Febrile seizure is one of the most common convulsive disorders in children. The neuromodulator adenosine exerts anticonvulsant actions through binding adenosine receptors. Here, the impact of hyperthermia‐induced seizures on adenosine A1 and A2A receptors and 5′‐nucleotidase activity has been studied at different periods in the cerebral cortical area by using radioligand binding, real‐time PCR, and 5′‐nucleotidase activity assays. Hyperthermic seizures were induced in 13‐day‐old rats using a warmed air stream from a hair dryer. Neonates exhibited rearing and falling over associated with hindlimb clonus seizures (stage 5 on Racine scale criteria) after hyperthermic induction. A significant increase in A1 receptor density was observed using [3H]DPCPX as radioligand, and mRNA coding A1 was observed 48 h after hyperthermia‐induced seizures. In contrast, a significant decrease in A2A receptor density was detected, using [3H]ZM241385 as radioligand, 48 h after hyperthermia‐evoked convulsions. These short‐term changes in A1 and A2A receptors were also accompanied by a loss of 5′‐nucleotidase activity. No significant variations either in A1 or A2A receptor density or 5′‐nucleotidase were observed 5 and 20 days after hyperthermic seizures. Taken together, both regulation of A1 and A2A receptors and loss of 5′‐nucleotidase in the cerebral cortex suggest the existence of a neuroprotective mechanism against seizures.
The role of physical exercise as a neuroprotective agent against ischemic injury has been extensively discussed. Nevertheless, the mechanisms underlying the effects of physical exercise on cerebral ischemia remain poorly understood. Here, we investigate the hypothesis that physical exercise increases ischemic tolerance by decreasing the induction of cellular apoptosis and glutamate release. Rats (n = 50) were submitted to a swimming exercise protocol for 8 weeks. Hippocampal slices were then submitted to oxygen and glucose deprivation. Cellular viability, pro‐apoptotic markers (Caspase 8, Caspase 9, Caspase 3, and apoptosis‐inducing factor), and glutamate release were analyzed. The percentage of cell death, the amount of glutamate release, and the expression of the apoptotic markers were all decreased in the exercise group when compared to the sedentary group after oxygen and glucose deprivation. Our results suggest that physical exercise protects hippocampal slices from the effects of oxygen and glucose deprivation, probably by a mechanism involving both the decrease of glutamatergic excitotoxicity and apoptosis induction.
Kiss1, a neuropeptide predominantly expressed in the habenula, modulates the serotonin (5‐HT) system to decrease odorant cue [alarm substance (AS)]‐evoked fear behaviour in the zebrafish. The purpose of this study was to assess the interaction of Kiss1 with the 5‐HT system as well as to determine the involvement of the 5‐HT receptor subtypes in AS‐evoked fear. We utilized 0. 28 mg/kg WAY 100635 (WAY), a selective 5‐HT1A receptor antagonist, to observe the effects of Kiss1 administration on AS‐evoked fear. We found WAY significantly inhibited the anxiolytic effects of Kiss1 (p <0.001) with an exception of freezing behaviour. Based on this, we utilized 92.79 mg/kg methysergide, a 5‐HT1 and 5‐HT2 receptor antagonist, and found that methysergide significantly blocked the anxiolytic effects of Kiss1 in the presence of the AS (p <0.001). From this, we conclude that Kiss1 modulates AS‐evoked fear responses mediated by the 5‐HT1A and 5‐HT2 receptors.
Soluble N‐ethylmaleimide sensitive factor attachment protein receptors (SNAREs) are crucial for exocytosis, trafficking, and neurite outgrowth, where vesicular SNAREs are directed toward their partner target SNAREs: synaptosomal‐associated protein of 25 kDa and syntaxin. SNARE proteins are normally membrane bound, but can be cleaved and released by botulinum neurotoxins. We found that botulinum proteases types C and D can easily be transduced into endocrine cells using DNA‐transfection reagents. Following administration of the C and D proteases into normally refractory Neuro2A neuroblastoma cells, the SNARE proteins were cleaved with high efficiency within hours. Remarkably, botulinum protease exposures led to cytotoxicity evidenced by spectrophotometric assays and propidium iodide penetration into the nuclei. Direct delivery of SNARE fragments into the neuroblastoma cells reduced viability similar to botulinum proteases' application. We observed synergistic cytotoxic effects of the botulinum proteases, which may be explained by the release and interaction of soluble SNARE fragments. We show for the first time that previously observed cytotoxicity of botulinum neurotoxins/C in neurons could be achieved in cells of neuroendocrine origin with implications for medical uses of botulinum preparations.
