Changes in FDB and soleus muscle activity after a train of stimuli during upright stance

Background: Evidence of self-sustained muscle activation following a brief electrical stimulation has been reported in the literature for certain muscles. Objectives: This report shows that the foot muscle (Flexor Digitorum Brevis - FDB) shows a self-sustained increase in muscle activity during upright stance in some subjects following a train of stimuli to the tibial nerve. Methods: Healthy subjects were requested to stand upright and surface EMG electrodes were placed on the FDB, Soleus and Tibialis Anterior muscles. After background muscle activity (BGA) acquisition, a 50 Hz train of stimuli was applied to the tibial nerve at the popliteal fossa. The root mean square values (RMS) of the BGA and the post-stimulus muscle activation were computed. Results: There was a 13.8% average increase in the FDB muscle EMG amplitude with respect to BGA after the stimulation was turned off. The corresponding post-stimulus Soleus EMG activity decreased by an average of 9.2%. We hypothesize that the sustained contraction observed in the FDB following stimulus may be evidence of persistent inward currents (PIC) generated in FDB spinal motoneurons. The post-stimulus decrease in soleus activity may have occurred due to the action of inhibitory interneurons caused by the PICs, which were triggered by the stimulus train. Conclusions: These sustained post-stimulation changes in postural muscle activity, found in different levels in different subjects, may be part of a set of possible responses that contribute to overall postural control.

Shape and function of the perineal muscles of viscacha (Lagostomus maximus)

Pieri N.C.G., Alicia M. Flamini A.M., Barbeito C.G., Casals J.B., Roque K.B., Favaron P.O., Miglino M.A. & Martins D.S. 2012. [Shape and function of the perineal muscles of viscacha (Lagostomus maximus).] Forma e funcao dos musculos perineais da viscacha (Lagostomus maximus). Pesquisa Veterinaria Brasileira 32(2):183-187. Departamento de Zootecnia, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de Sao Paulo, Av. Duque de Caxias Norte 225, Pirassununga, SP 13635-900, Brazil. E-mail: daniele@usp.br. Among the rodent species studied we can highlight the wide variation in the morphology of the male reproductive system. Thus, considering the ecological importance of rodents, and the large number and geographical representation of this animal, as well as shortages regarding the reproductive anatomy, we developed this study with viscacha, a South American histricomorph rodent. As this species has some very peculiar reproductive features, we described the gross anatomy of the perineal muscles and the role of copulatory behavior. The perineal region of viscacha is composed of five muscles, three of which are arranged in the superficial genitourinary diaphragm, as Musculus ischiocavernosus, M. bulbocavernosus and M. bulbospongiosus, and the muscles that lie at the pelvic diaphragm, M. levator ani and M. retractor penis. Therefore, we emphasize that the study of the pelvic floor in wild animals is of great value, then contribute to a better understanding of the mechanisms related to erection and ejaculation or collaborate with studies on the reproduction of animals.

Models of passive and active dendrite motoneuron pools and their differences in muscle force control

Motoneuron (MN) dendrites may be changed from a passive to an active state by increasing the levels of spinal cord neuromodulators, which activate persistent inward currents (PICs). These exert a powerful influence on MN behavior and modify the motor control both in normal and pathological conditions. Motoneuronal PICs are believed to induce nonlinear phenomena such as the genesis of extra torque and torque hysteresis in response to percutaneous electrical stimulation or tendon vibration in humans. An existing large-scale neuromuscular simulator was expanded to include MN models that have a capability to change their dynamic behaviors depending on the neuromodulation level. The simulation results indicated that the variability (standard deviation) of a maintained force depended on the level of neuromodulatory activity. A force with lower variability was obtained when the motoneuronal network was under a strong influence of PICs, suggesting a functional role in postural and precision tasks. In an additional set of simulations when PICs were active in the dendrites of the MN models, the results successfully reproduced experimental results reported from humans. Extra torque was evoked by the self-sustained discharge of spinal MNs, whereas differences in recruitment and de-recruitment levels of the MNs were the main reason behind torque and electromyogram (EMG) hysteresis. Finally, simulations were also used to study the influence of inhibitory inputs on a MN pool that was under the effect of PICs. The results showed that inhibition was of great importance in the production of a phasic force, requiring a reduced co-contraction of agonist and antagonist muscles. These results show the richness of functionally relevant behaviors that can arise from a MN pool under the action of PICs.

