The role of TRPV1 in different subtypes of dorsal root ganglion neurons in rat chronic inflammatory nociception induced by complete Freund's adjuvant

Background

The aberrant release of the neurotransmitters, glutamate and calcitonin-gene related peptide (CGRP), from trigeminal neurons has been implicated in migraine. The voltage-gated P/Q-type calcium channel has a critical role in controlling neurotransmitter release and has been linked to Familial Hemiplegic Migraine. Therefore, we examined the importance of voltage-dependent calcium channels in controlling release of glutamate and CGRP from trigeminal ganglion neurons isolated from male and female rats and grown in culture. Serotonergic pathways are likely involved in migraine, as triptans, a class of 5-HT₁ receptor agonists, are effective in the treatment of migraine and their effectiveness may be due to inhibiting neurotransmitter release from trigeminal neurons. We also studied the effect of serotonin receptor activation on release of glutamate and CGRP from trigeminal neurons grown in culture.

Results

P/Q-, N- and L-type channels each mediate a significant fraction of potassium-stimulated release of glutamate and CGRP. We determined that 5-HT significantly inhibits potassium-stimulated release of both glutamate and CGRP. Serotonergic inhibition of both CGRP and glutamate release can be blocked by pertussis toxin and NAS-181, a 5-HT_1B/1D antagonist. Stimulated release of CGRP is unaffected by Y-25130, a 5-HT₃ antagonist and SB 200646, a 5-HT_2B/2C antagonist.

Conclusion

These data suggest that release of both glutamate and CGRP from trigeminal neurons is controlled by calcium channels and modulated by 5-HT signaling in a pertussis-toxin dependent manner and probably via 5-HT₁ receptor signaling. This is the first characterization of glutamate release from trigeminal neurons grown in culture.     

Hot receptors in the brain

Background

The complex neuronal circuitry of the dorsal horn of the spinal cord is as yet poorly understood. However, defining the circuits underlying the transmission of information from primary afferents to higher levels is critical to our understanding of sensory processing. In this study, we have examined phosphodiesterase 1C (Pde1c) BAC transgenic mice in which a green fluorescent protein (GFP) reporter gene reflects Pde1c expression in sensory neuron subpopulations in the dorsal root ganglia and spinal cord.

Results

Using double labeling immunofluorescence, we demonstrate GFP expression in specific subpopulations of primary sensory neurons and a distinct neuronal expression pattern within the spinal cord dorsal horn. In the dorsal root ganglia, their distribution is restricted to those subpopulations of primary sensory neurons that give rise to unmyelinated C fibers (neurofilament 200 negative). A small proportion of both non-peptidergic (IB4-binding) and peptidergic (CGRP immunoreactive) subclasses expressed GFP. However, GFP expression was more common in the non-peptidergic than the peptidergic subclass. GFP was also expressed in a subpopulation of the primary sensory neurons immunoreactive for the vanilloid receptor TRPV1 and the ATP-gated ion channel P2X₃. In the spinal cord dorsal horn, GFP positive neurons were largely restricted to lamina I and to a lesser extent lamina II, but surprisingly did not coexpress markers for key neuronal populations present in the superficial dorsal horn.

Conclusion

The expression of GFP in subclasses of nociceptors and also in dorsal horn regions densely innervated by nociceptors suggests that Pde1c marks a unique subpopulation of nociceptive sensory neurons.     

Chronic morphine administration enhances nociceptive sensitivity and local cytokine production after incision

Background

Recent evidence suggests that oxytocin (OT), secreted in the superficial spinal cord dorsal horn by descending axons of paraventricular hypothalamic nucleus (PVN) neurons, produces antinociception and analgesia. The spinal mechanism of OT is, however, still unclear and requires further investigation. We have used patch clamp recording of lamina II neurons in spinal cord slices and immunocytochemistry in order to identify PVN-activated neurons in the superficial layers of the spinal cord and attempted to determine how this neuronal population may lead to OT-mediated antinociception.

Results

We show that OT released during PVN stimulation specifically activates a subpopulation of lamina II glutamatergic interneurons which are localized in the most superficial layers of the dorsal horn of the spinal cord (lamina I-II). This OT-specific stimulation of glutamatergic neurons allows the recruitment of all GABAergic interneurons in lamina II which produces a generalized elevation of local inhibition, a phenomenon which might explain the reduction of incoming Aδ and C primary afferent-mediated sensory messages.

