Caught in the act: the first record of copulating fossil vertebrates.

The behaviour of fossil organisms can typically be inferred only indirectly, but rare fossil finds can provide surprising insights. Here, we report from the Eocene Messel Pit Fossil Site between Darmstadt and Frankfurt, Germany numerous pairs of the fossil carettochelyid turtle Allaeochelys crassesculpta that represent for the first time among fossil vertebrates couples that perished during copulation. Females of this taxon can be distinguished from males by their relatively shorter tails and development of plastral kinesis. The preservation of mating pairs has important taphonomic implications for the Messel Pit Fossil Site, as it is unlikely that the turtles would mate in poisonous surface waters. Instead, the turtles initiated copulation in habitable surface waters, but perished when their skin absorbed poisons while sinking into toxic layers. The mating pairs from Messel are therefore more consistent with a stratified, volcanic maar lake with inhabitable surface waters and a deadly abyss.

Persistence of meal-entrained circadian rhythms following area postrema lesions in the rat.

Rats anticipate daily meals with increased approaches to a feeder and an increase in core body temperature. Food-anticipatory activity (FAA) is thought to be under the control of a feeding-entrained circadian oscillator (FEO). Ibotenic acid and electrolytic lesions in the region of the parabrachial nuclei (PBN) in the rat severely disrupt FAA (feeder approaches) and temperature rhythms. The PBN receive dense input from the area postrema (AP), which lacks a blood-brain barrier and thus has access to humoral factors in the systemic circulation. The present study assesses development and maintenance of FAA in rats with cautery lesions of the AP. The results demonstrate that AP lesions do not alter FAA. This experiment does not support the hypothesis that the AP in the caudal brainstem detects and relays circulating signals from the periphery that trigger FAA.

Food-anticipatory activity persists after olfactory bulb ablation in the rat.

Restricted daily food access acts as an entraining stimulus (zeitgeber) for a circadian clock, the feeding-entrainable oscillator (FEO). There are many properties of a daily meal that could potentially convey timing information to the FEO. Olfactory cues associated with feeding are one such property. In order to rule out olfaction as a necessary entraining stimulus, olfactory bulbectomized and sham-operated male Sprague--Dawley rats had access to food for 2 h each day. Food bin approach behavior was monitored as an index of food-anticipatory activity (FAA). Both groups entrained to the daily meal with an increase in feeder approach time several hours before meal onset. There were no significant differences in the timing or the amount of FAA between groups. Furthermore, FAA was maintained during 3 days of food deprivation in both groups. In accordance with previous studies, the results show that olfactory cues are not necessary for the entrainment of FAA.

Feeding-entrained circadian rhythms are attenuated by lesions of the parabrachial region in rats.

Rats anticipate daily restricted meals with increased approaches to a feeder and an increase in core body temperature. Food anticipatory activity (FAA) is thought to be under the control of a feeding-entrained circadian oscillator. Although numerous forebrain lesions have failed to permanently abolish FAA, the hindbrain has not been investigated. The parabrachial nuclei (PBN) integrate information from visceral and gustatory afferents. This region is also innervated by neurons in the area postrema that have access to the peripheral circulation. Therefore, it is possible that this region plays a role in triggering FAA. In two experiments, a total of 19 rats were given ibotenic acid or electrolytic lesions targeted at the PBN. The PBN-lesioned animals showed a marked attenuation in anticipatory approaches to the food bin relative to sham-operated controls. Some animals did not anticipate the meal at all. In addition, the expected increase in core body temperature was severely attenuated in the PBN-lesioned animals compared with controls. The most likely interpretation of these data is that the PBN serve as a relay for information about the zeitgeber (food in the gut) or as a clock output pathway, but not as the site of the feeding-entrained circadian oscillator.

Feeding-entrained circadian rhythms in hypophysectomized rats with suprachiasmatic nucleus lesions.

Several pituitary hormones are important in the regulation of metabolism, and their release appears to be controlled by a circadian clock. Consequently, they may be involved in feeding-entrained circadian rhythms. Hypophysectomized (Hypox) and sham-operated male Sprague-Dawley rats had access to food for 4 h each day. Food-anticipatory activity (FAA) and core body temperature (T(b)) were monitored. Both groups entrained to the daily meal with an increase in activity in the 4 h preceding meal access and quickly reentrained after an 8-h phase advance of food access. FAA was not disrupted in either group after suprachiasmatic lesions were added. Core T(b) increased in the sham-operated subjects before mealtime, but Hypox rats failed to show this effect. Rather, T(b) declined during anticipation and throughout the food access period. Respiratory quotient (RQ), an indirect measure of metabolic rate, was measured for 24 h in some subjects. Sham-operated rats showed a dramatic downturn in RQ 1 h before mealtime, whereas Hypox rats showed a steadily decreasing RQ throughout the day. The results show that the pituitary hormones are not necessary for FAA and that in Hypox rats the anticipatory rise in T(b) and changes in RQ become dissociated from anticipatory behavior, indicating that these functions are separate outputs of the food-entrainable circadian oscillator.