Assessment of a Neurophysiological Model of the Mandibular Branch of the Facial Nerve in Rats by Electromyography

Objectives: Our objective was to develop an experimental model for the noninvasive and objective evaluation of facial nerve regeneration in rats using a motor nerve conduction test (electromyography). Methods: Twenty-two rats were submitted to neurophysiological evaluation using motor nerve conduction of the mandibular branch of the facial nerve to obtain the compound muscle action potentials (CMAPs). To record the CM APs, we used two needle electrodes that were inserted into the lower lip muscle of the rat. A supramaximal electrical stimulus was applied, and the values of CMAP latency, amplitude, length, area, and stimulus intensity obtained from each side were compared by use of the Wilcoxon test. Results: There was no significant difference (all p > 0.05) in latency, amplitude, duration, area, or intensity of stimuli between the two sides. The amplitudes ranged between 1.61 and 8.30 mV, the latencies between 1.03 and 1.97 ms, and the stimulus intensities between 1.50 and 2.90 mA. Conclusions: This is a noninvasive, easy, and highly reproducible method that contributes to an improvement of the techniques previously described and may contribute to future studies of the degeneration and regeneration of the facial nerve.

Differential cellular FGF-2 upregulation in the rat facial nucleus following axotomy, functional electrical stimulation and corticosterone: a possible therapeutic target to Bell's palsy

Abstract Background The etiology of Bell's palsy can vary but anterograde axonal degeneration may delay spontaneous functional recovery leading the necessity of therapeutic interventions. Corticotherapy and/or complementary rehabilitation interventions have been employed. Thus the natural history of the disease reports to a neurotrophic resistance of adult facial motoneurons leading a favorable evolution however the related molecular mechanisms that might be therapeutically addressed in the resistant cases are not known. Fibroblast growth factor-2 (FGF-2) pathway signaling is a potential candidate for therapeutic development because its role on wound repair and autocrine/paracrine trophic mechanisms in the lesioned nervous system. Methods Adult rats received unilateral facial nerve crush, transection with amputation of nerve branches, or sham operation. Other group of unlesioned rats received a daily functional electrical stimulation in the levator labii superioris muscle (1 mA, 30 Hz, square wave) or systemic corticosterone (10 mgkg-1). Animals were sacrificed seven days later. Results Crush and transection lesions promoted no changes in the number of neurons but increased the neurofilament in the neuronal neuropil of axotomized facial nuclei. Axotomy also elevated the number of GFAP astrocytes (143% after crush; 277% after transection) and nuclear FGF-2 (57% after transection) in astrocytes (confirmed by two-color immunoperoxidase) in the ipsilateral facial nucleus. Image analysis reveled that a seven days functional electrical stimulation or corticosterone led to elevations of FGF-2 in the cytoplasm of neurons and in the nucleus of reactive astrocytes, respectively, without astrocytic reaction. Conclusion FGF-2 may exert paracrine/autocrine trophic actions in the facial nucleus and may be relevant as a therapeutic target to Bell's palsy.

Motor recovery and synaptic preservation after ventral root avulsion and repair with a fibrin sealant derived from snake venom

