Cyclic AMP imaging in neurones in brain slice preparations.
The second messenger cascade of cyclic AMP (cAMP) plays an important physiological role in neurones, modulating neuronal excitability and synaptic transmission. The fluorescent probe FlCRhR allows real time ratiometric imaging of cAMP changes inside cells (Nature 349 (1991) 694). Until now, the only way to introduce FlCRhR into cells was microinjection, which restricted the use of FlCRhR to large invertebrate neurones. This report describes the use of the patch-clamp technique to deliver FlCRhR into the cytosol of several types of neurones in brain slice preparations. Direct activation of adenylate cyclase by forskolin produced marked increases in fluorescence ratio, confirming that the probe can report cAMP increases. However, some neurones failed to exhibit a cAMP response and this lack of response was related to the nucleus integrity. Stimulation of membrane receptors positively coupled to adenylate cyclase elicited cAMP increases in various neuronal cell types. This is the first report of a cAMP response to neuromodulators measured by an imaging technique in neurones in brain slices. The method described here could find many applications such as testing the ability of agonists to specifically activate the cAMP cascade in identified neurones, studying the kinetics of the cAMP response and determining the subcellular localisation of cAMP changes.
Vincent
P
P
Equipe Neurobiologie Cellulaire, Neurobiologie des Processus Adaptatifs FRE 2371, CNRS Université Paris VI, Mailbox #16, 9, quai St. Bernard, F-75005 Paris, France. pvincent@ccr.jussieu.fr
Brusciano
D
D
D000818:Animals; D001921:Brain / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000032:analysis* / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000493:pharmacokinetics; D003712:Dendrites / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000494:pharmacology; D019793:Fluorescein; D005456:Fluorescent Dyes / Q000493:pharmacokinetics*; D019204:GTP-Binding Proteins / Q000187:drug effects / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008839:Microelectrodes / Q000592:standards; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009924:Organ Culture Techniques / Q000295:instrumentation / Q000379:methods*; D018408:Patch-Clamp Techniques / Q000295:instrumentation / Q000379:methods*; D011874:Radiometry / Q000295:instrumentation / Q000379:methods; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism; D012235:Rhodamines
D005456:Fluorescent Dyes; D011956:Receptors, Cell Surface; D012235:Rhodamines; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D005576:Colforsin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D019204:GTP-Binding Proteins; D019793:Fluorescein