Ammonia is considered to be the main neurotoxin responsible for hepatic encephalopathy resulting from liver failure. Liver failure has been reported to alter expression and activity of P‐glycoprotein (P‐gp) and multidrug resistance‐associated protein 2 (Mrp2) at the blood–brain barrier (BBB). The aim of this study was to investigate whether ammonia is involved in abnormalities of expression and activity of P‐gp and Mrp2 at the BBB. Hyperammonemic rats were developed by an intraperitoneal injection of ammonium acetate (NH4Ac, 4.5 mmol/kg). Results showed that Mrp2 function markedly increased in cortex and hippocampus of rats at 6 h following NH4Ac administration. Significant increase in function of P‐gp was observed in hippocampus of rats. Meanwhile, such alterations were in line with the increase in mRNA and protein levels of P‐gp and Mrp2. Significant increase in levels of nuclear amount of nuclear factor‐κB (NF‐κB) p65 was also observed. Primarily cultured rat brain microvessel endothelial cells (rBMECs) were used for in vitro study. Data indicated that 24 h exposure to ammonia significantly increased function and expression of P‐gp and Mrp2 in rBMECs, accompanied with activation of NF‐κB. Furthermore, such alterations induced by ammonia were reversed by NF‐κB inhibitor. In conclusion, this study demonstrates that hyperammonemia increases the function and expression of P‐gp and Mrp2 at the BBB via activating NF‐κB pathway.
Dopaminergic signaling pathways are conserved between mammals and Drosophila, but the factors important for maintaining the functional pool of synaptic dopamine are not fully understood in Drosophila. In this study, we characterized the releasable and reserve dopamine pools in Drosophila larvae using ATP/P2X2‐mediated stimulation. Dopamine release was stable with stimulations performed at least every 5 min but decayed with stimulations performed 2 min apart or less, indicating the replenishment of the releasable pool occurred on a time scale between 2 and 5 min. Dopamine synthesis or uptake was pharmacologically inhibited with 3‐iodotyrosine and cocaine, respectively, to evaluate their contributions to maintain the releasable dopamine pool. We found that both synthesis and uptake were needed to maintain the releasable dopamine pool, with synthesis playing a major part in long‐term replenishment and uptake being more important for short‐term replenishment. These effects of synthesis and uptake on different time scales in Drosophila are analogous to mammals. However, unlike in mammals, cocaine did not activate a reserve pool of dopamine in Drosophila when using P2X2 stimulations. Our study shows that both synthesis and uptake replenish the releasable pool, providing a better understanding of dopamine regulation in Drosophila.
Acyl‐CoA‐binding protein (ACBP) is a ubiquitously expressed protein that binds intracellular acyl‐CoA esters. Several studies have suggested that ACBP acts as an acyl‐CoA pool former and regulates long‐chain fatty acids (LCFA) metabolism in peripheral tissues. In the brain, ACBP is known as Diazepam‐Binding Inhibitor, a secreted peptide acting as an allosteric modulator of the GABAA receptor. However, its role in central LCFA metabolism remains unknown. In the present study, we investigated ACBP cellular expression, ACBP regulation of LCFA intracellular metabolism, FA profile, and FA metabolism‐related gene expression using ACBP‐deficient and control mice. ACBP was mainly found in astrocytes with high expression levels in the mediobasal hypothalamus. We demonstrate that ACBP deficiency alters the central LCFA‐CoA profile and impairs unsaturated (oleate, linolenate) but not saturated (palmitate, stearate) LCFA metabolic fluxes in hypothalamic slices and astrocyte cultures. In addition, lack of ACBP differently affects the expression of genes involved in FA metabolism in cortical versus hypothalamic astrocytes. Finally, ACBP deficiency increases FA content and impairs their release in response to palmitate in hypothalamic astrocytes. Collectively, these findings reveal for the first time that central ACBP acts as a regulator of LCFA intracellular metabolism in astrocytes.
