Fronto-parietal alpha ERD and visuo-spatial attention in pregnant women.

Many pregnant women report impairments in their attentional capacities. However, comparative studies between pregnant and non-pregnant women using standardised attention paradigms are rare and inconsistent. During attention tasks alpha activity is known to suppress irrelevant sensory inputs and previous studies show that a large event-related desynchronisation (ERD) in the alpha range prior to target-onset predicts enhanced attentional processing. We quantified the relationship between performance (accuracy, response time) in a standardised visuo-spatial attention task and alpha ERD (∼6-12 Hz) as well as saliva estradiol level in fifteen pregnant women (M = 26.6, SD = 3.0 years) compared to fifteen non-pregnant, naturally cycling women (M = 23.1, SD = 4.3 years). Compared to non-pregnant women, alpha frequency was increased in pregnant women. Furthermore, pregnant women showed a greater magnitude of the alpha ERD prior to target-onset and a moderate increase in accuracy compared to non-pregnant women. In addition, accuracy correlated negatively with estradiol in pregnant women as well as with frontal alpha ERD in all women. These correlational findings indicate that pregnancy-related enhancement in alpha desynchronisation in a fronto-parietal network might modulate accuracy during a visuo-spatial attention task.

Associations of endogenous 17-ß-estradiol with theta amplitude and performance in semantic categorization in young women.

In semantic categorization processes, individuals form a relation between perceived or imagined objects and knowledge about these objects. In the present semantic categorization study, we correlated endogenous 17-ß-estradiol levels (E2) with performance as well as amplitude of theta oscillations in young women (age 23.1±3.4 years). The semantic categorization task consisted of nouns representing either living or non-living items. Each item was characterized either by many or by few features. We identified parameters associated or not associated with menstrual cycle phases. Irrespective of the menstrual cycle phase, women (1) responded faster to living items as well as to nouns characterized by many features compared to non-living items and items characterized by few features, (2) showed higher accuracy to non-living items and items having many features, and (3) showed negative correlation between response time (RT) and theta amplitude. RT, accuracy and post-stimulus theta amplitude were not statistically significantly different among early follicular, late follicular or luteal women. In early follicular but not in late follicular or luteal women, we observed (1) a positive correlation between E2 and latency in RT, (2) a negative correlation between E2 and accuracy, and (3) a negative correlation between E2 and post-stimulus theta amplitude. A mosaic of menstrual cycle phase-dependent and -independent associations may indicate that a similar performance in each menstrual cycle phase is related to different modulation of synaptic activity by hormones.

Nitric oxide up-regulates ferritin mRNA level in snail neurons.

We cloned and sequenced the ferric ion-binding protein, ferritin, from the nervous system of the pulmonate snail, Helix pomatia. Helix H-ferritin cDNA contains a 519-bp open reading frame (ORF) and predicts an iron-responsive element (IRE) at the 5'-untranslated region (5'-UTR) of the ferritin mRNA. The deduced amino acid sequence revealed 86% similarity with Lymnaea stagnalis ferritin and about 70% similarity with vertebrate H-ferritin. While secreted ferritin isoforms contain a signalling sequence at their N-terminal end, Helix ferritin does not contain this sorting signal indicating that it is restricted to the cytoplasm. The amino acid ligands at positions Glu25, Tyr30, Glu59, Glu60, His63, Glu105 and Gln139 indicate an active ferroxidase site in Helix ferritin. In situ hybridization visualized ferritin mRNA in neuronal cell bodies but not in the neuropil. In contrast, ferritin-immunoreactive protein was localized in cell bodies and neurites. We further demonstrate that the NO donors S-nitroso-N-acetylpenicillamine (SNAP), or hydroxylamine (HA), increase the intracellular ferritin mRNA level by about 55%. In conclusion, our findings show that Helix neurons express an intracellular H-ferritin isoform and suggest that iron and NO metabolism are coupled.

Nitric oxide increases excitability by depressing a calcium activated potassium current in snail neurons.

