Spinal neurons bursting in phase with fictive scratching are not related to spontaneous cord dorsum potentials.

Spontaneous cord dorsum potentials (spontaneous CDPs) are produced by the activation of dorsal horn neurons distributed along the L4 to S1 spinal cord segments, in Rexed's laminae III-VI, in the same region in which there are interneurons rhythmically bursting during fictive scratching in cats. An interesting observation is that spontaneous CDPs are not rhythmically superimposed on the sinusoidal CDPs generated during fictive scratching episodes, thus suggesting that the interneurons producing both types of CDPs belong to different spinal circuits. In order to provide experimental data to support this hypothesis, we recorded unitary activity of neurons in the L6 spinal cord segment. We found that the neurons firing rhythmically during the sinusoidal CDPs associated with the extensor, flexor or intermediate phases of scratching were not synchronized with the spontaneous CDPs. Moreover, we found that the neurons firing during the spontaneous CDPs were not synchronized with the sinusoidal CDPs. These results suggest that the neurons involved in the occurrence of spontaneous CDPs are not part of the spinal cord central pattern generators (CPGs). This study will be relevant for understanding the relationships between the spinal cord neuronal populations firing spontaneously and the CPGs, in the intact and injured spinal cord.

GABAA receptors mediate motoneuron tonic inhibition in the turtle spinal cord.

GABA(A) receptors mediating tonic inhibitory currents are present in neurons from hippocampus, cerebellum, sensory cortex and thalamus. These receptors located at peri- and extra-synaptic sites are constituted mainly by α(4/6) and α(5) subunits which confer them high affinity for GABA and low desensitization. Immunohistochemical and in vitro hybridization studies have shown the expression of these subunits, while functional studies have reported the presence of GABAergic tonic currents in spinal dorsal horn neurons. However, the presence of this inhibitory current has not been documented in motoneurons. In addition, we previously reported that the monosynaptic reflex is facilitated by furosemide, an antagonist of the α(4/6) GABA(A) receptors, without affecting the dorsal root potential, which suggests the presence of a GABAergic tonic inhibitory current in motoneurons. The aim of this work was to investigate the presence of high affinity GABA(A) receptors in motoneurons. By intracellular recordings made with sharp electrodes and the whole-cell patch clamp recording technique we show here that the membrane input resistance and the monosynaptic excitatory post-synaptic potential (EPSPs) are significantly increased by bicuculline. Likewise, the depression of the EPSPs and the input membrane resistance normally induced by muscimol was partially reverted by 20 µM bicuculline and abolished when the concentration of the antagonist was raised to 100 µM. Last, bicuculline at low concentration did not affect the holding current as occur with the high concentration that block the tonic inhibitory GABAergic current. Together these results suggest that the excitability in motoneurons may be tonically inhibited by high affinity GABA(A) receptors.

Antigens from Leishmania amastigotes inducing clinical remission of psoriatic arthritis.

A first generation vaccine (AS100-1) was manufactured with protein from four cultured Leishmania species, which proved to be effective in the treatment of psoriasis. A single blind trial on 3,132 psoriasis patients revealed 508 (16.2%) subjects with psoriatic arthritis (PsA) that received AS100-1 antigens. The study group was distributed according to percent psoriasis area and severity index (PASI) reduction from PASI 10 to PASI 100. All groups decreased in arthritis score (AS), tender joints counts and nail changes after treatment; the highest decreased in the PASI 100 group. Relapses of psoriasis and PsA had PASI and AS lower than initial values before treatment. Clinical remissions were at lower doses and less time, after the second course of treatment. Peripheral blood mononuclear cells (PBMC) lymphocyte subsets (LS) varied with PASI range (1-10, 11-20 and 21-72). Pre-treatment, absolute values of gated LS: CD4+, CD8+HLA-, CD8+HLA+, CD8+CD3-, CD8+CD3+ decreased in PBMC as PASI increased, suggesting migration from the blood to the skin. In contrary to the previous finding, the following LS: CD8+CD4-, CD3+CD8-, HLA+CD8-, CD19, CD8+CD4+ and membrane surface immunoglobulin IgA+, IgD+, IgM+, IgE+, and IgG+ increased in PBMC as PASI increased suggesting activation and proliferation by unknown antigens creating a homeostatic cycle between skin/joints and peripheral blood. After nine doses of AS100-1, the following LS: CD8+CD3+, CD8+HLA+, CD3+CD8-, CD4+CD8-, CD8+HLA-, HLA+CD8-, CD8+CD3-, CD19+, CD8+CD4-, CD8+CD4+, IgA+, IgD+, IgM+, IgE+, and IgG+ decreased significantly as compared with values before treatment. The LS decreased stops the vicious cycle between skin/joints and blood explaining clinical remission of lesions.

