Jakob Jankowski

Characterization of the neuronal marker NeuN as a multiply phosphorylated antigen with discrete subcellular localization

NeuN (neuronal nuclei) is an antigen used widely in research and diagnostics to identify postmitotic neurons. The present study aims at an initial understanding of the molecular nature and functional significance of this as yet ill‐defined antigen. Using isoelectric focusing, both the 46‐ and 48‐kDa isoforms of NeuN can be separated in multiple spots spanning a pH range of 8–10.5, suggesting that they might be phosphorylated. Enzymatic dephosphorylation abolishes NeuN immunoreactivity, confirming that NeuN is indeed a phosphoprotein, and establishing that binding of the defining antibody depends on its state of phosphorylation. Combined biochemical and immunohistochemical analysis show that both the 46‐ and the 48‐kDa NeuN isoforms can be localized to the cell nucleus as well as in the neuronal cytoplasm. Their relative concentration in these compartments is distinct, however, with the 48‐kDa isoform being the predominant isoform in the cytoplasm. Within the nucleus, NeuN is found preferentially in areas of low chromatin density and virtually excluded from areas containing densely packed DNA. The present identification of multiple differentially phosphorylated isoforms of NeuN, together with recent reports on the dependence of NeuN immunoreactivity levels on a variety of physiologic or pathologic signals, suggests a previously unappreciated level of complexity in the regulation of this enigmatic, neuron‐specific antigen.

A role of the basal ganglia and midbrain nuclei for initiation of motor sequences.

The mesial premotor cortex is crucial for planning sequential procedures and movement initiation. With event-related (ER) functional magnetic resonance imaging (fMRI) it has been possible to separate mesial premotor activation before, during, and after self-initiated movements and, thereby, to distinguish advance planning from execution. The mesial premotor cortex is part of distributed cortico-basal ganglia-thalamo-cortical networks but, to date, the subcortical contributions to self-initiated movements are far less well understood. Using ER fMRI at 3T in 12 right-handed male volunteers, we studied the subcortical activation preceding an automated four-digit finger sequence that was either self-initiated or triggered externally by a visual cue. Beyond typical cortical activation increases in fronto-parietal regions, both initiation modes induced consistent subcortical activation in basal ganglia, midbrain (substantia nigra), and ipsilateral cerebellum. The planning phase of the internally initiated condition, when contrasted with the externally triggered condition, was associated with enhanced activity in frontal regions (mesial premotor cortex/rostral cingulate zone, dorsolateral prefrontal cortex), parietal regions (precuneus, inferior parietal cortex, encroaching onto V5/MT), insula, contralateral anterior putamen and midbrain (bilateral red nucleus/subthalamic nucleus). These data demonstrate the impact of initiation mode on planning-related activity in the ventral basal ganglia and interconnected midbrain nuclei, thereby stressing the crucial role of distributed cortico-basal ganglia-thalamo-cortical networks for self-initiated automated motor repertoires. Involvement of the substantia nigra during planning, as shown here, indicates dopaminergic gating of motor sequences.

Distinct striatal regions for planning and executing novel and automated movement sequences

The basal ganglia-thalamo-cortical circuits are viewed as segregated parallel feed back loops crucially involved in motor control, cognition, and emotional processing. Their role in planning novel, as compared to overlearned movement patterns is as yet not well defined. We tested for the involvement of the associative striatum (caudate/anterior putamen) in the generation of novel movement patterns, which is a critical cognitive requirement for non-routine motor behavior. Using event related functional MRI in 14 right-handed male subjects, we analyzed brain activity in the planning phase of four digit finger sequences. Subjects either executed a single overlearned four digit sequence (RECALL), or self-determined four digit sequences of varying order (GENERATE). In both conditions, RECALL and GENERATE, planning was associated with activation in mesial/lateral premotor cortices, motor cingulate cortex, superior parietal cortex, basal ganglia, insula, thalamus, and midbrain nuclei. When contrasting the planning phase of GENERATE with the planning phase of RECALL, there was significantly higher activation within this distributed network. At the level of the basal ganglia, the planning phase of GENERATE was associated with differentially higher activation located specifically within the associative striatum bilaterally. On the other hand, the execution phase during both conditions was associated with a shift of activity towards the posterior part of the putamen. Our data show the specific involvement of the associative striatum during the planning of non-routine movement patterns and illustrate the propagation of activity from rostral to dorsal basal ganglia sites during different stages of motor processing.

An fMRI study on the acute effects of exercise on pain processing in trained athletes.

