Kv7.5 is the primary Kv7 subunit expressed in C-fibers

Kv7 (KCNQ) potassium channel openers (enhancers) decrease neuropathic pain in experimental models. Here we show that C-fibers, and their associated small-diameter neurons in the dorsal root ganglia (both IB4- and TrkA-positive), expressed Kv7.5. In contrast, C-fibers did not express detectable levels of Kv7.2 or Kv7.3, which are instead localized to nodes of Ranvier and the cell bodies of large sensory neurons. These data suggest that Kv7.5 provides the primary M current in nociceptive neurons.     

Distinct cellular expression and subcellular localization of Kv2 voltage-gated K+ channel subtypes in dorsal root ganglion neurons conserved between mice and humans

The distinct organization of Kv2 voltage-gated potassium channels on and near the cell body of brain neurons enables their regulation of action potentials and specialized membrane contact sites. Somatosensory neurons have a pseudounipolar morphology and transmit action potentials from peripheral nerve endings through axons that bifurcate to the spinal cord and the cell body within ganglia including the dorsal root ganglia (DRG). Kv2 channels regulate action potentials in somatosensory neurons, yet little is known about where Kv2 channels are located. Here, we define the cellular and subcellular localization of the Kv2 paralogs, Kv2.1 and Kv2.2, in DRG somatosensory neurons with a panel of antibodies, cell markers, and genetically modified mice. We find that relative to spinal cord neurons, DRG neurons have similar levels of detectable Kv2.1 and higher levels of Kv2.2. In older mice, detectable Kv2.2 remains similar, while detectable Kv2.1 decreases. Both Kv2 subtypes adopt clustered subcellular patterns that are distinct from central neurons. Most DRG neurons co-express Kv2.1 and Kv2.2, although neuron subpopulations show preferential expression of Kv2.1 or Kv2.2. We find that Kv2 protein expression and subcellular localization are similar between mouse and human DRG neurons. We conclude that the organization of both Kv2 channels is consistent with physiological roles in the somata and stem axons of DRG neurons. The general prevalence of Kv2.2 in DRG as compared to central neurons and the enrichment of Kv2.2 relative to detectable Kv2.1 in older mice, proprioceptors, and axons suggest more widespread roles for Kv2.2 in DRG neurons.     

Serotonin- and proton-induced and modified ionic currents in frog sensory neurons

Using the whole-cell patch-clamp technique, the effects of serotonin (5-HT) and increased acidity to produce membrane currents and to modify high threshold voltage-dependent calcium currents were studied in isolated dorsal root ganglion (DRG) cells of the frog maintained in short-term culture. DRG cells were classified by morphology into two types: (1) cells with a large number of dark rusty brown granules, and (2) cells devoid of these granules or with few scattered pale granules. Fast application of 5-HT (10–30 μM) induced a rapidly desensitizing inward current with a reversal potential at about 0 mV in 38 of 50 granule-containing neurons (76%) which was never observed (0/35) in “clear” neurons. This current was blocked by 10 nM (+)-tubocurarine. In addition, a small noninactivating outward current was also observed in most DRG neurons during 5-HT superfusion. A sudden decrease of pH from 7.4 to 6 or 5.8 induced a fast inactivating inward current of 100–300 pA in 74% of the “clear” neurons and only 24% of the granule-containing neurons. Small noninactivating membrane currents induced by lowering pH were observed in all neurons. Both 5-HT and increased extracellular H⁺ reduced the magnitude of high threshold calcium currents in all DRG neurons. It is suggested that the 5-HT receptors are expressed on a morphologically distinct population of neurons while the cells with channels responsible for the fast inactivating proton-induced current cannot be related to any distinct morphological cell type. © 1995 Wiley-Liss, Inc.     

