Hippocampal Membrane Fractions Research Articles

Possible role of transcriptional regulation of 5-HT1A receptor in the midbrain on unadaptation to stress in mice

The ability to adapt to stress is an essential defensive function of a living body, and disturbance of this ability in the brain may contribute to the development of affective illness. Previously, we reported that mice exposed to unadaptable restraint stress show emotional abnormality. Moreover, this emotional abnormality was alleviated by chronic treatment with flesinoxan, a serotonin (5-HT)1A receptor agonist. 5-HT1A receptor expression is regulated by several transcription factors such as nuclear deformed epidermal autoregulatory factor (NUDR/Deaf-1) and five prime repressors under dual repression binding protein 1 (Freud-1). The present study was designed to investigate the expression levels of 5-HT1A receptor and its transcription factors in the midbrain and hippocampus of stress-adaptive and -unadaptive mice. Mice were exposed to 14 days of repeated adaptable (1 h/day) or repeated unadaptable (4 h/day) restraint stress, or were left in their home cage (non-stressed groups). In a western blot analysis, a significant increase in the expression levels of 5HT1A receptor protein were observed in the hippocampal membrane fraction in stress-adaptive mice. In contrast, the expression levels of 5-HT1A receptor protein in stress-unadaptive mice were significantly increased in both cytoplasmic and membrane fraction of the midbrain. Furthermore, real-time PCR analysis revealed that, in the midbrain of stress-unadaptive mice, the expression levels of 5-HT1A receptor mRNA and Freud-1 or NUDR mRNA were significantly increased and decreased, respectively. These results suggest that increased expression of 5-HT1A receptor due to decrease in the expression of Freud-1 and NUDR in the midbrain may play a pivotal role in the emotional abnormality of stress-unadaptive mice.

Characterization of 5-HT1A receptor and transport protein KIF13A expression in the hippocampus of stress-adaptive and -maladaptive mice

The ability to adapt to stress is an essential defensive function of a living body, and disturbance of this ability in the brain may contribute to the development of affective illness including major depression and anxiety disorders. A growing body of evidence suggests that brain serotonin (5-HT)1A receptors may be involved, at least in part, in the development of adaptation to stress. 5-HT1A receptor was reported to be transported by KIF13A, a motor protein and a member of the kinesin superfamily, from the golgi apparatus to the plasma membrane. The aim of the present study was to characterize the expression pattern of 5-HT1A receptor and KIF13A in the hippocampus of stress-adaptive and -maladaptive mice. Mice were either exposed to repeated adaptable (1 h/day) or unadaptable (4 h/day) restraint stress, or left in their home cage for 14 days. The levels of 5-HT1A receptor and KIF13A expression were assessed by western blot analysis. To confirm the formation of a 5-HT1A receptor and KIF13A complex, we performed blue native-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (BN-SDS-PAGE). Western blotting showed that neither 5-HT1A receptor nor KIF13A expression changed significantly in the hippocampal total extract of stress-adaptive and -maladaptive mice. In contrast, expression of 5 H T1A receptor and KIF13A in the hippocampal membrane fraction was increased in stress-adaptive mice, but not in stress-maladaptive mice. BN-SDS-PAGE analysis revealed that the bands of 5-HT1A receptor and KIF13A were both observed at a molecular weight of approximately 70 kDa, which indicated that 5-HT1A receptor and KIF13A form a complex. The present findings suggest that translocation of 5-HT1A receptor in complex with KIF13A to the plasma membrane of the hippocampus may play an important role in the formation of stress adaptation.

Redistribution of Monocarboxylate 1 and 4 in Hippocampus and Spatial Memory Impairment Induced by Long-term Ketamine Administration.

The monocarboxylate transporters (MCTs) MCT1, MCT2, and MCT4 are essential components of the astrocyte-neuron lactate shuttle (ANLS), which is a fundamental element of brain energetics. Decreased expression of MCTs can induce cognitive dysfunction of the brain. In the present study, we established a mouse model of long-term ketamine administration by subjecting mice to a 6-month course of a daily intraperitoneal injection of ketamine. These mice demonstrated learning and memory deficits and a significant decline in MCT1 and MCT4 proteins in the hippocampal membrane fraction, while cytoplasmic MCT1 and MCT4 protein levels were significantly increased. In contrast, the levels of global MCT2 protein were significantly increased. Analysis of mRNA levels found no changes in MCT1/4 transcripts, although the expression of MCT2 mRNA was significantly increased. We suggest that redistribution of hippocampal MCT1 and MCT4, but not MCT2 up-regulation, may be related to learning and memory deficits induced by long-term ketamine administration.

