A novel target for analgesic substances: physiological role of Na,K-ATPase as the signal transducer

A possible molecular mechanism of the ligand–receptor binding of Ac-Lys-Lys-Lys-NH2 (Ac-KKK-NH2) to the NaV1.8 channel that is responsible for nociceptive signal coding in the peripheral nervous system is investigated by a number of experimental and theoretical techniques. Upon Ac-KKK-NH2 application at 100 nM, a significant decrease in the effective charge carried by the NaV1.8 channel activation gating system Zeff is demonstrated in the patch-clamp experiments. A strong Ac-KKK-NH2 analgesic effect at both the spinal and supraspinal levels is detected in vivo in the formalin test. The distances between the positively charged amino groups in the Ac-KKK-NH2 molecule upon binding to the NaV1.8 channel are 11–12 Å, as revealed by the conformational analysis. The blind docking with the NaV1.8 channel has made it possible to locate the Ac-KKK-NH2 binding site on the extracellular side of the voltage-sensing domain VSDI. The Ac-KKK-NH2 amino groups are shown to form ionic bonds with Asp151 and Glu157 and a hydrogen bond with Thr161, which affects the coordinated movement of the voltage sensor up and down, thus modulating the Zeff value. According to the results presented, Ac-KKK-NH2 is a promising candidate for the role of an analgesic medicinal substance that can be applied for pain relief in humans.

Intracerebroventricular injection of trazodone produces 5-HT receptor subtype mediated anti-nociception at the supraspinal and spinal levels

Aim:Clitoria ternatea Linn. (C. ternatea) is an Ayurvedic herb traditionally used as medicine to relieve inflammatory, rheumatism, ear diseases, fever, arthritis, eye ailments, sore throat and body ache. This study aims to evaluate and elucidate the possible mechanism underlying the antinociceptive action of methanolic extracts of C. ternatea leaf and root using several antinociception models.Materials and Methods:The different antinociception models such as hot plate, tail-flick and formalin tests were used along with naloxone (a non-selective opioid antagonist) to establish the antinociceptive activity of both leaf and root extracts.Results:Both C. ternatea leaf and root extracts markedly demonstrated antinociceptive action in experimental animals. Results of formalin test showed that the antinociceptive activity of the extracts may be mediated at both central and peripheral level. Moreover, the results of hot plate and tail-flick tests further implies that C. ternatea root extract mediates antinociceptive activity centrally at supraspinal and spinal levels whereas, the C. ternatea leaf extract's antinociceptive activity is mediated centrally at supraspinal level only. It is believed that the opioid receptors are probably involved in antinociceptive activity of both C. ternatea root extract.Conclusions:Our studies support the traditional use of C. ternatea leaf and root against pain. The extracts can also be utilised as a new source of central analgesics in treatment of pain.

Mechanisms of the influence of midazolam on morphine antinociception at spinal and supraspinal levels in rats

We report here studies of the long-term effects of combined stress in the prenatal and prepubertal periods of development on measures of tonic inflammatory pain in the formalin test and the severity of depression-like behavior, and also the stress reactivity of the hormonal response in adult rats. In addition, the effects of the serotonin (5-HT) reuptake inhibitor fluoxetine and the 5-HT1A receptor agonist buspirone, given chronically to stressed mothers during pregnancy, on various types of adaptive behavior impaired by prenatal stress were assessed in rats of both sexes. The results showed that in rats of both sexes prenatal stress increased the pain response organized at the spinal and supraspinal levels of the central nervous system and that fluoxetine and buspirone normalized responses. Stress during the prepuberal period of development eliminated the effects of prenatal stress on the inflammatory pain responses integrated at the supraspinal level in adult rats; in these conditions, fluoxetine and buspirone had no effects, in contrast to their antinociceptive actions on the pain response integrated at the spinal level. Stress at prepubertal age eliminated sex-related differences seen in depression-like behavior in prenatally unstressed and prenatally stressed rats given physiological saline. Control adult females and adult females exposed to prenatal stress in the prepubertal period showed increases in the plasma corticosterone level after forced swimming as compared with the basal hormone level, though there were no significant differences in the level of stress reactivity of the hormonal response after forced swimming. Thus, the conditions for stress actions increasing stress resistance in adult rats were identified. Stress in the critical period of development formed a phenotype with increased stress resistance to inflammatory pain, which was seen in responses organized at the supraspinal level in adult individuals.

