Combining Motion Analysis and Microfluidics – A Novel Approach for Detecting Whole-Animal Responses to Test Substances

Small, early life stages, such as zebrafish embryos are increasingly used to assess the biological effects of chemical compounds in vivo. However, behavioural screens of such organisms are challenging in terms of both data collection (culture techniques, drug delivery and imaging) and data evaluation (very large data sets), restricting the use of high throughput systems compared to in vitro assays. Here, we combine the use of a microfluidic flow-through culture system, or BioWell plate, with a novel motion analysis technique, (sparse optic flow - SOF) followed by spectral analysis (discrete Fourier transformation - DFT), as a first step towards automating data extraction and analysis for such screenings. Replicate zebrafish embryos housed in a BioWell plate within a custom-built imaging system were subject to a chemical exposure (1.5% ethanol). Embryo movement was videoed before (30 min), during (60 min) and after (60 min) exposure and SOF was then used to extract data on movement (angles of rotation and angular changes to the centre of mass of embryos). DFT was subsequently used to quantify the movement patterns exhibited during these periods and Multidimensional Scaling and ANOSIM were used to test for differences. Motion analysis revealed that zebrafish had significantly altered movements during both the second half of the alcohol exposure period and also the second half of the recovery period compared to their pre-treatment movements. Manual quantification of tail flicking revealed the same differences between exposure-periods as detected using the automated approach. However, the automated approach also incorporates other movements visible in the organism such as blood flow and heart beat, and has greater power to discern environmentally-driven changes in the behaviour and physiology of organisms. We suggest that combining these technologies could provide a highly efficient, high throughput assay, for assessing whole embryo responses to various drugs and chemicals.     

Stress-Induced Changes in t-[35S]Butylbicyclophosphorothionate Binding to γ-Aminobutyric Acid-Gated Chloride Channels Are Mimicked by In Vitro Occupation of Benzodiazepine Receptors

The allosteric modulation of t-[35S]butylbicyclophosphorothionate binding by flunitrazepam was studied in well-washed brain membranes prepared from control and swim-stressed rats. Swim stress has been reported to decrease the KD and increase the Bmax of this radioligand. Flunitrazepam increased radioligand binding with equal potency (EC50 approximately 11 nM) in both groups, but the maximal enhancement (efficacy) produced by this drug was significantly greater in control than in swim-stressed rats. Ro 15-1788 (a benzodiazepine receptor antagonist) blocked the effect of flunitrazepam on t-[35S]butylbicyclophosphorothionate binding in both groups. This increase in t-[35S]butylbicyclophosphorothionate binding resulted from a significant reduction in KD with no alteration in Bmax. The KD values obtained in cortical membranes of control rats after addition of flunitrazepam were not significantly different from those in the swim-stressed group. Preincubation of cortical homogenates from control animals with flunitrazepam prior to extensive tissue washing resulted in Bmax and KD values of t-[35S]butylbicyclophosphorothionate similar to those obtained in stressed animals. These findings suggest that stress and flunitrazepam may share a common mechanism in regulating t-[35S]butylbicyclophosphorothionate binding and support the concept that stress-induced modification of gamma-aminobutyric acid (GABA)-gated chloride channels in the CNS results from the release of an endogenous modulator (with benzodiazepine-like properties) of the benzodiazepine-GABA receptor chloride ionophore receptor complex.     

A study of cytoplasmic and nuclear estrogen and progestin receptors in gynecologic neoplasms

A rapid method for simultaneous preparation of cytosol and nuclear estrogen (E) and progestin (P) receptors and their in vitro determination is described. The method was applied to several uterine or ovarian surgical specimens to evaluate their steroid hormone "dependence". The results suggest that low cytoplasmic E receptor levels (ERc) are associated with higher nuclear E receptor (ERn) levels but no apparent correlation was observed between PRc and ERn levels. The method appeared to be suitable for screening steroid hormone receptor content in tumor tissues and may provide better estimation of steroid dependence since both cytoplasmic and nuclear compartments can be studied simultaneously.     

Characterization of benzodiazepine receptors with fluorescent ligands

Fluorescein conjugates of the high-affinity benzodiazepine receptor ligands Ro 15-1788 and Ro 7-1986 were synthesized. The binding of these fluorescent ligands (BD 621 and BD 607) to benzodiazepine receptors was characterized by direct fluorescence measurement. Both the equilibrium dissociation constants (KD) of BD 621 and BD 607 and the maximum number of binding sites (Bmax) estimated by fluorescence monitoring were consistent with values obtained by using radioligand binding techniques. The binding of BD 621 and BD 607 assessed by fluorescence measurement was reversible, abolished by photoaffinity labeling with Ro 15-4513, and unaffected by a variety of substances that do not bind to benzodiazepine receptors. The potencies of chemically diverse benzodiazepine receptor compounds to inhibit fluorescent ligand binding were highly correlated (r = 0.94, P less than 0.001), with potencies obtained from radioligand binding techniques. These findings demonstrate the feasibility of using direct fluorescence measurement techniques to quantitate ligand-receptor interactions.     

