Patricia J. Sollars

Modulation of IL-1β gene expression in the rat CNS during sleep deprivation

We hypothesize that sleep homeostasis involves, at least in part, the immune system modulator interleukin-1β (IL-1β). Using the reverse transcription-polymerase chain reaction, IL-1β mRNA levels in the rat CNS were evaluated after a period of sleep deprivation. In addition, IL-1β gene expression was analyzed before the projected onset of activity and rest phase in free-running animals. No changes in IL-1β mRNA were observed in the circadian cycle, but 24 h of sleep deprivation resulted in a 2-fold increase in the level of IL-1β mRNA in the hypothalamus and in the brain stem compared with controls (p< 0.0002 and (p < 0.0001 respectively). The alteration in IL-1β mRNA levels following sleep deprivation supports the hypothesis that modulation of IL-1β gene expression is involved in the sleep homeostatic process.

Mutagenesis and behavioral screening for altered circadian activity identifies the mouse mutant, Wheels.

The molecular processes underlying the generation of circadian behavior in mammals are virtually unknown. To identify genes that regulate or alter circadian activity rhythms, a mouse mutagenesis program was initiated in conjunction with behavioral screening for alterations in circadian period (tau), a fundamental property of the biological clock. Male mice of the inbred BALB/c strain, treated with the potent mutagen N-ethyl-N-nitrosourea were mated with wild-type hybrids. Wheel-running activity of approximately 300 male progeny was monitored for 6-10 weeks under constant dark (DD) conditions. The tau DD of a single mouse (#187) was longer than the population mean by more than three standard deviations (24.20 vs. 23.32 +/- 0.02 h; mean +/- S.E.M.; n = 277). In addition, mouse #187 exhibited other abnormal phenotypes, including hyperactive bi-directional circling/spinning activity and an abnormal response to light. Heterozygous progeny of the founder mouse, generated from outcrossings with wild-type C57BL/6J mice, displayed lengthened tau DD although approximately 20% of the animals showed no wheel-running activity despite being quite active. Under light:dark conditions, all animals displaying circling behavior that ran in the activity wheels exhibited robust wheel-running activity at lights-ON and these animals also showed enhanced wheel-running activity in constant light conditions. The genetic dissection of the complex behavior associated with this mutation was facilitated by the previously described genetic mapping of the mutant locus causing circling behavior, designated Wheels (Whl), to the subcentromeric portion of mouse chromosome 4. In this report, the same locus is shown to be responsible for the abnormal responses to light and presumably for the altered circadian behavior. Characterization of the gene altered in the novel Whl mutation will contribute to understanding the molecular elements involved in mammalian circadian regulation.

Vasoactive intestinal peptide efferent projections of the suprachiasmatic nucleus in anterior hypothalamic transplants : correlation with functional restoration of circadian behavior

Circadian rhythmicity can be restored by transplantation of fetal anterior hypothalamic (AH) tissue containing the suprachiasmatic nucleus (SCN) into hosts rendered arrhythmic by SCN ablation. However, the nature of the SCN effector pathways mediating functional recovery has remained elusive. To examine implant-derived SCN innervation of the host, AH homografts (hamster-to-hamster) and heterografts (mouse- or rat-to-hamster) were employed and the distribution of vasoactive intestinal peptide (VIP) within the SCN terminal fields was evaluated. A comparison was made between cases where circadian locomotor activity was restored and cases where circadian rhythmicity remained disrupted following AH transplantation. A dense aggregation of VIP neurons and processes was identified in each transplant that restored behavioral rhythmicity in the host. In these cases, SCN-derived VIP fibers were integrated with the host brain and could be identified in host terminal fields typically innervated by SCN-VIP fibers. A correlation was noted between VIP innervation of the host paraventricular thalamic nucleus (PVT) and restoration of circadian rhythmicity. Neither qualitative nor quantitative differences in transplant VIP projections were noted between AH homografts and heterografts. These results demonstrate that SCN VIP neurons in AH transplants send an appropriately restricted set of efferent projections to the host brain and suggest that SCN efferent projections to the PVT may participate in mediating the functional recovery of circadian locomotor activity.

Altered circadian rhythmicity in the Wocko mouse, a hyperactive transgenic mutant

INDUCED changes in the level of daily activity can alter the period of the mammalian circadian clock. In this report, we examined the period of the circadian rhythm of wheel-running activity in a transgenic neurological mouse mutant, Wocko. Wocko mice display a dominant behavioral phenotype that consists of hyperactivity, circling and head tossing. The period of the circadian rhythm of wheel-running activity in constant dark conditions was significantly shorter in mice expressing the Wocko mutation than in their normal littermates. Total activity, monitored by the interruption of an array of infrared beams, was significantly elevated in Wocko mice. These findings support the view that spontaneous exercise can modulate the circadian timekeeping system.

Time course of fiber outgrowth from fetal anterior hypothalamic heterografts

Fetal anterior hypothalamic (AH) heterografts can restore circadian rhythmicity to animals rendered arrhythmic following ablation of the suprachiasmatic nucleus (SCN). Behavioral restoration of circadian activity typically begins between two and six weeks post-implantation. The time course of fiber outgrowth from fetal AH heterografts was examined to determine whether neuronal outgrowth from the implants precedes the typically observed effects of such implants upon circadian behavior. Fetal mouse or rat AH tissue containing the SCN was implanted into the third ventricle of SCN-lesioned hamsters. Using species-specific monoclonal antibodies generated against mouse or rat neuronal elements, fiber outgrowth into the host hypothalamus was examined at 2, 4, 7, 14, 30 and 45 days after implantation. Fibers were observed to have emerged from the implant at the earliest time point examined. Four days after surgery, individual fibers had extended up to 0.6 mm into the host neuropil. By 14 days post-implantation, outgrowth from the implant had formed a dense fiber plexus in the host hypothalamus. This observation demonstrates that neuronal integration of the implant with the host brain begins within 48 hours of implantation, and is extensively established well before a restoration of rhythmicity is typically observed. Thus, on the basis of the time course of fiber outgrowth, it is clear that neuronal contact between graft and host may mediate the observed restoration of circadian rhythmicity.

Thyrotropin-releasing hormone phase shifts circadian rhythms in hamsters

The role of thyrotropin-releasing hormone (TRH) in regulating circadian rhythms was investigated by assessing the ability of TRH microinjections into the suprachiasmatic nucleus (SCN) to induce phase shifts in hamster wheel-running behavior. TRH injected into the SCN at 10 and 100 nM doses produced phase advances in wheel-running activity of 18.3 +/- 1.9 and 34.8 +/- 2.9 minutes, respectively, when administered at circadian time (CT) 6. Injections at CT 18 produced no effects. The temporal sensitivity of the SCN to TRH administration was examined by administering TRH at specific circadian times. TRH produced significant phase advances at CT 4, 6, and 8, while no significant changes in wheel-running onset were observed at other CT times. These studies represent the first evidence of TRHs ability to affect circadian function.