Antagonism of ORLI receptor produces an algesic effect in the rat formalin test

The authors investigated the role of endogenously released nociceptin (also known as orphanin FQ) spinal and supraspinal nociceptive transmission during the rat formalin test by examining the effect of intrathecal and intracerebroventricular injection of J-113397, a non-peptidyl ORL1 receptor selective antagonist. When J-113397 was injected intrathecally or intracerebroventricularly 10 min before the formalin injection, it enhanced the agitation behavior induced by paw formalin injection. This suggested that paw formalin injection induced nociceptin release in the spinal cord and the supraspinal brain sites, that this endogenously released nociceptin produced an analgesic effect and that J-113397 antagonized this analgesic effect of nociceptin and produced an algesic effect in the rat formalin test.     

Noxious peripheral stimulation produces antinociception mediated via substance P and opioid mechanisms in the rat tail-flick test

Physiological experiments were run to examine the effects of noxious thermal stimulation of one hindpaw on the tail-flick reflex in the lightly anesthetized rat. Male Sprague-Dawley rats were anesthetized with an i.p. injection of a mixture of Na-pentobarbital (20 mg/kg) and chloral hydrate (120 mg/kg). After baseline readings were taken in the tail-flick test, either a non-noxious or a noxious stimulus was applied which consisted of immersion of one hindpaw in water at 40, 45, 50 or 55°C for 1.5 min. After immersion, tail-flick readings were taken at 3-min intervals for at least 16 min. Paw immersion in water at 55°C induced an antinociceptive response, consisting of an increase in the reaction time, at 0.5 min after immersion. Recovery to baseline levels occurred over the next 3–6 min. Immersion at lower temperatures provoked smaller antinociceptive responses, except at 40°C, where readings remained around the baseline values. The increase in reaction time in response to immersion at 55°C was attenuated or blocked by the novel, nonpeptide substance P (NK-1) receptor antagonist, CP-96,345, administered s.c. 30 or 60 min, respectively, prior to paw immersion. Similar injection of CP-96,344, the inactive stereoisomer, had no effect on the response, while another NK-1 receptor antagonist, CP-99,994, also attenuated the antinociceptive effect of the immersion. The increase in reaction time induced by immersion at 55°C was absent in animals treated neonatally with capsaicin. This nociceptive response was blocked by the broad spectrum opiate antagonist, naloxone (10 mg/kg i.p.) given 20 min before paw immersion. The data provide evidence that a noxious stimulus produces an intensity-related heterosegmental antinociceptive effect and that C-fibers, and NK-1 and opiate receptors are involved in mediating this effect.     

Association study of EP1 gene polymorphisms with suicide completers in the Japanese population

Background

Both environmental and genetic factors have been reported to be involved in suicidal behaviors. Considerable evidence indicates that impulsive aggression is one of the important risk factors that contribute to suicide. A recent study has shown that prostaglandin E2 type 1 receptor (EP1) signaling regulates impulsive-aggressive behaviors in mice under both social and environmental stresses. To test the possible involvement of the EP1 gene in suicide, we carried out an association study of EP1 gene polymorphisms with suicide completers in the Japanese population.

Methods

We studied 5 SNPs including one SNP in exon 2 (rs3745459) and four SNPs in the potential promoter region of the EP1 gene (rs3810255, rs3810254, rs3810253 and rs10416814) in 374 healthy control and 287 completed suicide victims using standard Taqman probe genotyping assays.

Results

No significant differences of the genotypic distribution, allelic frequency or haplotype distribution between controls and suicide completers were found. Gender based analysis revealed that genotypic, allelic and haplotypic distributions of rs3810255, rs3810254, rs3810253 and rs10416814 SNPs were significantly different between the female control and female suicide groups, although the differences did not withstand correction for multiple comparisons.

Conclusion

We could not find an association of EP1 gene with suicide in the Japanese population. Because several SNPs in the promoter region of the EP1 gene were nominally significantly associated with suicide in the female, further studies with a larger sample size and different population are needed to confirm this result.     