K(+) current regulates calcium-activated chloride current-induced after depolarization in axotomized sensory neurons

One of the major electrophysiological effects of axotomy is a hyperexcitability of injured afferents that is thought to be involved in peripheral neuropathic pain. The molecular determinants of injured sensory neuron excitability are complex and not all have been identified. We have previously shown that sciatic nerve section upregulates the Ca(2+)-activated Cl(-) current in subsets of medium and large sensory neurons. In the peripheral nervous system, the Ca(2+)-activated Cl(-) current can promote after depolarization (ADP) and may therefore be involved in excitability. In this study, we set the conditions for Ca(2+)-activated Cl(-) current activation during the electrical activity of axotomized sensory neurons. We used the whole-cell patch-clamp technique and Ca(2+) fluorescence measurements to record electrical activity or ionic currents associated with intracellular Ca(2+) transients. An analysis of Ca(2+) fluorescence variation under Ca(2+)-activated Cl(-) current activation showed that the Ca(2+) sensitivity of the Ca(2+)-activated Cl(-) current did not allow activation upon one action potential (AP) but instead necessitated intracellular Ca(2+) loading under high-frequency electrical activity or AP lengthening. Nevertheless, ADP was exclusively recorded under AP lengthening following K(+) current inhibition with either extracellular tetraethylammonium or intracellular Cs(+). The measurement of APs and ionic currents associated with the use of niflumic acid to inhibit Cl(-) currents showed that the Ca(2+)-activated Cl(-) current was responsible for the ADP observed during K(+) current inhibition. Thus, the Ca(2+)-activated Cl(-) current-induced ADP in axotomized sensory neurons is regulated by K(+) current density.     

Kv1.2-immunoreactive primary sensory neurons in the rat trigeminal ganglion

Immunohistochemistry for Kv1.2, a subunit of voltage-gated K(+) channels, was performed on the trigeminal ganglion (TG). Immunoreactivity (ir) was detected in half (48%) the TG neurons. These neurons were mostly medium-sized to large (range 137.6-2664.8 microm(2), mean+/-S.D. 892.6+/-413.3 microm(2)). A double immunofluorescence method also revealed co-expression of Kv1.2 and parvalbumin. Half (54%) the Kv1.2-immunoreactive (ir) neurons exhibited parvalbumin-ir, and parvalbumin-ir neurons mostly showed Kv1.2-ir (95%). Kv1.2-ir neurons which co-expressed CGRP-ir were rare in this ganglion. Some 40% of TG neurons retrogradely labeled from the facial skin exhibited Kv1.2-ir, whereas ir was detected in 16% of those labeled from the tooth pulp. The present study indicates that Kv1.2-ir TG neurons include low-threshold mechanoreceptors and nociceptors which innervate the facial skin and tooth pulp, respectively.     

A Kv7.2 mutation associated with early onset epileptic encephalopathy with suppression‐burst enhances Kv7/M channel activity

Mutations in the KCNQ2 gene encoding the voltage‐gated potassium channel subunit Kv7.2 cause early onset epileptic encephalopathy (EOEE). Most mutations have been shown to induce a loss of function or to affect the subcellular distribution of Kv7 channels in neurons. Herein, we investigated functional consequences and subcellular distribution of the p.V175L mutation of Kv7.2 (Kv7.2^V175L) found in a patient presenting EOEE. We observed that the mutation produced a 25–40 mV hyperpolarizing shift of the conductance–voltage relationship of both the homomeric Kv7.2^V175L and heteromeric Kv7.2^V175L/Kv7.3 channels compared to wild‐type channels and a 10 mV hyperpolarizing shift of Kv7.2^V175L/Kv7.2/Kv7.3 channels in a 1:1:2 ratio mimicking the patient situation. Mutant channels also displayed faster activation kinetics and an increased current density that was prevented by 1 μm linopirdine. The p.V175L mutation did not affect the protein expression of Kv7 channels and its localization at the axon initial segment. We conclude that p.V175L is a gain of function mutation. This confirms previous observations showing that mutations having opposite consequences on M channels can produce EOEE. These findings alert us that drugs aiming to increase Kv7 channel activity might have adverse effects in EOEE in the case of gain‐of‐function variants.     