Regulation of γ-Aminobutyric Acid/Barbiturate Receptor-Gated Chloride Ion Flux in Brain Vesicles by Phospholipase A2: Possible Role of Oxygen Radicals

Preincubation of brain membranes with phospholipase A2 (PLA2) has been shown previously to affect the binding characteristics of various recognition sites associated with the gamma-aminobutyric acid (GABA) receptor complex. In the present study, we have investigated the effects of PLA2 (from Naja naja siamensis venom) on the functional activity of the GABA receptor/chloride ion channel. PLA2 (0.001-0.02 U/mg protein) preincubation decreased pentobarbital-induced 36Cl- efflux and muscimol-induced 36Cl- uptake in rat cerebral cortical synaptoneurosomes. The effect of PLA2 was prevented by EGTA and two nonselective PLA2 inhibitors, mepacrine and bromophenacyl bromide. The removal of free fatty acids by addition of bovine serum albumin both prevented and reversed the effect of PLA2. Products of the catalytic activity of PLA2, such as the unsaturated free fatty acids, arachidonic and oleic acids, mimicked the effect of PLA2. However, the saturated fatty acid, palmitic acid, and lysophosphatidyl choline had no effect on pentobarbital-induced 36Cl- efflux. Because unsaturated free fatty acids are highly susceptible to peroxidation by oxygen radicals, the role of oxygen radicals was investigated. Xanthine plus xanthine oxidase, a superoxide radical generating system, mimicked the effect of PLA2, whereas the superoxide radical scavenger, superoxide dismutase, diminished the effects of PLA2 and arachidonic acid on pentobarbital-induced 36Cl- efflux. Similarly, the effect of PLA2 was also inhibited by methanol (1 mM), a scavenger of the hydroxyl radical, and by catalase. These data indicate that exogenously added PLA2 induces alterations in membrane phospholipids, possibly promoting the generation of oxygen radicals and fatty acid peroxides which can ultimately modulate GABA/barbiturate receptor function in brain.     

Differences in the Biophysical Properties of the Benzodiazepine/γ-Aminobutyric Acid Receptor Chloride Channel Complex in the Long-Sleep and Short-Sleep Mouse Lines

Significant differences in the thermal stability of benzodiazepine receptors were found in cerebral cortical membranes prepared from the long-sleep (LS) and short-sleep (SS) selected mouse lines. Thus, benzodiazepine receptors from LS mice were heat inactivated (55 degrees C) at a significantly faster rate than those from SS mice. Although gamma-aminobutyric acid (GABA) reduced the rate of heat inactivation in both lines, the more rapid rate of inactivation in the LS line was maintained. Furthermore, the potency of GABA to enhance [3H]flunitrazepam binding decreased threefold in membranes from LS mice as the incubation temperature was increased from 0 degrees to 37 degrees C, but was unaltered in membranes from SS mice. These differences in the biophysical properties of the benzodiazepine/GABA receptor chloride channel complex ("supramolecular complex"), together with a higher KD for t-[35S]butylbicyclophosphorothionate in membranes from LS compared to SS mice, suggest that the supramolecular complex may modulate the differential sensitivity to some depressants and convulsants in these lines.     

Tissue specific regulation of "peripheral-type" benzodiazepine receptor density after chemical sympathectomy

The characteristics of [3H]Ro 5-4864 binding to "peripheral" benzodiazepine receptors (PBR) in the central nervous system and peripheral tissues were examined after chemical sympathectomy with 6-hydroxydopamine (6-OHDA). One week after the intracisternal administration of 6-OHDA, the number of [3H]Ro 5-4864 binding sites (Bmax) in the hypothalamus and striatum increased 41 and 50%, respectively, concurrent with significant reductions in catecholamine content. An increase (34%) in the Bmax of [3H]Ro 5-4864 to cardiac ventricle was observed one week after parenteral 6-OHDA administration. In contrast, the Bmax of [3H]Ro 5-4864 to pineal gland decreased 48% after 6-OHDA induced reduction in norepinephrine content. The Bmax values for [3H]Ro 5-4864 binding to other tissues (including lung, kidney, spleen, cerebral cortex, cerebellum, hippocampus and olfactory bulbs) were unaffected by 6-OHDA administration. The density of pineal, but not cardiac PBR was also reduced after reserpine treatment, an effect reversed by isoproterenol administration. These findings demonstrate that alterations in sympathetic input may regulate the density of PBR in both the central nervous system and periphery in a tissue specific fashion.     

Stress modulates calcium mobilization in immune cells

Both acute and chronic restraint stress modulated mitogen-induced increases in cytoplasmic free-calcium concentrations ([Ca2+]i) in mouse spleen cells. Dual-color analysis of lymphocyte subpopulations demonstrated that acute (2 hour) restraint stress suppressed mitogen-stimulated increases in [Ca2+]i in CD4+ T cells, but enhanced [Ca2+]i in CD8+ T cells. Chronic restraint stress (2 hours daily for up to 21 days) resulted in a significant suppression of mitogen-stimulated increases in [Ca2+]i in CD4+ T cells at 3 and 7 days, but not at 21 days. CD8+ T cells were unaffected by chronic stress. Chronic stress (for 7 days) had a modest suppressive effect on mitogen-induced Ca2+ responses in B cells. Within T lymphocyte subpopulations, both acute and chronic stress predominantly affected CD4+ T cells, which may induce a functional reversal of the CD4/CD8 ratios in vivo. Such a reversal could result in suppression of a variety of immune responses such as lymphocyte proliferation and antigen-specific antibody production. These findings indicate that the inhibitory effects of stress on calcium mobilization in lymphocytes may be an early event mediating stress-induced immunosuppression.     

Genetic Controls over Activities of Tyrosinase and Dopachrome Conversion Factor in Murine Melanocytes

We evaluated the three catalytic activities of tyrosinase and one activity of dopachrome conversion factor (DCF) in extracts made from skins of 6-day-old yellow and nonyellow mice. At least one of the catalytic activities of tyrosinase and of DCF correlate with the color of pigment being produced in the hair follicles of the mice. We use these data to evaluate existing hypotheses about the mechanism of the interacting genetic controls over melanogenesis.