Phospho-regulation of synaptic and extrasynaptic N-methyl-d-aspartate receptors in adult hippocampal slices.

Recent evidence demonstrates that N-methyl-d-aspartate receptor (NMDAR) trafficking contributes to synaptic plasticity in the hippocampus. Phosphorylation of tyrosine residues, especially NR2B tyrosine 1472, appears to be a mechanism by which NMDAR endocytosis is prevented, suggesting that the tyrosine phosphorylation and surface expression of NMDARs are positively correlated. Previous work from our laboratory and others has confirmed that modulation of tyrosine phosphatase and kinase activity alters the surface expression of NMDARs. However, the changes in NMDAR surface expression described in those studies were in terms of total surface membrane versus intracellular receptors. Within the plasma membrane of glutamatergic synapses, distinct populations of NMDARs exist. Namely, receptors at the surface can be differentiated into synaptic and extrasynaptic pools based on their association with the post-synaptic density (PSD) and availability to glutamate. In the present study, we utilized a subcellular fractionation approach coupled with detergent extraction to prepare synaptic and extrasynaptic NMDARs from adult rat hippocampal slices. Using this method, we examined how tyrosine phosphatase and Src-family tyrosine kinase (SFK) inhibitors modulate the phosphorylation and localization of these different pools of NMDARs. We found that both synaptic and extrasynaptic NMDARs were modulated by tyrosine phosphatase and SFK inhibitors; however subunit- and residue-specific effects were observed. Specifically, phosphorylation of NR2B tyrosine 1472 was associated with enrichment of synaptic NMDARs, whereas phosphorylation of NR2B tyrosine 1336 was associated with enrichment of extrasynaptic NMDARs. Using electrophysiological methods, we also reveal that the biochemical modifications produced by these inhibitors were associated with corresponding changes in NMDAR function.

Isolation and characterization of a regulated form of actin depolymerizing factor.

Actin depolymerizing factor (ADF) is an 18.5-kD protein with pH-dependent reciprocal F-actin binding and severing/depolymerizing activities. We previously showed developing muscle down-regulates ADF (J. R. Bamburg and D. Bray. 1987. J. Cell Biol. 105: 2817-2825). To further study this process, we examined ADF expression in chick myocytes cultured in vitro. Surprisingly, ADF immunoreactivity increases during the first 7-10 d in culture. This increase is due to the presence of a new ADF species with higher relative molecular weight which reacts identically to brain ADF with antisera raised against either brain ADF or recombinant ADF. We have purified both ADF isoforms from myocytes and have shown by peptide mapping and partial sequence analysis that the new isoform is structurally related to ADF. Immunoprecipitation of both isoforms from extracts of cells prelabeled with [32P]orthophosphate showed that the new isoform is radiolabeled, predominantly on a serine residue, and hence is called pADF. pADF can be converted into a form which comigrates with ADF on 1-D and 2-D gels by treatment with alkaline phosphatase. pADF has been quantified in a number of cells and tissues where it is present from approximately 18% to 150% of the amount of unphosphorylated ADF. pADF, unlike ADF, does not bind to G-actin, or affect the rate or extent of actin assembly. Four ubiquitous protein kinases failed to phosphorylate ADF in vitro suggesting that ADF phosphorylation in vivo is catalyzed by a more specific kinase. We conclude that the ability to regulate ADF activity is important to muscle development since myocytes have both pre- and posttranslational mechanisms for regulating ADF activity. The latter mechanism is apparently a general one for cell regulation of ADF activity.

Protein kinase C enhances recombinant bovine alpha 1 beta 1 gamma 2L GABAA receptor whole-cell currents expressed in L929 fibroblasts.

The beta 1 and gamma 2L subunits of the gamma-aminobutyric acid type A receptor (GABAR) contain phosphorylation sites for PKC. To determine the effect of PKC on GABAR function, whole-cell recordings were obtained from mouse fibroblasts expressing recombinant alpha 1 beta 1 gamma 2L receptors, and catalytically active PKC (PKM) was applied via the recording pipette. The first experiment was a population study. Intracellular application of PKM increased GABAR currents, and the enhancement was antagonized by coapplication of the PKC inhibitory peptide. No acceleration or deceleration of GABAR desensitization was observed. The second experiment was a reimpalement study in which paired recordings were made successively from individual cells. Enhancement of GABAR currents by PKM was again obtained. PKM increased GABAR currents at high (> 10 microM) but not at low (< 10 microM) GABA concentrations, resulting in increases in both EC50 and maximal GABAR current. Thus, PKC phosphorylation enhanced recombinant alpha 1 beta 1 gamma 2L GABAR current by increasing maximal current without increasing the affinity of GABA for the GABARs.

