Afferent Excitation Research Articles

ETHOLOGICAL AND VEGETATIVE CORRELATES OF EXPERIMENTAL NEUROGENIC STRESS

The study aimed to comprehensively investigate the ethological and vegetative mechanisms involved in the development of experimental emotional stress, establish the relationship between these correlates, and identify an indicator or group of indicators with prognostic value for the occurrence of cardiac dysfunction under neurogenic stress conditions. Materials and Methods. The study presents the results of research on the ethological and autonomic correlates of emotional stress in 254 sexually mature male Wistar rats, using the "afferent excitation conflict" model. Results. Heart rhythm was studied electrocardiographically as an autonomic correlate of emotional stress. The results of the research have shown that ethological and vegetative reactions that perform regulatory and adaptive functions are constant and natural correlates of emotional stress states. The data obtained allow us to note that animals of all three typological groups responded to the onset of stress with a tendency to increase heart rate. Prolonged exposure to the conflict situation was associated with a return of heart rate to baseline values in animals of groups I and II, and a significant decrease in heart rate in animals of group III. Qualitative analysis of the variation curves showed that vagotonic (right position of the variation curve – right graph) prevailed in animals of group I before stress exposure, and sympathotonic (left graph) regulatory influences on the sinus node prevailed in the animals of group III. The onset of stress exposure led to a leftward shift of the variation curves in animals of all groups. However, in animals of group I, this shift was accompanied by a widening of the variation curves (83%), and in animals of group III – by a narrowing (67%). Conclusion. As a result of the studies, four types of heart rate changes were identified during prolonged (3-hour) exposure to stress. A correlation was established between the ethological and typological characteristics of the animals and the occurrence of cardiac rhythm disorders. In animals with a high frequency of cardiac arrhythmias under stress, there was also an increased influence of the sympathetic nervous system on the sinus node, accompanied by a decrease in heart rate variability. Animals with a high frequency of cardiac arrhythmias under stress also showed an increase in the influence of the sympathetic nervous system on the sinus node but with a decrease in heart rate variability.

Modelling of impulse activity of afferent fibers of antagonist muscles during transcutaneous electrical stimulation of the spinal cord during walking

The article describes the results of studies on the impulse activity of various groups of afferent fibers and EMG patterns of lower leg antagonist muscles when walking without, during and after transcutaneous electrical stimulation of the dorsal roots of the lower thoracic spinal cord of a person. Using a mathematical model based on the prediction of the triggering of muscle spindles, variability in the manifestation of impulse activity of various afferents tibialis anterior muscle (TA) and gastrocnemius medialis muscle (GM) when walking under different experimental conditions is shown. It was found that walking on a movable treadmill tape in the absence of spinal cord stimulation was accompanied by strong impulse activity of afferents I (Ia and Ib) and II groups GM, increased excitability of its motorneuron pool and weakening of afferent activity and excitability of TA. On the contrary, electrical stimulation of the spinal cord during walking caused strong impulsive activity of group II TA afferents and moderate — GM, while the activity of Ia fibers TA and GM decreased to moderate impulsivity, Ib afferents of the same muscles had the weakest activity, and the excitability of the GM motorneuron pool was greater than TA. During the postactivation period, walking was accompanied by increased impulses of afferent fibers of group Ib and II GM, weakening of afferent flows of Ib TA and Ia afferents GM, but along with this, afferent signals of group Ia and II to the motorneuron nucleus TA decreased to moderate impulses, and excitability of the motorneuron pool GM was higher than TA. The supposed reflex mechanisms of locomotion regulation are discussed on the basis of well-known phenomena associated with the interaction of various afferent inputs to the spinal cord neuronal apparatus in the system of lower leg antagonist muscles.

Non-drug therapies for patients with vibration disease associated with exposure to local vibration

