Cyclosporin-A differentially affects apoptosis during in vivo rat thymocyte maturation

Maturation arrest and interference with selection are two well-documented effects of cyclosporin-A (CsA) on the thymus. We recently hypothesized that these effects are related and owing to the reduced T-cell receptor (TCR)-CD3 complex-mediated signal transduction in thymocytes upon CsA treatment. In this hypothesis, the maturation arrest is the result of the additional depletion of thymocytes that normally survive by positive selection, whereas the impaired self-tolerance induction is caused by an increased survival of thymocytes that normally undergo negative selection. In this view, it is anticipated that CsA differentially affects thymocyte apoptosis during in vivo thymocyte maturation. Indeed, we report in this study a strong increase in apoptotic cells in the thymic cortex on in situ analysis. Simultaneously, the number of apoptotic cells had decreased at the cortico-medullary zone which is held to be the site for negative selection. Rapamycin (Rapa) also interferes with thymocyte maturation by inhibiting cytokine-driven proliferation. Hence, Rapa preferentially affects the early maturational stages of thymocyte development and is considered not to alter thymocyte selection and subsequent apoptotic events. Indeed, the number of apoptotic events appears not to be altered. However, possibly owing to the decrease in cortical macrophages, the apoptotic cells revealed an atypical enumeration around blood vessels. Taken together, our results favour the hypothesis that the dominant effect of CsA on the thymus is the reduction of the TCR-CD3 complex-mediated signal transduction in thymocytes upon interaction with stromal cells. Furthermore, the preferential localization of apoptotic cells next to blood vessels upon Rapa administration may indicate that endothelial cells are a back-up system for the removal of apoptotic cells.     

Acute bladder inflammation differentially affects rat spinal visceral nociceptive neurons

The present investigation examined the effect of inflammation produced by intravesical zymosan on spinal dorsal horn neuronal responses to urinary bladder distension (UBD). Extracellular single-unit recordings of neurons excited by UBD were obtained in spinalized female Sprague–Dawley rats. Neurons were classified as Type I—inhibited by heterotopic noxious conditioning stimuli (HNCS) or as Type II—not inhibited by a HNCS. In Experiment 1—following neuronal characterization, 1% zymosan was infused into the bladder and after 2 h spinal units were recharacterized. Control rats received intravesical saline or subcutaneous zymosan. In Experiment 2—rats were pretreated with intravesical zymosan 24 h prior to surgical preparation. Control rats received anesthesia only. 137 spinal dorsal horn neurons excited by UBD were characterized. In comparison with controls, Type II neurons demonstrated increased spontaneous and UBD-evoked activity following intravesical zymosan treatment (both Experiments 1 and 2) whereas Type I neurons demonstrated either no change (Experiment 1) or decreased activity (Experiment 2) following bladder inflammation. No significant changes were noted in neuronal activity in control experiments. Inflammation differentially affects subpopulations of spinal dorsal horn neurons excited by UBD that can be differentiated according to the effect of HNCS. This results in an altered pattern of spinal sensory transmission that may serve as the mechanism for the generation of visceral nociception.     

Characterization of the uptake of adenosine by cultured rat hippocampal cells and inhibition of the uptake by xanthine derivatives

Hippocampal cells were cultured in 24-well culture plates with enriched populations of neuron or glial cells. The [3H]adenosine uptake by 7-10-day cultures of these cells was dependent on temperature, but independent of extracellular Na+. The uptake of adenosine (10 microM) for 15 s was greatly blocked by addition of 100 microM dipyridamole, 50-200 microM propentofylline or 50 microM of 2-chloroadenosine or nitrobenzylthioinosine in both cells and by 100 microM pentoxifylline in neuron. Either caffeine or theophylline (50 microM each) had no effect on the uptake by these cells. Inhibition of the adenosine uptake by propentofylline was demonstrated to be competitive in both cells.     