Conclusion

Our results obtained in lamina II of the spinal cord provide the first clear evidence of a specific local neuronal network that is activated by OT release to induce antinociception. This OT-specific pathway might represent a novel and interesting therapeutic target for the management of neuropathic and inflammatory pain.     

Delayed functional expression of neuronal chemokine receptors following focal nerve demyelination in the rat: a mechanism for the development of chronic sensitization of peripheral nociceptors

Background

Animal and clinical studies have revealed that focal peripheral nerve axon demyelination is accompanied by nociceptive pain behavior. C-C and C-X-C chemokines and their receptors have been strongly implicated in demyelinating polyneuropathies and persistent pain syndromes. Herein, we studied the degree to which chronic nociceptive pain behavior is correlated with the neuronal expression of chemokines and their receptors following unilateral lysophosphatidylcholine (LPC)-induced focal demyelination of the sciatic nerve in rats.

Results

Focal nerve demyelination increased behavioral reflex responsiveness to mechanical stimuli between postoperative day (POD) 3 and POD28 in both the hindpaw ipsilateral and contralateral to the nerve injury. This behavior was accompanied by a bilateral increase in the numbers of primary sensory neurons expressing the chemokine receptors CCR2, CCR5, and CXCR4 by POD14, with no change in the pattern of CXCR3 expression. Significant increases in the numbers of neurons expressing the chemokines monocyte chemoattractant protein-1 (MCP-1/CCL2), Regulated on Activation, Normal T Expressed and Secreted (RANTES/CCL5) and interferon γ-inducing protein-10 (IP-10/CXCL10) were also evident following nerve injury, although neuronal expression pattern of stromal cell derived factor-1α (SDF1/CXCL12) did not change. Functional studies demonstrated that acutely dissociated sensory neurons derived from LPC-injured animals responded with increased [Ca²⁺]_i following exposure to MCP-1, IP-10, SDF1 and RANTES on POD 14 and 28, but these responses were largely absent by POD35. On days 14 and 28, rats received either saline or a CCR2 receptor antagonist isomer (CCR2 RA-[R]) or its inactive enantiomer (CCR2 RA-[S]) by intraperitoneal (i.p.) injection. CCR2 RA-[R] treatment of nerve-injured rats produced stereospecific bilateral reversal of tactile hyperalgesia.

Conclusion

These results suggest that the presence of chemokine signaling by both injured and adjacent, uninjured sensory neurons is correlated with the maintenance phase of a persistent pain state, suggesting that chemokine receptor antagonists may be an important therapeutic intervention for chronic pain.     

Protein phosphatase 2A regulates central sensitization in the spinal cord of rats following intradermal injection of capsaicin

Background

The latero-capsular part of the central nucleus of the amygdala (CeLC) is the target of the spino-parabrachio-amygdaloid pain pathway. Our previous studies showed that CeLC neurons develop synaptic plasticity and increased neuronal excitability in the kaolin/carrageenan model of arthritic pain. These pain-related changes involve presynaptic group I metabotropic glutamate receptors (mGluRs) and postsynaptic NMDA and calcitonin gene-related peptide (CGRP1) receptors. Here we address the role of group II mGluRs.

Results

Whole-cell current- and voltage-clamp recordings were made from CeLC neurons in brain slices from control rats and arthritic rats (>6 h postinjection of kaolin/carrageenan into the knee). Monosynaptic excitatory postsynaptic currents (EPSCs) were evoked by electrical stimulation of afferents from the pontine parabrachial (PB) area. A selective group II mGluR agonist (LY354740) decreased the amplitude of EPSCs more potently in CeLC neurons from arthritic rats (IC₅₀ = 0.59 nM) than in control animals (IC₅₀ = 15.0 nM). The inhibitory effect of LY354740 was reversed by a group II mGluR antagonist (EGLU) but not a GABA_A receptor antagonist (bicuculline). LY354740 decreased frequency, but not amplitude, of miniature EPSCs in the presence of TTX. No significant changes of neuronal excitability measures (membrane slope conductance and action potential firing rate) were detected.

Conclusion

Our data suggest that group II mGluRs act presynaptically to modulate synaptic plasticity in the amygdala in a model of arthritic pain.     

