Dorsal Root Stimulation Research Articles

Synergistic interaction between sensory inputs and propriospinal signalling underlying quadrupedal locomotion.

Stimulation of hindlimb afferent fibres can both stabilize and increase the activity of fore- and hindlimb motoneurons during fictive locomotion. The increase in motoneuron activity is at least partially due to the production of doublets of action potentials in a subpopulation of motoneurons. These results were obtained using an in vitro brainstem/spinal cord preparation of neonatal rat. Quadrupedal locomotion relies on a dynamic coordination between central pattern generators (CPGs) located in the cervical and lumbar spinal cord, and controlling the fore- and hindlimbs, respectively. It is assumed that this CPG interaction is achieved through separate closed-loop processes involving propriospinal and sensory pathways. However, the functional consequences of a concomitant involvement of these different influences on the degree of coordination between the fore- and hindlimb CPGs is still largely unknown. Using an in vitro brainstem/spinal cord preparation of neonatal rat, we found that rhythmic, bilaterally alternating stimulation of hindlimb sensory input pathways elicited coordinated hindlimb and forelimb CPG activity. During pharmacologically induced fictive locomotion, lumbar dorsal root (DR) stimulation entrained and stabilized an ongoing cervico-lumbar locomotor-like rhythm and increased the amplitude of both lumbar and cervical ventral root bursting. The increase in cervical burst amplitudes was correlated with the occurrence of doublet action potential firing in a subpopulation of motoneurons, enabling the latter to transition between low and high frequency discharge according to the intensity of DR stimulation. Moreover, our data revealed that propriospinal and sensory pathways act synergistically to strengthen cervico-lumbar interactions. Indeed, split-bath experiments showed that fully coordinated cervico-lumbar fictive locomotion was induced by combining pharmacological stimulation of either the lumbar or cervical CPGs with lumbar DR stimulation. This study thus highlights the powerful interactions between sensory and propriospinal pathways which serve to ensure the coupling of the fore- and hindlimb CPGs for effective quadrupedal locomotion.

Abstract 1450: Neural communication to peripheral tumors regulates cancer cell activity

Abstract Transcriptomics and sequencing analyses of tumors from patients provide invaluable information about the cells present in the tumor microenvironment (TME); however, nerves, whose cell bodies are absent from most peripheral TMEs, have eluded such approaches. Recent emerging data highlight the functional importance of innervation in the TME and its contribution to tumor progression, metastasis, and treatment resistance. The Exoneural Platform developed at Cygnal Therapeutics allows for investigation of complex biology in the context of multi-cell culture conditions in vitro and in vivo. As part of this platform, we have developed tools for rapid and selective manipulation of different cell types with the ability to monitor multiple cell specific read-outs. Here, we used a combination of approaches including RNA-seq, bioinformatics, immunohistochemistry, and microscopy to investigate the role of synaptic function in cancer biology. Transcriptomics analyses on co-cultures revealed a direct interaction between nerves and cancer cells based on cell type specific gene expression and ligand - receptor interaction assessment. Both neurons and cancer cells showed a profound shift in their gene expression profile when cultured together. This differential gene expression was reversed if neurons were ablated, pointing to the reversibility of this change and, potentially, the disease state itself. Examination for presence and function of synaptic proteins showed that synaptic proteins, such as PSD95 and synapsin1, were expressed in co-cultures, and in many cases, a co-localization of pre - and post - synaptic markers was observed. Selective stimulation of dorsal ganglion root (DRG) neurons, co-cultured with cancer cells, resulted in a robust and acute increase of cytosolic calcium in cancer cells. Bioinformatic image analysis confirmed that this calcium influx in cancer cells directly correlated with proximity and density of neurites to cancer cells. A calcium response was not observed if DRGs and cancer cells were cultured in two separate chambers between which media can freely travel, confirming that close proximity is required for this interaction. Pharmacological and genetic knock down of key synaptic proteins resulted in changes in cancer cell calcium influx and affected proliferation and gene expression. Bioinformatics analyses, based on public and proprietary data, have identified the synaptic pathway as a major contributing node that influences cancer cell biology in the context of several cancer types. These data collectively point to the importance of exoneural biology, and more specifically, synaptic biology in cancer. More importantly, these results are likely to suggest new oncology targets that have not been identified or pursued previously. Citation Format: Monica Thanawala, Chih-Chieh Wang, Jesse G. Turner, Kai-Chih Huang, Lexiang Ji, Alison Miller, Alexandria Fink, Shan Lou, Alexandra B. Lantermann, Hongyue Dai, John A. Wagner, Grazia Piizi, Jonathan B. Hurov, Pearl Huang, Amir M. Sadaghiani. Neural communication to peripheral tumors regulates cancer cell activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1450.