We studied the long-term effect of the combination of stress in the prenatal and prepubertal periods of development on indicators of tonic inflammatory pain in the formalin test and the depressive-like behavior, as well as on the stress reactivity of the hormonal response in adult rats. In addition, in rats of both sexes, the effect of serotonin reuptake inhibitor (5-HT) fluoxetine and 5-HT1A agonist buspirone, chronically administered to their stressed mothers during pregnancy, on the studied types of adaptive behavior disturbed by prenatal stress was evaluated. It was found that in rats of both sexes, prenatal stress intensified the pain response organized at the spinal and supraspinal levels of the central nervous system, fluoxetine and buspirone normalized them. Stress in the prepubertal period of development leveled the effect of prenatal stress on the inflammatory pain response integrated at the supraspinal level in adult rats; under these conditions, fluoxetine and buspirone did not act, unlike their antinociceptive effect on the pain response integrated at the spinal level. Stress in prepubertal age leveled the sex differences found in the depressive-like behavior in prenatally non-stressed and prenatally stressed rats with saline. In control adult females and adult females with prenatal effects, prepubertal stress increased plasma corticosterone after forced swimming compared to basal hormone levels, but no significant differences in the level of stress reactivity of the hormonal response after forced swimming were found. Thus, the conditions of stressful impacts that increase resistance to stress in adult rats are identified. Stress in critical periods of development forms a phenotype with increased stress resistance to inflammatory pain, which was found in the response organized at the supraspinal level in adults.

The effect of immobilization of pregnant rats was studied on parameters of the specific biphasic behavioral response (BBR) (patterns of flexion, shaking, licking, duration of the phases and of the interphase interval), of which the first phase characterizes the acute, while the second, he long-term pain in a nociceptive formalin test in the 40-day old female and male off-spring. The following was found: (1) an increase of intensity of patterns of flexion and shaking in the extremity injected with formalin at the second response phase and of the phase duration both in males and in females, (2) an increase of the licking pattern during the second phase and of the phase duration in males. Thus, the prenatal stress produced an increase of the pain sensitivity only at the long-term BBR phase; this increase was revealed in males from the patterns organized at the spinal and supraspinal levels, whereas in females, only at the spinal level. It was concluded that at the period of sex maturation, before the onset of sex maturity, the prenatally stressed males had more expressed damages in the behavioral parameters of the long-term pain in the formalin test, as compared with the prenatally stressed females. The comparative analysis of the response parameters allows suggesting the greater damage in males, then in females, of the inhibition process in the descending inhibitory system modulating nociceptive signals at the spinal cord level.

The aim of the present study was to determine if phenobarbital affects the nociception threshold. Systemic (1-20 mg/kg) phenobarbital administration dose dependently induced hyperalgesia in the tail-flick, hot-plate and formalin tests in rats and in the abdominal constriction test in mice. Formalin and abdominal constriction tests were the most sensitive procedures for the detection of hyperalgesia in response to phenobarbital compared with the tail-flick and hot-plate tests. The hyperalgesia induced by systemic phenobarbital was blocked by previous administration of 1 mg/kg ip picrotoxin or either 1-2 mg/kg sc or 10 ng icv bicuculline. Intracerebroventricular phenobarbital administration (5 microg) induced hyperalgesia in the tail-flick test. In contrast, intrathecal phenobarbital administration (5 microg) induced antinociception and blocked systemic-induced hyperalgesia in this test. We suggest that phenobarbital may mediate hyperalgesia through GABA-A receptors at supraspinal levels and antinociception through the same kind of receptors at spinal levels.

