Cultured Rat Spinal Cord Research Articles

Pharmacology of PACAP and VIP receptors in the spinal cord highlights the importance of the PAC1 receptor.

The spinal cord is a key structure involved in the transmission and modulation of pain. Pituitary adenylate cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP), are expressed in the spinal cord. These peptides activate G protein-coupled receptors (PAC1, VPAC1 and VPAC2) that could provide targets for the development of novel pain treatments. However, it is not clear which of these receptors are expressed within the spinal cord and how these receptors signal. Dissociated rat spinal cord cultures were used to examine agonist and antagonist receptor pharmacology. Signalling profiles were determined for five signalling pathways. The expression of different PACAP and VIP receptors was then investigated in mouse, rat and human spinal cords using immunoblotting and immunofluorescence. PACAP, but not VIP, potently stimulated cAMP, IP1 accumulation and ERK and cAMP response element-binding protein (CREB) but not Akt phosphorylation in spinal cord cultures. Signalling was antagonised by M65 and PACAP6-38. PACAP-27 was more effectively antagonised than either PACAP-38 or VIP. The patterns of PAC1 and VPAC2 receptor-like immunoreactivity appeared to be distinct in the spinal cord. The pharmacological profile in the spinal cord suggested that a PAC1 receptor is the major functional receptor subtype present and thus likely mediates the nociceptive effects of the PACAP family of peptides in the spinal cord. However, the potential expression of both PAC1 and VPAC2 receptors in the spinal cord highlights that these receptors may play differential roles and are both possible therapeutic targets.

Modulation of Glutamate Transporter EAAT1 and Inward-Rectifier Potassium Channel Kir4.1 Expression in Cultured Spinal Cord Astrocytes by Platinum-Based Chemotherapeutics.

Platinum-based chemotherapeutics still play an essential role in cancer treatment. Despite their high effectiveness, severe side effects such as chemotherapy-induced neuropathy (CIPN) occur frequently. The pathophysiology of CIPN by platinum-based chemotherapeutics is not fully understood yet, but primarily the disturbance of dorsal root ganglion cells is discussed. However, there is increasing evidence of central nervous system involvement with activation of spinal cord astrocytes after treatment with chemotherapeutics. We investigated the influence of cis- or oxaliplatin on the functionality of cultured rat spinal cord astrocytes by using immunocytochemistry and patch-clamp electrophysiology. Cis- or oxaliplatin activated spinal astrocytes and led to downregulation of the excitatory amino acid transporter 1 (EAAT1) expression. Furthermore, the expression and function of potassium channel Kir4.1 were modulated. Pre-exposure to a specific Kir4.1 blocker in control astrocytes led to a reduced immune reactivity (IR) of EAAT1 and a nearly complete block of the current density. When spinal astrocytes were pre-exposed to antibiotic minocycline, all effects of cis- or oxaliplatin were abolished. Taken together, the modulation of Kir4.1 and EAAT1 proteins in astrocytes could be linked to the direct impact of cis- or oxaliplatin, identifying spinal astrocytes as a potential target in the prevention and treatment of chemotherapy-induced neuropathy.

Melatonin receptor-mediated attenuation of excitotoxic cell death in cultured spinal cord slices

Recent studies suggest ex vivo modeling of neuronal injury is a robust approach for the mechanistic study of neurodegeneration. Melatonin, an indolamine, is a versatile molecule with antioxidative, antiapoptotic, neuroprotective, and anti-inflammatory properties. While melatonin has been studied as a therapeutic agent for spinal cord injury (SCI) related neuronal cell loss, its actions in organotypic slice cultures approximating SCI effects are less well understood. The actions of melatonin were therefore examined following exposure of cultured rat spinal cord slices to glutamate excitotoxicity. Exposure to glutamate (500 μM) for 4 hours induced neuronal degeneration that was prevented by 0.5 μM melatonin (applied immediately or 4 hours following glutamate exposure). Decreased internucleosomal DNA fragmentation, Bax:Bcl-2 and calpain:calpastatin ratios, caspase 8, 9 and 3 activities in slice cultures were measured following melatonin treatment. Melatonin receptor (MTR1, MTR2) mRNA levels were increased in the melatonin treated spinal cord slices. To confirm melatonin receptor-mediated protection, slice cultures were treated with 10 or 25 μM luzindole (melatonin receptor antagonist) at 0 and 4 hours, respectively, after glutamate exposure. Luzindole significantly decreased the ability of melatonin to prevent cell death in the sliced culture model. These results suggest melatonin receptors may provide a pathway for therapeutic applications to prevent penumbral neuron loss following SCI.

