Electrophysiological and immunocytochemical characterization of DRG neurons on an organosilane surface in serum-free medium

We are attempting to recreate a stretch reflex circuit on a patterned Bio-MEMS (bio-microelectromechanical systems) chip with deflecting micro-cantilevers. The first steps to recreate this system is to be able to grow individual components of the circuit (sensory neuron, motoneuron, skeletal muscle, and muscle spindle) on a patternable, synthetic substrate coating the MEMS device. Sensory neurons represent the afferent portion of the stretch reflex arc and also play a significant role in transmitting the signal from the muscle spindle to the spinal cord motoneurons. We have utilized a synthetic silane substrate N-1[3-(trimethoxysilyl) propyl) diethylenetriamine (DETA) on which to grow and pattern the cells. DETA forms a self-assembled monolayer on a variety of silicon substrates, including glass, and can be patterned using photolithography. In this paper, we have evaluated the growth of sensory neurons on this synthetic silane substrate. We have investigated the immunocytochemical and electrophysiological properties of the sensory neurons on DETA and compared the resultant properties with a biological control substrate (ornithine/laminin). Immunocytochemical studies revealed the survival and growth of all three subtypes of sensory neurons: trkA, trkB, and trkC on both surfaces. Furthermore, whole-cell patch clamp recordings were used to study the electrophysiological properties of the sensory neurons on the two surfaces. There were no significant differences in the electrical properties of the neurons grown on either surface. This is the first study analyzing the immunocytochemical and electrophysiological properties of sensory neurons grown long-term in a completely defined environment and on a nonbiological substrate.     

Tremor and other oscillations in neuromuscular systems

A model has been analyzed which is based on recent experimental evidence concerning the properties of muscles and the sensory feedback pathways from muscles. Damped oscillations can arise in the absence of sensory feedback due to the interaction of a muscle with inertial loads. These mechanical oscillations can have a wide range of frequencies depending on the inertial and elastic loads that are attached to the muscle. Small amounts of sensory feedback will tend to reduce deviations from a steady muscle length, but larger amounts of feedback can produce oscillations. The frequency of these reflex oscillations is determined by the properties of the muscle and feedback pathway, and is rather independent of load. If the strength of the sensory feedback is sufficient, either the mechanical oscillations or the reflex oscillations or both can grow, rather than decay, with time. The growth of these oscillations is limited by saturation non-linearities in the muscle receptors and the muscle itself, so that the oscillations approach a steady amplitude and frequency. Using typical properties of muscles and spinal reflex pathways, the frequency of reflex oscillations will be within the range 8–12 Hz found for physiological tremor. With the longer latency found for supraspinal reflexes, oscillations will occur in the range 4–6 Hz which is characteristic of Parkinson's and cerebellar diseases. The role of longer latency reflexes in the generation of these tremors is discussed.     

The use of hormonally induced mosaics to study alterations in the synaptic connections made by persistent sensory neurons during insect metamorphosis

During the initial phase of metamorphosis in the hawkmoth, Manduca sexta, persistent mechanosensory neurons expand their terminal arborizations within the CNS and evoke a reflex response in the pupa which is different than in the larva. In an effort to determine the contribution of sensory neuron modifications to the difference in reflex responses, manipulations of juvenile hormone and 20-hydroxyecdysone were used to generate mosaic animals in which the sensory neurons were advanced or delayed developmentally with respect to the rest of the animal, including circuit components within the CNS. In the larval stage electrical stimulation of the sensory axons evokes a slow depolarization and a prolonged burst of action potentials in the ipsilateral intersegmental muscle motor neurons. By contrast, in pupal preparations the same motor neurons respond to an identical stimulus with a larger, more rapid depolarization which leads to a relatively brief, high-frequency burst of action potentials. Motor responses on the contralateral side of the body are also altered during pupal development. In mosaic animals where larval-like sensory neurons interact with a pupal CNS, a larval reflex response is generated. In the converse situation, pupal-like sensory neurons interacting with a larval or prepupal CNS evoke a motor response that is typical of larvae or prepupae. We conclude, therefore, that pupal development of the sensory neurons is necessary, but not sufficient, for the production of the pupal reflex.     

