Gelatinosa Research Articles

Chronic pain models amplify transient receptor potential vanilloid 1 (TRPV1) receptor responses in adult rat spinal dorsal horn

Persistent pain is associated with negative affect originating from hypersensitivity and/or allodynia. The spinal cord is a key area for nociception as well as chronic pain processing. Specifically, the dorsal horn neurons in lamina II (substantia gelatinosa: SG) receive nociceptive inputs from primary afferents such as C fibers and/or Aδ fibers. Transient receptor potential vanilloid 1 (TRPV1) is a major receptor to sense heat as well as nociception. TRPV1 are expressed in the periphery and the central axon terminals of C fibers and/or Aδ fibers in the spinal cord. Activating TRPV1 enhances the release of glutamate in the spinal cord from naïve rodents.Here, we studied whether or not chronic pain could alter the response of TRPV1 channels to exogenous, capsaicin through study of synaptic transmission and neural activity in rat SG neurons. Using in vitro whole-cell patch-clamp recording, we found that bath application of capsaicin facilitated both the frequency and amplitude of miniature and spontaneous excitatory postsynaptic currents beyond a nerve injury and a complete Freund's adjuvant injection observed in the naïve group. Strikingly, capsaicin produced larger amplitudes of inward currents in pain models than compared to the naïve group. By contrast, the proportions of neurons that show capsaicin-induced inward currents were similar among naïve and pain groups. Importantly, the capsaicin-induced inward currents were conducted by TRPV1 and required calcium influx that was independent of voltage-gated calcium channels.Our study provides fundamental evidence that chronic inflammation and neuropathic pain models amplify the release of glutamate through the activation of TRPV1 in central axon terminals, and that facilitation of TRPV1 function in rat spinal SG neurons may contribute to enhanced capsaicin-induced inward currents.

Involvement of metabotropic glutamate receptor 5 in ethanol regulation of NMDA receptor activity in rat substantia gelatinosa neurons

AimsGlutamatergic receptors are important targets of ethanol. Intake of ethanol may produce analgesic effects. The present study examined the effects of ethanol on the activity of ionotropic glutamate receptors in spinal cord substantia gelatinosa (SG) neurons, critical neurons involved in nociceptive transmission. Main methodsWhole-cell recordings were made from SG neurons of the lumbar spinal cord slices from 15 to 20-day-old rats. Ethanol and glutamate receptor agonists or antagonists were applied by superfusion. Key findingEthanol (50 and 100 mM) applied by superfusion for 5 min dose-dependently decreased the amplitude of evoked excitatory postsynaptic potential in SG neurons. Superfusion of ethanol (100 mM) for 15 min consistently inhibited NMDA- or AMPA-induced depolarizations in SG neurons. Ethanol (100 mM) also inhibited the depolarizations induced by glutamate. However, ethanol inhibition of glutamate-induced responses significantly decreased at 10–15 min following continuous superfusion, suggesting the development of acute tolerance to the inhibition during prolonged exposure. Application of MPEP hydrochloride (an antagonist of metabotropic glutamate receptor [mGluR] 5) or GF109203X (a protein kinase C [PKC] inhibitor), together with ethanol significantly blocked the tolerance. The inhibition by ethanol of the NMDA-induced, but not AMPA-induced, depolarizations significantly decreased at 15 min during continuous superfusion while ACPD (a mGluR agonist) was co-applied with ethanol. SignificanceThe results suggest that (1) ethanol exposure may inhibit ionotropic glutamate receptor-mediated neurotransmission; (2) regulation of NMDA receptor function by mGluR5/PKC pathways may be involved in the development of the tolerance to ethanol inhibition of glutamate-induced responses during prolonged exposure in SG neurons.

