p11 is up-regulated in the forebrain of stressed rats by glucocorticoid acting via two specific glucocorticoid response elements in the p11 promoter.

Posttraumatic stress disorder (PTSD) is one of the most common psychiatric disorders. Despite the extensive study of the neurobiological correlates of this disorder, the underlying mechanisms of PTSD are still poorly understood. Recently, a study demonstrated that dexamethasone (Dex), a synthetic glucocorticoid, can up-regulate p11, known as S100A10-protein which is down-regulated in patients with depression, (Yao et al., 1999; Huang et al., 2003) a common comorbid disorder in PTSD. These observations led to our hypothesis that traumatic stress may alter expression of p11 mediated through a glucocorticoid receptor. Here, we demonstrate that inescapable tail shock increased both prefrontal cortical p11 mRNA levels and plasma corticosterone levels in rats. We also found that Dex up-regulated p11 expression in SH-SY5Y cells through glucocorticoid response elements (GREs) within the p11 promoter. This response was attenuated by either RU486, a glucocorticoid receptor (GR) antagonist or mutating two of three glucocorticoid response elements (GRE2 and GRE3) in the p11 promoter. Finally, we showed that p11 mRNA levels were increased in postmortem prefrontal cortical tissue (area 46) of patients with PTSD. The data obtained from our work in a rat model of inescapable tail shock, a p11-transfected cell line and postmortem brain tissue from PTSD patients outline a possible mechanism by which p11 is regulated by glucocorticoids elevated by traumatic stress.

Characterization of extracellular accumulation of Zn2+ during ischemia and reperfusion of hippocampus slices in rat.

The mammalian CNS contains an abundance of chelatable zinc that is sequestered in the vesicles of glutamatergic presynaptic terminals and co-released with glutamate. Considerable Zn(2+) is also released during cerebral ischemia and reperfusion (I/R) although the mechanism of this release has not been elucidated. We report here the real time observation of increase of the concentration of extracellular Zn(2+) ([Zn(2+)](o)), accompanied by a rapid increase of intracellular free Zn(2+)concentration, in the areas of dentate gyrus (DG), CA1 and CA3 in acute rat hippocampus slices during ischemia simulated by deprivation of oxygen and glucose (OGD) followed by reperfusion with normal artificial cerebrospinal fluid. A brief period of OGD caused a sustained increase of [Zn(2+)](o). Subsequent reperfusion with oxygenated medium containing glucose resulted in a further increase of [Zn(2+)](o). Longer periods of OGD caused greater increases of [Zn(2+)](o,) and subsequent reperfusion caused still further increases of [Zn(2+)](o,) regardless of OGD duration. The Zn(2+) chelator CaEDTA (10 mM) significantly reduced the increase of [Zn(2+)] induced by OGD and reperfusion. Significant regional differences of [Zn(2+)](o) over the areas of the DG, CA1 and CA3 were not observed during I/R. Neither sodium channel blockade by tetrodotoxin (2 microM), perfusion with nominally calcium-free medium nor anatomical disassociation of the DG, CA1 and CA3 regions from one another by lesioning affected the increase of [Zn(2+)](o). The non-specific nitric oxide synthase (NOS) inhibitor, Nomega-nitro-l-arginine methyl ester (1 mM), however, blocked the increase of [Zn(2+)](o) during ischemia and reperfusion. The data indicate the important role of NO in causing the release of Zn(2+) during I/R and suggest that NOS inhibitors may be used to reduce Zn(2+)-induced neuronal injury.

Serotonin type II receptor activation facilitates synaptic plasticity via N-methyl-D-aspartate-mediated mechanism in the rat basolateral amygdala.

