Purkinje cell-specific knockout of the protein phosphatase PP2B impairs potentiation and cerebellar motor learning.

Cerebellar motor learning is required to obtain procedural skills. Studies have provided supportive evidence for a potential role of kinase-mediated long-term depression (LTD) at the parallel fiber to Purkinje cell synapse in cerebellar learning. Recently, phosphatases have been implicated in the induction of potentiation of Purkinje cell activities in vitro, but it remains to be shown whether and how phosphatase-mediated potentiation contributes to motor learning. Here, we investigated its possible role by creating and testing a Purkinje cell-specific knockout of calcium/calmodulin-activated protein-phosphatase-2B (L7-PP2B). The selective deletion of PP2B indeed abolished postsynaptic long-term potentiation in Purkinje cells and their ability to increase their excitability, whereas LTD was unaffected. The mutants showed impaired "gain-decrease" and "gain-increase" adaptation of their vestibulo-ocular reflex (VOR) as well as impaired acquisition of classical delay conditioning of their eyeblink response. Thus, our data indicate that PP2B may indeed mediate potentiation in Purkinje cells and contribute prominently to cerebellar motor learning.

Echinoderm microtubule-associated protein like protein 4, a member of the echinoderm microtubule-associated protein family, stabilizes microtubules.

Echinoderm microtubule-associated protein (EMAP) is the major microtubule binding protein in dividing sea urchin (Strongylocentrotus purpuratus) eggs. Echinoderm microtubule-associated protein like protein 4 (Eml4, restrictedly overexpressed proliferation-associated protein 120 kDa (Ropp120)) is one of the five mammalian EMAP homologues, the cellular function of which remains to be elucidated. In our first set of experiments we determined the spatio-temporal expression pattern of Eml4 in mouse brain. Our results demonstrate that Eml4 is a highly developmentally regulated gene with high expression levels in the developing nervous system of E11 embryos declining to low levels in adult. Spatially, Eml4 expression becomes restricted to the olfactory bulb, hippocampus and cerebellum. Transient transfection of a fusion construct of full-length mouse Eml4 with green fluorescent protein (GFP-Eml4) into Cos7 and HeLa cells resulted in colocalization of GFP-Eml4 with microtubules. This colocalization was observed both with microtubules of non-dividing cells and with the mitotic spindle of dividing cells. In addition, transient overexpression of GFP-Eml4 in Cos7 cells resulted in microtubules that were resistant to nocodazole treatment suggesting that Eml4 stabilizes microtubules. A consequence of microtubule stabilization is a net reduction in the amount of free tubulin. Microtubule stabilizing proteins therefore are expected to indirectly decrease the microtubule growth rate. Indeed, transient transfection of GFP-Eml4 resulted in a marked decrease in the microtubule growth rate, which is in line with our hypothesis that Eml4 functions as a microtubule stabilizing protein. In summary, our results suggest that Eml4 is a developmentally regulated protein that colocalizes with and stabilizes microtubules.

Monitor-induced tachycardia in a patient with a rate-responsive pacemaker.

A patient with a ventricle-paced, ventricle-sensed and inhibited, respiratory rate-responsive pacemaker presented for orthopaedic surgery. She had been operated upon recently without any adverse effects. In the recovery unit her electrocardiograph showed a paced ventricular tachycardia of about 140 beats x min(-1) without any other adverse sign or symptom. Although this tachycardia initially decreased to 70 beats x min(-1) after about 30 min, it later rose again to 140 beats x min(-1). A pacemaker technician was consulted, who suggested that there may be interference between the bioelectric impedance signals of the patient monitoring system that was being used to measure respiratory rate and the pacemaker's own measurement systems.

Steroid receptor coactivator-1 splice variants differentially affect corticosteroid receptor signaling.

