Rostral migratory stream neuroblasts turn and change directions in stereotypic patterns

Neuroblasts generated in the adult subventricular zone (SVZ) migrate through the rostral migratory stream (RMS) to the olfactory bulb (OB). Previous work uncovered motility ranging from straight to complex, but it was unclear if directional changes were stochastic or exhibited stereotypical patterns. Here, we provide the first in-depth two-photon time-lapse microscopy study of morphological and dynamic features that accompany turning and direction reversals in the RMS. We identified three specific kinds of turning (30–90 degrees): bending of the leading process proximal to the cell body (P-bending 47% of cases), bending of the distal leading process (D-bending 30%) or branching of the leading process or lamellipodium (23%). Bending and branching angles were remarkably constrained and were significantly different from one another. Cells reversed direction (>90 degrees) through D-bendings (54%), branching (11%) or de novo growth of processes from the soma (23%), but not P-bending. Direction reversal was often composed of several iterations of D-bending or branching as opposed to novel modalities. Individual neuroblasts could turn or change direction in multiple patterns suggesting that the patterns are not specific for different lineages. These findings show that neuroblasts in the RMS use a limited number of distinct and constrained modalities to turn or reverse direction.Key words: neurogenesis, subventricular zone, migration, direction, motility     

Drebrin Regulates Neuroblast Migration in the Postnatal Mammalian Brain

After birth, stem cells in the subventricular zone (SVZ) generate neuroblasts that migrate along the rostral migratory stream (RMS) to become interneurons in the olfactory bulb (OB). This migration is crucial for the proper integration of newborn neurons in a pre-existing synaptic network and is believed to play a key role in infant human brain development. Many regulators of neuroblast migration have been identified; however, still very little is known about the intracellular molecular mechanisms controlling this process. Here, we have investigated the function of drebrin, an actin-binding protein highly expressed in the RMS of the postnatal mammalian brain. Neuroblast migration was monitored both in culture and in brain slices obtained from electroporated mice by time-lapse spinning disk confocal microscopy. Depletion of drebrin using distinct RNAi approaches in early postnatal mice affects neuroblast morphology and impairs neuroblast migration and orientation in vitro and in vivo. Overexpression of drebrin also impairs migration along the RMS and affects the distribution of neuroblasts at their final destination, the OB. Drebrin phosphorylation on Ser142 by Cyclin-dependent kinase 5 (Cdk5) has been recently shown to regulate F-actin-microtubule coupling in neuronal growth cones. We also investigated the functional significance of this phosphorylation in RMS neuroblasts using in vivo postnatal electroporation of phosphomimetic (S142D) or non-phosphorylatable (S142A) drebrin in the SVZ of mouse pups. Preventing or mimicking phosphorylation of S142 in vivo caused similar effects on neuroblast dynamics, leading to aberrant neuroblast branching. We conclude that drebrin is necessary for efficient migration of SVZ-derived neuroblasts and propose that regulated phosphorylation of drebrin on S142 maintains leading process stability for polarized migration along the RMS, thus ensuring proper neurogenesis.     

Sensory-evoked turning locomotion in red-eared turtles: kinematic analysis and electromyography

We examined the limb kinematics and motor patterns that underlie sensory-evoked turning locomotion in red-eared turtles. Intact animals were held by a band-clamp in a water-filled tank. Turn-swimming was evoked by slowly rotating turtles to the right or left via a motor connected to the shaft of the band-clamp. Animals executed sustained forward turn-swimming against the direction of the imposed rotation. We recorded video of turn-swimming and computer-analyzed the limb and head movements. In a subset of turtles, we also recorded electromyograms from identified limb muscles. Turning exhibited a stereotyped pattern of (1) coordinated forward swimming in the hindlimb and forelimb on the outer side of the turn, (2) back-paddling in the hindlimb on the inner side, (3) a nearly stationary, “braking” forelimb on the inner side, and (4) neck bending toward the direction of the turn. Reversing the rotation caused animals to switch the direction of their turns and the asymmetric pattern of right and left limb activities. Preliminary evidence suggested that vestibular inputs were sufficient to drive the behavior. Sensory-evoked turning may provide a useful experimental platform to examine the brainstem commands and spinal neural networks that underlie the activation and switching of different locomotor forms.     