Hypertriglyceridemia: a too long unfairly neglected major cardiovascular risk factor

Background

Cardiomyopathy and distal symmetrical polyneuropathy (DSPN), including sensory and autonomic dysfunction, often co-occur in diabetic mellitus (DM) patients. However, the temporal relationship and progression between these two complications has not been investigated. Using a streptozotocin DM animal model that develops insensate neuropathy, our aim was to examine in parallel the development of DSPN and DM-associated changes in cardiac structure and function as well as potential mechanisms, such as autonomic dysfunction, evaluated by changes in urinary and myocardial norepinephrine content and myocardial neuronal markers.

Methods

Sensory neuropathy was measured by behavioral tests using Von Frey filaments and Hargreaves methods. Echocardiography was used to evaluate myocardial structure and function. Autonomic function was evaluated by measuring urinary and myocardial norepinephrine (NE) levels by enzyme-linked immunosorbent assay and high-performance liquid chromatography/mass spectrometry. Quantitative immunohistochemistry was used to measure the myocardial neuronal markers, calcitonin gene-related peptide (CGRP) and general neuronal protein gene product 9.5 (PGP 9.5).

Results

The DM group developed tactile and thermal insensate neuropathy 4–5 weeks after DM onset. Cardiovascular changes were found between 4 and 12 weeks after DM onset and included bradycardia, diastolic and systolic dysfunction and cardiac dilation. There was a 2.5-fold reduction in myocardial NE levels and a 5-fold increase in urinary NE levels in the DM group. Finally, there was a 2.3-fold increase in myocardial CGRP levels in the DM group and no change in PGP9.5 levels.

Conclusions

Cardiovascular structural and functional changes developed early in the course of DM and in combination with insensate neuropathy. In parallel, signs of cardiac autonomic dysfunction were also found and included decreased myocardial NE levels and altered CGRP levels. These results may indicate the need for early cardiovascular evaluation in DM patients with insensate neuropathy.     

Glial cell line-derived neurotrophic factor family ligands and their therapeutic potential

Four glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) have been characterized: GDNF, neurturin (NRTN), artemin (ARTN) and persephin (PSPN). These proteins support and restore multiple neuronal populations such as dopaminergic, sensory, motor, hippocampal, basal forebrain, enteric, sympathetic and parasympathetic neurons. Therefore, GFLs attracted significant attention as a potential cure for the diseases caused by neuronal injury and degeneration. Results of multiple experiments indicate that GFLs can alleviate behavioral symptoms and restore affected neurons in animal models of several neurological disorders including, among others, Parkinson’s disease (PD). During the last decade, GDNF protein and NRTN gene therapy have been tested in several clinical trials in patients with PD. Although the results of phase I clinical trials were positive, phase II clinical trials failed to reach primary end-points. Poor pharmacokinetic properties of GFLs (inability to penetrate tissues barriers, high affinity for extracellular matrix, etc.) could contribute to the absence of clear clinical benefits of these proteins for the patients. The purpose of this paper was to review therapeutic potential of GFLs and discuss possibilities to overcome difficulties associated with pharmacokinetic properties and delivery of GFLs to target neurons.     

Inhibition of Neuronal Apoptosis and Axonal Regression Ameliorates Sympathetic Atrophy and Hemodynamic Alterations in Portal Hypertensive Rats

Background and Aim

A neuronal pathway participates in the development of portal hypertension: blockade of afferent sensory nerves in portal vein ligated (PVL) rats simultaneously prevents brain cardiovascular regularory nuclei activation, neuromodulator overexpression in superior mesenteric ganglia, sympathetic atrophy of mesenteric innervation and hemodynamic alterations. Here we investigated in PVL rats alterations in neuromodulators and signaling pathways leading to axonal regression or apoptosis in the superior mesenteric ganglia and tested the effects of the stimulation of neuronal proliferation/survival by using a tyrosine kinase receptor A agonist, gambogic amide.

Results

The neuronal pathway was confirmed by an increased neuronal afferent activity at the vagal nodose ganglia and the presence of semaphorin3A in sympathetic pre-ganglionic neurons at the intermediolateral nucleus of the spinal cord of PVL rats. Expression of the active form of tyrosine kinase receptor A (phosphorylated), leading to proliferation and survival signaling, showed a significant reduction in PVL comparing to sham rats. In contrast, the apoptotic and axonal retraction pathways were stimulated in PVL, demonstrated by a significant overexpression of semaphorin 3A and its receptor neuropilin1, together with increases of cleaved caspase7, inactive poly(ADP-ribose) polymerase and Rho kinase expression. Finally, the administration of gambogic amide in PVL rats showed an amelioration of hemodynamic alterations and sympathetic atrophy, through the activation of survival pathways together with the inhibition of apoptotic cascades and Rho kinase mediated axonal regression.

