TNFα contributes to excitotoxicity by increasing GluA2-lacking AMPA receptor (AMPAR) trafficking to the neuronal plasma membrane. In vitro, increased AMPAR on the neuronal surface after TNFα exposure is associated with a rapid internalization of GABAA receptors (GABAARs), suggesting complex timing and dose dependency of the CNS's response to TNFα. However, the effect of TNFα on GABAAR trafficking in vivo remains unclear. We assessed the effect of TNFα nanoinjection on rapid GABAAR changes in rats (N = 30) using subcellular fractionation, quantitative western blotting, and confocal microscopy. GABAAR protein levels in membrane fractions of TNFα and vehicle-treated subjects were not significantly different by Western Blot, yet high-resolution quantitative confocal imaging revealed that TNFα induces GABAAR trafficking to synapses in a dose-dependent manner by 60 min. TNFα-mediated GABAAR trafficking represents a novel target for CNS excitotoxicity.The proinflammatory cytokine TNF ARs) are a major source of fast inhibitory synaptic transmission in the CNS and thus play a crucial role in regulating neuronal networks. Altering the number of GABAARs in a synapse through rapid receptor trafficking therefore can have a major inhibitory impact on neuronal excitability [ARs to and from the plasma membrane has been shown to depend on neuronal activity [AR redistribution is also mediated by a variety of neuromodulatory substances such as hormones and cytokines. For example, insulin and PI3 kinase cause rapid insertion of GABAARs into the plasma membrane increasing the amplitude of miniature inhibitory postsynaptic currents (mIPSCs) [Gamma amino butyric acid type A receptors (GABAtability –3. Traffactivity –7. GABAA(mIPSCs) , 9.α), a proinflammatory cytokine that is constitutively expressed following spinal cord injury (SCI), has been shown to alter receptor trafficking of GABAARs to and from the cell surface in vitro [In vivo, TNFα levels are increased as part of the inflammatory response following SCI and various other nervous system disorders, contributing to secondary cell death [α has been shown to specifically promote trafficking of glutamate-receptor-2-(GluA2-) lacking AMPA receptors (AMPARs) to the plasma membrane of spinal neurons inducing excitotoxicity in vivo [α action pharmacologically reduces AMPAR trafficking and cell death after SCI, suggesting that modulation of AMPARs is a major mechanism by which TNFα promotes cell death in CNS disease [Tumor necrosis factor alpha  subunit as studies show that this subunit is critical for postsynaptic clustering of GABAARs and the vast majority of GABAARs are composed of subunits α and β combined with γ2 [AR levels in the plasma membrane and at synapses after TNFα nanoinjection into the spinal parenchyma. Whole membrane fractionation and quantitative western blotting showed a trend towards increased membrane GABAAR protein levels within 60 min of TNFα nanoinjection delivery, but these results did not reach significance. Confocal data revealed increased GABAARs at synaptic sites following TNFα nanoinjection, underscoring the subcellular specificity of GABAAR localization. Confocal image findings also suggest that there is a nonlinear dose-dependent relationship between TNFα and GABAAR trafficking.The aim of the current study is to assess  with γ2 , 15. We N = 30) with ad libitum access to food and water. All procedures were nonsurvival surgeries performed under deep anesthesia. All possible steps were taken to avoid unnecessary suffering. All experimental procedures followed the National Institutes of Health guidelines and were approved by the Institutional Animal Care and Use Committees at The Ohio State University and the University of California, San Francisco.Female Long-Evans rats, 77- to 87-d-old, were housed in pairs . An albumin injection served as a control due to its similarity in molecular weight to rat recombinant TNFα (R&D systems). For imaging experiments, subjects (n = 4) received a dose of TNFα  and an injection of albumin on the contralateral ventral horn to serve as a within-subject control. These doses of TNFα have been found sufficient to increase glutamate-mediated excitotoxicity in the spinal cord, but insufficient to cause cell death [α (1 μM) or albumin along a 750 μm length of spinal cord .Nanoinjections (35 nl) were delivered stereotactically into the T9-10 ventral horn as described in Hermann et al., 2001, by applying compressed air micropressure to pulled glass pipettes . The resulting suspension was then passed through a 22 gauge needle five times and centrifuged at 5,000 RCF for 5 min at 4°C. The supernatant (S1) was transferred to a new tube and centrifuged again at 13,000 RCF for 30 min at 4°C. The supernatant (S2) was transferred to a new tube, and the membrane-enriched pellet (P2) was resuspended in 50 μL of PBS containing protease inhibitor. P2 fractions were vortexed and sonicated, and all sample fractions were stored at −80°C.One hour after nanoinjection, the spinal cord was extracted under deep anesthesia; 7.5 mm of the cord was snap-frozen with dry ice and stored at −80°C for later processing. Fractionation procedures were based on prior work with rat spinal cord . The snaβ-mercaptoethanol (BioRad), and 20 μg of protein per lane was immediately loaded onto a precast 10% Tris-HCl polyacrylamide gel (BioRad). Sample loading on the gel was performed so that each subject had their own lane, loading order was counterbalanced across injection condition to account for regional variability within the gel, and the experimenter was blind to subject condition. A kaleidoscope ladder  was loaded to confirm molecular weights. The loaded gel was electrophoresed in SDS running buffer  for 1 h at 100 volts. The protein was then transferred to nitrocellulose membrane in cold tris-glycine buffer . The membrane was blocked for 1 h in Odyssey blocking buffer (Li-Cor) containing 0.1% Tween 20 and then incubated overnight (18 h) in the dark at 4°C in a primary antibody solution containing Odyssey blocking buffer, 0.05% Tween 20, and a rabbit polyclonal anti- GABAA receptor γ2 primary antibody . The membrane was washed 4 × 5 min with TBS containing 0.1% Tween 20 (TTBS) then incubated for 1 h in a fluorescently labeled secondary antibody solution containing Odyssey blocking buffer, 0.2% Tween 20, and 1 : 30,000 IRDye 680 goat anti-rabbit secondary antibody (Li-Cor) and subsequently washed 4 × 5 min in TTBS, 1 × 5 min in TBS. The membrane was immediately scanned for protein bands using the corresponding 680 nm laser at a scanning intensity of 4 on the Odyssey Infrared Imaging System (Li-Cor). The membrane was then reblocked and reincubated in a primary antibody solution containing 1 : 800 mouse anti-N-Cadherin primary antibody . The membrane was washed and reincubated in a secondary antibody solution containing 1 : 30,000 IRDye 800 goat anti-mouse secondary antibody (Li-Cor). The membrane was washed again and rescanned for protein bands in the 800 nm channel at a scanning intensity of 3.Sample protein concentration was assayed using BCA (Pierce) and quantified with a plate reader . P2 fractions from each subject were diluted 1 : 2 with cold Laemmli sample buffer containing 5%  R2, of protein band fluorescent intensity from a protein dilution curve of a control sample  to definitively quantify the intensity of fluorescently labeled protein bands. To ensure that our intensity measurements were truly quantitative, we generated linear ranges for each antibody by plotting band intensity measurement relative to the concentration of protein loaded for a protein dilution curve. Laser scanning intensities for each antibody were selected by determining the laser intensity which yielded the