MASTER’S THESIS IN
MOLECULAR MEDICAL BIOLOGY
45 hp
HT2011-VT2012
Differential gene expression profiling of
chromatin-modifying enzymes and
remodeling factors in the rat motor cortex
after motor skill training
Parisa Rabiei Far
parrah101@studentmail.oru.se
Abstract
Fine motor skills are learned through repetitive practice and once learned, last for a long time. Skilled reaching is linked to structural and functional changes in multiple brain regions including, in particular, the primary motor cortex. Previous studies demonstrated that fine motor skill learning is associated with cortical synaptogenesis and motor map reorganization. At present, studies have implicated an indispensable role of epigenetic alterations in both hippocampal- and striatal-dependent memory formations, while examinations into the epigenetic changes in the primary motor cortex are lacking. The current study was aimed to identify epigenetic changes in motor cortex as a result of extensive motor skill learning using the single pellet skilled reaching task. Male Wistar rats were trained in the single pellet skilled reaching task (n = 6) for 10 consecutive days or were, under similar conditions, given access to pellets that did not require skilled reaches (n = 6). Skilled motor trained rats exhibited a rapid increase in successful reaches during the first four days of motor training before reaching a plateau, indicative of the acquisition and consolidation of the learned task, respectively. Expression profiles of chromatin modifying enzymes were screened using epigenetic-targeted PCR arrays. Results suggest that gene expression levels of multiple chromatin regulatory enzymes were down-regulated in the motor cortex of trained animals compared to controls following 10 days of motor training in the skilled reaching task. Among the chromatin modifying enzymes, the transcription level of Smyd1 (SET and MYND domain containing 1; NM_001106595) was lower (-2.17 fold-change) in motor cortex after 10 days of training compared to controls. Our results point to an epigenetic regulation of chromatin modification markers in the primary motor cortex that possibly underlie the mechanisms of synaptic plasticity, synaptogenesis and the formation of procedural memory.
Introduction
Fine motor skills are learned after repetitive training and once learned can be retained for a long time. Many of daily life activities such as grabbing a pen, tying shoe and turning pages in the book are dependent up on well-developed fine motor skills which are reliant on cooperation of brain, central nervous system and muscles.
Motor learning is defined as “Set of processes associated with practice leading to permanent changes in the capability to response” (Schmidt, 1982). Motor learning development consists of , fast phase (acquisition) which involves rapid performance improvement that forms within/across a single training session, while second slow phase (Maintenance) which involves moderate gains in performance that progresses across multiple training sessions (Luft and Buitrago, 2005).
Different brains circuits are responsible to organize different courses of motor learning, explicitly Striatum, Cerebellum and motor cortical regions of the frontal lobe (Luft and Buitrago, 2005). Two distinct cortical-subcortical circuits: a cortico-striato-thalamo-cortical loop and a cortico-cerebello-thalamo-cortical loop organize the activity of these brain circuits (Doyon et al., 2003).
Any deficiencies occurring within aforementioned circuits can lead in to either neurodegenerative or neurodevelopmental disorders. Roles of these circuits in procedural memory formation was percept from deficiencies of patients with striatal dysfunction such as Parkinson’s or Huntington’s disease originating from damage to cerebellum, or within the frontal lobe involving motor cortical areas (Ackermann H et al., 1996; Doyon J et al., 1997; Doyon J et al., 1998; Gabrieli JD et al., 1997; Harrington DL et al., 1990). Furthermore, these are the same circuits associated with neurodevelopmental disorders such as ADHD (Attention Deficit/ Hyperactivity Disorder). Thus, having a good understanding of cellular and molecular mechanisms implicated in skilled motor learning is crucial in pursuit of a treatment for neurodevelopmental disorders.
Skilled reaching is highly linked to structural and functional changes in different brain regions including Motor cortex. The temporal relationship between Synaptogenesis and motor map reorganization as a result of motor training for 3, 7 and 10 consecutive days was investigated by (Kleim et al., 2004). Results confirmed that, although significant improvement in the performance was observed after 3 days of training, major increase in synaptogenesis was not observed until day 7 of experiment; moreover, wrist and digit movement expansion was only demonstrated after 10 days of training. These findings demonstrate learning-dependent
Plasticity as a temporally dynamic process by nature; besides, they confirmed that synaptogenesis precedes motor map reorganization but follows motor skill acquisition.
