Fiber Innervation Research Articles

Lack of Mucosal Cholinergic Innervation Is Associated With Increased Risk of Enterocolitis in Hirschsprung’s Disease

Background & AimsHirschsprung’s disease (HSCR) is a congenital intestinal motility disorder defined by the absence of enteric neuronal cells (ganglia) in the distal gut. The development of HSCR-associated enterocolitis remains a life-threatening complication. Absence of enteric ganglia implicates innervation of acetylcholine-secreting (cholinergic) nerve fibers. Cholinergic signals have been reported to control excessive inflammation, but the impact on HSCR-associated enterocolitis is unknown.MethodsWe enrolled 44 HSCR patients in a prospective multicenter study and grouped them according to their degree of colonic mucosal acetylcholinesterase-positive innervation into low-fiber and high-fiber patient groups. The fiber phenotype was correlated with the tissue cytokine profile as well as immune cell frequencies using Luminex analysis and fluorescence-activated cell sorting analysis of colonic tissue and immune cells. Using confocal immunofluorescence microscopy, macrophages were identified in close proximity to nerve fibers and characterized by RNA-seq analysis. Microbial dysbiosis was analyzed in colonic tissue using 16S-rDNA gene sequencing. Finally, the fiber phenotype was correlated with postoperative enterocolitis manifestation.ResultsThe presence of mucosal nerve fiber innervation correlated with reduced T-helper 17 cytokines and cell frequencies. In high-fiber tissue, macrophages co-localized with nerve fibers and expressed significantly less interleukin 23 than macrophages from low-fiber tissue. HSCR patients lacking mucosal nerve fibers showed microbial dysbiosis and had a higher incidence of postoperative enterocolitis.ConclusionsThe mucosal fiber phenotype might serve as a prognostic marker for enterocolitis development in HSCR patients and may offer an approach to personalized patient care and new therapeutic options.

Misalignment considerations in laser diode to single-mode graded index fiber coupling via upside down tapered parabolic microlens on the circular core fiber

We report the theoretical analysis of the coupling optics involving laser diode to single-mode circular core graded index fiber excitation via upside down tapered parabolic microlens on the fiber in presence of possible transverse and angular misalignments. We use the relevant ABCD matrix appropriate for such type of coupling device in order to prescribe analytical expressions of coupling efficiencies in presence of the said misalignments. The prescribed expressions are employed for estimation of the concerned coupling optics. The execution of our formalism involves little computation and as such the present formalism will prove user friendly to the system engineers. The results obtained will be useful to the designers and packagers in the context of maximum launch optics of such coupling device.

Clustering fibromyalgia patients: A combination of psychosocial and somatic factors leads to resilient coping in a subgroup of fibromyalgia patients

BackgroundCoping strategies and their efficacy vary greatly in patients suffering from fibromyalgia syndrome (FMS).ObjectiveWe aimed to identify somatic and psychosocial factors that might contribute to different coping strategies and resilience levels in FMS.Subjects and methodsStandardized questionnaires were used to assess coping, pain, and psychological variables in a cohort of 156 FMS patients. Quantitative real-time polymerase chain reaction (qRT-PCR) determined gene expression of selected cytokines in white blood cells of 136 FMS patients and 25 healthy controls. Data of skin innervation, functional and structural sensory profiles of peripheral nociceptive nerve fibers of a previous study were included into the statistics. An exploratory factor analysis was used to define variance explaining factors, which were then included into cluster analysis.Results54.9% of the variance was explained by four factors which we termed (1) affective load, (2) coping, (3) pain, and (4) pro-inflammatory cytokines (p < 0.05). Considering differences in the emerged factors, coping strategies, cytokine profiles, and disability levels, 118 FMS patients could be categorized into four clusters which we named “maladaptive”, “adaptive”, “vulnerable”, and “resilient” (p < 0.05). The adaptive cluster had low scores in disability and in all symptom categories in contrast to the vulnerable cluster, which was characterized by high scores in catastrophizing and disability (p < 0.05). The resilient vs. the maladaptive cluster was characterized by better coping and a less pro-inflammatory cytokine pattern (p < 0.05).ConclusionOur data suggest that problem- and emotion-focused coping strategies and an anti-inflammatory cytokine pattern are associated with reduced disability and might promote resilience. Additional personal factors such as low anxiety scores, ability of acceptance, and persistence further favor a resilient phenotype. Individualized therapy should take these factors into account.