Conclusion

The adrenergic alteration and sympathetic atrophy in mesenteric vessels during portal hypertension is caused by alterations on neuromodulation leading to post-ganglionic sympathetic regression and apoptosis and contributing to splanchnic vasodilation.     

Genetic reduction of chronic muscle pain in mice lacking calcium/calmodulin-stimulated adenylyl cyclases

Background

Numerous studies have implicated spinal extracellular signal-regulated kinases (ERKs) as mediators of nociceptive plasticity. These studies have utilized pharmacological inhibition of MEK to demonstrate a role for ERK signaling in pain, but this approach cannot distinguish between effects of ERK in neuronal and non-neuronal cells. The present studies were undertaken to test the specific role of neuronal ERK in formalin-induced inflammatory pain. Dominant negative MEK (DN MEK) mutant mice in which MEK function is suppressed exclusively in neurons were tested in the formalin model of inflammatory pain.

Results

Formalin-induced second phase spontaneous pain behaviors as well as thermal hyperalgesia measured 1 – 3 hours post-formalin were significantly reduced in the DN MEK mice when compared to their wild type littermate controls. In addition, spinal ERK phosphorylation following formalin injection was significantly reduced in the DN MEK mice. This was not due to a reduction of the number of unmyelinated fibers in the periphery, since these were almost double the number observed in wild type controls. Further examination of the effects of suppression of MEK function on a downstream target of ERK phosphorylation, the A-type potassium channel, showed that the ERK-dependent modulation of the A-type currents is significantly reduced in neurons from DN MEK mice compared to littermate wild type controls.

Conclusion

Our results demonstrate that the neuronal MEK-ERK pathway is indeed an important intracellular cascade that is associated with formalin-induced inflammatory pain and thermal hyperalgesia.     

Exosomal cell-to-cell transmission of alpha synuclein oligomers

Background

Neurotrophins and their receptors regulate several aspects of the developing and mature nervous system, including neuronal morphology and survival. Neurotrophin receptors are active in signaling endosomes, which are organelles that propagate neurotrophin signaling along neuronal processes. Defects in the Npc1 gene are associated with the accumulation of cholesterol and lipids in late endosomes and lysosomes, leading to neurodegeneration and Niemann-Pick type C (NPC) disease. The aim of this work was to assess whether the endosomal and lysosomal alterations observed in NPC disease disrupt neurotrophin signaling. As models, we used i) NPC1-deficient mice to evaluate the central cholinergic septo-hippocampal pathway and its response to nerve growth factor (NGF) after axotomy and ii) PC12 cells treated with U18666A, a pharmacological cellular model of NPC, stimulated with NGF.

Results

NPC1-deficient cholinergic cells respond to NGF after axotomy and exhibit increased levels of choline acetyl transferase (ChAT), whose gene is under the control of NGF signaling, compared to wild type cholinergic neurons. This finding was correlated with increased ChAT and phosphorylated Akt in basal forebrain homogenates. In addition, we found that cholinergic neurons from NPC1-deficient mice had disrupted neuronal morphology, suggesting early signs of neurodegeneration. Consistently, PC12 cells treated with U18666A presented a clear NPC cellular phenotype with a prominent endocytic dysfunction that includes an increased size of TrkA-containing endosomes and reduced recycling of the receptor. This result correlates with increased sensitivity to NGF, and, in particular, with up-regulation of the Akt and PLC-?? signaling pathways, increased neurite extension, increased phosphorylation of tau protein and cell death when PC12 cells are differentiated and treated with U18666A.

Conclusions

Our results suggest that the NPC cellular phenotype causes neuronal dysfunction through the abnormal up-regulation of survival pathways, which causes the perturbation of signaling cascades and anomalous phosphorylation of the cytoskeleton.     

Role of calcitonin gene-related peptide in bone repair after cyclic fatigue loading

Background

Calcitonin gene related peptide (CGRP) is a neuropeptide that is abundant in the sensory neurons which innervate bone. The effects of CGRP on isolated bone cells have been widely studied, and CGRP is currently considered to be an osteoanabolic peptide that has effects on both osteoclasts and osteoblasts. However, relatively little is known about the physiological role of CGRP in-vivo in the skeletal responses to bone loading, particularly fatigue loading.