Induction of microsomal prostaglandin E synthase in the rat brain endothelium and parenchyma in adjuvant-induced arthritis

Although central nervous symptoms such as hyperalgesia, fatigue, malaise, and anorexia constitute major problems in the treatment of patients suffering from chronic inflammatory disease, little has been known about the signaling mechanisms by which the brain is activated during such conditions. Here, in an animal model of rheumatoid arthritis, we show that microsomal prostaglandin E-synthase, the inducible terminal isomerase in the prostaglandin E(2)-synthesizing pathway, is expressed in endothelial cells along the blood-brain barrier and in the parenchyma of the paraventricular hypothalamic nucleus. The endothelial cells but not the paraventricular hypothalamic cells displayed a concomitant induction of cyclooxygenase-2 and expressed interleukin-1 type 1 receptors, which indicates that the induction is due to peripherally released cytokines. In contrast to cyclooxygenase-2, microsomal prostaglandin E synthase had very sparse constitutive expression, suggesting that it could be a target for developing drugs that will carry fewer side effects than the presently available cyclooxygenase inhibitors. These findings, thus, suggest that immune-to-brain communication during chronic inflammatory conditions involves prostaglandin E2-synthesis both along the blood-brain barrier and in the parenchyma of the hypothalamic paraventricular nucleus and point to novel avenues for the treatment of the brain-elicited disease symptoms during these conditions.     

福尔马林皮下注入引起大鼠脊髓背角表层神经元Fos蛋白和蛋白激酶Cβ亚型的共同表达:免疫细胞化学双标法研究

目的:研究慢性伤害性模型大鼠脊髓背角表层神经元中Fos蛋白和蛋白激酶C β亚型的表达间的关系.方法:用免疫细胞化学双标技术,观察了大鼠一侧后脚掌注射福尔马林1 h后,脊髓腰段背角神经元中Fos蛋白和蛋白激酶C β亚型(PKCβ)的表达及二者的共存情况.结果:1)注射侧背角表层中有大量Fos蛋白样免疫组化染色阳性神经元出现,且其中一半以上同时有PKCβ样免疫反应的增强,而背角表层中有PKCβ样免疫反应增强的神经元中,只有1/5同时有Fos蛋白样阳性反应出现;2)在注射侧背角Ⅴ～Ⅶ层中也有少量Fos蛋白样阳性反应神经元出现,但它们都不出现PKCβ-LI反应的增强.结论:背角表层中的双标免疫阳性神经元可能与慢性伤害性输入引起的脊髓过敏状态的形成有关.     

Eccentric exercise induces chronic alterations in musculoskeletal nociception in the rat

Eccentric muscle exercise is a common cause of acute and chronic (lasting days to weeks) musculoskeletal pain. To evaluate the mechanisms involved, we have employed a model in the rat, in which eccentric hind limb exercise produces both acute mechanical hyperalgesia as well as long‐term changes characterized by enhanced hyperalgesia to subsequent exposure to an inflammatory mediator. Eccentric exercise of the hind limb produced mechanical hyperalgesia, measured in the gastrocnemius muscle, which returned to baseline at 120 h post‐exercise. When nociceptive thresholds had returned to baseline, intramuscular injection of prostaglandin E₂ (PGE₂) induced hyperalgesia that was unattenuated 240 h later, much longer than PGE₂‐induced hyperalgesia in unexercised rats (4 h). This marked prolongation of PGE₂ hyperalgesia induced by eccentric exercise was prevented by the spinal intrathecal injection of oligodeoxynucleotide antisense to protein kinase Cε, a second messenger in nociceptors implicated in the induction of chronic pain. Exercise‐induced hyperalgesia and prolongation of PGE₂ hyperalgesia were inhibited by the spinal intrathecal administration of antisense for the interleukin‐6 but not the tumor necrosis factor α type 1 receptor. These findings provide further insight into the mechanism underlying exercise‐induced chronic muscle pain, and suggest novel approaches for the prevention and treatment of exercise‐ or work‐related chronic musculoskeletal pain syndromes.     