The mechanisms of receptor- and cell-specific effects of the adrenal corticosteroid hormones via mineralo- (MRs) and glucocorticoid receptors (GRs) are still poorly understood. Because the expression levels of two splice variants of the steroid receptor coactivator-1 (SRC-1) 1a and 1e, can differ significantly in certain cell populations, we tested the hypothesis that their relative abundance could determine cell- and receptor-specific effects of corticosteroid receptor-mediated transcription. In transient transfections, we demonstrate three novel types of SRC-1a- and SRC-1e-specific effects for corticosteroid receptors. One is promoter dependence: SRC-1e much more potently coactivated transcription from several multiple response element-containing promoters. Mammalian 1-hydrid studies indicated that this likely does not involve promoter-specific coactivator recruitment. Endogenous phenylethanolamine-N-methyltransferase mRNA induction via GRs was also differentially affected by the splice variants. Another type is receptor specificity: responses mediated by the N-terminal part of the MR, but not the GR, were augmented by SRC-1e at synergizing response elements. SRC fragment SRC(988-1240) by the MR but not the GR N-terminal fragment in a 1-hybrid assay. The last type, for GRs, is ligand dependence. Due to effects on partial agonism of RU486-activated GRs, different ratios of SRC-1a and 1e can lead to large differences in the extent of antagonism of RU486 on GR-mediated transcription. Furthermore, we show that SRC-1e but not SRC-1a mRNA expression was regulated in the pituitary by corticosterone. We conclude that the cellular differences in SRC-1a to SRC-1e ratio demonstrated in vivo might be involved in cell-specific responses to corticosteroids in a promoter- and ligand-dependent way.

Eml5, a novel WD40 domain protein expressed in rat brain.

We have isolated a novel transcript with homology to the major microtubule-associated protein in dividing sea urchin embryos, EMAP. The protein has a predicted MW of approximately 180 kDa and we have named it Eml5 (EMAP-like protein 5). Eml5 contains 11 putative WD40 domains and 3 hydrophobic stretches of 43 aa, HELP domains, which have been suggested to be involved in microtubule binding. Eml5 appears to consist of two tandem repeats of the complete EMAP protein separated by a putative dimerization domain. Eml5 mRNA and protein is expressed at high levels in the hippocampus, cerebellum and olfactory bulb, as determined by in situ hybridization and immunocytochemistry. Eml5 transcripts can be detected in fore- and hindbrain structures from embryonic day 13 onwards. Because other EMAP-like proteins are involved in regulating microtubule dynamics, it is likely that Eml5 plays a role in the regulation of cytoskeletal rearrangements during neuronal development and in adult brain

Changes in cerebral oxygenation and blood flow during LBNP in spinal cord-injured individuals.

Spinal cord-injured (SCI) individuals, having a sympathetic nervous system lesion, experience hypotension during sitting and standing. Surprisingly, they experience few syncopal events. This suggests adaptations in cerebrovascular regulation. Therefore, changes in systemic circulation, cerebral blood flow, and oxygenation in eight SCI individuals were compared with eight able-bodied (AB) individuals. Systemic circulation was manipulated by lower body negative pressure at several levels down to -60 mmHg. At each level, we measured steady-state blood pressure, changes in cerebral blood velocity with transcranial Doppler, and cerebral oxygenation using near-infrared spectroscopy. We found that mean arterial pressure decreased significantly in SCI but not in AB individuals, in accordance with the sympathetic impairment in the SCI group. Cerebral blood flow velocity decreased during orthostatic stress in both groups, but this decrease was significantly greater in SCI individuals. Cerebral oxygenation decreased in both groups, with a tendency to a greater decrease in SCI individuals. Thus present data do not support an advantageous mechanism during orthostatic stress in the cerebrovascular regulation of SCI individuals.

Sympathetic nervous system activity and cardiovascular homeostatis during head-up tilt in patients with spinal cord injuries.