Plasma glucagon, glucose, insulin, and motilin in rats anticipating daily meals.

The circadian food entrainable oscillator (FEO) mediates an increase in activity preceding access to periodic meals. The FEO is anatomically independent of the suprachiasmatic nucleus (SCN), but its locus remains to be established. Whether the FEO is located in the central nervous system (CNS) or in the periphery, it seems reasonable to assume that there is a link of communication between the digestive system and the CNS because only nutritive meals entrain the FEO. Subdiaphragmatic vagotomy and visceral deafferentation with capsaicin do not eliminate food-anticipatory activity (FAA), indicating that a neural signal is not necessary. The present study investigates the hypothesis that humoral signals from the digestive system act upon the CNS to trigger or entrain FAA. Intact rats and rats with SCN lesions were entrained to daily meals and then sacrificed prior to FAA or during FAA, but before meal access. Average plasma concentrations of glucose and insulin were nearly identical in both groups. Plasma and duodenal mucosal motilin concentrations also were not different between the two times. Corticosterone was elevated during anticipation, but the difference was not statistically reliable. Glucagon concentration was decreased during FAA compared to concentration prior to FAA in both intact and SCN-lesioned subjects. This difference was not observed in control rats fasted for the same number of hours, but not previously entrained to a daily meal. Although the decrease in glucagon could be a signal that initiates FAA, a causal role remains to be established.

Profound conditioned taste aversion induced by oral consumption of 2-deoxy-D-glucose.

In a previous experiment rats avidly avoided a solution of 2-deoxy-d-glucose in 0.2% saccharin (2-DG+S), drinking less than 1 mL over 3 days. The present study investigated taste avoidance and conditioned taste aversion (CTA) to orally presented 2-DG+S as well as CTA in response to i.p. injections of 2-DG. In Experiment 1, rats were given either a glucose/saccharin(G +S) solution or 2-DG+S for eight 30-s test periods. Four seconds into the first 30-s test, rats in the 2-DG+S group licked significantly less than rats in the G+S group, and licking almost totally ceased in the remaining seven tests. Overnight water intake was not different between the groups, but when offered a G+S solution, rats in the 2-DG+S group almost totally avoided the solution and still showed a significant aversion to G+S when retested 6 days later. In Experiment 2, rats were allowed to drink G +S, and were then injected i.p. with 500 mg 2-DG/kg, 500 mg D-glucose/kg body weight in 0.3 mL water, or with 0.3 mL saline. When tested with a G +S solution the next day, rats in the 2-DG group showed a highly significant avoidance, while rats in the glucose group were not different from those in the saline group. The results of the second experiment are consistent with earlier studies of CTA induced by i.p. injections of 2-DG. The present study indicates that small amounts of orally ingested 2-DG produce a CTA as strong or even stronger than that following injected 2-DG, most likely by inducing malaise. Whether the onset of malaise is fast enough to account for the rapid initial avoidance of this solution or a taste factor is also involved is not yet clear.

Glucose, but not fat, phase shifts the feeding-entrained circadian clock.

To study the ability of single macronutrients to entrain or phase shift the feeding entrainable circadian oscillator, rats with lesions of the suprachiasmatic nucleus were first maintained on a single daily meal of lab chow until robust anticipatory approaches to the feeder or anticipatory wheel running was established. The meal time was then delayed by 8 h and chow was replaced with a 25-mL solution of 0.2% saccharin or 25 mL of saccharin plus 15 g of glucose. For other phase shifts, rats received either 6 mL of vegetable oil or mineral oil for 2 consecutive days. Consumption of about 6 g (24 kcal) or more of glucose resulted in robust delaying transients on the days after ingestion, whereas saccharin induced only small delays consistent with the initiation of a free-running rhythm with a period greater than 24 h. Surprisingly, consumption of 5.5 g of vegetable oil (47 kcal) did not result in delays greater than those in rats receiving mineral oil. The introduction of oil also produced a severe reduction in approaches to the feeder which could be alleviated by placing inaccessible chow in the feeders between oil meals. Phase shifts with oil were repeated with rats housed in wheels using anticipatory wheel running as a phase marker to assess whether the lack of phase shifts with fat was apparatus dependent. As was the case with approach behavior, anticipatory wheel running was not significantly delayed by vegetable oil consumption. These results indicate that a simple monosaccharide, glucose, has zeitgeber properties for the feeding entrainable oscillator. Vegetable oil, despite a higher caloric content, may be ineffective because of slower gastric emptying and nutrient absorption or because fat is not a good zeitgeber for the feeding entrained circadian oscillator.

