The modulatory effect of crotoxin and its phospholipase A2 subunit from Crotalus durissus terrificus venom on dendritic cells interferes with the generation of effector CD4+ T lymphocytes.

Dendritic Cells (DCs) direct either cellular immune response or tolerance. The crotoxin (CTX) and its CB subunit (phospholipase A2) isolated from Crotalus durissus terrificus rattlesnake venom modulate the DC maturation induced by a TLR4 agonist. Here, we analyzed the potential effect of CTX and CB subunit on the functional ability of DCs to induce anti-ovalbumin (OVA) immune response. Thus, CTX and CB inhibited the maturation of OVA/LPS-stimulated BM-DCs from BALB/c mice, which means inhibition of costimulatory and MHC-II molecule expression and proinflammatory cytokine secretion, accompanied by high expression of ICOSL, PD-L1/2, IL-10 and TGF-ß mRNA expression. The addition of CTX and CB in cultures of BM-DCs incubated with ConA or OVA/LPS inhibited the proliferation of CD3+ or CD4+T cells from OVA-immunized mice. In in vitro experiment of co-cultures of purified CD4+T cells of DO11.10 mice with OVA/LPS-stimulated BM-DCs, the CTX or CB induced lowest percentage of Th1 and Th2 and CTX induced increase of Treg cells. In in vivo, CTX and CB induced lower percentage of CD4+IFNγ+ and CD4+IL-4+ cells, as well as promoted CD4+CD25+IL-10+ population in OVA/LPS-immunized mice. CTX in vivo also inhibited the maturation of DCs. Our findings demonstrate that the modulatory action of CTX and CB on DCs interferes with the generation of adaptive immunity and, therefore contribute for the understanding of the mechanisms involved in the generation of cellular immunity, which can be useful for new therapeutic approaches for immune disorders.

Decreased duration of Ca(2+)-mediated plateau potentials in striatal neurons from aged rats.

1. The influence of age on striatal neuron Ca2+ physiology was studied through an analysis of intracellularly recorded Ca(2+)-mediated plateau potentials. In vitro brain slices from young and aged rats were treated with the K+ channel blocker tetraethylammonium (30 mM) to facilitate the expression of plateau potentials. A sample of neurons was also filled with biocytin and post hoc correlations were performed between morphology and physiology. 2. Testing of sampling parameters in neurons from young rats revealed that tetrodotoxin did not affect the amplitude or duration of plateau potentials. The membrane potential induced during plateau testing and the rate of plateau potential generation, however, had to be held constant because these variables affected plateau potential duration. 3. A significant age-related decrease was found in the duration of Ca(2+)-mediated plateau potentials that could not be explained by alterations in the activation or inactivation properties of the plateau potential. Investigation into relationships between cell morphology and plateau potential duration revealed a number of correlations. Soma size and dendritic length were correlated with plateau potential duration, independent of age (hierarchical regression), and an age-related decrease in dendritic length but not in soma size was found. Spine density and plateau potential duration were also correlated, but the significance depended on the variance associated with age. These data indicate that the extent of somadendritic membrane (including spines) affects plateau potential duration in striatal neurons and that dendrite and spine loss in aged animals may contribute to age-related decreases in plateau potential duration. 4. The response to replacement of Ca2+ with Ba2+ was age dependent, with Ba2+ causing a greater increase in the duration of plateau potentials in young neurons. These data rule out an increase in Ca(2+)-mediated inactivation of Ca2+ channels as a primary cause for the shortening of plateau potentials in aged neurons. Our morphological findings suggest that dendritic regression in aged neurons may have reduced the number of Ca2+ channels participating in plateau potential generation, but other mechanisms related to changes in the type of Ca2+ channel expressed and possible differences in their inactivation kinetics may also contribute to the age-related change in plateau potential duration.

Synaptic activation of N-methyl-D-aspartate receptors induces short-term potentiation at excitatory synapses in the striatum of the rat.

We examined the effect of augmenting the activation of N-methyl-D-aspartate receptors during tetanic stimulation of the corpus callosum. Excitatory postsynaptic potentials were recorded from striatal neurons in an in vitro brain slice bathed in either normal or Mg(2+)-free artificial cerebrospinal fluid. Post-tetanic potentiation was followed by a long-lasting depression of the excitatory postsynaptic potential in normal artificial cerebral spinal fluid. Mg(2+)-free artificial cerebrospinal fluid increased the duration of the pretetanus excitatory postsynaptic potential and the amplitude and duration of the direct response to the tetanus. Mg(2+)-free artificial cerebrospinal fluid also enabled the expression of a short-term potentiation of the excitatory postsynaptic potential amplitude and duration. N-methyl-D-aspartate receptor antagonists blocked the induction of all tetanus-induced excitatory postsynaptic potential changes specific to the Mg(2+)-free artificial cerebral spinal fluid. These results indicate that maintained activation of N-methyl-D-aspartate receptors by synaptically released glutamate can produce a sustained enhancement of the excitatory postsynaptic potential. This use-dependent increase in synaptic efficacy may contribute to basal ganglia-related motor performance.