Methodology/Principal Findings

We used the rat ulna end-loading model to induce fatigue damage in the ulna unilaterally during cyclic loading. We postulated that CGRP would influence skeletal responses to cyclic fatigue loading. Rats were fatigue loaded and groups of rats were infused systemically with 0.9% saline, CGRP, or the receptor antagonist, CGRP_8–37, for a 10 day study period. Ten days after fatigue loading, bone and serum CGRP concentrations, serum tartrate-resistant acid phosphatase 5b (TRAP5b) concentrations, and fatigue-induced skeletal responses were quantified. We found that cyclic fatigue loading led to increased CGRP concentrations in both loaded and contralateral ulnae. Administration of CGRP_8–37 was associated with increased targeted remodeling in the fatigue-loaded ulna. Administration of CGRP or CGRP_8–37 both increased reparative bone formation over the study period. Plasma concentration of TRAP5b was not significantly influenced by either CGRP or CGRP_8–37 administration.

Conclusions

CGRP signaling modulates targeted remodeling of microdamage and reparative new bone formation after bone fatigue, and may be part of a neuronal signaling pathway which has regulatory effects on load-induced repair responses within the skeleton.     

IL-1β stimulates COX-2 dependent PGE₂ synthesis and CGRP release in rat trigeminal ganglia cells

Objective

Pro-inflammatory cytokines like Interleukin-1 beta (IL-1β) have been implicated in the pathophysiology of migraine and inflammatory pain. The trigeminal ganglion and calcitonin gene-related peptide (CGRP) are crucial components in the pathophysiology of primary headaches. 5-HT1B/D receptor agonists, which reduce CGRP release, and cyclooxygenase (COX) inhibitors can abort trigeminally mediated pain. However, the cellular source of COX and the interplay between COX and CGRP within the trigeminal ganglion have not been clearly identified.

Methods and Results

1. We used primary cultured rat trigeminal ganglia cells to assess whether IL-1β can induce the expression of COX-2 and which cells express COX-2. Stimulation with IL-1β caused a dose and time dependent induction of COX-2 but not COX-1 mRNA. Immunohistochemistry revealed expression of COX-2 protein in neuronal and glial cells. 2. Functional significance was demonstrated by prostaglandin E2 (PGE₂) release 4 hours after stimulation with IL-1β, which could be aborted by a selective COX-2 (parecoxib) and a non-selective COX-inhibitor (indomethacin). 3. Induction of CGRP release, indicating functional neuronal activation, was seen 1 hour after PGE₂ and 24 hours after IL-1β stimulation. Immunohistochemistry showed trigeminal neurons as the source of CGRP. IL-1β induced CGRP release was blocked by parecoxib and indomethacin, but the 5-HT1B/D receptor agonist sumatriptan had no effect.

Conclusion

We identified a COX-2 dependent pathway of cytokine induced CGRP release in trigeminal ganglia neurons that is not affected by 5-HT1B/D receptor activation. Activation of neuronal and glial cells in the trigeminal ganglion by IL-β leads to an elevated expression of COX-2 in these cells. Newly synthesized PGE₂ (by COX-2) in turn activates trigeminal neurons to release CGRP. These findings support a glia-neuron interaction in the trigeminal ganglion and demonstrate a sequential link between COX-2 and CGRP. The results could help to explain the mechanism of action of COX-2 inhibitors in migraine.     

Saturated free fatty acids and apoptosis in microvascular mesangial cells: palmitate activates pro-apoptotic signaling involving caspase 9 and mitochondrial release of endonuclease G

Background

In type 2 diabetes, free fatty acids (FFA) accumulate in microvascular cells, but the phenotypic consequences of FFA accumulation in the microvasculature are incompletely understood. Here we investigated whether saturated FFA induce apoptosis in human microvascular mesangial cells and analyzed the signaling pathways involved.

Methods

Saturated and unsaturated FFA-albumin complexes were added to cultured human mesangial cells, after which the number of apoptotic cells were quantified and the signal transduction pathways involved were delineated.

