Neural plasticity within the spinal nociceptive network may be fundamental to the chronic nature of neuropathic pain. The relation of growth-associated protein-43 (GAP-43), a protein involved in the nerve fiber growth and sprouting, to pain hypersensitivity has been investigated. Glial activation and inflammatory cytokines released by microglia and astrocytes are considered to be involved in the neural sprouting and plasticity. In the present study, the anti-nociception effect of propentofylline, a glial modulating agent, was investigated in a rat chronic constriction injury (CCI) model aiming to explore the role of GAP-43 expression. Our results demonstrated that propentofylline could attenuate the CCI-induced mechanical allodynia and thermal hyperalgesia and inhibit the astrocyte activation and production of IL-1β. GAP-43 expression was also down-regulated by intrathecal propentofylline. These findings suggest that astrocyte activation is involved in the regulation of GAP-43 expression and propentofylline might be used in the treatment of neuropathic pain.
Astrocytes; growth-associated protein-43; interleukin 1β; neuropathic pain
Clearance of synaptically released glutamate, and hence termination of glutamatergic neurotransmission, is carried out by glutamate transporters, most especially glutamate transporter-1 (GLT-1) and the glutamate-aspartate transporter (GLAST) that are located in astrocytes. It is becoming increasingly well appreciated that changes in the function and expression of GLT-1 and GLAST occur under different physiological and pathological conditions. Here we investigated the plasticity in expression of GLT-1 and GLAST in the spinal dorsal horn using immunohistochemistry following partial sciatic nerve ligation (PSNL) in rats.
Animals were confirmed to develop hypersensitivity to mechanical stimulation by 7 days following PSNL. Baseline expression of GLT-1 and GLAST in naive animals was only observed in astrocytes and not in either microglia or neurons. Microglia and astrocytes showed evidence of reactivity to the nerve injury when assessed at 7 and 14 days following PSNL evidenced by increased expression of OX-42 and GFAP, respectively. In contrast, the total level of GLT-1 and GLAST protein decreased at both 7 and 14 days after PSNL. Importantly, the cellular location of GLT-1 and GLAST was also altered in response to nerve injury. Whereas activated astrocytes showed a marked decrease in expression of GLT-1 and GLAST, activated microglia showed de novo expression of GLT-1 and GLAST at 7 days after PSNL and this was maintained through day 14. Neurons showed no expression of GLT-1 or GLAST at any time point.
These results indicate that the expression of glutamate transporters in astrocytes and microglia are differentially regulated following nerve injury.
Valproate produces analgesia in animals and humans, however, its mechanisms of action are yet unknown. The present study examined effects of repeated administration of valproate on behavioral hypersensitivity and expression of glutamate transporter-1 (GLT-1) and glutamate-aspartate transporter (GLAST) in the spinal dorsal horn in rats after L5–L6 spinal nerve ligation (SNL). SNL significantly reduced mechanical withdrawal threshold and expression of GLT-1 and GLAST in the spinal dorsal horn. Repeated oral administration of valproate reduced hypersensitivity, restored down-regulated expression of GLT-1 and GLAST in the spinal dorsal horn, and enhanced analgesia from the glutamate transporter activator riluzole. This analgesia from valproate was blocked by the selective GLT-1 blocker dihydrokainic acid (DHK). These data suggest that valproate restores down-regulated expression of glutamate transporters in the spinal cord to presumably reduce glutamate signaling and to reduce hypersensitivity after nerve injury, and that combination of valproate with riluzole produces enhanced analgesia which relies on the spinal glutamate transporters.
valproate; neuropathic pain; glutamate transporter; dihydrokainic acid; riluzole
In this study, we evaluated whether propentofylline, a methylxanthine derivative, modulates spinal glial activation and GABAergic inhibitory tone by modulation of glutamic acid decarboxylase (GAD)65, the GABA synthase enzyme, in the spinal dorsal horn following spinal cord injury (SCI). Sprague–Dawley rats (225–250 g) were given a unilateral spinal transverse injury, from dorsal to ventral, at the T13 spinal segment. Unilateral spinal injured rats developed robust bilateral hindlimb mechanical allodynia and hyperexcitability of spinal wide dynamic range (WDR) neurons in the lumbar enlargement (L4–L5) compared to sham controls, which was attenuated by intrathecal (i.t.) administration of GABA, dose-dependently (0.01, 0.1, 0.5 μg). Western blotting and immunohistochemical data demonstrated that the expression level of GAD65 protein significantly decreased on both sides of the lumbar dorsal horn (L4/5) after SCI (p < 0.05). In addition, astrocytes and microglia showed soma hypertrophy as determined by increased soma area and increased GFAP and CD11b on both sides of the lumbar dorsal horn compared to sham controls, respectively (p < 0.05). Intrathecal treatment with propentofylline (PPF 10 mM) significantly attenuated the astrocytic and microglial soma hypertrophy and mechanical allodynia (p < 0.05). Additionally, the Western blotting and immunohistochemistry data demonstrated that i.t. treatment of PPF significantly prevented the decrease of GAD65 expression in both sides of the lumbar dorsal horn following SCI (p < 0.05). In conclusion, our present data demonstrate that propentofylline modulates glia activation and GABAergic inhibitory tone by modulation of GAD65 protein expression following spinal cord injury.