CCP1, a Tubulin Deglutamylase, Increases Survival of Rodent Spinal Cord Neurons following Glutamate-Induced Excitotoxicity.

Microtubules (MTs) are cytoskeletal elements that provide structural support and act as roadways for intracellular transport in cells. MTs are also needed for neurons to extend and maintain long axons and dendrites that establish connectivity to transmit information through the nervous system. Therefore, in neurons, the ability to independently regulate cytoskeletal stability and MT-based transport in different cellular compartments is essential. Posttranslational modification of MTs is one mechanism by which neurons regulate the cytoskeleton. The carboxypeptidase CCP1 negatively regulates posttranslational polyglutamylation of MTs. In mammals, loss of CCP1, and the resulting hyperglutamylation of MTs, causes neurodegeneration. It has also long been known that CCP1 expression is activated by neuronal injury; however, whether CCP1 plays a neuroprotective role after injury is unknown. Using shRNA-mediated knock-down of CCP1 in embryonic rat spinal cord cultures, we demonstrate that CCP1 protects spinal cord neurons from excitotoxic death. Unexpectedly, excitotoxic injury reduced CCP1 expression in our system. We previously demonstrated that the CCP1 homolog in Caenorhabditis elegans is important for maintenance of neuronal cilia. Although cilia enhance neuronal survival in some contexts, it is not yet clear whether CCP1 maintains cilia in mammalian spinal cord neurons. We found that knock-down of CCP1 did not result in loss or shortening of cilia in cultured spinal cord neurons, suggesting that its effect on survival of excitotoxicity is independent of cilia. Our results support the idea that enzyme regulators of MT polyglutamylation might be therapeutically targeted to prevent excitotoxic death after spinal cord injuries.

Hydrogen sulfide increases intracellular Ca<sup>2+</sup> concentration by inhibition of mitochondrial respiration in cultured rat spinal cord astrocytes.

Hydrogen sulfide increases intracellular Ca<sup>2+</sup> concentration by inhibition of mitochondrial respiration in cultured rat spinal cord astrocytes.

Fibroblast growth factor 2 upregulates ecto-5'-nucleotidase and adenosine deaminase via MAPK pathways in cultured rat spinal cord astrocytes.

Adenosine triphosphate (ATP) and adenosine are neurotransmitters and neuromodulators in the central nervous system. Astrocytes regulate extracellular concentration of purines via ATP release and its metabolisms via ecto-enzymes. The expression and activity of purine metabolic enzymes in astrocytes are increased under pathological conditions. We previously showed that fibroblast growth factor 2 (FGF2) upregulates the expression and activity of the enzymes ecto-5'-nucleotidase (CD73) and adenosine deaminase (ADA). Here, we further demonstrate that this occurs in concentration- and time-dependent manners in cultured rat spinal cord astrocytes and is suppressed by inhibitors of the FGF receptor as well as the mitogen-activated protein kinases (MAPKs). We also found that FGF2 increased the phosphorylation of MAPKs, including extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 MAPK, leading to the increased expression and activity of CD73 and ADA. Our findings reveal the involvement of FGF2/MAPK pathways in the regulation of purine metabolic enzymes in astrocytes. These pathways may contribute to the control of extracellular purine concentrations under physiological and pathological conditions.

