Eating breakfast and avoiding late-evening snacking sustains lipid oxidation.

Circadian (daily) regulation of metabolic pathways implies that food may be metabolized differentially over the daily cycle. To test that hypothesis, we monitored the metabolism of older subjects in a whole-room respiratory chamber over two separate 56-h sessions in a random crossover design. In one session, one of the 3 daily meals was presented as breakfast, whereas in the other session, a nutritionally equivalent meal was presented as a late-evening snack. The duration of the overnight fast was the same for both sessions. Whereas the two sessions did not differ in overall energy expenditure, the respiratory exchange ratio (RER) was different during sleep between the two sessions. Unexpectedly, this difference in RER due to daily meal timing was not due to daily differences in physical activity, sleep disruption, or core body temperature (CBT). Rather, we found that the daily timing of nutrient availability coupled with daily/circadian control of metabolism drives a switch in substrate preference such that the late-evening Snack Session resulted in significantly lower lipid oxidation (LO) compared to the Breakfast Session. Therefore, the timing of meals during the day/night cycle affects how ingested food is oxidized or stored in humans, with important implications for optimal eating habits.

Zeta inhibitory peptide (ZIP) erases long-term memories in a cockroach.

Recent efforts to identify the molecules that are involved in the maintenance of long-term memories in mammals have focused attention on atypical isoforms of protein kinase C (PKC). Inhibition of these kinases by either the general PKC inhibitor, chelerythrine, or the more specific inhibitor, zeta inhibitory peptide (ZIP), can abolish both long-term potentiation in the hippocampus and as well as spatial, fear, appetitive, and sensorimotor memories. These inhibitors can also abolish long-term facilitation and long-term sensitization in the mollusk Aplysia californica. We have extended these results to an insect, the cockroach Leucophaea maderae. We show that systemic injections of either chelerythrine or ZIP erase long-term olfactory memories in the cockroach, but have no effect on memory acquisition during conditioning. We also show that inhibition of either protein kinase A (PKA) or protein synthesis can block memory acquisition but neither has an effect on the memory once it is formed. The results suggest that sustaining memories in insects requires the persistent activity of one or more isoforms of PKC and point to a strong evolutionary conservation of the molecular mechanisms that underlie the persistence of long-term memories in the central nervous system.

In vivo neuronal function of the fragile X mental retardation protein is regulated by phosphorylation.

Fragile X syndrome (FXS), caused by loss of the Fragile X Mental Retardation 1 (FMR1) gene product (FMRP), is the most common heritable cause of intellectual disability and autism spectrum disorders. It has been long hypothesized that the phosphorylation of serine 500 (S500) in human FMRP controls its function as an RNA-binding translational repressor. To test this hypothesis in vivo, we employed neuronally targeted expression of three human FMR1 transgenes, including wild-type (hFMR1), dephosphomimetic (S500A-hFMR1) and phosphomimetic (S500D-hFMR1), in the Drosophila FXS disease model to investigate phosphorylation requirements. At the molecular level, dfmr1 null mutants exhibit elevated brain protein levels due to loss of translational repressor activity. This defect is rescued for an individual target protein and across the population of brain proteins by the phosphomimetic, whereas the dephosphomimetic phenocopies the null condition. At the cellular level, dfmr1 null synapse architecture exhibits increased area, branching and bouton number. The phosphomimetic fully rescues these synaptogenesis defects, whereas the dephosphomimetic provides no rescue. The presence of Futsch-positive (microtubule-associated protein 1B) supernumerary microtubule loops is elevated in dfmr1 null synapses. The human phosphomimetic restores normal Futsch loops, whereas the dephosphomimetic provides no activity. At the behavioral level, dfmr1 null mutants exhibit strongly impaired olfactory associative learning. The human phosphomimetic targeted only to the brain-learning center restores normal learning ability, whereas the dephosphomimetic provides absolutely no rescue. We conclude that human FMRP S500 phosphorylation is necessary for its in vivo function as a neuronal translational repressor and regulator of synaptic architecture, and for the manifestation of FMRP-dependent learning behavior.

Circadian regulation of olfactory receptor neurons in the cockroach antenna.

