Locomotion grows up: The neuromechanical control of interlimb coordinating mechanisms in crayfish

Locomotion requires many dynamic interactions between organism and environment at several levels. It is not known how the nervous system controls all of these relationships to ultimately produce and guide locomotor behavior. Furthermore, it is not known whether the nervous system needs to recognize and control all of the possible body-environment interactions. In this study the crayfish (Procambarus clarkii) is used as a model system to test how size influences locomotor behavior and how a single, simplified neuromechanical system can accommodate these changes.;A set of behavioral experiments was conducted to characterize kinematics of freely walking juvenile crayfish to compare with adults. The purpose of these studies was to determine how crayfish adapt to a great change in size during their ontogeny. Juvenile and adult crayfish show differences in limb function and coordination. Although crayfish are decapods, the juveniles predominantly use the posterior legs and behave more like four-legged walkers. The difference in locomotor behavior can best be explained by differences in chelae size. Allometric relationships between juveniles and adults show limb and body morphologies scale proportionately. Adult chelae, or claws, are twice as long and contribute ∼20% more to the total body mass in fully mature crayfish. This increase in chelae size shifts the location of the center of mass anterior as crayfish grow. The result is a change in relative load distribution that appears to affect individual limb behavior and interlimb coordination. Shifting the center of mass in adults by amputating the chelae resulted in limb behavior and interlimb coordination more similar to that observed in juveniles. Likewise, applying load to the rostrum of juveniles altered behavior and changed limb function in the posterior legs similar to adults with large chelae. The results of these experiments suggest that crayfish of all sizes adapt to changes in load distribution by adjusting behavior of individual legs.;To test whether developmental influences have an effect on walking behavior, juveniles were induced to walk on a treadmill at various speeds. The animals showed more consistent limb coordination as walking speed increased, similar to adults. Selected legs were then amputated to test how gait was affected. Amputating legs removes sensory feedback from the distal leg to the central nervous system. The behavior of the stump is therefore more representative of the endogenous rhythmicity of the central pattern generator (CPG). Juveniles showed no differences in coordination in individual legs. Coordination between adjacent ipsilateral legs was also the same as that observed in adults following amputation. Furthermore, intact legs acquired new interlimb coordination similar to adults. These results suggested that juvenile and adult crayfish have functionally similar nervous systems controlling walking.;Finally, a 3-D virtual crayfish was built to test whether differences in walking between juveniles and adults could be due to mechanical influences alone. The model crayfish lacked direct connections between legs. The model responded to shifts in the center of mass by showing more consistent limb coordination in those legs nearest the center of mass. This was achieved through indirect mechanical coupling of the legs through the environment and body of the crayfish. This mechanism also produced realistic adaptive behavior when limbs were amputated. This showed that differences between adult and juvenile walking are due solely to mechanical influences associated with the changing center of mass as the animals grow. These results suggest further that organisms do not need high levels of control to produce coordinated behavior. Locomotor behavior arises through interactions between body, limb, and environment that are a function of the spatio-temporal dynamics of body morphology. The results may be applicable to a large number of walking systems

Enriched job design, high involvement management and organizational performance: The mediating roles of job satisfaction and well-being

The relationship between organizational performance and two dimensions of the 'high performance work system' - enriched job design and high involvement management (HIM) - is widely assumed to be mediated by worker well-being. We outline the basis for three models: mutual-gains, in which employee involvement increases well-being and this mediates its positive relationship with performance; conflicting outcomes, which associates involvement with increased stress for workers, accounting for its positive performance effects; and counteracting effects, which associates involvement with increased stress and dissatisfaction, reducing its positive performance effects. These are tested using the UK's Workplace Employment Relations Survey 2004. Job satisfaction mediates the relationship between enriched job design and four performance indicators, supporting the mutual gains model; but HIM is negatively related to job satisfaction and this depresses a positive relationship between HIM and the economic performance measures, supporting a counteracting effects model. Finally, HIM is negatively related to job-related anxiety-comfort and enriched job design is unrelated to it. © The Author(s) 2012

Ataxin-2 regulates RGS8 translation in a new BAC-SCA2 transgenic mouse model.

Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant disorder with progressive degeneration of cerebellar Purkinje cells (PCs) and other neurons caused by expansion of a glutamine (Q) tract in the ATXN2 protein. We generated BAC transgenic lines in which the full-length human ATXN2 gene was transcribed using its endogenous regulatory machinery. Mice with the ATXN2 BAC transgene with an expanded CAG repeat (BAC-Q72) developed a progressive cellular and motor phenotype, whereas BAC mice expressing wild-type human ATXN2 (BAC-Q22) were indistinguishable from control mice. Expression analysis of laser-capture microdissected (LCM) fractions and regional expression confirmed that the BAC transgene was expressed in PCs and in other neuronal groups such as granule cells (GCs) and neurons in deep cerebellar nuclei as well as in spinal cord. Transcriptome analysis by deep RNA-sequencing revealed that BAC-Q72 mice had progressive changes in steady-state levels of specific mRNAs including Rgs8, one of the earliest down-regulated transcripts in the Pcp2-ATXN2[Q127] mouse line. Consistent with LCM analysis, transcriptome changes analyzed by deep RNA-sequencing were not restricted to PCs, but were also seen in transcripts enriched in GCs such as Neurod1. BAC-Q72, but not BAC-Q22 mice had reduced Rgs8 mRNA levels and even more severely reduced steady-state protein levels. Using RNA immunoprecipitation we showed that ATXN2 interacted selectively with RGS8 mRNA. This interaction was impaired when ATXN2 harbored an expanded polyglutamine. Mutant ATXN2 also reduced RGS8 expression in an in vitro coupled translation assay when compared with equal expression of wild-type ATXN2-Q22. Reduced abundance of Rgs8 in Pcp2-ATXN2[Q127] and BAC-Q72 mice supports our observations of a hyper-excitable mGluR1-ITPR1 signaling axis in SCA2, as RGS proteins are linked to attenuating mGluR1 signaling

Decreased steady-state levels of <i>Rgs8</i> message and protein in BAC-Q72 mice.