Background: Ventral root avulsion is an experimental model of proximal axonal injury at the central/peripheral nervous system interface that results in paralysis and poor clinical outcome after restorative surgery. Root reimplantation may decrease neuronal degeneration in such cases. We describe the use of a snake venom-derived fibrin sealant during surgical reconnection of avulsed roots at the spinal cord surface. The present work investigates the effects of this fibrin sealant on functional recovery, neuronal survival, synaptic plasticity, and glial reaction in the spinal motoneuron microenvironment after ventral root reimplantation. Methodology/Principal Findings: Female Lewis rats (7 weeks old) were subjected to VRA and root replantation. The animals were divided into two groups: 1) avulsion only and 2) replanted roots with fibrin sealant derived from snake venom. Post-surgical motor performance was evaluated using the CatWalk system twice a week for 12 weeks. The rats were sacrificed 12 weeks after surgery, and their lumbar intumescences were processed for motoneuron counting and immunohistochemistry (GFAP, Iba-1 and synaptophysin antisera). Array based qRT-PCR was used to evaluate gene regulation of several neurotrophic factors and receptors as well as inflammatory related molecules. The results indicated that the root reimplantation with fibrin sealant enhanced motor recovery, preserved the synaptic covering of the motoneurons and improved neuronal survival. The replanted group did not show significant changes in microglial response compared to VRA-only. However, the astroglial reaction was significantly reduced in this group. Conclusions/Significance: In conclusion, the present data suggest that the repair of avulsed roots with snake venom fibrin glue at the exact point of detachment results in neuroprotection and preservation of the synaptic network at the microenvironment of the lesioned motoneurons. Also such procedure reduced the astroglial reaction and increased mRNA levels to neurotrophins and anti-inflammatory cytokines that may in turn, contribute to improving recovery of motor function.

Plantar flexion force induced by amplitude-modulated tendon vibration and associated soleus V/F-waves as an evidence of a centrally-mediated mechanism contributing to extra torque generation in humans

Background: High-frequency trains of electrical stimulation applied over the human muscles can generate forces higher than would be expected by direct activation of motor axons, as evidenced by an unexpected relation between the stimuli and the evoked contractions, originating what has been called “extra forces”. This phenomenon has been thought to reflect nonlinear input/output neural properties such as plateau potential activation in motoneurons. However, more recent evidence has indicated that extra forces generated during electrical stimulation are mediated primarily, if not exclusively, by an intrinsic muscle property, and not from a central mechanism as previously thought. Given the inherent differences between electrical and vibratory stimuli, this study aimed to investigate: (a) whether the generation of vibration-induced muscle forces results in an unexpected relation between the stimuli and the evoked contractions (i.e. extra forces generation) and (b) whether these extra forces are accompanied by signs of a centrally-mediated mechanism or whether intrinsic muscle properties are the redominant mechanisms. Methods: Six subjects had their Achilles tendon stimulated by 100 Hz vibratory stimuli that linearly increased in amplitude (with a peak-to-peak displacement varying from 0 to 5 mm) for 10 seconds and then linearly decreased to zero for the next 10 seconds. As a measure of motoneuron excitability taken at different times during the vibratory stimulation, short-latency compound muscle action potentials (V/F-waves) were recorded in the soleus muscle in response to supramaximal nerve stimulation. Results: Plantar flexion torque and soleus V/F-wave amplitudes were increased in the second half of the stimulation in comparison with the first half. Conclusion: The present findings provide evidence that vibratory stimuli may trigger a centrally-mediated mechanism that contributes to the generation of extra torques. The vibration-induced increased motoneuron excitability (leading to increased torque generation) presumably activates spinal motoneurons following the size principle, which is a desirable feature for stimulation paradigms involved in rehabilitation programs and exercise training.

Funktionelle Analyse der beiden Zinkfinger-Homöodomänentranskriptionsfaktoren Zfh-1 und Zfh-2 während der Entwicklung des Nervensystems von Drosophila melanogaster