Identification and characterization of neuroblasts in the subventricular zone and rostral migratory stream of the adult human brain

It is of great interest to identify new neurons in the adult human brain, but the persistence of neurogenesis in the subventricular zone (SVZ) and the existence of the rostral migratory stream (RMS)-like pathway in the adult human forebrain remain highly controversial. In the present study, we have described the general configuration of the RMS in adult monkey, fetal human and adult human brains. We provide evidence that neuroblasts exist continuously in the anterior ventral SVZ and RMS of the adult human brain. The neuroblasts appear singly or in pairs without forming chains; they exhibit migratory morphologies and co-express the immature neuronal markers doublecortin, polysialylated neural cell adhesion molecule and βIII-tubulin. Few of these neuroblasts appear to be actively proliferating in the anterior ventral SVZ but none in the RMS, indicating that neuroblasts distributed along the RMS are most likely derived from the ventral SVZ. Interestingly, no neuroblasts are found in the adult human olfactory bulb. Taken together, our data suggest that the SVZ maintains the ability to produce neuroblasts in the adult human brain.     

Changes in surface EMG and acoustic myogram parameters during static fatiguing contractions until exhaustion: influence of elbow joint angles

The purpose of this study was to evaluate muscle fatigue using electromyogram (EMG) and acoustic myogram (AMG) signals of the shoulder and arm muscles during sustained holding tasks, with the elbow at different angles and at different levels of maximum voluntary contraction (MVC). The EMG and AMG of four muscles, including the upper trapezius (TP), anterior deltoid (DL), biceps brachii (BB), and brachioradialis (BR), were recorded during experiments using 10 healthy young males. The experiments were conducted under 9 pairs of conditions: 3 elbow angles (120 degrees, 90 degrees, and 60 degrees) and *3 levels of %MVC (20%, 40%, and 60%). Subjects were instructed to hold a weight equal to the designated %MVC at designated joint angles and asked to maintain that condition for as long as possible until exhaustion. Joint angles were also recorded by the electrogoniometers. The analysis of variance revealed that there was no significant effect of elbow angle on the mean MVC or on the endurance time. Elbow angle showed a significant effect on mean power frequency (MPF) of EMG in DL, BB, and BR, and a significant effect on root mean square (RMS) of EMG in four muscles. In BB and BR, MPF of EMG at 120 degrees was found to be significantly lower than 90 degrees and 60 degrees, respectively. There was a significant main effect of elbow angle on MPF of AMG for TP at 20% MVC; for DL at 20% and 40% MVC; for BB at 40% and 60% MVC; and for BR at the three levels of %MVC. The results showed that the range MPF of AMG for DL, BB, and BR was between 32 to 46 Hz, whereas that for TP was from 49 to 83 Hz. There was a significant effect of elbow angle on RMS of AMG in all four muscles in all experiments. At 20% MVC, a progressive increase in RMS of AMG was observed with time. In contrast, at 40% and 60% MVC, RMS showed very different behavior; specifically, it was found that RMS of AMG at 20% MVC significantly increased with increase of elbow angle. We conclude that RMS of AMG has a good and clear correlation with elbow angle at a low level of contraction.     

Immunohistochemical Evidence for the Presence of Synaptic Connections of Nitrergic Neurons in the Rat Rostral Migratory Stream

The rostral migratory stream (RMS) is a migration route for neuroblasts originating in the richest neurogenic niche of the adult mammalian brain—the subventricular zone. Most studies are focused on cellular dynamics of migrating neuroblasts and interactions between neuroblasts and astrocytes which both represent the major cellular component of the RMS. Our previous experiments have brought evidence about the existence of a small population of mature neurons in the adult rat RMS with capacity to produce nitric oxide (NO). In order to further support functional significance of nitrergic cells, the aim of the present study was to determine whether NO producing neurons could form synapses. Sagittal sections from the adult rat brain were processed for simultaneous immunohistochemical detection of neuronal nitric oxide synthase (nNOS), the enzyme present in NO producing cells and synaptophysin, a glycoprotein found in synaptic vesicles. Synaptophysin positivity in the RMS was significantly lower in comparison with other brain areas, but its colocalization with nNOS-positive neurons was obvious. Our results suggest that nitrergic neurons in the RMS could be involved in a neuronal circuitry with potential impact on regulation of neurogenesis in the RMS.     