Summary Endurance exercise modulates affective pain ratings and pain‐evoked responses in distinct areas of the human pain matrix, presumably via central opioidergic modulation. Abstract Endurance exercise is known to promote sustained antinociceptive effects, and there is evidence that the reduction of pain perception mediated by exercise is driven by central opioidergic neurotransmission. To directly investigate the involved brain areas and the underlying neural mechanisms in humans, thermal heat‐pain challenges were applied to 20 athletes during 4 separate functional magnetic resonance imaging (fMRI) scans, i.e., before and after 2 hours of running (exercise condition) and walking (control condition), respectively. Imaging revealed a reproducible pattern of distributed pain‐related activation in all 4 conditions, including the mesial and lateral pain systems, and the periaqueductal gray (PAG) as a key region of the descending antinociceptive pathway. At the behavioral level, running as compared with walking decreased affective pain ratings. The influence of exercise on pain‐related activation was reflected in a significant time × treatment interaction in the PAG, along with similar trends in the pregenual anterior cingulate cortex and the middle insular cortex, where pain‐induced activation levels were elevated after walking, but decreased or unchanged after running. Our findings indicate that enhanced reactive recruitment of endogenous antinociceptive mechanisms after aversive repeated pain exposure is attenuated by exercise. The fact that running, but not walking, reproducibly elevated β‐endorphin levels in plasma indicates involvement of the opioidergic system in exercise. This may argue for an elevated opioidergic tone in the brain of athletes, mediating antinociceptive mechanisms. Our findings provide the first evidence using functional imaging to support the role of endurance exercise in pain modulation.

Engrailed-2 negatively regulates the onset of perinatal Purkinje cell differentiation

The transcription factor Engrailed‐2 is expressed in cerebellar Purkinje cells (PCs) throughout embryonic development but is downregulated in PCs after birth. Since the onset of PC differentiation coincides with this change of gene expression, we asked whether downregulation of Engrailed‐2 is necessary for proper timing of PC differentiation. To investigate this, we used an L7En‐2 transgenic mouse model in which Engrailed‐2 expression in PCs is maintained beyond the day of birth. In these L7En‐2 mice the onset of parvalbumin expression was delayed in all PCs by about 3 days; the spatial expression pattern, however, remained comparable to wildtype cerebella. Furthermore, parvalbumin expression resembled the known pattern of normal PC maturation, suggesting a direct link between parvalbumin expression and PC differentiation. Consistent with a delay of PC differentiation, we found that PCs of L7En‐2 cerebella displayed a reduced tendency to align in the typical monolayer. The average size of L7En‐2 PCs was reduced and the dendritic arbor developed more slowly than in wildtype PCs. In contrast, major morphological features of PCs were comparable in L7En‐2 and wildtype cerebella after postnatal day 11. In addition, we observed a transient reduction of PC survival in organotypic slice cultures of L7En‐2 cerebella in comparison with wildtype slice cultures. Since PC survival parallels PC differentiation in vitro, we propose that the observed delay in PC differentiation upon Engrailed‐2 overexpression is an intrinsic property of Engrailed‐2 activity, and that downregulation of Engrailed‐2 in wildtype PCs around the day of birth is critical for the timing of distinct steps of PC differentiation. J. Comp. Neurol. 472:87–99, 2004.

Physiological purkinje cell death is spatiotemporally organized in the developing mouse cerebellum.

Physiological cell death is crucial for matching defined cellular populations within the central nervous system. Whereas the time course of developmental cell death in the central nervous system is well analyzed, information about its precise spatial patterning is scarce. Yet, the latter one is needed to appraise its contribution to circuit formation and refinement. Here, we document that during normal cerebellar development, dying Purkinje cells were highly localized within the vermal midline and in a lobule specific, parasagittal pattern along the whole mediolateral axis. In addition, single hot spots of cell death localized to the caudal declive and ventral lobule IX within the posterolateral fissure. These hot spots of dying Purkinje cells partly overlapped with gaps within the Purkinje cell layer which supports the classification of different gaps based on histological and molecular criteria, i.e., midline gap, patchy gaps, and raphes. Areas characterized by a high incidence of Purkinje cell death and gaps colocalize with known molecular and functional boundaries within the cerebellar cortex. Physiological cell death can thus be considered to serve as an important regulator of cerebellar histogenesis.

Engrailed-2 regulates genes related to vesicle formation and transport in cerebellar Purkinje cells.

Engrailed transcription factors regulate survival, cell fate decisions and axon pathfinding in central neurons. En-2 can also attenuate Purkinje cell (PC) maturation. Here, we use array analysis to scrutinize gene expression in developing PCs overexpressing Engrailed-2 (L7En-2). The majority (70%) of regulated genes was found down-regulated in L7En-2 cerebella, consistent with the known repressive function of Engrailed-2. Differential gene expression, verified by in situ hybridization or Western blotting, was particularly evident during the first postnatal week, when L7En-2 PCs display conspicuous deficits in dendritogenesis. Functional classification revealed clusters of genes linked to vesicle formation and transport. Consistently, Golgi stacks located at the axonal pole of wild type PC somata were rarely detected in L7En-2 PCs. In addition, long continuous stretches of endoplasmic reticulum typically found around the axonal pole of wild type PCs were less frequently observed in transgenic cells. Engrailed-2 might therefore orchestrate PC survival and process formation as a regulator of subcellular organization.

