Fresh Rat Brain Research Articles

Global analysis of qualitative and quantitative metabolism of Notoginsenoside R1 in rat liver-brain-gut axis based on LC-IT-TOF/MS combing mMDF strategy

The metabolism study of active components for traditional Chinese medicine (TCM) in target organs is conducive to clarify the authentic active ingredients. Notoginsenoside R1 (NG-R1), one of the bioactive components of Panax notoginsenoside (PNS), is commonly acknowledged as the characteristic marker of PNS. However, the metabolism of NG-R1 in target organs has not been clarified yet due to the lack of robust technique and approach. The present study aimed to develop a reliable and efficient strategy and technology for revealing the qualitative and quantitative metabolism of active components of TCMs in target organs, and to clarify the biotransformation of NG-R1 in liver-brain-intestinal axis. The metabolic transformation of NG-R1 in the brain gut axis was investigated in the in vitro incubation system of fresh rat brain, liver homogenate, and intestinal flora. To quickly lock the target metabolites, we set the mass defect filter (MDF) in different ranges to screen metabolites with different molecular weight (MW). This strategy was defined as multi-stage MDF (mMDF). In addition, we performed relative quantitative analysis on all metabolites according to the peak area acquired by LC-IT-TOF/MS to overcome the challenge that metabolites are difficult to be quantified due to the lack of standards. When MDF was set at 0.50 to 0.65 to screen metabolites with MW of 900 to 1200 Da, 6 novel metabolites were quickly found, and then identified as glucuronic acid binding, oxidation, dehydrogenation, methylation and hydrogenation products according to their LC and MS characteristics. When setting MDF at 0.42 - 0.52, 6 metabolites with MW of 600 to 900 Da were effectively screened and identified as Rg1, NG-R2, Rh1, Rg1+CH2+2H and Rg1+CH2. To screen the metabolites with MW of 300 to 600 Da, MDF was set at 0.25 - 0.42, and 4 novel metabolites were screened rapidly. The results of quantitative metabolism suggested that intestinal flora was the main metabolic site of NG-R1 in rat, and more than 60% of NG-R1 was converted to Rg1 by deglycosylation in the intestinal flora. The mMDF strategy can significantly improve the research efficiency of qualitative metabolism of saponins. Although NG-R1 could be transformed into a variety of metabolites in rat liver and brain homogenate, it still existed mainly in prototype form. In the rat flora, NG-R1 mainly existed in the form of deglycosylated metabolite Rg1.

Ganglioside Detection from Formalin-Fixed Human Brain Tissue Utilizing MALDI Imaging Mass Spectrometry.

Matrix assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) is used to perform mass spectrometric analysis directly on biological samples providing visual and anatomical spatial information on molecules within tissues. A current obscuration of MALDI-IMS is that it is largely performed on fresh frozen tissue, whereas clinical tissue samples stored long-term are fixed in formalin, and the fixation process is thought to cause signal suppression for lipid molecules. Studies have shown that fresh frozen tissue sections applied with an ammonium formate (AF) wash prior to matrix application in the MALDI-IMS procedure display an increase in observed signal intensity and sensitivity for lipid molecules detected within the brain while maintaining the spatial distribution of molecules throughout the tissue. In this work, we investigate the viability of formalin-fixed tissue imaging in a clinical setting by comparing MALDI data of fresh frozen and postfixed rat brain samples, along with postfixed human brain samples washed with AF to assess the capabilities of ganglioside analysis in MALDI imaging of formalin-fixed tissue. Results herein demonstrate that MALDI-IMS spectra for gangliosides, including GM1, were significantly enhanced in fresh frozen rat brain, formalin-fixed rat brain, and formalin-fixed human brain samples through the use of an AF wash. Improvements in MALDI-IMS image quality were demonstrated, and the spatial distribution of molecules was retained. Results indicate that this method will allow for the analysis of gangliosides from formalin-fixed clinical samples, which can open additional avenues for neurodegenerative disease research.