Basal Ganglia Output Has a Permissive Non-Driving Role in a Signaled Locomotor Action Mediated by the Midbrain.

The basal ganglia are important for movement and reinforcement learning. Using mice of either sex, we found that the main basal ganglia GABAergic output in the midbrain, the substantia nigra pars reticulata (SNr), shows movement-related neural activity during the expression of a negatively reinforced signaled locomotor action known as signaled active avoidance; this action involves mice moving away during a warning signal to avoid a threat. In particular, many SNr neurons deactivate during active avoidance responses. However, whether SNr deactivation has an essential role driving or regulating active avoidance responses is unknown. We found that optogenetic excitation of SNr or striatal GABAergic fibers that project to an area in the pedunculopontine tegmentum (PPT) within the midbrain locomotor region abolishes signaled active avoidance responses, while optogenetic inhibition of SNr cells (mimicking the SNr deactivation observed during an active avoidance behavior) serves as an effective conditioned stimulus signal to drive avoidance responses by disinhibiting PPT neurons. However, preclusion of SNr deactivation, or direct inhibition of SNr fibers in the PPT, does not impair the expression of signaled active avoidance, indicating that SNr output does not drive the expression of a signaled locomotor action mediated by the midbrain. Consistent with a permissive regulatory role, SNr output provides information about the state of the ongoing action to downstream structures that mediate the action.SIGNIFICANCE STATEMENT During signaled active avoidance behavior, subjects move away to avoid a threat when directed by an innocuous sensory stimulus. Excitation of GABAergic cells in the substantia nigra pars reticulata (SNr), the main output of the basal ganglia, blocks signaled active avoidance, while inhibition of SNr cells is an effective stimulus to drive active avoidance. Interestingly, many SNr cells inhibit their firing during active avoidance responses, suggesting that SNr inhibition could be driving avoidance responses by disinhibiting downstream areas. However, interfering with the modulation of SNr cells does not impair the behavior. Thus, SNr may regulate the active avoidance movement in downstream areas that mediate the behavior, but does not drive it.

Next-generation sequencing application to investigate skeletal muscle channelopathies in a large cohort of Italian patients

Non-dystrophic myotonias and periodic paralyses are a heterogeneous group of disabling diseases classified as skeletal muscle channelopathies. Their genetic characterization is essential for prognostic and therapeutic purposes; however, several genes are involved. Sanger-based sequencing of a single gene is time-consuming, often expensive; thus, we designed a next-generation sequencing panel of 56 putative candidate genes for skeletal muscle channelopathies, codifying for proteins involved in excitability, excitation-contraction coupling, and metabolism of muscle fibres. We analyzed a large cohort of 109 Italian patients with a suspect of NDM or PP by next-generation sequencing. We identified 24 patients mutated in CLCN1 gene, 15 in SCN4A, 3 in both CLCN1 and SCN4A, 1 in ATP2A1, 1 in KCNA1 and 1 in CASQ1. Eight were novel mutations: p.G395Cfs*32, p.L843P, p.V829M, p.E258E and c.1471+4delTCAAGAC in CLCN1, p.K1302R in SCN4A, p.L208P in ATP2A1 and c.280–1G>C in CASQ1 genes. This study demonstrated the utility of targeted next generation sequencing approach in molecular diagnosis of skeletal muscle channelopathies and the importance of the collaboration between clinicians and molecular geneticists and additional methods for unclear variants to make a conclusive diagnosis.

Spinal motor mapping by epidural stimulation of lumbosacral posterior roots in humans.

SummaryEpidural electrical stimulation of the spinal cord is an emergent strategy for the neurological recovery of lower-extremity motor function. Motoneuron pools are thought to be recruited by stimulation of posterior roots. Here, we linked electromyographic data of epidurally evoked lower-extremity responses of 34 individuals with upper motoneuron disorders to a population model of the spinal cord constructed using anatomical parameters of thousands of individuals. We identified a relationship between segmental stimulation sites and activated spinal cord segments, which made spinal motor mapping from epidural space possible despite the complex anatomical interface imposed by the posterior roots. Our statistical approach provided evidence for low-threshold sites of posterior roots and effects of monopolar and bipolar stimulation previously predicted by computer modeling and allowed us to test the impact of different upper motoneuron disorders on the evoked responses. Finally, we revealed a statistical association between intraoperative and postoperative mapping of the spinal cord.