Endogenous TRPC channels mediate Ca2+ signals and trigeminal synaptic plasticity induced by mGluR5

AimsMetabotropic glutamate receptor 5 (mGluR5), a member of group I mGluR, exerts its effect via elevation of intracellular Ca2+ level. We here characterized Ca2+ signals in the tsA201 cells transfected with mGluR5 and investigated the role of passages for mGluR5-induced Ca2+ signals in synaptic plasticity. Main methodsUsing a genetically encoded Ca2+ indicator, GCamp2, Ca2+ signals were reliably induced by bath application of (S)-3,5-dihydroxyphenylglycine, the group I mGluR agonist, in the tsA201 cells transfected with mGluR5. Using whole-cell recordings in the substantia gelatinosa (SG) neurons of the spinal trigeminal subnucleus caudalis (Vc), excitatory postsynaptic currents were recorded by stimulating the trigeminal tract. Key findingsCa2+ signals were mediated by “classical” or “canonical” transient receptor potential (TRPC) channels, particularly TRPC1/3/4/6, but not TRPC5, naturally existing in the tsA201 cells. Interestingly, the induction of Ca2+ signals was independent of the phospholipase C signaling pathway; instead, it critically involves the cyclic adenosine diphosphate ribose/ryanodine receptor-dependent signaling pathway and only partially protein kinase C. On the other hand, both TRPC3 and TRPC4 mediated mGluR1/5-induced long-lasting potentiation of excitatory synaptic transmission from the trigeminal primary afferents to the SG neurons of the Vc. SignificanceThis study demonstrates that endogenous TRPC channels contribute to mGluR5-induced Ca2+ signals in tsA201 cells and synaptic plasticity at excitatory synapses.

The CCL2 elevation in primary afferent fibers produces zymosan-induced hyperalgesia through microglia-mediated neuronal activation in the spinal dorsal horn

Although C–C motif chemokine ligand 2 (CCL2) plays a critical role in the pathogenesis of neuropathic pain through neuron-microglia interactions, its pronociceptive function underlying inflammatory pain remains to be fully understood. The present study aimed to elucidate the potential role of CCL2 in pain sensitization following zymosan-induced inflammation. Intraplantar injection of zymosan into the rat hind paw significantly increased the expression of CCL2 in both dorsal root ganglions and the superficial dorsal horn. The expression of CCL2 was exclusively present in the isolectin B4-positive unmyelinated primary afferent fibers, but no in other cells of the spinal cord. Intrathecal administration of RS504393 (a CCR2 antagonist) markedly reduced the zymosan-induced thermal and mechanical hyperalgesia accompanied with reduced expression in the spinal cord of c-Fos, CD11b, phosphorylated p38 mitogen activated protein kinases (p-p38), and interleukin-1β. Whole cell patch-clamp recordings on spinal cord slices further revealed that the incubation of CCL2 evoked an evident inward current in substantia gelatinosa neurons and increased level of p-p38 in microglia. Moreover, co-incubation with minocycline (an inhibitor of microglial activation) prevented CCL2-mediated activation in the spinal cord slice. Taken together, we propose that the increased CCL2 expression from primary afferent fibers following zymosan-induced inflammation activates nociceptive neurons in the spinal dorsal horn via a microglia-dependent mechanism.

Action of Norepinephrine on Lamina X of the Spinal Cord

Lamina X is localized in the spinal cord within the region surrounding the central canal and receives descending projections from the supraspinal nuclei. Norepinephrine (NE) is a neurotransmitter in descending pathways emanating from the brain stem; NE-containing fibers terminate in the spinal dorsal cord, particularly in the substantia gelatinosa (SG). NE enhances inhibitory synaptic transmission in SG neurons by activating presynaptic α1-receptors and hyperpolarizes the membranes of SG neurons by acting on α2-receptors; NE may thus act directly on SG neurons of the dorsal spinal cord and inhibit nociceptive transmission at the spinal level. NE-containing fibers also reportedly terminate in lamina X, suggesting that NE also modulates synaptic transmission in lamina X. However, the cellular mechanisms underlying such action have not been investigated. We hypothesized that NE might directly act on lamina X and enhance inhibitory synaptic transmission therein. Using rat spinal cord slices and in vitro whole-cell patch-clamps, we found that the bath-application of NE to lamina X does not affect the excitatory interneurons but enhances GABAergic and glycinergic miniature inhibitory postsynaptic currents (mIPSCs) and induces an outward current. NE-induced enhancement of mIPSCs was blocked by α1A-receptor antagonists, and NE-induced outward current was blocked by α2-receptor antagonists. NE did not affect GABA- or glycine- induced outward currents. These findings are similar to those obtained from SG neurons: NE may act at presynaptic terminals of GABAergic and glycinergic interneurons on lamina X to facilitate inhibitory-transmitter release through α1A-receptor activation and directly induce inhibitory interneuron membrane hyperpolarization through α2-receptors activation.

