed 1.5-fold vs. 2-fold expression differences (FDR 5%). Polymerase chain reaction validated the expression of selected gene transcripts. The LCM method unavoidably captures glial cells in close apposition to FG+ neurons, thus affecting the specificity of the transcriptome attributed to GABA neurons.
Classification by gene ontology showed that these 322 strain-dependent transcripts lacked categorical enrichment. This wide-ranging alteration in the transcriptome from enriched NAcc GABA neurons is most consistent with a global change in neuronal function manifest throughout the cellular machinery. As literature mining by Chilibot (Chen & Sharp 2004) did not find networks amongst these genes, all 322 were analyzed for relationships to cellular processes pivotal to drug abuse (i.e. synaptic plasticity, signaling and regulation of gene transcription). Chilibot found genes with documented relationships to these three processes (Table 3). Almost all genes related to plasticity were common to signaling, whereas only 4 of 18 transcription genes were common to signaling/synaptic plasticity. Thirty-three of 38 genes related to plasticity, signaling and/or gene transcription have no association with addiction or nicotine. On the basis of novelty in the addiction literature, magnitude of the difference in strain-dependent expression and prior knowledge of protein function, the following genes are highlighted.
Mint-1 (i.e. APBA1) has a relatively large number of associations with signaling, synaptic plasticity and transcription, but only one with addiction. Mint, an adaptor protein containing isoform-specific N-terminal sequences and common PTB and PDZ domains, is found in soma, axons and dendrites of excitatory and inhibitory neurons throughout the brain (Duclos & Koenig 1995; Okamoto & Sudhof 1997; Tanahashi & Tabira 1999). Mints bind calcium channels, potentially affecting presynaptic calcium influx (Maximov & Bezprozvanny 2002; Maximov et al. 1999). Only the N-terminus of Mint-1 binds Cask (Borg et al. 1998; Butz et al. 1998), which itself is a synaptic membrane-associated guanylate kinase that interacts with membrane receptors affecting GABA release (Ho et al. 2003). Cask is associated with all three cellular processes, but not addiction/nicotine. In hippocampal interneurons, expressing high levels of Mint-1 vs. Mint-2, Mint-1 deficiency is associated with impaired regulation of GABA release (Ho et al. 2003). Therefore, in Lewis vs. Fisher rats, the release of GABA by NAcc neurons regulating VP motivational functions might be affected by increased Mint-1-Cask heterodimers, reflecting increased basal levels of both transcripts (Lewis/Fisher ratios of Mint-1 and Cask: 1.7 and 1.6, respectively).
Cask associates with synaptic vesicles through interactions with neurexins, Mints and synaptotagmin (Biederer & Sudhof 2000; Hata et al. 1993). Neurexins are presynaptic transmembrane cell-adhesion proteins (Nam & Chen 2005) involved in initiating postsynaptic specializations at glutamate and GABA synapses (Graf et al. 2004). Cask binds and phosphorylates the cytoplasmic tail of neurexin-1, which may be essential for regulation of the presynaptic cytoskeleton (Mukherjee et al. 2008). Cask kinase activity is suppressed by neuronal activity and increased by silencing synapses. Two reports suggest that inhibitory synapses may undergo silencing (Bekkers 2005; Kilman et al. 2002). In Lewis rats, the enhanced basal expression of Cask may promote phosphorylation of neurexin concomitant to the silencing of GABA synapses involving NAcc GABA projections.
In contrast to Mint-1 and Cask, Ca2+/calmodulin-dependent protein kinase IIδ (CamkIIδ) is related to all three cellular processes and addiction/nicotine. Its expression ratio in Lewis vs. Fisher NAcc GABA neurons was 1.76 (P = 0.03, FDR 5%). CamkIIδ is present throughout rat brain (Takeuchi et al. 1999). Nuclear CamkIIδ is implicated in gene transcription, exemplified by mPer1 (Nomura et al. 2003). Dopamine D2 receptors (D2R) activate CamkIIδ to increase brain-derived neurotropic factor (BDNF) (Takeuchi et al. 2002), which is required for expression of activity-dependent inhibitory synapses (Hong et al. 2008; Yamada et al. 2002). These observations and the strain-dependent differences in basal CamkIIδ expression indicate that neuronal plasticity, involving BDNF and inhibitory synapses encoding aspects of behavioral reward, may be facilitated in Lewis rats.
Table 3 shows that neural cell adhesion molecule 1 (Ncam1) has the strongest associations with signaling and synaptic plasticity and is also associated with addiction/nicotine. Its expression ratio in Lewis vs. Fisher NAcc projection neurons was 1.54 (P = 0.023, FDR 5%). Neural cell adhesion molecules mediate extracellular interactions with matrix and cells. Membrane clustering of Ncams initiate intracellular signaling cascades implicated in synaptic plasticity (Luthl et al. 1994; Maness & Schachner 2007). By direct interaction with D2R, Ncam1 promotes internalization and degradation, regulating D2R signaling (Xiao et al. 2009). Increased Ncam1 expression appears to reduce surface D2R levels, which inhibit NAcc GABA neuron activity. Across 21 strains of recombinant inbred BXD mice, D2R and Ncam expression were associated with models of ethanol preference (Hitzemann et al. 2003). In Lewis rats, increased Ncam1 expression in NAcc GABA projection neurons is likely to enhance synaptic plasticity and alter surface expression of D2R, reducing dopamine-dependent inhibition.