highest linear range,  Figures . The intP = 0.883) nor actin (P = 0.610) intensity varied between conditions. We therefore used NCad as a control both for variability in protein loading and in plasma membrane enrichment through subcellular fractionation [AR band intensities from each sample were normalized with respect to NCad by dividing GABAAR intensity by NCad intensity. All biochemistry was performed in a blinded, counterbalanced fashion. Two independent replications were performed, and the normalized densitometry results were averaged across runs.Prior experiments have used the plasma membrane protein N-Cadherin (NCad) to characterize the degree of membrane enrichment in each fraction generated by spinal cord subcellular fractionation , 16. In μm thick horizontal slices. The 30 mm length of spinal cord centered on the injection site was isolated and postfixed overnight (<18 h) in 4% paraformaldehyde followed by cryoprotection of the tissue in 30% sucrose for 2d. The tissue was then cut into 10 mm blocks, flash-frozen on dry ice, embedded in OCT, and sectioned into 20 A receptor γ2 primary antibody  overnight at room temperature. Slides were washed with 2 mL PBS then incubated for 1 h at room temperature in a solution containing 1 : 100 Alexa 488 goat anti-rabbit and 1 : 100 Alexa 633 goat anti-mouse secondary antibodies. After washing with 2 mL PBS, slides were coverslipped with Vectashield containing DAPI . Negative immunolabel control conditions consisted of no primary antibody, and each individual primary with the incorrect secondary. Confocal microscopy of the negative control slides showed no detectable label exceeding threshold.Fixed tissue sections from a full set of experimental conditions were antibody-labeled using a high-throughput staining station . Tissue was blocked and permeabilized with 5% normal goat serum and 0.3% Triton X-100 for 1 h. Sections were incubated in a solution consisting of mouse monoclonal antibody against presynaptic synaptophysin  and rabbit polyclonal antibody against GABAμm) were selected using wide-field fluorescence based on the distinctive synaptophysin outline surrounding the plasma membrane  was used to generate confocal stacks for large motor neurons. Control tissue was used to optimize filter and laser settings, which were then held constant throughout the experiment. These settings allowed for virtually complete distinction between immunolabels GABAARs (Alexa 488), synaptophysin (Alexa 633), and FluoroRuby (Texas Red). Confocal z-stacks consisted of 1 μm slices which were oversampled at 0.5 μm z-intervals. AutoQuant software was used to deblur these confocal stacks through the process of 3D blind iterative deconvolution. An iteration number of 3 was determined for GABAAR labeling based on a random subset of images and held constant during the experiment. Performing deconvolution on confocal image stacks allowed for greater resolution of receptor puncta than was possible with either technique alone.Large ventral motor neurons (characterized by a diameter >40 membrane . One motAR receptor pixels (intra- and extracellular puncta) as well as colocalization of synaptophysin and GABAAR pixels  in each field of the z-stack. This macro allowed for measurement of GABAAR puncta at the level of the neuropil. Another macro quantified fluorescently labeled GABAAR puncta on the plasma membrane of motor neurons. First, the macro identified the plane in the z-series with the highest amount of GABAAR/synaptophysin colocalization. A blinded researcher supervised the automated plane selection to prevent selection based on staining artifacts. Once a single plane was selected, a blinded researcher identified the plasma membrane of the motor neuron by tracing the synaptophysin-labeled outline of the cell. A 2 μm-thick “image-based subcellular fraction” was produced containing the plasma membrane of the cell from the single plane. From the plasma membrane subcellular image fraction, MetaMorph quantified GABAAR pixels (extrasynaptic receptors) and colocalized GABAAR/synaptophysin pixels (synaptic receptors).Images underwent automated image analysis to quantify the number of fluorescently labeled receptor puncta on the plasma membrane exceeding a predetermined pixel threshold based on control tissue. Automated image analysis was performed using custom designed MetaMorph (Molecular Devices) macros. One macro was designed to measure the amount of total GABAAR protein levels were accounted for by ANCOVA, which allowed for the distinction between receptor trafficking versus an increase in total GABAAR puncta as a result of receptor synthesis. Significance was established as P < 0.05.Quantitative Western Blot data were analyzed using an analysis of variance (ANOVA). Immunofluorescence data were analyzed using ANOVA, and the experiment was a mixed design (injection side and distance from injection site served as within-subject variables). When appropriate, Tukey's post hoc analyses were used to determine significant differences amongst multiple outcomes within a variable. Total GABAAR intensity ratios (GABAAR : NCad) for the membrane-enriched fraction of spinal cord homogenate (AR between injection condition (P = 0.315), yet group means reflect a nonsignificant trend towards a higher concentration of GABAARs in TNFα-treated subjects  60 minutes following TNFα nanoinjection (P < 0.001). Total GABAAR in the neuropil increased in a dose-dependent manner (P < 0.001) as well (AR puncta from the middle to the lowest TNFα dose (P < 0.02)  revealed a dose-dependent increase in synaptic GABA as well . Additio Figures . Studiesreceptor , 17, whiα-mediated increase in total GABAAR protein in the neuropil, thus isolating the dose-dependent changes in synaptic GABAAR attributable to receptor trafficking. After correcting for changes in total GABAAR protein, the dose-dependent effect of TNFα on synaptic GABAARs in the neuropil was robustly maintained (P < 0.001) indicating that the dose-dependent increase in synaptic GABAAR does not depend solely on wholesale protein changes but also relies on receptor trafficking to the synapse. Correcting for variance in total GABAAR protein in the neuropil with ANCOVA, revealed no significant interaction of dose by side for synaptic GABAAR (P = 0.325). Overall, results reveal that TNFα dose significantly influenced GABAAR receptor trafficking to synaptic sites within the neuropil containing spinal motor neurons.An ANCOVA was used to statistically account for the TNFAR 60 minutes after TNFα injection . There was significantly more extrasynaptic GABAARs associated with the lowest TNFα dose relative to the middle dose (P < 0.003). After statistically correcting for TNFα-mediated changes in total GABAAR level with ANCOVA, there remained a significant dose-dependent effect of TNFα on synaptic GABAAR indicating that TNFα increases receptor trafficking to synapses on the motor neurons' somatic surface in addition to increasing extrasynaptic receptors (P < 0.05). The effect of TNFα was widespread and extended onto the contralateral side of the spinal cord (the site of vehicle injection) eliciting a dose-dependent increase in synaptic and total GABAARs at the level of the plasma membrane and the neuropil, which spanned both sides of the cord . The extensive effect of TNFα on spinal tissue is also evidenced by the fact that there was no main effect of side of the cord in synaptic, nor total GABAAR measures (P > 0.05). There was an interaction of dose by side for extrasynaptic GABAAR on the plasma membrane (P = 0.037), but there was not a significant interaction for any other GABAAR measures (P > 0.05) (AR in the plasma membrane with ANCOVA showed no higher order interactions between dose and side