Fibroblast growth factor 2 modulates extracellular purine metabolism by upregulating ecto-5′-nucleotidase and adenosine deaminase in cultured rat spinal cord astrocytes

Purinergic signaling via ATP and adenosine produced by astrocytes is one pathway underlying neuron–glia interactions in the central nervous system (CNS). In production of purines, extracellular metabolism of released purines via ecto-enzymes is important. The expression and activities of these enzymes are altered under pathological conditions. Production of fibroblast growth factor 2 (FGF2) is increased under pathological conditions, and this has various effects on astrocytes. Here, we investigated the effects of FGF2 on purine metabolism in cultured rat spinal cord astrocytes. Astrocytes rapidly metabolized purines added to the extracellular solution. FGF2 increased extracellular metabolism of AMP to adenosine and of adenosine to inosine by upregulating ecto-5′-nucleotidase and adenosine deaminase (ADA), respectively. ADA activity and protein were detected both in the cytosol and external solution of astrocytes, and their levels were markedly increased by FGF2. FGF2 also increased metabolism of endogenously released ATP, resulting in a transient increase in adenosine and substantial accumulation of extracellular inosine. Moreover, FGF2 increased ATP release by upregulating the activity of gap junction hemichannels. These data show that FGF2 regulates purine production in astrocytes and suggest that extracellular ADA released by astrocytes plays an important role in extracellular purine metabolism in the CNS.

Therapeutic time window for the effects of erythropoietin on astrogliosis and neurite outgrowth in an in vitro model of spinal cord injury.

Background:The objective of this study was to investigate the underlying molecular mechanisms and the therapeutic time window for preventing astrogliosis with erythropoietin (EPO) treatment after in vitro modeled spinal cord injury (SCI).Methods:Cultured rat spinal cord astrocytes were treated with kainate and scratching to generate an in vitro model of SCI. EPO (100U/mL or 300U/mL) was added immediately or 2, 4, or 8 hours after injury. Some cultures were also treated with AG490, an inhibitor of the EPO-EPO receptor (EpoR) pathway mediator Janus kinase 2 (JAK2). To evaluate neurite extension, rat embryonic spinal cord neurons were seeded onto astrocyte cultures and treated with EPO immediately after injury in the presence or absence of anti-EpoR antibody.Results:EPO treatment at up to 8 hours after injury reduced the expression of axonal growth inhibiting molecules (glial fibrillary acidic protein, vimentin, and chondroitin sulfate proteoglycan), cytoskeletal regulatory proteins (Rho-associated protein kinase and ephephrin A4), and proinflammatory cytokines (tumor necrosis factor-alpha, transforming growth factor-beta, and phosphorylated-Smad3) in a dosedependent manner (P < .001). Most effects peaked with EPO treatment 2–4hours after injury. Additionally, EPO treatment up to 4 hours after injury promoted expression of the EpoR (>2-fold) and JAK2 (>3-fold) in a dose-dependent manner (P < .001), whereas co-treatment with AG490 precluded these effects (P < .001). EPO treatment up to 4hours after injury also enhanced axonal b-III tubulin-immunoreactivity (>12-fold), and this effect was precluded by co-treatment with an anti-EpoR antibody (P < .001).Conclusions:EPO treatment within 8 hours after injury reduced astrogliosis, and EPO treatment within 4 hours promoted neurite outgrowth. EPO therapy immediately after spinal cord injury may regulate glia to generate an environment permissive of axonal regeneration.

Sequences Flanking the Gephyrin-Binding Site of GlyRβ Tune Receptor Stabilization at Synapses.