Zinc protoporphyrin IX, an inhibitor of the enzyme that produces carbon monoxide, blocks spinal nociceptive transmission evoked by formalin injection in the rat

Carbon monoxide (CO) is now thought to act as a neural messenger, but the role of CO is poorly understood. In the present study, we investigated the role of CO in both the spinal thermal nociceptive transmission and spinal nociceptive transmission during peripheral inflammation using zinc protoporphyrin-IX (Zn-P), a potent inhibitor of hemoxygenase inhibitor (HO). Peripheral inflammation was induced by the paw formalin injection (formalin test). It is thought that CO acts as a second messenger for metabotropic glutamate receptor. We also investigated the effect of metabotropic glutamate receptor antagonists,l-2-amino-3-phosphonopropionic acid (d(+)AP3) and (RS)-α-methyl-4-carboxyphenylglycine (MCPG), on the formalin test. Drugs were administered intrathecally 5 min before (pre-treatment) or 7 min after (post-treatment) the formalin injection. In both the pre-treatment and the post-treatment study, Zn&z.sbnd;P inhibited the formalin induced flinching behavior in a dose-dependent manner. Bothl(+)AP3 and MCPG had no effect on the formalin induced flinching behavior in the pre-treatment study. Zn&z.sbnd;P had no effect on the thermal nociceptive test. These data suggest that CO plays an important role in spinal nociceptive transmission during the formalin test, but not during the thermal nociceptive test, and that, during the formalin test, CO may not act as a second messenger for metabotropic glutamate receptor in the spinal cord.     

Interaction between GSTM1-null and CYP2D6-deficient alleles in the pathogenesis of Parkinson''s disease

The present study was conducted to examine the interaction between cytochrome p450 2D6: CYP2D6 (phase I) poor metabolizer (PM) and glutathione S-transferase M1: GSTM1 (phase II) null genotypes, among 103 unrelated French Parkinson's disease (PD) patients. Both genes are involved in the biotransformation process, and the main objective of that work is to assess synergic effect between CYP2D6 PM and GSTM1 null genotypes in PD patients. Patients with both GSTM1 null genotype and poor metabolizer CYP2D6 have shown a strong dependency of multiplicative interaction (9.50; P = 0.016); this have also been observed when combining GSTM1 null with CYP2D6*4 deficient alleles, but were at the limit of significance (2.18; P = 0.076). Such a combination of polymorphic peculiarities in studied metabolic genes might represent additional risk factor for development of sporadic PD.     

Vesicular glutamate transporter 2 is required for the respiratory and parasympathetic activation produced by optogenetic stimulation of catecholaminergic neurons in the rostral ventrolateral medulla of mice in vivo

Catecholaminergic neurons of the rostral ventrolateral medulla (RVLM‐CA neurons; C1 neurons) contribute to the sympathetic, parasympathetic and neuroendocrine responses elicited by physical stressors such as hypotension, hypoxia, hypoglycemia, and infection. Most RVLM‐CA neurons express vesicular glutamate transporter (VGLUT)2, and may use glutamate as a ionotropic transmitter, but the importance of this mode of transmission in vivo is uncertain. To address this question, we genetically deleted VGLUT2 from dopamine‐β‐hydroxylase‐expressing neurons in mice [DβH^Cre/0;VGLUT2^flox/flox mice (cKO mice)]. We compared the in vivo effects of selectively stimulating RVLM‐CA neurons in cKO vs. control mice (DβH^Cre/0), using channelrhodopsin‐2 (ChR2–mCherry) optogenetics. ChR2–mCherry was expressed by similar numbers of rostral ventrolateral medulla (RVLM) neurons in each strain (~400 neurons), with identical selectivity for catecholaminergic neurons (90–99% colocalisation with tyrosine hydroxylase). RVLM‐CA neurons had similar morphology and axonal projections in DβH^Cre/0 and cKO mice. Under urethane anesthesia, photostimulation produced a similar pattern of activation of presumptive ChR2‐positive RVLM‐CA neurons in DβH^Cre/0 and cKO mice. Photostimulation in conscious mice produced frequency‐dependent respiratory activation in DβH^Cre/0 mice but no effect in cKO mice. Similarly, photostimulation under urethane anesthesia strongly activated efferent vagal nerve activity in DβH^Cre/0 mice only. Vagal responses were unaffected by α₁‐adrenoreceptor blockade. In conclusion, two responses evoked by RVLM‐CA neuron stimulation in vivo require the expression of VGLUT2 by these neurons, suggesting that the acute autonomic responses driven by RVLM‐CA neurons are mediated by glutamate.     