for synaptic GABAAR (P > 0.05). Taken together, results indicate that there is a significant effect of TNFα dose that is driving GABAAR trafficking to synapses on the plasma membrane and that this effect was widespread throughout the tissue.Image analysis of the plasma membrane also revealed a dose-dependent increase in synaptic and extrasynaptic GABA Figures   . AccountAR that occurs in spinal cord neurons in response to TNFα. TNFα nanoinjection in vivo allowed us to model rapid changes in synaptic efficacy that result from the inflammatory response following SCI. Biochemical fractionation methods that have previously been shown to be sufficient to measure rapid trafficking of the excitatory AMPA receptors [ARs. However, quantitative high-resolution confocal microscopy on morphologically preserved tissue sections revealed significant changes in GABAARs 60 minutes after TNFα injection. Analysis of the total neuropil in confocal z-series revealed significant increases in GABAAR positive puncta. By statistically correcting confocal images for total GABAAR puncta, we were able to isolate the effect of TNFα on synaptic GABAAR trafficking, revealing a specific increase in synaptic GABAAR levels in the neuropil. By applying algorithmic postprocessing of confocal images, we were able to generate image-based subcellular fractionation, isolating the plasma membrane of large ventral motor neurons. Analysis of this confocal subcellular fraction revealed a U-shaped TNFα dose-response effect on GABAAR levels at both extrasynaptic and synaptic sites on the motor neuron plasma membrane.Our present findings suggest that there is an increase in synaptic GABAeceptors , 16 did α-mediated, dynamic receptor changes at synapses following the inflammatory response in SCI. TNFα tissue levels have been shown to peak 60 minutes after SCI [α has potential to contribute to excitotoxic cell death in the spinal cord [In vivo studies have shown that TNFα causes GluA2-lacking AMPARs to be trafficked to synapses of spinal neurons [++. Inhibitory synaptic strength is directly correlated to the number of synaptic GABAARs, thus any mechanism that controls the quantity of synaptic GABAARs can have a profound effect on neuronal excitability [AR trafficking to the synapse may serve as a homeostatic mechanism that combats the excitotoxic effect of TNFα following SCI in vivo. An increase in extrasynaptic GABAAR as well as total GABAAR is consistent with research showing that GABAAR are first exocytosed onto the plasma membrane before trafficking laterally to synapses [AR may be necessary in order to ensure a sufficient availability of GABAARs for trafficking to synaptic sites. Increased GABAAR synthesis may occur in this system to meet the demands placed on intracellular receptor stores imposed by trafficking.The present results contribute to a more complete understanding of TNFfter SCI . This innal cord . In vivo neurons , therebytability –3. Givensynapses , 19. An ARs decreased following the middle dose of TNFα. This phenomenon could be due to a compensatory homeostatic degradation of GABAAR at the middle TNFα dose. At a low dose, GABAAR synthesis could increase to maintain cellular reserves of the receptor for trafficking to the membrane, yet an increase in GABAAR at a low dose could be slight enough to go undetected by regulatory mechanisms that would otherwise decrease GABAAR accumulation [α dose, GABAAR synthesis could increase to a level that would elicit a rapid homeostatic degradation or ubiquitination of these receptors so that there is a drop in total GABAAR protein following the middle dose. A large and rapid GABAAR accumulation following a high dose of TNFα could conceivably overwhelm homeostatic compensatory degradation of GABAAR resulting in a global increase in the protein.An unexpected finding was that extrasynaptic and total GABAmulation , 17. Howα, there was a widespread effect of synaptic and total GABAAR protein that extended onto the contralateral side of the spinal cord into the region of albumin nanoinjection, as previously reported for AMPAR receptor trafficking effects [AR measures on the TNFα-injected side and the contralateral albumin side. Yet, at lower doses, TNFα does not influence GABAAR measures at the site of albumin injection. Our results demonstrate that the TNFα-affected area increases with the size of the dose. This result could be explained by evidence that TNFα promotes a heightened inflammatory response, which causes an increase in glial activation leading to the release of a variety of inflammatory cytokines including TNFα [α could elicit a greater immune response leading to further release of TNFα through a positive feedback loop, which has been demonstrated in vitro [α elicits a higher excitatory effect in the neural circuitry at the injection site that could be transmitted across the midline of the spinal cord by commissural fibers [α also are known to elicit a greater localized excitation which is propagated further relative to lower doses of TNFα [AR trafficking may counteract this TNFα-induced excitotoxicity, so increasing the radius of excitation would likewise increase the tissue area affected by GABAAR trafficking.Results also showed that at high doses of TNF effects . At a hiing TNFα –22. Therin vitro , 24. Addl fibers , 26. Hig of TNFα . Our resin vitro findings of Stellwagen et al. that TNFα decreases surface GABAARs through endocytosis while simultaneously increasing surface GluA2-lacking AMPARs [in vivo that GABAARs and AMPARs are both trafficked to the membrane after TNF nanoinjection . An interesting finding was that the middle dose of TNFα used in our study elicited a lower amount of total GABAAR relative to our highest and lowest drug doses  close to the neuronal resting potential. Physiological conditions that cause the membrane potential to become more negative than EGABA reverse the direction of ion flow through GABAARs [GABA in hippocampal CA1 pyramidal cells in vitro. GABAergic synapses become depolarizing after a high-frequency train of stimulation causing an accumulation of Cl− inside the cell and high K+ outside, in the interstitial fluid [ARs have been implicated in pathological pain conditions and maladaptive spinal plasticity [α-mediated GABAAR trafficking. A better understanding of the link between TNFα-induced synaptic GABAAR trafficking and neural excitability is a clear target for further research that may lead to a novel therapeutic target for combating the spread of excitotoxicity after SCI and other CNS diseases.Further studies are necessary in order to corroborate our current findings and determine their functional consequences. It has been shown that mIPSC strength is directly correlated with the number of postsynaptic GABAtability . Electroreceptor , 27, 28. GABAARs . For exaal fluid , 31. In asticity –34. In l