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.

Noradrenaline modulates mechanically evoked responses in the rat spinal dorsal horn: an in vivo patch-clamp study.

Purpose: We investigated the effects of noradrenaline (NA) on physiologically evoked synaptic responses of substantia gelatinosa (SG) neurons using anesthetized animals.Methods: Male Sprague–Dawley rats (6–8 weeks, 200–300 g, n=21) were anesthetized. The lumbar spinal cord was exposed from L3 to L5; subsequently, the rats were fixed to a stereotaxic apparatus. The electrode was advanced at an angle of 30–45 degrees into the SG using a micromanipulator. We recorded excitatory post-synaptic currents (EPSC). Under these conditions, innocuous or noxious mechanical stimuli were applied to the receptive field of the ipsilateral hindlimb with or without NA, respectively.Results: NA (50 μM) pre-application induced three types of responses for pinch-evoked EPSCs. The number of neurons showing inhibition, facilitation, and no-effect was 15 (71.4%), 2 (9.5%), and 4 (19%), respectively (n=21). Pre-treatment with NA also induced three different types of responses for puff-evoked EPSC (n=21). The number of neurons showing inhibition, facilitation, and no-effect was 9 (42.9%), 9 (42.9%), and 3 (14.2%), respectively. Further, there was a significant difference in the rate distribution (inhibition, facilitation, and no change) between puff- and pinch-evoked responses.Conclusion: Our present data indicate that NA acts on noxious and innocuous mechanical transmission in the SG. Considering the distinct sensory inputs to the SG, the different actions of NA on the transmission of sensory information imply that NA exerts its analgesic effects in a manner more complicated than previously believed.