Contribution of dorsal horn CGRP-expressing interneurons to mechanical sensitivity.

Primary sensory neurons are generally considered the only source of dorsal horn calcitonin gene-related peptide (CGRP), a neuropeptide critical to the transmission of pain messages. Using a tamoxifen-inducible CalcaCreER transgenic mouse, here we identified a distinct population of CGRP-expressing excitatory interneurons in lamina III of the spinal cord dorsal horn and trigeminal nucleus caudalis. These interneurons have spine-laden, dorsally directed, dendrites, and ventrally directed axons. As under resting conditions, CGRP interneurons are under tonic inhibitory control, neither innocuous nor noxious stimulation provoked significant Fos expression in these neurons. However, synchronous, electrical non-nociceptive Aβ primary afferent stimulation of dorsal roots depolarized the CGRP interneurons, consistent with their receipt of a VGLUT1 innervation. On the other hand, chemogenetic activation of the neurons produced a mechanical hypersensitivity in response to von Frey stimulation, whereas their caspase-mediated ablation led to mechanical hyposensitivity. Finally, after partial peripheral nerve injury, innocuous stimulation (brush) induced significant Fos expression in the CGRP interneurons. These findings suggest that CGRP interneurons become hyperexcitable and contribute either to ascending circuits originating in deep dorsal horn or to the reflex circuits in baseline conditions, but not in the setting of nerve injury.

Spinal GABAergic neurons are under feed-forward inhibitory control driven by Aδ and C fibers in Gad2 td-Tomato mice.

BackgroundSpinal GABAergic neurons act as a critical modulator in sensory transmission like pain or itch. The monosynaptic or polysynaptic primary afferent inputs onto GABAergic neurons, along with other interneurons or projection neurons make up the direct and feed-forward inhibitory neural circuits. Previous research indicates that spinal GABAergic neurons mainly receive excitatory inputs from Aδ and C fibers. However, whether they are controlled by other inhibitory sending signals is not well understood.MethodsWe applied a transgenic mouse line in which neurons co-expressed the GABA-synthesizing enzyme Gad65 and the enhanced red fluorescence (td-Tomato) to characterize the features of morphology and electrophysiology of GABAergic neurons. Patch-clamp whole cell recordings were used to record the evoked postsynaptic potentials of fluorescent neurons in spinal slices in response to dorsal root stimulation.ResultsWe demonstrated that GABAergic neurons not only received excitatory drive from peripheral Aβ, Aδ and C fibers, but also received inhibitory inputs driven by Aδ and C fibers. The evoked inhibitory postsynaptic potentials (eIPSPs) mediated by C fibers were mainly Glycinergic (66.7%) as well as GABAergic mixed with Glycinergic (33.3%), whereas the inhibition mediated by Aδ fibers was predominately both GABA and Glycine-dominant (57.1%), and the rest of which was purely Glycine-dominant (42.9%).ConclusionThese results indicated that spinal GABAergic inhibitory neurons are under feedforward inhibitory control driven by primary C and Aδ fibers, suggesting that this feed-forward inhibitory pathway may play an important role in balancing the excitability of GABAergic neurons in spinal dorsal horn.

Muscle responses to radicular stimulation during lumbo-sacral dorsal rhizotomy for spastic diplegia: Insights to myotome innervation

ObjectiveMost of knowledge on muscle radicular innervation was from explorations in root/spinal cord pathologies. Direct and individual access to each of the lumbar-sacral -ventral and dorsal- nerve roots during dorsal rhizotomy for spastic diplegia allows precise study of the corresponding muscle innervation. Authors report the lumbo-sacral segmental myotomal organization obtained from recordings of muscle responses to root stimulation in a 20-children prospective series. MethodsSeven key-muscles in each lower limb and anal sphincter were Electromyography (EMG)-recorded and clinically observed by physiotherapist during L2-to-S2 dorsal rhizotomy. Ventral roots (VR), for topographical mapping, and dorsal roots (DR), for segmental excitability testing, were stimulated, just above threshold for eliciting muscular response. ResultsIn 70% of the muscles studied, VR innervation was pluri-radicular, from 2-to-4 roots, with 1 or 2 roots being dominant at each level. Overlapping was important. Muscle responses to DR stimulation were 1.75 times more extended compared to VR stimulation. Inter-individual variability was important. ConclusionsAccuracy of root identification and stimulation with the used method brings some more precise information to radicular functional anatomy. SignificanceThose neurophysiological findings plead for performing Intra-Operative Neuromonitoring when dealing with surgery in the lumbar-sacral roots.

