A cholinergic receptor 12-hour interval-timer displaying "circa" periodicities

The finding of brief pulses of receptor escape from atropine blocking of acetylcholine paralleling moonset and moonset-plus-12-hr led to a search for similar pulses paralleling sunset. Pulses occurring in the hour before sunset as well as in the hour before sunset-plus-12-hr were found. The pulses of receptor escape associated with sunset and sunset-plus-12-hr were in the opposite direction from those associated with moonset and moonset-plus-12-hr. The direction of receptor escape in all cases reversed with the winter solstice. These findings appear to provide evidence for an endogenous 12-hr interval-timer responding to external 24- and 24.8-hr oscillating systems which interact to yield a family of frequencies including those designated "circa".     

The cholinergic reaction of the amnion in the chick embryo

Studies have been made of the spontaneous contractions of the amnion and acetylcholine sensitivity of amniotic membrane in 8--14-day chick embryos. In 12--14-day embryos, the spontaneous rhythmic contractions were rather rare as compared to those in 8--9-day ones, their frequency being also lower. On the basis of kinetic analysis, it was concluded that both the dissociation constant (K) and the value of Pmax do not exhibit significant changes for tonotropic reaction from the 8th to the 14th day and for chronotropic reaction--from the 8th to the 10th day of incubation. After the 10th day of incubation, dose-effect chronotropic reaction not expressed. The spontaneous activity of the amnion and acetylcholine sensitivity of the amniotic membrane depend on the temperature being maximal at 38 degrees C. Possible regulatory mechanisms of contractile activity in chick amnion are discussed.     

Progesterone levels measured every two hours in the cyclic hamster.

Four groups of unanesthetized hamsters were bled by cardiac puncture three times a day at 4-hr intervals for each day of the 4-day estrous cycle. Serum progesterone (P) was determined by RIA. On day 1 of the cycle (day of ovulation) there was a trend for excursions in P at 4- to 6-hr intervals; this was followed on day 2 by a relatively steady level of P of 6-8 ng/ml. P dropped drastically after 0200 of day 3, indicating the onset of luteolysis. A significant increase in P occurred at 1600 of day 3 which was presumably nonluteal in origin. A series of cyclic fluctuations in P began at 1600 of day 4 (proestrus) comparable to the pattern observed on day 1 of the cycle.     

Control of Botrytis cinerea and Botrytis squamosa in overwintered salad onions by fungicide sprays

In 1974/75, 13 sprays of 0·2% a.i. thiram applied at 14 day intervals to overwintered salad onions reduced the incidence of Botrytis cinerea and significantly increased onion yields. In 1977/78 both B. cinerea and B. squamosa occurred, and 12 iprodione sprays at 0·1% a.i. applied at 14-day intervals or 6 sprays at 0·2% a.i. applied at 28-day intervals gave good control of B. cinerea and B. squamosa and significantly increased onion yields. Benomyl (0·1% a.i. at 14-day intervals, or 0·2% a.i. at 28-day intervals) failed to control either pathogen because of the development of carbendazim-insensitive strains of the fungi. Effective control of both pathogens and increased yields were obtained with an application of 0·4% a.i. thiram in October and November followed by an application of 0·2% a.i. iprodione in December and January.     

Germination characteristics ofDiamorpha cymosa seeds and an ecological interpretation