The modulation of synaptic plasticity by serotonin type II (5-hydroxytryptamine type II (5-HT(2)))-receptor stimulation was explored using intracellular, field potential and Fura-2 fluorescence image recordings in a rat amygdala slice preparation. Bath application of 5HT(2) receptor agonist 1-(2,5)-dimethoxy-4-iodophen-2-aminopropane (DOI) transformed theta-burst-stimulated (TBS) synaptic plasticity from short-term potentiation to long-term potentiation. DOI enhanced N-methyl-D-aspartate (NMDA) receptor-mediated potentials and calcium influx without affecting the resting membrane potential or input resistance of the neurons. In contrast, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainate receptor-mediated excitatory synaptic responses were unaffected by DOI. The facilitating effects of DOI were blocked by the 5-HT(2) receptor antagonist, ketanserin, and by the 5-HT(2C)-receptor selective antagonist, RS102221. These results indicate that 5-HT(2)-receptor activation enhances NMDA receptor-mediated synaptic function in the basolateral amygdala (BLA).

Rapid translocation of Zn(2+) from presynaptic terminals into postsynaptic hippocampal neurons after physiological stimulation.

Zn(2+) is found in glutamatergic nerve terminals throughout the mammalian forebrain and has diverse extracellular and intracellular actions. The anatomical location and possible synaptic signaling role for this cation have led to the hypothesis that Zn(2+) is released from presynaptic boutons, traverses the synaptic cleft, and enters postsynaptic neurons. However, these events have not been directly observed or characterized. Here we show, using microfluorescence imaging in rat hippocampal slices, that brief trains of electrical stimulation of mossy fibers caused immediate release of Zn(2+) from synaptic terminals into the extracellular microenvironment. Release was induced across a broad range of stimulus intensities and frequencies, including those likely to induce long-term potentiation. The amount of Zn(2+) release was dependent on stimulation frequency (1-200 Hz) and intensity. Release of Zn(2+) required sodium-dependent action potentials and was dependent on extracellular Ca(2+). Once released, Zn(2+) crosses the synaptic cleft and enters postsynaptic neurons, producing increases in intracellular Zn(2+) concentration. These results indicate that, like a neurotransmitter, Zn(2+) is stored in synaptic vesicles and is released into the synaptic cleft. However, unlike conventional transmitters, it also enters postsynaptic neurons, where it may have manifold physiological functions as an intracellular second messenger.

Induction of mossy fiber --> Ca3 long-term potentiation requires translocation of synaptically released Zn2+.

The mammalian CNS contains an abundance of chelatable Zn(2+) sequestered in the vesicles of glutamatergic terminals. These vesicles are particularly numerous in hippocampal mossy fiber synapses of the hilar and CA3 regions. Our recent observation of frequency-dependent Zn(2+) release from mossy fiber synaptic terminals and subsequent entry into postsynaptic neurons has prompted us to investigate the role of synaptically released Zn(2+) in the induction of long-term potentiation (LTP) in field CA3 of the hippocampus. The rapid removal of synaptically released Zn(2+) with the membrane-impermeable Zn(2+) chelator CaEDTA (10 mm) blocked induction of NMDA receptor-independent mossy fiber LTP by high-frequency electrical stimulation (HFS) in rat hippocampal slices. Mimicking Zn(2+) release by bath application of Zn(2+) (50-100 microm) without HFS induced a long-lasting potentiation of synaptic transmission that lasted more than 3 hr. Moreover, our experiments indicate the effects of Zn(2+) were not attributable to its interaction with extracellular membrane proteins but required its entry into presynaptic or postsynaptic neurons. Co-released glutamate is also essential for induction of LTP under physiological conditions, in part because it allows Zn(2+) entry into postsynaptic neurons. These results indicate that synaptically released Zn(2+), acting as a second messenger, is necessary for the induction of LTP at mossy fiber-->CA3 synapses of hippocampus.

Inhibition of excessive neuronal apoptosis by the calcium antagonist amlodipine and antioxidants in cerebellar granule cells.