Flexor and Extensor Ankle Afferents Broadly Innervate Locomotor Spinal Shox2 Neurons and Induce Similar Effects in Neonatal Mice.

Central pattern generators (CPGs) in the thoracolumbar spinal cord generate the basic hindlimb locomotor pattern. The locomotor CPG integrates descending commands and sensory information from the periphery to activate, modulate and halt the rhythmic program. General CPG function and response to sensory perturbations are well described in cat and rat models. In mouse, roles for many genetically identified spinal interneurons have been inferred from locomotor alterations following population deletion or modulation. However, the organization of afferent input to specific genetically identified populations of spinal CPG interneurons in mouse remains comparatively less resolved. Here, we focused on a population of CPG neurons marked by the transcription factor Shox2. To directly test integration of afferent signaling by Shox2 neurons, sensory afferents were stimulated during patch clamp recordings of Shox2 neurons in isolated spinal cord preparations from neonatal mice. Shox2 neurons broadly displayed afferent-evoked currents at multiple segmental levels, particularly from caudal dorsal roots innervating distal hindlimb joints. As dorsal root stimulation may activate both flexor- and extensor-related afferents, preparations preserving peripheral nerves were used to provide more specific activation of ankle afferents. We found that both flexor- and extensor-related afferent stimulation were likely to evoke similar currents in a given Shox2 neuron, as assessed by response polarity, latency, duration and amplitude. It has been proposed that Shox2 neurons can be divided into neurons which contribute to rhythm generation and neurons that are premotor by the absence and presence of the V2a marker Chx10, respectively. Response to afferent stimulation did not differ based on Chx10 expression. Although currents evoked in response to flexor and extensor afferent activation did not follow expected functional antagonism, they were consistent with the observation that stimulation of flexor- and extensor-related afferents both reset the phase of ongoing fictive locomotion to flexion in neonatal mice. Together, the data suggest that Shox2 neurons are interposed in multiple sensory pathways and low threshold proprioceptive input reinforces sensory perturbation of ongoing locomotion by similarly activating or inhibiting both the rhythm and patterning layers of the CPG.

Recruitment gain of spinal motor neuron pools in cat and human.

The output from a motor nucleus is determined by the synaptic input to the motor neurons and their intrinsic properties. Here, we explore whether the source of synaptic inputs to the motor neurons (cats) and the age or post-stroke conditions (humans) may change the recruitment gain of the motor neuron pool. In cats, the size of Ia EPSPs in triceps surae motor neurons (input) and monosynaptic reflexes (MSRs; output) was recorded in the soleus and medial gastrocnemius motor nerves following graded stimulation of dorsal roots. The MSR was plotted against the EPSP thereby obtaining a measure of the recruitment gain. Conditioning stimulation of sural and peroneal cutaneous afferents caused significant increase in the recruitment gain of the medial gastrocnemius, but not the soleus motor neuron pool. In humans, the discharge probability of individual soleus motor units (input) and soleus H-reflexes (output) was performed. With graded stimulation of the tibial nerve, the gain of the motor neuron pool was assessed as the slope of the relation between probability of firing and the reflex size. The gain in young subjects was higher than in elderly subjects. The gain in post-stroke survivors was higher than in age-matched neurologically intact subjects. These findings provide experimental evidence that recruitment gain of a motor neuron pool contributes to the regulation of movement at the final output stage from the spinal cord and should be considered when interpreting changes in reflex excitability in relation to movement or injuries of the nervous system.

The Mechanistic Basis for Successful Spinal Cord Stimulation to Generate Steady Motor Outputs.