Summary Laboratory-stored seeds ofDiamorpha cymosa (Nutt.) Britton (Crassulaceae) were germinated at monthly intervals starting shortly after maturity in late May and ending at approximately the time germination is completed in the field (November). Seeds were placed at 5, 10, 15, 20, 25, 30, 15/6, 20/10, 30/15 and 35/20°C at a 14-hr photoperiod (12/12 hr thermoperiods at the alternating temperature regimes) and in constant darkness. In June, seeds were almost completely dormant and thus germinated poorly or not at all under all conditions. As seeds aged from late May to November 1. germination at the 14-hr photoperiod increased in rate and total percentage, 2. the maximum germination temperature increased from 15 to 25°C at constant temperatures and from 20/10 to 30/15°C at the alternating temperature regimes and 3. the optimum temperature for germination increased from 15 to 15–20°C at constant temperatures but remained at20/10°C at alternating temperature regimes throughout the study. During the same period germination in constant darkness was negligible at constant and alternating temperature regimes. This pattern of physiological after-ripening apparently is an adaptation to summer-dry,winter-wet habitats such as rock outcrops of southeastern United States.A short period of illumination with white light given after a 12-hr imbibition period in darkness promoted germination in the dark at 25/10°C but not at 15 or 25°C. A short period of illumination given during the imbibition period was much less effective in promoting germination in the dark. Drying up to 7 days did not cause light-stimulated seeds to lose their ability to germinate in darkness. The light requirement for seed germination probably does not play a role in restrictingD. cymosa to its well-lighted habitats on granite and sandstone outcrops.This research was supported by funds from the University of Kentucky Research Foundation and by an NIH Biomedical Sciences Support Grant to the University of Kentucky.     

Prostaglandins F in uterine and ovarian venous plasma from nonpregnant and pregnant ewes collected by cannulation

Periodic collections of uterine venous blood were obtained from three nonmated, three pregnant and two mated but nonpregnant ewes in which uterine veins were cannulated with polyvinyl tubing on day 11 postestrus. Frequent sampling was achieved in three of these ewes with additional cannulae in the ovarian veins. Blood samples were collected at 3-hr intervals from 0600 on day 12 to 1800 on day 13 and then at 6-hr intervals through day 15. On day 13, three additional samples at 30-min intervals were collected between 1400 and 1530. Prostaglandins F (PGF) in plasma were quantified by radioimmunoassay.     

Atretogenic action of estrogen in rhesus monkeys: Effects of repeated treatment

The effects of 17β-estradiol (E₂), administered in Silastic capsules for 24 hours at intervals of 10 or 14 days, on follicular development and menstrual cycle characteristics were studied in 13 rhesus monkeys. In seven monkeys receiving E₂ at l0-day intervals for 50 treatment periods, new follicles frequently developed between treatments but usually regressed. In seven instances, the follicles persisted longer than expected but were steroidogenically suppressed and regressed spontaneously. Ovulation occurred in only two instances. In six monkeys receiving E₂ at 14-day intervals, new follicles developed regularly, with seven ovulations occurring in 37 treatment periods. A persistent anovulatory follicle was noted in only one instance. Menstruation occurred with equal frequency, and the interval from treatment to onset of menstruation was not significantly different regardless of treatment or the occurrence of ovulation; the intervals between menstruation approximated those of normal menstrual cycles. In general, following termination of treatment, menstrual cycles returned to normal quickly. These data indicate that E₂ administered intermittently at 10-day intervals effectively suppresses ovulation, and they provide new insight into the actions of E₂ on folliculogenesis in primates.     

Temporal response of the fetal pulmonary circulation to pharmacologic vasodilators

To determine the temporal response of the fetal pulmonary circulation to pharmacologic vasodilators and to assess vasoreactivity following vasodilation, we infused either acetylcholine, histamine, or bradykinin directly into the left pulmonary artery of 21 chronically prepared fetal sheep. Blood flow (Q) to the left lung was measured by electromagnetic flow transducer. Left pulmonary artery infusion of acetylcholine at 1.5 micrograms.min-1 for 2 hr produced an increase in Q from 59 +/- 8 ml.min-1 to a peak of 113 +/- 10 ml.min-1 at 20 min into the infusion (P less than 0.001). After the peak at 20 min, Q steadily declined toward baseline to 66 +/- 7 ml.min-1 at the end of the 2-hr infusion period (P less than 0.01). Q in the 1/2-hr period following infusion was significantly less than the baseline period (47 +/- 6; P less than 0.04) with no change in pulmonary artery pressure. Similar patterns were seen with 2-hr infusions of histamine (150 ng.min-1) and bradykinin (100 ng.min-1). After a 2-hr infusion of one of the agents, a repeat infusion with that agent or a different one resulted in a diminished response. We conclude that fetal pulmonary vasodilation in response to local infusion of acetylcholine, histamine, or bradykinin is not sustained over a 2-hr period, and that following 2-hr exposure to vasodilators, pulmonary vascular resistance is increased and pulmonary vasoreactivity to pharmacologic vasodilators is decreased.     