Diese Arbeit charakterisiert die Funktion und das Expressionmuster der beiden Zinkfinger-Homöodomänentranskriptionsfaktoren zfh1 und zfh2 von Drosophila melanogaster. Das zfh2 Gen wurde hierbei vor allem molekular charakterisiert. Es wurden eine Vielzahl möglicher Spleißformen identifiziert, welche das regulatorische Potential von Zfh2 enorm erweitern. Für Überexpressionsexperimente wurde zudem erstmalig die cDNA des längsten zfh2-Transkriptes kloniert. Durch Analysen an zfh1 Mutanten konnte gezeigt werden, dass zfh1 sowohl notwendig ist für die embryonale Entwicklung von Motoneuronen, als auch das larvale Wachstum motoneuronaler Endplatten reguliert. Wegen weit reichender pleiotroper Effekte, die zfh1 Funktionsverlustmutanten haben, war es notwendig, neben dem Einsatz hypomorpher Allele auf die Analyse genetischer Mosaike auszuweichen. Die als MARCM-Technik (Lee und Luo, 1999) bezeichnete Methode zur Erzeugung genetischer Mosaike wurde modifiziert um in dieser Arbeit erstmals für die Analyse mutanter larvaler Motoneurone eingesetzt werden zu können. Weitergehend konnte gezeigt werden, dass Zfh1 notwendig ist für die larvale Expression des Neuropeptides FMRFamid. Anhand von Sequenzvergleichen und durch Verwendung eines fmrfamid-Promoterkonstruktes (Benveniste et al., 1998) konnten Hinweise dafür gesammelt werden, dass die Zfh1-abhängige Regulation sehr wahrscheinlich direkter Natur ist. Bei fmrfamid handelt es sich somit um das erste identifizierte neurale Zielgen von Zfh1, an dem sich zudem modellhaft der molekulare Wirkmechanismus von Zfh1 erforschen lässt.

Hox genes and the regulation of programmed cell death in the embryonic central nervous system of Drosophila melanogaster

This study deals with the function and regulation of programmed cell death, or apoptosis, in the development of the embryonic central nervous system of Drosophila melanogaster. The first part provides a description of apoptosis-deficient embryos, which showed that preventing apoptosis does not cause gross morphological defects in the CNS, as it appears well organized despite the presence of too many cells. An analysis of the incidence and pattern of apoptosis over the course of development discloses a partly very orderly pattern suggesting tight spatio-temporal control, but also reveals random apoptotic cells, which suggests a certain amount of plasticity in the embryo. This analysis also allowed precise identification of some of the dying neural cells in the embryo, and establishment of single cell models for studying regulation of segment-specific apoptosis in the embryonic CNS. In the second part of the work, further investigations into mechanisms controlling segment-specific apoptosis revealed the involvement of two Hox genes, Antennapedia (Antp) and Ultrabithorax (Ubx), in this process. Hox genes control the formation of segment-specific structures in their domains of expression, but also regulate organ and tissue morphogenesis. The study presented here shows that Antp and Ubx play antagonistic roles in motoneuron survival in the embryo. Ubx expression in the CNS is strongly upregulated at a late point in development, when most cells have begun to differentiate. This upregulation shortly precedes Ubx-dependent, segment-specific apoptosis of two differentiated motoneurons. It could further be demonstrated that Antp is required for proper development of the NB7-3 lineage and for survival of the NB7-3 motoneuron in the anterior thoracic segments. In segments where Antp and Ubx expression overlaps, Ubx counteracts the anti-apoptotic function of Antp, resulting in cell death. Thus, these two Hox genes play opposing roles in the survival of differentiated neurons in the late developing nervous system. They thereby contribute to establishment of correct connections between outward-projecting neurons and their targets, which is crucial for the assembly of functional neural circuits, as these have to fulfill region-specific locomotion and sensory requirements along the antero-posterior body axis.

Ultrastructural changes in diaphragm neuromuscular junctions in a severe mouse model for Spinal Muscular Atrophy and their prevention by bifunctional U7 snRNA correcting SMN2 splicing

In Spinal Muscular Atrophy (SMA), the SMN1 gene is deleted or inactivated. Because of a splicing problem, the second copy gene, SMN2, generates insufficient amounts of functional SMN protein, leading to the death of spinal cord motoneurons. For a "severe" mouse SMA model (Smn -/-, hSMN2 +/+; with affected pups dying at 5-7 days), which most closely mimicks the genetic set-up in human SMA patients, we characterise SMA-related ultrastructural changes in neuromuscular junctions (NMJs) of two striated muscles with discrete functions. In the diaphragm, but not the soleus muscle of 4-days old SMA mice, mitochondria on both sides of the NMJs degenerate, and perisynaptic Schwann cells as well as endoneurial fibroblasts show striking changes in morphology. Importantly, NMJs of SMA mice in which a modified U7 snRNA corrects SMN2 splicing and delays or prevents SMA symptoms are normal. This ultrastructural study reveals novel features of NMJ alterations - in particular the involvement of perisynaptic Schwann cells - that may be relevant for human SMA pathogenesis.