Electrical stimulation of the spinal cord is a promising rehabilitation intervention to restore/augment motor function after spinal cord injury (SCI). Combining sensory feedback with stimulation of remaining motor circuits has been shown to be a prerequisite for the functional improvement of SCI patients. However, little is known about the cellular mechanisms potentially underlying this functional benefit in the injured spinal cord. Here, we combine computer simulations with an isolated whole-tissue adult mouse spinal cord preparation to examine synaptic, cellular, and system potentials measured from single motoneurons and ventral roots. The stimulation protocol included separate and combined activation of the sensory inputs (evoked by dorsal root stimulation) and motor inputs (evoked by stimulation of spinal cord tissue) at different frequencies, intensities, and neuromodulatory states. Our data show that, while sensory inputs exhibit short-term depression in response to a train of stimulation, motor inputs exhibit short-term facilitation. However, the concurrent activation of both inputs elicits a stronger and steadier motor output. This effect is enhanced by the application of pharmacological neuromodulators. Furthermore, sensorimotor excitatory postsynaptic potentials (EPSPs) summate sublinearly (i.e., their combination produces an excitatory potential smaller than the sum of the excitatory potentials they would individually produce). However, ventral root compound action potentials (CoAPs) summate supralinearly generating much higher outputs. Computer simulations revealed that the contrasting summation and disproportionality in plasticity between the excitatory postsynaptic potentials (EPSPs) and CoAPs result from the motoneuronal firing threshold acting as an amplitude-selective filter. Together, these results provide the mechanistic basis for the cellular processes contributing to the generation of steady motor outputs using spinal stimulation. This data has great potential to guide the design of more effective stimulation protocols in SCI patients.

Minocycline Inhibits the Enhanced Antidromic Stimulation-induced Sensitization of C-Fibers Following Intradermal Capsaicin Injection

Background: Our previous studies indicated that retrograde signaling initiating from the spinal cord was mediated by the plasticity of Dorsal Root Ganglion (DRG) neurons. Both retrograde signaling and neuronal plasticity contributed to neurogenic inflammation, which were modulated by the activity of Satellite Glial Cells (SGCs). Thus, we want to know whether retrograde signaling is involved in the hypersensitivity of nociceptive afferents, and whether this process is affected by the plasticity of DRG neurons and glia. Objective: The study aims to examine if retrograde signaling can induce hypersensitivity of primary afferent nociceptors and if hypersensitivity involves glial modulation. Methods: Antidromic Electrical Stimulation (ES) of dorsal roots was used to mimic retrograde signaling activity. C- primary nociceptive afferent activity was recorded for testing the effect of antidromic ES. In a separate group, intradermal capsaicin injection to the ipsilateral hindpaw was used to prime DRG nociceptive neurons. For the third group, a glial inhibitor, minocycline, was pre-administered to test glial modulation in this process. Results: Antidromic ES sensitized the responses of C-fibers to nociceptive mechanical stimuli. For rats subjected to intradermal capsaicin injection, C fibers experienced more drastic sensitization induced by antidromic ES, shown as a greater response and longer duration, implying that sensitization correlates with the activation of DRG neurons. Minocycline pretreatment significantly blocked the priming effect of capsaicin on C-fiber sensitization induced by antidromic ES, indicating the involvement of SGCs in DRG neuronal sensitization. Conclusion: Retrograde signaling may be one of the important mechanisms in neurogenic inflammation-mediated nociception, and this process is subjected to satellite glial modulation.

Implication of Kv7 Channels in the Spinal Antinociceptive Actions of Celecoxib.

Celecoxib is a nonsteroidal anti-inflammatory drug (NSAID) commonly used to treat pain conditions in humans. In addition to its blocking activity on cyclooxygenase (COX) enzymes, several other targets could contribute to its analgesic activity. Here we explore the spinal antinociceptive actions of celecoxib and the potential implication of Kv7 channels in mediating its effects. Spinal cord in vitro preparations from hind paw-inflamed animals were used to assess the segmental sensory-motor and the early sensory processing of nociceptive information. Electrophysiological recordings of ventral roots and dorsal horn neurones were obtained, and the effects of celecoxib and Kv7 modulators on responses to repetitive dorsal root stimulation at C-fiber intensity were assessed. Celecoxib applied at clinically relevant concentrations produced depressant effects on responses to dorsal root stimulation recorded from both ventral roots and individual dorsal horn neurones; by contrast, the non-nociceptive monosynaptic reflex was unaffected. The NSAID indomethacin had no effect on spinal reflexes, but further coapplication of celecoxib still produced depressant effects. The depressant actions of celecoxib were abolished after Kv7 channel blockade and mimicked by its structural analog dimethyl-celecoxib, which lacks COX-blocking activity. The present results identify Kv7 channels as novel central targets for celecoxib, which may be relevant to its analgesic effect. This finding contributes to better understand the pharmacology of celecoxib and reinforces both the role of Kv7 channels in modulating the excitability of central pain pathways and its validity as target for the design of analgesics.