DARPP‐32 and Cdk5 Expression in the Ventral Cervical Spinal Cord of Rats

Dopamine‐ and cAMP‐regulated phosphoprotein, 32 kDa (DARPP‐32) is a phosphatase that integrates complex neuronal information, including dopamine responsive neurons in the striatum. Differential regulation of DARPP‐32 phosphorylation is an important CNS mechanism integrating neuronal responses to concurrent activation by diverse neurochemicals, including neurotransmitters, neuromodulators and hormones. When Gs protein‐coupled metabotropic receptors activate adenylyl cyclase and increase cAMP levels, PKA activation phosphorylates DARPP‐32 at Threonine 34; in this state, DARPP‐32 inhibits protein phosphatase‐1 (PP‐1). Conversely, when DARPP‐32 is phosphorylated at Threonine 75 by Cyclin‐dependent kinase 5 (Cdk5), DARPP‐32 becomes a PKA inhibitor. Thus, DARPP‐32 is a dual‐function enzyme that inhibits PP‐1 or PKA, depending on prevailing conditions. Since PKA (Hoffman & Mitchell, Neuroscience, 2013) and PP‐1 (Huxtable et. al., J Appl. Physiol., 2018) are both key regulators of plasticity in the phrenic motor system, we performed preliminary studies to determine DARPP‐32 and Cdk5 expression in phrenic motor neurons or nearby cells. Since cervical spinal injury and/or intermittent hypoxia preconditioning trigger plasticity in the phrenic motor system, we explored if they change expression patterns of DARPP‐32 (n = 3) and/or Cdk5 (n = 15). We used immunofluorescence to detect DARPP‐32 and Cdk5 expression in the cervical spinal cords of rats that received intrapleural cholera toxin B subunit (CtB) injections to identify phrenic motor neurons. We studied intact and chronic (~8 week) C2 spinal hemisection (C2Hx) rats exposed to 4 weeks of normoxia or repetitive acute intermittent hypoxia (10, 5 min episodes of 10.5% O2; 5 min intervals; 4 days/week, 4 weeks; rAIH). Spinal cords were harvested, sectioned, and processed with antibodies for CtB and either DARPP‐32 or Cdk5 (C3‐C5; 40 μm). In all rats, DARPP‐32 was not expressed in motor nuclei, including phrenic motor neurons, or in the substantia gelatinosa of the dorsal spinal cord. Dense labeling was observed in distinct cells distributed throughout the medial grey matter; the size, shape, and distribution of these cells is consistent with tanycytes. Cdk5 expression was observed in many cell types throughout the grey matter, including phrenic motor neurons. Using a custom MATLAB algorithm, phrenic motor neuron somatic and total ventral horn expression was quantified; there were no significant effects of C2Hx or rAIH on staining intensity or distribution. In separate rats (n = 12), daily AIH for 28 days had no detectable effect on Cdk5 mRNA levels in ventral spinal cord homogenates, as assessed by qPCR (p=0.116). Thus, DARPP‐32 is not expressed in motor nuclei of the mid‐cervical spinal cord, making a role in phrenic motor plasticity unlikely; the role of DARPP‐32 in putative spinal tanycytes is unknown. Conversely, Cdk5 expression in phrenic motor neurons suggests potential for roles in plasticity or other motor neuron functions.Support or Funding InformationSupported by: University of Florida Graduate School Preeminence Award (MNK), NIH T32 HD043730 (LLA), NIH OT2OD023854 (SPARC; GSM), NIH R01 69064 (GSM), and the McKnight Brain Institute.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Characterization on Responsiveness of Excitatory Synaptic Transmissions to α1-Adrenoceptor Blockers in Substantia Gelatinosa Neurons Isolated From Lumbo-Sacral Level in Rat Spinal Cords.

PurposeThe aim of this study was to characterize the responsiveness of miniature excitatory postsynaptic currents (mEPSCs) to α1-adrenoceptor blockers in substantia gelatinosa (SG) neurons from the spinal cord to develop an explanation for the efficacy of α1-adrenoceptor blockers in micturition dysfunction.MethodsMale adult Sprague-Dawley rats were used. Blind whole-cell patch-clamp recordings were performed using SG neurons in spinal cord slices. Naftopidil (100μM), tamsulosin (100μM), or silodosin (30μM), α1-adrenoceptor blockers, was perfused. The frequency of mEPSCs was recorded in an SG neuron to which the 3 blockers were applied sequentially with wash-out periods. Individual frequencies in a pair before naftopidil and tamsulosin perfusion were plotted as baseline, and the correlation between them was confirmed by Spearman correlation coefficient; linear regression was then performed. The same procedure was performed before naftopidil and silodosin perfusion. Frequencies of pairs after naftopidil and tamsulosin perfusion and after naftopidil and silodosin perfusion were similarly analyzed. The ratios of the frequencies after treatment to before were then calculated.ResultsAfter the treatments, Spearman ρ and the slope were decreased to 0.682 from 0.899 at baseline and 0.469 from 1.004 at baseline, respectively, in the tamsulosin group relative to the naftopidil group. In the silodosin group, Spearman ρ and the slope were also decreased to 0.659 from 0.889 at baseline and 0.305 from 0.989 at baseline, respectively, relative to the naftopidil group. Naftopidil significantly increased the ratio of the frequency of mEPSCs compared to tamsulosin and silodosin (P=0.015 and P=0.004, respectively).ConclusionsThere was a difference in responsiveness in the frequency of mEPSCs to α1-adrenoceptor blockers, with the response to naftopidil being the greatest among the α1-adrenoceptor blockers. These data are helpful to understand the action mechanisms of α1-adrenoceptor blockers for male lower urinary tract symptoms in clinical usage.

