Cerebrospinal fluid, brain, and spinal cord levels of L-aspartate signal excitatory neurotransmission abnormalities in multiple sclerosis patients and experimental autoimmune encephalomyelitis mouse model.

The neuroinflammatory process characterizing multiple sclerosis (MS) is associated with changes in excitatory synaptic transmission and altered central concentrations of the primary excitatory amino acid, L-glutamate (L-Glu). Recent findings report that cerebrospinal fluid (CSF) levels of L-Glu positively correlate with pro-inflammatory cytokines in MS patients. However, to date, there is no evidence about the relationship between the other primary excitatory amino acid, L-aspartate (L-Asp), its derivative D-enantiomer, D-aspartate, and the levels of pro-inflammatory and anti-inflammatory cytokines in the CSF of MS. In the present study, we measured by HPLC the levels of these amino acids in the cortex, hippocampus, cerebellum, and spinal cord of mice affected by experimental autoimmune encephalomyelitis (EAE). Interestingly, in support of glutamatergic neurotransmission abnormalities in neuroinflammatory conditions, we showed reduced L-Asp levels in the cortex and spinal cord of EAE mice and increased D-aspartate/total aspartate ratio within the cerebellum and spinal cord of these animals. Additionally, we found significantly decreased CSF levels of L-Asp in both relapsing-remitting (n = 157) MS (RR-MS) and secondary progressive/primary progressive (n = 22) (SP/PP-MS) patients, compared to control subjects with other neurological diseases (n = 40). Importantly, in RR-MS patients, L-Asp levels were correlated with the CSF concentrations of the inflammatory biomarkers G-CSF, IL-1ra, MIP-1ß, and Eotaxin, indicating that the central content of this excitatory amino acid, as previously reported for L-Glu, reflects a neuroinflammatory environment in MS. In keeping with this, we revealed that CSF L-Asp levels were positively correlated with those of L-Glu, highlighting the convergent variation of these two excitatory amino acids under inflammatory synaptopathy occurring in MS.

Enhanced FGFR3 activity in postmitotic principal neurons during brain development results in cortical dysplasia and axonal tract abnormality.

Abnormal levels of fibroblast growth factors (FGFs) and FGF receptors (FGFRs) have been detected in various neurological disorders. The potent impact of FGF-FGFR in multiple embryonic developmental processes makes it challenging to elucidate their roles in postmitotic neurons. Taking an alternative approach to examine the impact of aberrant FGFR function on glutamatergic neurons, we generated a FGFR gain-of-function (GOF) transgenic mouse, which expresses constitutively activated FGFR3 (FGFR3K650E) in postmitotic glutamatergic neurons. We found that GOF disrupts mitosis of radial-glia neural progenitors (RGCs), inside-out radial migration of post-mitotic glutamatergic neurons, and axonal tract projections. In particular, late-born CUX1-positive neurons are widely dispersed throughout the GOF cortex. Such a cortical migration deficit is likely caused, at least in part, by a significant reduction of the radial processes projecting from RGCs. RNA-sequencing analysis of the GOF embryonic cortex reveals significant alterations in several pathways involved in cell cycle regulation and axonal pathfinding. Collectively, our data suggest that FGFR3 GOF in postmitotic neurons not only alters axonal growth of postmitotic neurons but also impairs RGC neurogenesis and radial glia processes.

Marine Excitatory Amino Acids: Structure, Properties, Biosynthesis and Recent Approaches to Their Syntheses.

This review considers the results of recent studies on marine excitatory amino acids, including kainic acid, domoic acid, dysiherbaine, and neodysiherbaine A, known as potent agonists of one of subtypes of glutamate receptors, the so-called kainate receptors. Novel information, particularly concerning biosynthesis, environmental roles, biological action, and syntheses of these marine metabolites, obtained mainly in last 10-15 years, is summarized. The goal of the review was not only to discuss recently obtained data, but also to provide a brief introduction to the field of marine excitatory amino acid research.

The glutamatergic system in the preoptic area is involved in the retention of maternal behavior in maternally experienced female rats.