Results

The saturated FFA palmitate and stearate were apoptotic unlike equivalent concentrations of the unsaturated FFA oleate and linoleate. Palmitate-induced apoptosis was potentiated by etomoxir, an inhibitor of mitochondrial β-oxidation, but was prevented by an activator of AMP-kinase, which increases fatty acid β-oxidation. Palmitate stimulated an intrinsic pathway of pro-apoptotic signaling as evidenced by increased mitochondrial release of cytochrome-c and activation of caspase 9. A caspase 9-selective inhibitor blocked caspase 3 activation but incompletely blocked apoptosis in response to palmitate, suggesting an additional caspase 9-independent pathway. Palmitate stimulated mitochondrial release of endonuclease G by a caspase 9-independent mechanism, thereby implicating endonuclease G in caspase 9-indpendent regulation of apoptosis by saturated FFA. We also observed that the unsaturated FFA oleate and linoleate prevented palmitate-induced mitochondrial release of both cytochrome-c and endonuclease G, which resulted in complete protection from palmitate-induced apoptosis.

Conclusions

Taken together, these results demonstrate that palmitate stimulates apoptosis by evoking an intrinsic pathway of proapoptotic signaling and identify mitochondrial release of endonuclease G as a key step in proapoptotic signaling by saturated FFA and in the anti-apoptotic actions of unsaturated FFA.     

Lentiviral gene transfer into the dorsal root ganglion of adult rats

Background

Neuronal hyperexcitability is a crucial phenomenon underlying spontaneous and evoked pain. In invertebrate nociceptors, the S-type leak K⁺ channel (analogous to TREK-1 in mammals) plays a critical role of in determining neuronal excitability following nerve injury. Few data are available on the role of leak K_2P channels after peripheral axotomy in mammals.

Results

Here we describe that rat sciatic nerve axotomy induces hyperexcitability of L4-L5 DRG sensory neurons and decreases TRESK (K2P18.1) expression, a channel with a major contribution to total leak current in DRGs. While the expression of other channels from the same family did not significantly change, injury markers ATF3 and Cacna2d1 were highly upregulated. Similarly, acute sensory neuron dissociation (in vitro axotomy) produced marked hyperexcitability and similar total background currents compared with neurons injured in vivo. In addition, the sanshool derivative IBA, which blocked TRESK currents in transfected HEK293 cells and DRGs, increased intracellular calcium in 49% of DRG neurons in culture. Most IBA-responding neurons (71%) also responded to the TRPV1 agonist capsaicin, indicating that they were nociceptors. Additional evidence of a biological role of TRESK channels was provided by behavioral evidence of pain (flinching and licking), in vivo electrophysiological evidence of C-nociceptor activation following IBA injection in the rat hindpaw, and increased sensitivity to painful pressure after TRESK knockdown in vivo.

Conclusions

In summary, our results clearly support an important role of TRESK channels in determining neuronal excitability in specific DRG neurons subpopulations, and show that axonal injury down-regulates TRESK channels, therefore contributing to neuronal hyperexcitability.     

Superoxide dismutase 1 encoding mutations linked to ALS adopts a spectrum of misfolded states

Background

Oxidative stress is a key pathophysiological mechanism contributing to degenerative processes in many neurodegenerative diseases and therefore, unraveling molecular mechanisms underlying various stages of oxidative neuronal damage is critical to better understanding the diseases and developing new treatment modalities. We previously showed that protein kinase C delta (PKCδ) proteolytic activation during the late stages of oxidative stress is a key proapoptotic signaling mechanism that contributes to oxidative damage in Parkinson's disease (PD) models. The time course studies revealed that PKCδ activation precedes apoptotic cell death and that cells resisted early insults of oxidative damage, suggesting that some intrinsic compensatory response protects neurons from early oxidative insult. Therefore, the purpose of the present study was to characterize protective signaling pathways in dopaminergic neurons during early stages of oxidative stress.

Results

Herein, we identify that protein kinase D1 (PKD1) functions as a key anti-apoptotic kinase to protect neuronal cells against early stages of oxidative stress. Exposure of dopaminergic neuronal cells to H₂O₂ or 6-OHDA induced PKD1 activation loop (PKD1S744/748) phosphorylation long before induction of neuronal cell death. Blockade of PKCδ cleavage, PKCδ knockdown or overexpression of a cleavage-resistant PKCδ mutant effectively attenuated PKD1 activation, indicating that PKCδ proteolytic activation regulates PKD1 phosphorylation. Furthermore, the PKCδ catalytic fragment, but not the regulatory fragment, increased PKD1 activation, confirming PKCδ activity modulates PKD1 activation. We also identified that phosphorylation of S916 at the C-terminal is a preceding event required for PKD1 activation loop phosphorylation. Importantly, negative modulation of PKD1 by the RNAi knockdown or overexpression of PKD1^S916A phospho-defective mutants augmented oxidative stress-induced apoptosis, while positive modulation of PKD1 by the overexpression of full length PKD1 or constitutively active PKD1 plasmids attenuated oxidative stress-induced apoptosis, suggesting an anti-apoptotic role for PKD1 during oxidative neuronal injury.