The efficacy of synaptic transmission is determined by the number of neurotransmitter receptors at synapses. Their recruitment depends upon the availability of postsynaptic scaffolding molecules that interact with specific binding sequences of the receptor. At inhibitory synapses, gephyrin is the major scaffold protein that mediates the accumulation of heteromeric glycine receptors (GlyRs) via the cytoplasmic loop in the β-subunit (β-loop). This binding involves high- and low-affinity interactions, but the molecular mechanism of this bimodal binding and its implication in GlyR stabilization at synapses remain unknown. We have approached this question using a combination of quantitative biochemical tools and high-density single molecule tracking in cultured rat spinal cord neurons. The high-affinity binding site could be identified and was shown to rely on the formation of a 310-helix C-terminal to the β-loop core gephyrin-binding motif. This site plays a structural role in shaping the core motif and represents the major contributor to the synaptic confinement of GlyRs by gephyrin. The N-terminal flanking sequence promotes lower affinity interactions by occupying newly identified binding sites on gephyrin. Despite its low affinity, this binding site plays a modulatory role in tuning the mobility of the receptor. Together, the GlyR β-loop sequences flanking the core-binding site differentially regulate the affinity of the receptor for gephyrin and its trapping at synapses. Our experimental approach thus bridges the gap between thermodynamic aspects of receptor-scaffold interactions and functional receptor stabilization at synapses in living cells.

Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-\u03baB signaling in an IL-6-dependent manner

BackgroundSpinal cord astrocyte swelling is an important component to spinal cord edema and is associated with poor functional recovery as well as therapeutic resistance after spinal cord injury (SCI). High mobility group box-1 (HMGB1) is a mediator of inflammatory responses in the central nervous system and plays a critical role after SCI. Given this, we sought to identify both the role and underlying mechanisms of HMGB1 in cellular swelling and aquaporin 4 (AQP4) expression in cultured rat spinal cord astrocytes after oxygen-glucose deprivation/reoxygenation (OGD/R).MethodsThe post-natal day 1–2 Sprague-Dawley rat spinal cord astrocytes were cultured in vitro, and the OGD/R model was induced. We first investigated the effects of OGD/R on spinal cord astrocytic swelling and HMGB1 and AQP4 expression, as well as HMGB1 release. We then studied the effects of HMGB1 inhibition on cellular swelling, HMGB1 and AQP4 expression, and HMGB1 release. The roles of both toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) signaling pathway and interleukin-6 (IL-6) in reducing cellular swelling resulting from HMGB1 inhibition in spinal cord astrocytes after OGD/R were studied. Intergroup data were compared using one-way analysis of variance (ANOVA) followed by Dunnett’s test.ResultsThe OGD/R increased spinal cord astrocytic swelling and HMGB1 and AQP4 expression, as well as HMGB1 release. Inhibition of HMGB1 using either HMGB1 shRNA or ethyl pyruvate resulted in reduced cellular volume, mitochondrial and endoplasmic reticulum swelling, and lysosome number and decreased upregulation of both HMGB1 and AQP4 in spinal cord astrocytes, as well as HMGB1 release. The HMGB1 effects on spinal cord astrocytic swelling and AQP4 upregulation after OGD/R were mediated—at least in part—via activation of TLR4, myeloid differentiation primary response gene 88 (MyD88), and NF-κB. These activation effects can be repressed by TLR4 inhibition using CLI-095 or C34, or by NF-κB inhibition using BAY 11-7082. Furthermore, either OGD/R or HMGB1 inhibition resulted in changes in IL-6 release. IL-6 was also shown to mediate AQP4 expression in spinal cord astrocytes.ConclusionsHMGB1 upregulates AQP4 expression and promotes cell swelling in cultured spinal cord astrocytes after OGD/R, which is mediated through HMGB1/TLR4/MyD88/NF-κB signaling and in an IL-6-dependent manner.

Simvastatin protects ischemic spinal cord injury from cell death and cytotoxicity through decreasing oxidative stress: in vitro primary cultured rat spinal cord model under oxygen and glucose deprivation-reoxygenation conditions