The present work continues our recent series of articles that aim to elucidate the ligand–receptor binding mechanism of short cationic peptides to the NaV1.8 channel in the nociceptive neuron. The applied methodological approach has involved several methods: the patch-clamp experimental evaluation of the effective charge of the NaV1.8 channel activation gating system, the organotypic tissue culture method, the formalin test, and theoretical conformational analysis. The lysine-containing short peptide Ac-KEKK-NH2 has been shown to effectively modulate the NaV1.8 channel activation gating system. As demonstrated by the organotypic tissue culture method, the studied short peptide does not trigger the downstream signaling cascades controlling neurite outgrowth and should not be expected to evoke adverse side effects. Conformational analysis of the Ac-KEKK-NH2 molecule has revealed that the distances between the positively charged amino groups of the lysine side chains are equal to 11–12 Å. According to the previously suggested mechanism of ligand–receptor binding of short peptides to the NaV1.8 channel molecule, Ac-KEKK-NH2 should exhibit an analgesic effect, which has been confirmed by the formalin test. The data obtained unequivocally indicate that the studied lysine-containing short peptide is a promising candidate for the role of a novel analgesic medicinal substance.

Background The process of pain perception includes activation of peripheral nociceptors,transduction of nociceptive signals via the dorsal horn and activation of brain regions involved pain.But pain related molecular circuitry involving transduction of nociceptive signals is still unclear.Objective Review the application of novel optogenetics in pain research.Content Optogenetics can be used in characterizing pain related genes in model organism,studying the connection between peripheral nociceptors and spinal cord pain circuitry,and researching regulatory circuitry in pain involved brain regions.Trend Optogenetic techniques have advantage in selective activation of regional neuronal subpopulations which can provide a new tool to explore the complexity of pain pathway at the peripheral,spinal and supraspinal levels. Key words: Optogenetics; Pain; Dorsal horn; Amygdala; Prefrontal cortex ; Genetics

We have previously reported that methamphetamine (METH) interacts with nicotinic acetylcholine receptor (nAChR) subtypes. This study investigated the involvement of nAChR in the effects of METH locomotion and pain. Chronic, but not acute, nicotine pretreatment potentiated METH-induced hyperlocomotion. This potentiation was abolished by pretreatment with methyllycaconitine, an antagonist of α-7nAChR, or dihydrobetaerythroidine, an antagonist of α-4/β-2nAChR. The mechanism by which amphetamines induce analgesia is not well understood. We investigated the analgesic effects of METH in the writhing, hot-plate and formalin tests and found that methyllycaconitine antagonized METH-induced analgesia in the writhing and formalin tests but not in the hot-plate test. Conversely, dihydrobetaerythroidine was only effective in the hot-plate test. We conclude that α-7nAChR activation by METH is involved in the analgesic effects induced by METH at both spinal and supraspinal levels, whereas METH activation of α-4/β-2nAChR is responsible for the analgesic effect elicited at the supraspinal level. These results show that nAChRs are involved in several actions of amphetamine derivatives and are an important target for the pharmacology of these drugs of abuse.

