Effects of the duper mutation on circadian responses to light

The circadian mutation duper in Syrian hamsters shortens the free-running circadian period (τ(DD)) by 2 hours when expressed on a tau mutant (τ(ss)) background and by 1 hour on a wild-type background. We have examined the effects of this mutation on phase response curves and entrainment. In contrast to wild types, duper hamsters entrained to 14L:10D with a positive phase angle. Super duper hamsters (expressing duper on a τ(ss) background) showed weak entrainment, while τ(ss) animals either completely failed to entrain or showed sporadic entrainment with episodes of relative coordination. As previously reported, wild-type and τ(ss) hamsters show low amplitude resetting in response to 15-minute light pulses after short-term (10 days) exposure to DD. In contrast, super duper hamsters show high amplitude resetting. This effect is attributable to the duper allele, as hamsters carrying duper on a wild-type background also show large phase shifts. Duper mutants that were born and raised in DD also showed high amplitude resetting in response to 15-minute light pulses, indicating that the effect of the mutation on PRC amplitude is not an aftereffect of entrainment to 14L:10D. Hamsters that are heterozygous for duper do not show amplified resetting curves, indicating that for this property, as for determination of free-running period, the mutant allele is recessive. In a modified Aschoff type II protocol, super duper and duper hamsters show large phase shifts as soon as the second day of DD. Despite the amplification of the PRC in super duper hamsters, the induction of Period1 gene expression in the SCN by light is no greater in these mutants than in wild-type animals. Period2 expression in the SCN did not differ between super duper and wild-type hamsters exposed to light at CT15, but albumin site D-binding protein (Dbp) mRNA showed higher basal levels and greater light induction in the SCN of super duper compared to wild-type animals. These results indicate that the duper mutation alters the amplitude of the circadian oscillator and further distinguish it from the tau mutation.     

Does the precision of a biological clock depend upon its period? Effects of the duper and tau mutations in Syrian hamsters

Mutations which alter the feedback loops that generate circadian rhythms may provide insight into their insensitivity to perturbation robustness) and their consistency of period (precision). I examined relationships between endogenous period, activity and rest (τ(DD), α and ρ) in Syrian hamsters using two different mutations, duper and tau, both of which speed up the circadian clock. I generated 8 strains of hamsters that are homozygous or heterozygous for the tau, duper, and wild type alleles in all combinations. The endogenous period of activity onsets among these strains ranged from 17.94+0.04 to 24.13 ± 0.04 h. Contrary to predictions, the variability of period was unrelated to its absolute value: all strains showed similar variability of τ(DD) when activity onsets and acrophase were used as phase markers. The τ(DD) of activity offsets was more variable than onsets but also differed little between genotypes. Cycle variation and precision were not correlated with τ(DD) within any strain, and only weakly correlated when all strains are considered together. Only in animals homozygous for both mutations (super duper hamsters) were cycle variation and precision reduced. Rhythm amplitude differed between strains and was positively correlated with τ(DD) and precision. All genotypes showed negative correlations between α and ρ. This confirms the expectation that deviations in the duration of subjective day and night should offset one another in order to conserve circadian period, even though homeostatic maintenance of energy reserves predicts that longer intervals of activity or rest would be followed by longer durations of rest or activity. Females consistently showed greater variability of the period of activity onset and acrophase, and of α, but variability of the period of offset differed between sexes only in super duper hamsters. Despite the differences between genotypes in τ(DD), ρ was consistently more strongly correlated with the preceding than the succeeding α.     