BackgroundIschemia and the following reperfusion damage are critical mechanisms of spinal cord injury. Statins have been reported to decrease ischemia–reperfusion injury in many organs including the spinal cord. Anti-oxidative effect is one of the main protective mechanisms of statin against neuronal death and cytotoxicity. We hypothesized that statins’ anti-oxidative property would yield neuroprotective effects on spinal cord ischemia–reperfusion injuryMethodsPrimary cultured spinal cord motor neurons were isolated from Sprague–Dawley rat fetuses. Ischemia–reperfusion injury model was induced by 60 min of oxygen and glucose deprivation (OGD) and 24 h of reoxygenation. Healthy and OGD cells were treated with simvastatin at concentrations of 0.1, 1, and 10 μM for 24 h. Cell viability was assessed using water-soluble tetrazolium salt (WST)-8, cytotoxicity with LDH, and production of free radicals with DCFDA (2′,7′-dichlorofluorescein diacetate).ResultsOGD reduced neuronal viability compared to normoxic control by 35.3%; however, 0.1–10 μM of simvastatin treatment following OGD improved cell survival. OGD increased LDH release up to 214%; however, simvastatin treatment attenuated its cytotoxicity at concentrations of 0.1–10 μM (p < 0.001 and p = 0.001). Simvastatin also reduced deteriorated morphological changes of motor neurons following OGD. Oxidative stress was reduced by simvastatin (0.1–10 μM) compared to untreated cells exposed to OGD (p < 0.001).ConclusionsSimvastatin effectively reduced spinal cord neuronal death and cytotoxicity against ischemia–reperfusion injury, probably via modification of oxidative stress.

Reactive oxygen species trigger motoneuron death in non-cell-autonomous models of ALS through activation of c-Abl signaling.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease in which pathogenesis and death of motor neurons are triggered by non-cell-autonomous mechanisms. We showed earlier that exposing primary rat spinal cord cultures to conditioned media derived from primary mouse astrocyte conditioned media (ACM) that express human SOD1G93A (ACM-hSOD1G93A) quickly enhances Nav channel-mediated excitability and calcium influx, generates intracellular reactive oxygen species (ROS), and leads to death of motoneurons within days. Here we examined the role of mitochondrial structure and physiology and of the activation of c-Abl, a tyrosine kinase that induces apoptosis. We show that ACM-hSOD1G93A, but not ACM-hSOD1WT, increases c-Abl activity in motoneurons, interneurons and glial cells, starting at 60 min; the c-Abl inhibitor STI571 (imatinib) prevents this ACM-hSOD1G93A-mediated motoneuron death. Interestingly, similar results were obtained with ACM derived from astrocytes expressing SOD1G86R or TDP43A315T. We further find that co-application of ACM-SOD1G93A with blockers of Nav channels (spermidine, mexiletine, or riluzole) or anti-oxidants (Trolox, esculetin, or tiron) effectively prevent c-Abl activation and motoneuron death. In addition, ACM-SOD1G93A induces alterations in the morphology of neuronal mitochondria that are related with their membrane depolarization. Finally, we find that blocking the opening of the mitochondrial permeability transition pore with cyclosporine A, or inhibiting mitochondrial calcium uptake with Ru360, reduces ROS production and c-Abl activation. Together, our data point to a sequence of events in which a toxic factor(s) released by ALS-expressing astrocytes rapidly induces hyper-excitability, which in turn increases calcium influx and affects mitochondrial structure and physiology. ROS production, mediated at least in part through mitochondrial alterations, trigger c-Abl signaling and lead to motoneuron death.

Proteomic Analysis of Glycine Receptor β Subunit (GlyRβ)-interacting Proteins: EVIDENCE FOR SYNDAPIN I REGULATING SYNAPTIC GLYCINE RECEPTORS

Glycine receptors (GlyRs) mediate inhibitory neurotransmission in spinal cord and brainstem. They are clustered at inhibitory postsynapses via a tight interaction of their β subunits (GlyRβ) with the scaffolding protein gephyrin. In an attempt to isolate additional proteins interacting with GlyRβ, we performed pulldown experiments with rat brain extracts using a glutathione S-transferase fusion protein encompassing amino acids 378-455 of the large intracellular loop of GlyRβ as bait. This identified syndapin I (SdpI) as a novel interaction partner of GlyRβ that coimmunoprecipitates with native GlyRs from brainstem extracts. Both SdpI and SdpII bound efficiently to the intracellular loop of GlyRβ in vitro and colocalized with GlyRβ upon coexpression in COS-7 cells. The SdpI-binding site was mapped to a proline-rich sequence of 22 amino acids within the intracellular loop of GlyRβ. Deletion and point mutation analysis disclosed that SdpI binding to GlyRβ is Src homology 3 domain-dependent. In cultured rat spinal cord neurons, SdpI immunoreactivity was found to partially colocalize with marker proteins of inhibitory and excitatory synapses. When SdpI was acutely knocked down in cultured spinal cord neurons by viral miRNA expression, postsynaptic GlyR clusters were significantly reduced in both size and number. Similar changes in GlyR cluster properties were found in spinal cultures from SdpI-deficient mice. Our results are consistent with a role of SdpI in the trafficking and/or cytoskeletal anchoring of synaptic GlyRs.