High-threshold primary afferent supply of spinal lamina X neurons.

The spinal gray matter region around the central canal, lamina X, is critically involved in somatosensory processing and visceral nociception. Although several classes of primary afferent fibers terminate or decussate in this area, little is known about organization and functional significance of the afferent supply of lamina X neurons. Using the hemisected ex vivo spinal cord preparation, we show that virtually all lamina X neurons receive primary afferent inputs, which are predominantly mediated by the high-threshold Aδ- fibers and C-fibers. In two-thirds of the neurons tested, the inputs were monosynaptic, implying a direct targeting of the population of lamina X neurons by the primary nociceptors. Beside the excitatory inputs, 48% of the neurons also received polysynaptic inhibitory inputs. A complex pattern of interactions between the excitatory and inhibitory components determined the output properties of the neurons, one-third of which fired spikes in response to the nociceptive dorsal root stimulation. In this respect, the spinal gray matter region around the central canal is similar to the superficial dorsal horn, the major spinal nociceptive processing area. We conclude that lamina X neurons integrate direct and indirect inputs from several types of thin primary afferent fibers and play an important role in nociception.

Peculiarities of the interneuronal pool influence on the spinal motoneurons under the conditions of prolonged hypoestrogenemia

The purpose of research is to study the peculiarities of the interneuronal networks influence on the activity of spinal anterior horns motoneurons under the conditions of experimental menopause. Materials and methods. The study was performed on 51 mature Wistar rats (females). Menopause was induced by a minimally invasive ovariohysterectomy. L5 dorsal root was stimulated by single and paired square-wave impulses. The response was recorded on the dorsal surface of the spinal cord and the ventral root at the same level. Results. An increase in the excitation threshold of the spinal cord afferent fibers and the latent period of 102.14 % and 94.12 %, respectively, were established in animals after ovariectomy. Supramaximal stimulation of the dorsal root L5 induced an increase in the amplitude of components N1 by 10.14 %, N2 by 11.82 %, N3 by 48.28 %, and P-wave by 31.58 %, and the duration of N3 component was increased by 26.54 % in the experimental group. In stimulation by paired pulses over a time interval of 2 ms to 3 ms, a significant increase was observed, and from 6 ms to 100 ms there was a suppression of the N1 component of the second PDP in the group of animals with experimental menopause. The threshold for excitation of motoneurons was decreased to 54.17 ± 19.80 %, the latent period was decreased to 85.39 ± 2.63 %, and the response amplitude was reduced to 127.59 ± 3.78 %. When paired stimuli were applied, there was delayed recovery of evoked response amplitude in the same range (2–100 ms) as in the interneuronal pool study and matching the positive component of potential duration of the spinal cord dorsal surface. Conclusions. Thus, the overall effect of the long-term estrogen deficiency is an increase in the spinal cord motoneurons excitability, facilitation of nerve transmission in the structures of the anterior and posterior horns at high-frequency of dorsal root stimulation, and marked inhibition of low-frequency input due to deepening of the presynaptic inhibition processes mediated by significantly increased activity of substantia gelatinosa neurons.

Computational Study of the Effect of Electrode Polarity on Neural Activation Related to Paresthesia Coverage in Spinal Cord Stimulation Therapy