Circadian and ultradian rhythms of proliferation in human ovarian cancer

Synchronous waves of proliferation in tumor cells taken from patients with ovarian cancer were observed using flow cytometry to measure the fraction of cells undergoing DNA replication and displaying tumor-cell-specific immunofluorescence. When saline washings of the abdominal cavity were analyzed at 2-4 hr intervals round-the-clock, the percentage of cells in the chromosome replication cycle (S + G2 percentage) showed 12-hr and often higher frequency rhythms in proliferation. These higher frequency rhythms in DNA replication show a relatively constant phase relationship to the patient's circadian clock with peak proliferation occurring most commonly at 10 a.m. to 12 noon and again at 10 p.m. This proliferation rhythm is therefore partially out of phase with the 24-hr rhythms in proliferation seen in normal cells. The findings on human cancer reveal a fundamental difference in the temporal organization of normal and tumor cell growth that should be exploited for therapeutic benefit.     

Correlation of nitric oxide (NO) activity and gonadal function in Japanese quail, Coturnix coturnix japonica following temporal phase relation of serotonergic and dopaminergic oscillations

Nitric oxide (NO), a highly reactive and short-lived radical, is considered to be an important trigger molecule for several physiological mechanisms including gonadotrophin releasing hormone (GnRH) secretion in mammals, although there is no such information in avian literature. On the other hand, specific temporal phase relation of circadian neural (serotonergic and dopaminergic) oscillations is reported to modulate reproductive activity in many avian species including Japanese quail. The present study was undertaken to investigate the correlation of NO activity and gonadal function of Japanese quail. In experiment I, the effect of serotonin and dopamine precursors, (5-hydroxytryptophan (5-HTP) and L-dihydroxyphenyalanine (L-DOPA) respectively; 5 mg per 100g body weight) administered at intervals of 8 or 12h over a period of 13 days, was studied on reproductive responses and NO activity. Measurements of body weight, cloacal gland size, testosterone concentration, spermatogenesis, nitrite-nitrate concentration in plasma, hypothalamus and testes, and NADPH-diaphorase (NADPH-d) activity in testes were made on the 2nd, 3rd, 6th and 11th days of treatment and 2nd and 30th day post-treatment. In experiment II, quail were divided into five groups including the control. One experimental group received 13 daily injections of 5-HTP and L-DOPA at intervals of 8h along with 0.1 ml of normal saline administered orally (8-hr+Veh), while another group of 8-hr quail received NO donor (sodium nitroprusside (SNP), 5 mg per 100 g body weight) orally (8-hr+SNP). The third experimental group received 5-HTP and L-DOPA at intervals of 12h along with normal saline (12-hr+Veh), while the fourth group of quail along with 5-HTP and L-DOPA at intervals of 12h also received the NOS inhibitor (N-nitro-L-arginine methyl ester, L-NAME, 25 microg per 100 g body weight) intraperitoneally (12-hr+L-NAME) for 13 days. This experiment was terminated after 21 days of the treatment. Results indicate that 5-HTP and L-DOPA administered 8h apart (8-hr) suppressed but if given 12h apart (12-hr) stimulated the reproductive system and NO activity compared to the control. These effects were apparent on the 6th day of injections and were maintained 30 days following the termination of the treatment. A significant decrease in nitrite and nitrate concentration and NADPH-d activity in reproductively inhibited 8-hr group and an increase in reproductively stimulated 12-hr quail was also evident. In contrast, these activities were stimulated in 8-hr+SNP quail and were suppressed in 12-hr+L-NAME group quail. It is concluded that activity of the reproductive system and NO activity waxes and wanes simultaneously in Japanese quail. Moreover, experimental modulation of gonadal activity (following changes in the phase relation of serotonergic and dopaminergic activity) or NO activity (following the administration of NO modulator or inhibitor) affects each other maintaining a parallel relation between the two systems. Further, it is interesting to note that the gonado-stimulatory effect of SNP overpowers the gonado-inhibitory effects of the 8-hr time interval and inhibitory effects of L-NAME mask the stimulatory effects of 12-hr temporal relation of 5-HTP and L-DOPA administration. These findings strongly suggest that reproductive effects may be induced via changes in NO activity, however the exact mechanism by which NO drives gonadal axis needs to be ascertained.     