Activation of Cdk2-pRB-E2F1 cell cycle pathway by repeated electroconvulsive shock in the rat frontal cortex.

BACKGROUND: Recent reports indicate that repeated electroconvulsive shock (ECS) induces cortical cell proliferation, suggesting the possibility that ECS may activate cell cycle progression in the rat brain cortex. METHODS: Sprague-Dawley rats (150-200g) were divided into four treatment groups and then given sham treatment or ECS treatment for 1, 5, and 10 days, respectively. The activity of cyclin-dependent kinase 2 (Cdk2), phosphorylation, and total protein amount of cyclin D1, cyclin E, pocket retinoblastoma family of protein (pRB), and E2F1 were analyzed in the rat cerebral cortex. RESULTS: The activity of Cdk2, the protein amount of pRB, Ser795 phosphorylation of pRB, and the protein amount of E2F1 were all increased compared with the sham-treated control subjects, and these increases were enhanced with the increasing number of ECS. In contrast, the protein amounts of Cdk2, cyclin D1, and cyclin E were not changed by repeated ECS. CONCLUSIONS: The Cdk2-pRB-E2F1 cell cycle pathway is activated by repeated ECS in the rat frontal cortex.     

Differential effects of endocannabinoids on [(3)H]-GABA uptake in the rat globus pallidus

In the globus pallidus, cannabinoid CB(1) receptors are localized pre-synaptically on GABAergic neurons. We assessed the influence of the endocannabinoids, anandamide, 2-arachidonoyl-glycerol (2-AG) and noladin ether, on the uptake of [(3)H]-GABA in pallidal slices from rat. Both 2-AG and noladin ether increased [(3)H]-GABA uptake (by 40.8 +/- 8.0% and 38.4 +/- 12.5%). The effect of 2-AG was blocked by the cannabinoid CB(1) receptor antagonist AM 251. In contrast, neither anandamide nor the agonist WIN 55,212-2 had an effect on [(3)H]-GABA uptake. Different roles might be played by different endocannabinoids, both physiologically and in basal ganglia disorders, such as Parkinson's disease.     

LPS-induced Fos expression in oxytocin and vasopressin neurons of the rat hypothalamus

The aim of this study was to examine the involvement of the hypothalamic oxytocin (OXT) and vasopressin (AVP) neurons in acute phase reaction using quantitative dual-labeled immunostaining with Fos and either OXT and AVP in several hypothalamic regions. Administration of low dose (5 μg/kg) and high dose (125 μg/kg) of LPS induced intense nuclear Fos immunoreactivity in many OXT and AVP neurons in all the observed hypothalamic regions. The percentage of Fos-positive nuclei in OXT magnocellular neurons was higher than that of AVP magnocellular neurons in the supraoptic nucleus (SON), the magnocellular neurons in the paraventricular nucleus (magPVN), rostral SON (rSON), and nucleus circularis (NC), whose axons terminate at the posterior pituitary for peripheral release. The percentage of Fos-positive nuclei in AVP parvocellular neurons in the paraventricular nucleus (parPVN) was higher than that of OXT parvocellular neurons, whose axons terminate within the brain for central release. Moreover, the percentage of Fos-positive nuclei in AVP magnocellular neurons of the SON and rSON was significantly higher than that of the magPVN and NC when animals were given LPS via intraperitoneal (i.p.)-injection. This regional heterogeneity was not observed in OXT magnocellular neurons of i.p.-injected rats or in either OXT or AVP magnocellular neurons of intravenous (i.v.)-injected rats. The present data suggest that LPS-induced peripheral release of AVP and OXT is due to the activation of the magnocellular neurons in the SON, magPVN, NC, and rSON, and the central release of those hormones is in part derived from the activation of parvocellular neurons in the PVN. It is also suggested that the activation of AVP magnocellular neurons is heterogeneous among the four hypothalamic regions, but that of OXT magnocellular neurons is homogenous among these brain regions in response to LPS administration.     