Isolation of Leishmania amastigote protein fractions which induced lymphocyte stimulation and remission of psoriasis.

A first generation polyvalent vaccine (AS100(1)) was manufactured with protein from several cultured leishmania species, which proved to be effective in the treatment of psoriasis. To determine the effective factor, a single blind trial with four monovalent second generation vaccines (AS100(2)) was done in 26 subjects, which also resulted in remission of psoriasis. AS100(2) vaccines were further purified, resulting in seven chromatography fractions (AS200) per species. In vitro testing of the fractions on blood lymphocytes resulted in subjects being categorized as low or high responders before treatment. Both responder groups had no statistical difference in clinical outcome after AS100(1) treatment. Subsequently, a single-blind trial in 55 subjects treated with AS200 fractions from Leishmania brasiliensis also induced remission of Psoriasis. Two HIV +/- subjects with plaque psoriasis experienced remission after treatment with AS100(1). There are factors in leishmania species which induce remission of psoriasis by stimulating lymphocytes.

Antigens from Leishmania amastigotes induced clinical remission of psoriasis.

While injecting volunteers in Venezuela with a vaccine for prevention of leishmaniasis, we observed 100% clinical remission of a psoriatic lesion in one subject. Subsequently, the vaccine (AS100) was evaluated in psoriatic patients with an open label, single center study. The study was conducted in 2,770 subjects and included plaque (79%), guttate (10%), plaque and guttate (10%), palm/plantar, erythrodermia, inverse, plaque and arthritis and nail psoriasis. Baseline PASI compared with post-treatment values were: PASI 100, 23%; PASI 75, 45%; PASI 50, 13%; PASI 10, 9% and

Thrombin increases hyposmotic taurine efflux and accelerates ICI-swell and RVD in 3T3 fibroblasts by a src-dependent EGFR transactivation.

The present study in Swiss3T3 fibroblasts examines the effect of thrombin on hyposmolarity-induced osmolyte fluxes and RVD, and the contribution of the src/EGFR pathway. Thrombin (5 U/ml) added to a 30% hyposmotic medium markedly increased hyposmotic 3H-taurine efflux (285%), accelerated the volume-sensitive Cl- current (ICI-swell) and increased RVD rate. These effects were reduced (50-65%) by preventing the thrombin-induced intracellular Ca2+ [Ca2+]i rise with EGTA-AM, or with the phospholipase C (PLC) blocker U73122. Ca2+calmodulin (CaM) and calmodulin kinase II (CaMKII) also participate in this Ca2+-dependent pathway. Thrombin plus hyposmolarity increased src and EGFR phosphorylation, whose blockade by PP2 and AG1478, decreased by 30-50%, respectively, the thrombin effects on hyposmotic taurine efflux, ICI-swell and RVD. Ca2+- and src/EGFR-mediated pathways operate independently as shown by (1) the persistence of src and EGFR activation when [Ca2+]i rise is prevented and (2) the additive effect on taurine efflux, ICI-swell or RVD by simultaneous inhibition of the two pathways, which essentially suppressed these events. PLC-Ca2+- and src/EGFR-signaling pathways operate in the hyposmotic condition and because thrombin per se failed to increase taurine efflux and ICI-swell under isosmotic condition it seems that it is merely amplifying these previously activated mechanisms. The study shows that thrombin potentiates hyposmolarity-induced osmolyte fluxes and RVD by increasing src/EGFR-dependent signaling, in addition to the Ca2+-dependent pathway.

Tyrosine kinases and osmolyte fluxes during hyposmotic swelling.

Recent evidence documents the involvement of protein tyrosine kinases (TK) in the signalling network activated by hyposmotic swelling and regulatory volume decrease. Both receptor type and cytosolic TK participate as signalling elements in the variety of cell adaptive responses to volume changes, which include adhesion reactions, reorganization of the cytoskeleton, temporal deformation/remodelling of the membrane and stress-detecting mechanisms. The present review refers to the influence of TK on the activation/operation of the osmolyte efflux pathways, ultimately leading to cell volume recovery, i.e. the osmosensitive Cl- channel (Cl-swell), the K+ channels activated by swelling in the different cell types and the taurine efflux pathway as representative of the organic osmolyte pathway.

Effect of Plantago major on cell proliferation in vitro.