The relationship between sympathetic nervous system activity and cardiovascular responses to head-up tilt in patients with spinal cord injuries and in able-bodied subjects was studied. Twenty-seven adults, nine in each of the three groups (tetraplegia, paraplegia, and able-bodied subjects) were tilted 70 degrees, head up, for 12 minutes after 20 minutes supine rest. Differences between steady-state measurements of mean arterial pressure, stroke volume, and sympathetic nervous system activity were estimated in both positions. Sympathetic nervous system activity was reflected by the low-frequency peak of the blood pressure variability spectrum. From supine rest to head-up tilt, low-frequency power increased in able-bodied subjects (median, 0.42 mm Hg2, p = 0.003), which was different (p = 0.015) from patients with tetraplegia and paraplegia (-0.15 and -0.10 mm Hg2, respectively). Stroke volume and mean arterial pressure decreased in patients with tetraplegia (-40% and -9 mm Hg, respectively; p = 0.008, both variables) more than in able-bodied subjects (-33%, 11 mm Hg, respectively) or patients with paraplegia (-24%, 8 mm Hg, respectively). Results indicated increased sympathetic nervous system activity during head-up tilt in able-bodied subjects, but not in patients with paraplegia or tetraplegia, whereas patients with tetraplegia, but not paraplegia, showed poorer cardiovascular homeostasis than able-bodied subjects. This suggests that patients with paraplegia maintained cardiovascular homeostasis during head-up tilt without increased sympathetic nervous system activity.

Systemic circulation and cerebral oxygenation during head-up tilt in spinal cord injured individuals.

OBJECTIVE: To compare tilt-induced alterations in cardiovascular homeostasis and cerebral oxygenation of spinal cord-injured (SCI) to able-bodied (AB) individuals. DESIGN: Subjects underwent 10 min supine rest followed by 10 min 70 degrees head-up tilt. The last 5 min of supine rest and head-up tilt were analyzed, provided a steady state existed. SUBJECTS: SCI individuals (n= 11), with lesions between C4 and T4, and AB individuals (n= 10), all males and balanced for age and weight. MAIN OUTCOME MEASURES: Calf circumference, mean arterial pressure (MAP), stroke volume, heart rate and cerebral oxygenated ([O2Hb]), deoxygenated ([HHb]) and total ([tHb]) haemoglobin concentration changes were measured. RESULTS: Head-up tilt evoked a greater fall in MAP (mean (SD): -9 (12) vs 2 (6) mmHg P=0.02) and stroke volume (-43 (12) vs -22 (10)%, P=0.005), and a greater increase in heart rate (27 (12) vs 18 (6) beats, P=0.04) in SCI than AB. Cardiac output decreased during head-up tilt in SCI but not in AB (-17 (15) vs 1 (15)%, P=0.01). The change in cerebral oxygenation ([HHb]: 3.9 (2.8) vs 2.8 (1.4) micromol x l(-1), P=0.1 and [O2Hb]: -6.1 (5.0) vs -2.1 (5.5) micromol x l(-1), P=0.1) was similar in SCI and AB. All variables mentioned showed a change significantly different from zero in both groups, apart from [O2Hb] in AB and [tHb] in both groups. CONCLUSION: SCI demonstrated a greater decrease of MAP and stroke volume with a similar decrease in cerebral oxygenation compared to AB. This suggests that although systemic circulation was less well regulated in SCI compared with AB, cerebral circulation in SCI was maintained as in AB.

Blood volume and hemoglobin after spinal cord injury.

OBJECTIVE: The main purpose of this study was to investigate the difference in total blood volume and hemoglobin mass between spinal cord-injured and able-bodied individuals. DESIGN: Total blood volumes of 13 able-bodied and 10 spinal cord-injured individuals (lesion >T4) were determined using the carbon monoxide method. The reproducibility of the total blood volume determination in our setting and the effect of increased physical activity were assessed. RESULTS: Comparison of groups showed a significantly higher hemoglobin mass in able-bodied compared with spinal cord-injured individuals. The total blood volume expressed per kilogram of body mass in able-bodied individuals was significantly greater than in spinal cord-injured individuals. CONCLUSIONS: These results suggest that total blood volume and hemoglobin mass are decreased in spinal cord-injured individuals with a lesion above T4, which may be related to their inactive lifestyle, because total blood volume increased with increased physical activity in these subjects.

Non-invasive assessment of autonomic nervous system integrity in able-bodied and spinal cord-injured individuals.

The purpose of this study is to identify features of the Valsalva maneuver (VM) that differentiate between able-bodied (AB, n = 14) and spinal cord-injured individuals with high (T, n = 11) and low (P, n = 10) lesions. The systolic blood pressure (BP) recovery during the VM was used as a measure of sympathetic and vagal activity, whereas from the BP rise in phase IV of the VM baroreflex sensitivity was derived. After a similar initial BP decrease in all groups, BP recovered faster and more completely in AB compared to P and T during the VM. After release of the VM, the BP overshoot was very similar in AB and P, suggesting that the BP overshoot reflects sympathetic activity onto the heart. The baroreflex sensitivity was smaller in P, but not in T, compared to AB. However, 36% of the observations of T had such a low correlation between BP and RR interval that a slope could not be determined, which caused loss of observations, but suggested a low baroreflex sensitivity as well.

