isoforms present in the same samples to examine the correlation of these proteins with one another and with the MHC and Tm mRNAs. These analyses revealed significant correlations among the different myofibrillar proteins, with the S1 and S2 fibers being characterized by a whole assemblage of myofibrillar isoforms. However, they also showed that small muscle samples, and more importantly single fibers, existed as a continuum from one phenotype to another. Most fibers possessed mixtures of mRNA for MHC isoforms that were unexpected based on protein analysis. These findings illustrate that muscle fibers in general may possess a phenotype that is intermediate between the extremes of 'pure' fiber types, not only at the MHC level but also in terms of whole myofibrillar assemblages. This study supports and extends our recent observations of mixed phenotype fibers in lobster claw and leg muscles. The existence of single fiber polymorphism in an invertebrate species underscores the generality of the phenomenon in skeletal muscles and emphasizes the need for an understanding of the proximal causes and physiological consequences of these intermediate fiber types.

The suprachiasmatic nucleus (SCN) is required for the daily rhythm of plasma glucocorticoids; however, the independent contributions from oscillators within the different subregions of the SCN to the glucocorticoid rhythm remain unclear. Here, we use genetically and neurologically intact, forced desynchronized rats to test the hypothesis that the daily rhythm of the glucocorticoid, corticosterone, is regulated by both light responsive and light-dissociated circadian oscillators in the ventrolateral (vl-) and dorsomedial (dm-) SCN, respectively. We show that when the vlSCN and dmSCN are in maximum phase misalignment, the peak of the plasma corticosterone rhythm is shifted and the amplitude reduced; whereas, the peak of the plasma adrenocorticotropic hormone (ACTH) rhythm is also reduced, the phase is dissociated from that of the corticosterone rhythm. These data support previous studies suggesting an ACTH-independent pathway contributes to the corticosterone rhythm. To determine if either SCN subregion independently regulates corticosterone through the sympathetic nervous system, we compared unilateral adrenalectomized, desynchronized rats that had undergone either transection of the thoracic splanchnic nerve or sham transection to the remaining adrenal. Splanchnicectomy reduced and phase advanced the peak of both the corticosterone and ACTH rhythms. These data suggest that both the vlSCN and dmSCN contribute to the corticosterone rhythm by both reducing plasma ACTH and differentially regulating plasma corticosterone through an ACTH- and sympathetic nervous system-independent pathway.

The master circadian clock located within the hypothalamic suprachiasmatic nucleus (SCN) is necessary for the circadian rhythm of glucocorticoid (GC) release. The pathways by which the SCN sustains rhythmic GC release remain unclear. We studied the circadian regulation of cortisol release in the behaviorally split golden hamster, in which the single bout of circadian locomotor activity splits into two bouts approximately 12 h apart after exposing the animals to constant light conditions. We show that unsplit control hamsters present a single peak of cortisol release that is concomitant with a single peak of ACTH release. In contrast, split hamsters show two peaks of cortisol release that are approximately 12 h appart and are appropriately phased to each locomotor activity bout but surprisingly do not rely on rhythmic release of ACTH. Our results are consistent with a model in which the circadian pacemaker within the SCN regulates the circadian release of GC via input to the hypothalamo-pituitary-adrenal axis and via a second regulatory pathway, which likely involves sympathetic innervation of the adrenal and can operate even in the absence of ACTH circadian rhythmic release. Furthermore, we show that although the overall 24-h cortisol output in split hamsters is lower than in unsplit controls, split hamsters release constant low levels of ACTH. This result suggests that the timing, rather than the absolute amount, of cortisol release is more critical for the induction of negative feedback effects that regulate the hypothalamo-pituitary-adrenal axis.

Lobster claw muscles undergo a process of fiber switching during development, where isomorphic muscles containing a mixture of both fast and slow fibers, become specialized into predominantly fast, or exclusively slow, muscles. Although this process has been described using histochemical methods, we lack an understanding of the shifts in gene expression that take place. In this study, we used several complementary techniques to follow changes in the expression of a number of myofibrillar genes in differentiating juvenile lobster claw muscles. RNA probes complementary to fast and slow myosin heavy chain (MHC) mRNA were used to label sections of 7th stage (approximately 3 months old) juvenile claw muscles from different stages of the molt cycle. Recently molted animals (1-5 days postmolt) had muscles with distinct regions of fast and slow gene expression, whereas muscles from later in the molt cycle (7-37 days postmolt) had regions of fast and slow MHC expression that were co-mingled and indistinct. Real-time PCR was used to quantify several myofibrillar genes in 9th and 10th stages (approximately 6 months old) juvenile claws and showed that these genes were expressed at significantly higher levels in the postmolt claws, as compared with the intermolt and premolt claws. Finally, Western blot analyses of muscle fibers from juvenile lobsters approximately 3 to 30 months in age showed a shift in troponin-I (TnI) isoform expression as the fibers differentiated into the adult phenotypes, with expression of the adult fast fiber TnI pattern lagging behind the adult slow fiber TnI pattern. Collectively, these data show that juvenile and adult fibers differ both qualitatively and quantitative in the expression of myofibrillar proteins and it may take as much as 2 years for juvenile fibers to achieve the adult phenotype.

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