Simulation-Based Optimization of Figure-of-Eight Coil Designs and Orientations for Magnetic Stimulation of Peripheral Nerve.

Although magnetic neural stimulation has many advantages over electrical neural stimulation, its main disadvantages are higher energy requirement and poor stimulation selectivity. The orientation and location of the coil with respect to the stimulation site play a critical role in determining the stimulation threshold and stimulation selectivity. Utilizing numerical simulations in this work, we optimized the design parameters, orientation, and positioning of magnetic coils with respect to the peripheral nerve for improved stimulation efficacy. Specifically, we investigated different orientations and positions of the figure-of-eight coils for neural stimulation of the rat sciatic nerve. We also examined the effect of coil design parameters (number of layers and turns) and different coil electrical configurations (opposite vs. same direction of coil currents and series vs. parallel coil connections) on the stimulation threshold. We leveraged the multi-resolution impedance method and a heterogeneous multi-fascicular anatomical model of rat sciatic nerve to explore the possibility of selective stimulation as well. Neural excitation of a nerve fiber was implemented by an equivalent cable model and Frankenhaeuser-Huxley equations using NEURON software. Results suggest that inter-fascicular selectivity could be achieved by properly orienting and positioning the coil with respect to the nerve. Further, by orienting the figure-of-eight coil at an angle of 90° and 6 mm offset, we could switch between primarily activating one fascicle (and barely activating the other) and reversing those roles by merely switching the current direction in the two coils of the figure-of-eight coil.

Sensitivity Study of Neuronal Excitation and Cathodal Blocking Thresholds of Myelinated Axons for Percutaneous Auricular Vagus Nerve Stimulation.

Excitation of myelinated nerve fibers is investigated by means of numerical simulations, for the application of percutaneous auricular vagus nerve stimulation (pVNS). High sensitivity to axon diameter is of interest regarding the goal of targeting thicker fibers. Excitation and blocking thresholds for different pulse types, phase durations, axon depths, axon-electrode distances, temperatures and axon diameters are investigated. The used model consists of a 50mm long axon and a centrally located needle electrode in a layered medium representing the auricle. Neuronal excitation is simulated using the Frankenhaeuser-Huxley equations for all combinations of parameter values. Multiple modes and locations of excitation along the axon were observed, depending on the pulse type and amplitude. When increasing the axon-electrode distance from 1mm to 2mm, sensitivity of thresholds to axon depth decreased with ca. 50%, while sensitivity to axon-electrode distance, axon diameter and phase duration each increased with ca. 15% to 20%, except from monophasic anodal pulses, showing a 45% decrease for axon-electrode distance. These trends for axon diameter and axon-electrode distance allow for more selective stimulation of thicker target fibers using monophasic anodal pulses at higher axon-electrode distances. Cathodal monophasic pulses did not perform well due to blocking of the thicker fibers, which was only rarely seen for other pulse types. Sensitivities of stimulation thresholds to these parameters by numerical simulation reveal how the stimulation parameters can be changed in order to increase therapeutic effect and comfort during pVNS by enabling more selective stimulation.

Dendritic Degeneration of Human Auditory Nerve Fibers and Its Impact on the Spiking Pattern Under Regular Conditions and During Cochlear Implant Stimulation.

Due to limitations of human in vivo studies, detailed computational models enable understanding the neural signaling in the degenerated auditory system and cochlear implants (CIs). Four human cochleae were used to quantify hearing levels depending on dendritic changes in diameter and myelination thickness from type I of the auditory nerve fibers (ANFs). Type I neurons transmit the auditory information as spiking pattern from the inner hair cells (IHCs) to the cochlear nucleus. The impact of dendrite diameter and degree of myelination on neural signal transmission was simulated for (1) synaptic excitation via IHCs and (2) stimulation from CI electrodes. An accurate three-dimensional human cochlear geometry, along with 30 auditory pathways, mimicked the CI environment. The excitation properties of electrical potential distribution induced by two CI were analyzed. Main findings: (1) The unimodal distribution of control dendrite diameters becomes multimodal for hearing loss cases; a group of thin dendrites with diameters between 0.3 and 1 μm with a peak at 0.5 μm appeared. (2) Postsynaptic currents from IHCs excite such thin dendrites easier and earlier than under control conditions. However, this advantage is lost as their conduction velocity decreases proportionally with the diameter and causes increased spike latency and jitter in soma and axon. Firing probability reduces through the soma passage due to the low intracellular current flow in thin dendrites during spiking. (3) Compared with dendrite diameter, variations in myelin thickness have a small impact on spiking performance. (4) Contrary to synaptic excitation, CIs cause several spike initiation sites in dendrite, soma region, and axon; moreover, fiber excitability reduces with fiber diameter. In a few cases, where weak stimuli elicit spikes of a target neuron (TN) in the axon, dendrite diameter reduction has no effect. However, in many cases, a spike in a TN is first initiated in the dendrite, and consequently, dendrite degeneration demands an increase in threshold currents. (5) Threshold currents of a TN and co-stimulation of degenerated ANFs in other frequency regions depend on the electrode position, including its distance to the outer wall, the cochlear turn, and the three-dimensional pathway of the TN.