Expression of ezrin in subventricular zone neural stem cells and their progeny in adult and developing mice

Ezrin is a member of the ezrin–radixin–moesin (ERM) family of proteins, which link the cytoskeleton and cell membrane. ERM proteins are involved in pivotal cellular functions including cell–matrix recognition, cell–cell communication, and cell motility. Several recent studies have shown that ERM proteins are expressed in specific cell types of the adult rostral migratory stream (RMS). In this study, we found that ERM proteins are expressed highly in the early postnatal RMS and the ventricular zone of embryonic cerebral cortex, suggesting that these proteins may be expressed by neural progenitors. Furthermore, whereas ezrin previously was found to be expressed exclusively by astrocytes of the adult RMS, we found that ezrin-expressing cells also expressed the markers for indicating neuroblasts in vivo and in vitro, and that ezrin expression by neuroblasts decreases progressively as neuroblasts migrate. Using in vitro differentiation of adult neural stem cells, we found that ezrin is expressed by neural stem cells and their progeny (neuroblasts and astrocytes), but not by oligodendrocytic progeny. Collectively our findings demonstrate that adult neural stem cells and neuroblasts express ezrin and that ezrin may be involved in intracellular actin remodeling.     

Mechanomyogram and electromyogram responses of upper limb during sustained isometric fatigue with varying shoulder and elbow postures

To investigate the behavior of mechanomyogram (MMG) and electromyogram (EMG) signals in the time and frequency domains during sustained isometric contraction, MMG and surface EMG were obtained simultaneously from four muscles: upper trapezius (TP), anterior deltoid (DL), biceps brachii (BB), and brachioradialis (BR) of 10 healthy male subjects. Experimental conditions consisted of 27 combinations of 9 postures [3 shoulder angles (SA): 0 degree, 30 degrees, 60 degrees and 3 elbow angles (EA): 120 degrees, 90 degrees, 60 degrees] and 3 contraction levels: 20%, 40%, and 60% of maximum voluntary contraction (MVC). Subjective evaluations of fatigue were also assessed using the Borg scale at intervals of 60, 30, and 10 sec at 20%, 40%, and 60% MVC tests, respectively. The mean power frequency (MPF) and root mean square (RMS) of both signals were calculated. The current study found clear and significant relationships among physiological and psychological parameters on the one hand and SA and EA on the other. EA's effect on MVC was found to be significant. SA had a highly significant effect on both endurance time and Borg scale. In all experimental conditions, significant correlations were found between the changes in MPF and RMS of EMG in BB with SA and EA (or muscle length). In all four muscles, MMG frequency content was two or three times lower than EMG frequency content. During sustained isometric contraction, the EMG signal showed the well-known shift to lower frequencies (a continuous decrease from onset to completion of the contraction). In contrast, the MMG spectra did not show any shift, although its form changed (generally remaining about constant). Throughout the contraction, increased RMS of EMG was found for all tests, whereas in the MMG signal, a significant progressive increase in RMS was observed only at 20% MVC in all four muscles. This supports the hypothesis that the RMS amplitude of the MMG signal produced during contraction is highly correlated with force production. Possible explanations for this behavioral difference between the MMG and EMG signals are discussed.     

Changes in cell migration and survival in the olfactory bulb of the pcd/pcd mouse

Postnatally, the Purkinje cell degeneration mutant mice lose the main projecting neurons of the main olfactory bulb (OB): mitral cells (MC). In adult animals, progenitor cells from the rostral migratory stream (RMS) differentiate into bulbar interneurons that modulate MC activity. In the present work, we studied changes in proliferation, tangential migration, radial migration patterns, and the survival of these newly generated neurons in this neurodegeneration animal model. The animals were injected with bromodeoxyuridine 2 weeks or 2 months before killing in order to label neuroblast incorporation into the OB and to analyze the survival of these cells after differentiation, respectively. Both the organization and cellular composition of the RMS and the differentiation of the newly generated neurons in the OB were studied using specific markers of glial cells, neuroblasts, and mature neurons. No changes were observed in the cell proliferation rate nor in their tangential migration through the RMS, indicating that migrating neuroblasts are only weakly responsive to the alteration in their target region, the OB. However, the absence of MC does elicit differences in the final destination of the newly generated interneurons. Moreover, the loss of MC also produces changes in the survival of the newly generated interneurons, in accordance with the dramatic decrease in the number of synaptic targets available.     