Non-drug methods include acupuncture, the most effective way to regulate the body's energy system. Pulsed magnetic stimulation (PMS) is based on the application of the principles of electromagnetic induction, causes hyperpolarization or depolarization of nerve cells. The methods are practically not used in the clinic of occupational diseases, and their effectiveness in the treatment of vibration disease (VD) has not been established. The study aims to evaluate changes in the central nervous system and peripheral nerves, as well as in the psychological status of patients with vibration disease after sessions of acupuncture and pulsed magnetic stimulation. Using the acupuncture method, 24 male patients were treated with a diagnosis of VD associated with exposure to local vibration (average age 49.9±3.8 years, average length of service — 19.4±4.3 years), PMS — 24 people with a diagnosis of VD associated with exposure to local vibration (average age 48.8±3.4 years, average length of service — 18.1±3.4 years). The treatment methods performed made it possible to improve the quality of therapy, which was confirmed by a positive change in the indicators of visual and somatosensory evoked potentials, data from electroneuromyographic studies, vibration sensitivity and algesimetry, the state of the mnestic and attentional spheres of activity improved. The use of the acupuncture method allowed to improve the indicators of bioelectric activity of the brain: the amplitude of the peak N1 of visual evoked potentials increased (from 3.2 to 6.7 MV, p<0.05), the latency of the peak P200 decreased from 178.0 to 142.5 ms, p<0.05. The threshold of pain sensitivity on the zygomatic bone, on the phalanx of the 2nd finger of the hand and on the protruding part of the inner ankle decreased by 63, 125 and 250 Hz, p<0.05. Vibration sensitivity improved (on the ulnar process, phalanx of 2 fingers of the hand, p<0.01, on the tubercle of the tibia, p<0.05). Magnetic stimulation contributed to the improvement of cerebral changes in the treated patients — the latency of auditory evoked potentials decreased (p<0.03 for peak P1 and p<0.05 for peak P2), the amplitude of visual evoked potentials increased (p<0.05), the bioelectric activity of the occipital lobes of the brain increased. The duration of the afferent excitation wave at the level of the cervical region (latency N11 and N13, p<0.05) and thalamic structures (latency P25, p<0.05) increased. Non-drug therapy allowed to restore the time of excitation by the motor component of axons in patients on the upper extremities during acupuncture, on the upper and lower extremities during treatment with magnetic stimulation (p<0.05), improved indicators characterizing the mnestic-attentional and psychoemotional spheres of the treated. Ethics. Conclusion of the MEС of the Federal State Budgetary Scientific Institution "East Siberian Institute of Medical and Environmental Research" No. 32 dated 09/10/2019.

RdHSV-CA8 non-opioid analgesic gene therapy decreases somatosensory neuronal excitability by activating Kv7 voltage-gated potassium channels.

Chronic pain is common and inadequately treated, making the development of safe and effective analgesics a high priority. Our previous data indicate that carbonic anhydrase-8 (CA8) expression in dorsal root ganglia (DRG) mediates analgesia via inhibition of neuronal ER inositol trisphosphate receptor-1 (ITPR1) via subsequent decrease in ER calcium release and reduction of cytoplasmic free calcium, essential to the regulation of neuronal excitability. This study tested the hypothesis that novel JDNI8 replication-defective herpes simplex-1 viral vectors (rdHSV) carrying a CA8 transgene (vHCA8) reduce primary afferent neuronal excitability. Whole-cell current clamp recordings in small DRG neurons showed that vHCA8 transduction caused prolongation of their afterhyperpolarization (AHP), an essential regulator of neuronal excitability. This AHP prolongation was completely reversed by the specific Kv7 channel inhibitor XE-991. Voltage clamp recordings indicate an effect via Kv7 channels in vHCA8-infected small DRG neurons. These data demonstrate for the first time that vHCA8 produces Kv7 channel activation, which decreases neuronal excitability in nociceptors. This suppression of excitability may translate in vivo as non-opioid dependent behavioral- or clinical analgesia, if proven behaviorally and clinically.

Postsynaptic potentials of soleus motor neurons produced by transspinal stimulation: a human single-motor unit study.

Transspinal (or transcutaneous spinal cord) stimulation is a noninvasive, cost-effective, easily applied method with great potential as a therapeutic modality for recovering somatic and nonsomatic functions in upper motor neuron disorders. However, how transspinal stimulation affects motor neuron depolarization is poorly understood, limiting the development of effective transspinal stimulation protocols for rehabilitation. In this study, we characterized the responses of soleus α motor neurons to single-pulse transspinal stimulation using single-motor unit (SMU) discharges as a proxy given the 1:1 discharge activation between the motor neuron and the motor unit. Peristimulus time histogram, peristimulus frequencygram, and surface electromyography (sEMG) were used to characterize the postsynaptic potentials of soleus motor neurons. Transspinal stimulation produced short-latency excitatory postsynaptic potentials (EPSPs) followed by two distinct phases of inhibitory postsynaptic potentials (IPSPs) in most soleus motor neurons and only IPSPs in others. Transspinal stimulation generated double discharges at short interspike intervals in a few motor units. The short-latency EPSPs were likely mediated by muscle spindle group Ia and II afferents, and the IPSPs via excitation of group Ib afferents and recurrent collaterals of motor neurons leading to activation of diverse spinal inhibitory interneuronal circuits. Further studies are warranted to understand better how transspinal stimulation affects depolarization of α motor neurons over multiple spinal segments. This knowledge will be seminal for developing effective transspinal stimulation protocols in upper motor neuron lesions.NEW & NOTEWORTHY Transspinal stimulation produces distinct actions on soleus motor neurons: an early short-latency excitation followed by two inhibitions or only inhibition and doublets. These results show how transspinal stimulation affects depolarization of soleus α motor neurons in healthy humans.

Implications of high homocysteine levels in migraine pain: An experimental study of the excitability of peripheral meningeal afferents in rats with hyperhomocysteinemia.

