Associations of Hyperactivity and Inattention Scores with Theta and Beta Oscillatory Dynamics of EEG in Stop-Signal Task in Healthy Children 7-10 Years Old.

In the current study, we aimed to investigate the associations between the natural variability in hyperactivity and inattention scores, as well as their combination with EEG oscillatory responses in the Stop-Signal task in a sample of healthy children. During performance, the Stop-Signal task EEGs were recorded in 94 Caucasian children (40 girls) from 7 to 10 years. Hyperactivity/inattention and inattention scores positively correlated with RT variability. Hyperactivity/inattention and inattention scores negatively correlated with an increase in beta spectral power in the first 200 ms after presentation of the Go stimulus. Such results are in line with the lack of arousal model in ADHD children and can be associated with less sensory arousal in the early stages of perception in children with symptoms of inattention. The subsequent greater increase in theta rhythm at about 300 ms after presentation of the Go stimulus in children with higher inattention scores may be associated with increased attention processes and compensation for insufficient vigilance in the early stages of perception.

Intrinsic Connectivity Networks in the Self- and Other-Referential Processing.

Neuroimaging studies have revealed a multitude of brain regions associated with self- and other-referential processing, but the question how the distinction between self, close other, and distant other is processed in the brain still remains unanswered. The default mode network (DMN) is the primary network associated with the processing of self, whereas task-positive networks (TPN) are indispensable for the processing of external objects. We hypothesize that self- and close-other-processing would engage DMN more than TPN, whereas distant-other-processing would engage TPN to a greater extent. To test this hypothesis, we used functional magnetic resonance imaging (fMRI) functional connectivity data obtained in the course of a trait adjective judgment task while subjects evaluated themselves, the best friend, a neutral stranger, and an unpleasant person. A positive association between the degree of self-relatedness and the degree of DMN dominance was revealed in cortical midline structures (CMS) and the left lateral prefrontal cortex. Relative to TPN, DMN showed greater connectivity in me than in friend, in friend than in stranger, and in stranger than in unpleasant conditions. These results show that the less the evaluated person is perceived as self-related, the more the balance of activity in the brain shifts from the DMN to the TPN.

Muscle LIM Protein Is Expressed in the Injured Adult CNS and Promotes Axon Regeneration.

Muscle LIM protein (MLP) has long been regarded as a muscle-specific protein. Here, we report that MLP expression is induced in adult rat retinal ganglion cells (RGCs) upon axotomy, and its expression is correlated with their ability to regenerate injured axons. Specific knockdown of MLP in RGCs compromises axon regeneration, while overexpression in vivo facilitates optic nerve regeneration and regrowth of sensory neurons without affecting neuronal survival. MLP accumulates in the cell body, the nucleus, and in axonal growth cones, which are significantly enlarged by its overexpression. Only the MLP fraction in growth cones is relevant for promoting axon extension. Additional data suggest that MLP acts as an actin cross-linker, thereby facilitating filopodia formation and increasing growth cone motility. Thus, MLP-mediated effects on actin could become a therapeutic strategy for promoting nerve repair.

Boosting CNS axon regeneration by harnessing antagonistic effects of GSK3 activity.

Implications of GSK3 activity for axon regeneration are often inconsistent, if not controversial. Sustained GSK3 activity in GSK3S/A knock-in mice reportedly accelerates peripheral nerve regeneration via increased MAP1B phosphorylation and concomitantly reduces microtubule detyrosination. In contrast, the current study shows that lens injury-stimulated optic nerve regeneration was significantly compromised in these knock-in mice. Phosphorylation of MAP1B and CRMP2 was expectedly increased in retinal ganglion cell (RGC) axons upon enhanced GSK3 activity, but, surprisingly, no GSK3-mediated CRMP2 inhibition was detected in sciatic nerves, thus revealing a fundamental difference between central and peripheral axons. Conversely, genetic or shRNA-mediated conditional KO/knockdown of GSK3ß reduced inhibitory phosphorylation of CRMP2 in RGCs and improved optic nerve regeneration. Accordingly, GSK3ß KO-mediated neurite growth promotion and myelin disinhibition were abrogated by CRMP2 inhibition and largely mimicked in WT neurons upon expression of constitutively active CRMP2 (CRMP2T/A). These results underscore the prevalent requirement of active CRMP2 for optic nerve regeneration. Strikingly, expression of CRMP2T/A in GSK3S/A RGCs further boosted optic nerve regeneration, with axons reaching the optic chiasm within 3 wk. Thus, active GSK3 can also markedly promote axonal growth in central nerves if CRMP2 concurrently remains active. Similar to peripheral nerves, GSK3-mediated MAP1B phosphorylation/activation and the reduction of microtubule detyrosination contributed to this effect. Overall, these findings reconcile conflicting data on GSK3-mediated axon regeneration. In addition, the concept of complementary modulation of normally antagonistically targeted GSK3 substrates offers a therapeutically applicable approach to potentiate the regenerative outcome in the injured CNS.