Plantago major (Plantaginaceae) is popularly used to treat tumors, infections and as a blood purifier. Aqueous, methanol, chloroform and hexane extracts of the aerial parts (leaves and seeds) were added to CD(1) mice bone marrow and spleen cultures incubated at 37 degrees C for 72h, and also added to Escherichia coli, Bacillus subtilis and Candida albicans cultures, while methanol extract dilutions were added to HTC-15, OVCAR, UISO and KB cell line cultures. Doses of 0.4 and 0.2 mg/mL of aqueous and methanol extracts increased the bone marrow cell concentration by 2.70- and 3.15-fold, respectively, and increased the spleen cell concentration by 3.38- and 6.39-fold, respectively (p < 0.001). Aqueous extract inhibited Bacillus subtilis growth from 78 to 21%; hexane extract inhibited the growth of Escherichia coli, and methanol and chloroform extracts weakly inhibited the growth of Bacillus subtilis and Escherichia coli, respectively. Methanol extract (1 microg/mL) decreased the UISO and OVCAR cell concentrations to 59 and 82%, respectively. Data demonstrate for the first time that Plantago major has hematopoietic activity in vitro.

Epidermal growth factor receptor is a common element in the signaling pathways activated by cell volume changes in isosmotic, hyposmotic or hyperosmotic conditions.

Changes in external osmolarity, including both hyper- or hyposmotic conditions, elicit the tyrosine phosphorylation of a number of tyrosine kinase receptors (TKR). We show here that the epidermal growth factor receptor (EGFR) is activated by both cell swelling (hyposmolarity, isosmotic urea, hyperosmotic sorbitol) or shrinkage (hyperosmotic NaCl or raffinose) and discuss the mechanisms by which these apparently opposed conditions come to the same effect, i.e., EGFR activation. Evidence suggests that this results from early activation of integrins, p38 and tyrosine kinases of the Src family, which are all activated in the two anisosmotic conditions. TKR transactivation by integrins and p38 is likely occurring via an effect on the metalloproteinases. Information discussed in this review, points to TKR as elements in osmotransduction as a useful mechanism to amplify and diversify the initial response to anisosmolarity and cell volume changes, due to their privileged situation as convergence point for numerous intracellular signaling pathways. The variety of effector pathways connected to TKR is advantageous for the cell to cope with the changes in cell volume including adaptation to stress, cytoskeleton remodeling, adhesion reactions, cell survival and the adaptive mechanisms to ultimately restore the original cell volume.

Mechanisms of chloride influx during KCl-induced swelling in the chicken retina.

An increase in extracellular KCl ([KCl]o) occurs under various pathological conditions in the retina, leading to retinal swelling and possible neuronal damage. The mechanisms of this KCl o-induced retinal swelling were investigated in the present study, with emphasis on the Cl- transport mechanisms. Increasing [KCl]o (from 5 to 70 mM) led to concentration-dependent swelling in chicken retinas. The curve relating the degree of swelling to [KCl]o was biphasic, with one component from 5 to 35 mM [KCl]o and another from 35 to 100 mM. As Cl- omission prevented swelling in all conditions, the effect of cotransporter or Cl- channel blockers was examined to investigate the mechanisms of Cl- influx. The cotransporter blockers bumetanide and DIOA reduced swelling by 68% and 76%, respectively at [KCl]o 25 mM (K25), and by 14-17% at [KCl]o 54 mM (K54). The Cl- channel blockers NPPB and niflumic acid did not affect swelling at K25 but reduced it by 90-95% at K54 (NPPB IC50 60.7 microM). Furosemide showed an atypical effect, decreasing swelling by 14% at K25 and by 95% at K54 (IC50 173.9 microM). Na+ omission decreased swelling at K25 but not at K54. These results suggest the contribution of cotransporters to Cl- influx at K25 and of Cl- channels at K54. At K25, swelling was found in the ganglion cell layer and in the lower half of the inner nuclear layer. With K54, swelling occurred in all inner retinal layers. The ganglion cell layer swelling was due to both Muller cell end-foot and ganglion cell soma swelling. K54 also induced ganglion cell damage as shown by disorganized, pyknotic and refringent nuclei.

Cholinergic facilitation of erection and ejaculation in spinal cord-transected rats.

Penile reflexes (PRs) were monitored in chronic spinal cord-transected rats by identifying them visually, and at the same time they were recorded as the electromyographic activity of bulbospongiosus muscles. Intraperitoneal injection of the agonist muscarine (10 microg) produced a facilitation of PRs. A decrease in the latency, an increase in the number of clusters and often an increase in the duration of cups were found after muscarine. In addition, 66% (six out of nine) of the animals ejaculated after muscarine. These results suggest that cholinergic receptor stimulation may be involved in erectile and ejaculatory mechanisms mediated by the spinal cord.