For over the last 50 years, the molecular mechanism of anti‐psychotic drugs' action has been far from clear. While risperidone is very often used in clinical practice, the most efficient known anti‐psychotic drug is clozapine (CLO). However, the biochemical background of CLO's action still remains elusive. In this study, we performed comparative proteomic analysis of rat cerebral cortex following chronic administration of these two drugs. We observed significant changes in the expression of cytoskeletal, synaptic, and regulatory proteins caused by both antipsychotics. Among other proteins, alterations in collapsin response mediator proteins, CRMP2 and CRMP4, were the most spectacular consequences of treatment with both drugs. Moreover, risperidone increased the level of proteins involved in cell proliferation such as fatty acid‐binding protein‐7 and translin‐associated factor X. CLO significantly up‐regulated the expression of visinin‐like protein 1, neurocalcin δ and mitochondrial, stomatin‐like protein 2, the calcium‐binding proteins regulating calcium homeostasis, and the functioning of ion channels and receptors.
A major hallmark feature of Alzheimer's disease is the accumulation of amyloid β (Aβ), whose formation is regulated by the γ‐secretase complex and its activating protein (also known as γ‐secretase activating protein, or GSAP). Because GSAP interacts with the γ‐secretase without affecting the cleavage of Notch, it is an ideal target for a viable anti‐Aβ therapy. GSAP derives from a C‐terminal fragment of a larger precursor protein of 98 kDa via a caspase 3‐mediated cleavage. However, the mechanism(s) involved in its degradation remain unknown. In this study, we show that GSAP has a short half‐life of approximately 5 h. Neuronal cells treated with proteasome inhibitors markedly prevented GSAP protein degradation, which was associated with a significant increment in Aβ levels and γ‐secretase cleavage products. In contrast, treatment with calpain blocker and lysosome inhibitors had no effect. In addition, we provide experimental evidence that GSAP is ubiquitinated. Taken together, our findings reveal that GSAP is degraded through the ubiquitin–proteasome system. Modulation of the GSAP degradation pathway may be implemented as a viable target for a safer anti‐Aβ therapeutic approach in Alzheimer's disease.
Cellular interactions mediated by the neural cell adhesion molecule (NCAM) are critical in cell migration, differentiation and plasticity. Switching of the NCAM‐interaction mode, from adhesion to signalling, is determined by NCAM carrying a particular post‐translational modification, polysialic acid (PSA). Regulation of cell‐surface PSA‐NCAM is traditionally viewed as a direct consequence of polysialyltransferase activity. Taking advantage of the polysialyltransferase Ca2+‐dependent activity, we demonstrate in TE671 cells that downregulation of PSA‐NCAM synthesis constitutes a necessary but not sufficient condition to reduce cell‐surface PSA‐NCAM; instead, PSA‐NCAM turnover required internalization of the molecule into the cytosol. PSA‐NCAM internalization was specifically triggered by collagen in the extracellular matrix (ECM) and prevented by insulin‐like growth factor (IGF1) and insulin. Our results pose a novel role for IGF1 and insulin in controlling cell migration through modulation of PSA‐NCAM turnover at the cell surface.
In this study, in vitro and in vivo experiments were carried out with the high‐affinity multifunctional D2/D3 agonist D‐512 to explore its potential neuroprotective effects in models of Parkinson's disease and the potential mechanism(s) underlying such properties. Pre‐treatment with D‐512 in vitro was found to rescue rat adrenal Pheochromocytoma PC12 cells from toxicity induced by 6‐hydroxydopamine administration in a dose‐dependent manner. Neuroprotection was found to coincide with reductions in intracellular reactive oxygen species, lipid peroxidation, and DNA damage. In vivo, pre‐treatment with 0.5 mg/kg D‐512 was protective against neurodegenerative phenotypes associated with systemic administration of MPTP, including losses in striatal dopamine, reductions in numbers of DAergic neurons in the substantia nigra (SN), and locomotor dysfunction. These observations strongly suggest that the multifunctional drug D‐512 may constitute a novel viable therapy for Parkinson's disease.