Using computer simulation, we investigated the effect of electrode polarity on neural activation in spinal cord stimulation and propose a new strategy to maximize the activating area in the dorsal column (DC) and, thus, paresthesia coverage in clinical practice. A new three-dimensional spinal cord model at the T10 vertebral level was developed to simulate neural activation induced by the electric field distribution produced by different typical four-contact electrode polarities in single- and dual-lead stimulation. Our approach consisted of the combination of a finite element model of the spinal cord developed in COMSOL Multiphysics and a nerve fiber model implemented in MATLAB. Five evaluation parameters were evaluated, namely, the recruitment ratio, the perception and discomfort thresholds, and the activating area and depth. The results were compared quantitatively. The dual-guarded cathode presents the maximum activating area and depth in single- and dual-lead stimulation. However, the lowest value of the ratio between the perception threshold in DC and the perception threshold in the dorsal root (DR) is achieved when the guarded cathode is programmed. Although the two versions of bipolar polarity (namely bipolar 1 and bipolar 2) produce higher activating area and depth than the guarded cathode, they are suitable for producing DR stimulation. Similarly, dual-lead stimulation is likely to activate DR fibers because the electrodes are closer to these fibers. The results suggest that the activating area in the DC is maximized by using the dual-guarded cathode both in single- and dual-lead stimulation modes. However, DC nerve fibers are preferentially stimulated when the guarded cathode is used. According to these results, the new electrode programming strategy that we propose for clinical practice first uses the dual-guarded cathode, but, if the DR nerve fibers are activated, it then uses guarded cathode polarity.

(246) Determining the Role of Presynaptic Ca2+ Signaling in the Spinal Cord in Pain Sensitization

Chronic neuropathic pain affects approximately 10% of the population and only a minority of patients experience satisfactory relief of their pain with currently available pharmaceutics. As most previous reports focused primarily on the post-synaptic signaling of the first sensory synapse via patch-clamp recordings, we aim to address a gap in the current knowledge about the potential role of presynaptic Ca2+ signaling in central sensitization following neuropathic injury. Utilizing a novel ex vivo intact spinal cord preparation, a mouse line that expresses GCaMP specifically in the sensory neurons, and multiphoton microscopy to image presynaptic Ca2+ signaling, we are able to evaluate presynaptic Ca2+ signaling in the spinal cord isolated from mice that underwent either spared nerve injury (SNI) or sham surgery. Behavioral testing was used to confirm development of mechanical allodynia in SNI (but not sham) mice prior to presynaptic Ca2+ imaging. Presynaptic [Ca2+]cyt changes were induced by dorsal root stimulation, bradykinin, and capsaicin. The effects of modulatory neurotransmitters, such as dopamine, were also tested for their ability to regulate [Ca2+]cyt in central processes of sensory afferent neurons. Preliminary results quantifying frequency and duration of presynaptic [Ca2+]cyt transients indicate that neuropathic injury alters basal Ca2+ signaling and responsiveness to sensory modulators.

(225) Extrinsic Colon Afferents Influence Enteric Neuron Activity and Colon Motility by Engaging Spinal Reflexes

Abdominal pain and bowel dysmotility are hallmark symptoms of functional bowel disorders, yet it is unclear how pain and dysmotility are correlated mechanistically. Extrinsic colon afferents (EPANs) provide sensory input to the CNS, and visceral pain is thought to be due to hypersensitivity of these afferents. The enteric nervous system (ENS) acts autonomously to control motility reflexes, but extrinsic nerve pathways coordinate activity between distant regions of the GI tract, and importantly, allow the central nervous system (CNS) to regulate GI functioning. To test whether EPANs can directly influence ENS activity, we used an ex vivo colon-pelvic nerve-L6 DRG preparation, in which the roots were cut from the spinal cord, that allowed selective activation of EPANs via dorsal root stimulation and simultaneous calcium imaging (GCaMP6s) of myenteric neurons in the colon. Electrical stimulation of the dorsal root activated the majority of L6 DRG neurons, but produced no GCaMP responses in myenteric neurons of the colon in any of the visual fields tested (n=8). By contrast, ventral root stimulation led to GCaMP responses in ∼20% of myenteric neurons, followed by smooth muscle contraction, as indicated by movement of the imaging field. Interestingly, when the spinal cord was left intact, dorsal root stimulation produced responses in 12% of myenteric neurons (n=5), suggesting the presence of a sensory-parasympathetic spinal reflex. To elucidate this reflex in vivo, we used optogenetic stimulation to produce visceromotor responses and then processed spinal cord tissue for the immediate early gene, c-fos. There were significantly greater numbers of parasympathetic preganglionic neurons (PPNs) with c-fos staining in lumbosacral spinal segments from TRPV1-ChR2 mice (n=3) compared to controls (n=3), and TRPV1 central terminals were observed in close apposition to PPN. Overall, our data suggests that colon afferents influence ENS activity and colon motility by engaging parasympathetic pathways through a spinal reflex.