Circadian food anticipation persists in capsaicin deafferented rats.

The feeding-entrained circadian oscillator (FEO) organizes locomotor activity and other variables to anticipate daily timed meals. Whether the biological substrate for the FEO is in the central nervous system or in the periphery, there must be communication between the gut and the brain to result in a behavioral output. To investigate potential neural routes of communication, rats with suprachiasmatic lesions were given systemic capsaicin (total dose: 100 mg/kg, i.p.) to produce visceral deafferentation. Deafferentation was confirmed using the phenyl-p-benzoquinone stretch test and the corneal irritation test. A 3-h meal was made available at the same time each day while wheel running was recorded for several weeks. Results indicated that rats with capsaicin lesions were somewhat more active overall and during nonanticipation times, but the onset time and the amount of anticipatory wheel running did not differ from vehicle-treated controls. In addition, reentrainment following a phase delay of mealtime and the persistence of anticipatory activity during food deprivation were similar between the groups. Since capsaicin deafferentation and subdiaphragmatic vagotomy do not prevent food-anticipatory activity, it is likely that communication between the gut and the brain is accomplished via a humoral route.

The effects of food deprivation, nutritive and non-nutritive feeding and wheel running on gastric stress ulcers in rats.

Feeding and housing conditions that induce gastric lesions were investigated. Rats were housed in activity wheels or in hanging cages and exposed to food deprivation, ad lib cellulose or 6 g of cellulose per day for 5 days. Food-deprived rats in both housing conditions had ulcers in the rumen but many rats also had mucosal ulcers. Cellulose prevented rumenal ulcers but produced a tendency toward more severe mucosal ulcers. Ulcers in wheel-housed rats were somewhat larger but the difference was not significant. In a second experiment, rats were fed 6 g/day laboratory chow or 6 g/day chow + ad lib cellulose until b.wt. reached a preset criterion. On the average, about 10 days on the feeding regimen were required to induce ulcers in these groups. None of the rats had rumenal ulcers. Mucosal ulcers were reliably larger in rats that received cellulose in addition to 6 g of chow. There was no difference in ulcer area between wheel-housed and cage-housed rats. The results indicate that solid bulk, regardless of its caloric value or amount, protects the nonglandular stomach whereas noncaloric bulk tends to aggravate ulcers in the glandular stomach. A small amount of chow delays the rate of b.wt. loss and consequently ulcer formation. Furthermore, wheel running is not necessary to produce mucosal ulcers when food intake is insufficient to maintain b.wt. and b.wt. at sacrifice seems to be a good predictor of ulcer formation.

Calories affect zeitgeber properties of the feeding entrained circadian oscillator.

Rats with suprachiasmatic (SCN) lesions readily entrain to daily meals by increasing their activity prior to food access. Although previous experiments suggest that entrainment requires a nutritive meal, it is not yet clear what the parameters of the entraining stimulus are. The first experiment investigated the role of caloric content in resetting the feeding entrainable oscillator (FEO). Rats were entrained to 20 g chow/day until anticipatory activity was stable. The food access time was then delayed by 8 h and rats received either 0, 2, 6, or 16 g of chow for two days. Sixteen g of chow produced large delays on the next two cycles, while 0 and 2 g produced no delays. Two of 8 rats receiving 6 g showed delays, indicating that 22 kcal is near the threshold. In a second experiment, the effects of bulk were investigated. After an 8 h phase delay, rats received 0, 6, 10 or 16 g of chow mixed with cellulose for a total of 21 g for all groups. The 10 and 16 g chow groups delayed while the 0 g chow group did not. In the 6 g groups some animals phase shifted while others did not. Thus, the addition of non-nutritive bulk to the phase shifted meal had little or no effect on resetting the FEO and it appears that caloric content rather than gastric distention provides an effective zeitgeber for the FEO.

Photoperiod duration and energy balance in the pigeon.

