Imaging synaptic inhibition in transgenic mice expressing the chloride indicator, Clomeleon

We describe here a molecular genetic approach for imaging synaptic inhibition. The thy-1 promoter was used to express high levels of Clomeleon, a ratiometric fluorescent indicator for chloride ions, in discrete populations of neurons in the brains of transgenic mice. Clomeleon was functional after chronic expression and provided non-invasive readouts of intracellular chloride concentration ([Cl(-)](i)) in brain slices, allowing us to quantify age-dependent declines in resting [Cl(-)](i) during neuronal development. Activation of hippocampal interneurons caused [Cl(-)](i) to rise transiently in individual postsynaptic pyramidal neurons. [Cl(-)](i) increased in direct proportion to the amount of inhibitory transmission, with peak changes as large as 4 mM. Integrating responses over populations of pyramidal neurons allowed sensitive detection of synaptic inhibition. Thus, Clomeleon imaging permits non-invasive, spatiotemporally resolved recordings of [Cl(-)](i) in a large variety of neurons, opening up new opportunities for imaging synaptic inhibition and other forms of chloride signaling.     

Imaging synaptic inhibition throughout the brain via genetically targeted Clomeleon

Here we survey a molecular genetic approach for imaging synaptic inhibition. This approach is based on measuring intracellular chloride concentration ([Cl(-)](i)) with the fluorescent chloride indicator protein, Clomeleon. We first describe several different ways to express Clomeleon in selected populations of neurons in the mouse brain. These methods include targeted viral gene transfer, conditional expression controlled by Cre recombination, and transgenesis based on the neuron-specific promoter, thy1. Next, we evaluate the feasibility of using different lines of thy1::Clomeleon transgenic mice to image synaptic inhibition in several different brain regions: the hippocampus, the deep cerebellar nuclei (DCN), the basolateral nucleus of the amygdala, and the superior colliculus (SC). Activation of hippocampal interneurons caused [Cl(-)](i) to rise transiently in individual postsynaptic CA1 pyramidal neurons. [Cl(-)](i) increased linearly with the number of electrical stimuli in a train, with peak changes as large as 4 mM. These responses were largely mediated by GABA receptors because they were blocked by antagonists of GABA receptors, such as GABAzine and bicuculline. Similar responses to synaptic activity were observed in DCN neurons, amygdalar principal cells, and collicular premotor neurons. However, in contrast to the hippocampus, the responses in these three regions were largely insensitive to antagonists of inhibitory neurotransmitter receptors. This indicates that synaptic activity can also cause Cl(-) influx through alternate pathways that remain to be identified. We conclude that Clomeleon imaging permits non-invasive, spatiotemporally precise recordings of [Cl(-)](i) in a large variety of neurons, and provides new opportunities for imaging synaptic inhibition and other forms of neuronal chloride signaling.     

Requirements for maximal enrichment of viable intraerythrocytic Plasmodium falciparum rings by saponin hemolysis

The purpose of the present study was to confirm the effectiveness of saponin hemolysis for concentrating ring-infected erythrocytes in Plasmodium falciparum cultures and to determine the actual numbers of the enriched parasites, not just percentage parasitemia. This is important because various molecular biology and vaccine development against malaria require useable quantities of pure culture with minimal number of uninfected erythrocytes at all stages. Synchronized cultures of three P. falciparum strains were exposed to 0.015% isotonic saponin solution for 30 minutes on ice. They were centrifuged and the pellets were treated again with saponin solution for 3-7 minutes. Initially, most of the cultures contained approximately 10(10) erythrocytes and 1-7% parasitemia, but at the end of the enrichment up to 10(8) of erythrocytes containing 90-99.8% parasitemia were recovered (maximal enrichment). From microscopic examination of the cells it was calculated that the hemolysis rate of uninfected and infected erythrocytes was circa 27 to 1, which could account for the enrichment. Studies by other investigators have suggested that P. falciparum merozoite invasion decreases erythrocyte membrane lipids, and it has been reported that reduction of membrane cholesterol could make erythrocytes saponin-resistant. The possibility that merozoite invasion made erythrocytes partially resistant to saponin hemolysis was strengthened by the observation that the proportions of multiple infections increased significantly in the enriched cultures. However, mature asexual parasites could not be concentrated by this method, suggesting possible differences between the membranes of erythrocytes containing ring forms and those of trophozoites and schizonts. Ring-infected erythrocytes freshly from malaria patients could also not be concentrated by the method described here, suggesting that the ability to induce saponin resistance in erythrocytes was acquired by the parasites in vitro.     