Animal models of chronic pain increase spontaneous glutamatergic transmission in adult rat spinal dorsal horn in vitro and in vivo

The ability to detect noxious stimulation is essential to an organism's survival and wellbeing. Chronic pain is characterized by abnormal sensitivity to normal stimulation coupled with a feeling of unpleasantness. This condition afflicts people worldwide and severely impacts their quality of life and has become an escalating health problem.The spinal cord dorsal horn is critically involved in nociception and chronic pain. Especially, the substantia gelatinosa (SG) neurons of lamina II, which receives nociceptive inputs from primary afferents. Two major models are used to study chronic pain in animals, including nerve injury and the injection of a complete Freund's adjuvant (CFA) into the hind paw. However, how these models induce glutamatergic synaptic plasticity in the spinal cord is not fully understood.Here, we studied synaptic plasticity on excitatory transmissions in the adult rat SG neurons. Using in vitro and in vivo whole-cell patch-clamp recording methods, we analyzed spontaneous excitatory postsynaptic currents (sEPSCs) 2 weeks following nerve injury and 1 week following CFA injection. In the spinal slice preparation, these models increased both the frequency and amplitude of sEPSCs in SG neurons. The frequency and amplitude of sEPSCs in the nerve injury and the CFA group were reduced by the presence of tetrodotoxin (TTX). By contrast, TTX did not reduce the sEPSCs compared with miniature EPSCs in naïve rats. Next, we analyzed the active electrophysiological properties of neurons, which included; resting membrane potentials (RMPs) and the generation of action potentials (APs) in vitro. Interestingly, about 20% of recorded SG neurons in this group elicited spontaneous APs (sAPs) without changing the RMPs. Furthermore, we performed in vivo whole-cell patch-clamp recording in SG neurons to analyze active electrophysiological properties under physiological conditions. Importantly, in vivo SG neurons generated sAPs without affecting RMP in the nerve injury and the CFA group.Our study describes how animal models of chronic pain influence both passive and active electrophysiological properties of spinal SG neurons.

Effect of myocardial ischemic preconditioning on ischemia-reperfusion stimulation-induced activation in rat thoracic spinal cord with functional MRI

BackgroundMyocardial ischemia and reperfusion-evoked spinal reflexes involve nociceptive signals that trigger neuronal excitation through cardiac afferents, projecting into the thoracic spinal cord. Ischemic preconditioning (IPC) involves brief episodes of sublethal ischemia and reperfusion enhances the resistance of the myocardium to subsequent ischemic insults. This study investigated the effects of IPC on ischemia-reperfusion (I/R) stimulation-induced activation in the thoracic spinal cord of rats. MethodsA new remotely controlled I/R model was established. The infarct size was determined as a percentage of area at risk (IS/AAR) and arrhythmia scores were evaluated. Non-invasive in vivo fMRI was used for signal quantitative analysis of thoracic spinal spatiotemporal. The role of IPC on the excitability of substantia gelatinosa (SG) neurons was assessed by spinal patch clamp recording technique. The altered expressions of c-Fos, SP, and CGRP in T4 segment were detected by immunohistochemical staining. ResultsThe novel I/R model was induced successfully and reliably utilized, and IPC treatment markedly reduced the myocardial infarct size. fMRI analysis revealed that IPC reduced the increased BOLD signals induced by prolonged ischemia-reperfusion. Patch clamp recording showed that IPC treatment reversed the enhanced excitability of SG neurons during I/R treatment. The results of immunofluorescent staining indicated that IPC mitigated the I/R-induced dramatic increase of c-Fos, and reduced the release of SP and CGRP in dorsal horns of spinal cord. ConclusionsThese results suggested that IPC suppressed neuronal activation induced by I/R stimuli in rat thoracic spinal cord using spinal cord fMRI and patch clamp recording techniques.

Early neurophysiological biomarkers and spinal cord pathology in inherited prion disease.

A common presentation of inherited prion disease is Gerstmann-Sträussler-Scheinker syndrome, typically presenting with gait ataxia and painful dysaesthesiae in the legs evolving over 2-5 years. The most frequent molecular genetic diagnosis is a P102L mutation of the prion protein gene (PRNP). There is no explanation for why this clinical syndrome is so distinct from Creutzfeldt-Jakob disease, and biomarkers of the early stages of disease have not been developed. Here we aimed, first, at determining if quantitative neurophysiological assessments could predict clinical diagnosis or disability and monitor progression and, second, to determine the neuropathological basis of the initial clinical and neurophysiological findings. We investigated subjects known to carry the P102L mutation in the longitudinal observational UK National Prion Monitoring Cohort study, with serial assessments of clinical features, peripheral nerve conduction, H and F components, threshold tracking and histamine flare and itch response and neuropathological examination in some of those who died. Twenty-three subjects were studied over a period of up to 12 years, including 65 neurophysiological assessments at the same department. Six were symptomatic throughout and six became symptomatic during the study. Neurophysiological abnormalities were restricted to the lower limbs. In symptomatic patients around the time of, or shortly after, symptom onset the H-reflex was lost. Lower limb thermal thresholds were at floor/ceiling in some at presentation, in others thresholds progressively deteriorated. Itch sensation to histamine injection was lost in most symptomatic patients. In six patients with initial assessments in the asymptomatic stage of the disease, a progressive deterioration in the ability to detect warm temperatures in the feet was observed prior to clinical diagnosis and the onset of disability. All of these six patients developed objective abnormalities of either warm or cold sensation prior to the onset of significant symptoms or clinical diagnosis. Autopsy examination in five patients (including two not followed clinically) showed prion protein in the substantia gelatinosa, spinothalamic tracts, posterior columns and nuclei and in the neuropil surrounding anterior horn cells. In conclusion, sensory symptoms and loss of reflexes in Gerstmann-Sträussler-Scheinker syndrome can be explained by neuropathological changes in the spinal cord. We conclude that the sensory symptoms and loss of lower limb reflexes in Gerstmann-Sträussler-Scheinker syndrome is due to pathology in the caudal spinal cord. Neuro-physiological measures become abnormal around the time of symptom onset, prior to diagnosis, and may be of value for improved early diagnosis and for recruitment and monitoring of progression in clinical trials.

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons.

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms underlying sensory transmission, nociceptive regulation, and chronic pain or itch development. Implementations of electrophysiological recordings together with morphological studies based on the utility of acute spinal cord slices have further improved our understanding of neuronal properties and the composition of local circuitry in SG. Here, we present a detailed and practical guide for the preparation of spinal cord slices and show representative whole-cell recording and morphological results. This protocol permits ideal neuronal preservation and can mimic in vivo conditions to a certain extent. In summary, the ability to obtain an in vitro preparation of spinal cord slices enables stable current- and voltage-clamp recordings and could thus facilitate detailed investigations into the intrinsic membrane properties, local circuitry and neuronal structure using diverse experimental approaches.

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

Modulation of inhibitory and excitatory neurotransmissions by Zn2+ on the substantia gelatinosa neurons of the trigeminal subnucleus caudalis in mice.

