Dopamine loss alters glutamate synapses and transporters in the medial prefrontal cortex and anxiety-related behaviour in a male MPTP rodent model of Parkinson's disease.

Anxiety is a prominent non-motor symptom of Parkinson's disease (PD). Changes in the B-spectrum recordings in PD patients of the prefrontal cortex correlate with increased anxiety. Using a rodent model of PD, we reported alterations in glutamate synapses in the striatum and substantia nigra following dopamine (DA) loss. We hypothesize that DA loss will result in increased anxiety-related behaviours and that this will be associated with alterations in glutamate synapses and transporters within the medial prefrontal cortex (mPFC). Following 4 weeks of progressive 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration, there was an increase in anxiety-related behaviours and a 78% decrease in plasma corticosterone levels versus the vehicle (VEH)-treated mice. This was associated with a 30% decrease in the density of dendritic spines in Layers Il/Ill, and a 53% decrease in the density of glutamate immuno-gold labelling within vesicular glutamate transporter 1 (Vglut1)-labelled nerve terminals and spines, with no change within vesicular glutamate transporter 2 (Vglut2) positive terminals/spines in the MPTP versus VEH groups. Our prior work determined that a decrease in striatal glutamate terminal density was associated with an increase in extracellular glutamate levels. There was an increase in protein expression of Vglut1 (40%), Vglut2 (37%) and glutamate aspartate transporter (GLAST) (225%), and a decrease in glutamate transporter 1 (GLT-1) (50%) and excitatory amino acid carrier 1 (EAAC1) (51%), in the MPTP versus VEH groups within the mPFC. These data suggest that the decrease in dendritic spines within the mPFC following nigrostriatal DA loss may be due to increased extracellular glutamate levels (decrease in glutamate transporters), leading to an increase in anxiety-related behaviours.

Ceftriaxone and MC-100093 mitigate fentanyl-induced cardiac injury in mice: Preclinical investigation of its underlying molecular mechanisms.

Drug addiction is considered a worldwide concern and one of the most prevailing causes of death globally. Opioids are highly addictive drugs, and one of the most common opioids that is frequently used clinically is fentanyl. The potential harmful effects of chronic exposure to opioids on the heart are still to be elucidated. Although ß-lactam antibiotics are well recognized for their ability to fight bacteria, its protective effect in the brain and liver has been reported. In this study, we hypothesize that ß-lactam antibiotic, ceftriaxone, and the novel synthetic non-antibiotic ß-lactam, MC-100093, are cardioprotective against fentanyl induced-cardiac injury by upregulating xCT expression. Mice were exposed to repeated low dose (0.05 mg/kg, i.p.) of fentanyl for one week and then challenged on day 9 with higher dose of fentanyl (1 mg/kg, i.p.). This study investigated cardiac histopathology and target genes and proteins in serum and cardiac tissues in mice exposed to fentanyl overdose and ß-lactams. We revealed that fentanyl treatment induced cardiac damage as evidenced by elevated cardiac enzymes (troponin I). Furthermore, fentanyl treatment caused large aggregations of inflammatory cells and elevation in the areas and volumes of myocardial fibers, indicating hypertrophy and severe cardiac damage. Ceftriaxone and MC-100093 treatment, However, induced cardioprotective effects as evidenced by marked reduction in cardiac enzymes (troponin I) and changes in histopathology. Furthermore, ceftriaxone and MC-100093 treatment decreased the levels of hypertrophic genes (α-MHC & ß-MHC), apoptotic (caspase-3), and inflammatory markers (IL-6 & NF-κB). This study reports for the first time the cardioprotective effect of ß-lactams against fentanyl-induced cardiac injury. Further studies are greatly encouraged to completely identify the cardioprotective properties of ceftriaxone and MC-100093.

Astrocytic aryl hydrocarbon receptor mediates chronic kidney disease-associated mental disorders involving GLT1 hypofunction and neuronal activity enhancement in the mouse brain.