LTP leads to rapid surface expression of NMDA but not AMPA receptors in adult rat CA1.

In the CA1 region of the rat hippocampus, long-term potentiation (LTP) requires the activation of NMDA receptors (NMDARs) and leads to an enhancement of AMPA receptor (AMPAR) function. In neonatal hippocampus, this increase in synaptic strength seems to be mediated by delivery of AMPARs to the synapse. Here we studied changes in surface expression of native AMPA and NMDA receptors following induction of LTP in the adult rat brain. In contrast to early postnatal rats, we find that LTP in the adult rat does not alter membrane association of AMPARs. Instead, LTP leads to rapid surface expression of NMDARs in a PKC- and Src-family-dependent manner. The present study suggests a developmental shift in the LTP-dependent trafficking of AMPA receptors. Moreover, our results indicate that insertion of NMDA receptors may be a key step in regulating synaptic plasticity.

G-protein-coupled receptors act via protein kinase C and Src to regulate NMDA receptors.

The N-methyl-D-aspartate (NMDA) receptor contributes to synaptic plasticity in the central nervous system and is both serine-threonine and tyrosine phosphorylated. In CA1 pyramidal neurons of the hippocampus, activators of protein kinase C (PKC) as well as the G-protein-coupled receptor ligands muscarine and lysophosphatidic acid enhanced NMDA-evoked currents. Unexpectedly, this effect was blocked by inhibitors of tyrosine kinases, including a Src required sequence and an antibody selective for Src itself. In neurons from mice lacking c-Src, PKC-dependent upregulation was absent. Thus, G-protein-coupled receptors can regulate NMDA receptor function indirectly through a PKC-dependent activation of the non-receptor tyrosine kinase (Src) signaling cascade.

Analysis of glutamate receptor surface expression in acute hippocampal slices.

Trafficking of receptors to and from the cell surface is a powerful mechanism for regulating neuronal excitability. To date, the majority of studies concerning glutamate receptor trafficking have been performed in neuronal cultures in which surface expression can be readily assayed by immunofluorescence techniques. Results from such studies have had important implications in the field of synaptic plasticity. However, cultured neurons are, by necessity, prepared from very young animals. Moreover, although an enhancement of excitatory neurotransmission can be induced in such systems, classic long-term potentiation (LTP) can be produced only in acute slices or in vivo. To study trafficking in adult tissues, we have adapted two biochemical techniques, proteolysis and cross-linking. These techniques help define surface-expressed and intracellular pools of native receptors in acute hippocampal slices.

Ultrasonography and computed tomography in severe urinary tract infection.

A prospective study evaluated the utility of renal computed tomography (CT) and ultrasonography in 35 patients hospitalized for treatment of urinary tract infection. Renal computed tomograms were abnormal in 18 of 28 patients with acute pyelonephritis and three of four patients with urosepsis, showing findings consistent with pyelonephritis in 17 patients and intrarenal abscess or focal bacterial nephritis in four patients. Renal sonograms were abnormal in only eight patients, showing findings compatible with pyelonephritis in four and intrarenal abscess or focal bacterial nephritis in the other four. Flank tenderness was absent in only four patients with CT findings of pyelonephritis, of whom three were diabetic. We therefore found that (1) renal CT is a sensitive test for acute upper urinary tract infection, (2) ultrasonography detects focal bacterial nephritis and abscesses but is insensitive to uncomplicated upper urinary tract infection, and (3) painless pyelonephritis may be more common in patients with diabetes mellitus.

Significant reductions in synapsin but not synaptophysin specific activity in the brains of some schizophrenics.

The levels of the synaptic vesicle-associated proteins, synapsin and synaptophysin, were examined in human postmortem hippocampus from the brains of schizophrenics and age-matched controls using a quantitative western blot analysis. The schizophrenic samples had significantly lower levels of synapsin I than controls. In individual data, five of the seven schizophrenic samples had extremely low levels of synapsin, whereas two of the schizophrenic samples had normal levels of synapsin. This deficit in synapsin does not appear to be due to some non-specific neuronal loss as the levels of the other synaptic vesicle marker, synaptophysin, were near normal in all seven schizophrenics. Given that synapsin is thought to regulate neurotransmitter release, it is possible that this deficit in synapsin could result in abnormal processing of neuronal information as is seen in various sensory processing abnormalities associated with schizophrenia.