Microglia-like Cells Derived from Hematopoietic Stem and Progenitor Cells Are a Model System to Investigate Chronic Pain in Sickle Cell Disease

Background: Patients with sickle cell disease (SCD) often experience severe chronic pain. In chronic pain, microglia are readily activated, stimulating neurons to send a pain signal. Human microglia are difficult to obtain; we have cultured peripheral blood derived microglia-like cells (PBMLC) from human peripheral blood to develop a model system to investigate chronic pain in sickle cell disease. We have shown that our PB-MLC resemble microglia morphologically, have the same surface markers, and can be activated by lipopolysaccharide (LPS). Our PB-MLC are more easily activated and secrete more inflammatory cytokines if they are derived from a SCD patient with chronic pain compared to a HbAA individual without pain or a SCD patient with acute pain only. Now, we seek to assess the suitability of our model system to screen for compounds to treat chronic pain by using of a panel of compounds to reduce microglia and PB-MLC activation and cytokine release in response to LPS. Finally, we propose to validate our model system by comparing the morphology, surface markers, and cytokine release between PB-MLC and true in situ brain derived microglia (BDM), both from Sprague Dawley (SD) rats. Methods: Human peripheral blood mononuclear cells (HPB-MCs) were obtained from three individuals with SCD with chronic pain and three normal donors (WT). HPB-MCs were cultured with human GM-CSF (10 ng/ml) and human IL-34 (100 ng/ml) to induce peripheral blood derived microglia (HPB-MLC). TNF-alpha and IL-1beta secretion by HPB-MLCs in response to LPS with or without a panel of drugs were measured with ELISA. Rat BDM were isolated from fresh rat brain tissues using anti‐rat CD11b/c microbeads and cultured with murine IL-34(100 ng/ml) and murine GM-CSF (10 ng/ml). Rat PB-MLCs were developed from rat PB-MCs by culturing with murine IL-34 (100 ng/ml) and murine GM-CSF (10 ng/ml). On day 7 of culture, rat BDM and PB-MLCs were collected and morphology analyzed by phase contrast microscopy, phenotyped by flow cytometry and indirect immunofluorescence with anti-CX3CR1, TMEM119, CD68, Iba1 antibodies. Microglia morphology was evaluated by quantitative analysis of cell body roundness and branch length. TNF-alpha, IL-1beta, and IL-6 secretion by rat BDM and PB-MLCs in response to LPS with or without piceatannol was measured with ELISA. Results: To evaluate the possibility of using our PB-MLC model system to screen compounds to reduce abnormal microglia activation and treat chronic pain, we tested HPB-MLC cells from patients with SCD and chronic pain with the following drugs: gabapentin, metformin, piceatannol, and resveratrol, chosen based on published reports of their effect on microglia activation. All suppressed the release of proinflammatory cytokine from LPS-induced HPB-MLC in a dose-dependent manner (Figure 1), and reversed the deramification, or rounding of activated PB-MLC upon LPS stimulation by quantitative analysis of cell body roundness and branch length with immunostaining of Iba1. Gabapentin exhibited the smallest effect on reduction of HPB-MLC activation. Rat BDM and PBMLC both exhibited characteristic microglia branched morphology in culture, and rat PBMLC from SD rats expressed microglia specific marker TMEM119. Both PB-MLC and BDM from rats became amoeboid with LPS treatment, and showed increased expression of CD68, and Iba1 (Figure 2). In both BDM and PBMLC from rats, piceatannol reduced LPS activation and TNF-alpha, IL-1beta, and IL-6 secretion (Figure 3). Conclusions: We have established the microglia-like nature of PBMLC from patients with SCD and normal blood donors, and the preservation of the patient pain phenotype in culture. Here, we show that compounds reported to reduce microglia activation reduce the inflammatory cytokine release from HPB-MLCs from patients with SCD. BDM and PB-MLCs from SD rats have similar morphology, both quiescent and activated, and secrete inflammatory cytokines in the same manner in response to LPS. Piceatannol reduces activation and cytokine release in both. This comparison between peripheral blood derived and brain derived microglia supports our assertion that PB-MLC capture significant aspects of true in situ brain microglia biology, particularly that of activation and drug response. We therefore propose to use this model system to derive mechanistic insights into the development of chronic pain in SCD, and to screen pharmacologic agents to treat chronic pain. Disclosures No relevant conflicts of interest to declare.