Neuronal cell death as a result of apoptosis is associated with cerebrovascular stroke and various neurodegenerative disorders. Pharmacological agents that maintain normal intracellular Ca2+ levels and inhibit cellular oxidative stress may be effective in blocking abnormal neuronal apoptosis. In this study, a spontaneous (also referred to as age-induced) model of apoptosis consisting of rat cerebellar granule cells was used to evaluate the antiapoptotic activities of voltage-sensitive Ca2+ channel blockers and various antioxidants. The results of these experiments demonstrated that the charged, dihydropyridine Ca2+ channel blocker amlodipine had very potent neuroprotective activity in this system, compared with antioxidants and neutral Ca2+ channel blockers (nifedipine and nimodipine). Within its effective pharmacological range (10-100 nM), amlodipine attenuated intracellular neuronal Ca2+ increases elicited by KCl depolarization but did not affect Ca2+ changes triggered by N-methyl-D-aspartate receptor activation. Amlodipine also inhibited free radical-induced damage to lipid constituents of the membrane in a dose-dependent manner, independent of Ca2+ channel modulation. In parallel experiments, spontaneous neuronal apoptosis was inhibited in dose- and time-dependent manners by antioxidants (U-78439G, alpha-tocopherol, and melatonin), nitric oxide synthase inhibitors (N-nitro-L-arginine and N-nitro-D-arginine), and a nitric oxide chelator (hemoglobin) in the micromolar range. These results suggest that spontaneous neuronal apoptosis is associated with excessive Ca2+ influx, leading to further intracellular Ca2+ increases and the generation of reactive oxygen species. Agents such as amlodipine that block voltage-sensitive Ca2+ channels and inhibit cellular oxidative stress may be effective in the treatment of cerebrovascular stroke and neurodegenerative diseases associated with excessive apoptosis.

Chronic lithium treatment robustly protects neurons in the central nervous system against excitotoxicity by inhibiting N-methyl-D-aspartate receptor-mediated calcium influx.

Lithium is the most commonly used drug for the treatment of manic depressive illness. The precise mechanisms underlying its clinical efficacy remain unknown. We found that long-term exposure to lithium chloride dramatically protects cultured rat cerebellar, cerebral cortical, and hippocampal neurons against glutamate-induced excitotoxicity, which involves apoptosis mediated by N-methyl-D-aspartate (NMDA) receptors. This neuroprotection is long-lasting, occurs at therapeutically relevant concentrations of lithium with an EC50 of approximately 1.3 mM, and requires treatment for 6-7 days for complete protection to occur. In contrast, a 24-h treatment with lithium is ineffective. The protection in cerebellar neurons is specific for glutamate-induced excitotoxicity and can be attributed to inhibition of NMDA receptor-mediated calcium influx measured by 45Ca2+ uptake studies and fura-2 fluorescence microphotometry. The long-term effects of lithium are not caused by down-regulation of NMDA receptor subunit proteins and are unlikely related to its known ability to block inositol monophosphatase activity. Our results suggest that modulation of glutamate receptor hyperactivity represents at least part of the molecular mechanisms by which lithium alters brain function and exerts its clinical efficacy in the treatment for manic depressive illness. These actions of lithium also suggest that abnormality of glutamatergic neurotransmission as a pathogenic mechanism underlying bipolar illness warrants future investigation.

Antagonists have a greater selectivity for muscarinic receptor subtypes in intact cerebellar granule cells than in membranes.

A comparison of muscarinic acetylcholine receptor (mAChR) antagonist binding properties was made between intact cerebellar granule cell cultures and membranes prepared from these cells. [3H]quinuclidinyl benzylate (QNB) binding displacement by four mAChR antagonists was measured and the selectivities for m2- or m3-mAChRs estimated by curve fitting. For each antagonist, the preparation of membranes caused a subtype selective decrease in receptor affinity, as compared to intact cell binding. The m2-selective antagonists had lower affinities in membranes for m2- but not for m3-mAChR, while the m3-selective antagonists had lower affinities for m3- but not for m2-mAChR. As a result, the m2-mAChR selectivity of AF-DX 116 and methoctramine in membranes was 66- and 1.7-fold less than in intact cells, and the m3-mAChR selectivity of 4-DAMP and pFHHSiD was 2.4- and 3.9-fold less in membranes than in intact cells. The m3-mAChR selectivity of 4-DAMP in intact cells was unaffected by cytoskeletal depolymerization with cytochalasins and colchicine. We suggest that the changes in selectivity seen with cell disruption may be due to a loss of cellular factors which regulate receptor properties. Antagonists binding to receptors on intact cells may cause subtype-specific changes in the interaction of the mAChR with these factors. These data suggest that mAChR antagonist binding selectivity needs to be re-examined in intact cell systems.

