Improved genetically-encoded, FlincG-type fluorescent biosensors for neural cGMP imaging.

Genetically-encoded biosensors are powerful tools for understanding cellular signal transduction mechanisms. In aiming to investigate cGMP signaling in neurones using the EGFP-based fluorescent biosensor, FlincG (fluorescent indicator for cGMP), we encountered weak or non-existent fluorescence after attempted transfection with plasmid DNA, even in HEK293T cells. Adenoviral infection of HEK293T cells with FlincG, however, had previously proved successful.

Picomolar nitric oxide signals from central neurons recorded using ultrasensitive detector cells.

Nitric oxide (NO) is a widespread signaling molecule with potentially multifarious actions of relevance to health and disease. A fundamental determinant of how it acts is its concentration, but there remains a lack of coherent information on the patterns of NO release from its sources, such as neurons or endothelial cells, in either normal or pathological conditions. We have used detector cells having the highest recorded NO sensitivity to monitor NO release from brain tissue quantitatively and in real time.

Exquisite sensitivity to subsecond, picomolar nitric oxide transients conferred on cells by guanylyl cyclase-coupled receptors.

Nitric oxide (NO) functions as a diffusible transmitter in most tissues of the body and exerts its effects by binding to receptors harboring a guanylyl cyclase transduction domain, resulting in cGMP accumulation in target cells. Despite its widespread importance, very little is known about how this signaling pathway operates at physiological NO concentrations and in real time. To address these deficiencies, we have exploited the properties of a novel cGMP biosensor, named δ-FlincG, expressed in cells containing varying mixtures of NO-activated guanylyl cyclase and cGMP-hydrolyzing phosphodiesterase activity.

The mGlu1 antagonist CPCCOEt enhances the climbing fibre response in Purkinje neurones independently of glutamate receptors.

CPCCOEt (7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester) is frequently used to test for the involvement of mGlu1 receptors. Using whole-cell voltage recording from Purkinje cells in slices of rat cerebellum we find that CPCCOEt, at concentrations used to block mGlu1 receptors, causes an enhancement of the climbing fibre response. Application of alternative antagonists with activity at mGlu1 neither mimicked nor occluded the effects of CPCCOEt.

GABAB Receptors in the Parallel Fibre Pathway of Rat Cerebellum.

Binding studies indicate that the molecular layer of the cerebellum has a high concentration of gamma-aminobutyric acid type B (GABAB) receptors. In order to elucidate the function of these receptors we have recorded from Purkinje cells in biplanar slices of immature (14-day-old) and adult rat cerebellum using a low-noise, non-invasive, gap technique. The responses of Purkinje cells to parallel fibre stimulation in slices from both immature and adult rats contained a wave that could be inhibited by the GABAA antagonist, bicuculline.