Vicinal disulfide turns

The formation of a disulfide bond between adjacent cysteine residues is accompanied by the formation of a tight turn of the protein backbone. In nearly 90% of the structures analyzed a type VIII turn was found. The peptide bond between the two cysteines is in a distorted trans conformation, the omega torsion angle ranges from 159 to -133 degrees, with an average value of 171 degrees. The constrained nature of the vicinal disulfide turn and the pronounced difference observed between the oxidized and reduced states, suggests that vicinal disulfides may be employed as a 'redox-activated' conformational switch.     

Experimental Cerebral Malaria Spreads along the Rostral Migratory Stream

It is poorly understood how progressive brain swelling in experimental cerebral malaria (ECM) evolves in space and over time, and whether mechanisms of inflammation or microvascular sequestration/obstruction dominate the underlying pathophysiology. We therefore monitored in the Plasmodium berghei ANKA-C57BL/6 murine ECM model, disease manifestation and progression clinically, assessed by the Rapid-Murine-Coma-and-Behavioral-Scale (RMCBS), and by high-resolution in vivo MRI, including sensitive assessment of early blood-brain-barrier-disruption (BBBD), brain edema and microvascular pathology. For histological correlation HE and immunohistochemical staining for microglia and neuroblasts were obtained. Our results demonstrate that BBBD and edema initiated in the olfactory bulb (OB) and spread along the rostral-migratory-stream (RMS) to the subventricular zone of the lateral ventricles, the dorsal-migratory-stream (DMS), and finally to the external capsule (EC) and brainstem (BS). Before clinical symptoms (mean RMCBS = 18.5±1) became evident, a slight, non-significant increase of quantitative T2 and ADC values was observed in OB+RMS. With clinical manifestation (mean RMCBS = 14.2±0.4), T2 and ADC values significantly increased along the OB+RMS (p = 0.049/p = 0.01). Severe ECM (mean RMCBS = 5±2.9) was defined by further spread into more posterior and deeper brain structures until reaching the BS (significant T2 elevation in DMS+EC+BS (p = 0.034)). Quantitative automated histological analyses confirmed microglial activation in areas of BBBD and edema. Activated microglia were closely associated with the RMS and neuroblasts within the RMS were severely misaligned with respect to their physiological linear migration pattern. Microvascular pathology and ischemic brain injury occurred only secondarily, after vasogenic edema formation and were both associated less with clinical severity and the temporal course of ECM. Altogether, we identified a distinct spatiotemporal pattern of microglial activation in ECM involving primarily the OB+RMS axis, a distinct pathway utilized by neuroblasts and immune cells. Our data suggest significant crosstalk between these two cell populations to be operative in deeper brain infiltration and further imply that the manifestation and progression of cerebral malaria may depend on brain areas otherwise serving neurogenesis.     

Anomalous gravitropic response of Chara rhizoids during enhanced accelerations

Centrifugal accelerations of 50-250 g were applied to rhizoids of Chara globularis Thuill. at stimulation angles (alpha) of 5-90 degrees between the acceleration vector and the rhizoid axis. After the start of centrifugation, the statoliths were pressed asymmetrically onto the centrifugal flank of the apical cell wall. In contrast to the well-known bending (by bowing) under 1 g, the rhizoids responded in two distinct phases. Following an initial phase of sharp bending (by bulging), which is similar to the negatively gravitropic response of Chara protonemata, rhizoids stopped bending and, in the second phase, grew straight in directions clearly deviating from the direction of acceleration. These response angles (beta) between the axis of the bent part of the rhizoid and the acceleration vector were strictly correlated with the g-level of acceleration. The higher the acceleration the greater was beta. Except for the sharp bending, the shape and growth rate of the centrifuged rhizoids were not different from those of gravistimulated control rhizoids at 1 g. These results indicate that gravitropic bending of rhizoids during enhanced accelerations (5 degrees < or = alpha < or = 90 degrees) is caused not only by subapical differential flank growth, as it is the case at 1 g, but also by also by the centripetal displacement of the growth centre as was recently discussed for the negative gravitropism of Chara protonemata. A hypothesis for cytoskeletally mediated polar growth is presented based on data from positive gravitropic bending of Chara rhizoids at 1 g and from the anomalous gravitropic bending of rhizoids compared with the negatively gravitropic bending of Chara protonemata. The data obtained are also relevant to a general understanding of graviperception in higher-plant organs.     