Deficits in the expression of the NR2B subunit in the hippocampus of aged Fisher 344 rats.

The NMDA receptor (NMDAR) has been implicated in the induction of LTP at hippocampal synapses, and has been proposed to play a significant role in the involvement of the hippocampus with learning and memory. Aged rats are known to have deficits in LTP, learning and memory. We tested the hypothesis that aged rats might have deficits in expression of NMDAR subunits. Aged rats have significantly lower levels of NR2B mRNA and protein compared to young animals. This complements a recent report which showed improved learning and memory in mice which overexpress NR2B. No changes were seen in either the mRNA or the protein levels of the NMDAR subunit NR2A, nor in the alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionate receptor (AMPAR) subunit GluR2. Our data support the hypothesis that age related alterations in the expression of the NMDAR NR2B subunit might underlie deficits in LTP and learning and memory in aged animals.

Amelioration of age-related deficits in the stimulation of synapsin phosphorylation.

In a previous report we demonstrated that aged (24-26 month) rats have deficits in long-term potentiation, a form of synaptic enhancement that is dependent on protein phosphorylation (Moore et al., Hippocampus, 3:57-66; 1993). In the present study we demonstrate that aged rats have a deficit in the phosphorylation of the synaptic vesicle associated protein synapsin I. Specifically, aged animals exhibit defective phorbol ester-induced stimulation of synapsin phosphorylation at its calcium/calmodulin dependent protein kinase II sites. We also examined the effects of caloric restriction and antioxidant therapy on this age-related deficit. We found that either life-long caloric restriction or treatment with 16 mg/kg N-tert-butyl-alpha-phenylnitrone (PBN) for 2 weeks can at least partially ameliorate the age-related deficit in the phorbol ester stimulation of synapsin phosphorylation.

Ethanol sensitivity and subunit composition of NMDA receptors in cultured striatal neurons.

Assessment of ethanol (EtOH) sensitivity was combined with analysis of N-methyl-D-aspartate (NMDA) NR1-NR2 subunit composition in primary cultured striatal neurons. Subunit composition was determined by western blot analysis; assessment of ifenprodil and spermine sensitivity during whole-cell patch-clamp recordings. From 3-21 days in culture, NR2B was the only NR2 subunit detected using NR2 subunit specific antibodies; NMDA-induced currents were strongly inhibited by the NR2B-selective antagonist ifenprodil. Two populations of neurons were identified at all ages in culture: those in which NMDA-induced current was or was not potentiated by 100 microM spermine. This suggested that the striatal neurons expressed functional NMDARs which lacked or contained the NR1 N-terminal cassette. The EtOH sensitivity did not differ between these two populations of neurons nor did it change with age in culture at all concentrations of EtOH studied. Human embryonic kidney (HEK) 293 cells containing NR1-1a or NR1-1b with either the NR2A or NR2B subunits did not differ in their EtOH sensitivity. Thus, it would appear that the presence or absence of the N-terminal cassette does not affect the EtOH sensitivity of recombinant NMDARs and native NMDARs expressed in cultured striatal neurons.

Cell surface expression of NR1 splice variants and NR2 subunits is modified by prenatal ethanol exposure.

N-methyl-D-aspartate receptor dysfunction has been strongly suggested to link with the abnormalities seen in fetal alcohol syndrome. Thus, the effects of prenatal ethanol exposure on the total expression of NR1 splice variants and the cell surface expression of both NR1 and NR2 subunits in brain were investigated in rats. Western blot studies of membrane homogenates from cerebral cortices at postnatal days 1 through 21 indicate that prenatal ethanol treatment does not alter total NR1 expression or differential expression of NR1 splice variants during development. However, immunoprecipitation studies using PSD95 suggest that both C2'-terminal variants and NR2A subunits at the cortical postsynaptic membrane of postnatal day 21 were significantly reduced after prenatal ethanol treatment. Moreover, C1-terminal variants were decreased in both pair-fed and ethanol-treated groups, while no significant differences in the levels of total NR1 subunits, NR1 splice variants containing the N- or C2-terminal cassettes, or NR2B subunits were observed. Thus, these results suggest that prenatal exposure to ethanol may influence neuronal function by selective regulation of expression of C2'-terminal variants and NR2A subunits at the cell surface.

Evidence for a role for GABA(A) and NMDA receptors in ethanol inhibition of long-term potentiation.