In gastropods, the interneuronal messenger, nitric oxide (NO), modulates spike frequency and synaptic transmission. We have characterized the effect of NO on ion currents underlying neuronal excitability, using current-clamp and two-electrode voltage-clamp techniques. Identified neurons of the pulmonate snail, Helix pomatia, respond to the NO donor sodium nitroprusside (SNP) by increasing the firing frequency and decreasing the latency. Voltage-clamp experiments revealed that SNP or S-nitro-N-acetylpenicillamine (SNAP) depressed the macroscopic outward current, while the control compound N-acetylpenicillamine (NAP) had no effect. Current voltage curves generated from voltage steps to different membrane potentials ranging from -40 to +180 mV showed an N-shaped outward current. Superfusion of ganglia with Ca(2+) free Helix solution abolished the N-shape, indicating the contribution of a Ca(2+) activated K(+) current (I(K,Ca)). Exposure of neurons to SNP or SNAP diminished the N-shape, indicating that NO affects I(K,Ca). The depressing effect of SNP on the outward current was slow and reached steady state in about 5 min. In conclusion, our findings indicate that NO enhances excitability in Helix nervous system by decreasing I(K,Ca).

NADPH-diaphorase activity and nitric oxide synthase activity in the kidney of the clawed frog, Xenopus laevis.

Nitric oxide (NO) may play a central role in controlling renal hemodynamics and renal salt excretion. Thus, several investigations focused on localization and function of nitric oxide synthase (NOS) isoforms in the mammalian kidney. Although studies of amphibians have contributed significantly to the elucidation of renal physiology, NOS has not been investigated in the amphibian kidney. Therefore, we characterized NOS and reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase biochemically and, furthermore, visualized putative NO-producing cells in the kidney of the clawed frog, Xenopus laevis. Our results indicate that NADPH-diaphorase activity correlates with NOS activity. Both enzyme activities eluted at 225 mM NaCl on a diethylaminoethanol anion exchange column and had an apparent molecular weight of 235 kDa, as estimated on an S-300 Sephacryl column. In addition, these enzymes were sensitive to Ca2+ and NADPH, but insensitive to calmodulin antagonists (trifluoperazine, W-13) or omission of calmodulin from the reaction medium. The molecular identity of NOS in Xenopus kidney extract was estimated using polymerase chain reaction. Primers to Xenopus neuronal NOS hybridized to a transcript in Xenopus kidney homogenate. NADPH-diaphorase histochemistry revealed staining in the neck segment, distal tubules, collecting segment, and peritoneal funnels. NOS-immunoreactive material was visualized in distal tubules. These results indicate that Xenopus kidney contains at least neuronal NOS, but may contain an additional NOS isoform, which is less calmodulin sensitive.

Vanadate induces calcium signaling, Ca2+ release-activated Ca2+ channel activation, and gene expression in T lymphocytes and RBL-2H3 mast cells via thiol oxidation.

Using ratiometric Ca2+ imaging and patch-clamp measurement of Ca2+ channel activity, we investigated Ca2+ signaling induced by vanadium compounds in Jurkat T lymphocytes and rat basophilic leukemia cells. In the presence of external Ca2+, vanadium compounds produced sustained or oscillatory Ca2+ elevations; in nominally Ca2+-free medium, a transient Ca2+ rise was generated. Vanadate-induced Ca2+ signaling was blocked by heparin, a competitive inhibitor of the 1,4, 5-inositol trisphosphate (IP3) receptor, suggesting that Ca2+ influx is secondary to depletion of IP3-sensitive Ca2+ stores. In Jurkat T cells, vanadate also activated the Ca2+-dependent transcription factor, NF-AT. Intracellular dialysis with vanadate activated Ca2+ influx through Ca2+ release-activated Ca2+ (CRAC) channels with kinetics comparable to those of dialysis with IP3. Neither phosphatase inhibitors nor nonhydrolyzable nucleotide analogues modified CRAC channel activation. The action of vanadate, but not IP3, was prevented by the thiol-reducing agent DTT. In addition, the activation of CRAC channels by vanadate was mimicked by the thiol-oxidizing agent chloramine T. These results suggest that vanadate enhances Ca2+ signaling via thiol oxidation of a proximal element in the signal transduction cascade.