Nociceptive DRG neurons express muscle lim protein upon axonal injury.

Muscle lim protein (MLP) has long been regarded as a cytosolic and nuclear muscular protein. Here, we show that MLP is also expressed in a subpopulation of adult rat dorsal root ganglia (DRG) neurons in response to axonal injury, while the protein was not detectable in naïve cells. Detailed immunohistochemical analysis of L4/L5 DRG revealed ~3% of MLP-positive neurons 2 days after complete sciatic nerve crush and maximum ~10% after 4-14 days. Similarly, in mixed cultures from cervical, thoracic, lumbar and sacral DRG ~6% of neurons were MLP-positive after 2 days and maximal 17% after 3 days. In both, histological sections and cell cultures, the protein was detected in the cytosol and axons of small diameter cells, while the nucleus remained devoid. Moreover, the vast majority could not be assigned to any of the well characterized canonical DRG subpopulations at 7 days after nerve injury. However, further analysis in cell culture revealed that the largest population of MLP expressing cells originated from non-peptidergic IB4-positive nociceptive neurons, which lose their ability to bind the lectin upon axotomy. Thus, MLP is mostly expressed in a subset of axotomized nociceptive neurons and can be used as a novel marker for this population of cells.

Highly efficient transduction of primary adult CNS and PNS neurons.

Delivery and expression of recombinant genes, a key methodology for many applications in biological research, remains a challenge especially for mature neurons. Here, we report easy, highly efficient and well tolerated transduction of adult peripheral and central neuronal populations of diverse species in culture using VSV-G pseudo-typed, recombinant baculovirus (BacMam). Transduction rates of up to 80% were reliably achieved at high multiplicity of infection without apparent neuro-cytopathic effects. Neurons could be transduced either shortly after plating or after several days in culture. Co-incubation with two different baculoviruses attained near complete co-localization of fluorescent protein expression, indicating multigene delivery. Finally, evidence for functional protein expression is provided by means of cre-mediated genetic recombination and neurite outgrowth assays. Recombinant protein was already detected within hours after transduction, thereby enabling functional readouts even in relatively short-lived neuronal cultures. Altogether, these results substantiate the usefulness of baculovirus-mediated transduction of mature neurons for future research in neuroscience.

Boosting Central Nervous System Axon Regeneration by Circumventing Limitations of Natural Cytokine Signaling.

Retinal ganglion cells (RGCs) do not normally regenerate injured axons, but die upon axotomy. Although IL-6-like cytokines are reportedly neuroprotective and promote optic nerve regeneration, their overall regenerative effects remain rather moderate. Here, we hypothesized that direct activation of the gp130 receptor by the designer cytokine hyper-IL-6 (hIL-6) might induce stronger RGC regeneration than natural cytokines. Indeed, hIL-6 stimulated neurite growth of adult cultured RGCs with significantly higher efficacy than CNTF or IL-6. This neurite growth promoting effect could be attributed to stronger activation of the JAK/STAT3 and PI3K/AKT/mTOR signaling pathways and was also observed in peripheral dorsal root ganglion neurons. Moreover, hIL-6 abrogated axon growth inhibition by central nervous system (CNS) myelin. Remarkably, continuous hIL-6 expression upon RGC-specific AAV transduction after optic nerve crush exerted stronger axon regeneration than other known regeneration promoting treatments such as lens injury and PTEN knockout, with some axons growing through the optic chiasm 6 weeks after optic nerve injury. Combination of hIL-6 with RGC-specific PTEN knockout further enhanced optic nerve regeneration. Therefore, direct activation of gp130 signaling might be a novel, clinically applicable approach for robust CNS repair.