Maternally experienced female rats show high maternal behavior performance for a long time after acquisition of maternal experience, although the mechanisms responsible for the retention of maternal behavior are not well understood. The medial preoptic area (MPOA) plays an important role in the onset and maintenance of maternal behavior in female rats. We aimed to determine whether maternal experience affects the glutamatergic system in the MPOA for the retention of maternal behavior in female rats. First, to determine the effects of maternal experience in the postpartum period on dendritic spines, which are the postsynaptic component of excitatory glutamatergic neurotransmission, we examined the number of dendritic spines on MPOA neurons of primiparous mothers that had experienced mothering until weaning (sufficiently experienced mothers) and of primiparous mothers that were separated from their pups on the day of parturition (insufficiently experienced mothers). The number of mushroom spines, but not other types of spine, was significantly greater in the sufficiently experienced mothers compared with that in the insufficiently experienced mothers. Next, to determine the effects of maternal experience in the postpartum period on the expression of ionotropic glutamate receptors, we measured the mRNA levels of AMPA receptor subunits (GluA1-A4) and NMDA receptor subunits (GluN1, GluN2A-2D) in the MPOA of primiparous female rats that were kept with pups until brain sampling. As a result, we found that the mRNA levels of GluA3 and GluN2B were significantly higher in primiparous females on the day of weaning compared with those in primiparous females on the day of parturition. Additionally, we examined the effects of CNQX, an AMPA receptor antagonist, and MK-801, an NMDA receptor antagonist, injected into the MPOA on maternal behavior in maternally experienced primiparous female rats. Maternal behavioral activity was significantly reduced when CNQX or MK-801 was injected into the MPOA. These findings indicate that long-term maternal experience in the postpartum period up-regulates glutamatergic neurotransmission by increasing the number of mushroom spines and glutamate receptor expression, which may be involved in the retention of maternal behavior in maternally experienced female rats.

Effects of fluorescent glutamate indicators on neurotransmitter diffusion and uptake.

Genetically encoded fluorescent glutamate indicators (iGluSnFRs) enable neurotransmitter release and diffusion to be visualized in intact tissue. Synaptic iGluSnFR signal time courses vary widely depending on experimental conditions, often lasting 10-100 times longer than the extracellular lifetime of synaptically released glutamate estimated with uptake measurements. iGluSnFR signals typically also decay much more slowly than the unbinding kinetics of the indicator. To resolve these discrepancies, here we have modeled synaptic glutamate diffusion, uptake and iGluSnFR activation to identify factors influencing iGluSnFR signal waveforms. Simulations suggested that iGluSnFR competes with transporters to bind synaptically released glutamate, delaying glutamate uptake. Accordingly, synaptic transporter currents recorded from iGluSnFR-expressing astrocytes in mouse cortex were slower than those in control astrocytes. Simulations also suggested that iGluSnFR reduces free glutamate levels in extrasynaptic spaces, likely limiting extrasynaptic receptor activation. iGluSnFR and lower affinity variants, nonetheless, provide linear indications of vesicle release, underscoring their value for optical quantal analysis.

Ethanol-induced changes in synaptic amino acid neurotransmitter levels in the nucleus accumbens of differentially sensitized mice.

RATIONALE: Ethanol-induced behavioural sensitization (EBS) does not occur uniformly in mice exposed to the sensitization paradigm. This suggests innate differential responses to ethanol (EtOH) in the reward circuitry of individual animals. OBJECTIVES: To better characterize the adaptive differences between low-sensitized (LS) and high-sensitized (HS) mice, we examined excitatory amino acid (EAA) and inhibitory amino acid (IAA) neurotransmitter levels in the nucleus accumbens (NAc) during EBS expression. METHODS: Male DBA/2J mice received five ethanol (EtOH) (2.2 g/kg) or saline injections, and locomotor activity (LMA) was assessed during EBS induction. EtOH mice were classified as LS or HS on the basis of final LMA scores. Following an EtOH challenge (1.8 g/kg) 2 weeks later, LMA was re-evaluated and in vivo microdialysis samples were collected from the NAc. RESULTS: Most differences in amino acid levels were observed within the first 20 min after EtOH challenge. LS mice exhibited similar glutamate levels compared with acutely treated (previously EtOH naïve) mice, and generally increased levels of the IAAs GABA, glycine, and taurine. By contrast, HS mice exhibited increased glutamate and attenuated levels of GABA, glycine, and taurine. CONCLUSION: These data suggest that the profile of amino acid neurotransmitters in the NAc of LS and HS mice significantly differs. Elucidating these adaptive differences contributes to our understanding of factors that confer susceptibility/resilience to alcohol use disorder.