Endothelin-1 is over-expressed in amyotrophic lateral sclerosis and induces motor neuron cell death

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive loss of motor neurons (MNs) and astrogliosis. Recent evidence suggests that factors secreted by activated astrocytes might contribute to degeneration of MNs. We focused on endothelin-1 (ET-1), a peptide which is strongly up-regulated in reactive astrocytes under different pathological conditions. We show that ET-1 is abundantly expressed by reactive astrocytes in the spinal cord of the SOD1-G93A mouse model and sporadic ALS patients. To test if ET-1 might play a role in degeneration of MNs, we investigated its effect on MN survival in an in vitro model of mixed rat spinal cord cultures (MSCs) enriched of astrocytes exhibiting a reactive phenotype. ET-1 exerted a toxic effect on MNs in a time- and concentration-dependent manner, with an exposure to 100–200nM ET-1 for 48h resulting in 40–50% MN cell death. Importantly, ET-1 did not induce MN degeneration when administered on cultures treated with AraC (5μM) or grown in a serum-free medium that did not favor astrocyte proliferation and reactivity. We found that both ETA and ETB receptors are enriched in astrocytes in MSCs. The ET-1 toxic effect was mimicked by ET-3 (100nM) and sarafotoxin S6c (10nM), two selective agonists of endothelin-B receptors, and was not additive with that of ET-3 suggesting the involvement of ETB receptors. Surprisingly, however, the ET-1 effect persisted in the presence of the ETB receptor antagonist BQ-788 (200nM–2μM) and was slightly reversed by the ETA receptor antagonist BQ-123 (2μM), suggesting an atypical pharmacological profile of the astrocytic receptors responsible for ET-1 toxicity. The ET-1 effect was not undone by the ionotropic glutamate receptor AMPA antagonist GYKI 52466 (20μM), indicating that it is not caused by an increased glutamate release. Conversely, a 48-hour ET-1 treatment increased MN cell death induced by acute exposure to AMPA (50μM), which is indicative of two distinct pathways leading to neuronal death.Altogether these results indicate that ET-1 exerts a toxic effect on cultured MNs through mechanisms mediated by reactive astrocytes and suggest that ET-1 may contribute to MN degeneration in ALS. Thus, a treatment aimed at lowering ET-1 levels or antagonizing its effect might be envisaged as a potential therapeutic strategy to slow down MN degeneration in this devastating disease.

Protective effect of molecular hydrogen against H 2 O 2 -induced oxidative injury in cultured rat spinal cord neurons

Objective To investigate the protective effect of molecular hydrogen against hydrogen peroxide(H2O2)-induced oxidative injury in primary cultures of rat spinal cord neurons and the related mechanism.Methods Rat spinal cord neurons were cultured for 7days and were randomly assigned to four groups with different treatments-(1)Normal medium(NM)control:treated with NM;(2)Hydrogen-rich medium(HM):treated with HM;(3)NM+H2O2:treated with 100 μmol/L H2O2and 15μmol/L ferrous chloride(FeCl2)after pretreatment with NM for 2h;and(4)HM+H2O2:treated with 100μmol/L H2O2and 15μmol/L FeCl2after pretreatment with HM for 2h.The respective media were changed every 6hin each group,and 12hlater the cells were collected for assays of reactive oxygen species(ROS),hydroxyl radical(HO·), apoptosis,and the expression of glycogen synthase kinase-3β(GSK-3β)and p-GSK-3β.Results Compared with NM,HM significantly reduced the H2O2-induced intracellular production of ROS and HO·in purified neurons(P0.01),significantly decreased the number of apoptotic neurons(P0.01)and expression of caspase-3(P0.01),and significantly promoted phosphorylation of GSK-3β(P0.01).Conclusion Molecular hydrogen has protective effect against H2O2-induced oxidative injury in primary cultured neurons.The mechanisms might be related to reduction of intracellular production of ROS and HO·,inhibition of apoptosis in neurons and promotion of GSK-3βphosphorylation.