Identification of perineal sensory neurons activated by innocuous heat

C‐fiber sensory neurons comprise nociceptors and smaller populations of cells detecting innocuous thermal and light tactile stimuli. Markers identify subpopulations of these cells, aiding our understanding of their physiological roles. The transient receptor potential vanilloid 1 (TRPV1) cation channel is characteristic of polymodal C‐fiber nociceptors and is sensitive to noxious heat, irritant vanilloids, and protons. By using immunohistochemistry, in situ hybridization, and retrograde tracing, we anatomically characterize a small subpopulation of C‐fiber cells that express high levels of TRPV1 (HE TRPV1 cells). These cells do not express molecular markers normally associated with C‐fiber nociceptors. Furthermore, they express a unique complement of neurotrophic factor receptors, namely, the trkC receptor for neurotrophin 3, as well as receptors for neurturin and glial cell line‐derived neurotrophic factor. HE TRPV1 cells are distributed in sensory ganglia throughout the neuraxis, with higher numbers noted in the sixth lumbar ganglion. In this ganglion and others of the lumbar and sacral regions, 75% or more of such HE TRPV1 cells express estrogen receptor α, suggestive of their regulation by estrogen and a role in afferent sensation related to reproduction. Afferents from these cells provide innervation to the hairy skin of the perineal region and can be activated by thermal stimuli from 38°C, with a maximal response at 42°C, as indicated by induction of extracellular signal‐regulated kinase phosphorylation. We hypothesize that apart from participating in normal thermal sensation relevant to thermoregulation and reproductive functions, HE TRPV1 cells may mediate burning pain in chronic pain syndromes with perineal localization. J. Comp. Neurol. 518:137–162, 2010. © 2009 Wiley‐Liss, Inc.     

Adipocyte-derived angiopoietin-1 supports neurite outgrowth and synaptogenesis of sensory neurons

Sensory and sympathetic innervation of the white fat tissue (WAT) contributes to lipolysis. In addition, both fiber types adapt in density to weight gain and loss. Because these findings are indicative for a tight control of nerve fiber plasticity by adipokines, we tested whether adipocytes control neurite growth of sensory neurons through angiopoietin-1 (Ang-1). We further considered initial hints that Ang-1-induced neuritogenesis involves transactivation of the high-affinity nerve growth factor (NGF) receptor trkA. Coculturing dorsal root ganglion (DRG) cells with 3T3-L1 adipocytes supported neurite outgrowth. These neurotrophic effects were associated with the increased expression of Ang-1 (presumably in adipocytes) as well as of trkA. The effects were abolished upon inactivating Ang-1 in culture with selective antibodies. Likewise, neurite outgrowth was impaired in the presence of inactivating NGF antibodies as well as upon inhibition of the NGF high-affinity trkA receptor with the antagonist K252a, indicating a tight cooperation of Ang-1 and NGF in the control of neuritogenesis. DRG-adipipocyte cocultures were further used to establish whether sensory neurons would form synaptic contacts with adipocytes. Electron microscopy demonstrated that cultured sensory neurons develop predominantly neuroneuronal synapses but seem to affect adipocytes by synapses en passant. Comparably to the case for neuritogenesis, expression of the presynaptic protein synaptophysin as well of the postsynaptic protein PSD-95 correlated with Ang-1 levels in culture. It is concluded that adipocyte-secreted Ang-1 supports neurite outgrowth, which is involved in synaptogenesis. The novel function of Ang-1 appears to play a physiological role in WAT plasticity.     

Development of central projections of lumbosacral sensory neurons in the chick

The development of central projections of sensory neurons in lumbosacral dorsal root ganglia (DRGs) was examined by using horseradish peroxidase labeling techniques in chick embryos from stage 23 (E4) to stage 39 (E13). Our results show that primary afferents reach the spinal cord by stage 23. Afferent axons extend in the primordium of the dorsal funiculus for several segments rostral and caudal to their segment of entry for over 24 hours before invading the gray matter at stage 28 (E6). Sensory fibers grow into the vicinity of motoneuron dendrites by stage 32 (E7.5), about the time that reflexes and apparent monosynaptic EPSPs can first be elicited. Dense projections into the dorsal laminae of the spinal cord, presumably representing cutaneous afferents, appear somewhat later, at about stage 39 (E13), when the segmental projection pattern begins to resemble the mature pattern.     