Synapse formation and elimination during growth of the pectoral muscle in Xenopus laevis.

1. Synapse formation and synapse elimination were studied in the pectoral muscle of Xenopus laevis. 2. Histology showed that fibres were not added during postmetamorphic growth. Most fibres were innervated at two widely separated junctions and this number did not change as frogs grew. 3. Intracellular recording revealed that fibres with two junctions could be mononeuronally innervated, or innervated in one of three different polyneuronal patterns. A growth-related shift in innervation pattern was observed, with the polyneuronal patterns replaced by mononeuronal innervation. 4. Endplate potentials (EPPs) evoked by low-frequency nerve stimulation were simultaneously measured at both junctions on individual fibres. For each fibre, the ratio of EPP amplitudes (smaller/larger) was calculated. When the two junctions were innervated by different motoneurones (A-B), the median EPP ratio was smaller than when the two junctions were innervated by the same motoneurone (A-A), although the difference was not significant. 5. The difference in the ratio of EPP amplitudes became significant, however, if junctions were conditioned by a train of fifty stimuli at 10 Hz. Immediately after such a train, EPP ratios for A-B fibres were significantly smaller than ratios for A-A fibres. This difference was due to greater synaptic depression at one of the junctions on A-B fibres. 6. We concluded that enhanced depression of the EPP upon repetitive stimulation is a physiological correlate of the competition that underlies synapse elimination.

Temporal integration in an anuran auditory nerve.

We determined temporal integration in individual auditory nerve fibers of the arboreal frog, Eleutherodactylus coqui by measuring the change in rate threshold to tonal stimulation for tone burst durations from 20 to 450 ms. Temporal integration was quantified in two ways: (1) we calculated the temporal integration time constant of the fiber (tau), and (2) we calculated the shift in threshold per decade increment of the tone stimulus (dB/decade). In some cases, the procedure was repeated using a continuous, broadband noise masker (20-50 dB/Hz). Our results indicate that low frequency fibers (CF less than 0.50 kHz) have the longest mean integration time (274 ms), mid-frequency fibers (0.50 to 1.30 kHz) have the shortest mean integration time (183 ms) and high frequency fibers (CF greater than 1.3 kHz) have an intermediate mean integration time (235 ms). Continuous noise increased the integration times of some, but not all fibers, and caused some fibers which did not display temporal integration to do so. We investigated the possibility that these changes may be caused by a decrease in the slope of the rate-intensity function (measured between +5 and +15 dB re threshold) with the addition of the continuous noise masker. The slope of the rate-intensity function decreased (from 73 spikes/s/dB to 49 spikes/s/dB) with the addition of the continuous noise for those fibers (N = 28) that showed an increase in temporal integration with the addition of noise. However, the slopes of the rate-intensity functions also decreased by 30% for those fibers (N = 8) that did not show increasing temporal integration.

Temporal resolution in frog auditory-nerve fibers.

Sinusoidally amplitude-modulated (SAM) noise was monaurally presented to the neotropical frog, Eleutherodactylus coqui, while recording intracellularly from auditory-nerve fibers. Neuronal phase locking was measured to the SAM noise envelope in the form of a period histogram. The modulation depth was changed (in 10% steps) until the threshold modulation depth was determined. This was repeated for various modulation frequencies (20-1200 Hz) and different levels of SAM noise (34-64 dB/Hz). From these data, temporal modulation transfer functions (TMTFs) were produced and minimum integration time (MIT) for each auditory fiber was calculated. The median MIT was 0.42 ms (lower quartile 0.32, upper quartile 0.68 ms). A noise level-dependent effect was noted on the shape of the TMTF as well as the minimum integration time. The latter results may be explained as a loss in spectral resolution with increasing noise level, which is consistent with the correlation that was found between minimum integration time and bandwidth.

Tone-derived vs. tone-in-noise-derived filter functions of frog auditory nerve fibers: a comparison.

Intracellular recordings were made from 112 auditory nerve fibers of Rana pipiens pipiens. For each fiber, we determined its frequency-threshold curve (FTC), and its threshold to CF-tones simultaneously masked by either cosinusoidal or inverted comb-filtered noise (CFN). From the difference between the masked thresholds using cosinusoidal and inverted CFN, the filter function of the auditory fiber was calculated based on a constant signal-to-noise algorithm (Pick, 1980). The calculated filter functions and FTCs were then compared. Of the 63 comparisons made, 42 were statistically indiscriminable, 15 fibers produced a calculated filter function that differed significantly in shape from the FTC, and six fibers produced a flat filter function. We conclude by evaluating our methods, especially for the experimental conditions found in the frog, and compare and contrast our results with those observed in mammalian systems.