Interleukin-6 inhibits voltage-gated sodium channel activity of cultured rat spinal cord neurons.

Interleukin-6 (IL-6) is a pleiotropic proinflammatory cytokine that plays a key role in the injuries and diseases of the central nervous system (CNS). A voltage-gated Na+ channel (VGSC) is essential for the excitability and electrical properties of the neurons. However, there is still limited information on the role of IL-6 in voltage-gated sodium channels. Our study aimed to investigate the effects of IL-6 on Na+ currents in cultured spinal-cord neurons. VGSC currents were activated and recorded using whole-cell patch-clamp technique in the cultured rat spinal cord neurons. The effects of IL-6 concentration and exposure duration were examined. To determine whether any change in the number of channels in the plasma membrane can inhibit IL-6 on VGSC currents, we examined the expression of α1A (SCN1α) subunit mRNA level and protein level in the neurons before and after IL-6 induction using real-time polymerase chain reaction. We verified that IL-6, through a receptor-mediated mechanism, suppressed Na+ currents in a time- and dose-dependent manner, but did not alter the voltage-dependent activation and inactivation. Gp130 was involved in this inhibition. Furthermore, the spike amplitude was also inhibited by IL-6 in the doses that decreased the Na+ currents. VGSC currents are significantly inhibited by IL-6. Our findings reveal that the potential neuroprotection of IL-6 may result from the inhibitory effects on VGSC currents.

Differential Control of Thrombospondin over Synaptic Glycine and AMPA Receptors in Spinal Cord Neurons

Thrombospondin-1 (TSP-1) is a large extracellular matrix protein secreted by astrocytes during development and inflammation. In the developing CNS, TSP-1 is involved in neuronal migration and adhesion, neurite outgrowth, and synaptogenesis. We investigated the effects of TSP-1 on neurons with mature synapses using immunocytochemistry, single-particle tracking, surface biotinylation, and calcium imaging. We show that in cultured rat spinal cord neurons TSP-1 decreased neuronal excitability by reducing the accumulation of excitatory AMPA receptors (AMPARs) and increasing that of inhibitory glycine receptors (GlyRs) in synapses. The effects of TSP-1 on GlyRs were dependent on the activation of excitatory receptors. These changes were abolished by blocking β1-integrins and mimicked by blocking β3-integrins. In the presence of TSP-1, AMPARs were less stabilized at synapses, increasing their lateral diffusion and endocytosis. Interestingly, TSP-1 counteracted the increased neuronal excitability and neuronal death induced by TNFα. These results suggest a role of TSP-1 in controlling the balance between excitation and inhibition which could help the recovery of normal synaptic activity after injury responses.

Effects of remifentanil and fentanyl on N-methyl-D-aspartate receptor currents in rat spinal cord dorsal horn neurons