Investigation of chronic homocysteine action on the excitability and N-methyl-D-aspartate (NMDA) sensitivity of the peripheral trigeminovascular system of rats. Migraine is a neurological disease that affects 15%-20% of the general population. Epidemiological observations show that an increase of the sulfur-containing amino acid homocysteine in plasma-called hyperhomocysteinemia-is associated with a high risk of migraine, especially migraine with aura. In animal studies, rats with hyperhomocysteinemia demonstrated mechanical allodynia, photophobia, and anxiety, and higher sensitivity to cortical spreading depression. In addition, rats with hyperhomocysteinemia were more sensitive in a model of chronic migraine induced by nitroglycerin which indicated the involvement of peripheral nociceptive mechanisms. The present work aimed to analyze the excitability of meningeal afferents and neurons isolated from the trigeminal ganglion of rats with prenatal hyperhomocysteinemia. Experiments were performed on male rats born from females fed with a methionine-rich diet before and during pregnancy. The activity of meningeal afferents was recorded extracellularly in hemiskull preparations exvivo and action potentials were characterized using cluster analysis. The excitability of trigeminal ganglion neurons was assessed using whole-cell patch clamp recording techniques and calcium imaging studies. Meningeal mast cells were stained using toluidine blue. The baseline extracellular recorded electrical activity of the trigeminal nerve was higher in the hyperhomocysteinemia group with larger amplitude action potentials. Lower concentrations of KCl caused an increase in the frequency of action potentials of trigeminal afferents recorded in rat hemiskull exvivo preparations. In trigeminal ganglion neurons of rats with hyperhomocysteinemia, the current required to elicit at least one action potential (rheobase) was lower, and more action potentials were induced in response to stimulus of 2 × rheobase. In controls, short-term application of homocysteine and its derivatives increased the frequency of action potentials of the trigeminal nerve and induced Ca2+ transients in neurons, which are associated with the activation of NMDA receptors. At the same time, in rats with hyperhomocysteinemia, we did not observe an increased response of the trigeminal nerve to NMDA. Similarly, the parameters of Ca2+ transients induced by NMDA, homocysteine, and its derivatives were not changed in rats with hyperhomocysteinemia. Acute incubation of the meninges in homocysteine and homocysteinic acid did not change the state of the mast cells, whereas in the model of hyperhomocysteinemia, an increased degranulation of mast cells in the meninges was observed. Our results demonstrated higher excitability of the trigeminal system of rats with hyperhomocysteinemia. Together with our previous finding about the lower threshold of generation of cortical spreading depression in rats with hyperhomocysteinemia, the present data provide evidence of homocysteine as a factor that increases the sensitivity of the peripheral migraine mechanisms, and the control of homocysteine level may be an important strategy for reducing the risk and/or severity of migraine headache attacks.

Overactive bladder, inflammation and urinary tract infection: pathogenetic parallels

The review is devoted to modern ideas about the role of inflammation and urinary tract infection in the pathogenesis of overactive bladder. The molecular mechanisms of the mechanosensory function of the urothelium and the influence of bacterial colonization of the urothelium on it are described in detail. It has been shown that infectious inflammation, even in the absence of clinical symptoms, enhances the urothelial response to stretching and increases the excitability of afferent nerves. Bladder hypersensitivity and increased detrusor activity are pathogenetic basis for the development of overactive bladder. Data on the relationship between urinary infection and refractory overactive bladder are presented. The feasibility of conducting extended microbiological studies in patients with overactive bladder , especially when standard therapy is ineffective, has been demonstrated. A pathogenetic rationale for prescribing anti-inflammatory and immunoactive drugs to patients with overactive bladder is presented.

Contribution of oxytocin and dopamine to the formation of neural clusters in the neocortex representing multimodal sensory stimuli

We have previously proposed a unified mechanism for the formation of contrasted representations of multimodal sensory stimuli in the activity of neocortical neurons. Contrasting is based on the opposite sign of modification of the efficacy of strong and weak excitatory inputs to the spiny cells of the striatum (the input structure of the basal ganglia) and the subsequent dopamine-dependent activity reorganizations in parallel cortico – basal ganglia – thalamocortical loops. Oxytocin and dopamine (through D1 receptors) can improve the contrast of these representations, contributing to the induction of LTP of the efficacy of excitation of cortical, thalamic, and hippocampal neurons innervating spiny cells. In addition, oxytocin and dopamine can improve contrasting enhancement by increasing the signal-to-noise ratio in the neocortex, hippocampus, and striatum. A proposed mechanism for increasing the signal-to-noise ratio is based on the opposite sign of a long-term modification of the efficacy of monosynaptic excitatory and disynaptic inhibitory inputs, simultaneously affecting the postsynaptic neuron. The proposed mechanisms may underlie the contribution of oxytocin and dopamine to improving the formation and long-term maintenance of activity in neuronal groups with similar receptive fields that form columns in the primary visual cortex, a tonotopic map in the primary auditory cortex, a somatotopic map in the sensorimotor cortex, and distributed clusters in the olfactory piriform cortex. These mechanisms differ from the commonly accepted mechanisms of the formation of neuronal clusters in the neocortex with similar RPs, that are based on afferent and lateral excitation and inhibition, which does not allow providing the specificity and duration of effects. Understanding the mechanisms of involvement of oxytocin and dopamine in the processing of multimodal sensory information may be useful for developing treatments for some disorders of social behavior.