Therefore, finding an animal model for investigating the neural basis and the treatment of neurological diseases, including diseases that produce impairments in motor control, seems necessary. In between all animal models rat seems to fulfill these criteria (Whishaw IQ et al., 1986; Whishaw IQ et al., 1998; Whishaw IQ et al., 2002). Variety of behavioral methods has been implicated in animal models which among them Skilled reaching task is a well validated method in order to model neurological conditions involved in motor learning (Vergara-Aragon F et al., 2003).
Compelling evidence from several studies indicates that epigenetic mechanisms are critically involved in synaptic plasticity and memory formation (Sultan and Day, 2011; Alberini, 2009) . Previous studies have demonstrated that active chromatin remodeling happens in post-mitotic neurons, offering that these molecular processes are involved during the different stages of memory formation (from acquisition to consolidation). Examples of such chromatin modifications include the phosphorylation, acetylation and methylation of histones and the methylation of associated DNA to subsequently regulate gene expression throughout the stages of learning. While these mechanisms have been extensively investigated in hippocampal-dependent long-term memory (Gupta et al., 2010), studies directed specifically towards procedural memory have not been performed. In an effort to identify potential epigenetic targets underlying motor learning, we conducted the present study to establish a gene expression profile in the motor cortex subsequent to the consolidation of a complex motor skill (i.e., skilled-forelimb reaching task). We took advantage of the RT2 Profiler TM PCR Array System to screen 168 key genes encoding epigenetic-modifying enzymes and remodeling factors known to regulate chromatin accessibility and therefore gene expression. We aimed to advance the understanding of the basic molecular processes implicated in learning induced-motor plasticity following motor skill training.
Materials and Methods Subjects
All experiments were performed using male outbred Wistar rats from Charles River (Sulzfeld, Germany). Animals were 14 days old upon arrival and were housed in groups in standard plastic cages (Tecniplast GR 900 IVC) under controlled conditions of light: dark cycle (12:12 h, lights on at 07:00 h). Food and water were available ad libitum. The Animals were weaned from their mother on postnatal day 21. The following day, animals were randomly assigned into either control or trained groups (n=6 per group), and housed in pairs in the same type of cages. Each set of paired rats was separated by a clear Plexiglas partition (containing small holes; 10 mm diameter) that divided the home cage in half in order to monitor the daily food intake for each individual rat. All procedures were approved by the Local Committee on Ethics of Animal Experimentation, Stockholm, Sweden (N502/11).
Skilled forelimb reaching task
General behavioral procedure: The general experimental protocol for the skilled reaching
task is illustrated in Fig. 1. Training sessions were performed between 09:00 and 15:00 h under low illumination in order to reduce stress. Prior to any behavioral procedure, rats were brought in their home cages to the experimental testing room and allowed to habituate for at least 1 hour before the training sessions were started in order to minimize stress caused by environmental changes.
Single pellet reaching box: Single pellet reaching boxes were made of clear Plexiglas (14 cm
wide, 45 cm long and 35 cm high) as previously described (Qian et al., 2010). In the centre of each box’s front wall, there was an open vertical slot 1 cm wide that extended from the floor to a height of 16.5 cm (see Fig. 2). In front of the open slot, a shelf (width 4.5 cm and length 13 cm) was mounted 3 cm above the floor on the outside of the wall. Small indentations to hold the food pellets were located 2 cm from the inside of the front wall, aligned with the edges of the open slot (see Fig. 2B). This distance was sufficient to prevent rats from retrieving food pellets by use of their tongues.
Feeding and food restriction: Prior to and during skilled reaching training (see Fig. 1), rats
were placed on a restricted diet until they reached 85-90 % of the body weight of non food-restricted reference animals housed in pairs under the same conditions. To familiarize the rats with the food pellets used for the skilled reaching task, each rat received thirty pellets (Test Diet, 45 mg precision-weight, purified rodent tablets, UK) 8 hours prior to the daily Purina rat chow ratio the week preceding training. Once skilled reaching training began, and until the end of motor training, only rat chow was provided in the home cage since thirty food pellets were consumed during each training session.