Arrangement of Excitatory Synaptic Inputs on Dendrites of the Medial Superior Olive.

Neurons in the medial superior olive (MSO) detect 10 µs differences in the arrival times of a sound at the two ears. Such acuity requires exquisitely precise integration of binaural synaptic inputs. There is substantial understanding of how neuronal phase locking of afferent MSO structures, and MSO membrane biophysics subserve such high precision. However, we still lack insight into how the entirety of excitatory inputs is integrated along the MSO dendrite under sound stimulation. To understand how the dendrite integrates excitatory inputs as a whole, we combined anatomic quantifications of the afferent innervation in gerbils of both sexes with computational modeling of a single cell. We present anatomic data from confocal and transmission electron microscopy showing that single afferent fibers follow a single dendrite mostly up to the soma and contact it at multiple (median 4) synaptic sites, each containing multiple independent active zones (the overall density of active zones is estimated as 1.375 per μm2). Thus, any presynaptic action potential may elicit temporally highly coordinated synaptic vesicle release at tens of active zones, thereby achieving secure transmission. Computer simulations suggest that such an anatomic arrangement boosts the amplitude and sharpens the time course of excitatory postsynaptic potentials by reducing current sinks and more efficiently recruiting subthreshold potassium channels. Both effects improve binaural coincidence detection compared with single large synapses at the soma. Our anatomic data further allow for estimation of a lower bound of 7 and an upper bound of 70 excitatory fibers per dendrite.SIGNIFICANCE STATEMENT Passive dendritic propagation attenuates the amplitude of postsynaptic potentials and widens their temporal spread. Neurons in the medial superior olive, with their large bilateral dendrites, however, can detect coincidence of binaural auditory inputs with submillisecond precision, a computation that is in stark contrast to passive dendritic processing. Here, we show that dendrites can counteract amplitude attenuation and even decrease the temporal spread of postsynaptic potentials, if active subthreshold potassium conductances are triggered in temporal coordination along the whole dendrite. Our anatomic finding that axons run in parallel to the dendrites and make multiple synaptic contacts support such coordination since incoming action potentials would depolarize the dendrite at multiple sites within a brief time interval.

Laser diode to single-mode graded index fiber coupling via cylindrical microlens on the fiber tip: evaluation of coupling efficiency by ABCD matrix formalism

Abstract We use ABCD matrix formalism for estimation of the coupling optics involving laser diode to cylindrical lensed circular core graded index fiber excitation. The investigation has been carried on for some typical step, parabolic, and triangular index fibers as examples of graded index fiber. It is relevant to mention in this connection that we have shown that the coupling optics is excellent along the vertical plane only while the coupling along the horizontal plane produces poor performance. Analytic expressions for coupling efficiencies are prescribed. The investigations have been made for two commonly used wavelengths, namely 1.3 and 1.5 µm. Our formulations require little computation for the necessary execution. The wavelength of 1.3 µm has been found to be more efficient in respect of coupling. Our formalism takes care of the limited aperture allowed by the cylindrical lens and thus provides the realistic prediction of coupling optics. The results will prove extremely useful to the designers and packagers working with cylindrical microlens.

LGI1 downregulation increases neuronal circuit excitability.

