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  • Specific Na+ sensors are functionally expressed in a neuronal population of the median preoptic nucleus of the rat. 15102913

    Whole-cell patch-clamp recordings were performed on acute brain slices of male rats to investigate the ability of the neurons of the median preoptic nucleus (MnPO) to detect fluctuation in extracellular osmolarity and sodium concentration ([Na+]out). Local application of hypotonic and hypertonic artificial CSF hyperpolarized and depolarized the neurons, respectively. Similar responses obtained under synaptic isolation (0.5 microM TTX) highlighted the intrinsic ability of the MnPO neurons to detect changes in extracellular osmolarity and [Na+]out. Manipulating extracellular osmolarity, [Na+]out, and [Cl-]out showed in an independent manner that the MnPO neurons responded to a change in [Na+]out exclusively. The specific Na+ response was voltage insensitive and depended on the driving force for Na+ ions, indicating that a sustained background Na+ permeability controlled the membrane potential of the MnPO neurons. This specific response was not reduced by Gd3+, amiloride, or benzamil, ruling out the participation of mechanosensitive cationic channels, specific epithelial Na+ channels, and Phe-Met-Arg-Phe-gated Na+ channels, respectively. Combination of in situ hybridization, using a riboprobe directed against the atypical Na+ channel (Na(X)), and immunohistochemistry, using an antibody against neuron-specific nuclei protein, revealed that a substantial population of MnPO neurons expressed the Na(X) channel, which was characterized recently as a concentration-sensitive Na+ channel. This study shows that a neuronal population of the MnPO acts as functional Na+ sensors and that the Na(X) channel might represent the molecular basis for the extracellular sodium level sensing in these neurons.
    Document Type:
    Reference
    Product Catalog Number:
    MAB377
    Product Catalog Name:
    Anti-NeuN Antibody, clone A60

  • Optical sensors for measuring dynamic changes of cytosolic metabolite levels in yeast. 22036883

    Optical sensors allow dynamic quantification of metabolite levels with subcellular resolution. Here we describe protocols for analyzing cytosolic glucose levels in yeast using genetically encoded Förster resonance energy transfer (FRET) sensors. FRET glucose sensors with different glucose affinities (Kd) covering the low nano- to mid- millimolar range can be targeted genetically to the cytosol or to subcellular compartments. The sensors detect the glucose-induced conformational change in the bacterial periplasmic glucose/galactose binding protein MglB using FRET between two fluorescent protein variants. Measurements can be performed with a single sensor or multiple sensors in parallel. In one approach, cytosolic glucose accumulation is measured in yeast cultures in a 96-well plate using a fluorimeter. Upon excitation of the cyan fluorescent protein (CFP), emission intensities of CFP and YFP (yellow fluorescent protein) are captured before and after glucose addition. FRET sensors provide temporally resolved quantitative data of glucose for the compartment of interest. In a second approach, reversible changes of cytosolic free glucose are measured in individual yeast cells trapped in a microfluidic platform, allowing perfusion of different solutions while FRET changes are monitored in a microscope setup. By using the microplate fluorimeter protocol, 96 cultures can be measured in less than 1 h; analysis of single cells of a single genotype can be completed in <2 h. FRET-based analysis has been performed with glucose, maltose, ATP and zinc sensors, and it can easily be adapted for high-throughput screening using a wide spectrum of sensors.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple