Metformin attenuates increase of synaptic number in the rat spinal dorsal horn with painful diabetic neuropathy induced by type 2 diabetes: a stereological study

In our previous study, we have shown that number of synapses in the L5 segment of spinal dorsal horn increased significantly in a rat model of painful diabetic neuropathy (PDN) induced by high-dose of streptozotocin (an animal model of type 1 diabetes). The aims of this study were: (1) to determine whether high fat diet/low dose streptozotocin-diabetes, a rat model for type 2 diabetes, related PDN was also associated with this synaptic plasticity, (2) to reveal the range of this synaptic plasticity change occurred (in the whole length of spinal dorsal horn or only in the L5 lumbar segment of spinal dorsal horn) and (3) to discover whether treatment with metformin had effect on this synaptic plasticity. Male adult Sprague–Dawley rats were randomly allocated into the control group (n?=?7), the PDN group (n?=?6) and the PDN treated with metformin (PDN?+?M) group (n?=?7), respectively. 28 days after medication, synaptic and neuronal numbers in the whole length of spinal dorsal horn or in 1 mm length of the L5 segment of spinal dorsal horn were estimated by the optical disector (a stereological technique). Compared to the control group and the PDN?+?M group, number of synapses in the L5 segment of spinal dorsal horn increased significantly in the PDN group (P?<?0.05). There was no significant change between the control group and the PDN?+?M group in terms of the parameters in the L5 segment of the spinal dorsal horn (P?>?0.05). Parameters of the whole length of spinal dorsal horn showed no significant changes (P?>?0.05). Our results suggest that high fat diet/low dose streptozotocin diabetes related PDN is also associated with a numerical increase of synapses in the L5 segment of spinal dorsal horn but not in the whole length of spinal dorsal horn. Furthermore, the analgesic effect of metformin against PDN is related to its inhibition of numerical increase of synaptic number in the rat spinal dorsal horn.     

Three-Dimensional Distribution of Sensory Stimulation-Evoked Neuronal Activity of Spinal Dorsal Horn Neurons Analyzed by In Vivo Calcium Imaging

The spinal dorsal horn comprises heterogeneous populations of interneurons and projection neurons, which form neuronal circuits crucial for processing of primary sensory information. Although electrophysiological analyses have uncovered sensory stimulation-evoked neuronal activity of various spinal dorsal horn neurons, monitoring these activities from large ensembles of neurons is needed to obtain a comprehensive view of the spinal dorsal horn circuitry. In the present study, we established in vivo calcium imaging of multiple spinal dorsal horn neurons by using a two-photon microscope and extracted three-dimensional neuronal activity maps of these neurons in response to cutaneous sensory stimulation. For calcium imaging, a fluorescence resonance energy transfer (FRET)-based calcium indicator protein, Yellow Cameleon, which is insensitive to motion artifacts of living animals was introduced into spinal dorsal horn neurons by in utero electroporation. In vivo calcium imaging following pinch, brush, and heat stimulation suggests that laminar distribution of sensory stimulation-evoked neuronal activity in the spinal dorsal horn largely corresponds to that of primary afferent inputs. In addition, cutaneous pinch stimulation elicited activities of neurons in the spinal cord at least until 2 spinal segments away from the central projection field of primary sensory neurons responsible for the stimulated skin point. These results provide a clue to understand neuronal processing of sensory information in the spinal dorsal horn.     

Differential Changes in Neuronal Excitability in the Spinal Dorsal Horn After Spinal Nerve Ligation in Rats

Previous studies demonstrated that peripheral nerve injury induced excessive neuronal response and glial activation in the spinal cord dorsal horn, and such change has been proposed to reflect the development and maintenance of neuropathic pain states. The aim of this study was to examine neuronal excitability and glial activation in the spinal dorsal horn after peripheral nerve injury. We examined noxious heat stimulation-induced c-Fos protein-like immunoreactivity (Fos-LI) neuron profiles in fourth-to-sixth lumbar (L4–L6) level spinal dorsal horn neurons after fifth lumbar spinal nerve ligation (L5 SNL). Immunofluorescence labeling of OX-42 and GFAP was also performed in histological sections of the spinal cord. A significant increase in the number of Fos-LI neuron profiles in the spinal dorsal horn at the L4 level was found at 3 days after SNL, but returned to a level similar to that in sham-operated controls by 14 days after injury. As expected, a decrease in the number of Fos-LI neuron profiles in the spinal dorsal horn at the L5 level was found at 3 days after SNL. However, these profiles had reappeared in large numbers by 14 and 21 days after injury. Immunofluorescence labeling of OX-42 and GFAP indicated sequential activation of microglia and astrocytes in the spinal dorsal horn. We conclude that nerve injury causes differential changes in neuronal excitability in the spinal dorsal horn, which may coincide with glial activation. These changes may play a substantial role in the pathogenesis of neuropathic pain after peripheral nerve injury.     

福尔马林致痛后大鼠中脑导水管周围灰质甲啡肽及脊髓背角P物质含量的变化

本文用免疫组化方法结合计算机图像处理技术观察大鼠后脚掌皮下注射福尔马林后脊髓背角Ｐ物质免疫阳性反应（ＳＰＬＩ）变化的节段性分布及中脑导水管周围灰质（ＰＡＧ）内甲啡肽样免疫阳性反应（ＭＥＬＩ）的变化。结果显示,注射福尔马林后,脊髓腰段（Ｌ１－２,Ｌ４－５）背角ＳＰＬＩ显著增强（Ｐ＜．０５）,３０ｍｉｎ组与６０ｍｉｎ组相比较无显著变化（Ｐ＞０．０５）;胸脊髓（Ｔ８）无显著变化（Ｐ＞０．０５）;颈脊髓背角ＳＰＬＩ有增强趋势（０．０５＜Ｐ＜０．１）;ＰＡＧ中ＭＥＬＩ减弱,腹外侧部３０ｍｉｎ组比６０ｍｉｎ组变化更大（Ｐ＜０．０５）。ＰＡＧ中ＭＥＬＩ与脊髓背角ＳＰＬＩ变化的时相关系提示福尔马林致痛引起的脊髓背角Ｐ物质的增多可能与ＰＡＧ中甲啡肽及阿片受体活动有关。     

The glutamatergic nature of TRPV1-expressing neurons in the spinal dorsal horn

The transient receptor potential vanilloid receptor 1 (TRPV1) is expressed on primary afferent terminals and spinal dorsal horn neurons. However, the neurochemical phenotypes and functions of TRPV1-expressing post-synaptic neurons in the spinal cord are not clear. In this study, we tested the hypothesis that TRPV1-expressing dorsal horn neurons are glutamatergic. Immunocytochemical labeling revealed that TRPV1 and vesicular glutamate transporter-2 were colocalized in dorsal horn neurons and their terminals in the rat spinal cord. Resiniferatoxin (RTX) treatment or dorsal rhizotomy ablated TRPV1-expressing primary afferents but did not affect TRPV1- and vesicular glutamate transporter-2-expressing dorsal horn neurons. Capsaicin significantly increased the frequency of glutamatergic spontaneous excitatory post-synaptic currents and miniature excitatory post-synaptic currents in almost all the lamina II neurons tested in control rats. In RTX-treated or dorsal rhizotomized rats, capsaicin still increased the frequency of spontaneous excitatory post-synaptic currents and miniature excitatory post-synaptic currents in the majority of neurons examined, and this effect was abolished by a TRPV1 blocker or by non-NMDA receptor antagonist. In RTX-treated or in dorsal rhizotomized rats, capsaicin also produced an inward current in a subpopulation of lamina II neurons. However, capsaicin had no effect on GABAergic and glycinergic spontaneous inhibitory post-synaptic currents of lamina II neurons in RTX-treated or dorsal rhizotomized rats. Collectively, our study provides new histological and functional evidence that TRPV1-expressing dorsal horn neurons in the spinal cord are glutamatergic and that they mediate excitatory synaptic transmission. This finding is important to our understanding of the circuitry and phenotypes of intrinsic dorsal horn neurons in the spinal cord.     

Normal anatomy and physiology of the spinal cord dorsal horn

The dorsal horn of the spinal cord receives afferent input from innocuous primary afferent neurons via collaterals from the dorsal columns. This input is integrated and relayed primarily by neurons in laminae III-VI. Dorsal horn neurons which encode innocuous inputs project to the medulla and the cervical spinal cord via the dorsal columns and the dorsolateral funiculus. Nociceptive primary afferent neurons enter the spinal dorsal horn via collaterals from Lissauer's tract. Nociceptive input is integrated and relayed by neurons in laminae I, II and V which project to the reticular formation and thalamus via the anterolateral tract.     

Glial glutamate transporter and glutamine synthetase regulate GABAergic synaptic strength in the spinal dorsal horn

Decreased GABAergic synaptic strength ('disinhibition') in the spinal dorsal horn is a crucial mechanism contributing to the development and maintenance of pathological pain. However, mechanisms leading to disinhibition in the spinal dorsal horn remain elusive. We investigated the role of glial glutamate transporters (GLT-1 and GLAST) and glutamine synthetase in maintaining GABAergic synaptic activity in the spinal dorsal horn. Electrically evoked GABAergic inhibitory post-synaptic currents (eIPSCs), spontaneous IPSCs (sIPSCs) and miniature IPSCs were recorded in superficial spinal dorsal horn neurons of spinal slices from young adult rats. We used (2S,3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (TFB-TBOA), to block both GLT-1 and GLAST and dihydrokainic acid to block only GLT-1. We found that blockade of both GLAST and GLT-1 and blockade of only GLT-1 in the spinal dorsal horn decreased the amplitude of GABAergic eIPSCs, as well as both the amplitude and frequency of GABAergic sIPSCs or miniature IPSCs. Pharmacological inhibition of glial glutamine synthetase had similar effects on both GABAergic eIPSCs and sIPSCs. We provided evidence demonstrating that the reduction in GABAergic strength induced by the inhibition of glial glutamate transporters is due to insufficient GABA synthesis through the glutamate-glutamine cycle between astrocytes and neurons. Thus, our results indicate that deficient glial glutamate transporters and glutamine synthetase significantly attenuate GABAergic synaptic strength in the spinal dorsal horn, which may be a crucial synaptic mechanism underlying glial-neuronal interactions caused by dysfunctional astrocytes in pathological pain conditions.     

miR-9 Mediates CALHM1-Activated ATP-P2X7R Signal in Painful Diabetic Neuropathy Rats

In this study, we planned to illuminate the mechanisms of the expression and function of CALHM1 in painful diabetic neuropathy (PDN). PDN rat model was constructed. The expression of CALHM1 and miR-9 in rat spinal dorsal horn neurons was detected. The correlation between the level of CALHM1 mRNA and 50 % PWT and the relationship between the expression of CALHM1 and miR-9 in rat spinal dorsal horn neurons were statistically analyzed. The effect of miR-9 and CALHM1 on each other’s expression in PDN rat spinal dorsal horn neurons were tested by qRT-PCR or Western blot. The co-culture system of neurons and glias from PDN rat spinal dorsal horn was constructed. The concentration of calcium and ATP as well as the expression of P2X7 receptor regulated by CALHM1 and miR-9 in PDN rat spinal dorsal horn neurons was measured. The results showed that the expression of CALHM1 was increased in PDN rat compared with controls, while its mRNA level was negatively correlated with 50 % PWT. miR-9, which was also upregulated in the spinal dorsal horn neurons of PDN rats, was positively correlated with the expression of CALHM1. The concentration of calcium and ATP as well as the expression of P2X7 receptor in glias was also increased in PDN rats. These increases could be reverted by inhibiting CALHM1 and/or miR-9. CALHM1 is involved in miR-9-mediated ATP-P2X7 pathway between neurons and glias in PDN rat.     

Analysis of receptive fields revealed by in vivo patch-clamp recordings from dorsal horn neurons and in situ intracellular recordings from dorsal root ganglion neurons

It has been thought that spinal dorsal horn neurons receive convergent inputs from not only somatosensory but also visceral pathways. For instance, the referred pain is presumed to be due to the convergence of sensory inputs from cardiac and shoulder receptive fields. However, precise investigation has not been made from dorsal horn neurons yet, because of difficulty in studying the pathways from those regions by means of conventional electrophysiology. The purpose of this study is to clarify the convergent inputs to single dorsal horn neurons from wide receptive fields using an in vivo patch-clamp recording technique from the superficial spinal dorsal horn and an intracellular recording from dorsal root ganglion neurons that keep physiological connections with the peripheral sites. Identified dorsal root ganglion neurons received an input from a quite small area, about 1 x 1 mm in width of the skin. In contrast, substantia gelatinosa neurons in the spinal cord received inputs from an unexpectedly wide area of the skin. Previous extracellular recordings have, however, revealed that substantia gelatinosa neurons have small receptive field. This discrepancy is probably due mainly to an availability of the in vivo patch-clamp method to analyze sub-threshold synaptic responses. In contrast, the extracellular recording technique allows us to analyze predominantly the firing frequency of neurons. Thus, the in vivo patch-clamp recordings from dorsal horn neurons and the intracellular recordings from DRG neurons will be useful for well understanding the sensory processing in the spinal cord.     

刺激中缝背核对大鼠脊髓背角神经元伤害性反应的影响及其传出途径的分析

大量资料表明,中缝背核(DR)在痛觉调节中具有重要作用。本实验用电生理学方法研究DR在痛觉调制中的下行性抑制作用,主要观察刺激DR对清醒制动大鼠脊髓背角神经元伤害性放电的影响。其主要结果是:①刺激DR或电针可以抑制脊髓背角神经元的伤害性反应,吗啡可加强这种抑制效应;②损毁中缝大核(NRM)、纳洛酮、麦角酰二乙胺(LSD)、赛庚啶及对氯苯丙氨酸(PCPA)均能部分阻断DR对脊髓背角神经元伤害性反应的抑制,实验结果表明:刺激DR抑制脊髓背角神经元的伤害性反应,部分是通过NRM间接控制背角神经元的伤害性传入;还有一部分是不通过NRM,可能是DR直接对脊髓背角伤害性信息的调制。在这种下行性抑制通路中有5-HT和阿片样物质的参与。     

亨廷顿蛋白相关蛋白1在成年大鼠脊髓中的分布

目的观察亨廷顿蛋白相关蛋白1(huntingtin-associated protein 1, HAP1)在成年大鼠脊髓中的分布特点.方法采用免疫组织化学ABC法和免疫印迹(Western blotting)方法.结果免疫组织化学结果显示,在成年大鼠脊髓中,以背角灰质浅层(Rexed Ⅰ,Ⅱ层)的HAP1免疫反应性最强,阳性细胞最密集,免疫反应产物除分布在胞体外,还大量弥散分布于胞体间的神经毡内;背角深层有部分HAP1免疫反应阳性细胞呈散在分布,中央管周围灰质(Rexed X)内阳性胞体密度和免疫反应性强度仅次于后角浅层,而在脊髓腹角,偶见HAP1免疫反应阳性神经元.此外, Western blotting分析显示,脊髓背角内HAP1表达水平明显高于脊髓前角.结论 HAP1主要分布于大鼠脊髓背角灰质浅层和中央管周围灰质神经元内,提示其可能与痛觉信息一级传入和/或调控有关.     

A novel transverse push-pull microprobe: in vitro characterization and in vivo demonstration of the enzymatic production of adenosine in the spinal cord dorsal horn

Adenosine produces analgesia in the spinal cord and can be formed extracellularly through enzymatic conversion of adenine nucleotides. A transverse push-pull microprobe was developed and characterized to sample extracellular adenosine concentrations of the dorsal horn of the rat spinal cord. Samples collected via this sampling technique reveal that AMP is converted to adenosine in the dorsal horn. This conversion is decreased by the ecto-5'-nucleotidase inhibitor, alpha,beta-methylene ADP. Related behavioral studies demonstrate that AMP administered directly to the spinal cord can reverse the secondary mechanical hyperalgesia characteristic of the intradermal capsaicin model of inflammatory pain. The specific adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT) inhibits the antihyperalgesia produced by AMP. This research introduces a novel microprobe that can be used as an adjunct sampling technique to microdialysis and push-pull cannulas. Furthermore, we conclude that AMP is converted to adenosine in the dorsal horn of the spinal cord by ecto-5'-nucleotidase and subsequently may be one source of adenosine, acting through adenosine A(1) receptors in the dorsal horn of the spinal cord, which produce antihyperalgesia.     