Pre-training and training sessions: Two days prior to skilled reaching training, rats were
placed both days into the reaching box (Fig. 2) with food pellets on the shelf (in front of the indentations; see Fig. 2B) for 15 min. The objective was to introduce each animal to the testing box and to determine which forelimb was preferred for reaching/grasping the pellets through the open slot. By the end of the second day, all animals demonstrated a consistent preference for one forelimb in their attempts to reach/grasp the pellets (>80 % of the time). Once the forelimb preference was determined for each individual animal, food pellet per trial was placed into the indentation located 2 cm from the inside of the front wall contralateral to the preferred forelimb (see Fig. 2B). During the subsequent 10 days, animals in the trained group underwent daily 15-minute training sessions consisting of 30 discrete trials, 1 single pellet per trial. The first 10 trials were considered a “warm-up” period and the latter 20 trials were quantitatively scored. Each food pellet was immediately removed from the shelf when the pellet was displaced too far away from the indentation to prevent additional reaching attempts after an unsuccessful discreet trial. During inter-trial intervals, rats were trained to leave the open slot at the front of the box and walk to the rear wall of the cage to wait a few seconds before returning to the front of the cage for the subsequent trial. This was accomplished by occasionally placing a food pellet close to the rear wall of the cage. In addition, food pellets were not placed on the shelf on semi-randomly selected trials in order to teach the animals to reach only when a food pellet was present in the front shelf. To control for the potential effects of increased motor activity on gene expression, the control group was trained on a variation of the skilled reaching task. They were placed into identical training cages as the trained group, but all pellets during the 30 discreet trials were placed on the floor of the box close to the front wall. In this way, the control group had the exact amount of pellets and a similar patter of movement and motor activity between the front and back of the
skilled reaching task box as the trained group, but did not employ skilled reaching movements during the 30 discreet trials to acquire food pellets.
Video recording: Reaching performance was video recorded using a SAMSUNG
HMX-H100P high definition camcorder. Frame-by-frame analysis of skilled reaches was evaluated using the computer-based software (Windows Media Player).
Reaching behavior was analyzed by measuring. (1) Total success: A successful reach was
defined as one in which an animal grasped a food pellet, transported it in the paw into the cage, and placed it into its mouth regardless of the number of forelimb advances toward the food pellet required, and (2) First attempt success: First attempt success was the percentage of success in which a rat obtained a food pellet on the first advance of the forelimb toward the food.
Gene expression analysis
Tissue preparation: Thirty minutes after the last training session of the skilled reaching task
was completed (i.e., on the 10th day of training), animals were sacrificed by decapitation and specific brain regions (prefrontal cortex, motor cortex, striatum and cerebellum) were rapidly dissected on ice and frozen on dry ice and stored at -80°C until used. Only motor cortex was analyzed in the present study.
Extraction of RNA. Total RNA was isolated using RNeasy® Mini Kits (QIAGEN, USA) according to the manufacturers instructions (including the optional DNase digestion step for 15 min at room temperature) and quantified by spectrophotometry using a NanoDrop® ND-2000 spectrophotometer (NanoDrop Technologies, USA). The integrity and purity of the RNA preparations were analyzed by capillary electrophoresis with a Bio-Rad Experion automated electrophoresis system (BIO-RAD, Sweden) using Experion RNA StdSens Chip kit (BIO-RAD, Sweden). The extracted RNA from two animals was excluded due to technical problems during the isolation.
Complementary DNA synthesis for its use in PCR Arrays. RNA samples from the motor cortex, contralateral to the preferred forelimb (see above), were used for complementary
(cDNA) synthesis. The total RNA from four animals in each group (control vs. trained) were pooled and the cDNAs of these pooled samples were synthesized using the RT² First Strand Kit (SABiosciences) according to the manufacturer’s instructions. This kit includes a step to effectively eliminate genomic DNA and also a built-in External RNA Control that helps monitor reverse transcription efficiency (see below).
PCR array. The RT2 Profiler TM PCR Array System (SABiosciences), a high-throughput quantitative PCR method, was used to screen for differentially expressed gene transcripts in motor cortex between the trained and control groups. Two separate Pathway-Focused Arrays were used in the present study: the Rat Epigenetic Chromatin Modification Enzymes (PARN-085A) and the Rat Epigenetic Chromatin Remodeling Factors (PARN-086A), which profiles the expression of 84 key genes encoding enzymes known or predicted to modify genomic DNA and histones, and 84 genes involved in recognizing chromatin modifications and remodeling chromatin, respectively. In addition, both PCR arrays contained five
housekeeping genes (i.e., GUSB, HPRT1, HSP90ab1, GADPH and ACTB) as well as
multiple controls to monitor genomic DNA contamination, first strand synthesis and real-time PCR efficiency. Amplification, data acquisition, and melting curves were carried out by the
iCycler iQ5 detection system (BIO-RAD). The PCR cycling program was set as follows: step 1: 95°C for 10 min, step 2: 40 cycles of 95°C for 15 sec followed by 60°C for 1 minute, step 3: 95°C for 1 min, step 4: 55°C for 1 minute and step 5: 80 cycles for 10 sec each, beginning at 55°C and increasing 0.5°C with each subsequent cycle. The data was analyzed using an integrated web-based software package for the RT2 Profiler TM PCR Array System
(http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis/php). The data analysis was based on the ΔΔCt method with the normalization of the raw data to multiple housekeeping genes.