Energy balance, and daily rhythms in feeding activity, body temperature (Tb), metabolic rate (O2 consumption), and RQ (CO2/O2) that affect that balance, were studied in pigeons when the duration of the photophase gradually lengthened (LP group) or shortened (SP group) from an initial starting point at LD 12:12. The end point of change for the LP group was LD 21:3, and for the SP group was LD 3:21. Standard laboratory conditions were in effect (moderate ambient temperature; ad lib food and water). On LD 12:12, energy balance was positive (the ratio of gross energy intake to energy expenditure approximated 1.25). In the light phase, a bimodal pattern of feeding was accompanied by elevated levels in Tb, O2 consumption, and RQ; in the dark phase, Tb and O2 consumption fell at lights-off, and prior to lights-on there were anticipatory rises in both measures and a drop in RQ. Energy balance was remarkably constant over a wide range of photoperiods, but at the shortest photoperiods energy balance became more positive (approximately 1.45) because energy intake increased without much change in energy expenditure. Changes in the daily rhythms of the various measures provided some bases for understanding the changes in energy balance. Analysis of the Tb rhythms indicated that the circadian system of the pigeon appears to be capable of adjusting to a wide range of photoperiods. It is suggested that the increase in energy balance at short photoperiods may occur because of inadequate feedback from nutritional and metabolic signals, or may reflect anticipatory winter seasonal adjustments triggered by photoperiod duration.

Coupling between light- and food-entrainable circadian oscillators in pigeons.

Two experiments were conducted with a restricted feeding schedule to determine whether pigeons have a separate food-entrainable oscillator (FEO) and to assess the coupling strength between the FEO and the light-entrainable oscillator (LEO). In the baseline condition, the body temperature (Tb) and O2 consumption of two pigeons increased prior to light onset (LD 12:12 cycle) and food presentation (at the ninth hour of the light phase). In one experiment, when the LD cycle was phase delayed or advanced by 4 h while feeding time remained unchanged, the Tb and O2 rises prior to light-onset showed the expected delaying or advancing transients, but the rises prior to feeding also delayed or advanced for several days before returning to their proper phase position. In the second experiment, food was presented at 23.5-h intervals for 10 days while the LD cycle continued with a 24-h period. Entrainment to the LD cycle was unaffected, and Tb and O2 consumption continued to rise prior to the changing feeding time, but with a reduced lead time. When the feeding time was subsequently delayed by 5 h, Tb and O2 consumption with regard to the LD cycle were unaffected, but delaying transients occurred until both measures reentrained to the new feeding time. The results provide support for the existence of a separate FEO in pigeons and indicate asymmetrical coupling between the LEO and FEO, with the former having a stronger effect on the latter.

Body fat reserves attenuate gastric ulcers induced by restricted feeding in rats.

The purpose of this experiment was to explicitly test the hypothesis that the depletion of fat reserves is a critical factor in the generation of ulcers in the glandular stomach. Fat reserves were systematically manipulated by using chow vs. high-fat diets and by using rats of different ages (3, 7, and 17 months). The ulcerogenic procedure consisted of limiting food intake to 6 g of chow per day. For chow-fed rats, 20 days were required to induce ulcers in 3-month-old rats whereas more than 40 days were required in 7- and 17-month-old rats. In 7-month-old rats fed a high-fat diet, the ulcerogenic phase lasted 63 days. Virtually no ulcers were found in control groups with larger body weights that were yoked to the duration of the ulcerogenic phase. Measurement of retroperitoneal and epididymal fat pads indicated that these were nearly depleted in rats with ulcers whereas some fat remained in ulcer-free rats, regardless of age, diet, or the duration of the ulcerogenic phase. These results suggest that the depletion of fat reserves to a critical level triggers a metabolic stress response that then produces gastric ulcers.

Activity-stress ulcers in rats: the role of preentrainment to meal time.

Rats housed in activity wheels (A-W) or hanging cages (H-C) received food restriction to 4 h/day for 15 days. Food was given at fixed times to entrain the feeding entrainable oscillator or at irregular times to prevent entrainment. One half of the rats received food in the light phase, the other half in the dark phase of the light-dark cycle. Food was then reduced to 6 g/day for 4 days for A-W rats or 7 days for H-C rats. Stomachs were removed 24 h after the last meal and the ulcerated area was quantified. Twenty one of 32 A-W rats and 19 of 32 H-C rats had ulcers in the glandular stomach. Ulcers were significantly larger in A-W rats (p < 0.01). Activity levels (in A-W rats), % body weight loss and terminal body weight (in both groups) were significantly correlated with the area of ulcers. There was no significant time of day effect on the ulcer severity. However, an unexpected finding was that for A-W rats, those fed at fixed times had larger ulcers than those fed at irregular times (p < 0.01) while H-C rats showed an expected trend in the opposite direction. One possible explanation for this result is that for A-W rats, the reduced food supply at an expected time of day generated a greater stress response than in rats whose feeding time was unpredictable.