Visinin-like 1 (Vsnl1; aka Vilip-1) and hippocalcin-like 1 (Hpcal1) are members of the neuronal Ca2+ sensor subfamily of Vsnl proteins, which belong to the superfamily of EF-hand Ca2+ binding proteins (Braunewell & Gundelfinger 1999). Vsnl interacts with multiple intracellular signaling cascades and affects exocytosis, modulation of adenylyl cyclase activity and regulation of both ligand- and voltage-gated ion channels (Burgoyne 2007). Vsnl proteins are cytoplasmic at resting Ca2+ concentrations, and translocate to the plasma or Golgi membrane with increased intracellular Ca2+ concentration (Spilker et al. 2002). Vsnl1 has low to moderate associations with signaling and synaptic plasticity and minimal relationships to addiction/nicotine. Similarly, Hpcal1 has relatively few relationships with signaling and synaptic plasticity, and none with addiction/nicotine. The expression ratios for Vsnl1 and Hpcal1 in Lewis vs. Fisher were relatively high: 2.03 (P = 0.023, FDR 5%) and 1.75 (P = 0.043, FDR 5%), respectively. The Vsnl1 ratio was confirmed by RT-PCR.
Vsnl1 interacts directly with the alpha4 subunit of the most abundant brain nicotinic cholinergic receptor (nAChR), alpha4beta2, increasing the surface expression of functional receptors, depending on Ca2+ concentration (Zhao et al. 2009b). In contrast, alpha7-containing nAChRs elicit a calcium-dependent membrane localization of Vsnl1 (Zhao et al. 2009a). The inward Ca2+ current induced by nicotinic stimulation of alpha7 nAChRs (Berg et al. 2006) may drive the activation and membrane localization of Vsnl1 that is associated with upregulation of alpha4beta2 nAChRs. The brain-region-specific upregulation of alpha4beta2 nAChRs has been reported in rats that chronically self-administer nicotine (Moretti et al. 2010; Parker et al. 2004). Upregulation of nAChRs would likely increase neuronal sensitivity and responsiveness to nicotine (Nguyen et al. 2004). However, in NAcc GABA projection neurons, the effect of chronic nicotine exposure on the level of nAChRs is not, to our knowledge, known. Nevertheless, the twofold increased expression of Vsnl1 in Lewis vs. Fisher GABA projection neurons may contribute to the motivational effects of nicotine in Lewis rats and to their ability to acquire nicotine SA.
Carbonic anhydrase 8 (Car8) displayed the greatest difference in expression between Lewis and Fisher rats (3.36-fold; P = 0.024, FDR 5%). This was confirmed by RT-PCR. Car8, an acatalytic carbonic anhydrase, binds inositol trisphosphate [IP(3)] receptors, inhibiting binding to inositol 1,4,5-trisphosphate (Hirota et al. 2003). Thus, Car8 modulates IP(3) receptor-dependent mobilization of Ca2+ from intracellular stores, which is critical to propagation of cytosolic Ca2+ signals (Taylor & Tovey 2010). Car8 mutant mice show the essential role of Car8 in synaptogenesis and maintenance of synaptic function (Hirasawa et al. 2007). The difference in basal expression by Lewis vs. Fisher rats suggests that Car8 modulation of intracellular Ca2+ mobilization and signaling varies substantially between these inbred strains.
In summary, these studies show the feasibility of obtaining neuronal phenotype-enriched samples to ascertain strain-specific differences in gene transcript expression. On the basis of gene ontologies of 322 transcripts that differed between these two strains, there is a wide-ranging alteration in the transcriptome of GABA neurons projecting from NAcc shell to VP. This is most consistent with a global change in function. In Lewis rats, significant upregulation of Mint-1, Cask, CamkIIδ, Ncam1, Vsnl1, Hpcal1 and Car8, which participate in cellular signaling and synaptic plasticity, indicates that these gene transcripts may contribute to altered function of NAcc GABA neurons, potentially predisposing to nicotine SA in Lewis rats. However, data from a manifold of inbred strains and F1 crosses will be required to specify the potential role of individual genes.
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These studies were supported by DA-028962 (B.M.S., H.C., S.G.M.) from NIDA.
Additional Supporting Information may be found in the online version of this article:
Table S1: Genes differentially expressed in Lewis vs. Fisher nucleus accumbens shell GABA projection neurons.
Laser capture microdissection was used to obtain nucleus accumbens shell GABA neurons project to ventral pallidum labeled by Fluorogold. The gene expression profiles of these neurons were obtained using Affymetrix Rat Gene ST 1.0 microarrays. A total of 322 genes were differentially expressed at a Lewis vs Fisher ratio of >1.5-fold (P < 0.05 and FDR < 5%).
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