BACKGROUND: The analgesic properties of arginine vasopressin and its synthetic analogue, 1-deamino-8-D-arginine vasopressin, are known. However, it is difficult to use neuropeptides in clinical practice due to the possible development of side effects. The study of the molecular mechanisms and effects of 1-deamino-8-D-arginine-vasopressin in the management of various types of pain will allow us to identify new therapeutic targets and determine the conditions for the safe use of the peptide. AIM: The study aimed to evaluate the effect of intranasal 1-deamino-8-D-arginine-vasopressin administration on pain sensitivity, the content of monoamines and BDNF in the parietal cortex and spinal cord in thermal and electrocutaneous stimulation in rats. METHODS: 1-deamino-8-D-arginine-vasopressin was administered intranasally in a thermal pain model at 0.002 µg/day (0.01 µg/course) and 2 µg/day (10 µg/course); 0.02 µg/day (0.1 µg/course) and 2 µg/day (10 µg/course) for electrocutaneous stimulation. Serum corticosterone and brain-derived neurotrophic factor levels in the parietal cortex and spinal cord were determined using enzyme-linked immunosorbent assay. The levels of norepinephrine (NE), dopamine (DA), serotonin (5-HT), and their metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA), in the brain were determined using high-performance liquid chromatography. RESULT: 1-deamino-8-D-arginine vasopressin reduced pain sensitivity regardless of the type of pain and doses administered. The involvement of monoamines and brain-derived neurotrophic factor in the analgesic effects depended on the type of exposure and dose of the drug. NE and brain-derived neurotrophic factor at the supraspinal and spinal levels, 5-HT at the spinal cord level have been implicated in analgesia in the thermal pain model. During electrocutaneous stimulation, DA and 5-HT contributed to analgesia at the supraspinal level; 5-HT, DA and NE at the spinal level. 1-deamino-8-D-arginine-vasopressin did not affect the blood corticosterone in rats with different types of pain. CONCLUSION: The peptide caused reduced pain sensitivity under various influences. However, this effect in different conditions was due to different neurochemical mechanisms; in thermal pain, it was associated with the modulatory effect of the peptide on monoamine and BDNF levels in the brain or only monoamines in electrocutaneous stimulation.

In Wistar rats, a comparative study of pain sensitivity to the long-term stimulus in the formalin test was carried out at different age periods—the prepubertal (25 days), pubertal (40 days), and sex maturity (90 days) periods. The pain sensitivity was evaluated by standard indexes of the biphasic behavioral response (BBR)—patterns of flexing, shaking, licking of the leg injected by formalin, and by duration of the first and second response phases and of the interphase interval. It is found that with development of the central nervous system the pain sensitivity changed, with differences in the first, acute and the second, tonic BBR phases: in the tonic phase of response the pain sensitivity increased in males by the indexes organized at the spinal, while in females, at the supraspinal level, whereas in the acute phase it decreased essentially at the supraspinal level in individuals of both sexes. The greater number of age differences in the pain sensitivity is revealed in females by the response patterns organized at the supraspinal level. At the same level, essential readjustments in hormonal and neurotransmitter systems are reflected in the high BBR indexes. In adult individuals the sex dimorphism is detected in duration of the interphase intervals. Activity of the bulbospinal descending inhibitory monoaminergic systems is shown to continue increasing for the first three months of life, with predominance of this process in females. The obtained data allow concluding that the BBR characteristics depend on age and sex of individuals and are determined by the organization level in CNS of the response patterns characterizing the pain sensitivity in the formalin test.

Cardiac steroids (CS), an important class of naturally occurring compounds, are synthesized in plants and animals. The only established receptor for CS is the ubiquitous Na(+),K(+)-ATPase, a major plasma membrane transporter. The binding of CS to Na(+),K(+)-ATPase causes the inhibition of Na(+) and K(+) transport and elicits cell-specific activation of several intracellular signaling mechanisms. It is well documented that the interaction of CS with Na(+),K(+)-ATPase is responsible for numerous changes in basic cellular physiological properties, such as electrical plasma membrane potential, cell volume, intracellular [Ca(2+)] and pH, endocytosed membrane traffic, and the transport of other solutes. In the present study we show that CS induces the formation of dark structures adjacent to the nucleus in human NT2 and ACHN cells. These structures, which are not surrounded by membranes, are clusters of glycogen and a distorted microtubule network. Formation of these clusters results from a relocation of glycogen and microtubules in the cells, two processes that are independent of one another. The molecular mechanisms underlying the formation of the clusters are mediated by the Na(+),K(+)-ATPase, ERK1/2 signaling pathway, and an additional unknown factor. Similar glycogen clusters are induced by hypoxia, suggesting that the CS-induced structural change, described in this study, may be part of a new type of cellular stress response.