Enhancement of Lipid Signal in MALDI MS imaging obtained from Formalin Fixed Human Brain Tissue

Matrix assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) is used to perform mass spectrometric analysis directly on biological samples providing accurate visual/anatomical spatial information of molecules within the tissue. A current limitation of MALDI‐IMS is that it is largely performed on fresh frozen tissue whereas clinical tissue samples stored long term are fixed in formalin. It has been shown that fresh frozen tissue sections applied with an ammonium formate (AF) wash prior to matrix application in the MALDI‐IMS procedure display an increase in observed signal intensity and sensitivity for lipid molecules detected in the brain while maintaining the special distribution of molecules throughout the tissue. In this work we investigate the effectiveness of this AF wash on post‐fixed rat and human brain tissue sections in an effort to increase the viability of formalin fixed tissue imaging in a clinical setting. Results herein demonstrate that the AF wash significantly improved MALDI‐IMS spectra for gangliosides, including GM1 in fresh frozen rat brain, formalin‐fixed rat brain and formalin fixed human brain samples. AF wash also demonstrated improvements in MALDI‐IMS image quality while retaining the spatial distribution of molecules. Results indicate that this method will allow analysis of gangliosides from formalin‐fixed clinical samples, which can open additional avenues for neurodegenerative disease research.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Interference elimination in terahertz imaging based on inverse image processing

We propose a novel image restoration approach to eliminate the interference fringes in terahertz (THz) imaging. By making use of the information in the background of a THz image, inverse image processing is introduced under a priori knowledge about the degradation mechanism to suppress the interference. Based on a simulated interference image and the original THz image of a piece of paper coated with graphite powder, taken from our continuous THz imaging system, the interference fringes are successfully eliminated. The enhancement of the peak signal-to-noise ratio and structural similarity index is achieved, compared with the frequency domain filtering. Moreover, the inverse image processing is performed on a THz image of fresh rat brain tissue. It presents an excellent result with accurate information for the brain tissue after interference elimination.

Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue.

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific sets of sparsely distributed activated neurons called neuronal ensembles that mediate learned associations. Fluorescence Activated Cell Sorting (FACS) has recently been optimized for adult rat brain tissue and allowed isolation of activated neurons using antibodies against the neuronal marker NeuN and Fos protein, a marker of strongly activated neurons. Until now, Fos-expressing neurons and other cell types were isolated from fresh tissue, which entailed long processing days and allowed very limited numbers of brain samples to be assessed after lengthy and complex behavioral procedures. Here we found that yields of Fos-expressing neurons and Fos mRNA from dorsal striatum were similar between freshly dissected tissue and tissue frozen at -80 ºC for 3 - 21 days. In addition, we confirmed the phenotype of the NeuN-positive and NeuN-negative sorted cells by assessing gene expression of neuronal (NeuN), astrocytic (GFAP), oligodendrocytic (Oligo2) and microgial (Iba1) markers, which indicates that frozen tissue can also be used for FACS isolation of glial cell types. Overall, it is possible to collect, dissect and freeze brain tissue for multiple FACS sessions. This maximizes the amount of data obtained from valuable animal subjects that have often undergone long and complex behavioral procedures.

Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

Pathologically confirmed autoimmune encephalitis in suspected Creutzfeldt-Jakob disease.

Objective:To determine the clinical features and presence in CSF of antineuronal antibodies in patients with pathologically proven autoimmune encephalitis derived from a cohort of patients with suspected Creutzfeldt-Jakob disease (CJD).Methods:The Dutch Surveillance Centre for Prion Diseases performed 384 autopsies on patients with suspected CJD over a 14-year period (1998–2011). Clinical information was collected from treating physicians. Antineuronal antibodies were tested in CSF obtained postmortem by immunohistochemistry on fresh frozen rat brain sections, by Luminex assay for the presence of well-characterized onconeural antibodies, and by cell-based assays for antibodies against NMDAR, GABABR1/2, GABAAR GLUR1/2, LGI1, Caspr2, and DPPX.Results:In 203 patients, a diagnosis of definite CJD was made, while in 181 a variety of other conditions were diagnosed, mainly neurodegenerative. In 22 of these 181, the neuropathologist diagnosed autoimmune encephalitis. One patient was excluded because of lack of clinical information. Inflammatory infiltrates were predominantly perivascular and consisted mainly of T cells. The predominant locations were basal ganglia and thalamus (90%) and temporal lobes and hippocampus (81%). In 6 patients (29%), antineuronal antibodies were detected in postmortem CSF, directed against Hu, NMDAR, GABABR1/2, Caspr2, and an unidentified synaptic antigen in 2. The most frequent symptoms were dementia (90%), gait disturbance (86%), cerebellar signs (67%), and neuropsychiatric symptoms (67%). Immunopathologic and clinical findings did not differ between autoantibody-negative patients and patients with antineuronal antibodies.Conclusions:It is important to consider immune-mediated disorders in the differential diagnosis of rapidly progressive neurologic deficits.

