The neuronal activity-dependent change in the manner in which light is absorbed or scattered in brain tissue is called the intrinsic optical signal (IOS), and provides label-free, minimally invasive, and high spatial (~100 µm) resolution imaging for visualizing neuronal activity patterns. IOS imaging in isolated brain slices measured at an infrared wavelength (>700 nm) has recently been attributed to the changes in light scattering and transmittance due to aquaporin-4 (AQP4)-dependent astrocytic swelling. The complexity of functional interactions between neurons and astrocytes, however, has prevented the elucidation of the series of molecular mechanisms leading to the generation of IOS. Here, we pharmacologically dissected the IOS in the acutely prepared brain slices of the stratum radiatum of the hippocampus, induced by 1 s/20 Hz electrical stimulation of Schaffer-collateral pathway with simultaneous measurement of the activity of the neuronal population by field potential recordings. We found that 55% of IOSs peak upon stimulation and originate from postsynaptic AMPA and NMDA receptors. The remaining originated from presynaptic action potentials and vesicle fusion. Mechanistically, the elevated extracellular glutamate and K⁺ during synaptic transmission were taken up by astrocytes via a glutamate transporter and quinine-sensitive K2P channel, followed by an influx of water via AQP-4. We also found that the decay of IOS is mediated by the DCPIB- and NPPB-sensitive anion channels in astrocytes. Altogether, our results demonstrate that the functional coupling between synaptic activity and astrocytic transient volume change during excitatory synaptic transmission is the major source of IOS.
Action Potentials ; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid ; Amino Acid Transport System X-AG ; Astrocytes ; Brain ; Electric Stimulation ; Glutamic Acid ; Hippocampus ; Jupiter ; Neurons ; Receptors, N-Methyl-D-Aspartate ; Synaptic Transmission ; Water

Chronic immobilization stress (CIS) induces low levels of glutamate (Glu) and glutamine (Gln) and hypoactive glutamatergic signaling in the mouse prefrontal cortex (PFC), which is closely related to the Glu-Gln cycle. A Gln-supplemented diet ameliorates CIS-induced deleterious changes. Here, we investigated the effects of CIS and Gln supplementation on Glu-Gln cycle-related proteins to characterize the underlying mechanisms. Using the CIS-induced depression mouse model, we examined the expression of 11 proteins involved in the Glu-Gln cycle in the PFC. CIS decreased levels of glutamate transporter 1 (GLT1) and sodium-coupled neutral amino acid transporter (SNAT) 1, SANT2, SNAT3, and SNAT5. Gln supplementation did not affect the non-stressed group but significantly increased GLT1 and SNATs of the stressed group. By immunohistochemical analysis, we confirmed that SNAT1 and SNAT2 were decreased in neurons and GLT1, SNAT3, and SNAT5 were decreased in astrocytes in the medial PFC of the stressed group, but Gln-supplemented diet ameliorated these decrements. Collectively, these results suggest that CIS may cause depressive-like behaviors by decreasing Glu and Gln transportation in the PFC and that a Gln-supplemented diet could prevent the deleterious effects of CIS.
Amino Acid Transport System X-AG ; Amino Acid Transport Systems ; Animals ; Astrocytes ; Depression ; Depressive Disorder ; Diet ; Glutamic Acid ; Glutamine ; Immobilization ; Mice ; Neurons ; Prefrontal Cortex ; Transportation

Drosophila dEAAT2, a member of the excitatory amino-acid transporter (EAAT) family, has been described as mediating the high-affinity transport of taurine, which is a free amino-acid abundant in both insects and mammals. However, the role of taurine and its transporter in hearing is not clear. Here, we report that dEAAT2 is required for the larval startle response to sound stimuli. dEAAT2 was found to be enriched in the distal region of chordotonal neurons where sound transduction occurs. The Ca imaging and electrophysiological results showed that disrupted dEAAT2 expression significantly reduced the response of chordotonal neurons to sound. More importantly, expressing dEAAT2 in the chordotonal neurons rescued these mutant phenotypes. Taken together, these findings indicate a critical role for Drosophila dEAAT2 in sound transduction by chordotonal neurons.
Acoustic Stimulation ; Action Potentials ; genetics ; Animals ; Animals, Genetically Modified ; Auditory Pathways ; physiology ; Calcium ; metabolism ; Drosophila ; genetics ; Drosophila Proteins ; genetics ; metabolism ; Excitatory Amino Acid Transporter 2 ; genetics ; metabolism ; Hearing ; genetics ; Larva ; Luminescent Proteins ; genetics ; metabolism ; Mutation ; genetics ; Nervous System ; cytology ; Neurons ; metabolism