Motor learning is defined as the series of processes by which the performance of a task or skill is learned and refined through plastic reorganization within the nervous system. Although many studies have focused on deciphering the motor learning mechanisms within supraspinal structures, recent investigations have placed greater emphasis on understanding plastic responses at the spinal cord. The findings from these studies appear to suggest that reorganization at the spinal cord level is a unique response to skilled performance tasks as opposed to general motor activities and that the efficacy with which specificity is transferred may rely on a time component influenced by sleep or subsequent training sessions. In a recent study published in The Journal of Physiology, Giboin et al. (2020) further expand the motor learning literature by examining how the skill requirements of a balance task influence plastic changes at the spinal cord immediately and 24 h after practising the skill. Giboin et al. (2020) utilized a series of skill-graded balance performance tasks (i.e. sitting, unilateral standing, tilt board) to examine spinal cord plasticity via the Hoffmann reflex (H-reflex) amplitude relative to the M-wave of the soleus muscle. Eighteen healthy adults (10 women, eight men) underwent two assessment sessions separated by 24 h. During the first session, participants were tasked with learning how to perform a unilateral stance on a tilt board. Prior to practising the tilt board task (pre-acquisition), H-reflex amplitude was measured at rest, when standing unilaterally on the floor, and then when performing the tilt board task. Time to failure for the tilt board task was also assessed as a performance measure. Participants were then given 60 trials to practice the tilt board before H-reflex amplitude and performance were assessed across all three conditions again (post-acquisition). A second testing session, conducted 24 h later, was used to assess skill retention and determine any temporal effects of the skill acquisition (retention). Immediately following the skill acquisition session, H-reflex amplitude was decreased at rest. Interestingly, a similar decrease in H-reflex amplitude was observed during retention, but only during the tilt board task, despite no observed changes in performance from post-acquisition to retention. This provides compelling evidence that the plastic reorganization occurring within the spinal cord structures following skill training is probably both task- and time-dependent. This recent work by Giboin et al. (2020) brings to light several critical points for future investigations and interventions regarding motor learning. The findings of their study suggest that learning occurred at the motor neuron or spinal level as indicated by a down-regulation in H-reflex amplitude, potentially indicating a shift from a feedback-based motor command to feedforward. However, some issues regarding the validity of the H-reflex as a measure of plasticity at the spinal cord level are noted. Specifically, it was addressed that the H-reflex may not necessarily be indicative of plastic changes that occur exclusively at the spinal cord level and may reflect plastic changes that instead occur at supraspinal levels. Although it may be reasonable to hypothesize that changes in H-reflex amplitude observed at rest immediately after task acquisition may represent some short-term changes in plasticity at the spinal level, it may be argued that any conclusions drawn from these findings should be done so with caution. Prior studies have identified some limitations of the H-reflex. One such limitation is that the H-reflex is an electrically induced reflex that does not occur naturally in the human body, making it difficult to draw widely generalizable conclusions regarding dynamic muscle activity. Another limitation identified by prior studies is that the H-reflex may not accurately reflect motor neuron excitability considering synaptic connections between afferent and motor neurons may be subject to presynaptic modification. These limitations point towards the need for future research further investigating the validity of the H-reflex as an independent measure of plasticity at the spinal cord level. The decision by Giboin et al. (2020) to use a Bayesian linear mixed model in this investigation is prudent. When analysing data such as H-reflex amplitude, the inherent between-trial variability can present challenges determining where physiological phenomena actually occur. Linear mixed models will account for the high variability of the H-reflex and a Bayesian analysis will allow greater opportunity to discuss the credibility of results than frequentists statistics. The results of this analysis showed that H-reflex amplitude was higher during pre-acquisition than at retention with a strong evidence ratio during the tilt board task. Pre-acquisition H-reflex amplitude was also higher