A measurement and modeling study of temperature in living and fixed tissue during and after radiofrequency exposure

Fluorescent intensity of the dye Rhodamine-B (Rho-B) decreases with increasing temperature. We show that in fresh rat brain tissue samples in a custom-made radiofrequency (RF) tissue exposure device, temperature rise due to RF radiation as measured by absorbed dye correlates well with temperature measured nearby by fiber optic probes. Estimates of rate of initial temperature rise (using both probe measurement and the dye method) accord well with estimates of local specific energy absorption rate (SAR). We also modeled the temperature characteristics of the exposure device using combined electromagnetic and finite-difference thermal modeling. Although there are some differences in the rate of cooling following cessation of RF exposure, there is reasonable agreement between modeling and both probe measurement and dye estimation of temperature. The dye method also permits measurement of regional temperature rise (due to RF). There is no clear evidence of local differential RF absorption, but further refinement of the method may be needed to fully clarify this issue.

Region-specific causal mechanism in the effects of ammonia on cerebral glucose metabolism in the rat brain

Ammonia, which is considered to be the main agent responsible for hepatic encephalopathy, inhibits oxidative glucose metabolism in the brain. However, the effects of ammonia on cerebral glucose metabolism in different brain regions remains unclear. To clarify this issue, we added ammonia directly to fresh rat brain slices and measured its effects on glucose metabolism. Dynamic positron autoradiography with [(18)F]2-fluoro-2-deoxy-D-glucose and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1) colorimetric assay revealed that ammonia significantly increased the cerebral glucose metabolic rate and depressed mitochondrial function, as compared to the unloaded control in each of the brain regions examined (cerebral cortex, striatum, and cerebellum), reflecting increased glycolysis that compensates for the decrease in aerobic metabolism. Pre-treatment with (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801), a N-methyl-D-aspartate (NMDA) receptor antagonist, significantly attenuated these changes induced by ammonia in cerebellum, but not in cerebral cortex or striatum. The addition of ammonia induced an increase in cyclic guanosine monophosphate (cGMP) levels in cerebellum, but not in cerebral cortex or striatum, reflecting the activation of the NMDA receptor-nitric oxide-cGMP pathway. These results suggested that NMDA receptor activation is responsible for the impairment of glucose metabolism induced by ammonia specifically in cerebellum.

A simple desalting method for direct MALDI mass spectrometry profiling of tissue lipids

Direct MALDI-mass spectrometry (MALDI-MS) profiling of tissue lipids often observes isobaric phosphatidylcholine (PC) species caused by the endogenous alkali metal ions that bias the relative abundance of tissue lipids. Fresh rat brain cryosections were washed with 70% ethanol (EtOH), water (H₂O), or 150 mM ammonium acetate (NH₄Ac), and the desalting effectiveness of each fluid was evaluated by MALDI-MS profiling of PC and sphingomyelin (SM) species in tissue and in the washing runoff. The results indicated that EtOH and H₂O only partially desalted the tissue lipids, yet both substantially displaced the tissue lipids to the washing runoffs. On the other hand, NH₄Ac effectively desalted the tissue lipids and produced a runoff containing no detectable PCs or SMs. NH₄Ac wash also unveiled the underlying changes of PCs and SMs in the infarcted rat cortex previously masked by edema-caused increase of tissue sodium. The MS/MS of an isobaric PC in the infarcted cortex revealed the precursor change as the result of NH₄Ac wash and confirmed the desalting effectiveness of such wash. Other than desalting, NH₄Ac wash also removes contaminants in tissue, enhances the overall spectral quality, and benefits additionally in profiling of biological molecules in tissue.