Hippocampal GluA2 and GluA4 protein but not corresponding mRNA and promoter methylation levels are modulated at retrieval in spatial learning of the rat.

AMPA receptors mediate most fast excitatory synaptic transmission in the brain. Highly dynamic AMPA receptors are subjected to trafficking, recycling, and/or degradation and replacement. Changes in AMPA receptor abundance is an important mechanism involved in learning and memory formation. Results obtained with the Morris water maze (MWM), a paradigm for testing spatial memory in rodent, correlate with hippocampal synaptic plasticity and NMDA function. Different phases of spatial learning like acquisition and retrieval involve AMPA receptors. Long-term memory formation requires dynamic changes in gene transcription and protein synthesis. It is, however, not known so far if epigenetic marks such as DNA methylation and mRNA levels participate in regulation of AMPA receptors in hippocampus during memory retrieval. In the present study, rats were trained or untrained in the MWM. Steady state levels of hippocampal GluA1-4 mRNA were determined by RT-PCR and promoter methylation levels of GluA1-4 by in-house developed bisulfite pyrosequencing methods. GluA1-4 protein levels were determined in parallel in a membrane fraction by SDS-PAGE followed by Western blotting. Our results indicate that changes of hippocampal membrane AMPA receptors were modulated at the protein level, while no changes were observed at the mRNA and at the promoter methylation level of hippocampal GluA1-4. Training in the MWM at retrieval may, therefore, involve GluA2 and GluA4 subunits that may be regulated by protein stability or trafficking as protein determinations were carried out in a hippocampal membrane fraction.

Glutamate Delta-1 Receptor Regulates Metabotropic Glutamate Receptor 5 Signaling in the Hippocampus

The delta family of ionotropic glutamate receptors consists of glutamate delta-1 (GluD1) and glutamate delta-2 receptors. We have previously shown that GluD1 knockout mice exhibit features of developmental delay, including impaired spine pruning and switch in the N-methyl-D-aspartate receptor subunit, which are relevant to autism and other neurodevelopmental disorders. Here, we identified a novel role of GluD1 in regulating metabotropic glutamate receptor 5 (mGlu5) signaling in the hippocampus. Immunohistochemical analysis demonstrated colocalization of mGlu5 with GluD1 punctas in the hippocampus. Additionally, GluD1 protein coimmunoprecipitated with mGlu5 in the hippocampal membrane fraction, as well as when overexpressed in human embryonic kidney 293 cells, demonstrating that GluD1 and mGlu5 may cooperate in a signaling complex. The interaction of mGlu5 with scaffold protein effector Homer, which regulates mechanistic target of rapamycin (mTOR) signaling, was abnormal both under basal conditions and in response to mGlu1/5 agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) in GluD1 knockout mice. The basal levels of phosphorylated mTOR and protein kinase B, the signaling proteins downstream of mGlu5 activation, were higher in GluD1 knockout mice, and no further increase was induced by DHPG. We also observed higher basal protein translation and an absence of DHPG-induced increase in GluD1 knockout mice. In accordance with a role of mGlu5-mediated mTOR signaling in synaptic plasticity, DHPG-induced internalization of surface α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunits was impaired in the GluD1 knockout mice. These results demonstrate that GluD1 interacts with mGlu5, and loss of GluD1 impairs normal mGlu5 signaling potentially by dysregulating coupling to its effector. These studies identify a novel role of the enigmatic GluD1 subunit in hippocampal function.

Neurobiological and pharmacological validity of curcumin in ameliorating memory performance of senescence-accelerated mice