Quantitative morphologic studies on the myocardium in early stages of ontogenesis and after application of various cardioactive agents

The authors have morphometrically studied the differentiation of the myocardium in dynamic phases of the embryonic and postnatal development in chickens and Syrian Hamsters. Moreover, they investigated the action of the beta-adrenalytic substances Practolol and Trimepranol on ultrastructure of the cardiac muscle in adult animals. The volume of mitochondria in myocardial cells in 6-day old chicken embryos amounts to 5.65% of the total cell volume, in 12-day old embryos 14.35%, in 18-day old embryos 19.60%, in 1-day old chickens 23.24% which is nearly as much as in adult animals. The volume of myofibrils in 6-day old embryos is about 3.2%, in 12-day old embryos about 7.4%, in 18-day old embryos about 16.4% and in 1-day old chickens about 21.2%. The differences between individual groups are statistically significant. The dynamics of differentiation of the myocardium in Syrian Hamsters was studied in 5 phases, namely in 14-day old embryos and in postnatal phases on the 2nd, 5th, 14th and 21st days after birth. Most cells in 14-day old embryos are rather immature. Participation of the volume of mitochondria, myofibrils, equipment of mitochondria with cristae etc. considerably increase in postnatal phases. These findings suggest that the heart of mammals is rather immature at birth and will differentiate mainly in the postnatal developmental phases. Many morphometric findings, as regards the action of beta-adrenalytic drugs on the ultrastructure of the myocardium in adult rabbits, point to the fact that application of these substances will give rise to degenerative alterations in approximately 10% of myocardial cells. Theoretical explantation of these mechanisms is being discussed.     

Atypical 24-hour rhythms of serotonin N-acetyltransferase activity in the rat pineal gland

Previous long-term studies have shown that in the pineal gland of rats melatonin synthesis is subject to infradian rhythms with periods between 4 and 7 days. Since in these studies melatonin-related parameters were measured at one timepoint of a 24-hr cycle only, the aim of the present investigation was to extend these experiments by more frequent sampling, to characterize the infradian rhythmicity in more detail. Male Sprague-Dawley rats kept under a light schedule of LD 12:12 (lights on at 0700) were killed at 6-hr intervals on 8 consecutive days. After decapitation the pineal gland was rapidly dissected out, followed by measurements of one of the melatonin-forming enzymes, serotonin N-acetyltransferase (NAT) activity. It was found that pineal NAT activity exhibited the well known day/night rhythm, i.e. low activity during daytime and strikingly enhanced activity at night, during the first 4 days of the experiment. On the fifth night (from Saturday to Sunday) an unusually high NAT peak occurred at 2400 hr, followed by two atypical 24-hr cycles. In the first cycle the midnight and 0600 hr values were equal and in the second cycle the 0600 hr value was significantly higher than the midnight value. To investigate whether the unusually high NAT peak was a single event or not, four additional short-term experiments were carried out at 2400 hr on 4 consecutive weekends, from Friday to Monday. In each of the four 4-day experiments a distinctly higher peak of NAT activity was found on Saturday, but with time the peaks became less prominent.(ABSTRACT TRUNCATED AT 250 WORDS)     

In Vitro Induction of Altered Response of Rat Atria to Acetylcholine with Histamine-Sensitizing Factor of Bordetella pertussis