than post-acquisition but not retention during rest. This is interesting because there was an increase in performance from pre- to post-acquisition for the balance board task, yet no correlations were found between performance increases and changes in the H-reflex amplitude. To attempt to isolate H-reflex adaptations as a result of neural plasticity, Giboin et al. (2020) recorded M-wave and background electromyography (EMG) values. Background EMG values were higher during the floor and tilt board task than at rest, which is expected as a result of the increased muscle activity that occurs during task performance. Overall, the statistical approach used in this intervention was creative and appropriate. Bayesian approaches may prove to be an advantageous means of analysis in the fields of physiology or exercise science. Although no changes in performance on the tilt board task were observed, the decreased H-reflex amplitude during the balance task corresponds to a body of literature suggesting that motor learning and neuromuscular control is task-specific. The importance of task-specificity with respect to balance tasks has previously been emphasized by this group, reporting that slack-line training results in improved task-specific performance and neuromuscular control, without any improvements in general balance performance (Giboin et al. 2018; Ringhof et al. 2019). These findings may be clinically useful, especially in adults and children with neurological pathologies. Task-specific balance exercises, individualized to each patient's needs, may have more positive effects than general balance exercises on activities of daily living for post-stroke adults and children with neurological developmental disorders that affect coordination (Veerbeek et al. 2014). Task-specificity is also relevant in the context of strength-training adaptations and transferability. Among resistance-trained men, unstable variations of the chest-press exercise involving either dumbbells on a bench or a loaded barbell on a swiss ball resulted in greater task-specific strength gains compared to a stable variation of the chest-press using a Smith machine on a bench (Saeterbakken et al. 2016). This is probably because the unstable variations of the chest-press had greater potential for improvement in coordination compared to the fixed-path of a Smith machine. In the orthopaedic rehabilitation setting, one of the goals for common sports injuries, such as lateral ankle sprains, is to improve neuromuscular control of the muscles that help protect the joint. Based on the findings from Saeterbakken et al. (2016), it can be inferred that replicating similar, sport-specific scenarios and positions during rehabilitation with an added element of instability may help to improve neuromuscular control in a variety of unstable contexts. If applied appropriately, this approach may effectively lower the risk for re-injury and promote a safe return to sport. In addition to task-specificity, intensity of the practiced task contributes to motor learning. Although unmeasured, it is not unreasonable to suspect that the rate of perceived exertion was probably higher for the balance task on the tilt board compared to the balance task on the floor. Greater task intensity has been found to be associated with greater changes in neuroplasticity and corticospinal excitability, in agreement with the finding of Giboin et al. (2020) of decreased H-reflex amplitude for the balance tilt board task but not the floor balance task. Although conclusions cannot be drawn as to whether the down-regulated, task-specific H-reflex amplitude is resultant from adaptations at the spinal level or from supraspinal feedforward mechanisms, the findings support the use of task-specificity and intensity for addressing motor learning in both rehabilitation and performance settings. Clinicians who want to induce neuroplastic changes for improved motor learning in various populations (including neurological and orthopaedic) should consider individualizing the parameters that may affect neuroplasticity, including intensity and task-specificity. Although our current understanding of the exact mechanisms responsible for plastic reorganization at the spinal cord level during motor learning remains far from complete, the recent work by Giboin et al. (2020) presents valuable evidence regarding the specific task- and time-dependent nature of these adaptations. Furthermore, their investigation clarifies several avenues through which future studies can expand upon our understanding of spinal cord neuroplasticity at the same time as providing clinicians with several strategies to assist with improving motor performance. None. All authors have contributed towards and approved the final version. Each of the authors agree to be accountable for all aspects of the work. None. We thank Dr Matt S. Stock for his guidance in the preparation of this Journal Club submission.