Neuronal expression of muscle LIM protein in postnatal retinae of rodents.

Muscle LIM protein (MLP) is a member of the cysteine rich protein family and has so far been regarded as a muscle-specific protein that is mainly involved in myogenesis and the organization of cytoskeletal structure in myocytes, respectively. The current study demonstrates for the first time that MLP expression is not restricted to muscle tissue, but is also found in the rat naive central nervous system. Using quantitative PCR, Western blot and immunohistochemical analyses we detected MLP in the postnatal rat retina, specifically in the somas and dendritic arbors of cholinergic amacrine cells (AC) of the inner nuclear layer and the ganglion cell layer (displaced AC). Induction of MLP expression started at embryonic day 20 and peaked between postnatal days 7 and 14. It subsequently decreased again to non-detectable protein levels after postnatal day 28. MLP was identified in the cytoplasm and dendrites but not in the nucleus of AC. Thus, retinal MLP expression correlates with the morphologic and functional development of cholinergic AC, suggesting a potential role of this protein in postnatal maturation and making MLP a suitable marker for these neurons.

EEG-correlates of trait anxiety in the stop-signal paradigm.

The relationship between trait anxiety and event-related EEG oscillatory reactions in the stop-signal paradigm was studied in 15 non-clinical subjects with average age of 26 years (13 men). In the paradigm, subjects responded to target stimuli by pressing one of the two choice buttons. In 30 out of 130 trials, target presentation was followed by a stop-signal, indicating that subjects had to refrain from a prepared motor response. The subject's level of anxiety was assessed using the State Trait Anxiety Inventory. Wide-band desynchronization (8-25 Hz) was found before button-press. It was sustained after the subjects pressed the button at 7-14 Hz frequency range. Also, synchronization at 15-25 Hz band occurred in 400-1400 ms after the button-press. Synchronization at lower frequencies (1-7 Hz) was also found during 0-700 ms after the stop-signal onset. Also, desynchronization at 8-20 Hz was found in 300-800 ms after stop-signal onset. The group with higher anxiety showed desynchronization at 10-13 Hz in 0-800 ms after the button-press, whereas the group with lower anxiety showed synchronization at the same frequency range. In 0-600 ms after stop-signal onset, desynchronization at 8-13 Hz was observed in the group with higher anxiety, whereas the group with lower anxiety demonstrated synchronization or weak desynchronization. Our findings support the Eysenck et al. [M.W. Eysenck, N. Derakshan, R. Santos, M.G. Calvo, Anxiety and cognitive performance: attentional control theory, Emotion 7(2) (2007) 336-356] theory that subjects with higher anxiety have more attentional control over reaction and increased use of processing resources as compared with lower anxiety subjects.

The effects of prostaglandin F2alpha treatment on peripheral-type benzodiazepine receptors in the ovary and uterus during pseudopregnancy of rats.

A previous study by us indicated that peripheral-type benzodiazepine receptor (PBR) density may be increased in the ovaries and uterus of pregnant rats (Weizman R, Dagan E, Snyder SH, Gavish M. Impact of pregnancy and lactation on GABAA receptor and central-type and peripheral-type benzodiazepine receptors. Brain Res 1997;752:7-14). In the present study, the effects of prostaglandin F2alpha (PGF2alpha) on PBR density in the ovary and uterus of pseudopregnant rats were assayed. Pseudopregnancy was induced on day 29 post-partum (PP) by s.c. injection of 50IU pregnant mare serum gonadotropin (PMSG) and 3 days later by s.c. injection of 20IU human chorionic gonadotropin (hCG). PBR ligand binding density was assayed with the specific PBR ligand [3H]PK 11195. A two-fold increase in ovarian PBR density was observed 2 days after hCG administration compared with vehicle control rats and this effect was maintained for 3 weeks. In the uterus, a three-fold increase in PBR density was observed and this increase was maintained for 1 week after hCG administration. Pseudopregnancy did not appear to affect renal PBR density or affinity. Treatment with PGF2alpha, which causes luteolysis, resulted in an approximately 50% reduction of PBR density in the ovaries of pseudopregnant rats at day 53 PP compared to pseudopregnant control rats. Treatment with indomethacin, which prevents the formation of PGF2alpha, caused the PBR density in the uterus of pseudopregnant rats at day 53 PP to be twice as high as in pseudopregnant control rats. All the above treatments did not affect the affinity of [3H]PK 11195 to ovarian and uterine PBR. These data suggest that PBR density in corpora lutea and uterus during pseudopregnancy is regulated by PGF2alpha.