Conclusion

Collectively, our results demonstrate that PKCδ-dependent activation of PKD1 represents a novel intrinsic protective response in counteracting early stage oxidative damage in neuronal cells. Our results suggest that positive modulation of the PKD1-mediated compensatory protective mechanism against oxidative damage in dopaminergic neurons may provide novel neuroprotective strategies for treatment of PD.     

P2X7 receptors in satellite glial cells mediate high functional expression of P2X3 receptors in immature dorsal root ganglion neurons

Background

In addition to their central effects, opioids cause peripheral analgesia. There is evidence showing that peripheral activation of kappa opioid receptors (KORs) inhibits inflammatory pain. Moreover, peripheral ??-opioid receptor (MOR) activation are able to direct block PGE₂-induced ongoing hyperalgesia However, this effect was not tested for KOR selective activation. In the present study, the effect of the peripheral activation of KORs on PGE₂-induced ongoing hyperalgesia was investigated. The mechanisms involved were also evaluated.

Results

Local (paw) administration of U50488 (a selective KOR agonist) directly blocked, PGE₂-induced mechanical hyperalgesia in both rats and mice. This effect was reversed by treating animals with L-NMMA or N-propyl-L-arginine (a selective inhibitor of neuronal nitric oxide synthase, nNOS), suggesting involvement of the nNOS/NO pathway. U50488 peripheral effect was also dependent on stimulation of PI3K??/AKT because inhibitors of these kinases also reduced peripheral antinociception induced by U50488. Furthermore, U50488 lost its peripheral analgesic effect in PI3K?? null mice. Observations made in vivo were confirmed after incubation of dorsal root ganglion cultured neurons with U50488 produced an increase in the activation of AKT as evaluated by western blot analyses of its phosphorylated form. Finally, immunofluorescence of DRG neurons revealed that KOR-expressing neurons also express PI3K?? (? 43%).

Conclusions

The present study indicates that activation of peripheral KORs directly blocks inflammatory hyperalgesia through stimulation of the nNOS/NO signaling pathway which is probably stimulated by PI3K??/AKT signaling. This study extends a previously study of our group suggesting that PI3K??/AKT/nNOS/NO is an important analgesic pathway in primary nociceptive neurons.     

Aromatase and 5-alpha reductase gene expression: modulation by pain and morphine treatment in male rats

Background

Neuronal transduction by adeno-associated viral (AAV) vectors has been demonstrated in cortex, brainstem, cerebellum, and sensory ganglia. Intrathecal delivery of AAV serotypes that transduce neurons in dorsal root ganglia (DRG) and spinal cord offers substantial opportunities to 1) further study mechanisms underlying chronic pain, and 2) develop novel gene-based therapies for the treatment and management of chronic pain using a non-invasive delivery route with established safety margins. In this study we have compared expression patterns of AAV serotype 5 (AAV5)- and AAV serotype 8 (AAV8)-mediated gene transfer to sensory neurons following intrathecal delivery by direct lumbar puncture.

Results

Intravenous mannitol pre-treatment significantly enhanced transduction of primary sensory neurons after direct lumbar puncture injection of AAV5 (rAAV5-GFP) or AAV8 (rAAV8-GFP) carrying the green fluorescent protein (GFP) gene. The presence of GFP in DRG neurons was consistent with the following evidence for primary afferent origin of the majority of GFP-positive fibers in spinal cord: 1) GFP-positive axons were evident in both dorsal roots and dorsal columns; and 2) dorsal rhizotomy, which severs the primary afferent input to spinal cord, abolished the majority of GFP labeling in dorsal horn. We found that both rAAV5-GFP and rAAV8-GFP appear to preferentially target large-diameter DRG neurons, while excluding the isolectin-B4 (IB4) -binding population of small diameter neurons. In addition, a larger proportion of CGRP-positive cells was transduced by rAAV5-GFP, compared to rAAV8-GFP.

Conclusions

The present study demonstrates the feasibility of minimally invasive gene transfer to sensory neurons using direct lumbar puncture and provides evidence for differential targeting of subtypes of DRG neurons by AAV vectors.