The sodium concentration of the lateral intercellular spaces of MDCK cells: A microspectrofluorimetric study

MDCK cell monolayers grown on glass coverslips were used to examine the Na⁺ concentration in individual lateral intercellular spaces (LIS) by video fluorescence microscopy. The LIS was filled with the Na⁺-sensitive fluorescent dye SBFO by incubation of the monolayers for 75–90 min with 250 m of the membrane impermeant form of the dye. After dye loading, the monolayers were perfused at 37°C with solutions buffered with HEPES or bicarbonate/CO₂ containing 142 mm Na⁺. Ratios of the fluorescence images after sequential excitation with 340 nm and 380 nm light were performed and in situ calibration of LIS Na⁺ was accomplished after blocking the Na⁺ pump with 5 × 10^–4 m ouabain. Measurements of Na⁺ along the basolateral-to-apical axis of the LIS at 1.0 or 1.5 m intervals did not reveal a Na⁺ gradient when the perfusate was either HEPES or bicarbonate/CO₂ solutions. In bicarbonate solutions, the mean Na⁺ concentration (mm) was 157.2 ± 2.3, 15 mm higher than the bath Na⁺ concentration. In HEPES solutions, however, the Na⁺ concentration was not different from the bath concentration (142.7 ± 3.1 mm). The time course of Na⁺ changes in LIS was investigated by rapidly switching the perfusate from 142 to 80 mm Na⁺ and measuring the Na⁺ changes at one focal plane.We would like to thank P.H. Tran and C. Gibson for their technical and computational assistance as well as Dr. B.-E. Persson (University of Uppsala, Sweden) for his contribution in the early phases of the study.     

A Novel Optical Intracellular Imaging Approach for Potassium Dynamics in Astrocytes

Astrocytes fulfill a central role in regulating K⁺ and glutamate, both released by neurons into the extracellular space during activity. Glial glutamate uptake is a secondary active process that involves the influx of three Na⁺ ions and one proton and the efflux of one K⁺ ion. Thus, intracellular K⁺ concentration ([K⁺]_i) is potentially influenced both by extracellular K⁺ concentration ([K⁺]_o) fluctuations and glutamate transport in astrocytes. We evaluated the impact of these K⁺ ion movements on [K⁺]_i in primary mouse astrocytes by microspectrofluorimetry. We established a new noninvasive and reliable approach to monitor and quantify [K⁺]_i using the recently developed K⁺ sensitive fluorescent indicator Asante Potassium Green-1 (APG-1). An in situ calibration procedure enabled us to estimate the resting [K⁺]_i at 133±1 mM. We first investigated the dependency of [K⁺]_i levels on [K⁺]_o. We found that [K⁺]_i followed [K⁺]_o changes nearly proportionally in the range 3–10 mM, which is consistent with previously reported microelectrode measurements of intracellular K⁺ concentration changes in astrocytes. We then found that glutamate superfusion caused a reversible drop of [K⁺]_i that depended on the glutamate concentration with an apparent EC₅₀ of 11.1±1.4 µM, corresponding to the affinity of astrocyte glutamate transporters. The amplitude of the [K⁺]_i drop was found to be 2.3±0.1 mM for 200 µM glutamate applications. Overall, this study shows that the fluorescent K⁺ indicator APG-1 is a powerful new tool for addressing important questions regarding fine [K⁺]_i regulation with excellent spatial resolution.     

Repeated oral administration of capsaicin increases anxiety-like behaviours with prolonged stress-response in rats

This study was conducted to examine the psycho-emotional effects of repeated oral exposure to capsaicin, the principal active component of chili peppers. Each rat received 1 mL of 0.02% capsaicin into its oral cavity daily, and was subjected to behavioural tests following 10 daily administrations of capsaicin. Stereotypy counts and rostral grooming were significantly increased, and caudal grooming decreased, in capsaicin-treated rats during the ambulatory activity test. In elevated plus maze test, not only the time spent in open arms but also the percent arm entry into open arms was reduced in capsaicin-treated rats compared with control rats. In forced swim test, although swimming duration was decreased, struggling increased in the capsaicin group, immobility duration did not differ between the groups. Repeated oral capsaicin did not affect the basal levels of plasma corticosterone; however, the stress-induced elevation of plasma corticosterone was prolonged in capsaicin treated rats. Oral capsaicin exposure significantly increased c-Fos expression not only in the nucleus tractus of solitarius but also in the paraventricular nucleus. Results suggest that repeated oral exposure to capsaicin increases anxiety-like behaviours in rats, and dysfunction of the hypothalamic-pituitary-adrenal axis may play a role in its pathophysiology.     