Metformin inhibits Aß25-35 -induced apoptotic cell death in SH-SY5Y cells.

Metformin, a first-line drug for type-2 diabetes, plays a potentially protective role in preventing Alzheimer's disease (AD), but its underlying mechanism is unclear. In this study, Aß25-35 -treated SH-SY5Y cells were used as a cell model of AD to investigate the neuroprotective effect of metformin, as well as its underlying mechanisms. We found that metformin decreased the cell apoptosis rate and death, ratio of Bcl-2/Bax, and expression of NR2A and NR2B, and increased the expression of LC3 in Aß25-35 -treated SH-SY5Y cells. Metformin also reduced intracellular and extracellular Glu concentrations, as well as the intracellular concentration of Ca2+ and ROS in Aß25-35 -treated SH-SY5Y cells. These findings suggest that metformin inhibits Aß25-35 -treated SH-SY5Y cell death by inhibiting apoptosis, decreasing intracellular Ca2+ and ROS by reducing neurotoxicity of excitatory amino acids, and by possibly reversing autophagy disorder via regulating autophagy process.

Reduced excitatory neurotransmitter levels in anterior insulae are associated with abdominal pain in irritable bowel syndrome.

Irritable bowel syndrome (IBS) is a visceral pain condition with psychological comorbidity. Brain imaging studies in IBS demonstrate altered function in anterior insula (aINS), a key hub for integration of interoceptive, affective, and cognitive processes. However, alterations in aINS excitatory and inhibitory neurotransmission as putative biochemical underpinnings of these functional changes remain elusive. Using quantitative magnetic resonance spectroscopy, we compared women with IBS and healthy women (healthy controls [HC]) with respect to aINS glutamate + glutamine (Glx) and γ-aminobutyric acid (GABA+) concentrations and addressed possible associations with symptoms. Thirty-nine women with IBS and 21 HC underwent quantitative magnetic resonance spectroscopy of bilateral aINS to assess Glx and GABA+ concentrations. Questionnaire data from all participants and prospective symptom-diary data from patients were obtained for regression analyses of neurotransmitter concentrations with IBS-related and psychological parameters. Concentrations of Glx were lower in IBS compared with HC (left aINS P < 0.05, right aINS P < 0.001), whereas no group differences were detected for GABA+ concentrations. Lower right-lateralized Glx concentrations in patients were substantially predicted by longer pain duration, while less frequent use of adaptive pain-coping predicted lower Glx in left aINS. Our findings provide first evidence for reduced excitatory but unaltered inhibitory neurotransmitter levels in aINS in IBS. The results also indicate a functional lateralization of aINS with a stronger involvement of the right hemisphere in perception of abdominal pain and of the left aINS in cognitive pain regulation. Our findings suggest that glutaminergic deficiency may play a role in pain processing in IBS.

Neurometabolite changes in patients with complex regional pain syndrome using magnetic resonance spectroscopy: a pilot study.

