Extensive overlap of mu-opioid and nicotinic sensitivity in cortical interneurons.

We studied mu-opioid transmission in acute slices of rat neocortex using whole-cell recordings and single-cell reverse transcription-polymerase chain reaction. The mu-opioid receptor (MOR) was found in gamma-aminobutyric acidergic (GABAergic) interneurons that were either layer I cells frequently expressing neuropeptide Y or layers II-V cells expressing vasoactive intestinal peptide and enkephalin (Enk). We found that mu-opioid agonists inhibit these interneurons that are selectively excited by nicotinic agonists. The extensive overlap of mu-opioid and nicotinic responsiveness allowed mu-opioid agonists to inhibit nicotinic excitation of responsive interneurons and of their GABAergic output onto pyramidal cells. Finally, nicotinic stimulation resulted in a dynamic sequence where GABAergic transmission was first enhanced and then depressed below its baseline. This latter disinhibitory effect was prevented by a mu-opioid antagonist, indicating that excitation of nicotinic-responsive interneurons induced the release of endogenous Enk, which in turn led to MOR activation. Our results suggest that neocortical mu-opioid transmission acts as an inhibitory feedback onto nicotinic-responsive interneurons, which may change network excitability and inhibition patterns during cholinergic excitation.

Calcium dependence of aequorin bioluminescence dissected by random mutagenesis.

Aequorin bioluminescence is emitted as a rapidly decaying flash upon calcium binding. Random mutagenesis and functional screening were used to isolate aequorin mutants showing slow decay rate of luminescence. Calcium sensitivity curves were shifted in all mutants, and an intrinsic link between calcium sensitivity and decay rate was suggested by the position of all mutations in or near EF-hand calcium-binding sites. From these results, a low calcium affinity was assigned to the N-terminal EF hand and a high affinity to the C-terminal EF-hand pair. In WT aequorin, the increase of the decay rate with calcium occurred at constant total photon yield and thus determined a corresponding increase of light intensity. Increase of the decay rate was underlain by variations of a fast and a slow component and required the contribution of all three EF hands. Conversely, analyses of double EF-hand mutants suggested that single EF hands are sufficient to trigger luminescence at a slow rate. Finally, a model postulating that proportions of a fast and a slow light-emitting state depend on calcium concentration adequately described the calcium dependence of aequorin bioluminescence. Our results suggest that variations of luminescence kinetics, which depend on three EF hands endowed with different calcium affinities, critically determine the amplitude of aequorin responses to biological calcium signals.

Thermostable mutants of the photoprotein aequorin obtained by in vitro evolution.

Aequorin is a photoprotein that emits light upon binding calcium. Aequorin mutants showing increased intensity or slow decay of bioluminescence were isolated by in vitro evolution combining DNA shuffling and functional screening in bacteria. Luminescence decay mutants were isolated at the first round of screening and carried mutations located in EF-hand calcium binding sites or their vicinity. During in vitro evolution, the luminescence intensity of the population of mutants increased with the frequency of effective mutations whereas the frequency of other amino acid substitutions remained roughly stable. Luminescence intensity mutations neighbored the His-16 or His-169 coelenterazine binding residues or were located in the first EF-hand. None of the selected mutants exhibited an increase in photon yield when examined in a cell-free assay. However, we observed that two mutants, Q168R and L170I, exhibited an increase of the photoprotein lifetime at 37 degrees C that may underlie their high luminescence intensity in bacteria. Further analysis of Q168R and L170I mutations showed that they increased aequorin thermostability. Conversely, examination of luminescence decay mutants revealed that the F149S substitution decreased aequorin thermostability. Finally, screening of a library of random Gln-168 and Leu-170 mutants confirmed the involvement of both positions in thermostability and indicated that optimal thermostability was conferred by Q168R and L170I mutations selected through in vitro evolution. Our results suggest that Phe-149 and Gln-168 residues participate in stabilization of the coelenterazine peroxide and the triggering of photon emission by linking the third EF-hand to Trp-129 and His-169 coelenterazine binding residues.

Global/temporal gene expression in diaphragm and hindlimb muscles of dystrophin-deficient (mdx) mice.

The mdx mouse is a model for human Duchenne muscular dystrophy (DMD), an X-linked degenerative disease of skeletal muscle tissue characterized by the absence of the dystrophin protein. The mdx mice display a much milder phenotype than DMD patients. After the first week of life when all mdx muscles evolve like muscles of young DMD patients, mdx hindlimb muscles substantially compensate for the lack of dystrophin, whereas mdx diaphragm muscle becomes progressively affected by the disease. We used cDNA microarrays to compare the expression profile of 1,082 genes, previously selected by a subtractive method, in control and mdx hindlimb and diaphragm muscles at 12 time points over the first year of the mouse life. We determined that 1) the dystrophin gene defect induced marked expression remodeling of 112 genes encoding proteins implicated in diverse muscle cell functions and 2) two-thirds of the observed transcriptomal anomalies differed between adult mdx hindlimb and diaphragm muscles. Our results showed that neither mdx diaphram muscle nor mdx hindlimb muscles evolve entirely like the human DMD muscles. This finding should be taken under consideration for the interpretation of future experiments using mdx mice as a model for therapeutic assays.