Cellular and network mechanisms of rhythm generation in spinal cord circuits

The generation of rhythmic electrical activity is a prominent feature of spinal cord circuits that is used for locomotion and also for circuit refinement during development. The mechanisms involved in rhythm generation in spinal cord networks are not fully understood. It is for example not known whether spinal cord rhythms are driven by pacemaker neurons and if yes, which neurons are involved in this function. We studied the mechanisms involved in rhythm generation in slice cultures from fetal rats that were grown on multielectrode arrays (MEAs). We combined multisite extracellular recordings from the MEA electrodes with intracellular patch clamp recordings from single neurons. We found that spatially restricted oscillations of activity appeared in most of the cultures spontaneously. Such activity was based on intrinsic activity in a percentage of the neurons that could activate the spinal networks through recurrent excitation. The local oscillator networks critically involved NMDA, AMPA and GABA / glycine receptors at subsequent phases of the oscillation cycle. Intrinsic spiking in individual neurons (in the absence of functional synaptic coupling) was based on persistent sodium currents. Intrinsic firing as well as persistent sodium currents were increased by 5-HT through 5-HT2 receptors. Comparing neuronal activity to muscle activity in co-cultures of spinal cord slices with muscle fibers we found that a percentage of the intrinsically spiking neurons were motoneurons. These motoneurons were electrically coupled among each other and they could drive the spinal networks through cholinergic recurrent excitation. These findings open the possibility that during development rhythmic activity in motoneurons is not only involved in circuit refinement downstream at the neuromuscular endplates but also upstream at the level of spinal cord circuits.

'Neurons in Action' in Action - Educational settings for simulations and tutorials using NEURON

Neurons in Action (NIA1, 2000; NIA1.5, 2004; NIA2, 2007), a set of tutorials and linked simulations, is designed to acquaint students with neuronal physiology through interactive, virtual laboratory experiments. Here we explore the uses of NIA in lecture, both interactive and didactic, as well as in the undergraduate laboratory, in the graduate seminar course, and as an examination tool through homework and problem set assignments. NIA, made with the simulator NEURON (http://www.neuron.yale.edu/neuron/), displays voltages, currents, and conductances in a membrane patch or signals moving within the dendrites, soma and/or axon of a neuron. Customized simulations start with the plain lipid bilayer and progress through equilibrium potentials; currents through single Na and K channels; Na and Ca action potentials; voltage clamp of a patch or a whole neuron; voltage spread and propagation in axons, motoneurons and nerve terminals; synaptic excitation and inhibition; and advanced topics such as channel kinetics and coincidence detection. The user asks and answers "what if" questions by specifying neuronal parameters, ion concentrations, and temperature, and the experimental results are then plotted as conductances, currents, and voltage changes. Such exercises provide immediate confirmation or refutation of the student's ideas to guide their learning. The tutorials are hyperlinked to explanatory information and to original research papers. Although the NIA tutorials were designed as a sequence to empower a student with a working knowledge of fundamental neuronal principles, we find that faculty are using the individual tutorials in a variety of educational situations, some of which are described here. Here we offer ideas to colleagues using interactive software, whether NIA or another tool, for educating students of differing backgrounds in the subject of neurophysiology.

A large animal model of spinal muscular atrophy and correction of phenotype.