Single-channel recording of a store-operated Ca2+ channel in Jurkat T lymphocytes.

In T lymphocytes, a store-operated calcium ion (Ca2+) entry mechanism termed the calcium release-activated Ca2+ channel (CRAC channel) underlies the sustained or oscillatory intracellular calcium concentration signal required for interleukin-2 gene expression and cell proliferation. The use of sodium ions as a current carrier enabled single-channel recordings of CRAC channels during activation, inactivation, and blockade of current in the presence of divalent cations. A large conductance of 36 to 40 picosiemens indicates that 100 to 400 CRAC channels are present in T lymphocytes.

A nongenomic mechanism for progesterone-mediated immunosuppression: inhibition of K+ channels, Ca2+ signaling, and gene expression in T lymphocytes.

The mechanism by which progesterone causes localized suppression of the immune response during pregnancy has remained elusive. Using human T lymphocytes and T cell lines, we show that progesterone, at concentrations found in the placenta, rapidly and reversibly blocks voltage-gated and calcium-activated K+ channels (KV and KCa, respectively), resulting in depolarization of the membrane potential. As a result, Ca2+ signaling and nuclear factor of activated T cells (NF-AT)-driven gene expression are inhibited. Progesterone acts distally to the initial steps of T cell receptor (TCR)-mediated signal transduction, since it blocks sustained Ca2+ signals after thapsigargin stimulation, as well as oscillatory Ca2+ signals, but not the Ca2+ transient after TCR stimulation. K+ channel blockade by progesterone is specific; other steroid hormones had little or no effect, although the progesterone antagonist RU 486 also blocked KV and KCa channels. Progesterone effectively blocked a broad spectrum of K+ channels, reducing both Kv1.3 and charybdotoxin-resistant components of KV current and KCa current in T cells, as well as blocking several cloned KV channels expressed in cell lines. Progesterone had little or no effect on a cloned voltage-gated Na+ channel, an inward rectifier K+ channel, or on lymphocyte Ca2+ and Cl- channels. We propose that direct inhibition of K+ channels in T cells by progesterone contributes to progesterone-induced immunosuppression.

Monovalent permeability, rectification, and ionic block of store-operated calcium channels in Jurkat T lymphocytes.

We used whole-cell recording to characterize ion permeation, rectification, and block of monovalent current through calcium release-activated calcium (CRAC) channels in Jurkat T lymphocytes. Under physiological conditions, CRAC channels exhibit a high degree of selectivity for Ca2+, but can be induced to carry a slowly declining Na+ current when external divalent ions are reduced to micromolar levels. Using a series of organic cations as probes of varying size, we measured reversal potentials and calculated permeability ratios relative to Na+, PX/PNa, in order to estimate the diameter of the conducting pore. Ammonium (NH4+) exhibited the highest relative permeability (PNH4/PNa = 1.37). The largest permeant ion, tetramethylammonium with a diameter of 0.55 nm, had PTMA/PNa of 0.09. N-methyl-D-glucamine (0.50 x 0.64 x 1.20 nm) was not measurably permeant. In addition to carrying monovalent current, NH4+ reduced the slow decline of monovalent current ("inactivation") upon lowering [Ca2+]o. This kinetic effect of extracellular NH4+ can be accounted for by an increase in intracellular pH (pHi), since raising intracellular pH above 8 reduced the extent of inactivation. In addition, decreasing pHi reduced monovalent and divalent current amplitudes through CRAC channels with a pKa of 6.8. In several channel types, Mg2+ has been shown to produce rectification by a voltage-dependent block mechanism. Mg2+ removal from the pipette solution permitted large outward monovalent currents to flow through CRAC channels while also increasing the channel's relative Cs+ conductance and eliminating the inactivation of monovalent current. Boltzmann fits indicate that intracellular Mg2+ contributes to inward rectification by blocking in a voltage-dependent manner, with a z delta product of 1.88. Ca2+ block from the outside was also found to be voltage dependent with z delta of 1.62. These experiments indicate that the CRAC channel, like voltage-gated Ca2+ channels, achieves selectivity for Ca2+ by selective binding in a large pore with current-voltage characteristics shaped by internal Mg2+.