The senescence-accelerated mouse prone 8 (SAMP8 mice) is known as a neurodegenerative model and may show age-related deficits of cognition. Curcumin, a major active component of spic turmeric, could increase the capacity of learning and memory in the aged rat. However, it is not known whether curcumin could improve cognitive deficits in SAMP8 mice. The present study was undertaken to evaluate the effect of curcumin on the learning and memory of SAMP8 mice and its possible mechanisms. Subjects were randomly divided into four groups: SAMR1 mice, SAMP8 mice and two SAMP8 mice groups treated, intragastrically, with curcumin at the dose of 20 and 50mg/kg per day, respectively. After 25days, spatial memory, superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, p-calcium/calmodulin-dependent kinase II (p-CaMKII) and p-N-methyl-d-aspartate receptor subunit 1 (p-NMDAR1) expression in the hippocampus of mice were examined by using the Morris water maze, biochemical analysis, immunohistochemistry and Western blot. Compared with SAMR1 mice, SAMP8 mice had longer escape latency, higher MDA content, lower SOD activity in the hippocampus, and lower intensity of p-CaMKII in the stratum lucidum of hippocampal CA3 and p-NMDAR1 expression in the hippocampal membrane fraction. Both 20 and 50mg/kg curcumin administration significantly shortened the escape latencies and decreased the hippocampal MDA content in the SAMP8 mice. 50mg/kg curcumin administration significantly ameliorated the hippocampal SOD activity, and increased the intensity of p-CaMKII in the stratum lucidum of hippocampal CA3 and p-NMDAR1 expression in the hippocampal membrane fraction of the SAMP8 mice. The present study demonstrated that curcumin treatment could attenuate cognitive deficits of SAMP8 mice in a dose-dependent manner by decreasing the oxidative stress and improving the expression of p-CaMKII and p-NMDAR1 in the hippocampus. Thus treatment with curcumin may have a potential therapeutic agent for aging-related cognitive dysfunctions.

An electrophoretic approach to screen for glutamine deamidation

Protein deamidation is a posttranslational modification with important implications in physiology and medicine. There is, however, no simple technique for a rapid screening of protein deamidation. The deamidating activity of transglutaminase was applied to establish a simple method for the screen of protein deamidation using recombinant human growth hormone, a rat hippocampal membrane fraction, and a cell homogenate enriched in 5-hydroxytryptamine-1A receptor as model systems. Here we report a simple, economic, and fast approach to assess protein deamidation by two electrophoretic methods: differential cleavage on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) via in situ V8 protease digestion and the principle of spot shifting via blue native (BN)–PAGE/two-dimensional (2D)–SDS–PAGE/immunoblotting.

Activation of NADPH oxidase and extracellular superoxide production in seizure‐induced hippocampal damage

We sought to determine whether the extracellular compartment contributed to seizure-induced superoxide (O2*-) production and to determine the role of the NADPH oxidase complex as a source of this O2*- production. The translocation of NADPH oxidase subunits (p47phox, p67phox and rac1) was assessed by immunoblot analysis and NADPH-driven O2*- production was measured using 2-(4-hydroxybenzyl)-6-(4-hydroxyphenyl)-8-benzyl-3,7-dihydroimidazo [1,2-alpha] pyrazin-3-one-enhanced chemiluminescence. Kainate-induced status epilepticus resulted in a time-dependent translocation of NADPH oxidase subunits (p47phox, p67phox and rac-1) from hippocampal cytosol to membrane fractions. Hippocampal membrane fractions from kainate-injected rats showed increased NADPH-driven and diphenylene iodonium-sensitive O2*- production in comparison to vehicle-treated rats. The time-course of kainate-induced NADPH oxidase activation coincided with microglial activation in the rat hippocampus. Finally, kainate-induced neuronal damage and membrane oxygen consumption were inhibited in mice overexpressing extracellular superoxide dismutase. These results suggest that seizure activity activates the membrane NADPH oxidase complex resulting in increased formation of O2*-.

Localization and regulation of GLUTx1 glucose transporter in the hippocampus of streptozotocin diabetic rats.

We describe the localization of the recently identified glucose transporter GLUTx1 and the regulation of GLUTx1 in the hippocampus of diabetic and control rats. GLUTx1 mRNA and protein exhibit a unique distribution when compared with other glucose transporter isoforms expressed in the rat hippocampus. In particular, GLUTx1 mRNA was detected in hippocampal pyramidal neurons and granule neurons of the dentate gyrus as well as in nonprincipal neurons. With immunohistochemistry, GLUTx1 protein expression is limited to neuronal cell bodies and the most proximal dendrites, unlike GLUT3 expression that is observed throughout the neuropil. Immunoblot analysis of hippocampal membrane fractions revealed that GLUTx1 protein expression is primarily localized to the intracellular compartment and exhibits limited association with the plasma membrane. In streptozotocin diabetic rats compared with vehicle-treated controls, quantitative autoradiography showed increased GLUTx1 mRNA levels in pyramidal neurons and granule neurons; up-regulation of GLUTx1 mRNA also was found in nonprincipal cells, as shown by single-cell emulsion autoradiography. In contrast, diabetic and control rats expressed similar levels of hippocampal GLUTx1 protein. These results indicate that GLUTx1 mRNA and protein have a unique expression pattern in rat hippocampus and suggest that streptozotocin diabetes increases steady-state mRNA levels in the absence of concomitant increases in GLUTx1 protein expression.

