Pitch Memory in Nonmusicians and Musicians: Revealing Functional Differences Using Transcranial Direct Current Stimulation.

For music and language processing, memory for relative pitches is highly important. Functional imaging studies have shown activation of a complex neural system for pitch memory. One region that has been shown to be causally involved in the process for nonmusicians is the supramarginal gyrus (SMG). The present study aims at replicating this finding and at further examining the role of the SMG for pitch memory in musicians. Nonmusicians and musicians received cathodal transcranial direct current stimulation (tDCS) over the left SMG, right SMG, or sham stimulation, while completing a pitch recognition, pitch recall, and visual memory task. Cathodal tDCS over the left SMG led to a significant decrease in performance on both pitch memory tasks in nonmusicians. In musicians, cathodal stimulation over the left SMG had no effect, but stimulation over the right SMG impaired performance on the recognition task only. Furthermore, the results show a more pronounced deterioration effect for longer pitch sequences indicating that the SMG is involved in maintaining higher memory load. No stimulation effect was found in both groups on the visual control task. These findings provide evidence for a causal distinction of the left and right SMG function in musicians and nonmusicians.

Cortical activation associated with asterixis in manifest hepatic encephalopathy.

OBJECTIVES: Severe hepatic encephalopathy gives rise to asterixis, a striking motor symptom also called flapping tremor, which is characterized by a sudden ceasing of muscle tone in all muscles of a limb. In this study, we aimed at scrutinizing the cortical activation associated with asterixis and unraveling the underlying pathophysiological mechanisms. MATERIAL AND METHODS: We recorded simultaneously neural activity with magnetoencephalography (MEG) and muscle activity with surface EMG in nine patients with manifest hepatic encephalopathy showing asterixis. Asterixis events were detected semiautomatically and served as triggers for averaging MEG signals. Evoked responses averaged time-locked to asterixis events were subjected to equivalent current dipole (ECD) modeling. Additionally, we localized the strongest cortico-muscular coherence in the frequency of the co-occurring tremulousness. RESULTS: Evoked fields averaged time-locked to asterixis events were best explained by a single dipolar source in the contralateral primary motor cortex (M1, Talairach coordinates of mean localization: -40, -20, and 64; Brodmann area 4). This dipole showed a twofold field reversal, that is biphasic wave, with frontal dipole orientation at 49 ms before flap onset and 99 ms after flap onset. Conversely, two maxima with occipital dipole orientation were observed 2 ms and 160 ms after flap onset. Cortico-muscular coherence for the tremulousness was likewise localized in the contralateral M1 confirming earlier findings in the present patient cohort. CONCLUSIONS: Our results reveal an involvement of M1 in the generation of asterixis. As also tremulousness, also called mini-asterixis, was shown to originate in M1, asterixis and mini-asterixis may share common pathophysiological mechanisms.

Motor-cortical oscillations in early stages of Parkinson's disease.

Pathophysiological changes in basal ganglia-thalamo-cortical circuits are well established in idiopathic Parkinson's disease (PD). However, it remains open whether such alterations already occur at early stages representing a characteristic neurophysiological marker of PD. Therefore, the present study aims at elucidating changes of synchronised oscillatory activity in early PD patients. In this study, we performed whole-head magnetoencephalography (MEG) in a resting condition and during steady state contraction of the more severely affected forearm in 10 drug­naive, de novo patients, in 10 early-stage patients with chronic medication and in 10 age-matched control subjects. While cortico-muscular coherence (CMC) did not differ between groups, patients showed increased sensori-motor cortical power at beta frequency (13­30 Hz) during rest as well as during isometric contraction compared to controls. In healthy control subjects the power of the contralateral hemisphere was significantly suppressed during isometric contraction. By contrast, both hemispheres were activated equally strongly in de novo patients. In medicated patients, the pattern was found to be reversed. Contralateral beta power was significantly correlated with motor impairment during isometric contraction but not during rest. The present results suggest that the reduced ability of the primary motor cortex to disengage from increased beta band oscillations during the execution of movements is an early marker of PD.

Clonal diversity in the B cell repertoire of patients with X-linked agammaglobulinemia.