Nitric oxide-mediated cGMP synthesis in Helix neural ganglia.

The central nervous system of the mollusc Helix pomatia was stimulated with NO donors sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP) or hydroxylamine, in the presence of a phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine (IBMX). Radioimmunoassay revealed that all of the three NO donors significantly increased cGMP levels by 22-27-fold above basal levels. Compared with controls, strong cGMP immunoreactivity was observed in axons and cytoplasm of the stimulated neurons. About 80% of cGMP-immunoreactive neurons colocalized with NADPH-diaphorase activity. Some glial cells and giant neurons were not stained by NADPH-diaphorase histochemistry but were cGMP-immunoreactive. The results suggest the existence of a NO/cGMP pathway and indicate NO as an intra- and intercellular signaling molecule in the Helix central nervous system.

Biochemical characterization and histochemical localization of nitric oxide synthase in the nervous system of the snail, Helix pomatia.

Nitric oxide synthase (NOS) in the snail Helix pomatia was characterized by biochemical and molecular biological techniques and localized by histochemical methods. Central ganglia contained particulate paraformaldehyde-sensitive and cytosolic paraformaldehyde-insensitive NADPH-diaphorase. The cytosolic NADPH-diaphorase activity coeluted with NOS activity. The activity of NOS was dependent on Ca2+ and NADPH and was inhibited by N(G)-nitro-L-arginine (L-NNA). Proteins purified by 2',5'-ADP affinity chromatography were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and migrated at 150, 60, 40, and 30 kDa. An antibody to mammalian NOS exclusively labeled the 60-kDa protein. Characterization of the cDNA of the corresponding 60-kDa NOS-immunoreactive protein revealed no sequence homology with any known NOS isoform. The recombinant protein exhibited Ca2+- and NADPH-dependent NOS activity, which was partially inhibited by EGTA and L-NNA. Histochemistry showed NADPH-diaphorase activity in discrete regions of the central and peripheral nervous system. About 60% of the NADPH-diaphorase-positive neurons colocalize with immunoreactive material detected by antibodies to mammalian NOS. Comparison of organs showed the highest NADPH-diaphorase activity in the nervous system, whereas moderate activity was present in muscle tissue, digestive tract, and gonads. Our study suggests the presence of NOS and a putative NOS-associated/regulating protein in mollusk nervous tissue.

Ion channels, Ca2+ signaling, and reporter gene expression in antigen-specific mouse T cells.

Whole cell recordings were performed in parallel with measurements of intracellular Ca2+ ([Ca2+]i) and gene expression using the murine T cell hybridoma, B3Z, a cell line stably infected with a lacZ reporter gene, driven by the minimal IL-2 promoter (NF-AT, nuclear factor of activated T cells). The physiologic roles of ion channels in B3Z cells were investigated by correlating the pharmacology of channel block with [Ca2+]i, and expression of lacZ. In B3Z cells and activated human T cells, the major component of voltage-gated K+ (K(V)) current had biophysical and pharmacologic properties associated with type n channels encoded by Kv1.3; a minor K(V) component was charybdotoxin (CTX) resistant. Ca2+-activated K+ (K(Ca)) current was sensitive to CTX, but not to margatoxin (MgTX). Inwardly rectifying K+ (K(IR)) current was blocked completely by 200 microM of Ba2+. Outwardly rectifying Cl- currents were induced by cell swelling. An inwardly rectifying Ca2+ current (I(CRAC)) was activated by dialyzing the cell with 10 mM EGTA and 10 microM IP3. CTX reduced thapsigargin-stimulated [Ca2+]i signaling and gene expression by approximately 25%. Although the thapsigargin-stimulated [Ca2+]i signal was resistant to complete inhibition by K+ channel blockers, it was very sensitive to the K+ diffusion potential and Cl- removal, suggesting that drug-resistant K+ channels and perhaps Cl- channels can maintain a sufficiently negative membrane potential to drive Ca2+ influx. Neither [Ca2+]i signaling nor gene expression induced by stimulation of the CD3-epsilon subunit of the TCR was inhibited by ion channel blockers used in this study. We conclude that several channel types can contribute to maintenance of Vm, Ca2+ signals, and gene expression.