Carbamazepine inhibition of N-methyl-D-aspartate-evoked calcium influx in rat cerebellar granule cells.

The effect of carbamazepine (CBZ) on N-methyl-D-aspartate (NMDA)-stimulated CA++ influx in rat cerebellar granule cells was studied by use of fura-2 microfluorometry. CBZ inhibited the rise in intracellular free Ca++ concentration ([Ca++]i) induced by NMDA and glycine in a rapid reversible and concentration-dependent manner. CBZ's inhibition of the [Ca++]i increase was noncompetitive with respect to NMDA, glycine and the facilitatory neurosteroid pregnenolone sulfate. The degree of inhibition of the NMDA response produced by CBZ increased with increasing concentrations of extracellular KCl. Excluding non-NMDA receptor-mediated contributions to Ca++ influx, depolarization by 50 mM KCl resulted in a 20-fold decrease (from 723 to 33 microM) in the IC50 for CBZ blockade of the NMDA response. Thus, significant blockade of NMDA receptor responses in cerebellar granule cells can occur at concentrations of CBZ within the therapeutic range under conditions believed to accompany seizures. Moreover, the common toxic side effects of CBZ, which include signs of cerebellar dysfunction, may occur as a result of CBZ blockade of the NMDA receptors of cerebellar granule cells.

Norepinephrine induces expression of c-fos mRNA through the alpha-adrenoceptor in rat aortic rings.

We examined whether norepinephrine at pharmacologically relevant doses induces increased expression of c-fos mRNA in rat aortic rings. c-fos mRNA was expressed at norepinephrine concentrations known to cause minimum and maximum contraction of rat aorta in vitro. At the concentration known to cause maximum contraction, norepinephrine produced a marked and sustained increase of c-fos mRNA expression. Induction of c-fos was blocked completely by the alpha 1-adrenergic antagonist prazosin, partially by the alpha 2-adrenergic antagonist yohimbine, and not at all by the beta-adrenergic antagonist propranolol. A prazosin inhibition curve showed that 1 nmol/L prazosin inhibited 10 micromol/L norepinephrine induced c-fos expression by 40%. At the pharmacologic dose known to cause maximum contraction, norepinephrine induces c-fos mRNA expression through the alpha-adrenoceptor in rat aortic rings.

Fluoxetine modulates G protein alpha s, alpha q, and alpha 12 subunit mRNA expression in rat brain.

Signal-transducing G proteins are central to the coordination of receptor-effector communication. We have explored the effects of long-term fluoxetine administration of G alpha s, G alpha i1, G alpha i2, G alpha o, G alpha q and G alpha 12 mRNA expression in various rat brain regions using reverse transcriptase-polymerase chain reaction (RT-PCR)-mediated cross-species partial cDNA cloning. Northern blot analysis, and RNase protection assay techniques. Fluoxetine decreased G alpha s mRNA in midbrain, while mRNA expression of the novel G protein alpha subunits, G alpha q and G alpha 12, was increased in neostriatum and frontal cortex. We conclude that in addition to post-translational modification, regulation of G protein function by antidepressant drugs may occur at the level of gene expression.

Beta-endorphin's modulation of lymphocyte proliferation is dose, donor, and time dependent.

We find that beta-endorphin (Bend) can have, positive, negative, or neutral dose-dependent effects on the mitogen-stimulated proliferation of human peripheral blood lymphocytes. The distribution of positive, negative, or neutral responses was nonrandom. In studies carried out over a year, we show that an individual's mitogen-stimulated lymphocyte proliferative response to Bend can change with time. We show that the inhibition induced by cortisol can be, in part, relieved by Bend. On the basis of our results and those of others in the field, we put forward a model that can qualitatively account for many of the observations we and other investigators have made.