Specification of synaptic connections between sensory and motor neurons in the developing spinal cord

Experimental studies of mechanisms underlying the specification of synaptic connections in the monosynaptic stretch reflex of frogs and chicks are described. Sensory neurons innervating the triceps brachii muscles of bullfrogs are born throughout the period of sensory neurogenesis and do not appear to be related clonally. Instead, the peripheral targets of these sensory neurons play a major role in determining their central connections with motoneurons. Developing thoracic sensory neurons made to project to novel targets in the forelimb project into the brachial spinal cord, which they normally never do. Moreover, these foreign sensory neurons make monosynaptic excitatory connections with the now functionally appropriate brachial motoneurons. Normal patterns of neuronal activity are not necessary for the formation of specific central connections. Neuromuscular blockade of developing chick embryos with curare during the period of synaptogenesis still results in the formation of correct sensory-motor connections. Competitive interactions among the afferent fibers also do not seem to be important in this process. When the number of sensory neurons projecting to the forelimb is drastically reduced during development, each afferent still makes central connections of the same strength and specificity as normal. These results are discussed with reference to the development of retinal ganglion cells and their projections to the brain. Although many aspects of the two systems are similar, patterned neural activity appears to play a much more important role in the development of the visual pathway than in the spinal reflex pathway described here.     

Afferent feedback in the control of human gait.

Sensory activity contributes to motor control in two fundamentally different ways. It may mediate 'error signals' following sudden external perturbations and it may contribute to the pre-programmed motoneuronal drive. Here we review data, which illustrate these two functions of sensory feedback in relation to human walking. When ankle plantarflexors are unloaded in the stance phase there is a sudden decrease in the sensory activity in muscle and tendon afferents from the active muscles. This decrease in sensory activity results in a drop in EMG activity recorded from the soleus muscle, which demonstrates that the sensory activity contributes importantly to the activation of the muscles. Data suggests that a spinal pathway from gr. II muscle afferents is responsible for this positive feedback contribution to the motoneuronal drive during walking.When cutaneous nerves from the foot are stimulated in the early swing phase of walking a late reflex response may be observed in the tibialis anterior muscle. This reflex may help to ensure that the foot is lifted effectively over an obstacle. Data suggest that this reflex response is at least partly mediated by a transcortical reflex pathway. It seems to be important that reactions to external perturbations are integrated at a supraspinal level during human walking.     

From neuron to behavior: Sensory‐motor coordination of zebrafish turning behavior

Recent development of optogenetics brought non‐invasive neural activation in living organisms. Transparent zebrafish larva is one of the suitable animal models for this technique, which enables us to investigate neural circuits for behaviors based on a whole individual nervous system. In this article we review our recent finding that suggests sensory‐motor coordination in larval zebrafish escape behavior. When water vibration stimulates mechanosensory Rohon‐Beard (RB) neurons, intra‐spinal reflex circuit launches contralateral trunk muscle contraction that makes rapid body curvature for turning. In addition, positional information of the stimulus is conveyed to supra‐spinal circuits, and then regulates the curvature strength for appropriate escape pathway from the threat. Sensory‐motor coordination is a fundamental feature to adapt behaviors to environment, and zebrafish larvae would be an excellent model for elucidating its neural backbones.     

Relating reflex gain modulation in posture control to underlying neural network properties using a neuromusculoskeletal model

During posture control, reflexive feedback allows humans to efficiently compensate for unpredictable mechanical disturbances. Although reflexes are involuntary, humans can adapt their reflexive settings to the characteristics of the disturbances. Reflex modulation is commonly studied by determining reflex gains: a set of parameters that quantify the contributions of Ia, Ib and II afferents to mechanical joint behavior. Many mechanisms, like presynaptic inhibition and fusimotor drive, can account for reflex gain modulations. The goal of this study was to investigate the effects of underlying neural and sensory mechanisms on mechanical joint behavior. A neuromusculoskeletal model was built, in which a pair of muscles actuated a limb, while being controlled by a model of 2,298 spiking neurons in six pairs of spinal populations. Identical to experiments, the endpoint of the limb was disturbed with force perturbations. System identification was used to quantify the control behavior with reflex gains. A sensitivity analysis was then performed on the neuromusculoskeletal model, determining the influence of the neural, sensory and synaptic parameters on the joint dynamics. The results showed that the lumped reflex gains positively correlate to their most direct neural substrates: the velocity gain with Ia afferent velocity feedback, the positional gain with muscle stretch over II afferents and the force feedback gain with Ib afferent feedback. However, position feedback and force feedback gains show strong interactions with other neural and sensory properties. These results give important insights in the effects of neural properties on joint dynamics and in the identifiability of reflex gains in experiments.     