Chronic kidney disease (CKD)-associated mental disorders have been attributed to the excessive accumulation of hemodialysis-resistant indoxyl-3-sulfate (I3S) in the brain. I3S not only induces oxidative stress but is also a potent endogenous agonist of the aryl hydrocarbon receptor (AhR). Here, we investigated the role of AhR in CKD-induced brain disorders using a 5/6 nephrectomy-induced CKD mouse model, which showed increased I3S concentration in both blood and brain, anxiety and impaired novelty recognition, and AhR activation in the anterior cortex. GFAP+ reactive astrocytes were increased accompanied with the reduction of glutamate transporter 1 (GLT1) on perineuronal astrocytic processes (PAPs) in the anterior cingulate cortex (ACC) in CKD mice, and these alterations were attenuated in both neural lineage-specific and astrocyte-specific Ahr conditional knockout mice (nAhrCKO and aAhrCKO). By using chronic I3S treatment in primary astrocytes and glia-neuron (GN) mix cultures to mimic the CKD brain microenvironment, we also found significant reduction of GLT1 expression and activity in an AhR-dependent manner. Chronic I3S treatment induced AhR-dependent pro-oxidant Nox1 and AhR-independent anti-oxidant HO-1 expressions. Notably, AhR mediates chronic I3S-induced neuronal activity enhancement and synaptotoxicity in GN mix, not neuron-enriched cortical culture. In CKD mice, neuronal activity enhancement was observed in ACC and hippocampal CA1, and these responses were abrogated by both nAhrCKO and aAhrCKO. Finally, intranasal AhR antagonist CH-223191 administration significantly ameliorated the GLT1/PAPs reduction, increase in c-Fos+ neurons, and memory impairment in the CKD mice. Thus, astrocytic AhR plays a crucial role in the CKD-induced disturbance of neuron-astrocyte interaction and mental disorders.

Astrocytic purinergic signalling contributes to the development and maintenance of neuropathic pain via modulation of glutamate release.

Although activation of astrocytes is critical in developing neuropathic pain (NP) following nerve injury, the underlying mechanisms of NP and therapeutic management for NP are still vague. Importantly, the decreases in the levels of astrocytic glutamate transporter-1 (GLT-1) in the spinal dorsal horn result in enhanced excitatory transmission and cause persistent pain. P2Y1 purinergic receptor (P2Y1R) has been shown to enhance many inflammatory processes. The up-regulated expression of astrocytic P2Y1R is crucial to participate in pain transduction under conditions of nerve injury and peripheral inflammation considering that P2Y1R is potentially involved in glutamate release and synaptic transmission. This study indicates that the expression of P2Y1R in the spinal cord was increased accompanied by the activation of A1 phenotype astrocytes in the rat model of spinal nerve ligation (SNL). Astrocyte-specific knockdown of P2Y1R alleviated SNL-induced nociceptive responses and mitigated A1 reactive astrocytes, which subsequently increased GLT-1 expression. Conversely, in naïve rats, P2Y1R over-expression induced a canonical NP-like phenotype and spontaneous hypernociceptive responses and increased the concentration of glutamate in the spinal dorsal horn. Besides, our in vitro data showed that the proinflammatory cytokine tumour necrosis factor-alpha contributes to A1/A2 astrocyte reactivity and Ca2+ -dependent release of glutamate. Conclusively, our results provide novel insights that as a significant regulator of astrocytic A1/A2 polarization and neuroinflammation, P2Y1R may represent a potential target for the treatment of SNL-induced NP.

Repeated sevoflurane exposures inhibit neurogenesis by inducing the upregulation of glutamate transporter 1 in astrocytes.

Sevoflurane is a widely used general anaesthetic in paediatric patients. Although repeated sevoflurane exposure is known to cause neurodevelopmental disorders in children, the mechanism of this neurotoxicity remains largely unknown. Herein, we investigated the role of glutamate transporter 1 (GLT1) in sevoflurane-induced decreased neurogenesis. Neonatal rat pups (postnatal Day 7, PN7) were exposed to 3% sevoflurane for 2 h for three consecutive days. Neuron loss and decreased neurogenesis have been observed in the neonatal rat brain, along with decreased number of astrocytes. Apoptotic astrocytes were observed after repeated sevoflurane exposure in vitro, resulting in decreased levels of brain-derived neurotrophic factor (BDNF). Calcium overload was observed in astrocytes after repeated sevoflurane exposure, in addition to upregulation of GLT1. Inhibition of GLT1 activity ameliorates repeated sevoflurane exposure-induced cognitive deficits in adult rats. Mechanically, the upregulation of GLT1 was caused by the activation of mRNA translation. RNA-sequencing analysis further confirmed that translation-related genes were activated by repeated sevoflurane exposure. These results indicate that cognitive deficits caused by repeated sevoflurane exposure during PN7-9 are triggered decreased neurogenesis. The proposed underlying mechanism involves upregulation of apoptosis in astrocytes induced by GLT1; therefore, we propose GLT1 as a potential pharmacological target for brain injury in paediatric practice.