In the cockroach, olfactory sensitivity as measured by the amplitude of the electroantennogram (EAG) is regulated by the circadian system. We wished to determine how this rhythm in antennal response was reflected in the activity of individual olfactory receptor neurons. The amplitude of the EAG and the activity of olfactory receptor neurons (ORNs) in single olfactory sensilla were recorded simultaneously for 3 to 5 days in constant darkness from an antenna of the cockroach Leucophaea maderae. Both EAG amplitude and the spike frequency of the ORNs exhibited circadian rhythms with peak amplitude/activity occurring in the subjective day. The phases of the rhythms were dependent on the phase of the prior light cycle and thus were entrainable by light. Ablation of the optic lobes abolished the rhythm in EAG amplitude as has been previously reported. In contrast, the rhythm in ORN response persisted following surgery. These results indicated that a circadian clock outside the optic lobes can regulate the responses of olfactory receptor neurons and further that this modulation of the ORN response is not dependent on the circadian rhythm in EAG amplitude.

Circadian rhythms in the mating behavior of the cockroach, Leucophaea maderae.

Mating behavior of small populations of virgin males and females of the cockroach Leucophaea maderae were continuously monitored via time-lapse video recording in controlled laboratory conditions. The time of onset of copulation was found to be rhythmic in a light cycle of 12 h light alternated with 12 h of darkness, with the peak of mating behavior occurring near the light to dark transition. This rhythm persisted in constant dim red illumination and constant temperature. In constant conditions, the period of the rhythm was slightly less than 24 h, with a peak of copulation during the late subjective day. These data demonstrated that mating behavior is gated by a circadian clock. When males and females were taken from light cycles that were 12 h out of phase, a bimodal rhythm was observed with one peak in the males' late subjective day and a second peak of equal amplitude in the late subjective day of females. The results indicated that circadian systems in both males and females contribute to the circadian rhythm in copulation. Bilateral section of the optic tracts (OTX) of both males and females abolished the rhythm, but the rhythm persisted when OTX females were paired with intact males or when OTX males were paired with intact females. Furthermore, when OTX males or OTX females were paired with intact animals that were 12 h out of phase, a bimodal rhythm was still observed. These results suggested that the circadian pacemaker in the optic lobes of both male and female cockroaches participates in the control of mating, but that a pacemaker outside the optic lobes is also likely involved. Finally, it was shown that the female's olfactory response (measured by electroantennogram) to components of the male sex pheromone exhibited a circadian rhythm, but the data suggested the peripheral olfactory rhythm is not likely to be involved in the rhythm of mating behavior.

Tau-independent Phase Analysis: A Novel Method for Accurately Determining Phase Shifts.

Estimations of period and phase are essential in circadian biology. While many techniques exist for estimating period, comparatively few methods are available for estimating phase. Current approaches to analyzing phase often vary between studies and are sensitive to coincident changes in period and the stage of the circadian cycle at which the stimulus occurs. Here we propose a new technique, tau-independent phase analysis (TIPA), for quantifying phase shifts in multiple types of circadian time-course data. Through comprehensive simulations, we show that TIPA is both more accurate and more precise than the standard actogram approach. TIPA is computationally simple and therefore will enable accurate and reproducible quantification of phase shifts across multiple subfields of chronobiology.

Revealing Oft-cited but Unpublished Papers of Colin Pittendrigh and Coworkers.

Among the scientific resources that Colin Pittendrigh passed on to his colleagues after his death in 1996 were two unpublished papers. These manuscripts, developed first in the mid-1960s and continually updated and refined through the late 1970s, centered on the development and experimental exploration of a model of circadian entrainment combining aspects of the well-known parametric (continuous) and nonparametric (discrete) models of entrainment. These texts reveal the experimental work surrounding Pittendrigh's determination of the limits of entrainment and the explanation of the bistability phenomenon. These manuscripts are being made publicly available in their final format (February 1978) as supplementary material to this introduction.

The Optic Lobes Regulate Circadian Rhythms of Olfactory Learning and Memory in the Cockroach.