OBJECTIVES Spinal muscular atrophy (SMA) is caused by reduced levels of survival motor neuron (SMN) protein, which results in motoneuron loss. Therapeutic strategies to increase SMN levels including drug compounds, antisense oligonucleotides, and scAAV9 gene therapy have proved effective in mice. We wished to determine whether reduction of SMN in postnatal motoneurons resulted in SMA in a large animal model, whether SMA could be corrected after development of muscle weakness, and the response of clinically relevant biomarkers. METHODS Using intrathecal delivery of scAAV9 expressing an shRNA targeting pig SMN1, SMN was knocked down in motoneurons postnatally to SMA levels. This resulted in an SMA phenotype representing the first large animal model of SMA. Restoration of SMN was performed at different time points with scAAV9 expressing human SMN (scAAV9-SMN), and electrophysiology measurements and pathology were performed. RESULTS Knockdown of SMN in postnatal motoneurons results in overt proximal weakness, fibrillations on electromyography indicating active denervation, and reduced compound muscle action potential (CMAP) and motor unit number estimation (MUNE), as in human SMA. Neuropathology showed loss of motoneurons and motor axons. Presymptomatic delivery of scAAV9-SMN prevented SMA symptoms, indicating that all changes are SMN dependent. Delivery of scAAV9-SMN after symptom onset had a marked impact on phenotype, electrophysiological measures, and pathology. INTERPRETATION High SMN levels are critical in postnatal motoneurons, and reduction of SMN results in an SMA phenotype that is SMN dependent. Importantly, clinically relevant biomarkers including CMAP and MUNE are responsive to SMN restoration, and abrogation of phenotype can be achieved even after symptom onset.

Central and peripheral defects in motor units of the diaphragm of spinal muscular atrophy mice.

Spinal muscular atrophy (SMA) is characterized by motoneuron loss and muscle weakness. However, the structural and functional deficits that lead to the impairment of the neuromuscular system remain poorly defined. By electron microscopy, we previously found that neuromuscular junctions (NMJs) and muscle fibres of the diaphragm are among the earliest affected structures in the severe mouse SMA model. Because of certain anatomical features, i.e. its thinness and its innervation from the cervical segments of the spinal cord, the diaphragm is particularly suitable to characterize both central and peripheral events. Here we show by immunohistochemistry that, at postnatal day 3, the cervical motoneurons of SMA mice receive less stimulatory synaptic inputs. Moreover, their mitochondria become less elongated which might represent an early stage of degeneration. The NMJs of the diaphragm of SMA mice show a loss of synaptic vesicles and active zones. Moreover, the partly innervated endplates lack S100 positive perisynaptic Schwann cells (PSCs). We also demonstrate the feasibility of comparing the proteomic composition between diaphragm regions enriched and poor in NMJs. By this approach we have identified two proteins that are significantly upregulated only in the NMJ-specific regions of SMA mice. These are apoptosis inducing factor 1 (AIFM1), a mitochondrial flavoprotein that initiates apoptosis in a caspase-independent pathway, and four and a half Lim domain protein 1 (FHL1), a regulator of skeletal muscle mass that has been implicated in several myopathies.

THE ULTRASTRUCTURAL ORGANIZATION OF THE HYPOGLOSSAL NUCLEUS IN THE RAT (SYNAPTOLOGY, CRANIAL NERVES)

An ultrastructural study of the hypoglossal nucleus (XII) in the rat has revealed two distinct neuronal populations. Hypoglossal motoneurons comprised the largest population of neurons in XII and were identified following injection of horseradish peroxidase (HRP) into the tongue. Motoneurons were large (25-50(mu)m), multipolar in shape and distributed throughout XII. The nucleus was large, round and centrally located, and the cytoplasm was characterized by dense lamellar arrays of rough endoplasmic reticulum. In contrast, a second population of small (10-18(mu)m), round to oval shaped neurons was found restricted to the ventral and dorsolateral regions of XII. The nucleus was markedly invaginated and eccentric, the cytoplasm scant and filled with free ribosomes, and the absence of lamellar arrays of rough endoplasmic reticulum was conspicuous. Neurons of this type were never found to contain HRP reaction product. These results demonstrate that the hypoglossal nucleus does not consist solely of motoneurons, but includes a distinctly separate, presumably non-motoneuronal pool. Arguments are presented in favor of this second neuron population being interneurons. The functional significance of these findings in relation to tongue control is discussed. ^