The aim of this study was to investigate distinct neurometabolites in the right and left thalamus and insula of patients with complex regional pain syndrome (CRPS) compared with healthy controls using proton magnetic resonance spectroscopy. Levels of N-acetylaspartate (NAA), N-acetylaspartylglutamate (NAAG), myo-inositol (ml), glutamine (Gln), glycerophosphocholine (GPC), glutathione (GSH), and alanine (Ala) relative to total creatine (tCr) levels, including creatine and phosphocreatine, were determined in the right and left thalamus and insula in 12 patients with CRPS compared with 11 healthy controls using magnetic resonance spectroscopy. Levels of NAAG/tCr and Ala/tCr were higher in patients with CRPS than in controls in the left thalamus. NAAG/tCr, ml/tCr, and Gln/tCr levels were higher but NAA/tCr levels were lower in the right insula of patients with CRPS compared with controls. There were negative correlations between GSH/tCr and pain score (McGill Pain Questionnaire) in the left thalamus. These findings are paramount to understand and determine all aspects of the complex pathophysiological mechanisms that underlie CRPS, including involvement of the central and parasympathetic nervous systems as well as oxidative stress and antioxidants. Thus, the distinct metabolites presented herein may be essential to understand a strong diagnostic and prognostic potential for CRPS and to develop effective medical treatments.

Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats.

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain interstitial space with the behavior and/or with the specific outcome of a pathology (e.g., seizures for epilepsy). When studying epilepsy, the microdialysis technique is often combined with short-term or even long-term video-electroencephalography (EEG) monitoring to assess spontaneous seizure frequency, severity, progression and clustering. The combined microdialysis-EEG is based on the use of several methods and instruments. Here, we performed in vivo microdialysis and continuous video-EEG recording to monitor glutamate and aspartate outflow over time, in different phases of the natural history of epilepsy in a rat model. This combined approach allows the pairing of changes in the neurotransmitter release with specific stages of the disease development and progression. The amino acid concentration in the dialysate was determined by liquid chromatography. Here, we describe the methods and outline the principal precautionary measures one should take during in vivo microdialysis-EEG, with particular attention to the stereotaxic surgery, basal and high potassium stimulation during microdialysis, depth electrode EEG recording and high-performance liquid chromatography analysis of aspartate and glutamate in the dialysate. This approach may be adapted to test a variety of drug or disease induced changes of the physiological concentrations of aspartate and glutamate in the brain. Depending on the availability of an appropriate analytical assay, it may be further used to test different soluble molecules when employing EEG recording at the same time.

Regulation of Excitatory Amino Acid Transmission in the Retina: Studies on Neuroprotection.

Excitotoxicity occurs in neurons due to the accumulation of excitatory amino acids such as glutamate in the synaptic and extrasynaptic locations. In the retina, excessive glutamate concentrations trigger a neurotoxic cascade involving several mechanisms, including the elevation of intracellular calcium (Ca2+) and the activation of α-amino-3-hydroxy 5-methyl-4-iso-xazole-propionic acid/kainate (AMPA/KA) and N-methyl-d-aspartate (NMDA) receptors leading to retinal degeneration. Both ionotropic glutamate receptors (iGluRs) and metabotropic glutamate receptors (mGluRs) are present in the mammalian retina. Indeed, due to the abundant expression of GluRs, the mammalian retina is highly susceptible to excitotoxic neurodegeneration. Excitotoxicity has been postulated to present a common downstream mechanism for several stimuli, including hypoglycemia, hypoxia, ischemia, and chronic neurodegenerative diseases. Experimental approaches to the study of neuroprotection in the retina have utilized insults that trigger hypoxia, hypoglycemia, or excitotoxicity. Using these experimental approaches, the neuroprotective potential of GluR agents, including the NMDA receptor modulators (MK801, ifenprodil, memantine); AMPA/KA receptor antagonist (CNQX); Group II and III mGluR agonists (LY354740, quisqualate); and Ca2+-channel blockers (diltiazem, lomerizine, verapamil, ω-conotoxin), and others (pituitary adenylate cyclase activating polypeptide, neuropeptide Y, acetylcholine receptor agonists) have been elucidated. In addition to corroborating the exocytotic role of excitatory amino acids in retinal degeneration, these studies affirm that multiple mechanism/s contribute to the prevention of damage caused by excitotoxicity in the retina. Therefore, it is feasible that several pathways are involved in protecting the retina from toxic insults in ocular neurodegenerative conditions such as glaucoma and retinal ischemia. Furthermore, these experimental models are viable tools for evaluating therapeutic candidates in ocular neuropathies.