Neuropeptide Y immunoreactivity in cutaneous sympathetic and sensory neurons during development of the guinea pig

Different levels of the cutaneous vasculature are innervated selectively by subpopulations of sympathetic neurons distinguished by the presence or absence of immunoreactivity (-IR) for neuropeptide Y (NPY). This study used multiple-labelling immunohistochemistry to examine the appearance of NPY-IR in neurons innervating cutaneous vessels in the ear pinna of embryonic, fetal, and neonatal guinea pigs. NPY-immunoreactive axons were detected in the ear bud at embryonic day 25. However, these axons lacked IR for tyrosine hydroxylase (TH) and often ran in bundles with substance P (SP)-immunoreactive axons close to the epidermis. Many neuronal somata in the cervical dorsal root ganglia (DRG) at late embryonic stages contained NPY-IR with or without SP-IR, but no NPY-IR was detected in DRG or subepidermal axons by late fetal stages. IR for calcitonin gene-related peptide increased in DRG neurons from midfetal to late fetal stages, after the decrease in NPY-IR. Populations of TH-IR neurons with or without NPY-IR were present in the superior cervical ganglion (SCG) from midembryonic stages. TH-immunoreactive axons were not detected in the ear pinna until midfetal stages, when axons with TH-IR and NPY-IR innervated proximal arteries and TH-immunoreactive axons without NPY-IR innervated distal vessels. Vasoactive intestinal peptide-IR was detected transiently in most fetal SCG neurons with TH-IR and NPY-IR but was not detected in cutaneous axons. These results demonstrate that selective expression of NPY by subpopulations of sympathetic neurons occurs prior to innervation of their targets. This suggests that target contact is not required to establish appropriate patterns of expression of peptide neurotransmitters by cutaneous sympathetic neurons.     

Localization of renal sensory neurons using the fluorescent dye technique

The location of cell bodies of renal sensory neurons was studied. Small injections of a fluorescent dye (True Blue of Fast Blue) were placed into either the right or left kidney of male or female rats. Whereas no differences were detected in the labeling patterns of males vs females, right kidney injections did label slightly higher dorsal root ganglia than left injections. In all cases the labeling was confined to the T6-L2 ganglia ipsilateral to the injection.     

Neurochemical characterization of insulin receptor-expressing primary sensory neurons in wild-type and vanilloid type 1 transient receptor potential receptor knockout mice

The insulin receptor (IR) is expressed by a subpopulation of primary sensory neurons (PSN), including a proportion of cells expressing the nociceptive transducer vanilloid type 1 transient receptor potential receptor (TRPV1). Recent data suggest functional links between the IR and other receptors, including TRPV1, which could be involved in the development of PSN malfunctions in pathological insulin secretion. Here we used combined immunohistochemical labelling on sections from L4-5 dorsal root ganglia of wild-type (WT) and TRPV1 knockout (KO) mice to examine the neurochemical properties of IR-expressing PSN and the possible effect of deletion of TRPV1 on those characteristics. We found that antibodies raised against the high-molecular-weight neurofilament (NF-200) and the neurofilament protein peripherin distinguished between small and large neurons. We also found that the IR was expressed predominantly by the small peripherin-immunopositive cells both in the WT and in the KO animals. IR expression, however, did not show any preference between the major subpopulations of the small cells, the calcitonin gene-related peptide (CGRP)-expressing and Bandeiraea simplicifolia isolectin B4 (IB4)-binding neurons, either in the WT or in the KO mice. Nevertheless, a significant proportion of the IR-expressing cells also expressed TRPV1. Comparison of the staining pattern of these markers showed no difference between WT and KO animals. These findings indicate that the majority of the IR-expressing PSN are small neurons, which are considered as nociceptive cells. Furthermore, these data show that deletion of the TRPV1 gene does not induce any additional changes in neurochemical phenotype of nociceptive PSN.     