Distribution Patterns of Astrocyte Populations in the Human Cortex.

Astrocytes are a major class of glial cell in the central nervous system that have a diverse range of types and functions thought to be based on their anatomical location, morphology and cellular properties. Recent studies highlight that astrocyte dysfunction contributes to the pathogenesis of neurological conditions. However, few studies have described the pattern, distribution and density of astrocytes in the adult human cortex. This study mapped the distribution and density of astrocytes immunolabelled with a range of cytoskeletal and membrane markers in the human frontal cortex. Distinct and overlapping astrocyte populations were determined. The frontal cortex from ten normal control cases (75 ± 9 years) was immunostained with glial fibrillary acidic protein (GFAP), aldehyde dehydrogenase-1 L1 (ALDH1L1), connexin-43 (Cx43), aquaporin-4 (AQP4), and glutamate transporter 1 (GLT-1). All markers labelled populations of astrocytes in the grey and white matter, separate cortical layers, subpial and perivascular regions. All markers were informative for labelling different cellular properties and cellular compartments of astrocytes. ALDH1L1 labelled the largest population of astrocytes, and Cx43-immunopositive astrocytes were found in all cortical layers. AQP4 and GLT-1 labelled distal astrocytic process and end-feet in the same population of astrocytes (98% of GLT-1-immunopositive astrocytes contained AQP4). In contrast, GFAP, the most widely used marker, predominantly labelled astrocytes in superficial cortical layers. This study highlights the diversity of astrocytes in the human cortex, providing a reference map of the distribution of distinct and overlapping astrocyte populations which can be used for comparative purposes in various disease, inflammatory and injury states involving astrocytes.

Deletion of RE1-silencing transcription factor in striatal astrocytes exacerbates manganese-induced neurotoxicity in mice.

Chronic manganese (Mn) overexposure causes a neurological disorder, referred to as manganism, exhibiting symptoms similar to parkinsonism. Dysfunction of the repressor element-1 silencing transcription factor (REST) is associated with various neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and Mn-induced neurotoxicity, but its cellular and molecular mechanisms have yet to be fully characterized. Although neuronal REST is known to be neuroprotective, the role of astrocytic REST in neuroprotection remains to be established. We investigated if astrocytic REST in the striatal region of the mouse brain where Mn preferentially accumulates plays a role in Mn-induced neurotoxicity. Striatal astrocytic REST was deleted by infusion of adeno-associated viral vectors containing sequences of the glial fibrillary acidic protein promoter-driven Cre recombinase into the striatum of RESTflox/flox mice for 3 weeks, followed by Mn exposure (30 mg/kg, daily, intranasally) for another 3 weeks. Striatal astrocytic REST deletion exacerbated Mn-induced impairment of locomotor activity and cognitive function with further decrease in Mn-reduced protein levels of tyrosine hydroxylase and glutamate transporter 1 (GLT-1) in the striatum. Astrocytic REST deletion also exacerbated the Mn-induced proinflammatory mediator COX-2, as well as cytokines such as TNF-α, IL-1ß, and IL-6, in the striatum. Mn-induced detrimental astrocytic products such as proinflammatory cytokines on neuronal toxicity were attenuated by astrocytic REST overexpression, but exacerbated by REST inhibition in an in vitro model using primary human astrocytes and Lund human mesencephalic (LUHMES) neuronal culture. These findings indicate that astrocytic REST plays a critical role against Mn-induced neurotoxicity by modulating astrocytic proinflammatory factors and GLT-1.

Spinal NLRP3 inflammasome activation mediates IL-1ß release and contributes to remifentanil-induced postoperative hyperalgesia by regulating NMDA receptor NR1 subunit phosphorylation and GLT-1 expression in rats.