Ethanol suppresses neuronal Ca2+ currents by effects on intracellular signal transduction.

The mechanism of action of ethanol on voltage-activated Ca2+ currents in neurons of the mollusk, Helix pomatia, was studied focusing on intracellular signaling. Ethanol suppressed inward Ca2+ currents in a time- and voltage-dependent manner. Buffering of intracellular Ca2+ with bis(o-aminophenoxy)ethane-N,N,N',N-tetraacetic acid (BAPTA) abolished the ethanol effects on Ca2+ currents. Intracellular GTP-gamma-S injection decreased Ca2+ currents whereas GDP-beta-S injection was ineffective. Ethanol had no further blocking effect on Ca2+ currents in GTP-gamma-S injected cells. In the presence of dopamine, which is known to suppress Ca2+ currents by G0-protein activation, ethanol application was ineffective. The protein kinase C (PKC) blockers, staurosporine and chelerythrine, prevented the ethanol effects on Ca2+ currents. The PKC activators, 1,2-oleoylacetylglycerol (OAG) and beta-phorbol-12,13-dibutyrate (PdBu), both, after maximum stimulation, also occluded the effect of ethanol on Ca2+ currents, whereas in the presence of 4-alpha-phorbol-12,13-didecanoate (4-alpha-PDD), an ineffective phorbol ester, ethanol suppressed Ca2+ currents. Ethanol increased the threshold of Ca2+-dependent action potentials and decreased their duration. Our results indicate that the suppression of voltage-activated Ca2+ currents by ethanol and its effects on action potentials involve activation of a G-protein/protein kinase transduction pathway.

Annexin-immunoreactive proteins in the nervous system and eye of the gastropods, Aplysia and Helix.

We studied the distribution of annexin I- and annexin V-like proteins in the eye and central nervous system of the snails, Aplysia californica and Helix pomatia by immunocytochemistry. Annexin I-immunoreactive material in Aplysia californica was localized in sensory and corneal cells of the eye and in distinct neurons of the cerebral, buccal, and abdominal ganglia, where it was exclusively located in bag cells. Annexin V-immunoreactive neurons were restricted to the pleural ganglia of Aplysia californica. In Helix pomatia annexin I-immunoreactive neurons were present in the cerebral, buccal, visceral, and left and right parietal ganglia, whereas annexin V-immunoreactivity neurons were present in left and right pleural, left and right parietal, visceral, and buccal ganglia. Annexin VI-immunoreactivity was absent in both gastropods studied. Our study shows a cell specific localization of annexin-like proteins in the central nervous system and eye of molluscs. The cell types containing the immunoreactive proteins suggests that the annexin-like proteins may be involved in intracellular signaling mechanisms, which ultimately may modulate egg-laying and circadian rhythmicity.

Polarity of T cell shape, motility, and sensitivity to antigen.

T cell activation requires contact with APCs. We used optical techniques to demonstrate T cell polarity on the basis of shape, motility, and localized sensitivity to antigen. An intracellular Ca2+ clamp showed that T cell shape and motility are extremely sensitive to changes in [Ca2+]i (Kd = 200 nM), with immobilization and rounding occurring via a calcineurin-independent pathway. Ca2+ dependent immobilization prolonged T cell contact with the antigen-presenting B cell; buffering the [Ca2+]i signal prevented the formation of stable cell pairs. Optical tweezers revealed spatial T cell sensitivity to antigen by controlling placement on the T cell surface of either B cells or alpha-CD3 MAb-coated beads. T cells were 4-fold more sensitive to contact made at the leading edge of the T cell compared with the tail. We conclude that motile T cells are polarized antigen sensors that respond physically to [Ca2+]i signals to stabilize their interaction with APCs.