Dexras1 Interacts with FE65 to Regulate FE65-Amyloid Precursor Protein-dependent Transcription

FE65 is an adaptor protein that binds to and forms a transcriptionally active complex with the gamma-secretase-derived amyloid precursor protein (APP) intracellular domain. The regulatory mechanisms of FE65-APP-mediated transcription are still not clear. In this report, we demonstrate that Dexras1, a Ras family small G protein, binds to FE65 PTB2 domain and potently suppresses the FE65-APP-mediated transcription. The suppression is not via competition for binding of FE65 between Dexras1 and APP because the two proteins can simultaneously bind to the FE65 PTB2 domain. Phosphorylation of FE65 tyrosine 547 within the PTB2 domain has been shown to enhance FE65-APP-mediated transcription but not to influence binding to APP. Here we find that this phosphorylation event reduces the binding between Dexras1 and FE65. We also demonstrate that Dexras1 inhibits the FE65-APP-mediated transcription of glycogen synthase kinase 3beta (GSK3 beta). Moreover, small interfering RNA knockdown of Dexras1 enhances GSK3 beta expression and increases phosphorylation of Tau, a GSK3 beta substrate. Thus, Dexras1 functions as a suppressor of FE65-APP-mediated transcription, and FE65 tyrosine 547 phosphorylation enhances FE65-APP-mediated transcription, at least in part, by modulating the interaction between FE65 and Dexras1. These findings reveal a novel regulatory mechanism for FE65-APP-mediated signaling.

Active γ‐secretase is localized to detergent‐resistant membranes in human brain

Several lines of evidence suggest that polymerization of the amyloid beta-peptide (Abeta) into amyloid plaques is a pathogenic event in Alzheimer's disease (AD). Abeta is produced from the amyloid precursor protein as the result of sequential proteolytic cleavages by beta-secretase and gamma-secretase, and it has been suggested that these enzymes could be targets for treatment of AD. gamma-Secretase is an aspartyl protease complex, containing at least four transmembrane proteins. Studies in cell lines have shown that gamma-secretase is partially localized to lipid rafts, which are detergent-resistant membrane microdomains enriched in cholesterol and sphingolipids. Here, we studied gamma-secretase in detergent-resistant membranes (DRMs) prepared from human brain. DRMs prepared in the mild detergent CHAPSO and isolated by sucrose gradient centrifugation were enriched in gamma-secretase components and activity. The DRM fraction was subjected to size-exclusion chromatography in CHAPSO, and all of the gamma-secretase components and a lipid raft marker were found in the void volume (> 2000 kDa). Co-immunoprecipitation studies further supported the notion that the gamma-secretase components are associated even at high concentrations of CHAPSO. Preparations from rat brain gave similar results and showed a postmortem time-dependent decline in gamma-secretase activity, suggesting that DRMs from fresh rat brain may be useful for gamma-secretase activity studies. Finally, confocal microscopy showed co-localization of gamma-secretase components and a lipid raft marker in thin sections of human brain. We conclude that the active gamma-secretase complex is localized to lipid rafts in human brain.

Micro-dissection of Rat Brain for RNA or Protein Extraction from Specific Brain Region

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

Topological and chronological features of the impairment of glucose metabolism induced by 1-methyl-4-phenylpyridinium ion (MPP+) in rat brain slices

1-Methyl-4-phenylpyridinium (MPP(+)) was added directly to fresh rat brain slices and the dynamic changes in the cerebral glucose metabolic rate (CMRglc) were serially and two-dimensionally measured with [(18)F]2-fluoro-2-deoxy-D-glucose as a tracer. MPP(+) dose-dependently increased CMRglc, reflecting enhanced glycolysis compensating for the decrease in aerobic metabolism. While the CMRglc enhancement induced by MPP(+) (<10 microM) was restricted to the striatum, MPP(+) (>or=10 microM) induced a significant CMRglc enhancement in all brain regions. MPP(+) at high concentration (1 mM) eventually initiated rapid metabolic collapse, with failure to sustain anaerobic glycolysis.