<p><b>(A)</b> qRT-PCR analyses of cerebellar RNAs from wild-type and BAC-Q22 mice show unchanged <i>Rgs8</i> levels, whereas BAC-Q72 mice show significant and progressive reduction of <i>Rgs8</i> mRNA levels starting at 5 weeks of age. n: number of animals in each group. The data are means ± SD, **p<0.01, ***p<0.001. <b>(B)</b> Western blot analyses indicate reduction of Rgs8 steady-state levels in cerebella of BAC-Q72 mice, but no change in BAC-Q22 mice when compared with wild-type mice. The blot is a representative Western blot of 3 independently performed experiments with 2 animals each per BAC line. <b>(C)</b> SCA2 patient-derived LB cells demonstrate decreased <i>RGS8</i> transcripts. Total RNAs were isolated from LB cell lines derived from two normal control individuals and two SCA2 patients and subjected to RT-PCR analysis using primers specifically amplifying the human <i>ATXN2</i> CAG repeat. RT-PCR analyses indicate the expression of ATXN2 with expanded CAG repeats (46 or 52) (left panel). qRT-PCR analyses of synthesized cDNAs from LB cells show significant reduction of <i>RGS8</i> in both SCA2-LB cell lines. The data represent mean ± SD, **p<0.01 (right panel).</p

BAC derived- <i>hATXN2</i> mRNA is identified in multiple layers of the cerebellum and deep cerebellar nuclei.

<p>Expression of transgenic h<i>ATXN2</i> and murine <i>Atxn2</i> mRNAs in cerebellar fractions isolated by Laser Capture Microdissection (LCM): ML, molecular layer; PC, Purkinje cell layer; GCL, granule cell layer; DN, dentate nucleus. <b>(A-B)</b> Quantitative RT-PCR analyses of transgenic <i>hATXN2</i> (A) and endogenous <i>mAtxn2</i> (B). <b>(C-F)</b> Relative enrichment of cell-type specific marker genes; <i>Pcp2</i>, <i>Calb1</i>, <i>Neurod1</i> and <i>Spp1</i> for each fraction as determined by qRT-PCR. The error bars indicate ± SD.</p

Motor phenotype of <i>ATXN2</i> BAC transgenic mice on the accelerating rotarod.

<p><b>(A)</b> BAC-Q22 mice performed as well as wild-type mice at all ages. <b>(B)</b> BAC-Q72 mice performed significantly worse than wild-type littermates on the rotarod starting at 16 weeks of age. Data represent mean ± SEM of three trials on the test day (day 3). Number of animals tested are shown within the bars. Significance was determined using repeated measures ANOVA with post-hoc test correction. *p<0.05 and ***p<0.001.</p

Early expression changes of key cerebellar genes including several PC-specific genes measured by quantitative RT-PCR.

<p><b>(A)</b> No significant changes in BAC-Q22 mice compared with wild-type at 16 and 45 weeks of age. <b>(B)</b> In BAC-Q72 mice, a small reduction of <i>Pcp2</i> mRNA levels is seen at 5 weeks, but significant reductions in three genes are only seen at 9 weeks. Reductions in expression of <i>Grm1</i> occur late (weeks 24 and 36). Of note, mRNA levels of mouse <i>Atxn2</i> remain unchanged throughout. Genes tested: human transgene (h<i>ATXN2</i>), mouse Ataxin-2 (m<i>Atxn2</i>), calbindin 28-kDa (<i>Calb1</i>), PC protein 2 (<i>Pcp2</i>), glutamate receptor ionotropic delta-2 (<i>Grid2</i>) and metabotropic glutamate receptor 1 (<i>Grm1</i>). n: animal numbers for each genotype and age group are listed in brackets. Gene expression was normalized to beta-actin. Student’s two-tailed t-test compared expression in BAC transgenic mice with wild-type mice in each age group. *p<0.05, **p<0.01, ***p<0.001. Error bars represent ± SD.</p

Comparison of transcriptome changes in BAC-Q72 and Pcp2-ATXN2[Q127] mice.

<p><b>(A)</b> The Venn diagram of transcriptome changes using an FDR ≥15 and Log2 ratio of change ≥|0.3|. Class I transcripts are changed only in BAC-Q72 and class III transcripts changed only in Pcp2-ATXN2[Q127] cerebella. A total of 236 transcripts (class II) are significantly altered in both models. <b>(B)</b> Validation of six overlapping genes (class II) by qRT-PCR. Cerebellar RNAs from BAC-Q72 and WT littermates (both at 8 weeks of age) and Pcp2-ATXN2[Q127] and WT littermates (both at 6 weeks of age) show significant reductions of transcript expression. Genes tested are; <i>Rgs8</i>, <i>Calb1</i>, <i>Pcp2</i>, Purkinje cell protein 4 (<i>Pcp4</i>), Homer homolog 3 (Drosophila) (<i>Homer3</i>) and Centrosomal protein 76 (<i>Cep76</i>). Gene expression levels were normalized to beta-actin. Six animals from each group were used in this experiment. Data are means ± SD, *p<0.05 **p<0.01, ***p<0.001, Student t-test. <b>(C)</b> Fold change relation between RNA-seq data and observed experimental qRT-PCR data are tabulated.</p