Neuronal, astroglial and microglial cytokine expression after an excitotoxic lesion in the immature rat brain

Cytokines are important intercellular messengers involved in neuron-glia interactions and in the microglial-astroglial crosstalk, modulating the glial response to brain injury and the lesion outcome. In this study, excitotoxic lesions were induced by the injection of N-methyl-D-aspartate in postnatal day 9 rats, and the cytokines interleukin-1 beta (IL-1beta), interleukin-6 (IL-6), tumour necrosis factor alpha (TNFalpha) and transforming growth factor beta 1 (TGF-beta1) analysed by ELISA and/or immunohistochemistry. Moreover, cytokine-expressing glial cells were identified by means of double labelling with glial fibrillary acidic protein or tomato lectin binding. Our results show that both neurons and glia were capable of cytokine expression following different patterns in the excitotoxically damaged area vs. the nondegenerating surrounding grey matter (SGM). Excitotoxically damaged neurons showed upregulation of IL-6 and downregulation of TNFalpha and TGF-beta1 before they degenerated. Moreover, in the SGM, an increased expression of neuronal IL-6, TNFalpha and TGF-beta1 was observed. A subpopulation of microglial cells, located in the SGM and showing IL-1beta and TNFalpha expression, were the earliest glial cells producing cytokines, at 2-10 h postinjection. Later on, cytokine-positive glial cells were found within the excitotoxically damaged area and the adjacent white matter: some reactive astrocytes expressed TNFalpha and IL-6, and microglia/macrophages showed mild IL-1beta and TGF-beta1. Finally, the expression of all cytokines was observed in the glial scar. As discussed, this pattern of cytokine production suggests their implication in the evolution of excitotoxic neuronal damage and the associated glial response.     

Protein kinase CK2 enhances Mcl‐1 gene expression through the serum response factor‐mediated pathway in the rat hippocampus

The protein kinase CK2 (casein kinase 2) is a ubiquitous serine/threonine protein kinase that suppresses apoptosis. CK2 is composed of catalytic and regulatory subunits, and CK2‐dependent phosphorylation is a global mechanism in the inhibition of caspase signaling pathways. The serum response factor (SRF) is an important regulator of cell growth and differentiation. Although CK2 has been shown to phosphorylate SRF in vitro, the biological relevance of this interaction remains largely unclear. We observed increased SRF phosphorylation and increased Mcl‐1 gene expression in hippocampal CA1 neurons following transfection with a plasmid expressing the wild‐type CK2α (CK2αWT) protein, whereas transfection with a plasmid expressing a catalytically inactive mutant of CK2α (CK2α156A) reduced Mcl‐1 gene expression. Cotransfection with a plasmid expressing the inactive SRF99A mutant inhibited the CK2αWT‐induced upregulation of Mcl‐1 gene expression. The expression of either the CK2α156A or the SRF99A mutant also inhibited the glutamate‐induced upregulation of Mcl‐1 protein expression in PC12 cells. Our results suggest that CK2‐mediated signaling represents a cellular mechanism that may aid in the development of alternative therapeutic strategies to attenuate apoptosis in hippocampal neurons. © 2013 Wiley Periodicals, Inc.     

Increased expression of the close homolog of the adhesion molecule L1 in different cell types over time after rat spinal cord contusion