The present study investigated the role of spinal glutamate recycling in the development of orofacial inflammatory pain or trigeminal neuropathic pain. Experiments were carried out on male Sprague-Dawley rats weighing between 230 and 280 g. Under anesthesia, a polyethylene tube was implanted in the atlanto-occipital membrane for intracisternal administration. IL-1β-induced inflammation was employed as an orofacial acute inflammatory pain model. IL-1β (10 ng) was injected subcutaneously into one vibrissal pad. We used the trigeminal neuropathic pain animal model produced by chronic constriction injury of the infraorbital nerve. DL-threo-β -benzyloxyaspartate (TBOA) or methionine sulfoximine (MSO) was administered intracisternally to block the spinal glutamate transporter and the glutamine synthetase activity in astroglia. Intracisternal administration of TBOA produced mechanical allodynia in naïve rats, but it significantly attenuated mechanical allodynia in rats with interleukin (IL)-1 β-induced inflammatory pain or trigeminal neuropathic pain. In contrast, intracisternal injection of MSO produced anti-allodynic effects in rats treated with IL-1β or with infraorbital nerve injury. Intracisternal administration of MSO did not produce mechanical allodynia in naive rats. These results suggest that blockade of glutamate recycling induced pro-nociception in naïve rats, but it paradoxically resulted in anti-nociception in rats experiencing inflammatory or neuropathic pain. Moreover, blockade of glutamate reuptake could represent a new therapeutic target for the treatment of chronic pain conditions.
Amino Acid Transport System X-AG ; Anesthesia ; Animals ; Astrocytes ; Chronic Pain ; Constriction ; Glutamate-Ammonia Ligase ; Glutamic Acid* ; Humans ; Hyperalgesia ; Inflammation ; Interleukins ; Male ; Membranes ; Methionine Sulfoximine ; Models, Animal ; Neuralgia* ; Polyethylene ; Rats* ; Rats, Sprague-Dawley ; Recycling*

OBJECTIVE: Deficient brain-derived neurotrophic factor (BDNF) is one of the important mechanisms underlying the neuroplasticity abnormalities in schizophrenia. Aberration in BDNF signaling pathways directly or circuitously influences neurotransmitters like glutamate and gamma-aminobutyric acid (GABA). For the first time, this study attempts to construct and simulate the BDNF-neurotransmitter network in order to assess the effects of BDNF deficiency on glutamate and GABA. METHODS: Using CellDesigner, we modeled BDNF interactions with calcium influx via N-methyl-D-aspartate receptor (NMDAR)-Calmodulin activation; synthesis of GABA via cell cycle regulators protein kinase B, glycogen synthase kinase and β-catenin; transportation of glutamate and GABA. Steady state stability, perturbation time-course simulation and sensitivity analysis were performed in COPASI after assigning the kinetic functions, optimizing the unknown parameters using random search and genetic algorithm. RESULTS: Study observations suggest that increased glutamate in hippocampus, similar to that seen in schizophrenia, could potentially be contributed by indirect pathway originated from BDNF. Deficient BDNF could suppress Glutamate decarboxylase 67-mediated GABA synthesis. Further, deficient BDNF corresponded to impaired transport via vesicular glutamate transporter, thereby further increasing the intracellular glutamate in GABAergic and glutamatergic cells. BDNF also altered calcium dependent neuroplasticity via NMDAR modulation. Sensitivity analysis showed that Calmodulin, cAMP response element-binding protein (CREB) and CREB regulated transcription coactivator-1 played significant role in this network. CONCLUSION: The study presents in silico quantitative model of biochemical network constituting the key signaling molecules implicated in schizophrenia pathogenesis. It provides mechanistic insights into putative contribution of deficient BNDF towards alterations in neurotransmitters and neuroplasticity that are consistent with current understanding of the disorder.
Amino Acid Transport System X-AG ; Brain-Derived Neurotrophic Factor* ; Calcium ; Calmodulin ; Cell Cycle ; Computer Simulation* ; Cyclic AMP Response Element-Binding Protein ; gamma-Aminobutyric Acid* ; Glutamate Decarboxylase ; Glutamic Acid* ; Glycogen Synthase Kinases ; Hippocampus ; N-Methylaspartate ; Neuronal Plasticity ; Neurotransmitter Agents ; Proto-Oncogene Proteins c-akt ; Schizophrenia* ; Signal Transduction ; Transportation