Nitric oxide synthase and growth-associated protein are coexpressed in primary sensory neurons after peripheral injury

A possible role for nitric oxide in growth and regeneration of dorsal root ganglion (DRG) afferents has been explored in lesion experiments by comparing immunocytochemistry for nitric oxide synthase (NOS) with that for the growth-associated phosphoprotein 43 (GAP-43). Sciatic nerve ligature induced a progressive increase in the number of small DRG cell profiles immunopositive for NOS between 2 days and 4 weeks of survival. In the proximal stump of the ligature, NOS-immunopositive fibers began to appear 2 days after injury and their growth cones were especially evident after 7 days. NOS-immunopositive fibers appeared past (i.e., distal to) the ligature at 14 days of survival and extended for at least 6 mm in either direction 4 weeks after the lesion. Dorsal root ligature alone at L4–L5 did not result in expression of NOS in DRG neurons or in the appearence of NOS-immunopositive fibers. In rats with dorsal root ligature and nerve ligature, the results were similar to those with nerve ligature only. DRG cell profiles immunopositive for GAP-43 kept increasing from 2 days to 4 weeks after sciatic nerve ligature and included small neurons initially and large neurons subsequently. Numerous axons became GAP-43 immunopositive on both sides of the ligature from 2 days after injury. In double-labeled material, about 80% of DRG cell profiles immunopositive for NOS were also immunopositive for GAP-43. The two antigens co-occurred in peripheral nerve axons proximal to the ligature starting at about 7 days and distal to it at about 2 weeks after ligature. Thus, in response to nerve lesion, nitric oxide may not only provide an injury signal to the central nervous system but may also contribute to the growth and regeneration of injured axons. J. Comp. Neurol. 404:64–74, 1999. © 1999 Wiley-Liss, Inc.     

去甲肾上腺素对背根神经节神经元P物质释放的调节作用(英文)

目的观察激活或抑制α-肾上腺素受体是否影响体外培养的背根神经节(dorsal root ganglion,DRG)神经元P物质(substance P,SP)的释放。方法胎龄15天的Wistar大鼠DRG神经元培养2天后,分别用去甲肾上腺素(nora-drenaline,NA)(1×10-4mol/L)、α1-受体拮抗剂哌唑嗪(1×10-6mol/L)+NA(1×10-4mol/L)、α2-受体拮抗剂育亨宾(1×10-5mol/L)+NA(1×10-4mol/L)孵育4天。用RT-PCR法检测DRG神经元编码SP蛋白的PPTmRNA表达水平,用Western blot法检测DRG神经元SP蛋白的表达水平,用酶联免疫吸附测定法检测SP的基础释放量和辣椒素刺激后的释放量。结果 NA单独孵育显著增加了DRG神经元辣椒素刺激后的SP释放量,α1-受体拮抗剂哌唑嗪预处理可阻断NA的效应,而α2-受体拮抗剂育亨宾不产生此作用。在各种实验条件下,PPT mRNA水平、SP蛋白表达水平和SP的基础释放量没有显著性差异。结论 NA通过激活α1-受体增加了DRG神经元辣椒素刺激后的SP释放量,这一作用可能与去甲肾上腺素能的疼痛调...     

Activation of calcium channel currents in rat sensory neurons by large depolarizations: effect of Guanine nucleotides and (-)-baclofen

Calcium channel currents have been recorded from cultured rat sensory neurons at clamp potentials of between -30 and +120 mV. At large depolarizing potentials between +50 and +120 mV, the current was outward. This outward current was shown to be largely due to ions passing through calcium channels, because it was substantially although generally incompletely blocked by Cd2+ (1 mM) and omega-conotoxin (1 microM). Internal GTP-gamma-S (100 microM) and to a lesser extent GTP (1 mM) reduced the amplitude and slowed the activation of the outward, as well as the inward calcium channel current. Baclofen (100 microM) reversibly inhibited both the inward and outward currents. These results suggest that the effect of baclofen and G protein activation on calcium channel currents is not due to a shift in the voltage-dependence of channel availability.     