BACKGROUND: Trafficking and activation of N-methyl-D-aspartate (NMDA) receptors play an important role in initiating and maintaining postoperative remifentanil-induced hyperalgesia (RIH). Activation of the NOD-like receptor protein 3 (NLRP3) inflammasome has been linked to the development of inflammatory and neuropathic pain. We hypothesized that activation of NLRP3 inflammasome mediates IL-1ß release and contributes to RIH in rats by increasing NMDA receptor NR1 (NR1) subunit phosphorylation and decreasing glutamate transporter-1 (GLT-1) expression. METHODS: Acute exposure to remifentanil (1.2 µg/kg/min for 60 min) was used to establish RIH in rats. Thermal and mechanical hyperalgesia were tested at baseline (24 h before remifentanil infusion) and 2, 6, 24, and 48 h after remifentanil infusion. The levels of IL-1ß, GLT-1, phosphorylated NR1 (phospho-NR1), and NLRP3 inflammasome activation indicators [NLRP3, Toll-like receptor 4 (TLR4), P2X purinoceptor 7 (P2X7R), and caspase-1] were measured after the last behavioral test. A selective IL-1ß inhibitor (IL-1ß inhibitor antagonist; IL-1ra) or three different selective NLRP3 inflammasome activation inhibitors [(+)-naloxone (a TLR4 inhibitor), A438079 (a P2X7R inhibitor), or ac-YVADcmk (a caspase-1 inhibitor)] were intrathecally administered immediately before remifentanil infusion into rats. RESULTS: Remifentanil induced significant postoperative hyperalgesia, increased IL-1ß and phospho-NR1 levels and activated the NLRP3 inflammasome by increasing TLR4, P2X7R, NLRP3, and caspase-1 expression, but it decreased GLT-1 expression in the L4-L6 spinal cord segments of rats, which was markedly improved by intrathecal administration of IL-1ra, (+)-naloxone, A438079, or ac-YVADcmk. CONCLUSION: NLRP3 inflammasome activation mediates IL-1ß release and contributes to RIH in rats by inducing NMDA receptor NR1 subunit phosphorylation and decreasing GLT-1 expression. Inhibiting the activation of the NLRP3 inflammasome may be an effective treatment for RIH.

Intrathecal Administration of the α1 Adrenergic Antagonist Phentolamine Upregulates Spinal GLT-1 and Improves Mirror Image Pain in SNI Model Rats.

Mirror image pain (MIP) is a type of extraterritorial pain that results in contralateral pain or allodynia. Glutamate transporter-1 (GLT-1) is expressed in astrocytes and plays a role in maintaining low glutamate levels in the synaptic cleft. Previous studies have shown that GLT-1 dysfunction induces neuropathic pain. Our previous study revealed bilateral GLT-1 downregulation in the spinal cord of a spared nerve injury (SNI) rat. We hypothesized that spinal GLT-1 is involved in the mechanism of MIP. We also previously demonstrated noradrenergic GLT-1 regulation. Therefore, this study aimed to investigate the effect of an α1 adrenergic antagonist on the development of MIP. Rats were subjected to SNI. Changes in pain behavior and GLT-1 protein levels in the SNI rat spinal cords were then examined by intrathecal administration of the α1 adrenergic antagonist phentolamine, followed by von Frey test and western blotting. SNI resulted in the development of MIP and bilateral downregulation of GLT-1 protein in the rat spinal cord. Intrathecal phentolamine increased contralateral GLT-1 protein levels and partially ameliorated the 50% paw withdrawal threshold in the contralateral hind paw. Spinal GLT-1 upregulation by intrathecal phentolamine ameliorates MIP. GLT-1 plays a role in the development of MIPs.

Vascular endothelial growth factor receptor-3 regulates astroglial glutamate transporter-1 expression via mTOR activation in reactive astrocytes following pilocarpine-induced status epilepticus.

Recent evidence has shown that the vascular endothelial growth factor (VEGF) system plays a crucial role in several neuropathological processes. We previously reported an upregulation of VEGF-C and its receptor, VEGFR-3, in reactive astrocytes after the onset of status epilepticus (SE). However, it remains unknown, which molecules act as downstream signals following VEGFR-3 upregulation, and are involved in reactive astrogliosis after SE. Therefore, we investigated whether VEGFR-3 upregulation within reactive astrocytes is associated with the activation of mammalian target of rapamycin (mTOR) signaling, which we confirmed by assaying for the phosphorylated form of S6 protein (pS6), and whether VEGFR-3-mediated mTOR activation induces astroglial glutamate transporter-1 (GLT-1) expression in the hippocampus after pilocarpine-induced SE. We found that spatiotemporal expression of pS6 was consistent with VEGFR-3 expression in the hippocampus after SE, and that both pS6 and VEGFR-3 were highly expressed in SE-induced reactive astrocytes. Treatment with the mTOR inhibitor rapamycin decreased astroglial VEGFR-3 expression and GLT-1 expression after SE. Treatment with a selective inhibitor for VEGFR-3 attenuated astroglial pS6 expression as well as suppressed GLT-1 expression and astroglial reactivity in the hippocampus after SE. These findings demonstrate that VEGFR-3-mediated mTOR activation could contribute to the regulation of GLT-1 expression in reactive astrocytes during the subacute phase of epilepsy. In conclusion, the present study suggests that VEGFR-3 upregulation in reactive astrocytes may play a role in preventing hyperexcitability induced by continued seizure activity.