An Approach for Adaptive Limbless Locomotion Using a CPG-Based Reflex Mechanism

Animals＇ free movement in natural environments has attracted many researchers to explore control methods for bio-inspired robots. This paper presents a novel reflex mechanism based on a Central Pattern Generator （CPG） for adaptive locomotion of limbless robots. First, inspired by the concept of reflex arc, the reflex mechanism is designed on a connectionist CPG model. Since the CPG model inspired by the spinal cord of lampreys is developed at the neuron level, it provides a possible natural solution for sensory reflex integration. Therefore, sensory neurons that bridge the external stimuli and the CPG model, together with the concept of reflex arc, are utilized for designing the sensory reflex mechanism. Then, a border reflex and a body reflex are further developed and applied on the ends and the middle part of a limbless robot, respectively. Finally, a ball hitting scenario and a corridor passing scenario are designed to verify the proposed method. Results of simulations and on-site experiments show the feasibility and effectiveness of the reflex mechanism in realizing fast response and adaptive limbless locomotion.     

Tachykinins as modulators of the micturition reflex in the central and peripheral nervous system

In the normal urinary bladder, tachykinins (TKs) are expressed in a population of bladder nociceptors that is sensitive to the excitatory and desensitizing effects of capsaicin (i.e., capsaicin-sensitive primary afferent neurons (CSPANs)). Several endobiotics or xenobiotics excite CSPANs and release TKs and other mediators at both the peripheral and spinal cord level. The peripheral release of TKs determines a set of responses (known as neurogenic inflammation) that includes vasodilatation, plasma protein extravasation, smooth muscle contraction and stimulation of afferent nerves. Following chronic inflammation, both immune cells and capsaicin-resistant sensory neurons can de novo express TKs: whether these pools of TKs are releasable and contribute to inflammatory processes is presently unsettled. At the spinal cord level, the release of TKs contributes in determining an altered pattern of vesicourethral reflexes in response to nociceptive stimulation of the bladder by conveying: (a) the afferent transmission to supraspinal sites, and (b) descending or sensory inputs to the sacral parasympathetic nucleus (SPN). Recent evidence also attribute a synergetic role of TKs in the supraspinal modulation of the sensory arm of the micturition reflex.The overall available information suggests that TK receptor antagonists may affect bladder motility/reflexes which occur during different pathological states, while having little influence on the normal motor bladder function.     

Responses of efferent octopaminergic thoracic unpaired median neurons in the locust to visual and mechanosensory signals

Insect thoracic ganglia contain efferent octopaminergic unpaired median neurons (UM neurons) located in the midline, projecting bilaterally and modulating neuromuscular transmission, muscle contraction kinetics, sensory sensitivity and muscle metabolism. In locusts, these neurons are located dorsally or ventrally (DUM- or VUM-neurons) and divided into functionally different sub-populations activated during different motor tasks. This study addresses the responsiveness of locust thoracic DUM neurons to various sensory stimuli. Two classes of sense organs, cuticular exteroreceptor mechanosensilla (tactile hairs and campaniform sensilla), and photoreceptors (compound eyes and ocelli) elicited excitatory reflex responses. Chordotonal organ joint receptors caused no responses. The tympanal organ (Müller's organ) elicited weak excitatory responses most likely via generally increased network activity due to increased arousal. Vibratory stimuli to the hind leg subgenual organ never elicited responses. Whereas DUM neurons innervating wing muscles are not very responsive to sensory stimulation, those innervating leg and other muscles are very responsive to stimulation of exteroreceptors and hardly responsive to stimulation of proprioceptors. After cutting both cervical connectives all mechanosensory excitation is lost, even for sensory inputs from the abdomen. This suggests that, in contrast to motor neurons, the sensory inputs to octopaminergic efferent neuromodulatory cells are pre-processed in the suboesophageal ganglion.     