Tetraspanin-5 (Tm4sf9) mRNA expression parallels neuronal maturation in the cerebellum of normal and L7En-2 transgenic mice

Tetraspanin‐5 (Tspan‐5) mRNA was recently shown to be strongly expressed within the central nervous system. In order to address Tspan‐5 function during nervous system development, we performed a detailed expression analysis in the postnatal FVB/N mouse cerebellum using in situ hybridizations. Tspan‐5 mRNA was expressed within cerebellar Purkinje cells (PCs) throughout postnatal development. The expression level, however, changed significantly with ongoing development. At the day of birth (P0), Tspan‐5 mRNA was expressed at very low levels in PCs. At this time, PCs of the FVB/N strain are postmitotic and bear axons, but no dendrites. At P7, Tspan‐5 mRNA expression was visible in all PCs, but was more prominent in those of the posterior lobules as compared to those of the anterior lobules. After P7, high levels of Tspan‐5 mRNA were seen in all PCs, which is when PCs elaborate and maintain their typical dendritic tree. This demonstrates that the level of Tspan‐5 mRNA is related to the developmental status of PCs. Consistently, expression of Tspan‐5 mRNA was specifically reduced in PCs of L7En‐2 animals, which display a delay in PC maturation during postnatal cerebellar development. In addition, whereas no Tspan‐5 mRNA signal could be detected in the proliferating granule cell layer, low levels could be found in postmitotic, premigratory granule cells and high levels in settled and differentiated granule cells. Thus, the level of Tspan‐5 mRNA expression correlates very well with the differentiation status of particular neurons. The level of Tspan‐5 expression might therefore be important for distinct phases of neuronal maturation. J. Comp. Neurol. 483:318–328, 2005.

Abnormal movement preparation in task-specific focal hand dystonia.

Electrophysiological and behavioral studies in primary dystonia suggest abnormalities during movement preparation, but this crucial phase preceding movement onset has not yet been studied specifically with functional magnetic resonance imaging (fMRI). To identify abnormalities in brain activation during movement preparation, we used event-related fMRI to analyze behaviorally unimpaired sequential finger movements in 18 patients with task-specific focal hand dystonia (FHD) and 18 healthy subjects. Patients and controls executed self-initiated or externally cued prelearnt four-digit sequential movements using either right or left hands. In FHD patients, motor performance of the sequential finger task was not associated with task-related dystonic posturing and their activation levels during motor execution were highly comparable with controls. On the other hand reduced activation was observed during movement preparation in the FHD patients in left premotor cortex / precentral gyrus for all conditions, and for self-initiation additionally in supplementary motor area, left mid-insula and anterior putamen, independent of effector side. Findings argue for abnormalities of early stages of motor control in FHD, manifesting during movement preparation. Since deficits map to regions involved in the coding of motor programs, we propose that task-specific dystonia is characterized by abnormalities during recruitment of motor programs: these do not manifest at the behavioral level during simple automated movements, however, errors in motor programs of complex movements established by extensive practice (a core feature of FHD), trigger the inappropriate movement patterns observed in task-specific dystonia.

Advocating neuroimaging studies of transmitter release in human physical exercise challenges studies.

This perspective attempts to outline the emerging role of positron emission tomography (PET) ligand activation studies in human exercise research. By focusing on the endorphinergic system and its acclaimed role for exercise-induced antinociception and mood enhancement, we like to emphasize the unique potential of ligand PET applied to human athletes for uncovering the neurochemistry of exercise-induced psychophysiological phenomena. Compared with conventional approaches, in particular quantification of plasma beta-endorphin levels under exercise challenges, which are reviewed in this article, studying opioidergic effects directly in the central nervous system (CNS) with PET and relating opioidergic binding changes to neuropsychological assessments, provides a more refined and promising experimental strategy. Although a vast literature dating back to the 1980s of the last century has been able to reproducibly demonstrate peripheral increases of beta-endorphin levels after various exercise challenges, so far, these studies have failed to establish robust links between peripheral beta-endorphin levels and centrally mediated behavioral effects, ie, modulation of mood and/or pain perception. As the quantitative relation between endorphins in the peripheral blood and the CNS remains unknown, the question arises, to what extent conventional blood-based methods can inform researchers about central neurotransmitter effects. As previous studies using receptor blocking approaches have also revealed equivocal results regarding exercise effects on pain and mood processing, it is expected that PET and other functional neuroimaging applications in athletes may in future help uncover some of the hitherto unknown links between neurotransmission and psychophysiological effects related to physical exercise.