Involvement of Group II Metabotropic Glutamate Receptors in Modulation of Evoked Activity in Frog Spinal Motoneurons

The effect of the agonist of group II mGluRs, including mGluR2 and mGluR3 (mGluR 2/3) receptor subtypes, on evoked activity and electric membrane properties of frog spinal (lumbar) motoneurons was studied electrophysiologically on sections of the isolated spinal cord. Extracellular recordings revealed a decrease in the amplitude of short-latency components of spinal reflexes and in the overall response area under the effect of DCG-IV [2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine], a mGluR 2/3 agonist, within its concentration range of 0.05-5 μM. The half-maximal effective concentration (EC50) of the agonist for the suppression of short-latency response components was about 0.5 μM. Intracellular recordings of postsynaptic potentials from motoneurons upon DCG-IV application demonstrated a decrease in the number of spikes and the overall area of responses evoked by dorsal root stimulation. Most of motoneurons studied responded with hyperpolarization, increased amplitude of antidromic action potentials, altered afterpotential amplitude and increased excitability, indicative of the agonist effect on postsynaptic group II mGluRs in frog spinal motoneurons.

Percutaneous direct current stimulation - a new electroceutical solution for severe neurological pain and soft tissue injuries.

There is a high medical need to improve the effectiveness of the treatment of pain and traumatic soft tissue injuries. In this context, electrostimulating devices have been used with only sporadic success. There is also much evidence of endogenous electrical signals that play key roles in regulating the development and regeneration of many tissues. Transepithelial potential gradients are one source of the direct current (DC) electrical signals that stimulate and guide the migration of inflammatory cells, epithelial cells, fibroblasts and mesenchymal stem cells to achieve effective wound healing. Up to now, this electrophysiological knowledge has not been adequately translated into a clinical treatment. Here, we present a mobile, handheld electroceutical smart device based on a microcontroller, an analog front end and a battery, which generates DC electric fields (EFs), mimicking and modulating the patient’s own physiological electrical signals. The electrical stimulation is applied to percutaneous metal probes, which are located close to the inflamed or injured tissue of the patient. The treatment can be used in an ambulatory or stationary environment. It shows unexpectedly, highly effective treatment for certain severe neurological pain conditions, as well as traumatic soft tissue injuries (muscle/ligament ruptures, joint sprains). Without EF intervention, these conditions, respectively, are either virtually incurable or take several months to heal. We present three cases – severe chronic cluster headache, acute massive muscle rupture of the rectus femoris and an acute ankle sprain with a ruptured anterior talofibular ligament – to demonstrate clinical effectiveness and discuss the fundamental differences between mimicking DC simulation and conventional transcutaneous electric nerve stimulation (TENS) or TENS-like implanted devices as used for peripheral nerve cord, spinal cord or dorsal root stimulation.

Dorsal rhizotomy for children with spastic diplegia of cerebral palsy origin: usefulness of intraoperative monitoring.

OBJECTIVE The utility of intraoperative neuromonitoring (ION), namely the study of muscle responses to radicular stimulation, remains controversial. The authors performed a prospective study combining ventral root (VR) stimulation for mapping anatomical levels and dorsal root (DR) stimulation as physiological testing of metameric excitability. The purpose was to evaluate to what extent the intraoperative data led to modifications in the initial decisions for surgical sectioning established by the pediatric multidisciplinary team (i.e., preoperative chart), and thus estimate its practical usefulness. METHODS Thirteen children with spastic diplegia underwent the following surgical protocol. First, a bilateral intradural approach was made to the L2-S2 VRs and DRs at the exit from or entry to their respective dural sheaths, through multilevel interlaminar enlarged openings. Second, stimulation-just above the threshold-of the VR at 2 Hz to establish topography of radicular myotome distribution, and then of the DR at 50 Hz as an excitability test of root circuitry, with independent identification of muscle responses by the physiotherapist and by electromyographic recordings. The study aimed to compare the final amounts of root sectioning-per radicular level, established after intraoperative neuromonitoring guidance-with those determined by the multidisciplinary team in the presurgical chart. RESULTS The use of ION resulted in differences in the final percentage of root sectioning for all root levels. The root levels corresponding to the upper lumbar segments were modestly excitable under DR stimulation, whereas progressively lower root levels displayed higher excitability. The difference between root levels was highly significant, as evaluated by electromyography (p = 0.00004) as well as by the physiotherapist (p = 0.00001). Modifications were decided in 11 of the 13 patients (84%), and the mean absolute difference in the percentage of sectioning quantity per radicular level was 8.4% for L-2 (p = 0.004), 6.4% for L-3 (p = 0.0004), 19.6% for L-4 (p = 0.00003), 16.5% for L-5 (p = 0.00006), and 3.2% for S-1 roots (p = 0.016). Decreases were most frequently decided for roots L-2 and L-3, whereas increases most frequently involved roots L-4 and L-5, with the largest changes in terms of percentage of sectioning. CONCLUSIONS The use of ION during dorsal rhizotomy led to modifications regarding which DRs to section and to what extent. This was especially true for L-4 and L-5 roots, which are known to be involved in antigravity and pelvic stability functions. In this series, ION contributed significantly to further adjust the patient-tailored dorsal rhizotomy procedure to the clinical presentation and the therapeutic goals of each patient.