QUANTITATIVE HISTOCHEMISTRY OF γ-AMINOBUTYRIC ACID IN CAT SPINAL CORD WITH SPECIAL REFERENCE TO PRESYNAPTIC INHIBITION

Abstract— A method is described for quantifying the GABA distribution in cat spinal cord at 200–500 μn resolution. Isolated spinal cord (L5–S1) was frozen and sectioned at about 150 μm thickness. The frozen tissue section was cut into 200 or 500 μm square blocks. The GABA content of each square tissue block was determined by enzymic micromethods and GABA distribution was mapped quantitatively. Average GABA concentrations were: 0·4 mmol/l. in white matter, 1·2 mmol/l. in ventral horn and 1·7 mmol/l. in dorsal horn. The highest concentrations of GABA (2–3 mmol/l.) were found in the dorsolateral part of dorsal horn. In order to destroy the interneurons of dorsal horn, the blood vessels supplying the dorsal horn of the lumbar enlargement were unilaterally cauterized. Seven to 30 days after operation, both the size of dorsal root potential and the GABA level in the dorsal horn were markedly reduced on the cauterized side. These results suggest that GABA is highly concentrated in the interneurons of dorsal horn and functions as a transmitter of presynaptic inhibition.     

Alteration of sensorineural circuits in spinal cord by chronic contact dermatitis

In the present study, eczema-induced alteration of sensorineural circuits of the spinal dorsal horn was investigated. Eczematous lesions resembling atopic dermatitis were induced by repeated application of diphenylcyclopropenone (DCP) onto murine right hind paws. Immunohistochemical labeling of calcitonin gene-related peptide and substance P was increased in the dorsal horn on the DCP-treated side. Expression of calcium binding proteins, calretinin and calbindin-D28K, normally widely seen in dorsal horn interneurons, was up-regulated on the DCP-treated side. E-Cadherin and alpha-N-catenin, synapse-related molecules, were intensely expressed in the spinal dorsal horn of the DCP-treated side. Interestingly, c-Fos positive cells were also significantly increased in laminae I and III of the DCP-treated side. These results suggest an enhanced release of neuropeptides from peripheral afferents and alterations in the sensorineural circuitry of the dorsal horn. These changes may account for the enhanced sensory sensitivity recognized in patients with chronic eczema and atopic dermatitis.     

Interferon-γ receptors are expressed at synapses in the rat superficial dorsal horn and lateral spinal nucleus

Summary Interferon-γ can facilitate the spinal nociceptive flexor reflex and elicit neuropathic pain-related behavior in rats and mice. Immunoreactivity for the interferon-γ receptor (IFN-γR) occurs in the superficial layers of the dorsal horn and the lateral spinal nucleus in the rat and mouse spinal cord, as well as in subsets of neurons in the dorsal root ganglia. The aim of the present study was to examine the cellular localization and origin of the IFN-γR in the spinal cord. As viewed by confocal microscopy, the immunopositivity for the IFN-γR was co-localized with that of the presynaptic marker synaptophysin and with neuronal nitric oxide synthase in the lateral spinal nucleus, whereas only a minor overlap with these molecules was observed in laminae I and II of the dorsal horn. There was no co-localization of the IFN-γR with markers for astrocytes and microglial cells. Ultrastructurally, the IFN-γR was found predominantly in axon terminals in the lateral spinal nucleus but also at postsynaptic sites in dendrites in laminae I and II. The IFN-γR expressed in neurons in dorsal root ganglia was transported in axons both centrally and peripherally. Hemisection of the spinal cord caused no reduction in immunolabelling of the IFN-γR in the dorsal horn or the lateral spinal nucleus. Since rhizotomy does not effect the immunolabelling in the lateral spinal nucleus, our observation indicates that the presynaptic receptors in this nucleus are derived from intrinsic neurons. The localization of the IFN-γR in the spinal cord differed from that of the AMPA glutamate receptor subunits 2 and 3 and the substance P receptor (NK1). Our results, showing localization of IFN-γR to pre- and postsynaptic sites in the dorsal horn and lateral spinal nucleus indicate that IFN-γ can modulate nociception at the spinal cord level.     