Astrocytes; Central neuropathic pain; Glutamic acid decarboxylase; Microglia; Spinal cord injury
Neuropathic pain is an intractable clinical problem. Drug treatments such as tramadol have been reported to effectively decrease neuropathic pain by inhibiting the activity of nociceptive neurons. It has also been reported that modulating glial activation could also prevent or reverse neuropathic pain via the administration of a glial modulator or inhibitor, such as propentofylline. Thus far, there has been no clinical strategy incorporating both neuronal and glial participation for treating neuropathic pain. Therefore, the present research study was designed to assess whether coadministration of tramadol and propentofylline, as neuronal and glial activation inhibitors, respectively, would exert a synergistic effect on the reduction of rat spinal nerve ligation (SNL)-induced neuropathic pain. Rats underwent SNL surgery to induce neuropathic pain. Pain behavioral tests were conducted to ascertain the effect of drugs on SNL-induced mechanical allodynia with von-Frey hairs. Proinflammatory factor interleukin-1β (IL-1β) expression was also detected by Real-time RT-PCR. Intrathecal tramadol and propentofylline administered alone relieved SNL-induced mechanical allodynia in a dose-dependent manner. Tramadol and propentofylline coadministration exerted a more potent effect in a synergistic and dose dependent manner than the intrathecal administration of either drug alone. Real-time RT-PCR demonstrated IL-1β up-expression in the ipsilateral spinal dorsal horn after the lesion, which was significantly decreased by tramadol and propentofylline coadministration. Inhibiting proinflammatory factor IL-1β contributed to the synergistic effects of tramadol and propentofylline coadministration on rat peripheral nerve injury-induced neuropathic pain. Thus, our study provided a rationale for utilizing a novel strategy for treating neuropathic pain by blocking the proinflammatory factor related pathways in the central nervous system.
Glutamate neurotransmission is highly regulated, largely by glutamate transporters. In the spinal cord, the glutamate transporter GLT-1 is primarily responsible for glutamate clearance. Downregulation of GLT-1 can occur in activated astrocytes, and is associated with increased extracellular glutamate and neuroexcitation. Among other conditions, astrocyte activation occurs following repeated opioids and in models of chronic pain. If GLT-1 downregulation occurs in these states, GLT-1 could be a pharmacological target for improving opioid efficacy and controlling chronic pain. The present studies explored whether daily intrathecal treatment of rats with ceftriaxone, a β-lactam antibiotic that upregulates GLT-1 expression, could prevent development of hyperalgesia and allodynia following repeated morphine, reverse pain arising from central or peripheral neuropathy, and reduce glial activation in these models. Ceftriaxone pre-treatment attenuated the development of hyperalgesia and allodynia in response to repeated morphine, and prevented associated astrocyte activation. In a model of multiple sclerosis (experimental autoimmune encephalomyelitis; EAE), ceftriaxone reversed tactile allodynia and halted the progression of motor weakness and paralysis. Similarly, ceftriaxone reversed tactile allodynia induced by chronic constriction nerve injury (CCI). EAE and CCI each significantly reduced the expression of membrane-bound, dimerized GLT-1 protein in lumbar spinal cord, an effect normalized by ceftriaxone. Lastly, ceftriaxone normalized CCI- and EAE-induced astrocyte activation in lumbar spinal cord. Together, these data indicate that increasing spinal GLT-1 expression attenuates opioid-induced paradoxical pain, alleviates neuropathic pain, and suppresses associated glial activation. GLT-1 therefore may be a therapeutic target that could improve available treatment options for patients with chronic pain.