Training of the respiratory muscles in individuals with tetraplegia.

STUDY DESIGN: An experimental cross-sectional design. OBJECTIVES: To evaluate whether training of the innervated respiratory muscles in individuals with a (partial) cervical spinal cord injury will improve the strength and endurance capacity of these muscles and the exercise performance in these individuals. SETTING: Department of Physiology and Pulmonary diseases, Nijmegen, The Netherlands. METHOD: In this study nine individuals with tetraplegia (C3 C7) performed a target flow endurance training of the inspiratory muscles, twice a day for 15 min. First, the subjects performed a 'sham' training for 6 weeks with no appreciable resistance, after that they performed a 'real' training for 6 weeks with a resistance of 70% of the maximal endurance capacity of the inspiratory muscles. The training was evaluated at 0, 6 and 12 weeks by the following tests: (1) the slow Inspiratory Vital Capacity (IVC) and the Forced Inspiratory and Expiratory Volumes over 1 s (FIV1 and FEV1); (2) the Maximal Inspiratory Mouth Pressure (Pimax) and the Endurance Pressure (Pendu) and (3) a maximal arm-cranking exercise test. RESULTS: After the sham training, the Pendu was increased from 3.98 to 4.71 kPa with a P-value of 0.05. The sham training had no influence on any of the other variables. The real training had no effect on the IVC, FIV1, FEV1 and Pimax, however, increased the Pendu from 4.71 to 6.16 kPa (P=0.01), representing the respiratory muscle-endurance capacity. The oxygen consumption (VO2 peak) in the maximal exercise test improved from 0.87 to 0.98 l/min (P = 0.05). CONCLUSION: The results of the study indicate that training of the respiratory muscles results in an enhanced endurance capacity of these muscles and a concomitant increase in the aerobic exercise performance.

Non-invasive cardiac output assessment during moderate exercise: pulse contour compared with CO2 rebreathing.

The arterial pulse contour method called Modelflow 2.1 calculates stroke volume continuously, beat to beat, from the non-invasive blood pressure signal measured by Finapres or Portapres. Portapres is the portable version of Finapres. The purpose of this study was to compare cardiac output (CO) calculated using Modelflow 2.1 (COmf) with CO obtained by the CO2 rebreathing method (COre) during steady state at moderate exercise levels. Twelve subjects visited the laboratory twice and performed submaximal exercise on a bicycle ergometer at 20%, 40% and 60% of their individual peak power output (POpeak). The averaged correlation between COmf and COre gives an r-value of 0.69, whereas the slope and intercept of the regression line were 1.06 and 1.65 respectively. The averaged difference between COmf and COre was 2.27 +/- 3.91 min-1 (mean +/- standard deviation). However, the test-retest difference between COmf and COre was 2.5 +/- 3.1 and 0.5 +/- 1.31 min-1 respectively. These results suggest that Modelflow 2.1 is not an accurate method for estimating CO from non-invasive blood pressure data collected by Portapres during exercise at up to 60% of the individual POpeak corresponding with daily life activity.

Reproducibility of the alterations in circulation and cerebral oxygenation from supine rest to head-up tilt.