Benzodiazepine receptor inverse agonist-induced kindling of rats alters learning and glutamate binding

Kindling, recognized as a model of epilepsy, can be obtained by applications of repeated nonconvulsive stimulations that finally lead to generalized seizures. Epileptics often show cognitive impairments. The present work analyzed the learning performance of male Wistar rats kindled with a convulsant inverse agonist of the GABA A-benzodiazepine receptor complex, methyl β-carboline-3-carboxylate (β-CCM). This compound is also known to have an action on learning processes. It was thus interesting to verify if β-CCM kindling had the same impairing action on learning as other kindling agents, such as pentylenetetrazol (PTZ). A two-way active-avoidance shuttle-box learning task was chosen, because a deficit was found after PTZ kindling in this learning model. On the other hand, hippocampal glutamate binding, has previously been shown to be modified by both seizures and learning. Thus, the level of glutamate binding was also measured in the present study. Results showed that fully kindled rats had poorer learning performance after the third day of test than controls or not fully kindled animals. l-[ 3H] glutamate binding to hippocampal membrane fractions of the fully kindled animals was significantly higher when compared with controls, whereas l-[ 3H] glutamate binding of not fully kindled subjects did not differ from that of controls. Neuronal plasticity changes are a possible explanation for the correlation between kindling, learning deficits, and increased glutamate binding.

Phorbol ester- and glutamate-sensitive phosphorylation of hippocampal membrane proteins from adult and neonatal rats

The phosphoinositide (PI) second messenger system in the neonatal rat brain is differentially stimulated as compared to that of the adult by agonists such as glutamate. Among the factors that might contribute to the neonatal pattern is the nature of phosphorylated membrane-bound proteins which could regulate this receptor-mediated response. This study was undertaken to compare membrane protein phosphorylation under conditions that affect PI hydrolysis in neonatal and adult rat hippocampus. Two-dimensional gel analysis revealed enhanced basal phosphorylation of two membrane proteins ( M r; 46,000 and 80,000; pI: 4.4 and 4.2, respectively in the neonatal hippocampus when compared to the adult. The former phosphoprotein is present only in neonatal hippocampus. Phosphorylation of a 48,000 M r protein with a pI of 4.5 is prominent in hippocampal membranes from both neonatal and adult rats. After incubation of neonatal hippocampal slices with an active phorbol ester, 12- O-tetradecanoyl phorbol-13-acetate (TPA), all three proteins show decreased post-hoc phosphorylation. Slices from neonatal rats incubated with glutamate demonstrated no alteration in the phosphorylation of any of these proteins, while those from adult rats produced a marked change in phosphorylation of the 80,000 M r protein. The data suggest that phosphorylation of this protein from neonates is not yet as efficiently coupled to receptor stimulation as that from the adult. Immunoblot analysis revealed that the 48,000 M r protein is the growth-associated protein B-50/GAP-43 and that the 80,000 M r protein is a membrane-associated form of the MARCKS protein. Immunoblot analysis shows that the MARCKS protein is present in the hippocampal membrane fraction and is expressed at higher levels in the neonate than in the adult. These proteins, which are differentially regulated in neonates, may mediate developmental effects of phorbol esters on PI metabolism.

Hippocampal NCAM180 transiently increases sialylation during the acquisition and consolidation of a passive avoidance response in the adult rat.

Synaptic connectivity change is a consistent anatomical feature of memory formation and the possibility that this is mediated by a replay of neurodevelopmental events has been investigated by measuring change in neural cell adhesion molecule sialylation state during the acquisition and consolidation of a passive avoidance response in the adult rat. The avoidance response was always generated after two to three trials and the animals remained on the platform for the criterion time of 5 min. In all cases training was complete within 5-8 min. Change in sialylation state was monitored following intraventricular infusion of the 3H-ManNAc precursor at 4 hr prior to the reference point. No task-specific change in general glycoconjugate sialylation was apparent in hippocampal P2 pellets at increasing times following training. Increased sialylation state was observed only in neural cell adhesion molecule (NCAM) immunoprecipitates of hippocampal membrane fractions at 12 and 24 hr after training. Change in hippocampal sialylation state could not be attributed to an increased accumulation of NCAM as detected by an immunoabsorbent assay. Immunoblotting of antibody precipitated NCAM demonstrated the 3H-ManNAc to be incorporated into the synapse-specific, 180 kDa isoform of NCAM and a novel 210 kDa isoform. Immunoprecipitation and immunoblotting procedures with an antibody specific for a2-8-polysialic acid showed the 180 and 210 kDa isoforms to be polysialylated. The role of NCAM180 sialylation as a mechanism for synapse selection in information storage is discussed.