Purified histamine-sensitizing factor (HSF) of Bordetella pertussis induced in vitro an alteration in the pharmacologic response of rat atria to acetylcholine. Spontaneously beating atrial preparations isolated from rats were exposed to HSF at a concentration of 50 ng/ml at 37 C for 30 min to 4 hr and washed with Krebs-Ringer solution, and then tested at 1-hr intervals up to 28 hr during incubation for their responses to epinephrine and acetylcholine. At 13 hr after exposure to HSF, irrespective of the exposure period, the HSF-treated atria, in which positive-inotropic action of epinephrine was manifested, depressed the negative-inotropic response to acetylcholine. The activity of HSF was neutralized by anti-HSF serum only in the first 3 hr of exposure. Only 1.8% of the added ¹²⁵I-labeled HSF bound “specifically” to one pair of atria for the manifestation of the altered response.     

Comparative studies on the superovulatory effect of PMSG and FSH in water buffalo (Bubalus bubalis )

A total of thirty-eight lactating water buffalo cows were treated in four experiments simultaneously either with FSH (first group) or PMSG(second group). To the first group (half of the animals), a total dose of 40 mg FSH-P at 12-hr intervals was given i.m. within a 4-day period. The second group was treated i.m. with 3000 IU PMSG (Gestyl). Forty-eight hours after initiation of the superovulatory treatment all buffaloes were given 500 ug Cloprostenol. Fi seen buffaloes from the FSH-treated group (78.9%) and 17 from the second group (89.5%) came into heat at average PGF 2 alpha/standing heat intervals of 42.8+/-1.48 and 44.8+/-2.31, respectively. Superovulatory treatment resulted in meath number of 4.3+/-0.87 and 1.9+/-0.50 CL and 0.5+/-0.24 and 2.2+/-0.82 follicles for the first and second group. Twenty-five eggs were recovered after non-surgical flushing from 8 of 13 flushes in the first group and all except one were fertilized and classified as good embryos. Twelve eggs were recovered from 4 of 11 flushes in the second group and 11 of the eggs were fertilized and 10 of them classified as good ones.     

Development of glucose active transport in embryonic chick intestine. Influence of thyroxine and hydrocortisone

1. Glucose active transport is detectable in 12-day-old embryonic chick duodenum and increases at least 11-fold after 4 days of postnatal life. 2. Glucose active transport develops at the in vivo rate in 72-hr cultures of 14-day embryonic duodenum. 3. In the presence of either 1 nM thyroxine or 1 microM hydrocortisone in vitro, glucose active transport reaches levels approximately 200% of control values (equivalent to 18-19 day levels in vivo). 4. Thyroxine and hydrocortisone act by different mechanisms based on their antagonistic interaction and differences in time course of action, requirement for protein synthesis and modulation by extracellular calcium.     

Appetitive and consummatory sexual behaviors of female rats in bilevel chambers. II. Patterns of estrus termination following vaginocervical stimulation

Copulation with intromission or manual vaginocervical stimulation (VCS) shortens the duration that intact female rats maintain lordosis responding during estrus. The present study examined whether VCS could shorten the duration of both appetitive and consummatory measures of female sexual behavior, and whether these effects occur differentially in time and across different hormone priming intervals. Ovariectomized, sexually experienced female rats were administered subcutaneous injections of estradiol benzoate 48 h and progesterone 4 h, before receiving 50 manual VCSs with a lubricated glass rod distributed over 1 h. Control females received sham VCSs distributed over the same time. The females were then tested for sexual behavior in bilevel chambers with two sexually vigorous males (to one ejaculatory series or 10 min with each male, separated by 5 min) 12, 16, and 20 h after VCS. Prior to the final hormone treatment, different groups of females had been given the same hormone treatment either 28, 14, 7, or 4 days before. In females tested at 28- and 14-day hormone intervals, VCS induced both active and passive rejection responses at 12, 16, and 20 h. In contrast, females that received sham VCS displayed relatively normal sexual behavior at 12 h, although by 16 and 20 h these females displayed active and passive rejection. Females tested at 7- or 4-day intervals displayed normal levels of lordosis at all testing times, regardless of VCS treatment. These data indicate that VCS facilitates rejection responses that precede the decrease in lordosis responsiveness. However, the effects of VCS are dependent on the frequency of hormone priming, suggesting that hormone treatment may block some of the long-term inhibitory effects of VCS on female sexual behavior.     