Ig protein and mRNA expression was examined in a collection of 18 monoclonal EBV-transformed B cell lines derived from five patients with X-linked agammaglobulinemia (XLA). A diversity of H and L chain isotypes were synthesized by these lines: the majority (12 lines) expressed mu kappa chains, while mu lambda (two lines), gamma kappa (one), gamma lambda (one), delta lambda (one), and alpha kappa (one) isotype expression was also observed. For all the mu kappa-producing XLA B cell lines, the mu and kappa mRNA transcripts were of native size, and sequence analysis across the regions of VHDJH and V kappa J kappa gene joining showed that Ig gene rearrangements occurred in a typical manner. A variety of VHDJH and V kappa J kappa gene rearrangements were observed, not only within the set of mu kappa+ XLA B cells as a whole, but also among the cell lines derived from single patients. Southern blot analysis for genomic Ig H chain gene rearrangements was done to fully assess the extent of clonal heterogeneity among multiple mu kappa+ XLA B cell lines derived from two patients; all the B cell lines possessed distinct gene rearrangement patterns demonstrating their clonal unrelatedness. Our findings indicate that the B cell repertoire in individual XLA patients is clonally diverse and that it is unlikely that the defect in B cell differentiation in XLA is the result of inefficient or ineffective rearrangement of Ig H or L chain genes. Rather, this study provides support for the idea that the XLA defect relates to a more generalized cellular function, such as regulating the proliferation and/or clonal expansion of cells of the B lymphoid lineage.

Structural characterization of antiidiotypic antibodies. Evidence that Ab2s are derived from the germline differently than Ab1s.

We have found that syngeneic Ab2s in the antiarsonate system are serologically and structurally similar to one another. In contrast, the allogeneic Ab2 response is heterogeneous and derives from a large number of unrelated germline gene segments. The Ab2 response of the BALB/c strain to polyclonal A/J Ars A molecules can probably best be compared with a response to a foreign protein and might have been predicted in a strain that completely lacks the H chain V region gene from which the Ab1 derives. Partial variable region sequences of Ab2s from three other systems in addition to previously reported Ab2 structures indicates that this difference in allogeneic vs. syngeneic Ab2s may be a general phenomena. These data support Jerne's hypothesis of complementary V region genes existing in the germline. However, there is good evidence that these antiidiotypic antibodies are not derived directly from the germline, as somatic processes most likely play an important role in their generation. The D segments of Ab2s in the arsonate system as well as in other systems, are novel in structure and cannot easily be explained by previously described germline D segments. D-D fusion may play a role in the generation of the third hypervariable region in these antibodies.

Motor impairment in liver cirrhosis without and with minimal hepatic encephalopathy.

AIM: Manifest hepatic encephalopathy (HE) goes along with motor symptoms such as ataxia, mini-asterixis, and asterixis. The relevance of motor impairments in cirrhotics without and with minimal HE (mHE) is still a matter of debate. PATIENTS AND METHODS: We tested three different groups of patients with liver cirrhosis: no signs of HE (HE 0), mHE, and manifest HE grade 1 according to the West Haven criteria (HE 1). All patients (n = 24) and 11 healthy control subjects were neuropsychometrically tested including critical flicker frequency (CFF), a reliable measure for HE. Motor abilities were assessed using Fahn Tremor Scale and International Ataxia Rating Scale. Fastest alternating index finger movements were analyzed for frequency and amplitude. RESULTS: Statistical analyses showed an effect of HE grade on tremor and ataxia (P < 0.01). Additionally, both ratings yielded strong negative correlation with CFF (P < 0.01, R = -0.5). Analysis of finger movements revealed an effect of HE grade on movement frequency (P < 0.03). Moreover, decreasing movement frequency and increasing movement amplitude parallel decreasing CFF (P < 0.01, R = 0.6). CONCLUSION: Our results indicate that ataxia, tremor, and slowing of finger movements are early markers for cerebral dysfunction in HE patients even prior to neuropsychometric alterations becoming detectable.

Using GFP in FRET-based applications.