Reduced proliferation in the adult mouse subventricular zone increases survival of olfactory bulb interneurons

Neurogenesis in the adult brain is largely restricted to the subependymal zone (SVZ) of the lateral ventricle, olfactory bulb (OB) and the dentate subgranular zone, and survival of adult-born cells in the OB is influenced by factors including sensory experience. We examined, in mice, whether survival of adult-born cells is also regulated by the rate of precursor proliferation in the SVZ. Precursor proliferation was decreased by depleting the SVZ of dopamine after lesioning dopamine neurons in the substantia nigra compacta with 6-hydroxydopamine. Subsequently, we examined the effect of reduced SVZ proliferation on the generation, migration and survival of neuroblasts and mature adult-born cells in the SVZ, rostral migratory stream (RMS) and OB. Proliferating cells in the SVZ, measured by 5-bromo-2-deoxyuridine (BrdU) injected 2 hours prior to death or by immunoreactivity against Ki67, were reduced by 47% or 36%, respectively, 7 days after dopamine depletion, and by 29% or 31% 42 days after dopamine depletion, compared to sham-treated animals. Neuroblast generation in the SVZ and their migration along the RMS were not affected, neither 7 nor 42 days after the 6-hydroxydopamine injection, since the number of doublecortin-immunoreactive neuroblasts in the SVZ and RMS, as well as the number of neuronal nuclei-immunoreactive cells in the OB, were stable compared to control. However, survival analysis 15 days after 6-hydroxydopamine and 6 days after BrdU injections showed that the number of BrdU+ cells in the SVZ was 70% higher. Also, 42 days after 6-hydroxydopamine and 30 days after BrdU injections, we found an 82% increase in co-labeled BrdU+/γ-aminobutyric acid-immunoreactive cell bodies in the granular cell layer, while double-labeled BrdU+/tyrosine hydroxylase-immunoreactive cell bodies in the glomerular layer increased by 148%. We conclude that the number of OB interneurons following reduced SVZ proliferation is maintained through an increased survival of adult-born precursor cells, neuroblasts and interneurons.     

A computational algorithm for classifying step and spin turns using pelvic center of mass trajectory and foot position

Transient changes in direction during ambulation are typically performed using a step (outside) or spin (inside) turning strategy, often identified through subjective and time-consuming visual rating. Here, we present a computational, marker-based classification method utilizing pelvic center of mass (pCOM) trajectory and time-distance parameters to quantitatively identify turning strategy. Relative to visual evaluation by three independent raters, sensitivity, specificity, and overall accuracy of the pCOM-based classification method were evaluated for 90-degree turns performed by 3 separate populations (5 uninjured controls, 5 persons with transtibial amputation, and 5 persons with transfemoral amputation); each completed turns using two distinct cueing paradigms (i.e., laser-guided “freeform” and verbally-guided “forced” turns). Secondarily, we compared the pCOM-based turn classification method to adapted versions of two existing computational turn classifiers which utilize trunk and shank angular velocities (AV). Among 366 (of 486 total) turns with unanimous intra- and inter-rater agreement, the pCOM-based classification algorithm was 94.5% accurate, with 96.6% sensitivity (accuracy of spin turn classification), and 93.5% specificity (accuracy of step turn classification). The pCOM-based algorithm (vs. both AV-based methods) was more accurate (94.5% vs. 81.1–80.6%; P < 0.001) overall, as well as specifically in freeform (92.9 vs. 80.4–76.8%; P < 0.003) and forced (96.0 vs. 83.8–81.8%; P < 0.001) cueing, and among individuals with (92.4 vs. 80.2–78.8%; P < 0.001) and without (99.1 vs. 86.2–80.8%; P < 0.001) amputation. The pCOM-based algorithm provides an efficient and objective method to accurately classify 90-degree turning strategies using optical motion capture in a laboratory setting, and may be extended to various cueing paradigms and/or populations with altered gait.     