Statistical analysis
Statistical analysis was performed using the STATVIEW computer software (version 4.0). All behavioral experiments were analyzed using either repeated measures analysis of variance (ANOVA; Time as main factor) or factorial ANOVA, as indicated in the text and in the figure legends. All post hoc comparisons were made using a Bonferroni/Dunn test when significant ANOVA effects were determined. Data from gene expression studies were analyzed using unpaired Student’s t test (two-tailed). The threshold for statistical significance was set as P ≤ 0.05. All data are presented as the means ± S.E.M.
Results
Monitoring growth and food restriction of Wistar rats
The growths of young rats were monitored and their weights were recorded daily following weaning from their mothers. The normal growth was closely monitored in order to correctly food-restrict animals that were later trained in the skilled reaching task. Weights of the animals were targeted to be ~90 % of their normal body weight, and were calculated from the weights of non-food restricted rats (n=2). The growth curves of food-restricted animals used in the present study are presented in Fig. 3 and indicate that weights of food-restricted animals were maintained around 90 % of non-food restricted animals for the duration of the experiment. All animals in the food restricted group reached a body weight of no less than 85 % of non-food restricted group.
Skilled reaching performance
The performance of Wistar rats in the single pellet skilled reaching task during 10 consecutive training days is illustrated in Fig 4. All animals exhibited typical pattern of motor skill learning that is characterized by two distinct phases of learning. The first phase involves rapid improvements in performance that is observed during the first 3 to 4 days of training. The second slower phase involves moderate gains in performance that occurs between days 5 to 10 of training. Analysis of reaching performance by repeated ANOVA confirmed a significant effect of training day on total success (F (9, 63) = 6.5, P< 0.0001) and on success with the first reach attempt, referred to as a “hit” (F (9, 63) = 5.7, P< 0.0001). Subsequent post hoc analysis with Bonferroni/Dunn tests give a highly significant increase in total success and in “hits” after 4 days of training (P<0.001). At day 4 of training, “hits” had improved from 9.4 % (± 3.2 %) on day 1 to 40 % (±7.2 %). Similarly, total success improved from 19.0 % (± 11 %) on day 1 to 67 % (±8.6 %) at day 4 of training in the skilled reaching task. There we no significant changes in performance (total success or number of hits) between day 5 to day 10 of training.
Gene expression analyses
Results from an in-depth epigenetic-targeted PCR array screen indicated that a number of epigenetic-related transcripts in primary motor cortex were differentially expressed between trained and control groups. Among the chromatin-modifying enzymes, the transcription levels of Smyd1, a histone methyltransferase, was significantly lower (-2.17 fold change) in motor cortex of trained animals after 10 days of training compared to controls (Table 1 and Fig. 5). Other genes such as Ezh2 and Prdm2 (two histone methyltransferases), Ciita (a histone acetyltransferase), and Dot1l (a histone methyltransferase) were also down-regulated but not as robust as Smyd1 (Table 1 and Fig. 5). Among the chromatin remodeling factors included in our screen, only SPEN, a hormone-inducible transcriptional repressor, was found to be down-regulated in trained animals (albeit below the 2-fold threshold; see Table 2 and Fig. 6).
Discussion
Epigenetic mechanisms such as DNA methylation and histone modification (demethylation or acetylation) have been determined to be necessary for synaptic plasticity and memory formation (Feng et al., 2007). While these mechanisms have been extensively studied in hippocampal- and amygdala-dependent long-term memory (Bredy and Barad, 2008; Monsey et al., 2011; Yeh et al., 2004), similar studies for procedural memory are lacking. In the present study, we investigated the expression profile of 168 key genes involved in epigenetic mechanisms of DNA methylation and histone modification (i.e., chromatin-modifying enzymes and remodeling factors) in primary motor cortex of rats that have learned a complex motor task (e.g., skilled-forelimb reaching task). Our results point to the involvement of specific chromatin-modifying enzymes and remodeling factors in learning-dependent motor cortex plasticity (e.g., increase complexity and density of forelimb motor cortical dendritic processes and synaptogenesis) that supports the production and refinement of skilled movement sequences.