The use of green fluorescent protein (GFP) is a powerful technology that has recently enabled investigators to study dynamic molecular events within living cells. One method for detecting molecular interactions involves fluorescence resonance energy transfer (FRET) between two GFPs or between GFP and a second fluorophore. This review summarizes the use of GFP for FRET and illustrates the theme with specific examples on how GFP has been employed as an intracellular molecular sensor.

Differentiation of adipose derived stem cells to keratinocyte-like cells on an advanced collagen wound matrix.

Despite advances in tissue engineering and regenerative medicine, skin regeneration and cutaneous wound healing remains a significant medical challenge. A bioengineered skin that stimulates the body's natural regeneration capability is needed to address the current lack of treatment options. To this end, a biocompatible collagen wound matrix was developed using an electrochemical deposition fabrication process. The advanced collagen wound matrix has relatively high tensile strength compared to normal collagen matrix made by the heat gelation process and open porosity, and serves as an excellent platform for cellular growth and differentiation. Human adipose derived stem cells (hADSCs) were cultured on this collagen matrix and a co-culture system with primary keratinocytes and keratinocyte conditioned media was developed for differentiation of the hADSCs to keratinocyte-like cells. After fifteen days, hADSCs in co-culture began to exhibit a "cobblestone-like" morphology, indicating preliminary signs of differentiation to a keratinocyte-like cell. Based on morphological analysis at day 30, the co-culture with keratinocyte conditioned media system shows promising preliminary evidence of hADSC differentiation to a keratinocyte-like cell on an electrochemically aligned collagen wound matrix.

The coupling between enhancer activity and hypomethylation of kappa immunoglobulin genes is developmentally regulated.

Previous studies have indicated that immunoglobulin enhancers are essential for establishing transcriptional competence but not for maintaining the activity of constitutively transcribed genes. To understand the basis for this developmental shift away from dependence on enhancer function, we have investigated the relationship between transcriptional activity and methylation status of the immunoglobulin kappa light-chain genes (kappa genes) in mouse cell lines representing different stages of B-cell maturation. Using pre-B-cell lines in which the level of a critical kappa enhancer-binding factor, NF-kappa B, was controlled by the administration or withdrawal of lipopolysaccharide and plasmacytoma lines that either contain or lack this factor, we studied the properties of endogenous kappa genes and of transfected kappa genes which were stably integrated into the genomes of these cells. In the pre-B cells, the exogenous (originally unmethylated) kappa genes, as well as endogenous kappa genes, were fully methylated and persistently dependent on enhancer function, even after more than 30 generations in a transcriptionally active state. In plasmacytoma cells, the endogenous kappa genes were invariably hypomethylated, whereas exogenous kappa genes were hypomethylated only in cells that contain NF-kappa B and are thus permissive for kappa enhancer function. These results indicate that the linkage of hypomethylation to enhancer-dependent activation of kappa transcription occurs after the pre-B-cell stage of development. The change in methylation status, together with associated changes in chromatin structure, may suffice to eliminate or lessen the importance of the enhancer for the maintenance of the transcriptionally active state.

Interleukin 1 and cyclic AMP induce kappa immunoglobulin light-chain expression via activation of an NF-kappa B-like DNA-binding protein.

Interleukin 1 (IL-1) induces the synthesis of kappa immunoglobulin light chains and the expression of surface immunoglobulin in the murine pre-B-cell line 70Z/3 (J. G. Giri, P. W. Kincade, and S. B. Mizel, J. Immunol. 132:223-228, 1984). In the present study, we found that these effects of IL-1 are mimicked by cyclic AMP (cAMP) analogs and cAMP-elevating drugs. The induction of kappa immunoglobulin light-chain gene expression by IL-1 was associated with an increase in intracellular cAMP levels. Incubation of 70Z/3 cells with IL-1 or cAMP resulted in the activation of the kappa immunoglobulin enhancer, as detected by the induction of chloramphenicol acetyltransferase (CAT) in cells transfected with a kappa enhancer-CAT expression plasmid. In contrast, CAT plasmids lacking a kappa immunoglobulin enhancer were inactive in the presence of IL-1 or cAMP. Furthermore, IL-1 and cAMP analogs and inducers were found to induce the activation of a NF-kappa B-like DNA-binding protein that exhibited specificity for the kappa immunoglobulin enhancer. These results suggest that cAMP may play an important role as a second messenger for IL-1 in the induction of kappa immunoglobulin light-chain synthesis in pre-B cells via the activation of a DNA-binding protein that is similar or identical to NF-kappa B.