Biphasic mechanism of the toxicity induced by 1-methyl-4-phenylpyridinium ion (MPP +) as revealed by dynamic changes in glucose metabolism in rat brain slices

1-Methyl-4-phenylpyridinium (MPP +) is a well-known neurotoxin which causes a clinical syndrome similar to Parkinson's disease. The classical mechanism of MPP + toxicity involves its entry into cells through the dopamine transporter (DAT) to inhibit aerobic glucose metabolism, while recent studies suggest that an oxidative mechanism may contribute to the toxicity of MPP +. However, it has not been adequately determined what role these two mechanisms play in the development of neurotoxicity after MPP + loading in the brain. To clarify this issue, MPP + was added directly to fresh rat brain slices and the dynamic changes in the cerebral glucose metabolic rate (CMRglc) produced by MPP + were serially and two-dimensionally measured using the dynamic positron autoradiography technique with [ 18F]2-fluoro-2-deoxy- d-glucose as a tracer. MPP + dose-dependently increased CMRglc in each of the brain regions examined, reflecting enhanced glycolysis compensating for the decrease in aerobic metabolism. Treatment with DAT inhibitor GBR 12909 significantly attenuated the enhanced glycolysis induced by 10 μM MPP + in the striatum. Treatment with free radical spin trap α-phenyl- N-tert-butylnitrone (PBN) significantly attenuated the enhancement of glycolysis induced by 100 μM MPP + in all brain regions. These results suggest that the mechanism of the toxicity of MPP + is biphasic and consists of a DAT-mediated mechanism selective for dopaminergic regions at a lower concentration of MPP + (10 μM), and an oxidative mechanism that occurs at a higher concentration of MPP + (100 μM) and is not restricted to dopaminergic regions.

Effects of chlorpromazine on plasma membrane permeability and fluidity in the rat brain: A dynamic positron autoradiography and fluorescence polarization study

Antipsychotic drugs have been widely used in psychiatry for the treatment of various mental disorders, but the underlying biochemical mechanisms of their actions still remain unclear. Although phenothiazine antipsychotic drugs have been reported to directly interact with the peripheral plasma membrane, it is not known whether these drugs actually affect plasma membrane integrity in the central nervous system. To clarify these issues, we investigated the effect of chlorpromazine (CPZ), a typical phenothiazine antipsychotic drug, on plasma membrane permeability in fresh rat brain slices using a dynamic positron autoradiography technique and [ 18F]2-fluoro-2-deoxy- d-glucose ([ 18F]FDG) as a tracer. Treatment with CPZ (≥ 100 μM) resulted in the leakage of [ 18F]FDG-6-phosphate, but not [ 18F]FDG, suggesting that the [ 18F]FDG-6-phosphate efflux was not mediated by glucose transporters, but rather by plasma membrane permeabilization. The leakage of [ 18F]FDG-6-phosphate was followed by slower leakage of cytoplasmic lactate dehydrogenase, suggesting that CPZ could initially induce small membrane holes that enlarged with time. Furthermore, the addition of CPZ (≥ 100 μM) caused a decrease in 1,6-diphenyl-1,3,5-hexatriene fluorescence anisotropy, which implies an increase in membrane fluidity. CPZ loading dose-dependently increased both membrane permeability and membrane fluidity, which suggested the involvement of a perturbation of membrane order in the mechanisms of membrane destabilization induced by antipsychotic drugs.

Complex association and dissociation kinetics of brevetoxin binding to voltage-sensitive rat brain sodium channels.

Brevetoxins bind with high affinity to the voltage-sensitive sodium channel and cause nerve cell depolarization and increased sodium ion conductance. Using 0.6 nM tritium-labeled brevetoxin-3 and freshly prepared synaptosomes from fresh or frozen rat brain, binding results at 6 degrees C fit well to a curve for 2-phase association with 65% of the binding in the rapid phase and t1/2 values of 11 and 74 min for the rapid and slow phases, respectively. Both phases were accelerated at higher toxin concentrations, binding of 9 nM brevetoxin-3 (PbTx-3) was close to equilibrium within 1 hr. The slow phase was not apparent when binding was done at 20 degrees C or when binding was done at 6 degrees C after the membrane sample had been preincubated at 4 degrees C for 1 day or at 22 degrees C for 1 hr. The 2-phase nature of association was not affected by substitution of KCl for choline chloride in the assay medium to produce sodium channel in the inactive state. Dissociation kinetics at 6 degrees C were also complex; the results fit well to a 2-phase curve with 55% of the dissociation in the rapid phase and t1/2 values of 13 and 64 min for the rapid and slow phases, respectively. The 2-phase nature did not change significantly after preincubation at 4 degrees C for 1 day. However, dissociation at 20 degrees C was rapid and fit a curve for 1-phase dissociation with a t1/2 of 2-6 min. At higher concentrations of PbTx-3, the binding is further complicated by the presence of 2-4 low-affinity binding sites with Kd values near 700 nM. In conclusion the association and dissociation of PbTx-3 with sodium channel from rat brain are complex processes that may involve changes in sodium channel conformation or interactions with other membrane (or membrane-associated) components.