Activation of adenosine A1 and A2 receptors differentially affects acetylcholine release from electric organ synaptosomes by modulating calcium channels

Adenosine inhibited the release of acetylcholine (ACh) evoked by high K⁺ depolarization from synaptosomes isolated from the electric organ of the Japanese electric ray Narke japonica. The adenosine A₁ receptor agonist N⁶-cyclohexyladenosine was an effective inhibitor. Conversely, in the presence of an A₁ receptor antagonist, 8-cyclopentyltheophylline, adenosine potentiated the release of ACh. The A₂ receptor agonist N⁶-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl] adenosine also facilitated the evoked ACh release. Thus, adenosine inhibits the evoked release of ACh via the A₁ receptor while it facilitates the release via the A₂ receptor. The EC₅₀ for inhibition and facilitation by adenosine was about 1 and 41 μM, respectively. There are three known types of calcium channels (N-, P/Q- and L-type) in synaptosomes. The effects of Ca²⁺ channel type-specific blockers on the modulation of ACh release by adenosine A₁ or A₂ receptor activation revealed that inhibition by A₁ receptor activation was caused via inhibition of N-type calcium channels and the facilitative effects by A₂ receptor activation was mediated by potentiation of P-type calcium channels.     

[3H]thymidine uptake in cells of rat condylar cartilage

Weanling rats were injected intraperitoneally with [³H]thymidine and sacrificed from 5 min to 20 days later. Their mandibular condylar cartilages were examined histologically, by thin-layer autoradiography, and by using liquid scintillation and microscopic counting methods. Labeled DNA appeared in some of the chondrocytes of the resting zone as early as 10 min postinjection, and reached the proliferative zone by 24 hr and the hypertrophic zone by 4 days. The labeling pattern in the last zone was more disperse, being oriented toward the periphery of the cells as they became hypertrophic. The maximum number of labeled chondrocytes was reached by 2 hr postinjection. These amounted to approximately 11% of the total chondrocyte population, the majority of which were located in the resting zone (73%). It is concluded that, over this period, the mitotic index for these cells is 50–60 per thousand resulting in approximately 100 labeled chondrocytes. In addition, some of the chondroclasts at the erosion front contained labeled DNA as early as 5 min after [³H]thymidine administration. By 10 min, 65% of these cells exhibited one or more labeled nuclei, and the ratio of labeled cells remained high through 20 days. Chondroclasts were seen to contain a diffuse label within their cytoplasm after 5 days. This label was similar to that seen in hypertrophic chondrocytes that had reached the erosion front by that time. Clearly, chondroclasts exhibit nuclear division and do not form from fusion of hypertrophic chondrocytes, although which specific mononuclear cells may act as chondroclast progenitors is not clear. In addition, these multinucleate resorbing cells are capable of ingesting or phagocytizing nuclear remnants from hypertrophic chondrocytes at the eroding face of cartilage.     

Systemic cholecystokinin differentially affects baro-activated GABAergic neurons in rat caudal ventrolateral medulla

Cholecystokinin (CCK) is released after a meal to promote digestion and satiety. Circulating CCK inhibits splanchnic sympathetic nerve activity (sSNA), which may contribute to postprandial increases in mesenteric blood flow. The CCK-induced sympathoinhibition occurs by activation of vagal afferent nerves and inhibition of a subset of presympathetic rostral ventrolateral medullary (RVLM) neurons. The present study sought to determine whether the caudal ventrolateral medulla (CVLM) may also play a role in the CCK-induced changes in sSNA. Rats were anesthetized with chloralose, artificially ventilated, paralyzed, and prepared for recording arterial pressure (AP), heart rate (HR), sSNA, and activity of individual CVLM neurons. Injection of CCK-8 (8-10 microg/kg, iv) decreased sSNA, AP, and HR. Most baro-activated CVLM neurons were excited by CCK (n = 25, 3.4-fold increase), whereas other baro-activated CVLM neurons were not affected (n = 7) or were inhibited (n = 3). A subset of baro-activated CVLM neurons that were activated (n = 8) or unaffected (n = 2) was confirmed to be GABAergic by the presence of GAD67 mRNA. Bilateral inhibition of the CVLM by microinjections of muscimol reversed the decreases in sSNA and AP to a prominent sympathoactivation and increase in AP (n = 18). These data suggest that systemic injection of CCK leads to the activation of most baro-activated GABAergic CVLM neurons and that the CVLM is essential for the production of CCK-induced inhibition of sSNA. The differential responses of baro-activated GABAergic CVLM neurons to CCK may contribute to the diverse responses of presympathetic RVLM neurons and sympathetic outflows observed with systemic CCK.     