The peripheral-type benzodiazepine receptor and tumorigenicity: isoquinoline binding protein (IBP) antisense knockdown in the C6 glioma cell line.

Peripheral-type benzodiazepine receptors (PBR) are constituted by three protein components, the isoquinoline binding protein (IBP), the voltage-dependent anion channel (VDAC), and the adenine nucleotide transporter (ANT). Recently, we found that high levels of PBR ligand binding in glioma cell lines correlate with in vitro tumorigenicity. To study whether enhanced PBR expression is causative or in response to cancer, we genetically modified C6 glioma cells. Antisense knockdown of the IBP resulted in more than 50% reductions in PBR ligand binding both in the mitochondrial and whole cell fractions, accompanied by similar reductions in IBP levels in these respective fractions. The IBP knockdown was accompanied by a 25% increase in cell number in confluent cultures. This correlated with an 8-fold increase in in vitro tumorigenicity, as assessed by anchorage independent growth. Cell cycle analysis indicated that knockdown of the IBP resulted in a 60% reduction in the number of cells in the pre-G1 apoptosis phase. This paralleled the reduction seen in apoptosis and cell death shown by DNA fragmentation and Trypan blue assays, respectively. Furthermore, knockdown of the IBP appeared to prevent induction of apoptosis by the antineoplastic agent, erucylphosphocholine. In addition, IBP knockdown prevented processing of the caspase 3 component of the apoptosis cascade by the erucylphosphocholine congener, erucylphospho-N,N,N-trimethylammonium. In conclusion, our results suggest that enhanced IBP expression, including enhanced PBR ligand binding, such as occurring in untreated C6 glioma cells, may provide a mechanism to increase apoptotic rates of cancer cells.

Peripheral-type benzodiazepine receptor density and in vitro tumorigenicity of glioma cell lines.

The peripheral-type benzodiazepine receptor is found primarily on the outer mitochondrial membrane and consists of three subunits: the 18kDa isoquinoline binding protein, the 32kDa voltage-dependent anion channel, and the 30kDa adenine nucleotide transporter. The current study evaluates the potential importance of peripheral-type benzodiazepine receptor expression in glioma cell tumorigenicity. While previous studies have suggested that peripheral-type benzodiazepine receptor-binding may be relatively increased in tumor tissue and cells, so far, little is known about the relationships between peripheral-type benzodiazepine receptor density and factors underlying tumorigenicity. In the present study, we found in glioma cell lines (C6, U87MG, and T98G), that peripheral-type benzodiazepine receptor ligand-binding density is relatively high for C6 and low for T98G, while U87MG displays intermediate levels. Cell growth of these cell lines in soft agar indicated that high levels of peripheral-type benzodiazepine receptor-binding were associated with increased colony size, indicative of their ability to establish anchorage independent cell proliferation. Potential causes for differences in tumorigenicity between these cell lines were suggested by various cell death and proliferation assays. Cell death, including apoptosis, appeared to be low in C6, and high in T98G, while U87MG displayed intermediate levels in this respect. Cell proliferation appeared to be high in C6, low in T98G, and intermediate in U87MG. In conclusion, our study suggests that relatively high peripheral-type benzodiazepine receptor-binding density is associated with enhanced tumorigenicity and cell proliferation rate. In particular, apoptosis appears to be an important tumorigenic determinant in these glioma cell lines. Moreover, application of PBR-specific ligands indicated that PBR indeed are functionally involved in apoptosis in glioma cells.