A ubiquitin-based tagging system for controlled modulation of protein stability.

Many biotechnology applications depend on the expression of exogenous proteins in a predictable and controllable manner. A key determinant of the intracellular concentration of a given protein is its stability or "half-life." We have developed a versatile and reliable system for producing short half-life forms of proteins expressed in mammalian cells. The system consists of a series of destabilization domains composed of varying numbers of a mutant form of ubiquitin (UbG76V) that cannot be cleaved by ubiquitin hydrolases. We show that increasing the number of UbG76V moieties within the destabilization domain results in a graded decrease in protein half-life and steady-state levels when fused to heterologous reporter proteins as well as cellular proteins. Cells expressing a destabilized beta-lactamase reporter act as a robust, high-throughput screening (HTS)-compatible assay for proteasome activity within cells.

Transcranial direct current stimulation (tDCS) applied to the left dorsolateral premotor cortex (dPMC) interferes with rhythm reproduction.

Movement timing in the sub-second range engages a brain network comprising cortical and sub-cortical areas. The present study aims at investigating the functional significance of the left dorsolateral premotor cortex (dPMC) for precise movement timing as determined by sensorimotor synchronization and rhythm reproduction. To this end, 18 healthy volunteers performed an auditorily paced synchronization-continuation task with the right hand. A simple reaction time task served as control condition. Transcranial direct current stimulation (tDCS) was applied over the left dPMC in order to modulate cortical excitability either with anodal or cathodal polarity or as sham stimulation. TDCS was applied for 10 minutes, respectively on separate days. For the continuation task the analysis revealed significantly smaller inter-tap intervals (ITIs) following cathodal tDCS suggesting movement hastening as well as a trend towards larger ITIs following anodal stimulation suggesting movement slowing. No significant effect was found following sham stimulation. Neither for synchronization nor for reaction time tasks significant polarity-specific effects emerged. The data suggest the causal involvement of the dPMC in temporally precisereproduction of isochronous rhythms rather than sensorimotor synchronization. The present findings support the hypothesis that different cortical brain areas within the motor-control-network distinctively contribute to movement timing in the sub-second range.

Strategies in probabilistic feedback learning in Parkinson patients OFF medication.

Studies on classification learning suggested that altered dopamine function in Parkinson's Disease (PD) specifically affects learning from feedback. In patients OFF medication, enhanced learning from negative feedback has been described. This learning bias was not seen in observational learning from feedback, indicating different neural mechanisms for this type of learning. The present study aimed to compare the acquisition of stimulus-response-outcome associations in PD patients OFF medication and healthy control subjects in active and observational learning. 16 PD patients OFF medication and 16 controls were examined with three parallel learning tasks each, two feedback-based (active and observational) and one non-feedback-based paired associates task. No acquisition deficit was seen in the patients for any of the tasks. More detailed analyses on the learning strategies did, however, reveal that the patients showed more lose-shift responses during active feedback learning than controls, and that lose-shift and win-stay responses more strongly determined performance accuracy in patients than controls. For observational feedback learning, the performance of both groups correlated similarly with the performance in non-feedback-based paired associates learning and with the accuracy of observed performance. Also, patients and controls showed comparable evidence of feedback processing in observational learning. In active feedback learning, PD patients use alternative learning strategies than healthy controls. Analyses on observational learning did not yield differences between patients and controls, adding to recent evidence of a differential role of the human striatum in active and observational learning from feedback.

Development and application of a GFP-FRET intracellular caspase assay for drug screening.