Modeling the dual pacemaker system of the tau mutant hamster

Circadian pacemakers in many animals are compound. In rodents, a two-oscillator model of the pacemaker composed of an evening (E) and a morning (M) oscillator has been proposed based on the phenomenon of "splitting" and bimodal activity peaks. The authors describe computer simulations of the pacemaker in tau mutant hamsters viewed as a system of mutually coupled E and M oscillators. These mutant animals exhibit normal type 1 PRCs when released into DD but make a transition to a type 0 PRC when held for many weeks in DD. The two-oscillator model describes particularly well some recent behavioral experiments on these hamsters. The authors sought to determine the relationships between oscillator amplitude, period, PRC, and activity duration through computer simulations. Two complementary approaches proved useful for analyzing weakly coupled oscillator systems. The authors adopted a "distinct oscillators" view when considering the component E and M oscillators and a "system" view when considering the system as a whole. For strongly coupled systems, only the system view is appropriate. The simulations lead the authors to two primary conjectures: (1) the total amplitude of the pacemaker system in tau mutant hamsters is less than in the wild-type animals, and (2) the coupling between the unit E and M oscillators is weakened during continuous exposure of hamsters to DD. As coupling strength decreases, activity duration (alpha) increases due to a greater phase difference between E and M. At the same time, the total amplitude of the system decreases, causing an increase in observable PRC amplitudes. Reduced coupling also increases the relative autonomy of the unit oscillators. The relatively autonomous phase shifts of E and M oscillators can account for both immediate compression and expansion of activity bands in tau mutant and wild-type hamsters subjected to light pulses.     

Constant darkness restores entrainment to phase-delayed Siberian hamsters

Over 90% of Siberian hamsters (Phodopus sungorus) fail to reentrain to a 5-h phase delay of a 16:8-h photocycle. Because constant darkness (DD) restores rhythms disrupted by constant light, we tested whether DD could also restore entrainment. DD began 0, 5, or 14 days after a 5-h phase delay, and the light-dark cycle was reinstated 14 days later. All hamsters exposed to DD on day 0 reentrained, whereas 42% reentrained irrespective of whether DD began 5 or 14 days later. For these latter two groups, tau (tau) and alpha (alpha) in DD predicted reentrainment; animals that reentrained had a mean tau and alpha of 24.1 and 8.9 h, respectively, whereas those that failed to reentrain maintained a mean tau and alpha of 25.0 and of 7.1 h, respectively. Restoration of entrainment by DD is somewhat paradoxical because it suggests that reentrainment to the photocycle was prevented by continued exposure to that same photocycle. The dichotomy of circadian responses to DD suggests "entrainment" phenotypes that are similar to those of photoperiodic responders and nonresponders.     

皮肤组织模型中吸收和散射系数对单线态氧发光特性的影响

构建由脂肪乳剂和墨水组成的皮肤组织模型,定量研究皮肤组织模型的吸收系数μa和散射系数μs对光敏化单线态氧(singlet oxygen,~1O_2)发光特性的影响。利用~1O_2发光检测系统测量含光敏剂四硫磺基酞菁氯化铝的皮肤组织模型分别在中心波长为1 230,1 270和1 310 nm处的时间分辨发光光谱,对扣除背景信号后的时间分辨~1O_2发光光谱进行积分和拟合,获得~1O_2发光积分强度以及激发三重态寿命τ_T和~1O_2寿命τ_D。结果表明在皮肤组织模型中,~1O_2发光积分强度随着μ_a和μ_s的增大而减小,μ_a对τ_T和τ_D没有影响。τ_T随着μs的增加而增加,τ_D随μ_s的增加先骤降而后缓慢上升。当μ_a1.5 mm~(-1)和μ_s32 cm~(-1)时,~1O_2发光积分强度和τ_T、τ_D均趋于稳定,其中τ_T和τ_D分别为3.4±0.6μs和3.3±0.7μs。     