A model for the induction of aplastic anemia by subcutaneous administration of benzene in mice.

Long-term exposure to benzene vapors is associated with hematological diseases such as leukemia, lymphoma and aplastic anemia. CD(1) male mice were randomly assigned to six groups: 1B(10), 1B(15), 1B(20), 2B(10), 2B(15), and 2B(20.) 1B mice were administered 2 ml/kg (1940 mg/kg) subcutaneous injection (in the dorsal region) of benzene 5 days a week, and 2B mice were exposed 3 days a week (Monday, Wednesday and Friday) until a total of 10, 15 and 20 doses were completed. About 48 h after treatment completion, leukocyte, erythrocyte, and bone marrow cells were counted, and spleen histopathology was analyzed. 1B(15) and 1B(20) mice showed lethargy and irritability, 80% body and 42% spleen weight loss (P<0.001), while body and spleen weight loss were less severe in 2B mice (12 and 48%, respectively). After exposure to 20 benzene doses, 1B(20) and 2B(20) mice showed decreased hemoglobin concentrations, and erythrocyte, leukocyte and bone marrow cell counts (37, 34, 80 and 50%, respectively in group 1B(20); P<0.001; and 12, 48, 62 and 62%, respectively in group 2B(20)). Thrombocytopenia occurred only in group 2B. Both benzene-treatment schemes caused aplastic anemia, however, the disease was masked by spleen toxicity in group 1B. Scheme 2 allowed mice survival and caused less non-hematological effects. We establish here a reproducible and inexpensive experimental model to induce aplastic anemia in mice by subcutaneous injection of 2 ml/kg benzene, using two short-term treatment schemes.

Over-expression of betaI tubulin in MDCK cells and incorporation of exogenous betaI tubulin into microtubules interferes with adhesion and spreading.

Little is known about the presence and distribution of tubulin isotypes in MDCK cells although essential epithelial functions in these monolayers are regulated by dynamic changes in the microtubule architecture. Using specific antibodies, we show here that the betaI, betaII, and betaIV isotypes are differentially distributed in the microtubules of these cells. Microtubules in subconfluent cells radiating from the perinuclear region contain betaI and betaII tubulins, while those extending to the cell edges are enriched in betaII. Confluent cells contain similar proportions of betaI and betaII along the entire microtubule length. betaIV is the less abundant isotype and shows a similar distribution to betaII. The effect of modifying tubulin isotype ratios in the microtubules that could affect their dynamics and function was analyzed by stably expressing in MDCK cells betaI tubulin from CHO cells. Three recombinant clones expressing different levels of the exogenous betaI tubulin were selected and subcloned. Clone 17-2 showed the highest expression of CHO beta1 tubulin. Total betaI tubulin levels (MDCK+CHO) in the clones were approximately 1.8 to 1.1-fold higher than in mock-transfected cells only expressing MDCK beta1 tubulin. In all the cells, betaII tubulin levels remained unchanged. The cells expressing CHO beta1 tubulin showed defective attachment, spreading, and delayed formation of adhesion sites at short times after plating, whereas mock-transfected cells attached and spread normally. Analysis of cytoskeletal fractions from clone 17-2 showed a MDCK betaI/CHO betaI ratio of 1.89 at 2 h that gradually decreased to 1.0 by 24 h. The ratio of the two isotypes in the soluble fraction remained unchanged, although with higher values than those found for the polymerized betaI tubulin. By 24 h, the transfected cells had regained normal spreading and formed a confluent monolayer. Our results show that excess levels of total betaI tubulin, resulting from the expression of the exogenous beta1 isotype, and incorporation of it into microtubules affect their stability and some cellular functions. As the levels return to normal, the cells recover their normal phenotype. Regulation of betaI tubulin levels implies the release of the MDCK betaI isotype from the microtubules into the soluble fraction where it would be degraded.

Epileptogenesis after status epilepticus reflects age- and model-dependent plasticity.