The regulation of chromatin structure is complex. This is due in part to the N-terminal tails of histones that are highly accessible to enzymatic transformation and that they contain multiple sites for a number of covalent post-translational modifications, which include acetylation, phosphorylation, methylation, ubiquitination, and sumoylation (Berger, 2007; Peterson and Laniel, 2004). Epigenetic changes in chromatin structure as a result of acetylation, methylation and deacetylation have been implicated in a number of cellular processes such as the control of cell cycle in embryonic stem cells, cell differentiation, synaptic plasticity and memory formation (Barrett and Wood, 2008; Levenson and Sweatt, 2005; Roth and Sweatt, 2009; Sultan and Day, 2011). All of these modifications are capable of influencing the rate of gene transcription by either permitting DNA to be more, or less, accessible to transcriptional machinery [for a review, see (Rumbaugh and Miller, 2011)]. Results from the present study revealed that the gene expression of a number of epigenetic chromatin-modifying enzymes (e.g., Smyd1, Dot11, Prdm2, Ezh2, and Ciita; see Table 1) were down-regulated in the contralateral motor cortex to the preferred forelimb of trained animals after 10 days of motor training in the skilled reaching task, compared to control animals. These genes encode a family of histone-modifying enzymes that are functionally grouped as either histone methyltransferases or histone acetyltransferases.
DNA methylation plays a pivotal role among other transcriptional regulators since methylation can both activate and deactivate transcription. Covalent methylation of DNA involves conversion of lysine base pairs to one of three different states: mono-, di-, and tri-methylated. Gene silencing occurs as a result of either di- or tri methylation of the histone H3 subunit at lysine residue 9 (H3K9) whereas transcription activation occurs as a result of methylation of the histone H3 subunit at lysine residue 4 (H3K4) (Margueron et al., 2005; Martin and Zhang, 2005; Sims et al., 2003; Vermeulen et al., 2007). Numerous recent studies have implicated lysine methylation of histones in the regulation of transcription and chromatin structure reorganization in the central nervous system (Huang and Akbarian, 2007; Huang et al., 2007; Kim et al., 2007; Tsankova et al., 2006). To date, there has been no report of specific histone methylation regulation in learning-dependent motor cortex plasticity and formation of procedural memory (Gupta et al., 2010).
Many proteins are responsible for the methylation of specific lysine residues on histones and are classified into the superfamily of SET-domain containing or non-SET domain containing methyltransferases. Their function is to transfer a methyl group from the cofactor and methyl group donor, S-adenosyl-L-methionine or SAM, to the amino group of lysine. Our findings indicate that among all of the chromatin-modifying enzymes screened, the transcription levels of Smyd1, a SET-domain containing methyltransferase, was down-regulated in the contralateral motor cortex of the preferred forelimb as a result of skilled reaching (see Table1 and Fig. 5). Since this enzyme is down-regulated and given that Smyd1 is involved in the activation of gene transcription through the methylation of lysine residue K4, its down-regulation following motor skill learning reflects a reduction in gene transcription. Studies aimed at determining the specific genes in motor cortex that are silenced by the down-regulation of Smyd1 after rats have learned a complex motor task are in progress.
To date, Smyd1 has been determined to be involved in myofibril organization during embryonic development (Tan et al., 2006) but no studies have yet described its role in learning and memory. The putative role Smyd1 plays in learning-dependent motor cortex plasticity (e.g., synaptogenesis) is novel and warrants further investigation.
Another candidate genes identified by the PCR array screen was EzH2. This histone methyltransferase has been recently demonstrated to be highly expressed in proliferating neuronal stem cells (NSCs) from embryonic mice and its expression decreases after NSCs differentiate into neurons and astrocytes (Chou et al., 2011; Sher et al., 2011). Motor learning induces astrocytic hypertrophy in the cerebellar cortex (Kleim et al., 2007). Taking this into consideration, a down-regulation of EzH2 may be involved in this type of glial plasticity following motor skill training. Since total RNA extracted from motor cortex was used for the PCR array screen, studies are planned to use in situ hybridization to determine whether the down-regulation of EzH2 is a general phenomena or is specific to certain cell types.