Spinocerebellar ataxias (SCAs) are autosomal dominant neurodegenerative disorders which disrupt the afferent and efferent pathways of the cerebellum that cause cerebellar ataxia. Spectrin beta non-erythrocytic 2 (SPTBN2) gene encodes the β-III spectrin protein with high expression in Purkinje cells that is involved in excitatory glutamate signaling through stabilization of the glutamate transporter, and its mutation is known to cause spinocerebellar ataxia type 5. Three years and 5 months old boy with delayed development showed leukodystrophy and cerebellar atrophy in brain magnetic resonance imaging (MRI). Diagnostic exome sequencing revealed that the patient has heterozygous mutation in SPTBN2 (p.Glu1251Gln) which is a causative genetic mutation for spinocerebellar ataxia type 5. With the patient's clinical findings, it seems reasonable to conclude that p.Glu1251Gln mutation of SPTBN2 gene caused spinocerebellar ataxia type 5 in this patient.
Amino Acid Transport System X-AG ; Atrophy ; Brain ; Cerebellar Ataxia ; Cerebellum ; Efferent Pathways ; Exome ; Glutamic Acid ; Humans ; Magnetic Resonance Imaging ; Male ; Neurodegenerative Diseases ; Purkinje Cells ; Spectrin ; Spinocerebellar Ataxias*

The aim of the present study was to investigate the effects of minocycline on cognitive functions in neonatal rat after hypoxia exposure and the underlying mechanism. A model of hypoxic brain damage (HBD) was developed by exposing postnatal 1 day (P1) rats to systemic hypoxia. The rats were intraperitoneally injected with normal saline (Hy group) or minocycline (Hy + M group) 2 h after hypoxia exposure. Some other P1 rats that were not subjected to systemic hypoxia were used as normal control (NG group). The Y-maze test was used to evaluate learning and memory ability on postnatal day 30. Inflammatory mediators (Iba-1, IL-1β, TNF-α and TGF-β1), glutamate transporters (EAAT1 and EAAT2), total Tau and phosphorylated Tau (phosphorylation sites: Tyr18, Thr205, Thr231, Ser396 and Ser404) protein expressions in the hippocampus were detected by Western blot 7 d after hypoxic exposure. The results showed that hypoxia induced learning and memory impairments of the neonatal rats, and minocycline administration could reverse the effects of hypoxia. The protein expression levels of Iba-1, IL-1β, TNF-α, EAAT2 and Tau phosphorylated at T231 were increased, but the total Tau expression was decreased in the hippocampus of the rats from Hy group 7 d after hypoxia exposure. In the hypoxia-treated rats, minocycline down-regulated Iba-1, IL-1β, TNF-α and EAAT2 protein expressions significantly, but did not affect total Tau and phosphorylated Tau protein expressions. Our results suggest that minocycline can prevent cognitive deficits of rats with hypoxia exposure, and the underlying mechanism may involve the inhibition of neuroinflammation and dysfunctional glutamate transporters but not the regulation of the Tau hyperphosphorylation.
Amino Acid Transport System X-AG ; Animals ; Animals, Newborn ; Cognition ; Cognition Disorders ; Disease Models, Animal ; Glutamates ; Hippocampus ; Hypoxia ; Inflammation ; Learning ; Memory ; Memory Disorders ; Minocycline ; Phosphorylation ; Rats ; Transforming Growth Factor beta1 ; Tumor Necrosis Factor-alpha ; tau Proteins

OBJECTIVEThe present study was undertaken to design antisense oligodeoxynucleotides (AS-ODNs) of glial glutamate transporter-la (GLT-1a) and to evaluate the effectiveness of the designed AS-ODNs on the expression of GLT-1a.

METHODSFive sequences of GLT-1a AS-ODNs were designed according to the C terminus specific sequences of GLT-1a mRNA using antisense design software of IDT Com- pany. Western blot analysis was used to evaluate the inhibition effects of the five GLT-1a AS-ODNs on the expression of GLT-la.