Apoptosis is a crucial biological process, and activation of caspase endoproteases is essential for proper regulation and execution of apoptosis. Because caspases also appear to be central players in several pathological states, there is a practical need within the biopharmaceutical research community for facile, noninvasive cellular assays for the discovery of compounds that modulate caspase activity. Tandem molecules of green fluorescent protein (GFP) stably expressed within cells can serve as a genetically encoded sensor of protease activity. Using this technology, we have developed a stable cellular system for the screening of agents that modulate activation of the caspase cascade. This assay technology allows for the real-time monitoring of apoptosis in situ, using conventional fluorescent plate reader detection. By applying this assay system to an actual compound screen, small-molecule inducers of cell apoptosis were reliably identified. Follow-up pharmacology confirmed that the rank-order potency of primary hits using the intracellular GFP assay corresponded to that found using a conventional, cell lysis-based assay method.

Analysis of idiotypic heterogeneity in the anti-alpha 1-3 dextran and anti-phosphorylcholine responses using monoclonal anti-idiotype antibodies.

Panels of monoclonal anti-idiotype antibodies (MAIDs) specific for individual (IdI) and cross-reactive (IdX) idiotopes were prepared and used to study the expression of these idiotopes on anti-DEX and anti-PC antibodies produced in response to antigenic stimulation in vivo, clonal expression of idiotopes in an in vitro splenic focus assay, and the alterations in the idiotypic profile of these responses after in vivo administration of monoclonal anti-Id antibodies. Using these panels of MAIDs, it was possible to inactivate IdI-bearing B cells both in neonates and adult mice without affecting the responsiveness of IdI- B cells. By contrast, suppression with IdX-specific antibodies resulted in greatly reduced antibody responses. By studying the idiotypic profile of anti-DEX clones in the splenic focus assay, it was shown that IgM, IgG, and IgA antibody Id were diverse and paralleled those expressed in serum. Within some clones there was evidence that idiotope-isotype associations differed, suggesting that V region variants may have been generated within the progeny of a clone following stimulation by dextran. An anti-anti-Id antibody isolated from a BALB/c mouse undergoing a normal immune response to R36A was shown to have a T-cell independent highly idiotope-specific regulatory effect on the T15+ anti-PC response, apparently affecting induction of anti-idiotypic B cells.

Development of object permanence in food-storing magpies (Pica pica).

The development of object permanence was investigated in black-billed magpies (Pica pica), a food-storing passerine bird. The authors tested the hypothesis that food-storing development should be correlated with object-permanence development and that specific stages of object permanence should be achieved before magpies become independent. As predicted, Piagetian Stages 4 and 5 were reached before independence was achieved, and the ability to represent a fully hidden object (Piagetian Stage 4) emerged by the age when magpies begin to retrieve food. Contrary to psittacine birds and humans, but as in dogs and cats, no "A-not-B error" occurred. Although magpies also mastered 5 of 6 invisible displacement tasks, evidence of Piagetian Stage 6 competence was ambiguous.

Changes of motor-cortical oscillations associated with motor learning.

Motor learning results from practice but also between practice sessions. After skill acquisition early consolidation results in less interference with other motor tasks and even improved performance of the newly learned skill. A specific significance of the primary motor cortex (M1) for early consolidation has been suggested. Since synchronized oscillatory activity is assumed to facilitate neuronal plasticity, we here investigate alterations of motor-cortical oscillations by means of event-related desynchronization (ERD) at alpha (8-12 Hz) and beta (13-30 Hz) frequencies in healthy humans. Neuromagnetic activity was recorded using a 306-channel whole-head magnetoencephalography (MEG) system. ERD was investigated in 15 subjects during training on a serial reaction time task and 10 min after initial training. The data were compared with performance during a randomly varying sequence serving as control condition. The data reveal a stepwise decline of alpha-band ERD associated with faster reaction times replicating previous findings. The amount of beta-band suppression was significantly correlated with reduction of reaction times. While changes of alpha power have been related to lower cognitive control after initial skill acquisition, the present data suggest that the amount of beta suppression represents a neurophysiological marker of early cortical reorganization associated with motor learning.

Effects of 10 Hz and 20 Hz transcranial alternating current stimulation (tACS) on motor functions and motor cortical excitability.