Torsion angle based design of peptidomimetics: a dipeptidic template adopting beta-I turn (Ac-Aib-AzGly--NH(2)).

We have attempted to design a model dipeptide (acetyl dipeptide amide, Ac-CA1-CA2--NH(2)) that can adopt specifically typical torsion angles of the beta-I turn (phi(i+1), psi(i+1), phi(i+2), psi(i+2)=-60 degrees, -30 degrees, -90 degrees, 0 degrees ). The key of the design is the combination of constrained amino acids that prefer to adopt the desired torsion angles. We chose Aib (aminoisobutyric acid) as the first residue of which phi and psi angles must be -60 degrees and -30 degrees, respectively. Then, we selected an azaamino acid as the second residue since previous studies have indicated that they prefer to adopt +/-90 degrees of phi angle and 0 degrees or 180 degrees of psi angle. The conformational preference of the resulting Ac-Aib-AzGly--NH(2) is investigated using ab initio methods. The conformations implying beta-I and beta-I' turns are energetically most favorable, as we expected. Thus, we synthesized the designed molecule on the solid phase considering the future generation of combinatorial libraries using an automatic peptide synthesizer. Then, NMR spectroscopy was carried out to confirm their conformational preference in solution was carried out. The results indicated that the Ac-Aib-AzGly--NH(2) adopt beta-I or beta-I' turns in solution forming an intramolecular hydrogen bonding between Ac--C(O) and terminal NH(2). We believe that such a small peptidomimetic template is highly useful for the design of drug candidates and molecular devices.     

Gap junctions are involved in cell migration in the early postnatal subventricular zone

The massive migration of neuroblasts and young neurons through the anterior extension of the postnatal subventricular zone (SVZ), known as the rostral migratory stream (RMS) is still poorly understood on its molecular basis. In this work, we investigated the involvement of gap junctional communication (GJC) in the robust centrifugal migration from SVZ/RMS explants obtained from early postnatal (P4) rats. Cells were dye‐coupled in homocellular and heterocellular pairings and expressed at least two connexins, Cx 43 and 45. Treatment with the uncoupler agent carbenoxolone (CBX, 10–100 μM) reversibly reduced outgrowth from SVZ explants, while its inactive analog, glycyrhizinic acid (GZA), had no effect. Consistent with a direct effect on cell migration, time‐lapse video microscopy show that different pharmacological uncouplers cause an abrupt and reversible arrest of cell movement in explants. Our results indicate that GJC is positively involved in the migration of neuroblasts within the SVZ/RMS. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

Interaction between descending input and thoracic reflexes for joint coordination in cockroach. II Comparative studies on tethered turning and searching

Tethered cockroaches turn from unilateral antennal contact using asymmetrical movements of mesothoracic (T2) legs (Mu and Ritzmannin J Comp Physiol A 191:1037–1054, 2005). During the turn, the leg on the inside of the turn (the inside T2 leg) has distinctly different motor patterns from those in straight walking. The transformation from walking to inside leg turning could be triggered by descending commands that alter a few critical reflexes that start a cascade of physical changes in leg movement or posture, leading to further alterations. This hypothesis has two implications: First, the descending activities must be able to influence thoracic reflexes. Second, one should be able to initiate the turning motor pattern in the absence of descending signals by mimicking a point farther down in the reflex cascade. We addressed the first implication in the companion paper. To examine the second implication, we compared kinematics and motor activities of the T2 leg during searching with that of inside leg turning. The reaching movements made during searching were found to be similar to the movements made by the inside leg during turning. Moreover, even after disconnecting the brain from the thoracic ganglia the reaching movements were similar. This observation is consistent with the second implication from the hypothesis.