Circadian organization of tau mutant hamsters: aftereffects and splitting

Homozygous tau mutant (tau(ss)) hamsters show an extremely short (20 h) circadian period (tau) that is attributable to altered enzymatic activity of casein kinase 1epsilon. It has been proposed that coupling of constituent circadian oscillators is strengthened in tau(ss) hamsters, explaining their tendency to show strong resetting after prolonged exposure to constant darkness. To evaluate further the circadian organization of tau(ss) hamsters, the authors assessed the extent of shortening of period as an aftereffect of exposure to light:dark cycles whose period (T) is 91% of tau and the ability of constant light to induce splitting. They find that tau(ss) hamsters show aftereffects comparable to wild types, indicating that normal CK1epsilon activity is not required for T cycles to shorten tau. This finding also contradicts the proposal that circadian period is homeostatically conserved. However, the authors find that tau(ss) hamsters rarely show splitting in constant light. Furthermore, LL does not induce lengthening of tau or reduction of activity duration (alpha) in these mutants. The authors' findings support the conclusion that the tau mutation alters the coupling between constituent circadian oscillators.     

Effect of Light Intensity on the Phase and Period Response Curves in the Nocturnal Field Mouse Mus booduga

The effect of light intensity on the phase response curve (PRC) and the period response curve (τRC) of the nocturnal field mouse Mus booduga was studied. PRCs and τRCs were constructed by exposing animals free-running in constant darkness (DD), to fluorescent light pulses (LPs) of 100 lux and 1000 lux intensities for 15min duration. The waveform of the PRCs and τRCs evoked by high light intensity (1000 lux) stimuli was significantly different compared to those constructed using low light intensity (100 lux). Moreover, a weak but significant correlation was observed between phase shifts and period changes when light stimuli of 1000 lux intensity were used; however, the phase shifts and period changes in the 100 lux PRC and τRC were not correlated. This suggests that the intensity of light stimuli affects both phase and period responses in the locomotor activity rhythm of the nocturnal field mouse M. booduga. These results indicate that complex mechanisms are involved in entrainment of circadian clocks, even in nocturnal rodents, in which PRC, τRC, and dose responses play a significant role.     

Changes in the phase response curve of the circadian clock to a phase-shifting stimulus.

Experiments were conducted in hamsters to determine whether the phase response curve (PRC) to injections of the short-acting benzodiazepine triazolam is a fixed or a labile property of the circadian clock. The results indicated that (1) both the shape and the amplitude of the PRC to triazolam generated on the first day of transfer from a light-dark cycle (LD 14:10) to constant darkness (DD) (i.e., PRCLD) were different from those of the PRC generated after many days in DD (PRCDD); and (2) the phase-shifting effects of triazolam on the activity rhythms of hamsters transferred from LD 14:10 or 12:12 to DD changed dramatically within the first 8-9 days spent in DD. In an attempt to accelerate the resynchronization of the circadian clock of hamsters subjected to an 8-hr advance in the LD cycle, triazolam was given to the animals at a time selected on the basis of the characteristics of PRCLD. The activity rhythms of five of eight triazolam-treated animals were resynchronized to the new LD cycle within 2-4 days after the shift, whereas those of most of the control animals were resynchronized 21-29 days after the shift. These findings suggest that attempts to use pharmacological or nonpharmacological tools to phase-shift circadian clocks under entrained conditions should take into account information derived from PRCs generated at the time of transition from entrained to free-running conditions.     

Internal Desynchronisation in Blow Fly (Calliphora vicina) Locomotor Activity Rhythms: Evidence for a Complex Circadian Pacemaker

The majority of blow flies (Calliphora vicina) display circadian locomotor activity rhythms that free-run with an unchanging period (τ) in darkness (DD), or entrain to a light-dark cycle (LD 1:23). However, a minority produce more complex patterns (spontaneous changes in τ, arrhythmicity, or 'split' rhythms) in DD, or undergo rhythm dissociation ('internal desynchronisation') when the light pulses of LD 1:23 initially illuminate the subjective night. These patterns are interpreted as evidence for a complex, multioscillatory and multicellular, structure of the insects' circadian system, and this complexity is discussed in terms of the neuronal architecture of the fly's brain.     