Localization of NADPH-diaphorase-containing neurons in sensory ganglia of the rat

The presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity was studied histochemically in the sensory ganglia of the rat. Supraspinally, the trigeminal ganglion possessed only a few cells positively stained for NADPH-diaphorase, while a large number of positive neurons was found in the nodose ganglion. In the dorsal root ganglia, the distribution of positive cells showed a peculiar pattern in relation to spinal levels. Very minor populations (less than 2% of the total ganglionic cells) exhibited positive reaction in ganglia at levels ranging from the first cervical (C1) to fourth thoracic (T4) and from the second lumber (L2) through the entire sacral levels. In the middle to lower thoracic levels (from T5 to L1), however, abundant diaphorase-positive cells were observed. From these positive neurons it was possible to trace intensely stained nerve fibers. In the lower thoracic level, for example, dense positive fibers were seen in the ramus communicans. Retrograde tracing studies revealed that diaphorase-containing neurons in the lower thoracic level project at least partly to the gastric wall and the celiac ganglion. These results indicate that the diaphorase-positive ganglionic neurons in the thoracicolumbar levels may carry autonomic visceral afferent information. Double staining with NADPH-diaphorase histochemistry and peptide immunohistochemistry revealed that NADPH-diaphorase colocalizes with calcitonin gene-related peptide and substance P in many of these visceral afferent neurons.     

Expression of oncostatin M receptor beta in a specific subset of nociceptive sensory neurons

Oncostatin M belongs to the interleukin-6 family of cytokines and acts as a multifunctional cytokine during murine embryogenesis and in inflammatory reactions. Although it has been demonstrated that oncostatin M has biological activities on many types of cells, including hepatocytes, dermal fibroblasts and endothelial cells, the roles of oncostatin M in the murine peripheral nervous system remain unclear. Here, we investigated the expression of specific beta-subunit of oncostatin M receptor in the dorsal root ganglia of adult mice. In the adult dorsal root ganglia, beta-subunit of oncostatin M receptor was exclusively expressed in small-sized neurons. Approximately 13% of total dorsal root ganglia neurons in mice contained beta-subunit of oncostatin M receptor. The double-immunofluorescence method revealed that approximately 28% of beta-subunit of oncostatin M receptor-positive neurons contained TrkA immunoreactivity, 63% expressed Ret immunoreactivity and 58% bound isolectin B4. No neuropeptides, including substance P and calcitonin gene-related peptide, were contained in the neurons. In addition, all beta-subunit of oncostatin M receptor-positive neurons expressed both vanilloid receptor 1 and P2X3 purinergic receptor. These neurons projected to the inner portion of lamina II in the dorsal horn of spinal cord and the dermis of skin. Seven days after sciatic nerve axotomy, the expression of beta-subunit of oncostatin M receptor was down-regulated in the lumbar dorsal root ganglia of the injured side. Our study demonstrated that beta-subunit of oncostatin M receptor was expressed in both cell bodies and processes of nonpeptidergic nociceptive neurons in adult mice, suggesting that oncostatin M may affect the nociceptive function of the neurons through the modulation of vanilloid receptor 1 and P2X3 expression.     

Insulin induces cobalt uptake in a subpopulation of rat cultured primary sensory neurons

Previous findings show that both the vanilloid receptor 1 and the insulin receptor are expressed on small primary sensory neurons. As insulin evokes activity in second messengers which could induce opening of the vanilloid receptor 1, we examined, by using the cobalt-uptake technique, whether or not insulin can activate cultured rat primary sensory neurons through activating the vanilloid receptor 1. Capsaicin (50, 100 and 500 nm) induced concentration-dependent labelling in primary sensory neurons. Preincubation of cells in insulin (10 micromoles) for 10 min followed by a 2-min wash did not produce significant change in the capsaicin-induced labelling. Coapplication of insulin (10 micromoles) with capsaicin, however, potentiated the 50 and 100 nm capsaicin-evoked staining. Insulin itself also produced cobalt labelling in a concentration-dependent manner. The size-frequency distributions of neurons showing capsaicin- or insulin-induced cobalt accumulation were similar. The insulin-induced cobalt labelling was significantly reduced by the tyrosine kinase inhibitor, tyrphostin AG1024, the vanilloid receptor 1 antagonists, ruthenium red and capsazepine, the protein kinase inhibitor, staurosporine and the phospholipase C inhibitor neomycin. Double immunostaining of cultured primary sensory neurons and sections from dorsal root ganglia revealed that about one-third of the cells coexpress the insulin receptor and vanilloid receptor 1. These findings suggest that insulin activates a subpopulation of primary sensory neurons, probably through phosphorylation- and/or phosphatidylinositol(4,5)biphosphate hydrolysis-evoked activation of the vanilloid receptor 1. Although the insulin-induced activation of vanilloid receptor 1 seems to be a short-lived effect in vitro, in vivo it might play a role in the development of burning pain sensation in hyperinsulinism.     