A51 MICROBIALLY-MEDIATED IMPAIRMENT OF VAGAL AFFERENT NEURONAL EXCITABILITY IS OREXIN RECEPTOR DEPENDENT

Abstract Background An impairment of vagally-mediated satiety signalling has been implicated in the caloric imbalance that leads to weight gain during obesity. Previous studies have suggested that a reduction in the excitability of vagal afferent neurons with cell bodies in nodose ganglia (NG) was responsible, but the cellular mechanisms are unclear. Host and bacterially derived mediators present in the small intestine and stool provide a physiologically relevant model to help elucidate the role luminal mediators play in modulating vagal afferent neuronal excitability. Aims We hypothesize that the microbiota of obese individuals and mice produce mediators that impair NG neuron excitability and satiety in mice. Methods Perforated patch clamp was used to measure the excitability of NG neurons following exposure to human and mouse fecal supernatants (FS), mouse jejunal supernatants (JS), and mice serum samples. Human FS were from ampersand:003E 5 healthy human donors or FS from ampersand:003E 5 obese donors. Mice FS, JS and serum samples were collected from ampersand:003E 5 obese mice fed a high-fat diet and ampersand:003E 5 control mice fed a normal diet. Results NG neurons incubated in FS from obese participants were significantly less excitable (rheobase was 30% higher and action potential discharge at 2x rheobase was 50% lower) than NG neurons exposed to FS from non-obese participants. NG neurons incubated in FS or JS from obese mice were also significantly less excitable (rheobase was 65% higher and action potential discharge at 2x rheobase was 50% lower) than NG neurons incubated with FS or JS from control mice. Lastly, NG neurons incubated with obese mouse serum were significantly less excitable (rheobase was 50% higher and action potential discharge at 2x rheobase was 80% lower) than NG neurons incubated with serum from control mice. We then attempted to identify mediators that may account for this inhibitory effect by using receptor antagonists that block GABA, ghrelin, and orexin signalling. Ghrelin and GABA receptor antagonists did not block the inhibitory effect of obese patients’ FS on NG neurons but the orexin receptor 1 antagonist (SB-334867;10µM) did. Following this, we incubated the orexin receptor 1 antagonist (SB-334867;10µM) on NG neurons incubated with mouse FS and JS and observed a similar blocking of inhibitory effects back to control values. Conclusions These findings suggest that the gut luminal contents of obese mice and humans contain an orexin receptor agonist that inhibits satiety and may contribute to over-eating. Funding Agencies CIHRNSERC

Experimental colitis-induced visceral hypersensitivity is attenuated by GABA treatment in mice.

Ulcerative colitis (UC) is linked with inflammation of the large intestine due to an overactive response of the colon-immune system. UC is associated with weight loss, rectal bleeding, diarrhea, and abdominal pain. Given that γ-amino butyric acid (GABA) suppresses immune cell activity and the excitability of colonic afferents, and that there is a decrease in colonic GABA during UC, we hypothesized that UC pain is due to a decrease in the inhibition of colonic afferents. Thus, restoring GABA in the colon will attenuate inflammatory hypersensitivity. We tested this hypothesis in a mouse model of colitis. Colon inflammation was induced with seven days of dextran sodium sulfate (DSS, 3%) in the drinking water. GABA (40 mg/kg) was administered orally for the same period as DSS, and body weight, colon length, colon permeability, clinical progression of colitis (disease activity index or DAI), and colon histological score (HS) were assessed to determine the effects of GABA on colitis. A day after the end of GABA treatment, visceral sensitivity was assessed with balloon distention (of the colon)-evoked visceromotor response and colon samples were collected for the measurement of GABA and cytokines. Treatment with GABA reduced the DSS-induced increase in the colon permeability, DAI, HS, and decrease in body weight and colon length. Furthermore, GABA inhibited the DSS-induced increase in the proinflammatory cytokines tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-12 (IL-12), and increased the expression of the anti-inflammatory cytokine IL-10 in the colon tissue. Importantly, GABA reduced DSS-induced visceral hypersensitivity. These data suggest that increasing gastrointestinal levels of GABA may be useful for the treatment of colitis.NEW & NOTEWORTHY GABA treatment reduces the severity of colitis and inflammation and produces inhibition of visceral hypersensitivity in colon-inflamed mice. These results raise the promising possibility that GABA treatment may be an effective therapeutic strategy for the management of symptoms associated with colitis. However, clinical studies are required to corroborate whether this mouse-model data translates to human colon.