Although epilepsy often begins in childhood, factors that contribute to the development of epilepsy as a consequence of status epilepticus (SE) during early development are poorly understood. We investigated animal models in which seizure-induced epileptogenicity could be studied. Rats undergoing self-sustaining SE induced by perforant path stimulation (PPS) at the ages of postnatal day 21 (P21) and P35 were compared with those subjected to SE by lithium and pilocarpine (LiPC). Although only one animal subjected to PPS at P21 developed chronic spontaneous seizures by several months of observation, all the animals subjected to PPS at P35 became epileptic. In the LiPC model, however, most of the rat pups subjected to SE at P21 became epileptic. Animals with spontaneous seizures showed increased inhibition in the dentate gyrus, a characteristic of the epileptic brain, with evidence of mossy fiber synaptic reorganization. Examination of circuit recruitment by c-Jun immunohistochemistry showed activation restricted to the hippocampus in P21 animals subjected to PPS, although extensive activation of hippocampal and extrahippocampal structures was seen in pups subjected to PPS-induced self-sustaining SE at P35 or LiPC SE at P21. These results demonstrate that the appearance of epilepsy as a consequence of SE is influenced by the type of insult as well as by age-dependent circuit recruitment.

Dorsal root potential produced by a TTX-insensitive micro-circuitry in the turtle spinal cord.

1, The mechanisms underlying the dorsal root potential (DRP) were studied in transverse slices of turtle spinal cord. DRPs were evoked by stimulating one filament in a dorsal root and were recorded from another such filament. 2. The DRP evoked at supramaximal stimulus intensity was reduced but not eliminated after blockade of GABAA receptors. The remaining component was eliminated by blocking NMDA and AMPA receptors. 3. The DRP was reduced but not eliminated after blockade of AMPA receptors. The early component of the remaining DRP was dependent on GABAA receptors and the residual component on NMDA receptors. 4. The DRP was reduced but not eliminated by TTX. GABAA, NMDA and AMPA receptors contributed to the generation of the TTX-insensitive DRP. The early component of the DRP in the presence of TTX depended on GABAA receptor activation, and the late component mainly on the activation of NMDA receptors. 5. Our results show that part of the DRP is generated by a TTX-resistant, probably non-spiking micro-circuit with separate components mediated by GABA and glutamate.

Oscillatory interaction between dorsal root excitability and dorsal root potentials in the spinal cord of the turtle.

The response to dorsal root stimulation, at one to two times threshold, was investigated in the isolated cervical enlargement of the turtle spinal cord. At frequencies near 10 Hz the synaptic response in motoneurons and the cord dorsum potential, after an initial lag time, oscillated in amplitude with a period of more than 1 s. The mono- and polysynaptyic postsynaptic response in motoneurons, the pre- and postsynaptic component of the cord dorsum potential and the dorsal root potential oscillated in synchrony. These oscillations were only observed with stimulus frequencies in the range 9-11 Hz. The oscillating response could only be evoked from stimulus sites to which dorsal root potentials were conducted from the spinal cord (2-3 mm). At more distant stimulus sites cyclic variations in amplitude of the cord dorsum potential and the synaptic response in motoneurons were not observed. During an oscillating spinal response to a stimulus train in one dorsal root filament, the response evoked by a stimulus in another short filament (2-3 mm) from the same root varied in amplitude with the induced oscillation. The spinal response to a stimulus in a longer filament (i.e. more than 3 mm) did not oscillate. It is argued that the oscillating responses described rely on interactions between distributed elements rather than on unit oscillators. We also show that primary afferent transmission is unaffected by the substantial variations in dorsal root potentials during oscillations.

Local facilitation of plateau potentials in dendrites of turtle motoneurones by synaptic activation of metabotropic receptors.

1. The spatial distribution of synaptic facilitation of plateau potentials in dendrites of motoneurones was investigated in transverse sections of the spinal cord of the turtle using differential polarization by applied electric fields. 2. The excitability of motoneurones in response to depolarizing current pulses was increased following brief activation of either the dorsolateral funiculus (DLF) or the medial funiculus (MF) even when synaptic potentials were eliminated by antagonists of ionotropic receptors. 3. The medial and lateral compartments of motoneurones were differentially polarized by the electric field generated by passing current between two electrodes on either side of the preparation. In one direction of the field lateral dendrites were depolarized while the cell body and medial dendrites were hyperpolarized (S- configuration). With current in the opposite direction the cell body and medial dendrites were depolarized while lateral dendrites were hyperpolarized (S + configuration). 4. Following brief activation of the DLF the excitability and the generation of plateau potentials were facilitated during differential depolarization of the lateral dendrites but not during differential depolarization of the cell body and medial dendrites. Following brief activation of the MF the excitability and generation of plateau potentials were facilitated during differential depolarization of the cell body and medial dendrites but not during differential depolarization of the lateral dendrites. 5. It is concluded that the synaptic facilitation of the dihydropyridine-sensitive response to depolarization is compartmentalized in turtle motoneurones.