14-3-3 Binds to and Mediates Phosphorylation of Microtubule-associated Tau Protein by Ser9-phosphorylated Glycogen Synthase Kinase 3β in the Brain

In mammalian brain, tau, glycogen synthase kinase 3beta (GSK3beta), and 14-3-3, a phosphoserine-binding protein, are parts of a multiprotein tau phosphorylation complex. Within the complex, 14-3-3 simultaneously binds to tau and GSK3beta (Agarwal-Mawal, A., Qureshi, H. Y., Cafferty, P. W., Yuan, Z., Han, D., Lin, R., and Paudel, H. K. (2003) J. Biol. Chem. 278, 12722-12728). The molecular mechanism by which 14-3-3 connects GSK3beta to tau within the complex is not clear. In this study, we find that GSK3beta within the tau phosphorylation complex is phosphorylated on Ser(9). From extracts of rat brain and rat primary cultured neurons, Ser(9)-phosphorylated GSK3beta precipitates with glutathione-agarose beads coated with glutathione S-transferase-14-3-3. Similarly, from rat brain extract, Ser(9)-phosphorylated GSK3beta co-immunoprecipitates with tau. In vitro, 14-3-3 binds to GSK3beta only when the kinase is phosphorylated on Ser(9). In transfected HEK-293 cells, 14-3-3 binds to Ser(9)-phosphorylated GSK3beta and does not bind to GSK3beta (S9A). Tau, on the other hand, binds to both GSK3beta (WT) and GSK3beta (S9A). Moreover, 14-3-3 enhances the binding of tau with Ser(9)-phosphorylated GSK3beta by approximately 3-fold but not with GSK3beta (S9A). Similarly, 14-3-3 stimulates phosphorylation of tau by Ser(9)-phosphorylated GSK3beta but not by GSK3beta (S9A). In transfected HEK-293 cells, Ser(9) phosphorylation suppresses GSK3beta-catalyzed tau phosphorylation in the absence of 14-3-3. In the presence of 14-3-3, however, Ser(9)-phosphorylated GSK3beta remains active and phosphorylates tau. Our data indicate that within the tau phosphorylation complex, 14-3-3 connects Ser(9)-phosphorylated GSK3beta to tau and Ser(9)-phosphorylated GSK3beta phosphorylates tau.

Different mechanisms of hypoxic injury on white matter and gray matter as revealed by dynamic changes in glucose metabolism in rats

Fresh rat brain slices were incubated with [ 18F]2-fluoro-2-deoxy- d-glucose ([ 18F]FDG) in oxygenated Krebs–Ringer solution at 36 °C, and the fractional rate constant (= k 3*) of [ 18F]FDG proportional to the cerebral glucose metabolic rate in white matter and gray matter was investigated with positron autoradiography. In both white matter and gray matter, the k 3* value with ≥20 min hypoxia was markedly lower than the unloaded control value, indicating irreversible hypoxic injury. Next, the neuroprotective effect against hypoxia induced by the addition of an N-methyl- d-aspartate receptor antagonist or a free radical scavenger was assessed by determining whether a decrease in the k 3* value after hypoxia loading was prevented. In gray matter, both agents exhibited a neuroprotective effect against 20 min hypoxia. In white matter, however, only the free radical scavenger was effective. These results suggest a similarity in the degree of vulnerability to hypoxia between white matter and gray matter as well as a difference in the developmental mechanism of hypoxic injury, i.e. the involvement of both glutamate and free radicals in gray matter, and the more selective involvement of free radicals in white matter.