EAAT2 and the Molecular Signature of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a rapid and fatal neurodegenerative disease, primarily affecting upper and lower motor neurons. It is an extremely heterogeneous disease in both cause and symptom development, and its mechanisms of pathogenesis remain largely unknown. Excitotoxicity, a process caused by excessive glutamate signaling, is believed to play a substantial role, however. Excessive glutamate release, changes in postsynaptic glutamate receptors, and reduction of functional astrocytic glutamate transporters contribute to excitotoxicity in ALS. Here, we explore the roles of each, with a particular emphasis on glutamate transporters and attempts to increase them as therapy for ALS. Screening strategies have been employed to find compounds that increase the functional excitatory amino acid transporter EAAT2 (GLT1), which is responsible for the vast majority of glutamate clearance. One such compound, ceftriaxone, was recently tested in clinical trials but unfortunately did not modify disease course, though its effect on EAAT2 expression in patients was not measured.

Microtubule-associated protein 1B (MAP1B)-deficient neurons show structural presynaptic deficiencies in vitro and altered presynaptic physiology.

Microtubule-associated protein 1B (MAP1B) is expressed predominantly during the early stages of development of the nervous system, where it regulates processes such as axonal guidance and elongation. Nevertheless, MAP1B expression in the brain persists in adult stages, where it participates in the regulation of the structure and physiology of dendritic spines in glutamatergic synapses. Moreover, MAP1B expression is also found in presynaptic synaptosomal preparations. In this work, we describe a presynaptic phenotype in mature neurons derived from MAP1B knockout (MAP1B KO) mice. Mature neurons express MAP1B, and its deficiency does not alter the expression levels of a subgroup of other synaptic proteins. MAP1B KO neurons display a decrease in the density of presynaptic and postsynaptic terminals, which involves a reduction in the density of synaptic contacts, and an increased proportion of orphan presynaptic terminals. Accordingly, MAP1B KO neurons present altered synaptic vesicle fusion events, as shown by FM4-64 release assay, and a decrease in the density of both synaptic vesicles and dense core vesicles at presynaptic terminals. Finally, an increased proportion of excitatory immature symmetrical synaptic contacts in MAP1B KO neurons was detected. Altogether these results suggest a novel role for MAP1B in presynaptic structure and physiology regulation in vitro.

[Ischemic Stroke, Excitatory Amino Acids Toxicity and the Adjustment of Acupuncture Intervention].

Excitatory amino acids toxicity is an onset causation of cerebral ischemia injury cascade reaction, and eventually leading to brain cell necrosis and apoptosis. Acupuncture is reported to be effective for ischemic stroke in clinical practice and animal experiments, but its mechanism is still under exploring. In this paper the authors introduce the research status of antiexcitatory amino acids toxicity effect of acupuncture in ischemic stroke animals by summarizing its effects on subunits of ionotropic glutamate receptor (NMDA/AMPA) and metabotropic glutamate receptors (mGluRs), and on astrocyte activities. Results indicated that acupuncture intervention may down-regulate the expression levels of cerebral multi-types (NR 1, NR 2 B) of glutamate NMDA receptors, up-regulate expression of glutamate transporter-1, NR 2 A, cannabinoid receptor (CBR) type 1 and 2, and suppress activities of cerebral astrocytes, reduce the content of extracellular glutamate to lower its toxicity and to improve stroke at last. The present paper may provide a reference for acupuncture research on ischemic brain injury.

Coupling of glutamate and glucose uptake in cultured Bergmann glial cells.