Capsaicin treatment differentially affects feeding suppression by bombesin-like peptides

Peripheral administration of bombesin (BN) and the related mammalian peptides, gastrin-releasing peptide (GRP) and neuromedin B (NMB), suppress food intake in rats. To examine whether all BN-like peptides utilize the same neural pathways to reduce feeding, rats were treated on postnatal day 2 with the injection vehicle or capsaicin, a neurotoxin that damages a subset of visceral afferent fibers. When rats reached adulthood, we compared the ability of a dose range of systemically administered BN, GRP18-27 and NMB to reduce intake of a 0.5 kcal/ml glucose solution in a short-term feeding test. Our results demonstrate that capsaicin treatment abolished or attenuated the suppression of glucose intake produced by BN and NMB but had no effect on the ability of GRP to reduce feeding. These results suggest that different neural substrates underlie the anorexic effects of peripherally administered BN-like peptides.     

Chronic ethanol treatment differentially affects muscarinic receptor responses in rat hippocampus.

Sensitivity of hippocampal field potentials to local (iontophoretic) application of acetylcholine (ACh) was investigated in chronic ethanol treated (CET) and sucrose-fed (control) rats. CET and control rats were fed a liquid diet containing either ethanol or sucrose for 28 weeks. Five to six months after ethanol or sucrose was withdrawn, hippocampal slices were taken and ACh was applied in stratum pyramidale or stratum radiatum of CA1 to observe population spike facilitation or field EPSP inhibition, respectively. Population spikes were facilitated to a considerably lesser extent in CET slices relative to controls, while no treatment differences were observed for dendritic EPSP inhibition. These data suggest that ACh response properties in CA1 exhibit differential sensitivity to CET, and may reflect a distinct susceptibility of muscarinic receptor subtypes to the neurotoxic effects of ethanol.     

Maternal anesthesia via isoflurane or ether differentially affects pre-and postnatal behavior in rat offspring

Our understanding of prenatal behavior has been significantly advanced by techniques for direct observation and manipulation of unanesthetized, behaving rodent fetuses with intact umbilical connections to the mother. These techniques involve brief administration of an inhalant anesthesic, enabling spinal transection of the rat or mouse dam, after which procedures can continue with unanesthetized dams and fetuses. Because anesthetics administered to the mother can cross the placental barrier, it is possible that fetuses are anesthetized to varying degrees. We compared in perinatal rats the effects of prenatal maternal exposure to two inhalant anesthetics: ether and isoflurane. Fewer spontaneous fetal movements and first postpartum nipple attachments were observed following maternal exposure to ether as compared to isoflurane. Neonatal breathing frequencies and oxygenation did not account for group differences in nipple attachment. Our results provide evidence that the particular inhalant anesthetic employed in prenatal manipulation studies determines frequencies of perinatal behavior.     

Acute reduction of extracellular sodium differentially affects receptor-mediated and K+-induced calcium uptake in PC12 pheochromocytoma cells

Using a rapid-quench method to measure 45Ca2+ uptake into PC12 cells in suspension, we have studied basal, carbachol-stimulated and K+-induced Ca2+ uptake under control conditions [( Na+]o = 130 mM) and in the presence of acutely lowered extracellular sodium concentration [( Na+]o = 65 mM). Acute reduction of [Na+]o stimulates basal and K+-evoked Ca2+ uptake, but reduces net carbachol-stimulated uptake. Since total Ca2+ uptake measured in the presence of carbachol under control and low [Na+]o conditions is unchanged, the reduction in carbachol-stimulated uptake is due to the increase in basal uptake induced by low [Na+]o. These results reconcile apparently conflicting data regarding a specific Na+ requirement for nicotinic acetylcholine receptor-mediated responses in PC12 cells and adrenal chromaffin cells and suggest a mechanism for loss of nicotinic acetylcholine receptor (nAChR) responsiveness to agonists under low Na+ conditions.     

Development of Thy 1 positive rat thymocytes: analysis of fetal and neonatal thymocytes by flow microfluorometry

Rat thymocytes of embryos and neonates were analyzed for cell size and amount of Thy 1 by flow microfluorometry. The rat embryonic thymus of 18 days' gestation appeared to be composed of very large cells with relatively low levels of Thy 1. The Thy 1 content was intermediate between rat bone marrow cells (low Thy 1) and the majority of rat adult thymocytes (high Thy 1). As cells became small at the 19th and the 20th day, the Thy 1 content appeared to increase. In the early postnatal thymus of the rat large cells appeared with low Thy 1 levels which were comparable to those in the 18th day fetal thymus. These results indicate that immature thymocytes increase in Thy 1 content as they differentiate into more mature cells from low Thy 1 thymocyte precursors. This conclusion is in close agreement with our recent results regarding rat bone marrow prethymic lymphocyte progenitors and the rat immature thymocytes.     