Targeted overexpression of glutamate transporter-1 reduces seizures and attenuates pathological changes in a mouse model of epilepsy.

Astrocytic glutamate transporters are crucial for glutamate homeostasis in the brain, and dysregulation of these transporters can contribute to the development of epilepsy. Glutamate transporter-1 (GLT-1) is responsible for the majority of glutamate uptake in the dorsal forebrain and has been shown to be reduced at epileptic foci in patients and preclinical models of temporal lobe epilepsy (TLE). Current antiepileptic drugs (AEDs) work primarily by targeting neurons directly through suppression of excitatory neurotransmission or enhancement of inhibitory neurotransmission, which can lead to both behavioral and psychiatric side effects. This study investigates the therapeutic capacity of astrocyte-specific AAV-mediated GLT-1 expression in the intrahippocampal kainic acid (IHKA) model of TLE. In this study, we used Western blot analysis, immunohistochemistry, and long-term-video EEG monitoring to demonstrate that cell-type-specific upregulation of GLT-1 in astrocytes is neuroprotective at early time points during epileptogenesis, reduces seizure frequency and total time spent in seizures, and eliminates large behavioral seizures in the IHKA model of epilepsy. Our findings suggest that targeting glutamate uptake is a promising therapeutic strategy for the treatment of epilepsy.

Sulbactam improves binding property and uptake capacity of glutamate transporter-1 and decreases glutamate concentration in the CA1 region of hippocampus of global brain ischemic rats.

Glutamate transporter-1 (GLT-1) removes most glutamate in the synaptic cleft. Sulbactam confers neuronal protection against ischemic insults in the hippocampal CA1 region accompanied by the upregulation of GLT-1 expression in rats. The present study further investigates the effect of sulbactam on the binding property and uptake capacity of GLT-1 for glutamate, and the change in extracellular glutamate concentration in the hippocampal CA1 region of rats with global brain ischemia. The binding property and uptake capacity of GLT-1 were measured using a radioligand binding and uptake assay, respectively, with L-3H-glutamate. The extracellular glutamate concentration was detected using microdialysis and high-performance liquid chromatography-mass spectrometry. Neuropathological evaluation was performed based on thionin staining. It was shown that sulbactam pre-treatment changed GLT-1 binding property, including increased Bmax and decreased Kd values, increased GLT-1 uptake capacity for glutamate, and inhibited the elevation of extracellular glutamate concentration in rats with global cerebral ischemia. These effects of sulbactam were accompanied by its neuronal protection on the hippocampal CA1 neurons against delayed neuronal death resulted from ischemic insult. Furthermore, administration of GLT-1 antisense oligodeoxynucleotides, which inhibited the expression of GLT-1, blocked the aforementioned sulbactam-related effects, which suggested that GLT-1 upregulation mediated the above effect although other mechanisms independent of the upregulation of GLT-1 expression could not be excluded. It could be concluded that sulbactam improves the binding property and uptake capacity of GLT-1 for glutamate and then reduces the glutamate concentration and excitotoxicity during global cerebral ischemia, which contributes to the neuroprotection of sulbactam against brain ischemia.

Low corticosterone levels attenuate late life depression and enhance glutamatergic neurotransmission in female rats.