Deep brain stimulation (DBS) of the subthalamic nucleus (STN) is an efficient neurosurgical treatment for advanced Parkinson's disease. Non‐invasive metabolic neuroimaging during the course of DBS in animal models may contribute to our understanding of its action mechanisms. Here, DBS was adapted to in vivo proton magnetic resonance spectroscopy at 11.7 T in the rat to follow metabolic changes in main basal ganglia structures, the striatum, and the substantia nigra pars reticulata (SNr). Measurements were repeated OFF and ON acute and subchronic (7 days) STN‐DBS in control and parkinsonian (6‐hydroxydopamine lesion) conditions. Acute DBS reversed the increases in glutamate, glutamine, and GABA levels induced by the dopamine lesion in the striatum but not in the SNr. Subchronic DBS normalized GABA in both the striatum and SNr, and glutamate in the striatum. Taurine levels were markedly decreased under subchronic DBS in the striatum and SNr in both lesioned and unlesioned rats. Microdialysis in the striatum further showed that extracellular taurine was increased. These data reveal that STN‐DBS has duration‐dependent metabolic effects in the basal ganglia, consistent with development of adaptive mechanisms. In addition to counteracting defects induced by the dopamine lesion, prolonged DBS has proper effects independent of the pathological condition.
Mephedrone (4‐methylmethcathinone) is a synthetic cathinone designer drug that alters pre‐synaptic dopamine (DA) activity like many psychostimulants. However, little is known about the post‐synaptic dopaminergic impacts of mephedrone. The neuropeptide neurotensin (NT) provides inhibitory feedback for basal ganglia and limbic DA pathways, and post‐synaptic D1‐like and D2‐like receptor activity affects NT tissue levels. This study evaluated how mephedrone alters basal ganglia and limbic system NT content and the role of NT receptor activation in drug consumption behavior. Four 25 mg/kg injections of mephedrone increased NT content in basal ganglia (striatum, substantia nigra and globus pallidus) and the limbic regions (nucleus accumbens core), while a lower dosage (5 mg/kg/injection) only increased striatal NT content. Mephedrone‐induced increases in basal ganglia NT levels were mediated by D1‐like receptors in the striatum and the substantia nigra by both D1‐like and D2‐like receptors in the globus pallidus. Mephedrone increased substance P content, another neuropeptide, in the globus pallidus, but not in the dorsal striatum or substantia nigra. Finally, the NT receptor agonist PD149163 blocked mephedrone self‐administration, suggesting reduced NT release, as indicated by increased tissue levels, likely contributing to patterns of mephedrone consumption.
Chronic stress represents a major environmental risk factor for mood disorders in vulnerable individuals. The neurobiological mechanisms underlying these disorders involve serotonergic and endocannabinoid systems. In this study, we have investigated the relationships between these two neurochemical systems in emotional control using genetic and imaging tools. CB1 cannabinoid receptor knockout mice (KO) and wild‐type littermates (WT) were exposed to chronic restraint stress. Depressive‐like symptoms (anhedonia and helplessness) were produced by chronic stress exposure in WT mice. CB1 KO mice already showed these depressive‐like manifestations in non‐stress conditions and the same phenotype was observed after chronic restraint stress. Chronic stress similarly impaired long‐term memory in both genotypes. In addition, brain levels of serotonin transporter (5‐HTT) were assessed using positron emission tomography. Decreased brain 5‐HTT levels were revealed in CB1 KO mice under basal conditions, as well as in WT mice after chronic stress. Our results show that chronic restraint stress induced depressive‐like behavioral alterations and brain changes in 5‐HTT levels similarly to those revealed in CB1 KO mice in non‐stressed conditions. These results underline the relevance of chronic environmental stress on serotonergic and endocannabinoid transmission for the development of depressive symptoms.