opioid; spinal cord; multiple sclerosis; astrocyte; allodynia; hyperalgesia
Astrocytes are key cellular elements in both the tripartite synapse and the neurovascular unit. To fulfill this dual role in synaptic activity and metabolism, they express a panel of receptors and transporters that sense glutamate. Among them, the GLT-1 and GLAST transporters are known to regulate extracellular glutamate concentrations at excitatory synapses and consequently modulate glutamate receptor signaling. These major uptake systems are also involved in energy supply to neurons. However, the functional role of GLAST in concurrent regulation of metabolic and neuronal activity is currently unknown. We took advantage of the attractive structural and functional features of the main olfactory bulb to explore the impact of GLAST on sensory information processing while probing both glutamate uptake and neuronal activity in glomeruli and deeper cellular layers, respectively. Using odor-evoked 2-deoxyglucose imaging and local field potential recordings in GLAST knockout mice, we show in vivo that deletion of GLAST alters both glucose uptake and neuronal oscillations in olfactory bulb networks.
glutamate transporters; olfactory bulb; astrocytes; metabolic activity; neuronal oscillations
Multiple studies have shown that glial cells of the spinal cord, such as astrocytes and microglia, have close contact with neurons, suggesting the term tripartite synapse. In these synapses, astrocytes surrounding neurons contribute to neuronal excitability and synaptic transmission, thereby increasing nociception and thus the persistence of chronic pain. Conversely, the N-methyl-D-aspartate (NMDA) receptor is crucial in the generation and maintenance of chronic pain. It has multiple sites of modulation. One is the site of recognition of extracellular neurotransmitter (glutamate), which can be blocked by competitive antagonists such as (3-(2-carboxipiperazin-4)1-propyl phosphonic acid), (±)-CPP, resulting in a blockade of the calcium current and thus the intracellular transduction process. In the present study, we investigated whether the potential antinociceptive effect of glial inhibition produced by propentofylline (PPF) can be enhanced when combined with an NMDA-receptor inhibitor such as (±)-CPP.
We used Sprague-Dawley monoarthritic rats. The monoarthritis was induced by injection of complete Freund adjuvant in the right tibiotarsal joint. Four weeks later, rats were treated with PPF (1, 10, 30, and 100 μg/10 μl) intrathecally (i.t.) for 10 days, injected once with (±)-CPP (2.5, 5, 12.5, 25, 50, and 100 μg/10 μl, i.t.), or both treatments combined. The antinociceptive effect was evaluated on day 11 for PPF and immediately to (±)-CPP, by assessing the vocalization threshold to mechanical stimulation of the arthritic paw.
The data indicate that intrathecal administration of increasing concentrations of (±)-CPP or PPF produced a significant dose-dependent antinociceptive effect with respect to monoarthritic rats receiving saline. The linear regression analysis showed that the dose that produces 30% of maximal effect (ED30) for i.t. (±)-CPP was 3.97 μg, and 1.42 μg for i.t. PPF. The administration of the PPF and (±)-CPP combination in fixed proportions of ED30 produced a dose-dependent antinociceptive effect, showing an interaction of the supraadditive type.
The results suggest that glia inhibitors can synergically potentiate the effect of glutamate blockers for the treatment of chronic inflammatory pain.
The GLT-1 (EAAT2) subtype of glutamate transporter ensures crisp excitatory signaling and limits excitotoxicity in the CNS. Astrocytic expression of GLT-1 is regulated during development, by neuronal activity, and in neurodegenerative diseases. Although neurons activate astrocytic expression of GLT-1, the mechanisms involved have not been identified. In the present study, astrocytes from transgenic mice that express enhanced green fluorescent protein (eGFP) under the control of a bacterial artificial chromosome (BAC) containing a very large region of DNA surrounding the GLT-1 gene (BAC GLT-1 eGFP mice) were used to assess the role of nuclear factor-κB (NF-κB) in neuron-dependent activation of the GLT-1 promoter. We provide evidence that neurons activate NF-κB signaling in astrocytes. Transduction of astrocytes from the BAC GLT-1 eGFP mice with dominant-negative inhibitors of NF-κB signaling completely blocked neuron-dependent activation of a NF-κB reporter construct and attenuated induction of eGFP. Exogenous expression of p65 and/or p50 NF-κB subunits induced expression of eGFP or GLT-1 and increased GLT-1-mediated transport activity. Using wild type and mutant GLT-1 promoter reporter constructs, we found that NF-κB sites at −583 or −251 relative to the transcription start site eliminated neuron-dependent reporter activation. Electrophoretic mobility shift and supershift assays reveal that p65 and p50 interact with these same sites ex vivo. Finally, chromatin immunoprecipitation (ChIP) showed that p65 and p50 interact with these sites in adult cortex, but not in kidney (a tissue that expresses no detectable GLT-1). Together, these studies strongly suggest that NF-κB contributes to neuron-dependent regulation of astrocytic GLT-1 transcription.