The human “mirror neuron system” has been proposed to be the neural substrate that underlies understanding and, possibly, imitating actions. However, since the brain activity with mirror properties seems insufficient to provide a good description for imitation of actions outside one’s own repertoire, the existence of supplementary processes has been proposed. Moreover, it is unclear whether action observation requires the same neural mechanisms as the explicit access to their meaning. The aim of this study was two-fold as we investigated whether action observation requires different processes depending on 1) whether the ultimate goal is to imitate or understand the presented actions and 2) whether the to-be-imitated actions are familiar or unfamiliar to the subject. Participants were presented with both meaningful familiar actions and meaningless unfamiliar actions that they had to either imitate or discriminate later. Event-related Potentials were used as differences in brain activity could have been masked by the use of other techniques with lower temporal resolution. In the imitation task, a sustained left frontal negativity was more pronounced for meaningless actions than for meaningful ones, starting from an early time-window. Conversely, observing unfamiliar versus familiar actions with the intention of discriminating them led to marked differences over right centro-posterior scalp regions, in both middle and latest time-windows. These findings suggest that action imitation and action understanding may be sustained by dissociable mechanisms: while imitation of unfamiliar actions activates left frontal processes, that are likely to be related to learning mechanisms, action understanding involves dedicated operations which probably require right posterior regions, consistent with their involvement in social interactions. In recent years, the neural control of imitation has attracted a great deal of attention, with the particular aim of identifying its neural and cognitive mechanisms see  and funcThis network, known as the observation-execution matching system, has been proposed to be the human homologue of the monkey’s “mirror neuron system” (MNS) constituted of multimodal neurons in the ventral premotor and inferior parietal cortices that discharge not only when an animal executes a goal-directed gesture involving an object or a tool, but also when it observes or predicts the same gesture This execution-observation matching system has been identified as the neurophysiological correlate of a direct matching mechanism, whereby an observed action activates the stored motor-related representation Previous studies investigating the brain mechanisms subserving imitation of familiar and unfamiliar gestures differ greatly for the experimental procedures they adopted, thus making the comparison between them troublesome. Some used unfamiliar gestures The main activations in these studies were the following. The premotor cortex  has been found to be recruited when individuals imitate either familiar or unfamiliar actions across different studies Observation of actions recruits the same neural structures that are also involved in the actual execution of the observed actions e.g., . Thus, oDespite the general interest in imitation, not much investigation has been carried out to date on the time course in brain activation of the processes involved in action observation and action imitation except for a magnetoencephalographic study (MEG) ERPs have been used to study both imitation By using ERPs in the current study which adopted a full factorial design, we aimed at investigating the differences in the neural mechanisms underlying participants’ observation of familiar and unfamiliar actions with the intention to either imitate or discriminate them. On the one hand, this design allowed us to ascertain whether imitation of unfamiliar actions is accomplished by the same functional mechanisms engaged when imitating familiar actions or by different ones. The latter possibility is suggested by the studies showing that the areas with mirror properties seem insufficient to account for imitation of gestures that are not yet part of one’s own repertoire This study was previously approved by SISSA ethics committee.Eighteen right-handed Italian adults participated in the study . Handedness was assessed with the Edinburgh Handedness Inventory . All participants had normal or corrected-to-normal visual acuity. Participants gave written informed consent and received 20 Euros for their time.The stimuli used in both tasks comprised 12 meaningful (MF) and 12 meaningless (ML) hand gestures . The video-clips lasted 2000 msec each and were performed by a male model. The set of MF actions comprised 12 object-unrelated, symbolic gestures  was introduced to prevent participants from beginning to imitate before the critical time-window for ERPs, as movement artefacts are well known to severely affect EEG traces. Discrimination and imitation tasks differed only for the response slide. In the discrimination task, participants were prompted to answer the question ‘Does it have a meaning?’ , and required to press one of two additional buttons labelled ‘Yes’ (‘Si’) and ‘No’. In the imitation task, participants were prompted with ‘Now imitate the gesture’ (‘Ora imita l'azione!’) and were instructed to release the target button and imitate with the same hand as in the observed action. At the beginning of each task session, participants had a practice session of 12 trials, thus they were aware of which task they were going to perform afterwards while observing the video stimuli. Both sets of MF and ML actions were repeated 4 times in each task, for a total of 192 trials. The order of administration of the two different tasks (imitation versus discrimination) was counterbalanced across participants. In the middle of each task, a short pause was given to allow some rest and check electrode offsets. In order to exclude incorrect trials, participants’ performance was video-recorded and scored off-line by one experimenter.http://www.biosemi.com/) from a pre-cabled cap with 128 pin-type active Ag/AgCl electrodes (with a modified 10–20 system montage). Minimal skin preparation was necessary with active electrodes. Eight additional drop-down electrodes were attached around the participant’s face: horizontal electro-oculographic (EOG) signals were recorded at the left and right external canthi, vertical EOGs were recorded below the eyes, and 4 additional electrodes were placed bilaterally on the mastoids and peri-auricular sites. Individual electrodes were adjusted until the electrode offset was within the range of ±40 mV.EEG signal was continuously sampled at 256 Hz using a BioSemi ActiveTwo system  that captured the most prominent effects observed in our study, in order to maximize variations in the underlying cognitive processes of both tasks. Each ROI encompasses the electrode that shows the maximum MF versus ML difference in a given task and the three closest neighbouring electrodes see . For eacA clear dipole-like voltage pattern can be identified with negativity at the left frontal sites and positivity at right posterior sites in the ML-MF contrast for the imitation task at the first time-window (565–715 msec). This effect shows the maximum difference between MF and ML conditions at electrode F5’ around 640 msec after video stimuli onset. The left frontal ROI thus comprises the electrodes closest to F5’: AF5’, F7, AF7’ . The right counterpart is the region that includes electrodes F6’, AF6’, F8, AF8’.At a middle time-window (925–1075 msec), a central-parietal right-lateralized positivity was identifiable for the discrimination task see . The eleFor the discrimination task, a final component with negativity over right parieto-occipital scalp regions was observed much later (1475–1525 msec). To explore this effect, we selected the PO4’ electrode with maximum difference between MF and ML actions from inspection of the topographic map. The time-window for this effect was centered at 1450 msec after stimulus onset, the sample point where the difference reaches its maximum at PO4’ electrode. The right parieto-occipital region comprises PO4’ and its closest neighbouring electrodes: PO2’, PO6’, PO8. The left counterpart included electrodes PO1’, PO3’, PO5’, PO7.For each time-window, a repeated-measures ANOVA was performed on the mean amplitude values of correct trials for each participant, with Task , Meaning , ROI  and Lateralization  as independent variables. Post-hoc Tukey’s Honestly Significant Difference Test was used to detect the sources of the effects