BDNF increases release probability and the size of a rapidly recycling vesicle pool within rat hippocampal excitatory synapses

Exerting its actions pre-, post- and peri-synaptically, brain-derived neurotrophic factor (BDNF) is one of the most potent modulators of hippocampal synaptic function. Here, we examined the effects of BDNF on a rapidly recycling pool (RRP) of vesicles within excitatory synapses. First, we estimated vesicular release in hippocampal cultures by performing FM4-64 imaging in terminals impinging on enhanced green fluorescent protein (eGFP)-labelled dendritic spines – a hallmark of excitatory synapses. Consistent with a modulation of the RRP, BDNF increased the evoked destaining rate of FM4-64 only during the initial phase of field stimulation. Multiphoton microscopy in acute hippocampal slices confirmed these observations by selectively imaging the RRP, which was loaded with FM1-43 by hyperosmotic shock. Slices exposed to BDNF showed an increase in the evoked and spontaneous rates of FM1-43 destaining from terminals in CA1 stratum radiatum, mostly representing excitatory terminals of Schaffer collaterals. Variance-mean analysis of evoked EPSCs in CA1 pyramidal neurons further confirmed that release probability is increased in BDNF-treated slices, without changes in the number of independent release sites or average postsynaptic quantal amplitude. Because BDNF was absent during dye loading, imaging, destaining and whole-cell recordings, these results demonstrate that BDNF induces a long-lasting enhancement in the probability of transmitter release at hippocampal excitatory synapses by modulating the RRP. Since the endogenous BDNF scavenger TrkB-IgG prevented the enhancement of FM1-43 destaining rate caused by induction of long-term potentiation in acute hippocampal slices, the modulation of a rapidly recycling vesicle pool may underlie the role of BDNF in hippocampal long-term synaptic plasticity.     

Composition of the antigenic material removed from Campylobacter jejuni by heat

The antigenic material removed form Campylobacter jejuni by the boiling of whole cells in saline was examined biochemically. Analyses showed that the extracted material contained 3 micrograms of protein per ml per mg of wet cells and ca. 2.6 micrograms of carbohydrate per ml per mg of wet cells. Further extraction of the material with chloroform-methanol produced about 0.5 microgram of water-insoluble residue per ml per mg of wet cells, suggesting the presence of lipid as well. Additional analyses revealed the presence of hexose, pentose, heptose, hexosamine, and 2-keto-3-deoxyoctonic acid, and the extract was also positive by the Limulus amoebocyte lysate assay for lipopolysaccharide. An examination by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that at least 10 different protein bands could be detected. One of the major bands corresponded to the major outer membrane protein, as determined by comparison with an outer membrane protein preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Another major protein in the heated extract corresponded to a band previously shown to be flagellin. An analysis of the time course of the release of material showed that a significant amount was removed after 3 to 10 min at 100 degrees C, but the release of material seemed to be delayed at lower temperatures. These results show that the treatment of C. jejuni with heat produces a complex mixture of components, including cell wall lipopolysaccharide, the major outer membrane protein, and flagellin. It is likely that some cytoplasmic components are present as well. Blebs of outer membrane have been observed with this organism by electron microscopy. Our results confirm this and suggest that the heating of cells accelerates this blebbing process.     

Free choice ethanol intake of laboratory rats under different social conditions

To study the effects of different kinds of social deprivation on voluntary ethanol (ETOH) intake male Wistar rats were housed by (a) individual caging, (b) contact caging (partial social deprivation), and (c) group caging (four individuals per cage). In the latter condition the individuals were separated once a week from each other for 24 h. The rats simultaneously received water 5%, 10% and 20% ETOH for a period of 14 weeks. Additional control animals received water. Isolated individuals drank significantly more alcohol than group-housed or contact-caged rats. After a few days they preferred the 20% solution. Circadian measures revealed a discontinuous intake of high doses (> 0.5 g/kg/h) during short time periods. Contact-caged rats consumed much less ETOH, but both the preference for 20% ETOH and the circadian course of intake were similar to those occurring after isolation. ETOH intake of group-housed individuals was low. These individuals preferred the 5% solution and continuously consumed small ETOH doses. During the period of short-term isolation they drank even more ETOH than long-term isolated individuals. In contrast to the latter, the enhancement of intake decreased after some weeks. It is suggested that the differences between the housing groups not only reflect different degrees of isolation stress, but may also be explained by a contribution of different reinforcing or aversive psychotropic effects of ETOH. Reduction of isolation stress is probably most important in the situation of short term separation, whereas dose-dependent reinforcement via social stimulation or sedation may affect the drug taking behavior under the other social conditions.     