Splitting of the circadian rhythm of activity in hamsters: Effects of exposure to constant darkness and subsequent re-exposure to constant light

Summary The circadian rhythm of wheel running behavior was observed to dissociate into two distinct components (i.e. split) within 30 to 110 days in 56% of male hamsters exposed to constant light (Figs. 1–2). Splitting was abolished in all 16 animals that were transferred from constant light (LL) to constant darkness (DD) within 1–4 days of DD, and the components of the re-fused activity rhythm assumed a phase relationship that is characteristic of hamsters maintained in DD (Figs. 3–5). Re-fusion of the split activity rhythm was accompanied by a change in period (); in 14 animals increased while in the other 2 animals decreased after transfer to DD.After 10–30 days in DD, the hamsters were transferred back into LL at various time points throughout the circadian cycle. A few of these animals went through two or three LL to DD to LL transitions. The effect of re-exposure to LL was dependent on the phase relationship between the transition into LL and the activity rhythm. A rapid (i.e. 1–4 days) induction of splitting was observed in 7 of 9 cases when hamsters were transferred into LL 4–5 h after the onset of activity (Fig. 5). In the other 2 animals, the activity pattern was ultradian or aperiodic for 20 to 50 days before eventually coalescing into a split activity pattern. In contrast, transfer of animals (n = 13) from DD to LL at other circadian times did not result in the rapid induction of splitting and the activity rhythm continued to free-run with a single bout of activity (Fig. 5). Importantly, a transfer from DD to LL 4–5 h after the onset of activity did not induce splitting if the hamsters had not shown a split activity rhythm during a previous exposure to LL (n=10; Fig. 6).These studies indicate that transfer of split hamsters from LL to DD results in the rapid re-establishment of the normal phase relationship between the two circadian oscillators which underlie the two components of activity during splitting. In addition, there appears to be a history-dependent effect of splitting which renders the circadian system susceptible to becoming split again. The rapid re-initiation of the split condition upon transfer from DD to LL at only a specific circadian time is discussed in terms of the phase response curve for this species.Abbreviation PRC phase response curve This investigation was supported by NIH grants HD-09885 and HD-12622 from the National Institute of Child Health and Human Development and by a grant from the Whitehall FoundationRecipient of Research Career Development Award K04 HD-00249 from the National Institute of Child Health and Human Development     

Water oxidation by photosystem II: H(2)O-D(2)O exchange and the influence of pH support formation of an intermediate by removal of a proton before dioxygen creation

Understanding the chemistry of photosynthetic water oxidation requires deeper insight into the interrelation between electron transfer (ET) and proton relocations. In photosystem II membrane particles, the redox transitions of the water-oxidizing Mn complex were initiated by nanosecond laser flashes and monitored by absorption spectroscopy at 360 nm (A(360)). In the oxygen evolution transition (S(3) + hν → S(0) + O(2)), an exponential decrease in A(360) (τ(O(2)) = 1.6 ms) can be assigned to Mn reduction and O(2) formation. The corresponding rate-determining step is the ET from the Mn complex to a tyrosine radical (Y(Z)(ox)). We find that this A(360) decrease is preceded by a lag phase with a duration of 170 ± 40 μs (τ(lag) at pH 6.2), indicating formation of an intermediate before ET and O-O bond formation and corroborating results obtained by time-resolved X-ray spectroscopy. Whereas τ(O(2)) exhibits a minor kinetic isotope effect and negligible pH dependence, formation of the intermediate is slowed significantly both in D(2)O (τ(lag) increase of ～140% in D(2)O) and at low pH (τ(lag) of 30 ± 20 μs at pH 7.0 vs τ(lag) of 470 ± 80 μs at pH 5.5). These findings support the fact that in the oxygen evolution transition an intermediate is created by deprotonation and removal of a proton from the Mn complex, after Y(Z)(ox) formation but before the onset of electron transfer and O-O bond formation.     