Beta-endorphin modulates T-cell intracellular calcium flux and c-myc expression via a potassium channel.

To characterize the effect of beta-endorphin on T-lymphocyte activation, we examined its influence on membrane currents, intracellular calcium flux, and c-myc mRNA levels during mitogenic stimulation of Jurkat cells. While beta-endorphin weakly enhanced voltage-activated K+ currents of Jurkat cells by itself, it suppressed these currents in the presence of mitogen. Naloxone, by itself, also enhanced K+ current amplitude, but in the presence of mitogen partially reversed the suppressive effect of beta-endorphin. A 5-30 min exposure to beta-endorphin resulted in an increase in the rate of mitogen-stimulated intracellular calcium release and an increase in c-myc mRNA levels relative to controls. Longer exposure (1-2 h) to beta-endorphin retarded intracellular calcium release, and suppressed c-myc expression. The suppressive effects were reversed by naloxone and mimicked by the K+ channel blocker, tetraethylammonium ion. These data suggest that opiate receptors and K+ channels of Jurkat cells are functionally coupled in a way that modulates intracellular calcium release and c-myc expression - two key processes in T-cell mitogenesis.

Association of poly(adenosine diphosphate ribosylated) nucleosomes with transcriptionally active and inactive regions of chromatin.

We have investigated whether transcriptionally active or inactive gene sequences are associated in vivo with poly(adenosine diphosphate ribosylated) regions of chromatin. Soluble HeLa cell chromatin derived from nuclei treated either briefly or extensively with micrococcal nuclease was fractionated on an anti-poly(adenosine diphosphate ribose)-Sepharose column [Malik, N., Miwa, M., Sugimura, T., Thraves, P., & Smulson, M. E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2554-2558] to obtain fractions that were enriched or depleted in poly(ADP-ribosylated) chromatin. DNA obtained from these fractions was then probed for active and inactive gene sequences with a cDNA probe made from total cell mRNA and a probe for the beta-globin gene. Chromatin enriched in poly(ADP-ribosylated) nucleosomes contained both active and inactive gene sequences as detected by the probes and appeared to be more nuclease sensitive than that found in the fraction of chromatin depleted of poly(ADP-Rib). Poly(ADP-ribosylated) chromatin from nuclei digested briefly with nuclease showed an enrichment in both active and inactive genes while that treated extensively with nuclease showed either no enrichment or a depletion of active and inactive genes. Actively transcribed chromatin was digested at a rate several times that of the bulk or inactive chromatin. Nevertheless, the enrichment of active genes in poly(ADP-ribosylated) nucleosomes derived from brief nuclease digestion was greater than that of inactive genes. These results are interpreted as showing that some, but not all, of actively transcribed chromatin contains associated poly(ADP-ribosylated) proteins. However, since poly(ADP-ribosylated) proteins are also associated with inactive genes, the function of this modification cannot be assigned solely to transcription.

Characterization of the size and 5' cap of messenger RNA encoding neurophysin precursors.

The coding activity of bovine hypothalamic poly A+ mRNA for neurophysin I and II immunoreactive proteins was characterized with respect to size and 5' cap. The mRNA was fractionated by methylmercuric hydroxide agarose gel electrophoresis and subsequently translated in vitro in rabbit reticulocyte lysates. Alternatively, mRNA was fractionated by gel exclusion HPLC and translated in wheat germ extracts. Immunoprecipitated translation products were analyzed by gel exclusion HPLC. Neurophysin-immunospecific protein of approximately 17,000 daltons, the size expected for the neuropeptide hormone-neurophysin precursors, was encoded by mRNA species of two size classes. The smaller class of mRNA's was of the size expected from the size of the precursor proteins. The larger class was 5-10 times larger. The low K+ concentration optimum for translation of unfractionated mRNA encoding neurophysin I immunoreactive proteins and the inability of a cap analogue to inhibit this translation suggest that mRNA species encoding neurophysin I-immunoreactive translation products are incompletely capped. By contrast, the mRNA encoding neurophysin II-immunoreactive products appear to contain a normal cap structure.