Glutamate, the main excitatory neurotransmitter in the vertebrate brain, exerts its actions through specific membrane receptors present in neurons and glial cells. Over-stimulation of glutamate receptors results in neuronal death, phenomena known as excitotoxicity. A family of sodium-dependent, glutamate uptake transporters mainly expressed in glial cells, removes the amino acid from the synaptic cleft preventing neuronal death. The sustained sodium influx associated to glutamate removal in glial cells, activates the sodium/potassium ATPase restoring the ionic balance, additionally, glutamate entrance activates glutamine synthetase, both events are energy demanding, therefore glia cells increase their ATP expenditure favouring glucose uptake, and triggering several signal transduction pathways linked to proper neuronal glutamate availability, via the glutamate/glutamine shuttle. To further characterize these complex transporters interactions, we used the well-established model system of cultured chick cerebellum Bergmann glia cells. A time and dose-dependent increase in the activity, plasma membrane localization and protein levels of glucose transporters was detected upon d-aspartate exposure. Interestingly, this increase is the result of a protein kinase C-dependent signaling cascade. Furthermore, a glutamate-dependent glucose and glutamate transporters co-immunoprecipitation was detected. These results favour the notion that glial cells are involved in glutamatergic neuronal physiology.

Traumatic brain injury: a review of characteristics, molecular basis and management.

Traumatic brain injury (TBI) is a critical cause of hospitalization, disability, and death worldwide. The global increase in the incidence of TBI poses a significant socioeconomic burden. Guidelines for the management of acute TBI mostly pertain to emergency treatment. Comprehensive gene expression analysis is currently available for several animal models of TBI, along with enhanced understanding of the molecular mechanisms activated during injury and subsequent recovery. The current review focuses on the characteristics, molecular basis and management of TBI.

[Cerebral metabolic changes and chronic back pain: Study taking into consideration clinical and psychological parameters]. / Hirnmetabolische Veränderungen bei chronischem Rückenschmerz : Studie unter Berücksichtigung von klinischen und psychischen Parametern.

BACKGROUND: The manifestation of chronic pain and psychological impairments are related to alterations of neurotransmitter metabolism in cerebral pain processing regions, e.g., anterior cingular cortex (ACC), insula. Magnetic resonance spectroscopy ((1)H-MRS) enables in vivo quantification of neurotransmitters in the brain and was applied in this study to examine the hypothesized chronic pain-related imbalance between excitatory (glutamatergic) and inhibitory (GABA-ergic) neurotransmitter turnovers in the brain of patients with nonspecific chronic pain. MATERIALS AND METHODS: A total of 19 patients with nonspecific chronic (> 3 months) back pain and 19 age- and gender-matched healthy subjects participated in this study. Glutamate and GABA as well as glutamate/GABA ratios were determined in the ACC and insula using (1)H-MRS. Sociodemographic, psychological, and pain-related features were measured with standardized questionnaires. RESULTS: There was a strong variance of glutamate/GABA ratios for both patients and healthy subjects with no significant difference between the two groups. Regression analysis revealed certain significant predictors, such as anxiety as causal variable for reduced glutamate and depression and age as predictors for reduced GABA in ACC. In the patient group, intensity of pain was a significant predictor for glutamate and GABA levels in the insula. CONCLUSIONS: Despite the uniform diagnosis of nonspecific chronic back pain, we observed a strong variance of neurotransmitters in cerebral pain processing regions. It is necessary to include psychological as well as clinical parameters (e.g., intensity of pain or depression) for a proper interpretation of neurotransmitter turnovers.

[Effect of dexmedetomidine on the changes of EAA and the expression of NMDA NR1 protein in hippocampus in global cerebral ischemia/reperfusion rats].

OBJECTIVE: To observe the effects of dexmedetomidine (DEX) on glutamate (Glu), aspartic acid (Asp) release and NMDAR1 expression in hippocampus in global cerebral ischemia/reperfusion rats, and investigate the protective effect and the related mechanism of neurotransmitters. METHODS: Fifty-four male Wistar rats were randomly divided into three groups (n=18):sham group(A), ischemia/reperfusion group(B), dexmedetomidine pretreatment group(C). Total cerebral ischemia model was set up by four vessel occlusion in rats. Glu and Asp levels were measured with microdialysis at different time. Then the animals were decapitated and the brains were immediately removed to detect NMDAR1 expression in hippocampus area by immunohistochemistry and Western-blot. RESULTS: Compared with that in group B, the levels of Glu, Asp and NMDA NR1 protein were significantly decreased in the dexmedetomidine pretreatment group (P<0.05 or 0.01). CONCLUSIONS: Dexmedetomidine might has a protective effect on hippocampus in global cerebral ischemia/reperfusion animals. The protective mechanism might be involved in inhibiting excitatory amino acids(EAA) release and NMDAR1 expression.

