Calcyon mRNA expression in the frontal-striatal circuitry and its relationship to vesicular processes and ADHD

Prepubertal (three-week-old), adolescent (five-week-old) and young adult (ten-week-old) male SHR and WKY rats (Charles River Laboratories, Germany) were used. The animals arrived in the laboratory one week before the experiment and were housed in groups of the same strain in standard plastic cages (Type IV Makrolon^®), under controlled conditions of light: dark cycle (12:12 h, lights on at 07:00 h). Food and water were available ad libitum. The experiments were approved by the Animal Research Ethics Committee of Stockholm and the National Institute of Health Guide on Use of Laboratory Animals.

SHR (SHR/NCrI) were developed by Okamoto and Aoki at the Kyoto School of Medicine in 1963, from an outbred WKY male with marked elevated blood pressure mated to a female with slightly elevated blood pressure. Brother × sister matings with continued selection for spontaneous hypertension were then transferred to NIH in 1966 from Okamoto at F13, and to Charles River Laboratories from NIH in 1973 at F32 and were caesarean rederived in 1973. WKY (WKY/NCrI) rats originated from outbred Wistar stock transferred from Kyoto School of Medicine to NIH in 1971. This is the same stock from which the SHR strain was developed. They were transferred to Charles River Laboratories in 1974 from NIH, at F11 and caesarean rederived in 1974.

Antisense and sense cRNA probes for calcyon were prepared from a 500 base pair Bgl II fragment of rat calcyon cDNA cloned in vector pGEM7zf+ as previously described [8]. The plasmid was linearized with Sac I or EcoRI and then transcribed using T7 (antisense) and SP6 (sense) RNA polymerases, respectively. In vitro transcription was carried out using the MAXIscript™ SP6/T7 kit (Applied Biosystems, Sweden) and [α³⁵S]-UTP (SJ603, 20 mCi/ml; GE Healthcare, Sweden) according to the manufacture's instructions. The transcripts were purified using NucAway™ Spin Columns (Applied Biosystems, Sweden).

Expression of calcyon mRNA in the prefrontal cortex and striatum was investigated using in situ hybridization technique. Brains were rapidly dissected and frozen on dry ice. Coronal sections (14 μm) of the above areas were prepared on a cryostat and stored at -80°C until use. The in situ hybridization was performed as follows. The frozen tissue sections were fixed in cold 4% paraformaldehyde in 0.1 M sodium phosphate buffer pH 7.4 (PBS) for 10 min. After washing with PBS for 5 min, the sections were rinsed in DEPC-H₂0 (5 min) and deproteinated with 0.1 M HCl for 5 min. The sections were then rinsed twice with PBS (3 min each) and placed into 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0) for 20 min at room temperature; washed twice in PBS (3 min each) and dehydrated in 70%, 80% and 100% (2 min each). Sections were air dried and prehybridized [50% deionized formamide (pH 5), 50 mM Tris-HCl, pH 7.6, 25 mM ethylene-diamine-tetraacetate (EDTA), pH 8.0, 20 mM NaCl, 0.25 mg/ml yeast tRNA, 2.5 × Denhardt's solution] for 4 h at 55°C. After draining off the prehybridization buffer, sections were hybridized overnight (14–16 h) in a humidified chamber at 55°C. For hybridization, labeled probes were diluted to a final concentration of 1.0 × 10⁶ c.p.m./200 μL in a solution containing 50% deionized formamide (pH 5), 0.3 M NaCl, 20 mM Tris-HCl (pH 7.6), 5 mM EDTA (pH 8.0), 10 mM PBS, 0.2 mM dithiothreitol, 0.5 mg/ml yeast tRNA, 0.1 mg/ml poly-A-RNA, 10% dextran sulfate, and 1× Denhardt's solution. After hybridization, the slides were rinsed in 1 × standard saline citrate (SSC), 0.01% SDS (15 min); 1 × SSC, 0.01% SDS (30 min); 50% formamide/0.5 × SSC (1 h); 1 × SSC, and 0.01% SDS (15 min) at 55°C with continuous shaking. The sections were then treated with 1 μg/mL RNase A (Roche, Sweden) in RNase buffer (0.5 M NaCl, 10 mM Tris-HCl, 5 mM EDTA, ph 8.0) for 1 h at 37°C. After two additional washes in 1 × SSC, 0.01% SDS for 30 min, the sections were dehydrated in ascending alcohol series and air dried. Sections were placed against β-Max film (VWR, Sweden) and stored at room temperature for 3 to 5 days. Films were developed in D19 developer for 2 min and in 1:5 dilution of Amfix fixative for 5 min. Non-specific hybridization was determined by incubating sections with the respective ³⁵S-UTP-labelled sense cRNA probe for the above cDNA under identical conditions to that of the antisense RNA probe.