Leucine-rich glioma-inactivated 1 (LGI1) is a secreted transsynaptic protein that interacts presynaptically with Kv1.1 potassium channels and a disintegrin and metalloprotease (ADAM) protein 23, and postsynaptically influences α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors through a direct link with the ADAM22 cell adhesion protein. Haploinsufficiency of LGI1 or autoantibodies directed against LGI1 are associated with human epilepsy, generating the hypothesis that a subacute reduction of LGI1 is sufficient to increase network excitability. We tested this hypothesis in ex vivo hippocampal slices and in neuronal cultures, by subacutely reducing LGI1 expression with shRNA. Injection of shRNA-LGI1 in the hippocampus increased dentate granule cell excitability and low-frequency facilitation of mossy fibers to CA3 pyramidal cell neurotransmission. Application of the Kv1 family blocker, α-dendrotoxin, occluded this effect, implicating the involvement of Kv1.1. This subacute reduction of LGI1 was also sufficient to increase neuronal network activity in neuronal primary culture. These results indicate that a subacute reduction in LGI1 potentiates neuronal excitability and short-term synaptic plasticity, and increases neuronal network excitability, opening new avenues for the treatment of limbic encephalitis and temporal lobe epilepsies.

Small fiber pathology in autism and clinical implications.

To investigate small fiber innervation of the skin and its relationships with clinicometry of autism and peripheral afferents for contact heat-evoked potential (CHEP) and psychophysical measures of thermal thresholds. We recruited 32 men with autism (26.5 ± 5.9 years) and conducted small fiber assessments of skin biopsy with quantifying intraepidermal nerve fiber (IENF) density, CHEP, quantitative sensory testing, and large fiber physiology of nerve conduction studies. Results were compared with age-matched controls and analyzed with clinical measures of autism. Patients with autism showed a lower IENF density than controls (5.53 ± 2.09 vs 11.13 ± 3.49 fibers/mm, p < 0.0001). The IENF density was reduced in 17 (53.1%) men with autism classified as skin denervation group. On psychophysics, 9 (28%) men with autism had elevated thermal thresholds, and the warm threshold of the big toe was negatively correlated with IENF density (p = 0.0073), indicating functional impairments of small fiber sensory nerves. IENF density was negatively correlated with CHEP amplitude in autism (p = 0.003), in contrast to the pattern of positive correlation in controls, indicating different processing of nociceptive afferent in autism. Clinically, IENF density was related to distinct tactile symptom patterns in the skin denervation vs normal innervation group, respectively. Furthermore, IENF density was associated with autistic symptoms measured by the Autism Spectrum Quotient in a U-shaped model (p = 0.014). These observations indicated that a substantial portion of individuals with autism had small fiber pathology, which was associated with tactile and autistic symptoms, providing structural and physiologic evidence for the involvement of peripheral sensory nerves in autism.

Purinergic Modulation of Activity in the Developing Auditory Pathway.

Purinergic P2 receptors, activated by endogenous ATP, are prominently expressed on neuronal and non-neuronal cells during development of the auditory periphery and central auditory neurons. In the mature cochlea, extracellular ATP contributes to ion homeostasis, and has a protective function against noise exposure. Here, we focus on the modulation of activity by extracellular ATP during early postnatal development of the lower auditory pathway. In mammals, spontaneous patterned activity is conveyed along afferent auditory pathways before the onset of acoustically evoked signal processing. During this critical developmental period, inner hair cells fire bursts of action potentials that are believed to provide a developmental code for synaptic maturation and refinement of auditory circuits, thereby establishing a precise tonotopic organization. Endogenous ATP-release triggers such patterned activity by raising the extracellular K+ concentration and contributes to firing by increasing the excitability of auditory nerve fibers, spiral ganglion neurons, and specific neuron types within the auditory brainstem, through the activation of diverse P2 receptors. We review recent studies that provide new models on the contribution of purinergic signaling to early development of the afferent auditory pathway. Further, we discuss potential future directions of purinergic research in the auditory system.

MUSCLE FUNCTION & HOMEOSTASIS / MOLECULAR THERAPEUTIC APPROACHES: P.73 Depletion of ATP limits membrane excitability of skeletal muscle by increasing both ClC1-open probability and membrane conductance

Activation of skeletal muscle contractions require that action potentials can be excited and propagated along the muscle fibers. It is known that muscle fiber excitability is regulated during repeated firing of action potentials by cellular signalling systems that control the function of ion channels that determine the resting membrane conductance (Gm). In fast-twitch muscle, prolonged firing of action potentials triggers a marked increase in Gm, reducing muscle fiber excitability and causing action potential failure.