Prostaglandins F in uterine and ovarian venous plasma from nonpregnant and pregnant ewes collected by cannulation.

Periodic collections of uterine venous blood were obtained from three nonmated, three pregnant and two mated but nonpregnant ewes in which uterine veins were cannulated with polyvinyl tubing on day 11 postestrus. Frequent sampling was achieved in three of these ewes with additional cannulae in the ovarian veins. Blood samples were collected at 3-hr intervals from 0600 on day 12 to 1800 on day 13 and then 6-hr intervals through day 15. On day 13, three additional samples at 30-min intervals were collected between 1400 and 1530. Prostaglandins F (PGF) in plasma were quantified by radioimmunoassay. On day 12, one ewe in each group had at least one measurement which suggested an increased rate of release of PGF into the uterine vein. Seven of eight ewes on day 13 appeared to have increased rates of release of PGF from the uterus between 0900 and 1500. The highest level measured in each ewe during this period ranged from 2.7 to 11 ng per milliliter. Concentrations of PGF in ovarian venous plasma in two of three ewes were positively correlated (P less than .05) with concentrations of PGF in uterine venous plasma (r equals .64 in each ewe). No evidence was obtained that pregnant and nonpregnant ewes differ in rate or pattern of release of PGF from the uterus into the uterine vein on days 12 and 13. Comparisons could not be made with confidence concerning PGF either in uterine veins on days 14 and 15 or in ovarian veins on all days due to limited number of observations.     

Lack of Variation Over 24-Hours in Response to Stimulation of 5-HT1 Receptors in the Mouse Brain

Two behavioural measures of activity at 5HT₁ receptors in mice: RU 24969-induced hyperactivity and 80H-DPAT hypothermia, were measured at 3-hr intervals throughout the 12-12 L-D cycle. Neither parameter showed significant circadian variation.     

Rearing under different conditions results in different functional recoveries of giant interneurons in unilaterally cercus-ablated crickets, Gryllus bimaculatus

The effects of rearing conditions on the functional recovery of wind-sensitive giant interneurons (GIs) after unilateral cercal ablation were investigated in the cricket, Gryllus bimaculatus. Crickets were reared in a glass vials to prohibit free walking for 14 days after unilateral cercal ablation ("14-day vial" crickets). Other crickets were reared in an apparatus called a "walking inducer" (WI) to increase the walking distance during the same 14-day period ("14-day WI" crickets). In these crickets, the response properties of GIs 8-1, 9-1, 9-2, and 9-3 to air currents from various directions were investigated. From the intensity-response curves obtained, directionality curves expressed in terms of threshold velocity and response magnitude were made independently. To understand changes in the functional recovery of GIs more thoroughly, the directional characteristics of GIs in crickets 1 day after unilateral cercal ablation ("1-day free" crickets) were also compared. Between the 1-day free and 14-day vial crickets, all the GIs showed differences in both threshold velocity and response magnitude for some stimulus directions. Between the 14-day vial and 14-day WI crickets, differences in the threshold velocities of GIs 9-1, 9-2, and 9-3, and in the response magnitudes of GIs 8-1, 9-1, and 9-3 were detected. Because the rearing condition after unilateral cercal ablation largely affects the compensatory recovery in some parameters of wind-evoked escape behavior, such as relative occurrence and escape direction, we discuss the functional differences in GIs revealed here in relation to the roles of GIs in the neural system that controls escape behavior.     

Letter from the Editors

Two behavioural measures of activity at 5HT₁ receptors in mice: RU 24969-induced hyperactivity and 80H-DPAT hypothermia, were measured at 3-hr intervals throughout the 12-12 L-D cycle. Neither parameter showed significant circadian variation.