Neuropeptide Y Receptor in Vascular Smooth Muscle

125I-Bolton-Hunter (125I-BH) neuropeptide Y (NPY) was used to identify specific high-affinity NPY binding sites in porcine aortic smooth muscle membrane fractions and to characterize the binding sites in comparison with those in porcine hippocampal membrane fractions. Ca2+, but not Mg2+ or Mn2+, enhanced specific 125I-BH-NPY binding in aortic smooth muscle, while all of the cations did in hippocampus. The fast, saturable, and selective binding was to a single population of sites, with a KD of 0.99 +/- 0.11 nM and a Bmax of 0.35 +/- 0.06 fmol/mg protein. GTP and its non-hydrolyzable analogues reduced 125I-BH-NPY binding dose dependently. Neither calcium channel blockers nor noradrenaline had any effect on the binding. Among structurally related peptides, peptide YY displaced 125I-BH-NPY binding as potently as NPY, while human, avian, and rat pancreatic polypeptides displaced 125I-BH-NPY binding 100 times less potently than NPY. The C-terminal fragment NPY(13-36) for inhibiting 125I-BH-NPY binding in aortic smooth muscle was approximately 40 times less potent than in the hippocampus. When we examined the effect of these peptides on cytosolic free Ca2+ ([Ca2+]i) in cultured porcine aortic smooth muscle cells, only the peptides showing high affinity for 125I-BH-NPY binding sites increased [Ca2+]i. These results indicate that the NPY binding sites labeled by 125I-BH-NPY in aortic smooth muscle are functional receptors and have different properties from those in the hippocampus with respect to dependency on divalent cations, sensitivity to GTP analogues, and affinity for NPY(13-36) and pancreatic polypeptides.

Changes in [3H]nitrendipine binding and gamma-aminobutyric acid release in rat hippocampus following repeated morphine administration.

An antagonistic effect of calcium on the action of morphine was studied in rat hippocampal slices. The effect of repeated administration of morphine on gamma-aminobutyric acid (GABA) release and binding of [3H]nitrendipine, a calcium antagonist, was also examined. (1) In rat brain hippocampal slices, morphine enlarged the amplitude of the field potentials evoked in pyramidal neurons, disinhibiting them through basket cells. When the calcium concentration was elevated, potentiation of the field potentials by morphine was reduced. Decrease of the calcium concentration, on the other hand, enhanced the potentiating effect of morphine. Following repeated administration of morphine, its enhancing effect on the field potentials in slices was not observed. (2) In hippocampal membrane fractions obtained from rats repeatedly treated with morphine, enhancement of [3H]nitrendipine binding was observed. (3) In hippocampal slice preparations from rats receiving morphine repeatedly, K+ (45 mM)-stimulated [3H]GABA efflux was enhanced. The above results indicate that morphine antagonizes calcium, thereby reducing the release of transmitters. Furthermore, increase in calcium channels following repeated treatment of rats with morphine may explain the mechanism underlying development of tolerance.

Direct relation of long-term synaptic potentiation to phosphorylation of membrane protein F 1, a substrate for membrane protein kinase C

One hour after long-term potentiation (LTP) in the intact hippocampus, a selective increase in protein F 1 in vitro phosphorylation was observed in homogenate prepared from dorsal hippocampus. Protein F 1 phosphorylation was directly related to the magnitude and persistence of potentiation. No other phosphoprotein studied exhibited a relationship with synaptic enhancement. Low-frequency, non-potentiating stimulation did not increase protein F 1 phosphorylation, and phosphorylation of F 1 was not elevated when high-frequency stimulation did not produce potentiation. We also confirmed our earlier demonstration of a similar pattern of results 5 min after LTP. In related work we have previously observed: (1) that protein F 1 is a substrate for protein kinase C (PKC); (2) that membrane PKC activity was increased by translocation from the cytosol following LTP; and (3) that membrane PKC activity was directly related to the persistence of enhancement. We therefore predicted in the present study that protein F 1 phosphorylation in a dorsal hippocampal membrane fraction would be related to LTP. Hippocampal membrane protein F 1 was found to be directly related to both the magnitude and persistence of response enhancement. Thus the molecular events leading to prolonged potentiation may involve increased PKC/protein F 1 association. Persistence of potentiation may be related to synaptic growth processes involving the growth-associated function of protein F 1.