Immunohistochemical analysis of the vitellogenin response in the liver of Atlantic salmon exposed to environmental oestrogens

Induction of vitellogenin (Vtg) in oviparous vertebrates has been used as a biomarker of response for environmental oestrogens. This study reports the cellular localization of oestrogen- and xenoestrogen-induced Vtg synthesis in the liver of juvenile Atlantic salmon (Salmo salar). Paraffin-embedded liver sections were incubated with homologous monoclonal antibody against Atlantic salmon Vtg. Following intraperitoneal (ip) exposure of fish to estradiol-17beta E₂; 5 mg kg-1 or 4-nonylphenol NP; 125 mg kg-1, Vtg induction was primarily demonstrated immunohistochemically in the cytoplasm of hepatocytes, endothelial cells and within hepatic sinusoids. Vtg staining of hepatocytes was not evenly distributed, as there was a high degree of polarization toward the sinusoid. The intensity of positive Vtg staining was stronger in the liver sections of E₂-treated fish, compared with NP-treated fish. Hepatocytes of E₂-, NP- and vehicle (control)-treated fish showed normal cellular structures, thus showing no evidence of histopathological changes. In parallel, indirect enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis of plasma Vtg levels show significant induction of Vtg in E₂- and NP-treated fish, as compared with untreated (control) fish. The present study demonstrates the applicability of immunohistochemistry in studies of cellular structures, processes and responses of fish exposure to oestrogen and oestrogen-mimicking compounds.     

Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes

Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.     

A simple method for preparation of cultured cardiac fibroblasts from adult human ventricular tissue

Cardiac fibroblasts constitute greater than 90% of non-myocyte cells in the heart. Because they are responsible for synthesis of components of the extracellular matrix, growth factors and cytokines in the myocardium, they play an important role in normal and pathologic performance of the heart. An understanding of their biology requires in depth studies in a stable and reliable system in which the biological responses of cardiac fibroblasts to various stimuli can be determined. With the exception of few, all studies have been performed on cardiac fibroblasts obtained from rodent hearts. We present a method for isolation and subsequent culture of viable cardiac fibroblasts from ventricular tissue of adult human. This method allows rapid and reliable isolation and subsequent culture of cardiac fibroblasts from adult heart tissue without the need for cumbersome isolation techniques and complex nutrient-enriched and hormone-supplemented culture media for maintenance.     

Low-dose carbon monoxide reduces airway hyperresponsiveness in mice

Carbon monoxide (CO) in expired gas has been shown to be elevated with asthma; however, its function is not known, and there is some potential that it may serve a bronchoprotective role to decrease airway hyperresponsiveness (AHR). Thus the ability of CO to reverse methacholine (MCh)-induced bronchoconstriction was evaluated in C57BL/6 (C57) and A/J mice with and without airway inflammation produced by ovalbumin (OVA). Acutely administered CO (1% in air, 10 min) reduced MCh-driven increases in lung resistance in OVA-challenged C57 mice by an average of 50% (from 14.5 to 7.1 cmH2O.ml-1.s-1), whereas no effect was observed in na?ve C57 mice or OVA-challenged C57 mice inhaling air alone. Acutely inhaled CO (500 ppm = 0.05%, for 10 min) reduced MCh-induced airway reactivity (AR) by 20-60% in airway hyperresponsive na?ve A/J mice, whereas repeated 10-min administrations of 500 ppm CO over a 5-day period decreased AR by 50%. Repeated administration of low-dose CO [250 (0.025%) and (0.05%) 500 ppm, 1 h/day, 5 days] to A/J mice with airway inflammation likewise resulted in a drop of AR by 50%, compared with those not receiving CO. Inhibition of guanylyl cyclase/guanosine 3',5'-cyclic monophosphothioate (cGMP) using 1H-[1,2,4] oxydiazolo[4,3-a]quinoxalin-1-one or a competitive inhibitor, Rp diastereomers of 8-bromo-cGMP, resulted in inhibition of the effect of CO on AHR, suggesting that the effects of CO were mediated through this mechanism. These results indicate that low-dose CO can effectively reverse AHR in the presence and absence of airway inflammation in mice and suggest a potential role for CO in the modulation of AHR.     

Chasing coccidia--new tools enter the race

The 8th International Coccidiosis Conference, held on 9--13 July 2001 in Palm Cove, Australia, was a showcase of the latest studies on widely known coccidia, including Eimeria and Toxoplasma in addition to the emerging or re-emerging parasites such as Neospora, Cryptosporidium and Cyclospora. This meeting was staged in conjunction with the Annual Scientific Meeting of the Australian Society for Parasitology.