glutamate transport; NF-κB; astrocytes; p65; p50; EAAT2; GLT-1; IκBα
Astrocytes express the sodium-dependent glutamate transporters GLAST and GLT-1, which are critical to maintain low extracellular glutamate concentrations. Here, we analyzed changes in their expression and function following a mechanical lesion in the CA1 area of organotypic hippocampal slices. 6-7 days after lesion, a glial scar had formed along the injury site, containing strongly activated astrocytes with increased GFAP and S100β immunoreactivity, enlarged somata, and reduced capability for uptake of SR101. Astrocytes in the scar's periphery were swollen as well, but showed only moderate upregulation of GFAP and S100β and efficiently took up SR101. In the scar, clusters of GLT-1 and GLAST immunoreactivity colocalized with GFAP-positive fibers. Apart from these, GLT-1 immunoreactivity declined with increasing distance from the scar, whereas GLAST expression appeared largely uniform. Sodium imaging in reactive astrocytes indicated that glutamate uptake was strongly reduced in the scar but maintained in the periphery. Our results thus show that moderately reactive astrocytes in the lesion periphery maintain overall glutamate transporter expression and function. Strongly reactive astrocytes in the scar, however, display clusters of GLAST and GLT-1 immunoreactivity together with reduced glutamate transport activity. This reduction might contribute to increased extracellular glutamate concentrations and promote excitotoxic cell damage at the lesion site.
Astroglial glutamate transporter EAAT2/GLT1 prevents glutamate-induced excitotoxicity in the central nervous system. Expression of EAAT2/GLT1 is dynamically regulated by neurons. The pathogenesis of amyotrophic lateral sclerosis (ALS) involves astroglial dysfunction, including dramatic loss of EAAT2/GLT1. DNA methylation of gene promoters represents one of the most important epigenetic mechanisms in regulating gene expression. The involvement of DNA methylation in the regulation of astroglial EAAT2/GLT1 expression in different conditions, especially in ALS has not been explored. In this study, we established a procedure to selectively isolate a pure astrocyte population in vitro and in vivo from BAC GLT1 eGFP mice using an eGFP-based fluorescence-activated cell sorting approach. Astrocytes isolated from this procedure are GFAP+ and GLT1+ and respond to neuronal stimulation, enabling direct methylation analysis of GLT1 promoter in these astrocytes. To investigate the role of DNA methylation in physiological and pathological EAAT2/GLT1 expression, methylation status of the EAAT2/GLT1 promoter was analyzed in astrocytes from in vitro and in vivo paradigms or postmortem ALS motor cortex by bisulfite sequencing method. DNA demethylation on selective CpG sites of the GLT1 promoter was highly correlated to increased GLT1 mRNA levels in astrocytes in response to neuronal stimulation; however, low level of methylation was found on CpG sites of EAAT2 promoter from postmortem motor cortex of human amyotrophic lateral sclerosis patients. In summary, hypermethylation on selective CpG sites of the GLT1 promoter is involved in repression of GLT1 promoter activation, but this regulation does not play a role in astroglial dysfunction of EAAT2 expression in patients with ALS.
epigenetic; astrocyte; GLT1
Excitotoxicity has been implicated in the retinal neuronal loss in several ocular pathologies including glaucoma. Dysfunction of Excitatory Amino Acid Transporters is often a key component of the cascade leading to excitotoxic cell death. In the retina, glutamate transport is mainly operated by the glial glutamate transporter GLAST and the neuronal transporter GLT-1. In this study we evaluated the expression of GLAST and GLT-1 in a rat model of acute glaucoma based on the transient increase of intraocular pressure (IOP) and characterized by high glutamate levels during the reperfusion that follows the ischemic event associated with raised IOP. No changes were reported in GLAST expression while, at neuronal level, a reduction of glutamate uptake and of transporter reversal-mediated glutamate release was observed in isolated retinal synaptosomes. This was accompanied by modulation of GLT-1 expression leading to the reduction of the canonical 65 kDa form and upregulation of a GLT-1-related 38 kDa protein. These results support a role for neuronal transporters in glutamate accumulation observed in the retina following an ischemic event and suggest the presence of a GLT-1 neuronal new alternative splice variant, induced in response to the detrimental stimulus.