Synchronized oscillatory activity at alpha (8-12 Hz) and beta (13-30 Hz) frequencies plays a key role in motor control. Nevertheless, its exact functional significance has yet to be solved. Transcranial alternating current stimulation (tACS) allows the frequency-specific modulation of ongoing oscillatory activity. The goal of the present study was to investigate the effect of 10 and 20 Hz tACS over left primary motor cortex (M1) on motor functions and cortical excitability in healthy subjects. To this end, tACS was applied for 10 min. Sham stimulation served as control condition. Movement speed and accuracy of the right hand were assessed in 15 right-handed subjects before and after (0, 30 and 60 min) tACS of M1. Cortical silent period (CSP) and motor evoked potentials (MEPs) were determined as measures of M1 excitability. While 10 Hz tACS particularly increased movement variability, especially in tasks requiring internal pacing, 20 Hz tACS resulted in movement slowing. Behavioural effects occurred in distinct time windows. While 10 Hz effects developed over 30 min after stimulation, 20 Hz tACS effects were found immediately after stimulation. Following 10 Hz tACS these effects were significantly correlated with CSP duration, indicating interference with inhibitory pathways. The present findings suggest differential effects of stimulation frequency on motor behaviour and M1 excitability.

Task-dependent oscillations during unimanual and bimanual movements in the human primary motor cortex and SMA studied with magnetoencephalography.

The neural mechanisms subserving uni- and bimanual control of movements are not well understood. Nevertheless, recent studies indicate a functional role of oscillatory activity in movement control and point towards a hemispheric asymmetry in motor control. This study specifically addresses the issues of (i) task-relatedness, (ii) hemispheric symmetry, and (iii) frequency specificity of the measures power, cerebro-muscular coherence, and cerebro-cerebral coherence in bilateral primary motor cortex and supplementary motor area (SMA). We have studied 10 right-handed subjects with simultaneous recordings of magnetoencephalography and surface electromyography during different unimanual and bimanual tasks. Using the analysis technique Dynamic Imaging of Coherent Sources (DICS), left and right primary motor cortex and SMA were functionally localized. Power, cerebro-musclar coherence, and cerebro-cerebral coherence between these areas were computed for four frequency bands in each condition and subjected to ANOVA. Results show a task-specific modulation of power and coherence, and further indicate a hemispheric asymmetry in the control of unimanual and bimanual movements. In addition, different frequency bands showed different task-dependent variations. The gamma band (26-40 Hz) showed strongest modulation for cerebro-muscular coherence and was strongest for the isometric contraction conditions. In contrast, the beta band (13-24 Hz) showed the strongest variations between static and dynamic conditions, and seems to play a particular role in movement control. In summary, our results indicate a differential functional role of oscillatory activity and coupling in the motor system.

Identification and characterization of an apparent germline set of auto-anti-idiotypic regulatory B lymphocytes.

By fusing BALB/c splenic lymphocytes from mice immunized with phosphorylcholine (PC) to an immunoglobulin nonproducing plasmacytoma cell line, a B cell hybridoma was isolated (MM-60) that has been shown by multiple criteria to produce a bona fide auto-anti-(anti-T15 idiotype) antibody. In vivo administration of MM-60 antibody suppressed T15+ anti-PC antibody production in an idiotope-specific manner by activation of an intervening set of anti-T15 B cells. These T15-specific B cells i) appeared to express germline-encoded variable region gene products, ii) developed in parallel to, but independent of, T15+ B cells, and iii) suppressed the anti-PC response in a T cell-independent fashion. Variants of T15+ anti-PC B cells possessing aberrant immunoglobulin heavy chain D region structure escaped from the suppression imposed by this anti-T15 B cell set, suggesting that a function of the heavy chain D region may be to contribute to the formation of molecular target sites for idiotype-directed regulatory cells and/or antibodies. The indigenous nature of these particular populations of anti-idiotypic and anti-(anti-idiotypic) B cells and the ability of their immunoglobulin products to regulate antigen-specific B cells in vivo provides strong supportive evidence for the significant role idiotype-directed network interactions play in regulating specific antibody production during a normal immune response.