The substantia gelatinosa of the trigeminal subnucleus caudalis has been considered to be an essential location for the transference of orofacial sensory signals. The co-localization of inhibitory and excitatory neurotransmitters in the same substantia gelatinosa (SG) neurons has demonstrated their essential part in the modification of nociceptive transmission. Zn2+ is particularly numerous in the mammalian central nervous system. There are proofs demonstrating the role of Zn2+ in the modulation of voltage- and ligand-gated ion channels. However, little is known about what roles Zn2+ may play in the modulation of signal transmission in the SG neurons of the trigeminal subnucleus caudalis (Vc). Therefore, in this study, we used the whole-cell patch clamp technique to find out the effect of Zn2+ on the responses of three main neurotransmitters (glycine, GABA, and glutamate) on SG neurons of the Vc in mice. We have proved that Zn2+ induces a big potentiation of glycine receptor-mediated response but attenuates GABA- and glutamate-induced responses at micromolar concentrations, however, enhances glutamate-induced response at nanomolar concentration. Taken together, these data demonstrated that Zn2+ can modulate glycine, GABA and glutamate-mediated actions on the SG neurons of the Vc and support an important mechanism in spinal sensory information signaling.

Action of citral on the substantia gelatinosa neurons of the trigeminal subnucleus caudalis in juvenile mice.

The substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) is admitted as a pivotal site of integrating and regulating orofacial nociceptive inputs. Although citral (3,7-dimethyl-2,6-octadienal) is involved in antinociception, the action mechanism of citral on the SG neurons of the Vc has not been fully clarified yet. In this study, we examined the direct membrane effects of citral and how citral mediates responses on the SG neurons of the Vc in juvenile mice using a whole-cell patch-clamp technique. Under high chloride pipette solution, citral showed repeatable inward currents that persisted in the presence of tetrodotoxin, a voltage-gated Na+ channel blocker, and 6-cyano-7-nitro-quinoxaline-2,3-dione, a non-N-methyl-D-aspartate (NMDA) glutamate receptor antagonist, D-2-amino-5-phosphonopentanoic acid, an NMDA receptor antagonist. However, the citral-induced inward currents were partially blocked by picrotoxin, a gamma-aminobutyric acid (GABAA)-receptor antagonist, or by strychnine, a glycine receptor antagonist. Further, the citral-induced responses were almost blocked by picrotoxin with strychnine. We also found that citral exhibited additive effect with GABA-induced inward currents and glycine-induced inward currents were potentiated by citral. In addition, citral suppressed the firing activities by positive current injection on the SG neurons of the Vc. Taken together, these results demonstrate that citral has glycine- and/or GABA-mimetic actions and suggest that citral might be a potential target for orofacial pain modulation by the activation of inhibitory neurotransmission in the SG area of the Vc.

Inhibition by O-desmethyltramadol of glutamatergic excitatory transmission in adult rat spinal substantia gelatinosa neurons.

To reveal cellular mechanisms for antinociception produced by clinically used tramadol, we investigated the effect of its metabolite O-desmethyltramadol (M1) on glutamatergic excitatory transmission in spinal dorsal horn lamina II (substantia gelatinosa; SG) neurons. The whole-cell patch-clamp technique was applied at a holding potential of −70 mV to SG neurons of an adult rat spinal cord slice with an attached dorsal root. Under the condition where a postsynaptic action of M1 was inhibited, M1 superfused for 2 min reduced the frequency of spontaneous excitatory postsynaptic current in a manner sensitive to a μ-opioid receptor antagonist CTAP; its amplitude and also a response of SG neurons to bath-applied AMPA were hardly affected. The presynaptic effect of M1 was different from that of noradrenaline or serotonin which was examined in the same neuron. M1 also reduced by almost the same extent the peak amplitudes of monosynaptic primary-afferent Aδ-fiber and C-fiber excitatory postsynaptic currents evoked by stimulating the dorsal root. These actions of M1 persisted for >10 min after its washout. These results indicate that M1 inhibits the quantal release of L-glutamate from nerve terminals by activating μ-opioid but not noradrenaline and serotonin receptors; this inhibition is comparable in extent between monosynaptic primary-afferent Aδ-fiber and C-fiber transmissions. Considering that the SG plays a pivotal role in regulating nociceptive transmission, the present findings could contribute to at least a part of the inhibitory action of tramadol on nociceptive transmission together with its hyperpolarizing effect as reported previously.