involving more than two conditions.F = 22.5, p<.0001). Although we observed a mean amplitude potential lower for the right hemisphere than for the left hemisphere  = 16.1, p<.001), we observed a significant hemisphere × ROI  = 5.1, p = .011) interaction, with this effect being present at the frontal sites only (Tukey p<.001), and absent at the central parietal (Tukey p = .99), and at the parieto-occipital (Tukey p = .31) scalp sites. Overall ML actions had a lower mean amplitude than MF actions  = 6.6, p = .01), while this was not the case for the most posterior scalp region (Tukey p = .88), where this effect was slightly reverted  = 4.1, p = .025).At this time-window, a broadly distributed negativity was observed over the most anterior region with its inverted counterpart towards the most posterior regions  = 3.97, p = .028) was found at this time-window. Based on subsequent separate ANOVAs for each of the three ROIs, we were able to detect that the difference between imitation and discrimination task was present mainly at the left frontal region  = 8.87, p = .008); Task × Meaning × Hemisphere: F = 15.80, p = .001), while it did not occur in the Central-Parietal ROI  = 1.78, p>.1; Task × Meaning × Hemisphere: F = 1.79, p>.2), and it was reversed in the Parieto-occipital ROI  = 2.56, p>.1; Task × Meaning × Hemisphere: F = 6.22, p = .02).Critically, a significant Task × Meaning × ROI × Hemisphere interaction (p<.001); while the difference between MF and ML actions is not present in the discrimination task (Tukey p = .99). The 3-way interaction found at the parieto-occipital ROI is driven mostly by the absence of differences between the 2 hemispheres for ML action in the imitation task (Tukey p = .09), as overall the right hemisphere leads to a higher mean amplitude than the left hemisphere.Post-hoc comparisons further confirmed that this 3-way interaction found at the frontal ROI  is driveF = 25, p<.001). The meaning × ROI interaction  = 4.4, p = .02, see p<.001), while MF and ML actions did not differ significantly in the frontal region (Tukey p = .97) and parieto-occipital ones (Tukey p = .09).At this time-window overall MF actions show a higher mean amplitude than ML actions  = 11, p = .004, see p = .003) and the Imitation task (Tukey p = .001), at the right hemisphere this difference was even enhanced for the discrimination task (Tukey p = .0001) but tended to disappear for the imitation task (Tukey p = .071).Moreover, a Task × Meaning × Hemisphere interaction was found  = 11.76, p = .003), with the difference between MF and ML actions being present for the discrimination task in the right hemisphere (Tukey p<.001) but not in the left hemisphere (Tukey p = .7) (see p>.15).At the latest time-window a Task × Meaning × Hemisphere interaction was found ( .7) see . In the F = 4.49, p = .01) in which, for the discrimination task, Meaning did not differ in the most anterior region or in the central region  but in the parieto-occipital region only . For the imitation task, no significant differences between MF and ML actions were found .The difference between MF and ML actions found in the discrimination task in the right hemisphere is supported by a Task × Meaning × ROI interaction , suggests that it underlies imitation learning mechanisms. In imitating unfamiliar actions, a translation between the visual input and motor output is needed Moreover, according to the Hemispheric Encoding/Retrieval Asymmetry (HERA) model This frontal activity is also consistent with the idea of a left-lateralized and hand-independent network for planning and execution of both transitive and intransitive gestures, that comprises not only parietal regions but also temporal and frontal ones e.g., . Having The early recruitment of left lateralized frontal processes does not contradict the findings of Nishitani and Hari A dissociation between imitating and observing actions was also found both at a middle time window (around 1000 msec) and at the latest one (1550 msec). Earlier over right central-parietal and later on over right parieto-occipital scalp sites, an increased negativity was found for unfamiliar meaningless actions when participants prepared to performed the discrimination task. The specific participation of a right lateralized occipito-parietal network has been already reported for both imitation and perception of meaningless actions, as compared to meaningful ones Alternatively, this right lateralized component over parieto-occipital scalp regions could be partially driven by activity within the Superior Temporal Sulcus (STS). This structure has been implicated in visual perception of biological motion, as it is selectively active for biological-like motion as opposed to non-biological, random motion Besides the general role played by the STS in perception of biological motion, two studies have shown that this region is strongly engaged when participants observe an incorrect eye gaze movement st person versus 3rd person spatial transformations and perspective taking tasks st person versus 3rd person spatial transformations, or less conflict between them, thus explaining the reduction of sustained positivity in the discrimination task for ML actions.The notion of common coding and shared action representations raises the issues of how self versus others distinction comes about, and specifically which neural mechanisms are engaged in discriminating the representation of one self and those activated by external agents Ortigue and colleagues’ study In what concerns the relation between these two latest components, we believe that they need not be necessarily related. Nonetheless the fact that the putative engagement of areas that code for agency come only after processes that detect the violation of expectancies by the observation of unfamiliar actions could tell us otherwise. Our tentative interpretation of the latest component that was found for the discrimination of unfamiliar actions is based on the supposed involvement of brain areas that code for the need to distinguish between agents. Thus it could be the case that the STS activity, that we have interpreted as coding for actions that do not yet belong to the motor repertoire and that violate expectancies is feeding the TPJ with this information of the absence of stored motor representations.In summary, in the present study we show different ERP components during observation of unfamiliar actions, depending on whether participants had to later imitate the presented action or to extract its possible meaning. These components are indicative of distinct supplementary mechanisms recruited during action observation. An early but sustained negative component was found over the anterior left scalp sites when participants prepare to imitate ML unfamiliar actions, compatibly with the engagement of general associative learning mechanisms. On the other hand, an increased negativity was found when participants observed unfamiliar actions (for later discrimination) at a middle time window (around 1000 msec) over the right centro-parietal scalp sites, and later (around 1550 msec) over more posterior sites, suggesting the recruitment of distinct high-order perceptual mechanisms related to the function of right occipito-parietal regions.In conclusion, based on scalp topography, time course and sensitivity to experimental manipulations, our results suggest dissociable mechanisms for action imitation and action discrimination: imitating unfamiliar actions impose particular demands on a general learning mechanism, while discriminating unfamiliar actions calls for distinct high-order perceptual mechanisms.Appendix S1Complete list of meaningful actions used as stimuli.(DOC)Click here for additional data file.