Sustained elevation of corticosterone (CORT) is one of the common causes of aging and major depression disorder. However, the role of elevated CORT in late life depression (LLD) has not been elucidated. In this study, 18-month-old female rats were subjected to bilateral adrenalectomy or sham surgery. Their CORT levels in plasma were adjusted by CORT replacement and the rats were divided into high-level CORT (H-CORT), low-level CORT (L-CORT), and Sham group. We showed that L-CORT rats displayed attenuated depressive symptoms and memory defects in behavioral tests as compared with Sham or H-CORT rats. Furthermore, we showed that glutamatergic transmission was enhanced in L-CORT rats, evidenced by enhanced population spike amplitude (PSA) recorded from the dentate gyrus of hippocampus in vivo and increased glutamate release from hippocampal synaptosomes caused by high frequency stimulation or CORT exposure. Intracerebroventricular injection of an enzymatic glutamate scavenger system, glutamic-pyruvic transmine (GPT, 1 µM), significantly increased the PSA in Sham rats, suggesting that extracelluar accumulation of glutamate might be the culprit of impaired glutamatergic transmission, which was dependent on the uptake by Glt-1 in astrocytes. We revealed that hippocampal Glt-1 expression level in the L-CORT rats was much higher than in Sham and H-CORT rats. In a gradient neuron-astrocyte coculture, we found that the expression of Glt-1 was decreased with the increase of neural percentage, suggesting that impairment of Glt-1 might result from the high level of CORT contributed neural damage. In sham rats, administration of DHK that inhibited Glt-1 activity induced significant LLD symptoms, whereas administration of RIL that promoted glutamate uptake significantly attenuated LLD. All of these results suggest that glutamatergic transmission impairment is one of important pathogenesis in LLD induced by high level of CORT, which provide promising clues for the treatment of LLD.

Ginsenoside Rb1 attenuates lipopolysaccharide-induced neural damage in the brain of mice via regulating the dysfunction of microglia and astrocytes.

The purpose of our research was to evaluate whether ginsenoside Rb1 has neuroprotective effects against lipopolysaccharide (LPS)-induced brain injury. ICR mice were intraperitoneally (i.p.) injected with 20 or 40 mg/kg Rb1 or saline for 7 consecutive days. On the 7th day, 30 minutes after Rb1 or saline administration, a single dose of LPS (LPS group, Rb1+LPS group) or saline (control group) was injected i.p. into the mice. Results demonstrated that Rb1 treatment could significantly improve the behavior performance of LPS mice in both the open field test and the beam walking test. Rb1 can also markedly attenuate the neuronal lesion in both hippocampus and somatosensory cortex in the brain of LPS mice. In addition, Rb1 treatment also significantly inhibits the LPS-induced neuroinflammation in the brain, indicated by reduced reactive microglia and decreased IL-1ß production. Both immunostaining and western blot results suggest that Rb1 can further enhance the LPS-induced GLT-1 expression and alleviate LPS-induced GS reduction in the brain. Our findings show that Rb1 has a protective effect on LPS-induced neuronal damage in the CA1 of the hippocampus and in the somatosensory area of the cerebral cortex in mice, which is likely to be the basis for its improvement of locomotor and motor coordination. Rb1 regulating the function of astrocytes and microglia through GLT-1 and GS in astrocytes may be involved in its neuroprotective effects.

Astrocytic NDRG2 is critical in the maintenance of neuropathic pain.

Activation of astrocytes and abnormal synaptic glutamate metabolism are closely associated with the induction and maintenance of neuropathic pain (NP), but the exact mechanism underlying this association remains unclear. N-myc downstream-regulated gene 2 (NDRG2), a novel tumor-suppressor protein and stress-response gene, is involved in the pathogenesis of several neurodegenerative diseases. However, its role in nociceptive transduction has rarely been investigated. Here, we found that NDRG2, which was mainly expressed in the astrocytes in the central nervous system (CNS), was increased in the spinal cord of a spinal nerve ligation (SNL) rat model for NP. Suppression of NDRG2 by intrathecal injection of an NDRG2-RNAi-adenovirus significantly alleviated SNL-induced mechanical and thermal hypersensitivity, as well as elevated astrocytic glutamate transporter 1 (GLT-1) expression and downregulated pro-inflammatory cytokine levels, in the spinal dorsal horn of rats on Day 10 after SNL. Furthermore, in lipopolysaccharide (LPS)-stimulated primary astrocytic cultures derived from neonatal rats, inhibition of NDRG2 significantly reversed both the LPS-induced activation of astrocytes and decreased expression of GLT-1. By contrast, overexpression of NDRG2 by an adenoviral vector carrying NDRG2 resulted in astrocytic activation, aberrant glutamatergic neurotransmission, and spontaneous nociceptive responses in rats. Intrathecal injection of AG490, which is an inhibitor of the Janus tyrosine kinase and signal transducer and activator of the transcription 3 (JAK/STAT3) signaling pathway, significantly attenuated both mechanical and thermal hyperalgesia, as well as inhibited reactive astrocytes and restored normal expression levels of astrocytic GLT-1, in the spinal dorsal horn of NDRG2-overexpression rats. In conclusion, spinal astrocytic NDRG2 is critical in the maintenance of NP. Moreover, NDRG2 modulates astrocytic activation and inflammatory responses via regulating GLT-1 expression through the JAK/STAT3 signaling pathway. Our findings suggested that NDRG2 could be a novel therapeutic target for the treatment of NP.