Potentiation of the glycine response by serotonin on the substantia gelatinosa neurons of the trigeminal subnucleus caudalis in mice.

The lamina II, also called the substantia gelatinosa (SG), of the trigeminal subnucleus caudalis (Vc), is thought to play an essential role in the control of orofacial nociception. Glycine and serotonin (5-hydroxytryptamine, 5-HT) are the important neurotransmitters that have the individual parts on the modulation of nociceptive transmission. However, the electrophysiological effects of 5-HT on the glycine receptors on SG neurons of the Vc have not been well studied yet. For this reason, we applied the whole-cell patch clamp technique to explore the interaction of intracellular signal transduction between 5-HT and the glycine receptors on SG neurons of the Vc in mice. In nine of 13 neurons tested (69.2%), pretreatment with 5-HT potentiated glycine-induced current (IGly). Firstly, we examined with a 5-HT1 receptor agonist (8-OH-DPAT, 5-HT1/7 agonist, co-applied with SB-269970, 5-HT7 antagonist) and antagonist (WAY-100635), but 5-HT1 receptor agonist did not increase IGly and in the presence of 5-HT1 antagonist, the potentiation of 5-HT on IGly still happened. However, an agonist (α-methyl-5-HT) and antagonist (ketanserin) of the 5-HT2 receptor mimicked and inhibited the enhancing effect of 5-HT on IGly in the SG neurons, respectively. We also verified the role of the 5-HT7 receptor by using a 5-HT7 antagonist (SB-269970) but it also did not block the enhancement of 5-HT on IGly. Our study demonstrated that 5-HT facilitated IGly in the SG neurons of the Vc through the 5-HT2 receptor. The interaction between 5-HT and glycine appears to have a significant role in modulating the transmission of the nociceptive pathway.

Effects of naftopidil on inhibitory transmission in substantia gelatinosa neurons of the adult rat spinal dorsal horn.

Effects of naftopidil on inhibitory transmission in substantia gelatinosa neurons of the adult rat spinal dorsal horn.

Effects of High Concentrations of Naftopidil on Dorsal Root-Evoked Excitatory Synaptic Transmissions in Substantia Gelatinosa Neurons In Vitro.

PurposeNaftopidil ((±)-1-[4-(2-methoxyphenyl) piperazinyl]-3-(1-naphthyloxy) propan-2-ol) is prescribed in several Asian countries for lower urinary tract symptoms suggestive of benign prostatic hyperplasia. Previous animal experiments showed that intrathecal injection of naftopidil abolished rhythmic bladder contraction in vivo. Naftopidil facilitated spontaneous inhibitory postsynaptic currents in substantia gelatinosa (SG) neurons in spinal cord slices. These results suggest that naftopidil may suppress the micturition reflex at the spinal cord level. However, the effect of naftopidil on evoked excitatory postsynaptic currents (EPSCs) in SG neurons remains to be elucidated.MethodsMale Sprague-Dawley rats at 6 to 8 weeks old were used. Whole-cell patch-clamp recordings were made using SG neurons in spinal cord slices isolated from adult rats. Evoked EPSCs were analyzed in Aδ or C fibers. Naftopidil or prazosin, an α1-adrenoceptor blocker, was perfused at 100 μM or 10 μM, respectively.ResultsBath-applied 100 μM naftopidil significantly decreased the peak amplitudes of Aδ and C fiber-evoked EPSCs to 72.0%±7.1% (n=15) and 70.0%±5.5% (n=20), respectively, in a reversible and reproducible manner. Bath application of 10μM prazosin did not inhibit Aδ or C fiber-evoked EPSCs.ConclusionsThe present study suggests that a high concentration of naftopidil reduces the amplitude of evoked EPSCs via a mechanism that apparently does not involve α1-adrenoceptors. Inhibition of evoked EPSCs may also contribute to suppression of the micturition reflex, together with nociceptive stimulation.