Peripheral temperature dysregulation associated with functionally altered NaV1.8 channels.

The voltage-gated sodium channel NaV1.8 is prominently expressed in the soma and axons of small-caliber sensory neurons, and pathogenic variants of the corresponding gene SCN10A are associated with peripheral pain and autonomic dysfunction. While most disease-associated SCN10A variants confer gain-of-function properties to NaV1.8, resulting in hyperexcitability of sensory neurons, a few affect afferent excitability through a loss-of-function mechanism. Using whole-exome sequencing, we here identify a rare heterozygous SCN10A missense variant resulting in alteration p.V1287I in NaV1.8 in a patient with a 15-year history of progressively worsening temperature dysregulation in the distal extremities, particularly in the feet. Further symptoms include increasingly intensifying tingling and numbness in the fingers and increased sweating. To assess the impact of p.V1287I on channel function, we performed voltage-clamp recordings demonstrating that the alteration confers loss- and gain-of-function characteristics to NaV1.8 characterized by a right-shifted voltage dependence of channel activation and inactivation. Current-clamp recordings from transfected mouse dorsal root ganglion neurons further revealed that NaV1.8-V1287I channels broaden the action potentials of sensory neurons and increase their firing rates in response to depolarizing current stimulations, indicating a gain-of-function mechanism of the variant at the cellular level in a heterozygous setting. The data support the hypothesis that the properties of NaV1.8 p.V1287I are causative for the patient's symptoms and that nonpainful peripheral paresthesias should be considered part of the clinical spectrum of NaV1.8-associated disorders.

Intra- and inter-session reliability of electrical detection and pain thresholds of cutaneous and muscle primary afferents in the lower back of healthy individuals.

To advance evidence-based practice and targeted treatments of low back pain (LBP), a better pathophysiological understanding and reliable outcome measures are required. The processing of nociceptive information from deeper somatic structures (e.g., muscle, fascia) might play an essential role in the pathophysiology of LBP. In this study, we measured the intra- and inter-session reliability of electrical detection and pain thresholds of cutaneous and muscle primary afferents of the lower back. Twenty healthy participants attended two study visits separated by 27.7 ± 1.7days. To determine the location-specific electrical detection threshold (EDT) and pain threshold (EPT), needle electrodes were inserted in the epidermal layer over, and in the lumbar erector spinae muscle. Additionally, established quantitative sensory testing (QST) parameters were assessed. Reliability was determined by differences between measurements, intraclass correlation coefficients (ICC2,1), Bland-Altman plots, and standard error of measurement (SEM). Correspondence between QST parameters and electrical thresholds was assessed using Pearson's correlation. Except for cutaneous EPT, no significant (p ≤ 0.05) intra- and inter-session differences were observed. Excellent intra-session reliability was shown for cutaneous and intramuscular electrical stimulations and all QST parameters (ICC: 0.76-0.93). Inter-session reliabilities were good (ICC: 0.74-0.75) except for electrical stimulations (ICC: 0.08-0.36). Limits of agreement and SEM were higher for inter-session than intra-session. A medium to strong relationship was found between electrical and mechanical/pressure pain thresholds. In conclusion, cutaneous and intramuscular electrical stimulation will potentially close an important diagnostic gap regarding the selective examination of deep tissue afferents and provide location-specific information for the excitability of non-nociceptive and nociceptive afferents.

Synaptic and cellular endocannabinoid signaling mechanisms regulate stress-induced plasticity of nucleus accumbens somatostatin neurons

Interneuron populations within the nucleus accumbens (NAc) orchestrate excitatory-inhibitory balance, undergo experience-dependent plasticity, and gate-motivated behavior, all biobehavioral processes heavily modulated by endogenous cannabinoid (eCB) signaling. While eCBs are well known to regulate synaptic plasticity onto NAc medium spiny neurons and modulate NAc function at the behavioral level, how eCBs regulate NAc interneuron function is less well understood. Here, we show that eCB signaling differentially regulates glutamatergic and feedforward GABAergic transmission onto NAc somatostatin-expressing interneurons (NAcSOM+) in an input-specific manner, while simultaneously increasing postsynaptic excitability of NAcSOM+ neurons, ultimately biasing toward vHPC (ventral hippocampal), and away from BLA (basolateral amygdalalar), activation of NAcSOM+ neurons. We further demonstrate that NAcSOM+ are activated by stress in vivo and undergo stress-dependent plasticity, evident as a global increase in intrinsic excitability and an increase in excitation-inhibition balance specifically at vHPC, but not BLA, inputs onto NAcSOM+ neurons. Importantly, both forms of stress-induced plasticity are dependent on eCB signaling at cannabinoid type 1 receptors. These findings reveal eCB-dependent mechanisms that sculpt afferent input and excitability of NAcSOM+ neurons and demonstrate a key role for eCB signaling in stress-induced plasticity of NAcSOM+-associated circuits.