Glioblastoma multiforme (GBM) is the most common and aggressive primary brain cancer, with a median survival of less than 2 years after diagnosis. The tumor microenvironment plays a critical role in tumor invasion and progression. Microglia and infiltrating macrophages are the most abundant immune cells in the tumor. In the present study, we demonstrate that systemic propentofylline (PPF), an atypical methylxanthine with central nervous system (CNS) glial modulating and anti-inflammatory actions, significantly decreased tumor growth in a CNS-1 rat model of GBM by targeting microglia and not tumor cells. Rats received tumor injections of 1 × 105 CNS-1 cells in the right striatum with daily intraperitonial injections of PPF (50 mg/kg) or saline beginning the day of tumor injection. PPF did not cause apoptosis or decrease proliferation of CNS-1 tumor cells. Furthermore, we demonstrate, using in vitro methods, that PPF decreased microglial migration toward CNS-1 tumor cells and decreased MMP-9 expression. The effects of PPF were shown to be specific to microglia and not peripheral macrophages. These results support a differential functional role of resident microglia and infiltrating macrophages in the brain tumor environment. Our data highlight microglia as a crucial target for future therapeutic development and present PPF as a possible drug for treatment of human GBM.
Glioma; macrophages; microglia; migration; MMP-9
Glioblastoma multiforme (GBM) is the most common and aggressive primary brain cancer, with a median survival of less than 2 years after diagnosis with current available therapies. The tumor microenvironment serves a critical role in tumor invasion and progression, with microglia as a critical player. Our laboratory has previously demonstrated that propentofylline, an atypical methylxanthine with central nervous system glial modulating and anti-inflammatory actions, significantly decreases tumor growth in a GBM rodent model by preferentially targeting microglia. In the present study, we used the CNS-1 rat glioma model to elucidate the mechanisms of propentofylline. Here we demonstrate that propentofylline targets TROY, a novel signaling molecule up-regulated in infiltrating microglia, and not macrophages, in response to CNS-1 cells. We identify Pyk2, Rac1 and pJNK as the downstream signaling molecules of TROY through western blot analysis and siRNA transfection. We demonstrate that inhibition of TROY expression in microglia by siRNA transfection significantly inhibits microglial migration towards CNS-1 cells similar to 10 µM propentofylline treatment. These results identify TROY as a novel molecule expressed in microglia, involved in their migration and targeted by propentofylline. Furthermore, these results describe a signaling molecule that is differentially expressed between microglia and macrophages in the tumor microenvironment.
The cellular and molecular mechanisms that govern the response of the perinatal brain to injury remain largely unexplored. We investigated the role of white matter astrocytes in a rodent model of diffuse white matter injury produced by exposing neonatal mice to chronic hypoxia – a paradigm which mimics brain injury in premature infants. We demonstrate absence of reactive gliosis in the immature white matter following chronic hypoxia, as determined by astrocyte proliferation index and glial fibrillary acidic protein levels. Instead, Nestin expression in astrocytes is transiently increased, and the glial-specific glutamate transporters GLAST and GLT-1 are reduced. Finally, we demonstrate that JAK/STAT signaling - which is important in both astrocyte development and response to injury - is reduced in the white matter following hypoxia, as well as in primary astrocytes exposed to hypoxia in vitro. Hypoxia and JAK/STAT inhibition reduce glutamate transporter expression in astrocytes, but unlike hypoxia JAK/STAT inhibition downregulates GLAST expression without affecting GLT-1, as demonstrated in vitro by treatment with JAK Inhibitor I and in vivo by treatment with the JAK/STAT inhibitor AG490. Our findings: i) demonstrate specific changes in astrocyte function after perinatal hypoxia, which might contribute to the particular pathogenesis of perinatal white matter injury, ii) provide evidence that at least part of these changes result from a disturbance of the JAK/STAT pathway by hypoxia, and iii) identify JAK/STAT signaling as a potential therapeutic target to restore normal GLAST expression and uptake of glutamate after perinatal brain injury.