Cytoplasmic methionyl-tRNA synthetase from Bakers' yeast. A monomer with a post-translationally modified N terminus

Methionyl-tRNA synthetase has been purified from a yeast strain carrying the MES1 structural gene on a high copy number plasmid (pFL1). The purified enzyme is a monomer of Mr = 85,000 in contrast to its counterpart from Escherichia coli which is a dimer made up of identical subunits (Mr = 76,000; Dardel, F., Fayat, G., and Blanquet, S. (1984) J. Bacteriol. 160, 1115-1122). The yeast enzyme was not amenable to Edman's degradation indicating a blocked NH2 terminus. Its primary structure as derived from the DNA sequence (Walter, P., Gangloff, J., Bonnet, J., Boulanger, Y., Ebel, J.P., and Fasiolo, F. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2437-2441) has been confirmed using the fast atom bombardment-mass spectrometric method. This method was applied to tryptic digests of the carboxymethylated enzyme and the corresponding data provided extensive coverage of the translated DNA sequence, thus confirming its correctness. The ambiguity concerning which of the three NH2-terminally located methionine codons is the initiation codon was easily resolved from peptides identified in this region. It was possible to show that the first methionine had been removed and that the new NH2 terminus, serine, had been acetylated. A comparison between the yeast and E. coli sequences shows that the former has an N-terminal extension of about 200 residues as compared to the latter. It also lacks the C-terminal domain which is responsible for the dimerization of the E. coli methionyl-tRNA synthetase.     

Flash photolysis using a light emitting diode: an efficient, compact, and affordable solution

Flash photolysis has become an essential technique for dynamic investigations of living cells and tissues. This approach offers several advantages for instantly changing the concentration of bioactive compounds outside and inside living cells with high spatial resolution. Light sources for photolysis need to deliver pulses of high intensity light in the near UV range (300-380 nm), to photoactivate a sufficient amount of molecules in a short time. UV lasers are often required as the light source, making flash photolysis a costly approach. Here we describe the use of a high power 365 nm light emitting diode (UV LED) coupled to an optical fiber to precisely deliver the light to the sample. The ability of the UV LED light source to photoactivate several caged compounds (CMNB-fluorescein, MNI-glutamate, NP-EGTA, DMNPE-ATP) as well as to evoke the associated cellular Ca(2+) responses is demonstrated in both neurons and astrocytes. This report shows that UV LEDs are an efficient light source for flash photolysis and represent an alternative to UV lasers for many applications. A compact, powerful, and low-cost system is described in detail.     

Cell-Type-Specific Gene Expression Profiling in Adult Mouse Brain Reveals Normal and Disease-State Signatures

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Role of the JNK pathway in NMDA-mediated excitotoxicity of cortical neurons

Excitotoxic insults induce c-Jun N-terminal kinase (JNK) activation, which leads to neuronal death and contributes to many neurological conditions such as cerebral ischemia and neurodegenerative disorders. The action of JNK can be inhibited by the D-retro-inverso form of JNK inhibitor peptide (D-JNKI1), which totally prevents death induced by N-methyl-D-aspartate (NMDA) in vitro and strongly protects against different in vivo paradigms of excitotoxicity. To obtain optimal neuroprotection, it is imperative to elucidate the prosurvival action of D-JNKI1 and the death pathways that it inhibits. In cortical neuronal cultures, we first investigate the pathways by which NMDA induces JNK activation and show a rapid and selective phosphorylation of mitogen-activated protein kinase kinase 7 (MKK7), whereas the only other known JNK activator, mitogen-activated protein kinase kinase 4 (MKK4), was unaffected. We then analyze the action of D-JNKI1 on four JNK targets containing a JNK-binding domain: MAPK-activating death domain-containing protein/differentially expressed in normal and neoplastic cells (MADD/DENN), MKK7, MKK4 and JNK-interacting protein-1 (IB1/JIP-1).     

Long-term treadmill exercise improves spatial memory of male APPswe/PS1dE9 mice by regulation of BDNF expression and microglia activation

Increasing evidence suggests that physical activity could delay or attenuate the symptoms of Alzheimer''s disease (AD). But the underlying mechanisms are still not fully understood. To investigate the effect of long-term treadmill exercise on the spatial memory of AD mice and the possible role of β-amyloid, brain-derived neurotrophic factor (BDNF) and microglia in the effect, male APPswe/PS1dE9 AD mice aged 4 months were subjected to treadmill exercise for 5 months with 6 sessions per week and gradually increased load. A Morris water maze was used to evaluate the spatial memory. Expression levels of β-amyloid, BDNF and Iba-1 (a microglia marker) in brain tissue were detected by immunohistochemistry. Sedentary AD mice and wildtype C57BL/6J mice served as controls. The results showed that 5-month treadmill exercise significantly decreased the escape latencies (P < 0.01 on the 4th day) and improved the spatial memory of the AD mice in the water maze test. Meanwhile, treadmill exercise significantly increased the number of BDNF-positive cells and decreased the ratios of activated microglia in both the cerebral cortex and the hippocampus. However, treadmill exercise did not significantly alleviate the accumulation of β-amyloid in either the cerebral cortex or the hippocampus of the AD mice (P > 0.05). The study suggested that long-term treadmill exercise could improve the spatial memory of the male APPswe/PS1dE9 AD mice. The increase in BDNF-positive cells and decrease in activated microglia might underpin the beneficial effect.