Acid-sensing ion channel blocker diminazene facilitates proton-induced excitation of afferent nerves in a similar manner that Na+/H+ exchanger blockers do.

Tissue acidification causes sustained activation of primary nociceptors, which causes pain. In mammals, acid-sensing ion channels (ASICs) are the primary acid sensors; however, Na+/H+ exchangers (NHEs) and TRPV1 receptors also contribute to tissue acidification sensing. ASICs, NHEs, and TRPV1 receptors are found to be expressed in nociceptive nerve fibers. ASIC inhibitors reduce peripheral acid-induced hyperalgesia and suppress inflammatory pain. Also, it was shown that pharmacological inhibition of NHE1 promotes nociceptive behavior in acute pain models, whereas inhibition of TRPV1 receptors gives relief. The murine skin-nerve preparation was used in this study to assess the activation of native polymodal nociceptors by mild acidification (pH 6.1). We have found that diminazene, a well-known antagonist of ASICs did not suppress pH-induced activation of CMH-fibers at concentrations as high as 25 μM. Moreover, at 100 μM, it induces the potentiation of the fibers' response to acidic pH. At the same time, this concentration virtually completely inhibited ASIC currents in mouse dorsal root ganglia (DRG) neurons (IC50 = 17.0 ± 4.5 μM). Non-selective ASICs and NHEs inhibitor EIPA (5-(N-ethyl-N-isopropyl)amiloride) at 10 μM, as well as selective NHE1 inhibitor zoniporide at 0.5 μM induced qualitatively the same effects as 100 μM of diminazene. Our results indicate that excitation of afferent nerve terminals induced by mild acidification occurs mainly due to the NHE1, rather than acid-sensing ion channels. At high concentrations, diminazene acts as a weak blocker of the NHE. It lacks chemical similarity with amiloride, EIPA, and zoniporide, so it may represent a novel structural motif for the development of NHE antagonists. However, the effect of diminazene on the acid-induced excitation of primary nociceptors remains enigmatic and requires additional investigations.

Therapeutic effects of p38 mitogen-activated protein kinase inhibition on hyperexcitability of capsaicin sensitive bladder afferent neurons in mice with spinal cord injury

AimsNerve growth factor (NGF) has been implicated as a key molecule of pathology-induced changes in C-fiber afferent nerve excitability, which contributes to the emergence of neurogenic detrusor overactivity due to spinal cord injury (SCI). It is also known that the second messenger signaling pathways activated by NGF utilize p38 Mitogen-Activated Protein Kinase (MAPK). We examined the roles of p38 MAPK on electrophysiological properties of capsaicin sensitive bladder afferent neurons with SCI mice. Main methodsWe used female C57BL/6 mice and transected their spinal cord at the Th8/9 level. Two weeks later, continuous administration of p38 MAPK inhibitor (0.51 μg/h, i.t. for two weeks) was started. Bladder afferent neurons were labelled with a fluorescent retrograde tracer, Fast-Blue (FB), injected into the bladder wall three weeks after SCI. Four weeks after SCI, freshly dissociated L6-S1 dorsal root ganglion neurons were prepared and whole cell patch clamp recordings were performed in FB-labelled neurons. After recording action potentials or voltage-gated K+ currents, the sensitivity of each neuron to capsaicin was evaluated. Key findingsIn capsaicin-sensitive FB-labelled neurons, SCI significantly reduced the spike threshold and increased the number of action potentials during 800 ms membrane depolarization. Densities of slow-decaying A-type K+ (KA) and sustained delayed rectifier-type K+ (KDR) currents were significantly reduced by SCI. The reduction of KA, but not KDR, current density was reversed by the treatment with p38 MAPK inhibitor. SignificanceP38 MAPK plays an important role in hyperexcitability of capsaicin-sensitive bladder afferent neurons due to the reduction in KA channel activity in SCI mice.

Corticospinal excitability and reflex modulation in a contralateral non-stretched muscle following unilateral stretching.

Muscle stretching effect on the range of motion (ROM) and force deficit in non-stretched muscle, and the underlying mechanisms, is an ongoing issue. This study aimed to investigate crossover stretching effects and mechanisms on the plantar flexor muscles. Fourteen recreationally active females (n = 5) and males (n = 9) performed six sets of 45-s static stretching (SS) (15-s recovery) to the point of discomfort of the dominant leg (DL) plantar flexors or control (345-s rest). Participants were tested for a single 5-s pre- and post-test maximal voluntary isometric contraction (MVIC) with each plantar flexor muscle and were tested for DL and non-DL ROM. They were tested pre- and post-test (immediate, 10-s, 30-s) for the Hoffman (H)-reflex and motor-evoked potentials (MEP) from transcranial magnetic stimulation in the contralateral, non-stretched muscle. Both the DL and non-DL-MVIC force had large magnitude, significant (↓10.87%, p = 0.027, pƞ2 = 0.4) and non-significant (↓9.53%, p = 0.15, pƞ2 = 0.19) decreases respectively with SS. The SS also significantly improved the DL (6.5%, p < 0.001) and non-DL (5.35%, p = 0.002) ROM. The non-DL MEP/MMax and HMax/MMax ratio did not change significantly. Prolonged static stretching improved the stretched muscle's ROM. However, the stretched limb's force was negatively affected following the stretching protocol. The ROM improvement and large magnitude force impairment (statistically non-significant) were transferred to the contralateral muscles. The lack of significant changes in spinal and corticospinal excitability confirms that the afferent excitability of the spinal motoneurons and corticospinal excitability may not play a substantial role in non-local muscle's ROM or force output responses.