Axon guidance molecules determine the pattern of neuronal circuits. Accuracy of the process is ensured by unknown mechanisms that correct early guidance errors. Since the time frame of error correction in Sema3A null mice partly overlaps with the period of naturally occurring cell death in dorsal root ganglia (DRG) development, we tested the hypothesis that apoptosis of misguided neurons enables error correction. We crossed BAX null mice, in which DRG apoptosis is blocked, with Sema3A null mice to induce errors. Analyses of these double-null mouse embryos showed that the elimination of abnormal projections is not blocked in the absence of BAX. Surprisingly however, there are fewer surviving neurons in Sema3A null or Sema3A/BAX double-null newborn mice than in wild-type mice. These results suggest that guidance errors are corrected by a BAX-independent cell death mechanism. Thus, aberrant axonal guidance may lead to reductions in neuronal numbers to suboptimal levels, perhaps increasing the likelihood of neuropathological consequences later in life. Nervous system function is critically dependent on highly stereotypic and precise patterns of neuronal connectivity established during embryogenesis. The formation of these connections depends on the ability of axons to probe their environment and selectively navigate to their targets in an accurate manner The vertebrate nervous system undergoes massive cell death during development, with a loss of approximately half the neurons +/−:BAX+/− over Sema3A+/−:BAX+/−) were used to obtain wild-type, Sema3A+/+:BAX−/−, Sema3A−/−:BAX+/+, Sema3A−/−:BAX−/−, Sema3A+/−:BAX−/−, and Sema3A+/− :BAX+/+ animals. Animal handling adhered strictly to national and institutional guidelines for animal research and was approved by the Ethics Committee of the Hebrew University.BAX and Sema3A null mutant mice were used as previously described Neuronal number estimates were made using “the optical dissector method” previously described L5 DRG  of P0 mice were dissected as a whole, fixed and stained with anti-neurofilament antibody 2H3 to visualize neuronal somas and DAPI to visualize nuclei. Whole-mount immunostaining was performed as described Serial optical Z-sections (5 µm) of the entire ganglion were imaged using a laser-scanning confocal microscope (Olympus IX70). Two ganglia per embryo were imaged based on the integrity of the tissue and the staining quality. Adjacent optical sections were used as a dissector where the first optical section was the ‘sample’ section and the next section functioned as the ‘look up’ section. Nuclei of neurofilament-positive cells were counted manually if they did not appear in the ‘look up’ section, thereby eliminating over- or underestimation of neuronal counts (ImageJ). Cells were counted by an observer who was blind to the genotype of the embryos tested.In each ganglion, every 5th section  was used as a sample section and the average count per section was multiplied by the total section number to give the estimate of the total neuronal number per ganglion.+/+:BAX−/− embryos, three Sema3A−/−:BAX−/− embryos and five Sema3A−/−:Bax+/+ embryos).Only litters with a wild-type genotype as a reference were used. An average of total neuronal number per ganglion of each embryo was determined. Results represent the average total neuronal number per ganglion of each genotype . By E15.5, while the number of errors in Sema3A−/−:BAX+/+ is unchanged (6.61±0.79 errors per DRG), in Sema3A−/−:BAX−/− we observed a doubling of error number  of lumbar DRG axons in Sema3A−/−:BAX+/+ and Sema3A−/−:BAX+/+ axon errors are almost completely eliminated . This result is consistent with our previous study −/−:BAX−/− is reduced, albeit to a lesser degree , however two lines of evidence make this possibility unlikely. First, there is a very minor increase in the levels of abnormal projections in mice in which only BAX (Sema3A+/+ background) is mutated . To this end we compared the levels of cleaved caspase 3 between Sema3A+/+:BAX+/+ and Sema3A−/−:BAX+/+ mice at both E15.5 and E16.5. No significant increase in cleaved caspase 3 was detected  did not allow direct testing of this possibility. We therefore carried out direct counts of all DRG neurons to assess the involvement of cell death per se in the correction mechanism. In a previous study, Haupt et al. found no significant difference in the number of DRG neurons of wild-type and Sema3A mutant mice at brachial, thoracic or lumbar DRG levels at E15.5 +/+:BAX−/−, Sema3A−/−:BAX−/− and Sema3A−/− :BAX+/+ mutant mice . This increase in neuron number reflects a blockage of BAX-dependent apoptosis The results thus far demonstrate that guidance error correction occurs even in the absence of BAX activity, raising the possibility that a BAX-independent non-apoptotic cell death mechanism might be involved. Unfortunately, the lack of reliable tools to evaluate other non-apoptotic, programmed cell death ant mice . The ave−/−:BAX−/− the average number of neurons was reduced to 9,128 neurons , indicating that extensive cell death occurs in the absence of BAX and in association with path-finding error correction. The average number of DRG neurons in Sema3A−/−:BAX+/+ was only 6,860 (62% of wild type). This reduction in neuron number is associated with error correction and is likely due to the activity of BAX-independent cell-death mechanisms.In Sema3AWe have previously shown that extensive axon guidance abnormalities are largely eliminated during embryonic development by an unknown mechanism −/−:BAX+/+ and Sema3A−/−:BAX−/− mice. The possibility that this cell loss reflects a novel survival-promoting role Sema3A is highly unlikely, since it’s specific co-receptor Nrp1 is not expressed in DRG neurons during the critical period examined in vitro studies have reported that Sema3A functions as a death-inducing molecule for embryonic DRG neurons We found a reduction in postnatal neuronal cell number in both Sema3A−/−:BAX−/− mice is higher than in Sema3A−/− :BAX+/+ mice, although in both genotypes the number of errors is reduced proportionally . Recent emerging evidence also points to molecules such as presenilin, which play a pivotal role in neurodegenerative disease, as playing an essential role in axon guidance


DAT1) and catechol-O-methyltransferase genes (COMT) on the time-course of motor processing in a contingent negative variation (CNV) task.Dopamine plays an important role in orienting, response anticipation and movement evaluation. Thus, we examined the influence of functional variants related to dopamine inactivation in the dopamine transporter .64-channel EEG recordings were obtained from 195 healthy adolescents of a community-based sample during a continuous performance task (A-X version). Early and late CNV as well as motor postimperative negative variation were assessed. Adolescents were genotyped for the COMT and DAT1, indicating that COMT exerted stronger effects on lateralized motor post-processing  in homozygous carriers of a DAT1 haplotype increasing DAT1 expression. Source analysis showed that the time interval 500–1000 ms after the motor response was specifically affected in contrast to preceding movement anticipation and programming stages, which were not altered.The results revealed a significant interaction between Motor slow negative waves allow the genomic imaging of dopamine inactivation effects on cortical motor post-processing during response evaluation. This is the first report to point towards epistatic effects in the motor system during response evaluation, i.e. during the post-processing of an already executed movement rather than during movement programming. The perception-action-cycle involves different stages, from the orienting to a warning stimulus to the preparation of a reaction movement and the evaluation of the given response. Due to the high time resolution of electroencephalography, these stages can be excellently assessed in contingent negative variation (CNV) paradigms, which