Nonphotic enhancement of adjustment to new light-dark cycles: masking interpretation discounted

The adjustment of hamsters to advanced light-dark (LD) cycles can be greatly accelerated by scheduling a single 3-hr bout of extra activity in a novel running wheel, starting about 7 hr before the time when the animals become active in the preceding LD cycle. The present experiments were designed to provide stronger evidence that this effect depends on a shift in the pacemaker rather than on masking. It was shown that when hamsters were put into continuous darkness (DD) 1 day after the exercise-accelerated phase shift, their free-running rhythms took off from a time nearer to the onset of darkness in the new LD cycle than in the preceding LD cycle. An incidental finding was that in DD the free-running period of the hamsters with the accelerated phase shifts was longer than that of the control animals. Further evidence that the 3-hr exercise pulse had produced a greater phase advance than that occurring in undisturbed control animals was obtained by giving a light pulse at the same clock time to all animals after they had been in DD for 8 days. The animals that had previously exercised for the additional 3-hr phase-advanced in response to the light pulse, while the undisturbed control animals phase-delayed.     

Interaction of the retina with suprachiasmatic pacemakers in the control of circadian behavior

The suprachiasmatic nucleus (SCN) is the central circadian pacemaker governing the circadian rhythm of locomotor activity in mammals. The mammalian retina also contains circadian oscillators, but their roles are unknown. To test whether the retina influences circadian rhythms of locomotor behavior, the authors compared the activity of bilaterally enucleated hamsters with the activity of intact controls held in constant darkness (DD). Enucleated hamsters showed a broader range of free-running periods (tau) than did intact hamsters held for the same length of time in DD. This effect was independent of the age at enucleation (on postnatal days 1, 7, or 28). The average tau of intact animals kept in DD from days 7 or 28 was significantly longer than that of intact animals kept in DD from day 1 or any of the enucleated groups. This indicates that early exposure to light-dark cycles lengthens the tau and that the eye is required to maintain this effect even in DD. These data suggest that hypothalamic circadian pacemakers may interact continuously with the retina to determine the tau of locomotor activity. Enucleation caused a large decrease in glial fibrillary acidic protein in the SCN but has no (or slight) effects on calbindin, neuropeptide Y, vasopressin, or vasoactive intestinal polypeptide, which suggests that enucleation does not produce major damage to the SCN, an interpretation that is supported by the fact that enucleated animals retain robust circadian rhythmicity. The presence of an intact retina appears to contribute to system-level circadian organization in mammals perhaps as a consequence of interaction between its circadian oscillators and those in the SCN.     

Light induces c-fos and per1 expression in the suprachiasmatic nucleus of arrhythmic hamsters

Locomotor activity rhythms in a significant proportion of Siberian hamsters (Phodopus sungorus sungorus) become arrhythmic after the light-dark (LD) cycle is phase-delayed by 5 h. Arrhythmia is apparent within a few days and persists indefinitely despite the presence of the photocycle. The failure of arrhythmic hamsters to regain rhythms while housed in the LD cycle, as well as the lack of any masking of activity, suggested that the circadian system of these animals had become insensitive to light. We tested this hypothesis by examining light-induced gene expression in the suprachiasmatic nucleus (SCN). Several weeks after the phase delay, arrhythmic and re-entrained hamsters were housed in constant darkness (DD) for 24 h and administered a 30-min light pulse 2 h after predicted dark onset because light induces c-fos and per1 genes at this time in entrained animals. Brains were then removed, and tissue sections containing the SCN were processed for in situ hybridization and probed with c-fos and per1 mRNA probes made from Siberian hamster cDNA. Contrary to our prediction, light pulses induced robust expression of both c-fos and per1 in all re-entrained and arrhythmic hamsters. A separate group of animals held in DD for 10 days after the light pulse remained arrhythmic. Thus, even though the SCN of these animals responded to light, neither the LD cycle nor DD restored rhythms, as it does in other species made arrhythmic by constant light (LL). These results suggest that different mechanisms underlie arrhythmicity induced by LL or by a phase delay of the LD cycle. Whereas LL induces arrhythmicity by desynchronizing SCN neurons, phase delay-induced arrhythmicity may be due to a loss of circadian rhythms at the level of individual SCN neurons.     