Features of damage to the conductive structures of the nervous system in patients with vibration disease, burdened with diabetes mellitus and metabolic syndrome

Introduction. Of interest are the processes developing in vibration disease (VD) in combination with metabolic syndrome (MS) or diabetes mellitus (DM), combined forms of diseases worsen their course and prognosis.&#x0D; The study aims to assess the features of the state of central afferent conductive structures and peripheral nerves in patients with vibration disease, burdened with diabetes mellitus or metabolic syndrome.&#x0D; Materials and methods. The scientists have examined 155 workers with VD — group one with an established diagnosis of VD (86 people); group two — with VD combined with DM (30 people); group three — with VD burdened with MS (39 people). The authors have conducted the electroneuromyographic examination and registration of somatosensory evoked potentials, with stimulation of the median nerve in the wrist. Abduction points — Erb point, cervical spinal cord, from the scalp of the projection area of the precentral gyrus. The researchers have applied multivariate discriminant analysis from the STATISTICA — version 6 application software package of StatSoft Inc (USA).&#x0D; Results. With VD complicated by MS, changes were in the distal part of the motor axons, with VD burdened with DM, the amplitude of the motor component of the median and tibial nerves decreased, the speed of the pulse in the proximal part of the median nerve. Somatosensory evoked potentials in VD in combination with MS revealed an increase in latency of N13 and N20. The duration of N13-N18, N13-N20, latency N18, N20, N30 increased in the case of WB, burdened with DM.&#x0D; Limitations. The limitation of the study is the use of unrelated samples for statistical analysis. The study of the state of central and peripheral conductive structures in connected samples should be considered evidence-based: metabolic syndrome and further — diabetes mellitus.&#x0D; Conclusion. The patterns of peripheral nerve damage in VD associated with exposure to general and local vibration, burdened with DM or MS, were disorders of afferent axons of the upper and lower extremities, as well as changes in the distal parts of the motor component of the tibial and median nerves. The features of the peripheral nerve states in VD, burdened with DM, were the complex involvement of the motor component in the pathological process, characterized by changes in the amplitude, pulse transit time at the level of the terminal unmyelinated fibers of the median and tibial nerves and changes in the proximal part of the median nerve. In VD complicated by MS, pathological changes were characteristic of the distal part of the motor axons. The patterns of afferent conductive structures lesion in VD, burdened with DM or MS, consisted in a violation of the activity of neurons of the cervical spinal cord and the somatosensory cortex. The peculiarity of the state of the central afferent conducting structures in VD, burdened with DM, was the slowing down of the afferent excitation wave in the area of thalamic structures to the somatosensory zone of the cortex, a violation of the conduction from the trunk to the cortical projections of the somatosensory zone. In case of VD, complicated by MS — the time of the afferent excitation wave at the level of the cervical spine and the somatosensory cortex.&#x0D; Ethics. Conclusion of the MEC of the Federal State Budgetary Scientific Institution "East Siberian Institute of Medical and Environmental Research" No. 32 dated 09.10.2019.