The cockroach, Leucophaea maderae, can be trained in an associative olfactory memory task by either classical or operant conditioning. When trained by classical conditioning, memory formation is regulated by a circadian clock, but once the memory is formed, it can be recalled at any circadian time. In contrast, when trained via operant conditioning, animals can learn the task at any circadian phase, but the ability to recall the long-term memory is tied to the phase of training. The optic lobes of the cockroach contain a circadian clock that drives circadian rhythms of locomotor activity, mating behavior, sensitivity of the compound eye to light, and the sensitivity of olfactory receptors in the antennae. To evaluate the role of the optic lobes in regulating learning and memory processes, the authors examined the effects of surgical ablation of the optic lobes on memory formation in classical conditioning and memory recall following operant conditioning. The effect of optic lobe ablation was to "rescue" the deficit in memory acquisition at a time the animals normally cannot learn and "rescue" the animal's ability to recall a memory formed by operant conditioning at a phase where memory was not normally expressed. The results suggested that the optic lobe pacemaker regulates these processes through inhibition at "inappropriate" times of day. As a pharmacological test of this hypothesis, the authors showed that injections of fipronil, an antagonist of GABA and glutamate-activated chloride channels, had the same effects as optic lobe ablation on memory formation and recall. The data suggest that the optic lobes contain the circadian clock(s) that regulate learning and memory processes via inhibition of neural processes in the brain.

A fully automated Drosophila olfactory classical conditioning and testing system for behavioral learning and memory assessment.

BACKGROUND: Aversive olfactory classical conditioning has been the standard method to assess Drosophila learning and memory behavior for decades, yet training and testing are conducted manually under exceedingly labor-intensive conditions. To overcome this severe limitation, a fully automated, inexpensive system has been developed, which allows accurate and efficient Pavlovian associative learning/memory analyses for high-throughput pharmacological and genetic studies. NEW METHOD: The automated system employs a linear actuator coupled to an odorant T-maze with airflow-mediated transfer of animals between training and testing stages. Odorant, airflow and electrical shock delivery are automatically administered and monitored during training trials. Control software allows operator-input variables to define parameters of Drosophila learning, short-term memory and long-term memory assays. RESULTS: The approach allows accurate learning/memory determinations with operational fail-safes. Automated learning indices (immediately post-training) and memory indices (after 24h) are comparable to traditional manual experiments, while minimizing experimenter involvement. COMPARISON WITH EXISTING METHODS: The automated system provides vast improvements over labor-intensive manual approaches with no experimenter involvement required during either training or testing phases. It provides quality control tracking of airflow rates, odorant delivery and electrical shock treatments, and an expanded platform for high-throughput studies of combinational drug tests and genetic screens. The design uses inexpensive hardware and software for a total cost of ∼$500US, making it affordable to a wide range of investigators. CONCLUSIONS: This study demonstrates the design, construction and testing of a fully automated Drosophila olfactory classical association apparatus to provide low-labor, high-fidelity, quality-monitored, high-throughput and inexpensive learning and memory behavioral assays.

Circadian regulation of learning and memory.

The study of the relationship between biological clocks and learning and memory in insects goes back to work on Zeitgedachtnis (time memory) in bees originating in the early 1900s when it was shown that bees were able to remember the time of day a specific food source was available. More recent work has expanded on the role of circadian phase in memory acquisition, consolidation, and recall in additional insect species. The results show that the circadian system can modulate the ability of individuals to acquire memories or the ability to retrieve memories; however, questions remain both about the mechanisms by which the circadian system regulates these processes and about the functional/adaptive significance of this novel feature of insect circadian organization.

Circadian rhythm in olfactory response in the antennae controlled by the optic lobe in the cockroach.

The olfactory response in antennae of the cockroach, Leucophaea maderae, was investigated by measuring electroantennograms (EAGs) in restrained animals. The amplitude of the EAG response to pulses of ethyl acetate, octanol, or fenchone, exhibited a robust, light entrained, circadian rhythm that persisted at least 14 days in constant darkness. Dilution-response curves measured at the peak and trough of the rhythm indicated there was a 10-fold change in sensitivity. The EAG rhythm was abolished by severing the optic tracts, while entrainment was abolished by ablation of the compound eyes. The results indicate that the circadian system modulates olfactory sensitivity in the antennae and that the rhythm is driven by a circadian pacemaker in the optic lobes that is entrained by photoreceptors in the compound eyes.

Analysis of precurrent skills in solving mathematics story problems.

We conducted an analysis of precurrent skills (responses that increase the effectiveness of a subsequent or "current" behavior in obtaining a reinforcer) to facilitate the solution of arithmetic word (story) problems. Two students with developmental disabilities were taught four precurrent responses (identifying the initial value, change value, operation, and resulting value) in a sequential manner. Results of a multiple baseline design across behaviors showed that the teaching procedures were effective in increasing correct performance of each of the precurrent behaviors with untaught problems during probes and that once the precurrent behaviors were established, the number of correct problem solutions increased.

Effect of circadian phase on memory acquisition and recall: operant conditioning vs. classical conditioning.