Inhibitory effects of eugenol on putative nociceptive response in spinal cord preparation isolated from neonatal rats.

Eugenol is contained in several plants including clove and is thought to exert an analgesic effect. It has been suggested that the slow ventral root potential induced by ipsilateral dorsal root stimulation in the isolated (typically lumbar) spinal cord of newborn rats reflects the nociceptive response, and this in vitro experimental model is useful to assess the actions of analgesics. To further elucidate neuronal mechanisms of eugenol-induced analgesia, we examined the effects of extracellularly applied eugenol on the nociceptive spinal reflex response. To evaluate the effects of eugenol on putative nociceptive responses, the ipsilateral fifth lumbar (L5) dorsal root was stimulated using a glass suction electrode, and the induced reflex responses were recorded from the L5 and twelfth thoracic (Th12) ventral roots in spinal cord preparations (Th10-L5) from newborn rats (postnatal day 0-3). We found that eugenol (0.25-1.0mM) caused dose-dependent attenuation of the reflex response and also depressed spontaneous ventral root activity. We also found that the slow ventral root potential was further divided into two components: initial and late components. A lower concentration of eugenol selectively depressed the late component. The inhibitory effects by 1.0mM eugenol were not reversed by 10µM capsazepine (TRPV1 antagonist) or 40µM HC-030031 (TRPA1 antagonist). The depressive effect of eugenol on the reflex response was also confirmed by optical recordings using voltage-sensitive dye. Our report provides additional evidence on the basic neuronal mechanisms of eugenol to support its clinical use as a potential analgesic treatment.

Sacral neuromodulation blocks pudendal inhibition of reflex bladder activity in cats: insight into the efficacy of sacral neuromodulation in Fowler's syndrome.

This study tested the hypothesis that sacral neuromodulation, i.e., electrical stimulation of afferent axons in sacral spinal root, can block pudendal afferent inhibition of the micturition reflex. In α-chloralose-anesthetized cats, pudendal nerve stimulation (PNS) at 3-5 Hz was used to inhibit bladder reflex activity while the sacral S1 or S2 dorsal root was stimulated at 15-30 Hz to mimic sacral neuromodulation and to block the bladder inhibition induced by PNS. The intensity threshold (T) for PNS or S1/S2 dorsal root stimulation (DRS) to induce muscle twitch of anal sphincter or toe was determined. PNS at 1.5-2T intensity inhibited the micturition reflex by significantly ( P < 0.01) increasing bladder capacity to 150-170% of control capacity. S1 DRS alone at 1-1.5T intensity did not inhibit bladder activity but completely blocked PNS inhibition and restored bladder capacity to control level. At higher intensity (1.5-2T), S1 DRS alone inhibited the micturition reflex and significantly increased bladder capacity to 135.8 ± 6.6% of control capacity. However, the same higher intensity S1 DRS applied simultaneously with PNS, suppressed PNS inhibition and significantly ( P < 0.01) reduced bladder capacity to 126.8 ± 9.7% of control capacity. S2 DRS at both low (1T) and high (1.5-2T) intensity failed to significantly reduce PNS inhibition. PNS and S1 DRS did not change the amplitude and duration of micturition reflex contractions, but S2 DRS at 1.5-2T intensity doubled the duration of the contractions and increased bladder capacity. These results are important for understanding the mechanisms underlying sacral neuromodulation of nonobstructive urinary retention in Fowler's syndrome.