Selective neuronal vulnerability is involved in cerebellar lesions of Guinea pigs infected with bovine spongiform encephalopathy (BSE) prions: Immunohistochemical and electron microscopic investigations.

The cerebellar lesions of bovine spongiform encephalopathy (BSE)-infected guinea pigs were characterized as severe atrophy of the cerebellar cortex associated with the loss of granule cells, decrease in the width of the molecular layer, and intense protease-resistant prion protein (PrPSc ) accumulations that are similar to cerebellar lesions in kuru and the VV2 type of sporadic Creutzfeldt-Jakob disease. The aim of this study is to assess the relationships between the distribution and localization of PrPSc and synapses expressing neurotransmitter transporters in order to reveal the pathogenesis of the disease. We used cell-type-specific immunohistochemical makers recognizing glutamatergic and γ-aminobutylic acid (GABA)ergic terminals to identify terminals impaired with PrPSc accumulations. The distribution of PrPSc accumulations and immunoreactivity of synaptic vesicles were studied throughout the neuroanatomical pathways in cerebellar lesions. Time course study demonstrated that PrPSc accumulation showed a tendency to spread from granular layer to molecular layer. The immunoreactivity of vesicular glutamate transporter 1 (VGluT1) was localized in axon terminals of cerebellar granule cells, and decreased in association with the severity of PrPSc accumulations and loss of granule cells. Immunoreactivities of vesicular glutamate transporter 2 (VGluT2) and vesicular GABA transporter (VGAT) that exist in axon terminals of inferior olivary neurons and GABAergic synapses of Purkinje cells, respectively, were preserved well in these lesions. In brainstem, VGluT1 immunoreactivity decreased selectively in pontine nuclei that are a component of the pontocerebellar pathway, although other neurotransmitter immunoreactivities were preserved well. Our findings suggest that the selective loss of VGluT1-immunoreactive synapses subsequent to PrPSc accumulations can contribute to the pathogenesis of cerebellar lesions of BSE-infected guinea pigs.

Altered expression of glutamate transporter-1 and water channel protein aquaporin-4 in human temporal cortex with Alzheimer's disease.

AIMS: Glutamate neurotoxicity plays an important role in the pathogenesis of various neurodegenerative disorders. Many studies have demonstrated that glutamate transporter-1 (GLT-1), the dominant astrocytic glutamate transporter, is significantly reduced in the cerebral cortex of patients with Alzheimer's disease (AD), suggesting that glutamate-mediated excitotoxicity might contribute to the pathogenesis of AD. In a previous study, we have demonstrated marked alterations in the expression of the astrocytic water channel protein aquaporin-4 (AQP4) in relation to amyloid ß deposition in human AD brains. As a functional complex, GLT-1 and AQP4 in astrocytes may play a neuroprotective role in the progression of AD pathology. However, few studies have examined the correlation between the expression of GLT-1 and that of AQP4 in human AD brain. METHODS: Here, using immunohistochemistry with antibodies against GLT-1 and AQP4, we studied the expression levels and distribution patterns of GLT-1 in areas showing various patterns of AQP4 expression in autopsied temporal lobes from eight patients with AD and five controls without neurological disorders. RESULTS: GLT-1 staining in the control group was present throughout the neocortex as uniform neuropil staining with co-localized AQP4. The AD group showed a significant reduction in GLT-1 expression, whereas cortical AQP4 immunoreactivity was more intense in the AD group than in the control group. There were two different patterns of GLT-1 and AQP4 expression in the AD group: (i) uneven GLT-1 expression in the neuropil where diffuse but intense AQP4 expression was evident, and (ii) senile plaque-like co-expression of GLT-1 and AQP4. CONCLUSIONS: These findings suggest disruption of glutamate/water homoeostasis in the AD brain.

Dental malocclusion stimulates neuromuscular circuits associated with temporomandibular disorders.