Re-Entrainment Behavior of Djungarian Hamsters (phodopus sungorus) with Different Rhythmic Phenotype Following Light-Dark Shifts

Djungarian hamsters bred at the authors' institute reveal two distinct circadian phenotypes, the wild-type (WT) and DAO type. The latter is characterized by a delayed activity-onset, probably due to a deficient mechanism for photic entrainment. Experiments with zeitgeber shifts have been performed to gain further insight into the mechanisms underlying this phenomenon. Advancing and delaying phase shifts were produced by a single lengthening or shortening of the dark (D) or light (L) time by 6?h. Motor activity was recorded by passive infrared motion detectors. All WT hamsters re-entrained following various zeitgeber shifts and nearly always in the same direction as the zeitgeber shift. On the other hand, a considerable proportion of the DAO animals failed to re-entrain and showed, instead, diurnal, arrhythmic, or free-running activity patterns. All but one of those hamsters that re-entrained did so by delaying their activity rhythm independently of the direction of the LD shift. Resynchronization occurred faster following a delayed than an advanced shift and also after changes of D rather than L. WT animals tended to re-entrain faster, particularly following a zeitgeber advance (where DAO hamsters re-entrained by an 18-h phase delay instead of a 6-h phase advance). However, the difference between phenotypes was statistically significant only with a shortening of L. To better understand re-entrainment behavior, Type VI phase-response curves (PRCs) were constructed. To do this, both WT and DAO animals were kept under LD conditions, and light pulses (15 min, 100 lux) were applied at different times of the dark span. In WT animals, activity-offset always showed phase advances, whereas activity-onset was phase delayed by light pulses applied during the first half of the dark time and not affected by light pulses applied during the second half. When the light pulse was given at the beginning of D, activity-onset responded more strongly, but light pulses given later in D produced significant changes only in activity-offset. In accord with the delayed activity-onset in DAO hamsters, no or only very weak phase-responses were observed when light pulses were given during the first hours of D. However, the second part of the PRCs was similar to that of WT hamsters, even though it was compressed to an interval of only a few hours and the shifts were smaller. Due to these differences, the first light-on or light-off following an LD shift fell into different phases of the PRC and thus caused different re-entrainment behavior. The results show that it is not only steady-state entrainment that is compromised in DAO hamsters but also their re-entrainment behavior following zeitgeber shifts. (Author correspondence: weinert@zoologie.uni-halle.de)     

Re-entrainment behavior of Djungarian hamsters (Phodopus sungorus) with different rhythmic phenotype following light-dark shifts

Djungarian hamsters bred at the authors' institute reveal two distinct circadian phenotypes, the wild-type (WT) and DAO type. The latter is characterized by a delayed activity-onset, probably due to a deficient mechanism for photic entrainment. Experiments with zeitgeber shifts have been performed to gain further insight into the mechanisms underlying this phenomenon. Advancing and delaying phase shifts were produced by a single lengthening or shortening of the dark (D) or light (L) time by 6?h. Motor activity was recorded by passive infrared motion detectors. All WT hamsters re-entrained following various zeitgeber shifts and nearly always in the same direction as the zeitgeber shift. On the other hand, a considerable proportion of the DAO animals failed to re-entrain and showed, instead, diurnal, arrhythmic, or free-running activity patterns. All but one of those hamsters that re-entrained did so by delaying their activity rhythm independently of the direction of the LD shift. Resynchronization occurred faster following a delayed than an advanced shift and also after changes of D rather than L. WT animals tended to re-entrain faster, particularly following a zeitgeber advance (where DAO hamsters re-entrained by an 18-h phase delay instead of a 6-h phase advance). However, the difference between phenotypes was statistically significant only with a shortening of L. To better understand re-entrainment behavior, Type VI phase-response curves (PRCs) were constructed. To do this, both WT and DAO animals were kept under LD conditions, and light pulses (15 min, 100 lux) were applied at different times of the dark span. In WT animals, activity-offset always showed phase advances, whereas activity-onset was phase delayed by light pulses applied during the first half of the dark time and not affected by light pulses applied during the second half. When the light pulse was given at the beginning of D, activity-onset responded more strongly, but light pulses given later in D produced significant changes only in activity-offset. In accord with the delayed activity-onset in DAO hamsters, no or only very weak phase-responses were observed when light pulses were given during the first hours of D. However, the second part of the PRCs was similar to that of WT hamsters, even though it was compressed to an interval of only a few hours and the shifts were smaller. Due to these differences, the first light-on or light-off following an LD shift fell into different phases of the PRC and thus caused different re-entrainment behavior. The results show that it is not only steady-state entrainment that is compromised in DAO hamsters but also their re-entrainment behavior following zeitgeber shifts.     