γ-Aminobutyric acid (GABA) from satellite glial cells tonically depresses the excitability of primary afferent fibers

Primary afferent fibers express extrasynaptic GABAA and GABAB receptors in the axons and soma. However, whether these receptors are tonically activated by ambient GABA and the source of the neurotransmitter is presently unknown. Here, we show that GABA release from dorsal root ganglia (DRG) does not depend on extracellular calcium, but depends upon calcium released from intracellular stores, and is mediated by Best1 channels. Using a preparation consisting of the spinal nerve in continuity with the DRG and the dorsal root, we found that endogenous GABA tonically activates GABA receptors, depressing the excitability of the primary afferents. In addition, using HPLC we found that GABA is released in the DRG, and by immunofluorescence microscopy we show the presence of GABA, the Best1 channel, and some enzymes of the putrescine pathway of GABA biosynthesis, in glutamine synthase- and GFAP-positive satellite glial cells. Last, we found that the blockade of the Best1 channel activity reduced the excitability of primary afferents and prevented the activation of the GABA receptors. These results suggest that satellite glial cells may be the source of endogenous GABA released in the DRG via Best1 channels, which tonically activates extrasynaptic GABA receptors.

Pain-related behavior is associated with increased joint innervation, ipsilateral dorsal horn gliosis, and dorsal root ganglia activating transcription factor 3 expression in a rat ankle joint model of osteoarthritis.

Osteoarthritis (OA)-associated pain is often poorly managed, as our understanding of the underlying pain mechanisms remains limited. The known variability from patient to patient in pain control could be a consequence of a neuropathic component in OA. We used a rat monoiodoacetate model of the ankle joint to study the time-course of the development of pain-related behavior and pathological changes in the joint, dorsal root ganglia (DRG), and spinal cord, and to investigate drug treatments effects. Mechanical hypersensitivity and loss of mobility (as assessed by treadmill) were detected from 4 weeks after monoiodoacetate. Cold allodynia was detected from 5 weeks. Using histology and x-ray microtomography, we confirmed significant cartilage and bone degeneration at 5 and 10 weeks. We detected increased nociceptive peptidergic and sympathetic fiber innervation in the subchondral bone and synovium at 5 and 10 weeks. Sympathetic blockade at 5 weeks reduced pain-related behavior. At 5 weeks, we observed, ipsilaterally only, DRG neurons expressing anti-activating transcription factor 3, a neuronal stress marker. In the spinal cord, there was microgliosis at 5 and 10 weeks, and astrocytosis at 10 weeks only. Inhibition of glia at 5 weeks with minocycline and fluorocitrate alleviated mechanical allodynia. Besides a detailed time-course of pathology in this OA model, we show evidence of contributions of the sympathetic nervous system and dorsal horn glia to pain mechanisms. In addition, late activating transcription factor 3 expression in the DRG that coincides with these changes provides evidence in support of a neuropathic component in OA pain.

Divalent cations permeation in a Ca2+ non-conducting skeletal muscle dihydropyridine receptor mouse model

In response to excitation of skeletal muscle fibers, trains of action potentials induce changes in the configuration of the dihydropyridine receptor (DHPR) anchored in the tubular membrane which opens the Ca2+ release channel in the sarcoplasmic reticulum membrane. The DHPR also functions as a voltage-gated Ca2+ channel that conducts L-type Ca2+ currents routinely recorded in mammalian muscle fibers, which role was debated for more than four decades. Recently, to allow a closer look into the role of DHPR Ca2+ influx in mammalian muscle, a knock-in (ki) mouse model (ncDHPR) carrying mutation N617D (adjacent to domain II selectivity filter E) in the DHPRα1S subunit abolishing Ca2+ permeation through the channel was generated [Dayal et al., 2017]. In the present study, the Mn2+ quenching technique was initially intended to be used on voltage-clamped muscle fibers from this mouse to determine whether Ca2+ influx through a pathway distinct from DHPR may occur to compensate for the absence of DHPR Ca2+ influx. Surprisingly, while N617D DHPR muscle fibers of the ki mouse do not conduct Ca2+, Mn2+ entry and subsequent quenching did occur because Mn2+ was able to permeate and produce L-type currents through N617D DHPR. N617D DHPR was also found to conduct Ba2+ and Ba2+ currents were strongly blocked by external Ca2+. Ba2+ permeation was smaller, current kinetics slower and Ca2+ block more potent than in wild-type DHPR. These results indicate that residue N617 when replaced by the negatively charged residue D is suitably located at entrance of the pore to trap external Ca2+ impeding in this way permeation. Because Ba2+ binds with lower affinity to D, Ba2+ currents occur, but with reduced amplitudes as compared to Ba2+ currents through wild-type channels. We conclude that mutations located outside the selectivity filter influence channel permeation and possibly channel gating in a fully differentiated skeletal muscle environment.