involve a cue followed by a later imperative stimulus which requires a motor response The duration of dopaminergic action is limited by dopamine inactivation, i.e. mainly methylation by catechol-O-methyltransferase (COMT) in the prefrontal cortex COMT and DAT1 genes on the orientation reaction, movement programming and stimulus post-processing (indexed by the early and late components of the CNV as well as the motor postimperative negative variation component), during a continuous performance test with speeded button press response movements.In the current study, we investigated the effects of three functional polymorphisms in the COMT polymorphism, characterized by the substitution of valine for methionine at codon 158 DAT1 and the 6-repeat allele of a VNTR in intron 8 lead to greater DAT1 expression DAT expression DAT1-expression increasing VNTRs, the 6R–10R haplotype, has been reported to strengthen this effect DAT1 haplotype and its interaction with the COMT Val158Met polymorphism in relation to motor PINV.A widely studied functional COMT allele DAT1 allele Previous fMRI genomic imaging studies have demonstrated that higher prefrontal . Sintered silver/silver chloride electrodes were positioned by an equidistant electrode cap . Electrode impedances were kept below 10 kOhm. Vertical electrooculogram (VEOG) was recorded by electrodes 1 cm below and above the left eye. Horizontal electrooculogram (HEOG) was calculated by leads F9′ and F10′ next to the outer canthi. Small deviations of electrode positions from the international 10-10 system are indicated by apostrophes. The recording reference was placed near the left mastoid. Offline, data were transformed to average reference. The sampling rate was 500 Hz. An anti-aliasing low-pass filter with a cut-off frequency of 100 Hz was employed. The visual stimulation was presented by Gentask of the Neuroscan Stim software package. Reaction times were collected from response triggers from the response pad.Subjects performed a computerized A-X version of the continuous performance test  were employed as distractors. The distractor ‘H’ occurred 160 times (frequent distractor). The distractors ‘B’, ‘C’, ‘D’, ‘E’, ‘F’, ‘G’, ‘J’, and ‘L’ appeared 40 times each.For the analysis of early and late CNV, continuous recordings were segmented into stimulus-locked segments from 400 ms prior to the distractor before the warning stimulus ‘A’ to 1600 ms after the imperative stimulus ‘X’ . The 400 ms before the warning stimulus ‘A’ served as baseline.For the analysis of the initial motor potential peak during movement execution and the analysis of motor post-processing, response-locked epochs of 4 seconds were created, beginning 2800 ms before the response until 1200 ms afterwards. The first 400 ms of this interval served as baseline (2800 to 2400 ms before the response). Only trials with correct responses within 800 ms were included in the analysis. Taking into account the stimulus onset asynchrony of 1600 ms, this assured that the baseline interval was situated before the onset of the warning letter ‘A’ even for slow responses. The warning letter ‘A’ of a target sequence was never directly preceded by another target sequence. Thus, during the baseline, the subjects had processed a distractor letter and were waiting for the next stimulus to occur. Reaction times took at least approximately 150–200 ms, even for fast responses. We verified that there were no gene effects on the baseline time interval which could have influenced the results.Data were corrected for eye movements and blinks by the algorithm of Gratton and Coles . Average reference was calculated offline. Data were 30 Hz low-pass filtered by a zero-phase shift Butterworth filter with a slope of 48 dB/octave. Potentials exceeding 150 µV amplitude were rejected automatically as artifacts; remaining smaller artifacts were removed by an experienced EEG technician who was blind to the study hypotheses.We calculated lateralized movement-related potentials by subtracting the potentials at each electrode on the right hemisphere from the potentials measured at its homologue on the left side of the head (e.g. C3–C4). In this way, the resulting polarity reflects the polarity for potentials which were lateralized contralateral to the response movement with the right hand. In a previous study, we had found that lateralization in trials with the dominant right hand closely resembled the final lateralized movement-related potential Early CNV amplitude was measured at its topographical maximum at Fz during the time interval 600–900 ms The amplitude of the motor part of late CNV amplitude was determined as the mean potential 200 ms before the imperative stimulus (‘X’) over the left  motor  and the supplementary motor area  over the motor area  during the interval 400–800 ms after the unilateral index finger response movement, comparable to our previous studies DAT1 6R–10R/6R–10R+COMT Met/Met (highest motor PINV amplitudes) and the DAT1 other+COMT Val/Val group (low motor PINV amplitudes), because qualitative differences in motor PINV topography were found between the genetic groups.Source analysis was carried out on the group grand averages, which provide the best signal-to-noise ratio. Dipole source modelling with equivalent dipoles allows the examination of group differences in the time course of event-related potential components. According to our previous study In a second complementary approach with distributed sources instead of equivalent dipoles, sLORETA with the BESA default parameters  was performed on the same motor PINV time interval  in order to describe the extension of the distributed cortical sources of the lateralized motor PINV as exact as possible.EDTA anticoagulated venous blood samples were collected. Leukocyte genomic deoxyribonucleic acid (DNA) was isolated with the Qiamp DNA extraction kit .COMT single nucleotide polymorphism (SNP) was completed using TaqMan (SNP) Genotyping Assays . Amplification conditions for COMT rs4680 were: 3.0 µl TaqMan® Mastermix, 0.3 µl/0.15 µl TaqMan® oligonucleotide mix (20×/40×), 1.70 µl dH2O and 1 µl DNA solution (∼30 ng) in 96-well format in a 6 µl reaction. Amplification was performed by initial heating of 10 min–95°C, 40 cycles of 15 sec–95°C/1 min-60°C and final 10 min-4°C. TaqMan® assay-on-demand ID C_25746809_50 detected the alleles of rs4680 (hCV25746809) in the sequence context of CCAGCGGATGGTGGATTTCGCTGGC[A/G]TGAAGGACAAGGTGTGCATGCC.Genotyping of the DAT1 was genotyped with the primers and reaction conditions of Sano et al. DAT1 30 bp VNTR intron 8 p = 0.78; DAT1 40 bp VNTR 3′UTR p = 0.10; COMT p = 0.20).The 40-bp VNTR polymorphism in the 3′-untranslated-region (UTR) of DAT1 VNTRs were analyzed combined as haplotype. In accordance with the previous literature and in order to avoid small groups containing only a low number of subjects, with respect to the DAT1 haplotype, subjects were dichotomized into homozygous carriers of the 6R–10R haplotype, which have previously been demonstrated to increase the risk of psychiatric disorders Both DAT1 haplotype: 6R–10R/6R–10R (N = 79) versus at least one non-6R–10R haplotype (N = 95); and (2) COMT: Val/Val (N = 43) versus Val/Met (N = 96) versus Met/Met (N = 35).In detail, the following genotype groups were formed: (1) DAT1 haplotype  and COMT  on the target parameters , linear regression analyses were performed. In order to test for a significant epistasis between DAT1 and COMT, regression models with and without an interaction term were compared. Significant interactions were further examined by conducting separate regression analyses for each DAT1 haplotype level with COMT genotype as a predictor. All analyses included gender as a covariate. The same regression analysis as for EEG parameters was performed on behavioral measures, i.e. reaction time, reaction time variability, the number of omission and commission errors. In order to further examine the functional meaning of motor