Expression of the Immunoglobulin Superfamily Cell Adhesion Molecules in the Developing Spinal Cord and Dorsal Root Ganglion

Cell adhesion molecules belonging to the immunoglobulin superfamily (IgSF) control synaptic specificity through hetero- or homophilic interactions in different regions of the nervous system. In the developing spinal cord, monosynaptic connections of exquisite specificity form between proprioceptive sensory neurons and motor neurons, however, it is not known whether IgSF molecules participate in regulating this process. To determine whether IgSF molecules influence the establishment of synaptic specificity in sensory-motor circuits, we examined the expression of 157 IgSF genes in the developing dorsal root ganglion (DRG) and spinal cord by in situ hybridization assays. We find that many IgSF genes are expressed by sensory and motor neurons in the mouse developing DRG and spinal cord. For instance, Alcam, Mcam, and Ocam are expressed by a subset of motor neurons in the ventral spinal cord. Further analyses show that Ocam is expressed by obturator but not quadriceps motor neurons, suggesting that Ocam may regulate sensory-motor specificity in these sensory-motor reflex arcs. Electrophysiological analysis shows no obvious defects in synaptic specificity of monosynaptic sensory-motor connections involving obturator and quadriceps motor neurons in Ocam mutant mice. Since a subset of Ocam⁺ motor neurons also express Alcam, Alcam or other functionally redundant IgSF molecules may compensate for Ocam in controlling sensory-motor specificity. Taken together, these results reveal that IgSF molecules are broadly expressed by sensory and motor neurons during development, and that Ocam and other IgSF molecules may have redundant functions in controlling the specificity of sensory-motor circuits.     

Spinal and supraspinal effects of activity in ligament afferents.

In this paper available knowledge on effects from joint and ligament afferents on spinal neurones and pathways are briefly reviewed, and possible functional implications discussed. Ligament afferents may contribute to joint stability, muscle coordination and proprioception through direct polysynaptic reflex effects onto ascending pathways and skeletomotoneurones, and/or indirectly via reflex actions on the gamma-muscle spindle system. Theoretical and experimental evidence indicate that ligament afferents, together with afferents from other joint structures, muscles and the skin, provide the CNS with information on movements and posture through ensemble coding mechanisms, rather than via modality specific private pathways. The existence and functional relevance of ligamentomuscular protective reflexes, that are triggered when the ligament is threatened by potentially harmful loads, has been seriously questioned. It seems more likely that peripheral sensory inputs from ligament afferents participate in a continuous control of the muscle activity through feedforward, or preprogramming, mechanisms. In line with these ideas it has been suggested that ligament mechanoreceptors have an important role in muscle coordination and in the reflex regulation of the functional joint stability, by contributing to the preprogramming of the muscle stiffness through reflex modulation of the gamma-muscle spindle system.     

Effects of different vibration frequencies on spinal cord reflex circuits and thermoalgesic perception

Objectives:We studied the effect of different vibration frequencies on spinal cord excitability and heat pain perception. We hypothesized that the effects of vibration on spinal cord reflexes, and, also those on heat pain perception, depend on vibration frequency.Methods:In 9 healthy subjects, we applied vibration over the tibialis anterior muscle at three different frequencies (50, 150, or 250 Hz) on spinal cord reflex excitably, tested with the H reflex and the T wave in the soleus muscle, as well as on sensory and pain perception, tested by measuring warm perception (WT) and heat pain perception thresholds, (HPT) in sites rostral and caudal to vibration. Exams were carried out before, during, and after vibration.Results:The amplitude of the H reflex and T wave significantly decreased during vibration in comparison to baseline. Low frequencies (50 and 150Hz) induced greater reflex suppression than high frequency (250Hz). No significant changes were observed on WT and HPT.Conclusions:The effects of vibratory stimulation can be summarized as frequency-related suppression of the spinal cord excitability without an effect on warm and heat pain perception. The present results may help to design vibration-related interventions intended to diminish spinal cord reflex excitability in spastic patients.     