Function study of vasoactive intestinal peptide on chick embryonic bone development

Embryonic bone development is a complicated procedure and modulated by neuro-osteogenic interaction. Vasoactive intestinal peptide (VIP) was first identified as a neural vasodilator and further proved to possess multiple biological functions such as neurotransmitter and immune regulator. However, as a key peptide regulator presented in skeletal nerve fibers, the function of VIP on innervation and early bone development regulation has not fully been uncovered yet. In this study, the chick embryo has been used as an experimental model to address the effect of VIP on embryonic bone development. Our study results confirmed the innervation of peripheral nerve fibers into limb bone tissue, which was revealed by the detection of neurofilament (NF) and class III β-tubulin (TUJ-1) in bone tissue at various developing stages. The VIP mRNA and peptide expression level in bone tissue were also increased upon innervation progress. A chick embryonic chemical sympathectomy model was constructed by exposing chick embryos with neurotoxin 6-OHDA. The 6-OHDA exposure of the early chick embryo caused the reduction of neural crest formation and NF expression. 6-OHDA treatment also inhibited distal limb bone development as well as VIP expression. Furthermore, co-application of VIP with 6-OHDA exposure could rescue the inhibited osteogenesis activity and delayed bone development during embryogenesis. Taken together, these results reveal that VIP played an important role during innervation at early stage of bone development. VIP could restore chemical sympathectomy induced osteogenesis inhibition and bone development impair in chick embryos.

Unravelling the temporal properties of human eCAPs through an iterative deconvolution model

ObjectiveThe electrically evoked compound action potential (eCAP) has been widely studied for its clinical value in evaluating cochlear implants (CIs). However, to date, single-fiber recordings have not been recorded from the human auditory nerve, and many unknowns remain about the firing properties that underlie the eCAP in patients with CIs. In particular, the temporal properties of auditory nerve fiber firing might contain valuable information that may be used to estimate the condition of the surviving auditory nerve fibers. This study aimed to evaluate the temporal properties of neural firing underlying human eCAPs with a new deconvolution model. DesignAssuming that each auditory nerve fiber produces the same unitary response (UR), the eCAP can be seen as a convolution of a UR with a compound discharge latency distribution (CDLD). We developed an iterative deconvolution model that derived a two-component Gaussian CDLD and a UR from recorded eCAPs. The choices were based on a deconvolution fitting error minimization routine (DMR). The DMR iteratively minimized the error between the recorded human eCAPs and the eCAPs simulated by the convolution of a parameterised UR and CDLD model (instead of directly deconvolving recorded eCAPs). Our new deconvolution model included two separate steps. In step one, the underlying URs of all eCAPs were derived, and the average of these URs was called the human UR. In step two, the CDLD was obtained by using the DMR in combination with the estimated human UR. With this model, we investigated the temporal firing properties of eCAPs by analysing the CDLDs, including the amplitudes, widths, peak latencies, and areas of CDLDs. The differences of the temporal properties in eCAPs between children and adults were explored. Finally, we validated the two-Gaussian component CDLD model with a multiple-Gaussian component CDLD model. ResultsThe estimated human UR contained a sharper, narrower negative component and a wider positive phase, compared to the previously described guinea pig UR. Furthermore, the eCAPs from humans could be predicted by the convolution of the human UR with a two-Gaussian component CDLD. The areas under CDLD (AUCD) reflected the number of excited nerve fibers over time. Both the CDLD magnitudes and AUCDs were significantly correlated with the eCAP amplitudes. Furthermore, different eCAPs with the same amplitude could lead to greatly different AUCDs. Significant differences of the temporal properties of eCAPs between children and adults were found. At last, the two-Gaussian component CDLD model was validated as the most optimal CDLD model. ConclusionThis study described an iterative method that deconvolved human eCAPs into CDLDs, under the assumption that auditory nerve fibers had the same electrically evoked UR. Based on human eCAPs, we found a human UR that was different from the guinea pig UR. Furthermore, we found that CDLD characteristics revealed age-related temporal differences between human eCAPs. This temporal information may contain valuable clinical information on the survival and function of auditory nerve fibers. In turn, the surviving nerve condition might have prognostic value for speech outcomes in patients with CIs.