Recombinant vesicular stomatitis vaccine against Nipah virus has a favorable safety profile: Model for assessment of live vaccines with neurotropic potential

Nipah virus (NiV) disease is a bat-borne zoonosis responsible for outbreaks with high lethality and is a priority for vaccine development. With funding from the Coalition of Epidemic Preparedness Innovations (CEPI), we are developing a chimeric vaccine (PHV02) composed of recombinant vesicular stomatitis virus (VSV) expressing the envelope glycoproteins of both Ebola virus (EBOV) and NiV. The EBOV glycoprotein (GP) mediates fusion and viral entry and the NiV attachment glycoprotein (G) is a ligand for cell receptors, and stimulates neutralizing antibody, the putative mediator of protection against NiV. PHV02 is identical in construction to the registered Ebola vaccine (Ervebo) with the addition of the NiV G gene. NiV ephrin B2 and B3 receptors are expressed on neural cells and the wild-type NiV is neurotropic and causes encephalitis in affected patients. It was therefore important to assess whether the NiV G alters tropism of the rVSV vector and serves as a virulence factor. PHV02 was fully attenuated in adult hamsters inoculated by the intramuscular (IM) route, whereas parental wild-type VSV was 100% lethal. Two rodent models (mice, hamsters) were infected by the intracerebral (IC) route with graded doses of PHV02. Comparator active controls in various experiments included rVSV-EBOV (representative of Ebola vaccine) and yellow fever (YF) 17DD commercial vaccine. These studies showed PHV02 to be more neurovirulent than both rVSV-EBOV and YF 17DD in infant animals. PHV02 was lethal for adult hamsters inoculated IC but not for adult mice. In contrast YF 17DD retained virulence for adult mice inoculated IC but was not virulent for adult hamsters. Because of the inconsistency of neurovirulence patterns in the rodent models, a monkey neurovirulence test (MNVT) was performed, using YF 17DD as the active comparator because it has a well-established profile of quantifiable microscopic changes in brain centers and a known reporting rate of neurotropic adverse events in humans. In the MNVT PHV02 was significantly less neurovirulent than the YF 17DD vaccine reference control, indicating that the vaccine will have an acceptable safety profile for humans. The findings are important because they illustrate the complexities of phenotypic assessment of novel viral vectors with tissue tropisms determined by transgenic proteins, and because it is unprecedented to use a heterologous comparator virus (YF vaccine) in a regulatory-enabling study. This approach may have value in future studies of other novel viral vectors.     