PINV, Pearson correlation coefficients were calculated between motor PINV amplitude and the behavioral measures.To examine the effect of the DAT1 and COMT did not show any significant main effects or interactions on the number of commission errors, there was a significant interaction between the two functional polymorphisms with respect to omission errors . However, separate regression analyses for the two DAT1 haplotype groups with respect to COMT effects on omission errors did not yield significant results.Mean reaction time was 352±58 ms (± SD), with no significant effects of genetic variants. Trend level towards genetic influences on reaction time variability was not reached either . There wDAT1, COMT or their interaction on early and late CNV as well as on the initial motor potential peak for any examined target area . Descriptive data pointed towards a non-significant reduction of lateralized negativity over the motor area during the initial motor potential peak for those genotypes which were associated with an increased lateralized motor PINV  = 4.1; p = 0.008; each p = 0.01 for DAT1 and COMT; linear regression model without an interaction term, see The main finding of our study was that both DAT1 haplotype  reflected differences in lateralized motor system activation .aplotype  and the pe (COMT  genotypeDAT1 haplotype groups (6R–10R/6R–10R and at least one non-6R–10R haplotype) indicated that the COMT genotype only had a significant effect on motor PINV amplitude in carriers of the homozygous 6R–10R DAT1 haplotypes  but not for carriers of other haplotype combinations . The largest lateralized motor PINV amplitudes occurred in those subjects carrying the Met/Met COMT polymorphism and being homozygous for the 6R–10R DAT1 haplotype. The time course of the potential showed that the effects were specific for the motor PINV time interval because the early negative movement-related potentials around the button press trigger were unaffected by genetic variation.Separate linear regression analyses for the two DAT1 haplotype carrier groups ×3 COMT genotypes) presented mean handedness indexes ≥93. The highest handedness index was obtained for the group with at least one non-6R–10R and the Met/Met allele, i.e. the group that showed a smaller motor PINV lateralization than the 6R–10R/6R–10R+Met/Met group.In our sample, the degree of lateralization of the motor PINV was unrelated to differences in the degree of handedness between the subject groups: There was no correlation between the handedness index and motor PINV amplitude, as all six genetically defined groups , indicating that more lateralized negative motor PINV amplitudes were associated with longer reaction times. Trend level for an association of larger reaction time variability and more lateralized negative motor PINV amplitudes was almost reached .Moreover, there was a significant correlation between lateralized motor PINV amplitude and the number of omission errors , indicating that stronger motor post-processing during motor PINV on correct responses was associated with a higher number of omission errors. No such correlation of motor PINV with the number of commission errors was obtained .DAT1 non 6R–10/6R–10R+COMT Val/Val group, no spatial component near the central sulcus was found  near the central sulcus on the left hemisphere  was more lateralized in the subjects carrying the 6R–10R/6R–10R DAT1  and the enotypes , with stenotypes . Contralenotypes , 2 and 4DAT1 and COMT, which specifically affected the motor post-processing time interval. Interactions between these genes have been described so far by fMRI only with respect to other cognitive functions, namely reward sensitivity To the best of our knowledge, this is the first study to perform time-resolved genomic imaging of the influences of functional polymorphisms which are crucially involved in dopaminergic neurotransmission on orienting, movement preparation, execution and motor post-processing. Moreover, our study is the first to show an epistatic effect, i.e. an interaction between DAT1 allele and increased distractibility  were demonstrated to be associated with the diagnosis of attention deficit hyperactivity disorder (ADHD) DAT1 and COMT with regard to both the number of omission errors (an index of inattention) and lateralized motor PINV amplitude (which may be related to distraction as explained above).In addition, a small effect with respect to an association of larger motor PINV amplitudes with longer reaction times and increased reaction time variability was observed. Moreover, larger motor PINV amplitudes were linked with more omission errors. These findings are consistent with those from another study DAT1 and COMT related to lateralized motor post-processing indicated that COMT exerted significantly stronger effects in homozygous carriers of the 6R–10R DAT1 haplotype. In this case, individuals with the Met/Met genotype showed higher motor PINV amplitudes over contralateral motor areas than Val/Val carriers, while individuals with a heterozygous COMT exhibited intermediate motor PINV amplitudes.The significant interaction between DAT1 and COMT genotypes. This activity was reflected in equivalent dipole source analysis by a spatial component near the central sulcus, which illustrated that this activation was specific for the post-processing interval  involve different cortical and subcortical brain regions. While movement initiation involves the supplementary motor area and the basal ganglia DAT1 and COMT only on motor PINV without influences on preceding movement-related potentials is plausible as these potentials all reflect functionally different and separable processes, stages and networks. A differential modulation of motor PINV and contingent negative variation by dopamine antagonists (first generation antipsychotics) and in Parkinson's disease has been shown Thus, a selective influence of Although no electromyographic data were available, movement-related potential lateralization is mostly independent of muscle force COMT Met allele is likely to increase tonic dopamine levels and to reduce phasic dopamine responses COMT alleles are co-dominant COMT knockout mice show a prefrontal dopamine increase, with no similar effect in the striatum The COMT allele) and lower striatal  dopamine levels result in a more focused prefrontal cortical activation refers to the action via different types of dopamine receptors (D1/D2) and the nature of interactions in the cortico-striato-thalamo-cortical system The explanation of why higher prefrontal , which increases prefrontal cortex control of tonic dopamine level in the striatum DAT1 expression induced by the 6R–10R haplotype DAT1 6R–10R haplotype and the COMT Met/Met genotype exhibited the largest motor PINV amplitudes. However, functional responses in the striatum (e.g. reward cue-related BOLD responses or dopamine release) have been found to be larger for carriers of the 9R-allele in some but not all studies According to the tonic-phasic model amplitude rather than the duration of stimulus post-processing was mainly affected suggests that the effects were not directly related to the speed of dopamine inactivation by COMT or DAT1 At first glance, it seems highly plausible that genes which relate to dopamine inactivation affect stimulus post-processing. However, the finding that the post-processing Our findings suggest that tonic prefrontal and striatal dopamine levels interact. Specifically, in carriers of the 6R–10R/6R–10R+Met/Met genotype, a significant enhancement of motor post-processing could be shown in 15-year-old adolescents of a high-risk cohort. For the first time, dopaminergic genetic influences were observed on the time course of motor post-processing and were separated from preceding movement preparation and execution. Our results provide an example of how gene-gene interactions (apart from gene-environment interactions) can explain the limited effects of single genes on endophenotypes.