The inflammatory lesion of T cell line transferred experimental autoimmune encephalomyelitis of the Lewis rat: distinct nature of parenchymal and perivascular infiltrates

We have investigated the T cell receptor (TCR) repertoire in the inflammatory infiltrates of T line-transferred experimental autoimmune encephalomyelitis (EAE) of the Lewis rats. Using a panel of TCR V-specific monoclonal antibodies (mAbs) and immunocytochemistry, we studied the nature of the T cells entering the central nervous system (CNS) after transfer of either myelin basic protein (MBP)-reactive, or MBP-reactive but non-encephalitogenic T cell lines. All the MBP-specific T cell lines predominantely used the V8.2 TCR chain. T cell lines specific for the tuberculin purified protein derivative (PPD), using TCR V genes different from V8.2, served as controls. We first studied the time course of T cells entering the CNS. In all recipient rats, small, but significant numbers of -TCR-expressing infiltrate cells appeared in the CNS within the first 24 h after T cell transfer. In animals injected with either type of MBP-reactive T cells, the early infiltrate cells were preferentially located within the parenchyma of the spinal cord, while in PPD T lineinjected rats, the lymphocytes were mostly found in the meninges. TCR V gene usage was examined on the peak of clinical disease. Six days after T cell transfer, the TCR repertoire used by infiltrating lymphocytes in general seemed to be highly diverse. None of the V isotypes examined (i.e. V8.2, V8.5 or V10) was used by a major population of the -TCR-positive T cells. A more detailed, quantitative analysis of individual infiltrate compartments revealed, however, a preferential accumulation of V8.2-positive T cells within the parenchyma. In contrast, perivascular infiltrating cells used V genes randomly. Our results confirm first that activated T lymphocytes enter the brain rapidly irrespective of their antigen specificity. Second, the data show that most of the perivascular infiltrate T cells in the acute EAE lesion are host-derived, recruited presumably from the recirculating T cell pool, while the encephalitogenic, V8.2-positive T cells preferentially persist within the parenchyma.Abbreviations EAE experimental autoimmune encephalomyelitis - MBP myelin basic protein - TCL T cell line Supported by the Brazilian Research Council (CNPq)     

Mechanisms of delta-hexachlorocyclohexane toxicity: II. Evidence for Ca2+-dependent K+-selective ionophore activity

delta-Hexachlorocyclohexane (delta-HCH) interacts with cardiac ryanodine-sensitive Ca2+ channels (RyR2), accounting in part for altered Ca2+ transients and contractility (reported in companion report). Analysis of channel gating kinetics in the presence of delta-HCH also revealed a nonfluctuating membrane current that remained even after RyR2 channels were blocked. We further elucidated the nature of a direct interaction between delta-HCH and biological membranes by measuring ionic currents across planar lipid bilayers made from defined lipids lacking cellular protein using voltage-clamp. Dimethyl sulfoxide, in the presence or absence of 50 microM gamma-HCH (lindane) or delta-HCH, produced negligible steady-state current with symmetric 100 mM CsCl in the range of +/-50 mV. However, the addition of 50 microM Ca2+ to the bilayer chamber in the presence of delta-HCH induced a profound increase in ionic permeability that was not seen in the presence of gamma-HCH or dimethyl sulfoxide control. Significantly, the permeability increase 1) was proportional with increasing Ca2+ to approximately 600 microM and saturated between 1 and 2 mM Ca2+ regardless of holding potential, 2) occurred only when delta-HCH and Ca2+ were added to the same side of the membrane, and 3) was independent of the order of addition or of the side of the membrane to which delta-HCH and Ca2+ was added. The Ca2+-dependent current produced by delta-HCH was highly selective for monovalent cations (K+ > Cs+ > Na+), with negligible conductance for Ca2+ or Cl-. In symmetric 100 mM K+, the conductance induced with 50 microM concentration each of delta-HCH and Ca2+ was 4.25 pA/mV. The results show that delta-HCH increases the ionic permeability of phospholipid membranes by two distinct Ca2+-dependent mechanisms: one mediated through RyR and the other mediated by a unique ionophore activity.     

Abbreviated cyclosporine AUCs on Neoral – the search continues!

 Cyclosporine is a powerful immunosuppressant with a narrow therapeutic window and considerable inter- and intrapatient variability. The pre-dose trough concentration (C_min) is commonly used for therapeutic drug monitoring. With the new microemulsion (Neoral), intrapatient variability was reduced. However, the usefulness of Neoral C_min was questioned. Firstly, because of the improved and more-rapid absorption, accidental intake before blood sampling has a greater impact on C_min than with classic cyclosporine. Secondly, C_min may be low despite high drug exposure, due to rapid clearance in children. A full pharmacokinetic (PK) profile with determination of the area under the curve (AUC) is expensive and cumbersome, and therefore a search for an abbreviated AUC began. Here, we present a retrospective analysis of 84 PK profiles from 78 pediatric renal transplant recipients. By analysis of rejection episodes and toxicity, we estimated a target AUC above 5,000 ng×h/ml in the early post-transplant period and 3,900 ng×h/ml beyond 100 days. The abbreviated AUC using the 2- and 6-h concentrations (C2 and C6) and a simple estimate derived from the 3-h concentration (C3) were equally well correlated with the AUC. From our data, we recommend a target C3 at approximately 800 ng/ml early after transplantation and 450–550 ng/ml beyond 100 days. Received: 28 January 1998 / Revised: 10 June 1998 / Accepted: 15 June 1998