Endomorphin-2 is released from newborn rat primary sensory neurons in a frequency- and calcium-dependent manner

Recent evidence indicates that endomorphins, endogenous mu-opioid receptor (MOR) agonists, modulate synaptic transmission in both somatic and visceral sensory pathways. Here we show that endomorphin-2 (END-2) is expressed in newborn rat dorsal root ganglion (DRG) and nodose-petrosal ganglion complex (NPG) neurons, and rarely co-localizes with brain-derived neurotrophic factor (BDNF). In order to examine activity-dependent release of END-2 from neurons, we established a model using dispersed cultures of DRG and NPG cells activated by patterned electrical field stimulation. To detect release of END-2, we developed a novel rapid capture enzyme-linked immunosorbent assay (ELISA), in which END-2 capture antibody was added to neuronal cultures shortly before their electrical stimulation. The conventional assay was effective at reliably detecting END-2 only when the cells were stimulated in the presence of CTAP, a MOR-selective antagonist. This suggests that the strength of the novel assay is related primarily to rapid capture of released END-2 before it binds to endogenous MORs. Using the rapid capture ELISA, we found that stimulation protocols known to induce plastic changes at sensory synapses were highly effective at releasing END-2. Removal of extracellular calcium or blocking voltage-activated calcium channels significantly reduced the release. Together, our data provide the first evidence that END-2 is expressed by newborn DRG neurons of all sizes found in this age group, and can be released from these, as well as from NPG neurons, in an activity-dependent manner. These results point to END-2 as a likely mediator of activity-dependent plasticity in sensory pathways.     

Inflammation alters somatostatin mRNA expression in sensory neurons in the rat

Proinflammatory neuropeptides, such as substance P and calcitonin gene-related peptide, are up-regulated in primary afferent neurons in acute and chronic inflammation. While these neuropeptides have been intensively studied, potentially anti-inflammatory and/or anti-nociceptive neuropeptides such as somatostatin (SS) have been less widely investigated. Endogenous somatostatin is thought to exert a tonic antinociceptive effect. Exogenous SS is anti-inflammatory and antinociceptive and is thought to exert these actions through inhibition of proinflammatory neuropeptide release. In this study we have compared the expression of somatostatin in two inflammatory models: arthritis, a condition associated with increased nociception, and periodontitis, in which there is little evidence of altered nociceptive thresholds. In acute arthritis (< 24 h) SS mRNA was down-regulated in ipsilateral dorsal root ganglia (DRG; 52 +/- 7% of control, P < 0.05), and up-regulated in contralateral DRG (134 +/- 10% of control; P < 0.05). In chronic arthritis (14 days) this pattern of mRNA regulation was reversed, with SS being up-regulated ipsilaterally and down-regulated contralaterally. In chronic mandibular periodontitis (7-10 days), SS mRNA was up-regulated in only the mandibular division of the ipsilateral trigeminal ganglion (TG) (day 7, 219 +/- 9% and day 10, 217 +/- 12% of control; P < 0.02) but showed no change in other divisions of the trigeminal ganglion or in the mesencephalic nucleus. These data show that antinociceptive and anti-inflammatory neuropeptides are also regulated in inflammation. It is possible that the degree of inflammation and nociception seen may depend on the balance of pro- and anti-inflammatory and nociceptive peptide expression in a particular condition.