Peroxynitrite treatment reduces adenosine uptake via the equilibrative nucleoside transporter in rat astrocytes

In the oxidative stress-loaded brain, extracellular adenosine levels are elevated and thereby neuronal damage is attenuated, but mechanisms underlying alteration of the extracellular kinetics of adenosine remain unclear. Here we investigated whether oxidative stress might alter functional expression of nucleoside transporters (NTs), a predominant regulatory system for nucleoside kinetics, in cultured rat astrocytes. Treatment of astrocytes with 0.5 mM SIN-1 for 3 h caused apparent cellular accumulation of nitrotyrosine, but had no effect on their viability, indicating load of oxidative stress to astrocytes without any change in their viability. Under the condition, [³H]adenosine uptake was significantly less than that by control cells. This decreased uptake was due to decrease in adenosine uptake mediated by an equilibrative NT (ENT) 1 which was inhibited by low concentrations (≤0.1 μM) of nitrobenzylthioinosine (NBMPR), but not by sodium-dependent or high concentrations (≥1 μM) of NBMPR-inhibitable nucleoside transporters. The expression level of ENT1 was not altered, while the Michaelis constant, but not the maximum rate, of adenosine uptake was increased. These findings suggest that under oxidative stress-loaded conditions, decreased adenosine clearance via astrocytic ENT1 might involve, at least in part, in an elevated extracellular adenosine level in the brain.     

Transcranial magnetic stimulation differentially affects speed and direction judgments

This study was conducted to determine whether humans' judgments about the speed and direction of moving stimuli was differentially affected by transcranial magnetic stimulation (TMS). Subjects viewed two successively presented moving stimuli that differed from each other both in speed and direction of motion. Single-pulse TMS was applied either medially (approximately 2 cm above the inion) or laterally (approximately 5 cm lateral to and 4 cm above the inion), while subjects judged the speed and direction differences. The physical stimulation (visual and TMS) was identical on the two tasks, as was discriminability (d') when TMS was not applied. We found significant criterion (beta) shifts on the speed discrimination task at both stimulation sites. Specifically, on TMS trials the proportion of "slower" judgments increased significantly, consistent with subjective reports that stimuli often appeared to slow when TMS was applied. The subjective reports indicated no corresponding change in perceived direction. We also found that speed discriminability was impaired significantly more than direction discriminability, but only when TMS was applied medially. Indeed, after controlling for TMS-related changes in reaction time, speed discriminability was impaired significantly, while direction discriminability remained largely intact. This dissociation suggests that the sensory response constraining speed discrimination is at least partially independent from the sensory response constraining direction discrimination. Combined with previous psychophysical data, the present data suggest a double dissociation between speed and direction discrimination in humans.     

Cortisol differentially affects memory in young and elderly men

Nine young and 11 elderly men participated in this placebo-controlled, double-blind, crossover study (0.5 mg/kg cortisol or intravenous placebo). Participants learned a word list before cortisol administration, and delayed recall was then tested. A 2nd word list was learned and recalled after drug administration. In addition, the Paragraph Recall Test and tests measuring working memory (Digit Span), attention (timed cancellation), and response inhibition (Stroop Color and Word Test) were administered at 2 time points after drug administration. Cortisol reduced recall from the word list learned before treatment in both groups but did not influence recall of the list learned after treatment. In contrast, Digit Span performance was decreased by cortisol in young but not elderly participants. The possibility that differential age-associated brain changes might underlie the present results is discussed.     

K+ differentially affects the excitatory amino acids- and carbachol-elicited inositol phosphate formation in rat brain synaptoneurosomes

K+, excitatory amino acids (EAAs) and carbachol (Carb) were tested separately or in pairs for their ability to stimulate inositol phosphate (IPs) formation in rat forebrain synaptoneurosomes. K+ ions per se, stimulate IPs synthesis (158% of the control value) as well as EAAs and Carb. The glutamate (Glu)- and quisqualate (QA)-elicited IPs formation is not additive with that evoked by K+. Inversely, K+ ions (up to 30 mM) potentiate the Carb-induced IPs accumulation. These results indicate that QA (or Glu) and Carb enhance IPs formation independently and that QA- and K+ -induced IPs responses are interdependent. This suggests that they share a 'common intermediate' step in the multistep mechanism which leads from receptor activation to the IPs synthesis. This 'common intermediate' step may be depolarization and/or Na+ influx.