Stress ulcers in rats: the role of food intake, body weight, and time of day.

Rats were housed in activity wheels (n = 32) or in hanging cages (n = 32) for 2 weeks. Food intake of rats in hanging cages was curtailed to match body weight to that of the activity group. All rats then received 6 g of food at one of four different times of day (n = 8, each mealtime) for 4 consecutive days and were sacrificed 24 h after the last meal. Twenty-three rats in the activity group and 13 rats in the hanging cage group had ulcers in the glandular portion of the stomach. Ulcers were significantly larger and more numerous in the activity group, and activity levels were highly correlated with area of ulceration. In both groups, terminal body weight was also significantly correlated with area of ulceration. No statistically reliable mealtime effects were observed because of large within-group variability. Because many rats in hanging cages developed ulcers in the absence of wheel running, the results suggest that restricted food supply and loss of body weight are initiating factors in ulcer formation, while excessive wheel running contributes to the severity of ulcerations in the glandular stomach.

Diet-induced obesity attenuates anticipation of food access in rats.

Rats were housed in hanging cages and given ad lib access to food (control), approximately 20 g food/day (lean), or a palatable high-fat diet (fat). After body weights diverged, rats were transferred to activity wheels. When food access was reduced to 2 h/day, all control and lean rats displayed anticipatory activity (AA), while only two of eight fat rats anticipated the meals. Baseline activity levels and nonanticipatory wheel running were only marginally reduced in fat rats. In a second experiment, conducted entirely in activity wheels, rats were maintained on curtailed food or a high-fat diet until body weights diverged. Food access was then reduced to 2 h/day and the diets were reversed. Lean rats given restricted access to the high-fat diet gained weight and four of eight rats showed AA. Fat rats switched to chow lost weight and all eight rats displayed AA. The results indicate that body weight changes induced by diet manipulations result in a striking and rather selective reduction in the anticipation of daily meals. This effect is ascribed to a modulation of the output of a circadian pacemaker that entrains running-wheel activity to daily meals.

Evidence for a separate food-entrainable circadian oscillator in the pigeon.

In Experiment 1, four pigeons lived in a metabolic chamber on a 12h:12h LD cycle where they maintained a reduced body weight by consuming a daily ration of food presented at the eighth hour of the photophase. Body temperature (Tb) and oxygen consumption (Vo2) increased prior to the daily feeding. The possibility that a food-entrainable oscillator timed these anticipatory responses was tested by four manipulations, conducted in successive phases, each of which involved eliminating the regularly scheduled food presentation, which is the putative entraining stimulus for such an oscillator, while the 12h:12h LD cycle remained in effect. The manipulations, and their outcomes, were: when fasting was imposed for 3 days, the anticipatory responses continued to occur; when ad lib feeding was allowed for 11 days, the anticipatory responses were mostly eliminated; when fasting was reimposed for 5 days, there was evidence that the anticipatory responses reoccurred; and, when the time of the daily feeding was phase-shifted earlier in the photophase for 8 days, anticipatory responses persisted at the original feeding time and simultaneously developed at the new feeding time. In the first phase of Experiment 2, key pecking by two pigeons produced food only during hours 9-11 of the daily photophase (12h:12h LD). In this condition, Tb increased and key pecking occurred in anticipation of the daily period of food availability. Evidence for a food-entrained oscillator was sought in a second phase when constant dim light (LL) was imposed without changing the hours of food availability.(ABSTRACT TRUNCATED AT 250 WORDS)

Resetting of a feeding-entrainable circadian clock in the rat.

Reentrainment of anticipatory activity (AA) after phase shifts of food access was studied in rats with lesions of the suprachiasmatic nuclei. Eight- or 10-h phase delays of feeding time resulted in delaying transients of AA. Twelve-h phase shifts and some 10-h phase advances resulted in rapid reentrainment (2-3 days) without visible transients. Most phase advances of 10 h resulted in delaying transients while 8-h advances induced transients with advancing and delaying components in a number of rats. Split transients were not prevented by advancing mealtime in 1-h steps. Phase shifts of food in multiple steps failed to accelerate delay shifts but retarded advance shifts. After the first 8-h phase delay shift, increased activity reappeared at the preshift phase of AA, simultaneously with anticipation of the phase-shifted meal time. The observation of split transients indicates that two or more circadian oscillators mediate entrainment to mealtime, and the reappearance of AA at a previously established phase suggest the possibility that this system has a memory of phase displacement.