We have investigated the mechanisms by which acute ethanol inhibits the induction of long-term potentiation (LTP) in area CA1 of the rat hippocampal slice. In a previous report [Alcohol. Clin. Exp. Res. 21 (1997) 404] we demonstrated that ethanol produces only a modest inhibition of pharmacologically isolated N-methyl-D-aspartate receptors (NMDAR) in the CA1 region of the hippocampus. Moreover, this level of inhibition was not sufficient to account for ethanol's complete inhibition of LTP induction in this brain region. One possible explanation of these results is that we may have underestimated ethanol's ultimate effect on the NMDAR by focusing on pharmacologically isolated NMDAR responses. Ethanol might indirectly inhibit the NMDAR by, for example, potentiating the GABA(A)R. To explore this possibility, we first examined the effects of the GABA(A)R antagonist picrotoxin (PTX) and the allosteric GABA(A)R modulator flunitrazepam on NMDAR responses. We demonstrate that these modulators of GABA(A)R activity significantly affect the magnitude of synaptically evoked NMDAR responses. We next examined the effects of ethanol on NMDAR responses in the presence and absence of PTX. We see a significantly greater ethanol inhibition of the NMDAR when GABA(A)Rs are functional, i.e. in the absence of PTX. These data suggest that ethanol produces an inhibition of the NMDAR indirectly by affecting the GABA(A)R neurotransmission. Moreover, we found that ethanol inhibition of NMDAR activity, both directly through actions on the NMDAR, and indirectly, possibly through potentiation of GABA(A)R activity, is sufficient to account for ethanol's complete blockade of LTP induction.

Caloric restriction prevents age-related deficits in LTP and in NMDA receptor expression.

A major focus of aging research has been the search for treatments that will prevent or ameliorate the memory deficits associated with aging. One paradigm, lifelong caloric restriction, has been reported to reduce some of the effects of aging. In the current report, we examined the effects of this treatment on age-related deficits in LTP, a putative cellular building block for memory formation. We report here that lifelong caloric restriction completely prevents the age-related deficit in LTP. In addition, we report that there is a dramatic decrease in the expression of the NMDA receptor subunit NR1 in aged rats and this age-related defect is also prevented by caloric restriction. These data provide a molecular and cellular mechanism by which life long caloric restriction may ameliorate some of the cognitive deficits associated with the aging process.

Regional and subunit specific changes in NMDA receptor mRNA and immunoreactivity in mouse brain following chronic ethanol ingestion.

Chronic ethanol treatment of mice has been shown to result in increased binding of dizocilpine and glutamate to hippocampal NMDA receptors. These changes were suggested to reflect an increase in NMDA receptor number that may underlie certain signs of the ethanol withdrawal syndrome. However, there was no change in binding of a competitive NMDA receptor antagonist, or of ligand binding to the glycine co-agonist site on the receptor after chronic ethanol treatment. Differential changes in the binding of particular ligands at the NMDA receptor suggested the possibility that chronic ethanol ingestion might selectively affect the expression of particular NMDA receptor subunits. Our current work demonstrates that chronic ethanol ingestion by mice, which results in the generation of physical dependence, also produces increases in the NMDA receptor NR1 subunit protein in the hippocampus and cerebellum (approximately 50% and 95%, respectively), and produces increases in the NR2A subunit protein in the hippocampus and cortex (approximately 25% and 40%, respectively). However, the mRNA levels for these subunits were not increased in the respective brain areas by the same ethanol treatment. The changes in NMDA receptor subunit expression in discrete areas of the brain may contribute to the previously observed changes in ligand binding and, possibly, signs of ethanol withdrawal.

ABT-200, a norepinephrine uptake inhibitor, blocks Ca2+ signals in chromaffin cells.

We previously described inhibition by racemic (+/-)-(1'R*,3R*)-3-phenyl-1- [1',2',3',4'-tetrahydro-5',6'-methylenedioxy-1'- napthalenyl-methyl]-pyrrolidine methanesulfonate (ABT-200), and its two constituent enantiomers, SS,ABT-200 and RR,ABT-200, of nicotine-stimulated but not histamine-stimulated catecholamine release from bovine adrenal chromaffin cells. To test the hypothesis that this inhibition reflects a blockade of Ca2+ influx, we used fura-2 loaded chromaffin cells to investigate cytosolic Ca2+ signals. We found that SS,ABT-200 inhibited nicotine- and K(+)-stimulated Ca2+ signals, both of which depend on Ca2+ influx. However, the early phase of the histamine-stimulated Ca2+ signals, which depends on Ca2+ mobilization from intracellular stores, was unaffected. We also examined ion flux through the nicotinic receptor by measuring 86rubidium+ (86Rb+) efflux from preloaded mouse midbrain synaptosomes. We found that SS,ABT-200 partially inhibited nicotine-stimulated 86Rb+ efflux, suggesting that it blocks ion flux through the nicotinic receptor directly. These data support a model in which ABT-200 blocks nicotine-stimulated catecholamine release by inhibiting cation flux through multiple channels.