S-100-immunoreactivity in spontaneously active snail neurons.

The distribution of S-100-immunoreactive material was examined in the central nervous system of the gastropod snail. Helix pomatia, and electrophysiological properties of S-100-positive neurons were characterized. Immunocytochemical studies revealed S-100-like protein to be present in neurons localized in the cerebral, left parietal, and visceral ganglia, but not in glial cells. Among the immunoreactive neurons we identified the giant cells LPa3 and LPa4. Western blots showed a single S-100-immunoreactive band at 12-14 kDa. S-100-positive neurons are distinguished by spontaneous discharge activity in a beating or bursting mode and a prominent Ca(2+)-activated potassium outward current. Our result show that a S-100-like protein exclusively present in neurons of the Helix central nervous system is correlated with spontaneous discharge activity of these cells.

Distribution and seasonal variation of vasoactive intestinal (VIP)-like peptides in the nervous system of Helix pomatia.

The distribution of neuropeptides immunologically related to vasoactive intestinal peptide (VIP) and its precursor peptide preproVIP(111-122), as well as to other peptides of the VIP-family, was studied in the central and peripheral nervous and sensory system of the snail, Helix pomatia, by use of immunocytochemical methods. VIP and preproVIP immunoreactivity was present in somata and nerve fibres of all central ganglia. Hibernating snails contained on average a total of 670 VIP- and 763 preproVIP-immunoreactive neurons. The number of immunoreactive cells was substantially reduced by more than 50% in active snails during summer with an average of 289 VIP- and 356 preproVIP-immunoreactive neurons. Antiserum against VIP labelled nerve fibres next to blood vessels and smooth muscle cells, whereas preproVIP-like material was localized in nerve fibres and endocrine-like cells among dorsal body cells and in the connective tissue along fiber tracts. VIP-immunoreactive material was also found in accessory ganglia of small and large tentacles, ganglia of the lips, the sensory epithelium of the tentacles, free nerve endings between skin epithelial cells, neuronal cells in the retina and in the sensory epithelium of statocysts. The cell-specific distribution and the seasonal variation of VIP- and preproVIP-like peptides suggest that they may act as transmitters or modulators in the nervous and sensory system and may be involved in the physiological adaptation of central neurons during long-term resting periods of snails.

Lymphoid tissue in the kidney of the musk shrew, Suncus murinus.

We describe a lymphoid tissue in the kidney of the musk shrew, Suncus murinus. An anatomically well organised lymphoid tissue, resembling the mucosa-associated lymphoid tissue, is associated with the epithelium of the renal pelvis and the ureter, respectively. Lymphoid tissue distributed along the arcuate artery and arcuate vein is not structurally organised in centre and periphery. This tissue type is most prominently developed between blood vessels. Immunocytochemistry revealed S-100-immunoreactive dendritic cells in both, structurally organised and structurally non-organised lymphoid tissues. The lymphoid tissue is innervated by neurofilament-immunoreactive nerve fibres. Some of these nerve fibres are associated with glial fibrillary acidic protein-immunoreactive structures, indicating that they are myelinated.

Parvalbumin-immunoreactive proteins in the nervous system of planarians.

1. Using immunological methods, we have identified parvalbumin-like material in the triclade flatworms, Polycelis nigra, Polycelis auriculata, Crenobia alpina, Dugesia tahitiensis, and Dugesia polychroa. 2. Western immunoblot analysis of these five species revealed heat stable parvalbumin-immunoreactive proteins between 32 kD and 44 kD. 3. Proteins at 19 and 32 kD revealed intense labeling with 45Ca2+. 4. Double immunodiffusion of planarian supernatants showed complete fusion of precipitates, indicating immunological relatedness of the parvalbumin-like material among the species investigated. 5. Immunocytochemical studies exhibit parvalbumin-immunoreactive material exclusively in neurons supporting the notion of an early evolutionary appearance of these proteins in the nervous system.