Decreased GABAergic synaptic strength (“disinhibition”) in the spinal dorsal horn is a crucial mechanism contributing to the development and maintenance of pathological pain. However, mechanisms leading to disinhibition in the spinal dorsal horn remain elusive. We investigated the role of glial glutamate transporters (GLT-1 and GLAST) and glutamine synthetase in maintaining GABAergic synaptic activity in the spinal dorsal horn. Electrically evoked GABAergic inhibitory postsynaptic currents (eIPSCs), spontaneous IPSCs (sIPSCs) and miniature IPSCs (mIPSCs) were recorded in superficial spinal dorsal horn neurons of spinal slices from young adult rats. We used (2S, 3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (TFB-TBOA), to block both GLT-1 and GLAST and dihydrokainic acid (DHK) to block only GLT-1. We found that blockade of both GLAST and GLT-1 and blockade of only GLT-1 in the spinal dorsal horn decreased the amplitude of GABAergic eIPSCs, as well as both the amplitude and frequency of GABAergic sIPSCs or mIPSCs. Pharmacological inhibition of glial glutamine synthetase had similar effects on both GABAergic eIPSCs and sIPSCs. We provided evidence demonstrating that the reduction in GABAergic strength induced by the inhibition of glial glutamate transporters is due to insufficient GABA synthesis through the glutamate-glutamine cycle between astrocytes and neurons. Thus, our results indicate that deficient glial glutamate transporters and glutamine synthetase significantly attenuate GABAergic synaptic strength in the spinal dorsal horn, which may be a crucial synaptic mechanism underlying glial-neuronal interactions caused by dysfunctional astrocytes in pathological pain conditions.
The astrocyte glutamate transporter, GLT1, is responsible for the vast majority of glutamate uptake in the adult central nervous system (CNS), thereby regulating extracellular glutamate homeostasis and preventing excitotoxicity. Glutamate dysregulation plays a central role in outcome following traumatic spinal cord injury (SCI). To determine the role of GLT1 in secondary cell loss following SCI, mice heterozygous for the GLT1 astrocyte glutamate transporter (GLT1+/−) and wild-type mice received thoracic crush SCI. Compared to wild-type controls, GLT1+/− mice had an attenuated recovery in hindlimb motor function, increased lesion size, and decreased tissue sparing. GLT1+/− mice showed a decrease in intraspinal GLT1 protein and functional glutamate uptake compared to wild-type mice, accompanied by increased apoptosis and neuronal loss following crush injury. These results suggest that astrocyte GLT1 plays a role in limiting secondary cell death following SCI, and also show that compromise of key astrocyte functions has significant effects on outcome following traumatic CNS injury. These findings also suggest that increasing intraspinal GLT1 expression may represent a therapeutically relevant target for SCI treatment.
secondary injury; GLT1+/− mice; crush injury; glutamate uptake; excitotoxicity
In the present study, we examined whether activation of p-38α MAPK modulates mechanical allodynia and neuronal hyperexcitability, and if propentofylline (PPF, a glial modulator) modulates specifically localized activated p-38α MAPK expression in caudal regions remote from a low thoracic hemisection injury in rats. T13 spinal hemisection produces bilateral mechanical allodynia in hindpaws with evoked (in response to mechanical stimuli) neuronal hyperexcitability in lumbar spinal wide dynamic range (WDR) neurons compared to sham controls. The mechanical allodynia and the evoked activity of WDR neurons is attenuated by intrathecal and topical administration of SB203580, an inhibitor of p-38α MAPK activation, dose dependently (*p<0.05); however, the spontaneous activity showed no significant differences compared to sham controls. After T13 spinal hemisection, significantly increased phosphorylated (activated form) p-38α MAPK expression was present in both superficial and deep dorsal horn neurons as well as in microglia, but not in astrocytes, in the lumbar spinal cord compared to sham controls (*p<0.05). Intrathecal application of PPF significantly attenuated the expression of phosphorylated p-38α MAPK in superficial dorsal horn neurons (10 mM) and in microglia (1 and 10 mM) in the lumbar spinal cord compared to the hemisection group (*p<0.05). In conclusion, our present data demonstrate that activated neuronal and microglial, but not astrocytic, p-38α MAPK contributes to the maintenance of neuronal hyperexcitability in caudal regions following spinal cord injury.
Glia; Hyperexcitability; p38 MAPK; Propentofylline; Spinal cord injury
The glial glutamate transporter GLT-1 is abundantly expressed in astrocytes and is crucial for glutamate removal from the synaptic cleft. Decreases in glutamate uptake activity and expression of spinal glutamate transporters are reported in animal models of pathological pain. However, the lack of available specific inhibitors and/or activators for GLT-1 makes it difficult to determine the roles of spinal GLT-1 in inflammatory and neuropathic pain. In this study, we examined the effect of gene transfer of GLT-1 into the spinal cord with recombinant adenoviruses on the inflammatory and neuropathic pain in rats.