Position and size of the axon hillock in various groups of neurons

The origin of the axon was studied in Golgi-Kopsch impregnated specimens prepared from the spinal cord and brain of adult rats. Five types of neurons were sampled: large ventral horn neurons, neurons in the intermediate zone and ventral horn of the spinal cord, antenna-type neurons in the spinal dorsal horn, neurons in the thalamus, and neurons in the hypothalamus. The axon originated from the perikaryon in 76% of the large ventral horn neurons and in 64% of the neurons in the thalamus. In contrast, the axon emerged from one of the dendrites in 75% of the neurons in the intermediate zone and the ventral horn of the spinal cord and in 68% of the neurons in the hypothalamus. In the case of the antenna-type neurons in the spinal dorsal horn, the axon often originated from one of the dendrites, but never from a dorsally oriented dendrite. The mean distance of the axon hillock of dendritic origin was the longest in the neurons in the intermediate zone and the ventral horn of the spinal cord. The size of the axon hillock was proportional to the size of the perikaryon. The impregnated portion of the axon was longest in the large ventral horn neurons.     

Ablation of rat TRPV1-expressing Adelta/C-fibers with resiniferatoxin: analysis of withdrawal behaviors, recovery of function and molecular correlates

Microglia are the resident macrophages in the central nervous system. In the spinal cord dorsal horn, microglia stay in resting condition during physiological sensory processing, and are activated under pathological conditions such as peripheral nerve injury. In cases such as this, the nearby resting microglia increase their motility and accumulate at the site of injury. However, direct evidence to support that nerve activity can enhance the motility of microglia has not yet to be reported. In this study we investigated whether the activation of spinal microglia under in vivo nerve injury may be mimicked by neuronal activity in the spinal cord slice preparation. We found that local application of spinal excitatory neurotransmitters, such as glutamate and substance P did not cause any change in the motility of microglial cells in the spinal cord dorsal horn. The motility of microglial cells is unlikely modulated by other transmitters, neuromodulators and chemokines, because similar applications such as GABA, serotonin, noradrenaline, carbachol, fractalkine or interleukin did not produce any obvious effect. Furthermore, low or high frequency stimulation of spinal dorsal root fibers at noxious intensities failed to cause any enhanced extension or retraction of the microglia processes. By contrast, focal application of ATP triggered rapid and robust activation of microglial cells in the spinal dorsal horn. Our results provide the first evidence that the activation of microglia in the spinal cord after nerve injury is unlikely due solely to neuronal activity, non-neuronal factors are likely responsible for the activation of nerve injury-related microglial cells in the spinal dorsal horn.     

The homeodomain factor lbx1 distinguishes two major programs of neuronal differentiation in the dorsal spinal cord

Dorsal horn neurons in the spinal cord integrate and relay sensory information. Here, we show that the expression of the homeobox gene Lbx1 distinguishes two major neuronal classes generated in the dorsal spinal cord. The Lbx1(-) (class A) and Lbx1(+) (class B) neurons differ in their dependence on roof plate BMP signals for specification and settle in the deep and superficial dorsal horn, respectively. Lbx1 misexpression blocks the differentiation of class A neurons. Conversely, in Lbx1 mutant mice, class B neurons assume the identity of class A neurons. As a consequence, the morphology and neuronal circuitry of the dorsal horn are aberrant. We conclude that Lbx1 distinguishes two major neuronal classes in the dorsal spinal cord and is an important determinant of their distinct differentiation programs.