A17 EVIDENCE OF PROTEASE-MEDIATED PRO-NOCICEPTIVE EFFECTS OF FECAL SUPERNATANTS FROM CROHN'S DISEASE PATIENTS

Abstract Background Abdominal pain is a debilitating symptom of Crohn’s disease (CD). Despite the current treatment options for this disease, abdominal pain is an unresolved problem that commonly persists in the absence of active inflammation. This suggests that something other than inflammation is driving the pain during the quiescent phase. We have previously reported that microbial proteases can directly modulate the excitability of dorsal root ganglia (DRG) neurons, many of which are pain-sensing. We hypothesize that luminal proteases of CD patients are contributing to their abdominal pain. Purpose Determine whether luminal mediators in CD fecal samples induce changes in pain signalling. Method The effects of patient (active CD [n = 3] and healthy volunteer (HV) [n = 3]) fecal supernatant (FS) samples on pain-sensing neurons were assessed using ex-vivo single unit afferent nerve recordings from mouse colons. Each sample was tested in colonic preparations from a least 5 mice. To further examine cellular mechanisms, DRG neurons were isolated and incubated overnight in media containing CD FS or HV FS media. Changes in neuronal excitability were recorded by determining the rheobase (lower rheobase=increased excitability) using patch clamp recordings (n ≥ 9 DRG neurons/group). Protease inhibitors were applied in both bioassays to determine whether these inhibited the excitatory effect of FS. Lastly, total proteolytic activity in the CD and HV fecal samples was calculated using a casein colorimetric protease detection assay. Result(s) FS from HV had no effect on afferent nerve excitability (p = 0.8920). FS from active CD patients increased action potential discharge from colonic afferent nerves by 85% (p&amp;lt;0.0001) and selectively increased the activation of high-threshold units, which are putative nociceptors, by 44% (p=0.0074). A protease inhibitor cocktail (1:1000) and protease-activated receptor (PAR)-2 antagonist GB83 (10µM) both blocked the excitatory effects of CD FS (p&amp;lt;0.05). Overnight incubation with CD FS also had an excitatory effect on DRG neurons compared to HV FS (rheobase decreased by 46%, p&amp;lt;0.05). The effect of CD FS was blocked by GB83 (10µM) (p&amp;lt;0.001) and a serine protease inhibitor (FUT175; 100µM) (p&amp;lt;0.05) independently, but the activity was not blocked by E64 (30nM) a cysteine protease inhibitor. A 200-fold increase (p&amp;lt;0.0001) in total proteolytic activity was found in CD FS compared to HV FS. Conclusion(s) Luminal serine proteases, but not cysteine proteases, appear to be driving nociceptive signalling in CD patients. This provides insight into the generation of pain in CD patients and may be a potential target to mitigate this action. Further research is required to elucidate whether these pro-nociceptive proteases are of bacterial or host origin and their effects in the quiescent phase. Disclosure of Interest None Declared

Endocannabinoids, anandamide and 2-AG, regulate mechanosensitivity of mucosal afferents in the Guinea pig bladder

Bladder afferents play a crucial role in urine storage and voiding, and conscious sensations from the bladder. Endocannabinoids, anandamide (AEA) and 2-arachidonolylglycerol (2-AG), are endogenous ligands of G-protein coupled cannabinoid receptors 1 and 2 (CB1 and CB2) found in the CNS and peripheral organs. They also have off-target effects on some ligand- and voltage-gated channels. The aim of this study is to determine the role of AEA and 2-AG in regulation of mechanosensitivity of probable nociceptive neurons innervating the bladder - capsaicin-sensitive mucosal afferents. The activity of these afferents was determined by ex vivo single unit extracellular recordings in the guinea pig bladder. A stable analogue of anandamide, methanandamide (mAEA) evoked initial excitatory response of mucosal afferents followed by potentiation of their responses to mechanical stimulation. In the presence of TRPV1 antagonist (AMG9810), mAEA's effect on mechanosensitivity switched from excitatory to inhibitory. The inhibitory effect of mAEA is due to activation of both CB1 and CB2 cannabinoid receptors since it was abolished by combined application of selective CB1 (NESS0327) and CB2 (SR144528) antagonists. 2-AG application evoked a brief excitation of mucosal afferents, without potentiation of their mechanosensitivity, followed by the inhibition of their responses to mechanical stimulation. CB2 receptor antagonist, SR144528 abolished the inhibitory effect of 2-AG. Our data indicated that anandamide and 2-AG have opposite effects on mechanosensitivity of mucosal capsaicin-sensitive afferents in the guinea pig bladder; mAEA potentiated while 2-AG inhibited responses of mucosal afferents to mechanical stimulation. These findings are important for understanding of the role of endocannabinoids in regulating bladder sensation and function.

Cancer-nerve interplay in cancer progression and cancer-induced bone pain.

Cancer-induced bone pain (CIBP) is one of the most common and debilitating complications associated with bone metastasis. Although our understanding of the precise mechanism is limited, it has been known that bone is densely innervated, and that CIBP is elicited as a consequence of increased neurogenesis, reprogramming, and axonogenesis in conjunction with sensitization and excitation of sensory nerves (SNs) in response to the noxious stimuli that are derived from the tumor microenvironment developed in bone. Recent studies have shown that the sensitized and excited nerves innervating the tumor establish intimate communications with cancer cells by releasing various tumor-stimulating factors for tumor progression. In this review, the role of the interactions of cancer cells and SNs in bone in the pathophysiology of CIBP will be discussed with a special focus on the role of the noxious acidic tumor microenvironment, considering that bone is in nature hypoxic, which facilitates the generation of acidic conditions by cancer. Subsequently, the role of SNs in the regulation of cancer progression in the bone will be discussed together with our recent experimental findings. It is suggested that SNs may be a newly-recognized important component of the bone microenvironment that contribute to not only in the pathophysiology of CIBP but also cancer progression in bone and dissemination from bone. Suppression of the activity of bone-innervating SNs, thus, may provide unique opportunities in the treatment of cancer progression and dissemination, as well as CIBP.