Protein phosphorylation in isolated human adipocytes-adrenergic control of the phosphorylation of hormone-sensitive lipase.

The effect of adrenergic agents on protein phosphorylation in human adipocytes was examined. Freshly isolated human fat cells were incubated with 32PO4 in order to label intracellular ATP, then treated with a variety of adrenergic and other pharmacologic agents. Treatment with the beta-adrenergic agonist isoproterenol led to a significant increase in phosphate content of at least five protein bands (Mr 52, 53, 63, 67, 84 kDa). The increase in phosphorylation was partially inhibited by the alpha-2 agonist clonidine. Epinephrine, a combined alpha and beta agonist, was less effective at increasing phosphate content of the proteins than was isoproterenol. Neither insulin nor the alpha-1 agonist phenylephrine had any discernible effect on the pattern of protein phosphorylation. The 84 kDa phosphorylated peptide band appears to contain hormone-sensitive lipase, a key enzyme in the lipolytic pathway which is activated by phosphorylation. These results are somewhat different than previously reported results for rat adipocytes, and represent the first report of overall pattern and adrenergic modulation of protein phosphorylation in human adipocytes.

Microwave radiation, in the absence of hyperthermia, has no detectable effect on synapsin I levels or phosphorylation.

Recent reports have indicated that microwave radiation can produce effects on a variety of cell types in vitro. To determine whether microwave radiation might be neurotoxic, the effects of microwave radiation on synapsin I have been examined. Synapsin I is a neuron-specific phosphoprotein that is present in all neurons, where it is localized to the presynaptic terminal and is associated with synaptic vesicles. O'Callaghan and Miller have demonstrated that studies of such neuron-specific proteins can provide reliable indices of neurotoxicity. We have used a radioimmunoassay for synapsin I to determine whether microwave irradiation has any effect on the levels of synapsin I. Neither acute nor chronic exposure to microwave irradiation had any detectable effect on synapsin I levels. We have also examined the calcium-dependent phosphorylation of synapsin I in synaptosomes isolated from rats that had been subjected to microwave radiation. The phosphorylation of synapsin I in synaptosomes reflects numerous components of the presynaptic aspect of neuronal transmission. At intensities below that required to produce mild hyperthermia, no effects of microwave irradiation were seen on synapsin I phosphorylation.

Synapsin I, a phosphoprotein associated with synaptic vesicles: possible role in regulation of neurotransmitter release.

The data presented here provide evidence that the study of neuronal phosphoproteins can lead to the identification of previously unknown proteins and that these proteins may play important roles in neuronal communication. Specifically, in the case of synapsin I, direct evidence has been obtained that this phosphoprotein is involved in regulating neurotransmitter release. A tentative explanation of the results obtained in the micro-injection studies is as follows: synapsin I, in the dephosphostate, is bound to the cytoplasmic surface of synaptic vesicles and inhibits the ability of the vesicle to interact with the plasma membrane; increases in intracellular calcium activate calmodulin kinase II which in turn phosphorylates synapsin I and the phosphorylated synapsin I dissociates from the synaptic vesicle thus removing a constraint on the release of neurotransmitter. Clearly, more studies need to be done to rigorously test this hypothesis. Nevertheless these studies of synapsin I suggest that the study of previously unknown phosphoproteins will lead to the elucidation of previously unknown regulatory processes in neurons.

Comparisons of performance effectiveness among divers.

Using a criterion of performance effectiveness derived from actual dives made under operational conditions, comparisons were made between U.S. Navy divers identified as high and low in performance effectiveness. Comparison measures included intelligence, anxiety, disciplinary problems, and incidence of decompression sickness (DCS). As expected, the most effective divers made more frequent and more hazardous dives than the least effective divers. In addition they had fewer diving accidents and a lower incidence of DCS. While the most effective divers had lower intelligence scores than the least effective group, both groups were substantially above the Navy average. These findings indicate that intelligence appears to be a critical variable in the career retention of divers, as well as the frequency and types of dives to which divers are exposed. The higher incidence of diving accidents and complications, especially DCS, found among the least effective divers may also have been involved in the lower frequency of diving observed among the members of this group.