Intraspinal infusion of adenoviral vectors expressing the GLT-1 gene increased GLT-1 expression in the spinal cord 2–21 days after the infusion. Transgene expression was primarily localized to astrocytes. The spinal GLT-1 gene transfer had no effect on acute mechanical and thermal nociceptive responses in naive rats, whereas it significantly reduced the inflammatory mechanical hyperalgesia induced by hindlimb intraplantar injection of carrageenan/kaolin. Spinal GLT-1 gene transfer 7 days before partial sciatic nerve ligation recovered the extent of the spinal GLT-1 expression in the membrane fraction that was decreased following the nerve ligation, and prevented the induction of tactile allodynia. However, the partial sciatic nerve ligation-induced allodynia was not reversed when the adenoviruses were infused 7 or 14 days after the nerve ligation.
These results suggest that overexpression of GLT-1 on astrocytes in the spinal cord by recombinant adenoviruses attenuates the induction, but not maintenance, of inflammatory and neuropathic pain, probably by preventing the induction of central sensitization, without affecting acute pain sensation. Upregulation or functional enhancement of spinal GLT-1 could be a novel strategy for the prevention of pathological pain.
Astrocytes play a major role in the removal of glutamate from the extracellular compartment. This clearance limits the glutamate receptor activation and affects the synaptic response. This function of the astrocyte is dependent on its positioning around the synapse, as well as on the level of expression of its high-affinity glutamate transporters, GLT1 and GLAST. Using Western blot analysis and serial section electron microscopy, we studied how a change in sensory activity affected these parameters in the adult cortex. Using mice, we found that 24 h of whisker stimulation elicited a 2-fold increase in the expression of GLT1 and GLAST in the corresponding cortical column of the barrel cortex. This returns to basal levels 4 d after the stimulation was stopped, whereas the expression of the neuronal glutamate transporter EAAC1 remained unaltered throughout. Ultrastructural analysis from the same region showed that sensory stimulation also causes a significant increase in the astrocytic envelopment of excitatory synapses on dendritic spines. We conclude that a period of modified neuronal activity and synaptic release of glutamate leads to an increased astrocytic coverage of the bouton–spine interface and an increase in glutamate transporter expression in astrocytic processes.
After increased sensory activity in the somatosensory cortex, astrocytic processes expand to increase their coverage of the bouton-spine interface, and expression of astrocytic glutamate transporters is up-regulated as well.
Astrocytes remove glutamate from the synaptic cleft via specific transporters, and impaired glutamate reuptake may promote excitotoxic neuronal injury. In a model of viral encephalomyelitis caused by neuroadapted Sindbis virus (NSV), mice develop acute paralysis and spinal motor neuron degeneration inhibited by the AMPA receptor antagonist, NBQX. To investigate disrupted glutamate homeostasis in the spinal cord, expression of the main astroglial glutamate transporter, GLT-1, was examined. GLT-1 levels declined in the spinal cord during acute infection while GFAP expression was preserved. There was simultaneous production of inflammatory cytokines at this site, and susceptible animals treated with drugs that blocked IL-1β release also limited paralysis and prevented the loss of GLT-1 expression. Conversely, infection of resistant mice that develop mild paralysis following NSV challenge showed higher baseline GLT-1 levels as well as lower production of IL-1β and relatively preserved GLT-1 expression in the spinal cord compared to susceptible hosts. Finally, spinal cord GLT-1 expression was largely maintained following infection of IL-1β-deficient animals. Together, these data show that IL-1β inhibits astrocyte glutamate transport in the spinal cord during viral encephalomyelitis. They provide one of the strongest in vivo links between innate immune responses and the development of excitotoxicity demonstrated to date.
glutamate transporters; interleukin-1β; viral encephalomyelitis; motor neuron; excitotoxicity
Astrocyte heterogeneity remains largely unknown in the CNS due to lack of specific astroglial markers. In this study, molecular identity of in vivo astrocytes was characterized in BAC ALDH1L1 and BAC GLT1 eGFP promoter reporter transgenic mice. ALDH1L1 promoter is selectively activated in adult cortical and spinal cord astrocytes, indicated by the overlap of eGFP expression with ALDH1L1 and GFAP, but not with NeuN, APC, Olig2, IbaI, PDGFRα immunoreactivity in BAC ALDH1L1 eGFP reporter mice. Interestingly, ALDH1L1 expression levels (protein, mRNA, and promoter activity) in spinal cord were selectively decreased during postnatal maturation. In contrast, its expression was up-regulated in reactive astrocytes in both acute neural injury and chronic neurodegenerative (G93A mutant SOD1) conditions, similar to GFAP, but opposite of GLT1. ALDH1L1+ and GLT1+ cells isolated through fluorescence activated cell sorting (FACS) from BAC ALDH1L1 and BAC GLT1 eGFP mice share a highly similar gene expression profile, suggesting ALDH1L1 and GLT1 are co-expressed in the same population of astrocytes. This observation was further supported by overlap of the eGFP driven by the ALDH1L1 genomic promoter and the tdTomato driven by a 8.3kb EAAT2 promoter fragment in astrocytes of BAC ALDH1L1 eGFP X EAAT2-tdTomato mice. These studies support ALDH1L1 as a general CNS astroglial marker and investigated astrocyte heterogeneity in the CNS by comparing the molecular identity of the ALDH1L1+ and GLT1+ astrocytes from astroglial reporter mice. These astroglial reporter mice provide useful in vivo tools for the molecular analysis of astrocytes in physiological and pathological conditions.