Objective To investigate the effect of remifentanil and fentanyl on N-methyl-D-aspartate (NMDA) receptor currents in rat spinal cord dorsal horn neurons.Methods Whole-cell patch-clamp technique was used to record the NMDA receptor currents.The primary cultured E14SD rat spinal cord dorsal horn neurons (DH cells) were randomly divided into 3 groups (n =10 each):remifentanil group (group R),fentanyl group (group F) and control group (group C).DH cells were perfused with 4 nmol/L remifentanil (group R) or 10 μmol/L fentanyl (group F) for 60 min followed by washout.NMDA receptor currents were recorded immediately after administration (T0),at 15,30,45 and 60 min of action of drugs (T1-4),and at 15 and 30 min (T5-6) after washout.Results Compared with group C,no significant change in the peak NMDA receptor current was found at each time point in group F and at T0 and T1 in group R (P ＞ 0.05),and the peak NMDA receptor current was significantly increased at T2-6 in group R (P ＜ 0.01).The peak NMDA receptor current was significantly higher at T2-6 than at T0,while lower at T2-4 and T6 than at T5 in group R (P ＜ 0.01).Conclusion Remifentanil can increase NMDA receptor function in rat spinal cord horn neurons,and the peak effect is reached after washout,but fentanyl dose not have the effect. Key words: Piperidines; Receptors, N-methyl-D-aspartate; Drug tolerance

Functional Duality of Astrocytes in Myelination

Astrocytes undergo major phenotypic changes in response to injury and disease that directly influence repair in the CNS, but the mechanisms involved are poorly understood. Previously, we have shown that neurosphere-derived rat astrocytes plated on poly-L-lysine (PLL-astrocytes) support myelination in dissociated rat spinal cord cultures (myelinating cultures). It is hypothesized that astrocyte reactivity can affect myelination, so we have exploited this culture system to ascertain how two distinct astrocyte phenotypes influence myelination. Astrocytes plated on tenascin C (TnC-astrocytes), a method to induce quiescence, resulted in less myelinated fibers in the myelinating cultures when compared with PLL-astrocytes. In contrast, treatment of myelinating cultures plated on PLL-astrocytes with ciliary neurotrophic factor (CNTF), a cytokine known to induce an activated astrocyte phenotype, promoted myelination. CNTF could also reverse the effect of quiescent astrocytes on myelination. A combination of microarray gene expression analysis and quantitative real-time PCR identified CXCL10 as a potential candidate for the reduction in myelination in cultures on TnC-astrocytes. The effect of TnC-astrocytes on myelination was eliminated by neutralizing CXCL10 antibodies. Conversely, CXCL10 protein inhibited myelination on PLL-astrocytes. Furthermore, CXCL10 treatment of purified oligodendrocyte precursor cells did not affect proliferation, differentiation, or process extension compared with untreated controls, suggesting a role in glial/axonal ensheathment. These data demonstrate a direct correlation of astrocyte phenotypes with their ability to support myelination. This observation has important implications with respect to the development of therapeutic strategies to promote CNS remyelination in demyelinating diseases.

Evidence of endoplasmic reticular stress in the spinal motor neurons exposed to CSF from sporadic amyotrophic lateral sclerosis patients

We have earlier reported that intrathecal injection of cerebrospinal fluid (CSF) from sporadic Amyotrophic Lateral Sclerosis patients (ALS-CSF) into neonatal rats and supplementation of rat spinal cord cultures with ALS-CSF induces motor neuron degeneration via aberrant neurofilament phosphorylation and Golgi apparatus fragmentation. Intracellular aggregates immunoreactive to ubiquitin, phosphorylated neurofilaments and choline acetyl transferase (ChAT) were prominently seen in NSC-34 cells exposed to ALS-CSF. Protein aggregation could cause stress on endoplasmic reticulum (ER) and may precede Golgi fragmentation. Here we assessed the effect of ALS-CSF on the expression of GRP-78 and caspase-12 proteins, the markers of ER stress responses, in NSC-34 cells and rat spinal cords by immunochemistry and immunoblotting. Both in vitro and in vivo, increased expression of these proteins accompanied elevated active caspase-12 levels. Apoptotic nuclei and nuclear translocation of caspase-12 were noted in some cells. In vitro, the occurrence of ER stress was supported by electron microscopic observations of numerous free polyribosomes and fragmented ER cisternae. Aggregated mSOD1 protein causes ER stress in familial ALS. ER stress is also reported in the autopsy samples of sporadic ALS. Thus our observation of ER stress may be linked to the protein aggregation, viz. phosphorylated neurofilaments and ChAT, reported earlier.