(R)‐3‐[2,6‐cis‐Di(4‐methoxyphenethyl)piperidin‐1‐yl]propane‐1,2‐diol (GZ‐793A) inhibits methamphetamine‐evoked dopamine release from striatal slices and methamphetamine self‐administration in rats. GZ‐793A potently and selectively inhibits dopamine uptake at the vesicular monoamine transporter‐2 (VMAT2). This study determined GZ‐793A's ability to evoke [3H]dopamine release and inhibit methamphetamine‐evoked [3H]dopamine release from isolated striatal synaptic vesicles. Results show GZ‐793A concentration‐dependent [3H]dopamine release; nonlinear regression revealed a two‐site model of interaction with VMAT2 (High‐ and Low‐EC50 = 15.5 nM and 29.3 μM, respectively). Tetrabenazine and reserpine completely inhibited GZ‐793A‐evoked [3H]dopamine release, however, only at the High‐affinity site. Low concentrations of GZ‐793A that interact with the extravesicular dopamine uptake site and the High‐affinity intravesicular DA release site also inhibited methamphetamine‐evoked [3H]dopamine release from synaptic vesicles. A rightward shift in the methamphetamine concentration‐response was evident with increasing concentrations of GZ‐793A, and the Schild regression slope was 0.49 ± 0.08, consistent with surmountable allosteric inhibition. These results support a hypothetical model of GZ‐793A interaction at more than one site on the VMAT2 protein, which explains its potent inhibition of dopamine uptake, dopamine release via a High‐affinity tetrabenazine‐ and reserpine‐sensitive site, dopamine release via a Low‐affinity tetrabenazine‐ and reserpine‐insensitive site, and a low‐affinity interaction with the dihydrotetrabenazine binding site on VMAT2. GZ‐793A inhibition of the effects of methamphetamine supports its potential as a therapeutic agent for the treatment of methamphetamine abuse.
Mechanical perturbations can release ATP, which is broken down to adenosine. In this work, we used carbon‐fiber microelectrodes and fast‐scan cyclic voltammetry to measure mechanically stimulated adenosine in the brain by lowering the electrode 50 μm. Mechanical stimulation evoked adenosine in vivo (average: 3.3 ± 0.6 μM) and in brain slices (average: 0.8 ± 0.1 μM) in the prefrontal cortex. The release was transient, lasting 18 ± 2 s. Lowering a 15‐μm‐diameter glass pipette near the carbon‐fiber microelectrode produced similar results as lowering the actual microelectrode. However, applying a small puff of artificial cerebral spinal fluid was not sufficient to evoke adenosine. Multiple stimulations within a 50‐μm region of a slice did not significantly change over time or damage cells. Chelating calcium with EDTA or blocking sodium channels with tetrodotoxin significantly decreased mechanically evoked adenosine, signifying that the release is activity dependent. An alpha‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate receptor antagonist, 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione, did not affect mechanically stimulated adenosine; however, the nucleoside triphosphate diphosphohydrolase 1,2 and 3 (NTDPase) inhibitor POM‐1 significantly reduced adenosine so a portion of adenosine is dependent on extracellular ATP metabolism. Thus, mechanical perturbations from inserting a probe in the brain cause rapid, transient adenosine signaling which might be neuroprotective.
Most ingested ethanol is metabolized in the liver to acetaldehyde and then to acetate, which can be oxidized by the brain. This project assessed whether chronic exposure to alcohol can increase cerebral oxidation of acetate. Through metabolism, acetate may contribute to long‐term adaptation to drinking. Two groups of adult male Sprague–Dawley rats were studied, one treated with ethanol vapor and the other given room air. After 3 weeks the rats received an intravenous infusion of [2‐13C]ethanol via a lateral tail vein for 2 h. As the liver converts ethanol to [2‐13C]acetate, some of the acetate enters the brain. Through oxidation the 13C is incorporated into the metabolic intermediate α‐ketoglutarate, which is converted to glutamate (Glu), glutamine (Gln), and GABA. These were observed by magnetic resonance spectroscopy and found to be 13C‐labeled primarily through the consumption of ethanol‐derived acetate. Brain Gln, Glu, and, GABA 13C enrichments, normalized to 13C‐acetate enrichments in the plasma, were higher in the chronically treated rats than in the ethanol‐naïve rats, suggesting increased cerebral uptake and oxidation of circulating acetate. Chronic ethanol exposure increased incorporation of systemically derived acetate into brain Gln, Glu, and GABA, key neurochemicals linked to brain energy metabolism and neurotransmission.
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