Unilateral anterior crossbite (UAC) has been demonstrated to cause masseter hyperactivity via the periodontal trigeminal mesencephalic nucleus (Vme)-trigeminal motor nucleus circuit. Here, we studied activation of motor neurons of the facial nucleus (VII), hypoglossal nucleus (XII), nucleus ambiguus (Amb), and spinal nucleus of the accessory nerve (SNA) in rats with UAC via their similar connections with Vme. An anterograde tracer, biotinylated dextran amine (BDA), was injected into the Vme to identify the central axon terminals around the motor neurons of VII, XII, Amb, and SNA. The expression of vesicular glutamate transporter 1 (VGLUT1) in neurons of VII, XII, Amb, and SNA, and the expression of acetylcholinesterase (AChE) were measured in the stapedius, lingualis, palatopharyngeal, and sternocleidomastoid muscles. In BDA-treated rats, many BDA-labeled cell bodies in the Vme and terminals in VII, XII, Amb, and SNA were identified. Compared with control rats, rats with UAC showed higher expression of VGLUT1 in these nuclei, and statistically significantly higher expression of AChE in the stapedius, lingualis, and sternocleidomastoid muscles, but not in the palatopharyngeal muscle. These findings suggest that UAC activates orofacial, head, and cervical multimotor behaviors via connections between the Vme and the corresponding motor nuclei.

A computerized exposure system for animal models to optimize nicotine delivery into the brain through inhalation of electronic cigarette vapors or cigarette smoke.

Pre-clinical studies investigated the effects of chronic exposure to nicotine on lungs, kidneys and brains using animal models. Most of these studies delivered nicotine into the circulatory and central nervous systems (CNS) through intraperitoneal injection or oral consumption methods. Few studies used inhalation machine system for nicotine delivery into brains in rodents to mimic human exposure to cigarettes. However, finding a more accurate and clinically relevant method of nicotine delivery is critical. A computerized inhalation machine has been designed (SciReq) and is currently employed in several institutions. The computerized machine delivers electronic (e)-cigarette vapor as well as tobacco smoke to rodents using marketed e-cigarette devices or tobacco cigarettes. This provides evidence about clinical effects of nicotine delivery by traditional methods (combustible cigarettes) and new methodologies (e-cigarettes) in physiological systems. Potential neurobiological mechanisms for the development of nicotine dependence have been determined recently in mice exposed to e-cigarette vapors in our laboratory using SciReq system. In this review article, the discussion focuses on the efficiency and practical applicability of using this computerized inhalation exposure system in inducing significant changes in brain protein expression and function as compared to other nicotine delivery methods. The SciReq inhalation system utilized in our laboratory and others is a method of nicotine delivery to the CNS, which has physiological relevance and mimics human inhalant exposures. Translation of the effects of inhaled nicotine on the CNS into clinical settings could provide important health considerations.

Neurosteroid dehydroepiandrosterone enhances activity and trafficking of astrocytic GLT-1 via σ1 receptor-mediated PKC activation in the hippocampal dentate gyrus of rats.

Neurosteroid dehydroepiandrosterone (DHEA) has been reported to exert a potent neuroprotective effect against glutamate-induced excitotoxicity. However, the underlying mechanism remains to be elucidated. One of the possible mechanisms may be an involvement of astrocytic glutamate transporter subtype-1 (GLT-1) that can quickly clear spilled glutamate at the synapse to prevent excitotoxicity. To examine the effect of DHEA on GLT-1 activity, we measured synaptically induced glial depolarization (SIGD) in the dentate gyrus (DG) of adult rats by applying an optical recording technique to the hippocampal slices stained with voltage-sensitive dye RH155. Bath-application of DHEA for 10 min dose-dependently increased SIGD without changing presynaptic glutamate releases, which was sensitive to the GLT-1 blocker DHK. Patch-clamp recordings in astrocytes showed that an application of 50 µM DHEA increased glutamate-evoked inward currents (Iglu) by approximately 1.5-fold, which was dependent on the GLT-1 activity. In addition, the level of biotinylated GLT-1 protein in the surface of astrocytes was significantly elevated by DHEA. The DHEA-increased SIGD, Iglu, and GLT-1 translocation to the cell surface were blocked by the σ1 R antagonist NE100 and mimicked by the σ1 R agonist PRE084. DHEA elevated the phosphorylation level of PKC in a σ1 R-dependent manner. Furthermore, the PKC inhibitor chelerythrine could prevent the DHEA-increased SIGD, Iglu, and GLT-1 translocation. Collectively, present results suggest that DHEA enhances the activity and translocation to cell surface of astrocytic GLT-1 mainly via σ1 R-mediated PKC cascade.