RESULTSThe sequence of GLT-1a AS-ODNs with sequence of 5'-GGTTCTTCCTCAACACTGCA-3' could specifically inhibit the expression of GLT-1a in the hippocampal CA1 subfield of rats, while it had no effect on the expression of GLT-1b. This sequence showed similar inhibition on the expression of GLT-la in sham and ceftriaxone (Cef)-treated rats. It could also significantly inhibit the cerebral ischemic preconditioning (CIP)-induced up-regulation in the expression of GLT-1a. The magnitude of the inhibition in sham, Cef- or CIP-treated rats was similar by more than 60%.

CONCLUSIONFrom the designed five sequences of GLT-1a AS-ODNs, we obtained an effective sequence which can specifically inhibit the expression of GLT-1a.

Animals ; CA1 Region, Hippocampal ; metabolism ; Excitatory Amino Acid Transporter 2 ; antagonists & inhibitors ; metabolism ; Ischemic Preconditioning ; Oligonucleotides, Antisense ; genetics ; RNA, Messenger ; Rats ; Up-Regulation

In recent years, more attention has been paid to the role of the glutamate transporter 1 (GLT-1, EAAT2) in major depressive disorder (MDD). However, experimental data on brain GLT-1 levels are, to some extent, inconsistent in human postmortem and animal studies. These discrepancies imply that the role of GLT-1 in the pathophysiology of MDD and the action of antidepressants remain obscure. This work was designed to study the impact of chronic unpredictable stress (CUS) for 2 sessions per day for 35 days and four weeks of fluoxetine (FLX) on depressive-like behaviors in rats, as well as the concomitant expression of the GLT-1 protein in the hippocampus. Behavioral changes were assessed by the sucrose preference and open field tests. GLT-1 levels were detected by immunohistchemistry and Western blot analysis. Our study demonstrated that the animals exposed to CUS showed depressive-like behaviors and exhibited a significant decrease in GLT-1 expression in the hippocampus. Chronic FLX treatment reversed the behavioral deficits and the CUS-induced decrease in GLT-1 levels. Taken together, our results support the reduction of GLT-1 in human postmortem studies in MDD and suggest that GLT-1 may be involved in the antidepressant activity of FLX. Our studies further support the notion that GLT-1 is an attractive candidate molecule associated with the fundamental processes of MDD and may be a potential, and novel pharmacological target for the treatment of MDD.
Animals ; Antidepressive Agents, Second-Generation ; pharmacology ; Behavior, Animal ; drug effects ; Brain ; metabolism ; pathology ; Chronic Disease ; Depressive Disorder, Major ; drug therapy ; metabolism ; pathology ; Excitatory Amino Acid Transporter 2 ; metabolism ; Fluoxetine ; pharmacology ; Humans ; Male ; Rats ; Rats, Sprague-Dawley ; Stress, Psychological ; drug therapy ; metabolism ; pathology

This study was aimed to investigate the mechanisms underlying the modulation effect of Mas-related gene (Mrg) C receptors (MrgC) on morphine tolerance. Saline, morphine (20 μg), morphine plus bovine adrenal medulla 8-22 (BAM8-22, 1 nmol) or (Tyr(6))-2-MSH-6-12 (MSH, 5 nmol) were administered intrathecally in rats for 6 days. Pain-related molecules in the spinal cord and dorsal root ganglion (DRG) were examined using Western blot, immunocytochemistry and RT-PCR techniques. The results showed that intrathecal administration of the selective MrgC receptor agonists (BAM8-22 or MSH) remarkably attenuated or abolished chronic morphine-evoked reduction in glutamate transporters (GLAST, GLT-1 and EAAC1) in the spinal cord and increase in neuronal nitric oxide synthase (nNOS) in the spinal cord as well as DRG. In addition, MrgC receptor-like immunoreactivity (IR) was detected in superficial laminae of the spinal cord. Chronic morphine induced significant increases in MrgC receptor-IR in the spinal cord and MrgC receptor mRNA levels in DRG. These results suggest that the modulation of pro-nociceptive mediators in the spinal cord and DRG underlies the inhibition of morphine tolerance by MrgC receptor activation.
Amino Acid Transport System X-AG ; metabolism ; Animals ; Drug Tolerance ; Ganglia, Spinal ; metabolism ; Glutamates ; Morphine ; pharmacology ; Nitric Oxide Synthase Type I ; metabolism ; Pain ; Pain Measurement ; Peptide Fragments ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Receptors, G-Protein-Coupled ; metabolism ; Spinal Cord ; metabolism