There have been several studies on the role of circadian clocks in the regulation of associative learning and memory processes in both vertebrate and invertebrate species. The results have been quite variable and at present it is unclear to what extent the variability observed reflects species differences or differences in methodology. Previous results have shown that following differential classical conditioning in the cockroach, Rhyparobia maderae, in an olfactory discrimination task, formation of the short-term and long-term memory is under strict circadian control. In contrast, there appeared to be no circadian regulation of the ability to recall established memories. In the present study, we show that following operant conditioning of the same species in a very similar olfactory discrimination task, there is no impact of the circadian system on either short-term or long-term memory formation. On the other hand, ability to recall established memories is strongly tied to the circadian phase of training. On the basis of these data and those previously reported for phylogenetically diverse species, it is suggested that there may be fundamental differences in the way the circadian system regulates learning and memory in classical and operant conditioning.

Neuromodulation of olfactory sensitivity in the peripheral olfactory organs of the American cockroach, Periplaneta americana.

Olfactory sensitivity exhibits daily fluctuations. Several studies have suggested that the olfactory system in insects is modulated by both biogenic amines and neuropeptides. However, molecular and neural mechanisms underlying olfactory modulation in the periphery remain unclear since neuronal circuits regulating olfactory sensitivity have not been identified. Here, we investigated the structure and function of these signaling pathways in the peripheral olfactory system of the American cockroach, Periplaneta americana, utilizing in situ hybridization, qRT-PCR, and electrophysiological approaches. We showed that tachykinin was co-localized with the octopamine receptor in antennal neurons located near the antennal nerves. In addition, the tachykinin receptor was found to be expressed in most of the olfactory receptor neurons in antennae. Functionally, the effects of direct injection of tachykinin peptides, dsRNAs of tachykinin, tachykinin receptors, and octopamine receptors provided further support for the view that both octopamine and tachykinin modulate olfactory sensitivity. Taken together, these findings demonstrated that octopamine and tachykinin in antennal neurons are olfactory regulators in the periphery. We propose here the hypothesis that octopamine released from neurons in the brain regulates the release of tachykinin from the octopamine receptor neurons in antennae, which in turn modulates the olfactory sensitivity of olfactory receptor neurons, which house tachykinin receptors.

Social interactions and the circadian rhythm in locomotor activity in the cockroach Leucophaea maderae.

The role of social interactions in entrainment has not been extensively studied in the invertebrates. Leucophaea maderae is a gregarious species of cockroach that exhibits extensive social interactions. Social interactions associated with copulation between the sexes have been shown to be regulated by the circadian system. We show here that social interactions between males are also under circadian control. We examined the question of whether or not these rhythmic social contacts could function as zeitgebers capable of regulating circadian phase and period. Animals initially in phase that were housed as groups or pairs of single sex or mixed sex in constant darkness for 2-7 weeks were found to drift out of phase. Their behavior was not significantly different from individual animals maintained in isolation. Further, animals that were initially out of phase by 12 h housed as groups or pairs were not significantly different in phase from animals that were isolated. The results show the circadian clocks of cockroaches are remarkably insensitive to the extensive social interactions that occur between individuals.

Circadian regulation of insect olfactory learning.

Olfactory learning in insects has been used extensively for studies on the neurobiology, genetics, and molecular biology of learning and memory. We show here that the ability of the cockroach Leucophaea maderae to acquire olfactory memories is regulated by the circadian system. We investigated the effect of training and testing at different circadian phases on performance in an odor-discrimination test administered 30 min after training (short-term memory) or 48 h after training (long-term memory). When odor preference was tested by allowing animals to choose between two odors (peppermint and vanilla), untrained cockroaches showed a clear preference for vanilla at all circadian phases, indicating that there was no circadian modulation of initial odor preference or ability to discriminate between odors. After differential conditioning, in which peppermint odor was associated with a positive unconditioned stimulus of sucrose solution and vanilla odor was associated with a negative unconditioned stimulus of saline solution, cockroaches conditioned in the early subjective night showed a strong preference for peppermint and retained the memory for at least 2 days. Animals trained and tested at other circadian phases showed significant deficits in performance for both short- and long-term memory. Performance depended on the circadian time (CT) of training, not the CT of testing, and results indicate that memory acquisition rather than retention or recall is modulated by the circadian system. The data suggest that the circadian system can have profound effects on olfactory learning in insects.