Films were scanned with an Epson Perfection 1250 scanner as gray scale film, using 300 pixels and saved as high quality JPEG files. Optical density values were quantified using appropriate software (NIH Image J version 1.29, U.S. National Institutes of Health). A ¹⁴C step standard (GE Healthcare, Sweden) was included to calibrate optical density readings and convert measured values into nCi/g. Optical density measurements were averaged from two adjacent sections per animal and region of interest for statistical analyses. All comparisons between groups were made on sections hybridized together, under identical conditions and exposed for the same periods of time to β-Max film (VWR, Sweden).

Anatomical regions were identified and subdivided for densitometric analysis according to the stereotaxic atlas of Paxinos and Watson [18]. The rat prefrontal cortex consists of two spatially separated areas; namely, the medial and orbital regions [19]. The medial prefrontal cortex can be divided into infralimbic (IL), prelimbic (PrL), dorsal and ventral anterior cingulate, and medial precentral cortical area (PrCm). The orbital frontal cortex can be divided into medial, ventral and lateral orbital cortices, and agranular insular cortex. Our measurements of the orbital frontal cortex contained both the ventral and lateral orbital cortices, and for the medial prefrontal cortex contained the IL, PrL and cingulate (approximately + 2.6 mm posterior to bregma). The measurements of the motor cortex were taken from primary motor cortex (M1) (approximately +1.6 mm posterior to bregma). The measurements of the nucleus accumbens were taken from the shell region (approximately +1.6 mm posterior to bregma). The measurements from the rostral, middle, and caudal striatum were taken at approximately +1.6, +0.7, and -0.4 mm posterior to bregma, respectively. For further details see [20].

Statistical analysis of mRNA expression was performed using factorial ANOVA (STRAIN and AGE as main factors; each STRAIN × AGE group contained 5 animals as replicates) for each brain region. The pair-wise post-hoc comparisons were made using the Bonferroni/Dunn test. For all analyses, significance was assigned at the P < 0.05 level. All data are presented as means ± S.E.M.

The dataset from the Mouse Atlas of Gene Expression [21] was downloaded from Gene Expression Omnibus as GEO Series #4726. For each gene (a total of 11,328 genes from ENSEMBL mouse genome version) in each sample, its relative expression level was used, i.e. a log-transformed ratio of the SAGE tag abundance in the sample to its average abundance across all the samples. This normalization was needed to compute Pearson linear correlation coefficients. The correlation coefficients between mRNA expression profiles were computed across developing and adult brain tissue samples (missing observations excluded pair-wise). For comparison, alternative co-expression values were calculated identically across all the tissues of the Atlas. Pearson coefficients exceeding r > 0.55 (P < 0.001) for mRNA expression correlations were used to link genes with the tightest connections to each other or to calcyon. However, any two given genes may not show direct links in such a network. Indeed, even if A <-> B and B <-> C pairs are strongly correlated, A <-> C may be more weakly correlated and thus not discernable. Correlations between calcyon and genes of particular interest were calculated for each of the Mouse Atlas genes. The genes represented as color-marked nodes (see results section) are: dopamine receptors, clathrin chains, ADHD-associated genes from OMIM database, and a group of genes we found co-expressed with calcyon as human orthologs (found at InParanoid resource, [22]) in the Human Tissue Atlas ([23]; correlation values not shown). In total, more than 900,000 gene pairs were analyzed for functional linkage. Hence, even if a direct connection were not visible, it could be discovered via neighbors that share genes in the network.

A number of genes did not have valid data in the Mouse Atlas (e.g., the two clathrin light chains and dopamine receptors 3, 4, and 5). Some functional links may also exist but did not exceed the threshold we selected (r > 0.55) for determining the presence of mRNA co-expression. As has been shown, one of a pair of interacting genes can be expressed permanently but the joint activity can be regulated by transient expression of the other gene [24]. Such protein pairs would not be detected in our co-expression network analyses. Finally, the absence of some genes in sub-networks may be explained by low sensitivity (i.e., only a minority of functionally related gene pairs can usually be found by means of pure mRNA co-expression).

All mouse genes used in the co-expression network analysis are spelled according to the Mouse Genome Informatics (MGI) data base. For example, the mouse dopamine D1 receptor is abbreviated as Drd1a, while the human or rat dopamine D1 receptor is abbreviated as DRD1. Images were prepared with the network visualization tool Medusa [25].

All regional analyses consisted of two-way factorial ANOVAs with main factors STRAIN, DF = [1, 24] and AGE, DF = [2, 24] and STRAIN × AGE, DF = [2, 24]. ANOVA of the medial prefrontal cortex revealed a significant main effect of STRAIN [F = 130.3; P < 0.0001] and AGE [F = 32.9; P < 0.0001], but failed to reveal a STRAIN × AGE interaction. Post-hoc analysis with the Bonferroni/Dunn test showed that the three-, five- and ten-week old SHR expressed significantly (P < 0.0001) higher levels of calcyon mRNA when compared to WKY rats of the same age (see Figs. 1A and 2). In both strain of rats, the three-, and five-week old rats expressed significantly (P < 0.05) higher levels of calcyon mRNA than the ten-week-old rats.