astroglia; BAC; ALDH1L1; GLT1; GFAP; oligodendroglia; ALS
GLT-1 eGFP BAC reporter transgenic adult mice were used to detect GLT-1 gene expression in individual cells of CA1, CA3 and SI, and eGFP fluorescence was measured to analyze quantitatively GLT-1 promoter activity in different cells of neocortex and hippocampus. Virtually all GFAP+ astrocytes were eGFP+; we also found that about 80% of neurons in CA3 pyramidal layer, 10–70% of neurons in I-VI layers of SI and rare neurons in all strata of CA1 and in strata oriens and radiatum of CA3 were eGFP+. Analysis of eGFP intensity showed that astrocytes had a higher GLT-1 promoter activity in SI than in CA1 and CA3, and that neurons had the highest levels of GLT-1 promoter activity in CA3 stratum pyramidale and in layer VI of SI. Finally, we observed that the intensity of GLT-1 promoter activity in neurons is 1–20% of that measured in astrocytes. These results showed that in the hippocampus and neocortex GLT-1 promoter activity is observed in astrocytes and neurons, detailed the distribution of GLT-1 expressing neurons, and indicated that GLT-1 promoter activity in both astrocytes and neurons varies in different brain regions.
glutamate transporters; GLT-1/EAAT2; neurons; astrocytes; hippocampus; neocortex
Recent data suggest that excessive glutamatergic signaling in the prefrontal cortex may contribute to the pathophysiology of schizophrenia, and that promoting presynaptic glutamate modulation via group II metabotropic glutamate (mGlu2/3) receptor activation can exert antipsychotic efficacy. The glutamate transporter GLAST (EAAT1) regulates extracellular glutamate levels via uptake into glia, but the consequences of GLAST dysfunction for schizophrenia are largely unknown.
We examined GLAST knockout mice (KO) for behaviors thought to model positive symptoms in schizophrenia (locomotor hyperactivity to novelty, exaggerated locomotor response to N-methyl-D-aspartate receptor (NMDAR) antagonism), and the ability of haloperidol and the mGlu2/3 agonist LY379268 to normalize novelty-induced hyperactivity.
GLAST KO consistently showed locomotor hyperactivity to a novel but not familiar environment, relative to wild-type (WT). The locomotor hyperactivity-inducing effects of the NMDAR antagonist MK-801 was exaggerated in GLAST KO relative to WT. Treatment with haloperidol or LY379268 normalized novelty-induced locomotor hyperactivity in GLAST KO.
‘Schizophrenia-related’ abnormalities in GLAST KO raise the possibility that loss of GLAST-mediated glutamate clearance could be a pathophysiological risk factor for the disease. Our findings provide novel support for the hypothesis that glutamate dysregulation contributes to the pathophysiology of schizophrenia, and for the antipsychotic potential of mGlu2/3 agonists.
GLAST; glutamate; schizophrenia; NMDA; mGlu2/3
Neuroadapted Sindbis virus (NSV) is a neuronotropic virus that causes a fulminant encephalomyelitis in susceptible mice due to death of motor neurons in the brain and spinal cord. We and others have found that uninfected motor neurons die in response to NSV infection, at least in part due to disrupted astrocytic glutamate transport, resulting in excitotoxic motor neuron death. Here, we examined the mechanisms of astrocyte dysregulation associated with NSV infection. Treatment of organotypic slice cultures with NSV results in viral replication, cell death, altered astrocyte morphology, and the downregulation of the astrocytic glutamate transporter, GLT-1. We have found that TNF-α can mediate GLT-1 downregulation. Furthermore, TNF-α deficient mice infected with NSV exhibit neither GLT-1 downregulation nor neuronal death of brainstem and cervical spinal cord motor neurons and have markedly reduced mortality. These findings have implications for disease intervention and therapeutic development for the prevention of CNS damage associated with inflammatory responses.
Astrocyte; TNF-α; Motor neuron; GLT-1; Glutamate; Virus