Presently it has been reported that SPEN an epigenetic transcription modifier, is associated with diverse biological processes ranging from embryogenesis to aging (Yabe et al., 2007). Moreover it was demonstrated that Spen is a nuclear protein which regulates the expression of genes, which are critical for neuronal cell fate and morphology (Kuang et al., 2000). Our data suggest a possible role for this protein in learning-dependent motor cortex plasticity and its putative role is being investigated further.
The present study has certain limitations. First, the low number of rats (n=4 per group) used in this study, reduced our statistical power. In addition, the use of pooled RNA from individual animals in each group for the PCR arrays screen may have contributed to the few number of genes that were found to be differentially expressed between trained and control groups. A second major limitation of this study is that gene expression differences were only examined at a single time point, 10 days after training, which makes it difficult to speculate in this discussion about the nature and dynamics of epigenetic changes occurring during the different stages of motor skill learning.
The cellular and epigenetic mechanisms underlying the formation of procedural memory remain elusive; however, these results have clearly identified Smyd1, in spite of the limitations of the study as a candidate gene involved in epigenetic changes during the consolidation phase of motor skilled learning. Future studies are required to elucidate the specific role of Smyd1, as well as to investigate the other potential genes identified in the present study, in learning-induced motor cortex plasticity and formation of procedural
memory. This knowledge may provide a deeper understanding to the neurobiological basis of neurodevelopmental disorders with deficits in fine motor control (e.g., ADHD and autism spectrum disorders).
Acknowledgements
I would like to thank my supervisor Rochellys Diaz Heitjz for her excellent guidance. I also would like to express my deep gratitude to my co-supervisor Assistant Professor Daniel Marcellino and Professor Hans Forssberg for their professional guidance and valuable supports.
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Tables
Table 1. Fold-change in the expression levels of epigenetic chromatin-modifying enzymes in primary motor cortex after 10 days of motor training.
Ref. Seq Symbol Description Fold
Differences
NM_001106595 Smyd1 SET and MYND domain containing 1 -2,17
NM_001134979 Ezh2 Enhancer of zeste homolog 2 (Drosophila) -1,68
NM_053529 Ciita Class II, major histocompatibility complex,
transactivator -1,66
NM_001108733 Dot1l DOT1-like, histone H3 methyltransferase
(S. cerevisiae) -1,61
Table 2. Fold-change in the expression levels of chromatin remodeling factors in primary motor cortex after 10 days of motor training.
Ref. Seq Symbol Description Fold
Differences
XM_345315 Spen SPEN homolog, transcriptional regulator
Figure Legends
Figure 1. General overview of the experimental protocol of the skilled reaching task. See
the detailed description in Material and Methods.
Figure 2. The single pellet skill reaching box used to train rats in the skilled reaching task. (A) A photo of the single pellet reaching box indicating its dimensions. See Materials
and Methods for the detailed description of this box. (B) Photo taken of a rat performing a skilled reach to obtain a food pellet through the open slot at the front of the box.
Figure 3. Growth curves of food restricted and non-food restricted Wistar rats. Animals
were restricted to be 85-90 % of non-food restricted animals. For more details, see Material and Methods.
Figure 4. The performance of Wistar rats in the single pellet skilled reaching task during 10 consecutive training days. Endpoint measures of reaching behavior are presented:
(A) Total success in percentage and (B) Success on the first reach in percentage. The results are presented as the means ±S.E.M. (n=6 per group).
Figure 5. Results of the epigenetic chromatin modification enzymes PCR array performed after 10 days of motor training in the skilled reaching task. A heat map of 84
key transcripts assessed with the array was generated to illustrate changed levels of gene expression in the motor cortex of the trained group compared to controls in a 256-grade false color representation. The green areas display a decreased expression; the red areas display an increased expression of the analyzed transcript in comparison to their expression in controls. The change in the transcript expression after 10 days of motor training compared to the control is displayed as increasing intensities of red and green color. The array was performed with pooled samples (n=4 per group).
Figure 6. Results of the epigenetic chromatin remodeling factors PCR array performed after 10 days of motor training in the skilled reaching task. A heat map of 84 key
transcripts assessed with the array was generated to illustrate changed levels of gene expression in the motor cortex of the trained group compared to controls in a 256-grade false color representation. The green areas display a decreased expression; the red areas display an increased expression of the analyzed transcript in comparison to their expression in controls. The change in the transcript expression after 10 days of motor training compared to the control is displayed as increasing intensities of red and green color. The array was performed with pooled samples (n=4 per group).
Figures Fig. 1