The close homolog of the adhesion molecule L1 (CHL1) is important during CNS development, but a study with CHL1 knockout mice showed greater functional recovery after spinal cord injury (SCI) in its absence. We investigated CHL1 expression from 1 to 28 days after clinically relevant contusive SCI in Sprague-Dawley rats. Western blot analysis showed that CHL1 expression was significantly up-regulated at day 1 and further increased over 4 weeks after SCI. Immunohistochemistry of tissue sections showed that CHL1 in the intact spinal cord was expressed at low levels. By 1 day and through 4 weeks after SCI, CHL1 became highly expressed in NG2(+) cells. Hypertrophic GFAP(+) astrocytes also expressed CHL1 by 1 week after injury. The increase in CHL1 protein paralleled that of NG2 in the first week and GFAP between 1 and 4 weeks after injury. At 4 weeks, NG2(+) /CHL1(+) cells and GFAP(+) /CHL1(+) astrocytes were concentrated at the boundary between residual spinal cord tissue and the central lesion. NF200(+) spinal cord axons approached but did not penetrate this boundary. In contrast, CHL1(+) cells in the central lesion at 1 week and later colabeled with p75 and NG2 and were chronically associated with many NF200(+) axons, presumably axons that had sprouted in association with CHL1(+) Schwann cells infiltrating the cord after contusion. Thus, our study demonstrates up-regulation of CHL1 in multiple cell types and locations in a rat model of contusion injury and suggests that this molecule may be involved both in inhibition of axonal regeneration and in recovery processes after SCI.     

A deficit in astroglial organization causes the impaired reactive sprouting in human apolipoprotein E4 targeted replacement mice

The epsilon4 allele of apolipoprotein (apo)E associates with an increased risk of developing Alzheimer's disease (AD) as well as an earlier age of onset. However, the exact mechanisms by which apoE4 confers such susceptibility is currently unknown. We used a human apoE targeted replacement (hE-TR) mouse model to investigate the allele-specific response to entorhinal cortex lesion (ECL). We observed a marked impairment in reactive sprouting in hE4 mice compared to hE3 mice. ApoE expression was similar between genotypes at days post-lesion (DPL) 2 and 14. Thirty days post-lesion, hE4 mice had more reactive astrocytes as well as a defective outward migration pattern of the astrocytes in the dentate gyrus. The expression of the anti-inflammatory cytokine IL-1ra was delayed in hE4 mice compared to hE3 mice. ApoE and beta-amyloid (Abeta) 1-40 accumulated at 30 DPL in hE4 mice. These results suggest that the presence of apoE4 delays the astroglial repair process and indirectly compromises synaptic remodeling.     

Through the central V2, not V1 receptors influencing the endogenous opiate peptide system, arginine vasopressin, not oxytocin in the hypothalamic paraventricular nucleus involves in the antinociception in the rat

Our previous study has proven that hypothalamic paraventricular nucleus (PVN) played a role in the antinociception. The central bioactive substances involving in the PVN regulating antinociception were investigated in the rat. The results showed that electrical stimulation of the PVN increased the pain threshold, and L-glutamate sodium injection into the PVN elevated the pain threshold, but the PVN cauterization decreased the pain threshold; pain stimulation raised the arginine vasopressin (AVP), not oxytocin (OXT), leucine-enkephalin (L-Ek), beta-endorphin (beta-Ep) and DynorphinA1-13 (DynA1-13) concentrations in the PVN tissue using micropunch method, heightened AVP, L-Ek, beta-Ep and DynA1-13, not OXT concentrations in the PVN perfuse liquid, and reduced the number of AVP-, not OXT, L-Ek, beta-Ep and DynA1-13-immunoreactive neurons in the PVN especially in the posterior magnocellular part of the PVN using immunocytochemistry. There was a negative relationship between the PVN AVP concentration and the pain threshold; pain stimulation enhanced the AVP, not OXT mRNA expression in the PVN using in situ hybridization and RT-PCR; intraventricular injection of anti-AVP serum completely reversed L-glutamate sodium injection into the PVN-induced antinociception, and administration of naloxone - the opiate peptide antagonist, partly blocked this L-glutamate sodium effect, but anti-OXT serum pretreatment did not influence this L-glutamate sodium effect; L-glutamate sodium injection into the PVN-induced analgesia was inhibited by V2 receptor antagonist - d(CH2)5[D-Ile2, Ile4, Ala-NH2(9)]AVP, not V1 receptor antagonist - d(CH2)5Tyr(Me)AVP. The data suggested that the PVN was limited to the central AVP, not OXT, which was through V2, not V1 receptors influencing the endogenous opiate peptide system, to regulate antinociception.