Cardiovascular stability, as affected by several diseases, may be assessed by head-up tilt testing. Follow-up studies are essential in both evaluating interventions and assessing progression. However, data on the reproducibility of the changes in circulatory status and cerebral oxygenation provoked by head-up tilt testing are fundamental to follow-up studies. The aim of this study was, therefore, to assess the reproducibility of the alterations in stroke volume (SV), mean arterial pressure (MAP), as well as oxygenated ([O2Hb]) and deoxygenated haemoglobin ([HHb]) concentration in cerebral tissue from supine rest (SUP) to head-up tilt (HUT). SV was calculated by Modelflow, a pulse contour method, from the finger arterial pressure wave measured by Portapres, the portable version of Finapres. [O2Hb] and [HHb] were measured using near-infrared spectroscopy (NIRS). Ten healthy individuals visited the laboratory on two different days. On both days, they underwent 10 min SUP followed by 10 min 70 degrees HUT twice. SV decreased, which was (in part) compensated for by an increased heart rate, while MAP increased slightly during HUT compared with SUP. Although [HHb] increased during HUT, no presyncope symptoms were experienced. The circulatory variables (SV, HR and MAP) as well as [HHb] showed an acceptably small systematic and random error as well as reproducibility error compared with the observed difference between HUT and SUP and were similar between and within visits. Therefore, it is concluded that MAP measured by Portapres and SV calculated by Modelflow as well as HHb measured by NIRS seem to be reproducible and may therefore be used in follow-up studies.

Effect of a pulsating anti-gravity suit on peak exercise performance in individual with spinal cord injuries.

The aim of this study was to examine effects of a pulsating pressure anti-gravity suit on the peak values of oxygen uptake (VO2) and power during maximal arm exercise in spinal-cord-injured (SCI) individuals. Five well-trained SCI men (with lesions at levels between T6 and L1) and seven well-trained able-bodied men (ABC) performed two incremental (10 W x min(-1)) arm-cranking tests. During one test the pressure in the anti-G suit pulsated between 4.7 kPa (35 mmHg) and 9.3 kPa (70 mmHg) every 2 s (PPG+), during the other test (PPG-) all the subjects wore the anti-G suit in a deflated state. Tests were performed in a counter-balanced order. Peak VO2 in SCI was 1 ml x kg(-1) x min(-1) lower during PPG+ compared to PPG- (P = 0.05). Peak power and peak heart rate were not significantly different during PPG+ compared to PPG-. These results would suggest that no increase in work capacity can be obtained with a pulsating pressure anti-gravity suit in either SCI or ABC.

Muscle glycogen recovery after exercise during glucose and fructose intake monitored by 13C-NMR.

The purpose of this study was to examine muscle glycogen recovery with glucose feeding (GF) compared with fructose feeding (FF) during the first 8 h after partial glycogen depletion using 13C-nuclear magnetic resonance (NMR) on a clinical 1.5-TNMR system. After measurement of the glycogen concentration of the vastus lateralis (VL) muscle in seven male subjects, glycogen stores of the VL were depleted by bicycle exercise. During 8 h after completion of exercise, subjects were orally given either GF or FF while the glycogen content of the VL was monitored by 13C-NMR spectroscopy every second hour. The muscular glycogen concentration was expressed as percentage of the glycogen concentration measured before exercise. The glycogen recovery rate during GF (4.2 +/- 0.2%/h) was significantly higher (P < 0.05) compared with values during FF (2.2 +/- 0.3%/h). This study shows that 1) muscle glycogen levels are perceptible by 13 C-NMR spectroscopy at 1.5 T and 2) the glycogen restoration rate is higher after GF compared with after FF.

Physiological responses to asynchronous and synchronous arm-cranking exercise.

The purpose of this study was to examine mechanical efficiency (ME) and physiological responses during asynchronous (the pedal arms oriented in opposing directions) arm-cranking exercise (AACE) and compare these responses to those obtained during synchronous (the pedal arms oriented in the same direction) arm-cranking exercise (SACE). Ten male subjects participated in the study and performed two exercise tests, one AACE and the other SACE in counter-balanced order. Each test consisted of submaximal (30, 60 and 90 W) and maximal exercise. At 30 W, gross ME was significantly lower during SACE compared to AACE, whereas at 60 W and 90 W no differences between the two types of exercise could be observed. We found that at lower power output levels the flywheel mass and its moment of inertia may have induced more body movements for compensation, which may have been more pronounced during SACE than during AACE. At higher levels of power output this flywheel masseffect was less, which explained the lack of differences in ME at these levels. Physiological responses to maximal AACE or SACE exercise were not significantly different. The results indicated that there were no differences in physiological responses to AACE and SACE exercise at higher exercise intensities. However, at lower levels of power output ME seemed to decrease, most likely as a result of the flywheel-mass effect, which was more pronounced during SACE.