Experimentally induced ulcers and gastric sensory-motor function in rats

Prior studies have demonstrated that inflammation can sensitize visceral afferent neurons, contributing to the development of hyperalgesia. We hypothesized that both afferent and efferent pathways are affected, resulting in changes in motor and sensory function. Kissing ulcers (KU) were induced in the distal stomach by injecting 60% acetic acid for 45 s into a clamped area of the stomach. In controls, saline was injected into the stomach. A balloon catheter was surgically placed into the stomach, and electromyographic responses to gastric distension were recorded from the acromiotrapezius muscle at various times after ulcer induction. The accommodation reflex was assessed by slowly infusing saline into the distally occluded stomach. Gastric pressure changes in response to vagal stimulation were measured in anesthetized rats. Contractile function of circular muscle strips was examined in vitro using force-displacement transducers. KU caused gastric hypersensitivity that persisted for at least 14 days. Fluid distension of the stomach led to a rapid pressure increase in KU but not in control animals, consistent with an impaired accommodation reflex. Gastric ulcers enhanced the contractile response to vagal stimulation, whereas the effect of cholinergic stimulation on smooth muscle in vitro was not changed. These data suggest that inflammation directly alters gastric sensory and motor function. Increased activation of afferents will trigger vagovagal reflexes, thereby further changing motility and indirectly activating sensory neurons. Thus afferent and efferent pathways both contribute to the development of dyspeptic symptoms.     

Convergence of nociceptive information from temporomandibular joint and tooth pulp afferents on C1 spinal neurons in the rat

The aim of the present study was to test the hypothesis that there is a convergence of afferent inputs from the temporomandibular joint (TMJ) on C1 spinal neurons responding to electrical stimulation of the tooth pulp (TP). In 14 pentobarbital anesthetized rats, the extracellular single unit activity of 31 C1 spinal neurons and the amplitude in a digastric muscle electromyogram (n = 31) increased proportionally during 1.0-3.5 times the threshold for the jaw-opening reflex (JOR). Of 31 C1 spinal neurons responsive to TP afferents, 28 (approximately 90%) were also excited by electrical stimulation of the ipsilateral TMJ capsule. All neurons tested were divided into three categories of nociceptive specific, wide dynamic range and non-responsive as to their responsiveness to mechanical stimuli (pin prick and touch) of the somatic receptive field (skin of the face, neck, jaw and upper forearm) and TMJ capsule. Nineteen (68%) of 28 C1 spinal neurons received nociceptive information from C fibers of the TMJ capsule. These results suggest that there is a convergence of noxious information from the TMJ and TP afferents on the same C1 spinal neurons, which importantly contribute to pain perception from the TMJ region.     

Inflammation enhances reflex and spinal neuron responses to noxious visceral stimulation in rats

To improve understanding of sensory processes related to visceral inflammation, the effect of turpentine-induced inflammation on reflex (cardiovascular/visceromotor) and extracellularly recorded lumbosacral dorsal horn neuron responses to colorectal distension (CRD) was investigated. A 25% solution of turpentine, applied to the colorectal mucosa, produced inflammation, decreased compliance of the colonic wall, and enhanced reflex responses in unanesthetized rats within 2-6 h. At 24 h posttreatment, pressor responses to CRD (80 mmHg, 20 s) were 20% greater, and intraluminal pressures needed to evoke visceromotor reflexes were 30% lower than controls. Parallel electrophysiological experiments in spinal cord-transected, decerebrate rats demonstrated that two neuronal subgroups excited by CRD were differentially affected by turpentine administered 24 h before testing. During CRD, abrupt neurons were 70% less active and sustained neurons were 25% more active than similar neurons in controls. In summary, reflex and neuronal subgroup (sustained neurons) responses to CRD were both potentiated by chemical inflammation. This suggests that the neurophysiological basis for inflammation-induced increases in reflex responses to CRD is increased activity of this neuronal subgroup.