ACTIVITY RHYTHMS OF WILD AND LABORATORY GOLDEN HAMSTERS (MESOCRICETUS AURATUS) UNDER ENTRAINED AND FREE-RUNNING CONDITIONS

The golden hamster (Mesocricetus auratus) is one of the most frequently used laboratory animals, particularly in chronobiological studies. One reason is its very robust and predictable rhythms, although the question arises whether this is an inbreeding effect or rather is typical for the species. We compared the daily (circadian) activity rhythms of wild and laboratory golden hamsters. The laboratory hamsters were derived from our own outbred stock (Zoh:GOHA). The wild hamsters included animals captured in Syria and their descendants (F1). Experiments were performed under entrained (light: dark [LD] 14h:10h) and under free-running (constant darkness, DD) conditions. Locomotor activity was recorded using passive infrared detectors. Under entrained conditions, the animals had access to a running wheel for a certain time to induce additional activity. After 3 weeks in constant darkness, a light pulse (15 min, 100 lux) was applied at circadian time 14 (CT14). Both laboratory and wild hamsters showed well-pronounced and very similar activity rhythms. Under entrained conditions, all hamsters manifested about 80% of their total 24h activity during the dark portion of the LD cycle. The robustness of the daily rhythms was also similar. However, interindividual variability was higher in wild hamsters for both measures. All animals used the running wheels almost exclusively during the dark portion of the LD cycle, although the wild hamsters were three times more active. The period length, measured in constant darkness, was significantly shorter in wild (23.93h ± 0.10h) than in laboratory hamsters (24.06 ± 0.07h). The light-induced phase changes were not different (about 1.5h). In summary, these results indicate that the laboratory hamster is not much different from the wild type. (Chronobiology International, 18(6), 921-932, 2001)     

PHASE AND PERIOD RESPONSES OF THE JERBOA JACULUS ORIENTALIS TO SHORT LIGHT PULSES

The phase and period responses to short light pulses were studied in the jerboa, a seasonal, hibernating, nocturnal rodent from the Atlas region in Morocco. The jerboa, which is a saltatory species, showed precise activity onsets and offsets under a light-dark (LD) cycle using infrared captors to record locomotor activity. When released into constant darkness (DD), the majority of animals showed a circadian period (τ) <24?h (mean τ?=?23.89?±?0.13?h) and a lengthening of the activity span, α. Animals were subsequently exposed to up to eight 15-min light pulses, each separated by at least 2 wks, for up to 160 days in DD. During this span, most individuals maintained robust circadian rhythmicity, with clearly defined activity onsets and offsets, similar levels of total activity, duration of α, and percent activity occurring during the subjective night. The phase response curve (PRC) is typical of other nocturnal rodents, with light eliciting delays during late subjective day and early subjective night (CT8–CT19) and advances during late subjective night to early subjective day (CT19–CT2). A dead zone, when light had no effect on phase, is observed during mid-subjective day (CT3–CT8). A few individuals showed large (>9?h) Type 0 phase resetting near the singularity region (CT19) that resulted in a complete phase reversal, but otherwise displayed normal phase-shifting responses at other CT times. The τ response curve showed a decrease in period from early to late subjective night with increases at other times, but these changes were small (maximum <9?min) and highly variable. There was a distinct tendency for animals that had an initial short τ in DD to conserve a short τ during the series of light pulses and, inversely, for animals with long τ to conserve a long τ. This suggests possible constraints on the plasticity of variation of τ in relation to the endogenous period of the animal. (Author correspondence: howard.cooper@inserm.fr)     

The estrous cycle in golden hamsters: a circadian pacemaker times preovulatory gonadotropin release

In a population of cycling female hamsters entrained to an LD 6:18 light cycle (lights 1000-1600 hours), preovulatory release of luteinizing hormone and follicle-stimulating hormone occurred in some animals at 1300-1400 hours and in others at 1900 hours. In every case peak release was phase-locked (2-3-hour positive phase angle) to the circadian rhythm of locomotor activity. The pattern of entrainment of gonadotropin release on LD 6:18 is fully explicable in terms of the hamster's phase response curve to light. We conclude that periodic gonadotropin release in cycling females is timed by a circadian oscillator (biological clock) that is probably the same oscillator driving the circadian rhythm of locomotor activity.