Comparison of pre- and post-ischemic treatment of telmisartan and nimodipine combination in experimentally induced cerebral ischemia.

Time dependent intervention plays a crucial role in preventing neurodegeneration after ischemic insult. The intensity of excitotoxicity is greater in the secondary reperfusion phase (2-4 h) compared to the primary occlusion phase (2 h), which could be attributed to secondary elevation of excitatory amino acids (EAA) in cerebral ischemia. In the present study, we tried to assess the neuroprotective effects of telmisartan and nimodipine (TM-NM) combination on the secondary reperfusion phase. The drug treatments were made immediately after reperfusion and their effects were compared with pre-treatment. The neuroprotective effect was studied using middle cerebral artery occlusion (MCAo) transient ischemic model in rats. On the 7th day after reperfusion, the rats were subjected to behavioral studies. The brain was dissected out on the 9th day to measure neurobiochemical alterations and for histopathological observations. The results have shown that TM-NM (5 mg/kg) attenuated the EAA release in different brain regions with partial restoration of energy levels in secondary reperfusion phase. Similarly, it normalized the behavioral alteration and the effect was comparable to pre-ischemic treatment (2.5 mg/kg). Pre-ischemic treatment of TM-NM (2.5 mg/kg) protected the neurons from ischemic reperfusion injury by energy dependent EAA regulation. It can be concluded from the study that, even though the pre- and post-treatment of TM-NM show similar results, the post-ischemic treatment of TM-NM combination is beneficial due to better EAA control. Since hypertension is the primary risk factor for stroke, clinical incidents of stroke in hypertensive patients receiving angiotensin receptor blockers (ARBs) can be further investigated to understand the present study in the clinical situation.

Brain ischaemia induces shedding of a BDNF-scavenger ectodomain from TrkB receptors by excitotoxicity activation of metalloproteinases and γ-secretases.

Stroke remains a leading cause of death and disability in the world with limited therapies available to restrict brain damage or improve functional recovery after cerebral ischaemia. A promising strategy currently under investigation is the promotion of brain-derived neurotrophic factor (BDNF) signalling through tropomyosin-related kinase B (TrkB) receptors, a pathway essential for neuronal survival and function. However, TrkB and BDNF-signalling are impaired by excitotoxicity, a primary pathological process in stroke also associated with neurodegenerative diseases. Pathological imbalance of TrkB isoforms is critical in neurodegeneration and is caused by calpain processing of BDNF high affinity full-length receptor (TrkB-FL) and an inversion of the transcriptional pattern of the Ntrk2 gene, to favour expression of the truncated isoform TrkB-T1 over TrkB-FL. We report here that both TrkB-FL and neuronal TrkB-T1 also undergo ectodomain shedding by metalloproteinases activated after ischaemic injury or excitotoxic damage of cortical neurons. Subsequently, the remaining membrane-bound C-terminal fragments (CTFs) are cleaved by γ-secretases within the transmembrane region, releasing their intracellular domains (ICDs) into the cytosol. Therefore, we identify TrkB-FL and TrkB-T1 as new substrates of regulated intramembrane proteolysis (RIP), a mechanism that highly contributes to TrkB-T1 regulation in ischaemia but is minor for TrkB-FL which is mainly processed by calpain. However, since the secreted TrkB ectodomain acts as a BDNF scavenger and significantly alters BDNF/TrkB signalling, the mechanism of RIP could contribute to neuronal death in excitotoxicity. These results are highly relevant since they reveal new targets for the rational design of therapies to treat stroke and other pathologies with an excitotoxic component.