ANOVA of the orbital cortex revealed a significant main effect of STRAIN [F = 126.6; P < 0.0001] and AGE [F = 34.9; P < 0.0001], but failed to reveal a STRAIN × AGE interaction. Similar to the medial prefrontal cortex, the three-, five- and ten-week old SHR were found to express significantly (P < 0.0001) higher levels of calcyon mRNA when compared to WKY rats of the same age (see Figs. 1B and 2). Moreover, in both strain of rats, calcyon mRNA expression was significantly (P < 0.0001) higher in the three-, and five-week-old rats than in the ten-week old rats.

ANOVA of the primary motor cortex revealed a significant main effect of STRAIN [F = 10.0; P = 0.004] and AGE [F = 25.9; P < 0.0001], but failed to reveal a STRAIN × AGE interaction. Post-hoc analysis showed that the three-, and five-week-old SHR expressed a rather small but significant (P < 0.05) increase in calcyon mRNA when compared to the WKY rats of the same age (see Figs. 1C and 2). In both strains of rats, the three-, and five-week-old rats expressed significantly (P < 0.05) higher levels of calcyon mRNA than the ten-week-old rats.

ANOVA of the rostral, middle, and caudal striatum revealed a significant main effect of STRAIN ([F = 8.2; P = 0.009], [F = 8.4; P = 0.008], and [F = 14.3; P = 0.001], respectively) and AGE ([F = 9.5; P = 0.001], [F = 23.2; P < 0.0001], and [F = 21.4; P < 0.001], respectively), but failed to reveal a STRAIN × AGE interaction. In both the rostral and middle striatum, post-hoc analysis showed that the five-week-old SHR expressed a rather small but significant (P < 0.05) increase in calcyon mRNA when compared to WKY rats of the same age (see Figs. 1D and 2). In addition, in both strain of rats the three-week-old rats expressed significantly (P < 0.05) higher calcyon mRNA levels than the ten-week-old rats. In the caudal striatum, post-hoc analysis showed that the three-, and five-week-old SHR expressed a rather small but significant (P < 0.05) increase in calcyon mRNA when compared to WKY rats of the same age (see Figs. 1E and 2). In both strains of rats the three-, and five-week-old rats expressed significantly (P < 0.05) higher calcyon mRNA levels than the ten-week-old rats.

ANOVA of the nucleus accumbens (shell region) revealed a significant main effect of STRAIN [F = 9.3; P = 0.005] and AGE [F = 17.9; P < 0.0001], but failed to reveal a STRAIN × AGE interaction. Post-hoc analysis showed that the three-, and five-week-old SHR expressed a rather small but significant (P < 0.05) increase in calcyon mRNA when compared to WKY rats of the same age (see Figs. 1F and 2). In addition, in both strain of rats the three-, and five-week-old rats expressed significantly (P < 0.05) higher calcyon mRNA levels than the ten-week-old rats.

In order to shed more light on calcyon function, we explored its functional connections to other genes by performing network analyses. We reasoned that the most informative pattern would be revealed by analyzing a co-expression network in brain tissue. The Mouse Atlas of Gene Expression [21] was suitable for this purpose, as it contains the expression patterns of 11,328 genes. For a confident co-expression analysis, one should use datasets in which most of the genes have been observed in multiple expression conditions. We were aware of 13 large expression sets in human, mouse, and rat. Calcyon is a relatively novel and poorly studied gene. Thus apart from the Mouse Tissue Atlas, only one Human Atlas ([23]; considered in the paper as well) had a calcyon expression profile – but this atlas did not have enough various brain tissue conditions.

While analyzing functional links in the co-expression network, special attention was paid to genes previously suggested as being related to calcyon. Figure 3 shows the calcyon network and the potentially related genes in the developing and adult mouse brain tissues. No significant links were found between calcyon and dopamine receptors Drd2 and Drd1a (the latter previously having been shown to interact with calcyon), even when indirect connections via other genes were considered. The dopamine receptors Drd3, Drd4, and Drd5 were not present in the database. Several ADHD-associated genes with available mRNA profiles did not relate to calcyon either. However, multiple, and mostly strong, connections were revealed between calcyon and genes whose annotations suggested that they are involved in synaptic plasticity, endocytosis and/or vesicle formation (e.g. clathrin heavy chain, and the ionotropic glutamate receptor, AMPA1) (see Fig. 4 and Table 1). A number of genes in this calcyon-related cluster were novel, as indicated by absence of any annotation or gene names (0710005I19Rik, 2900002G04Rik, 2900011O08Rik, 5330410G16Rik, A030009H04Rik); others were only sparsely documented (Aplp1, Pja2, and Rusc1).