Synergistic inhibition by benzodiazepine and barbiturate drugs of the clock control of ovulation in hamsters.

A surge of pituitary luteinizing hormone (LH) into the bloodstream occurs in hamsters every 4 days between 1:30 p.m. and 3 p.m. in response to a signal from a biological clock. This surge initiates behavioral estrus approximately 2 h later and ovulation approximately 12 h later. Phenobarbital at a dose > or = 100 mg/kg consistently blocks LH release. Barbiturate and benzodiazepine drugs have separate binding sites in the GABAA receptor/chloride channel complex. Binding of either drug increases GABA-mediated chloride conductance, which suppresses the postsynaptic neuron. Barbiturate binding also increases benzodiazepine binding. This suggested that these drugs might synergize to inhibit LH release. A combination of triazolam and phenobarbital at doses of 10 mg/kg injected s.c. at 1:30 p.m. inhibited ovulation and extended the 4-day vaginal cycle in all treated hamsters. Either drug dose injected alone at 1:30 p.m., or the combination at 3 p.m., was completely ineffective. Bicuculline prevented inhibition by the combination at 1:30 p.m. The clock signal for LH release may act by antagonizing GABA transmission, which may be chronically inhibiting LH release. The combination delimited a 75-min period (1:30-2:45 p.m.) within which the clock signal for LH release occurred in all individuals (ET50 = 2:08 p.m.). This period appears to arise from individuals with different but constant clock settings rather than from a 75-min variation in the clock setting of the individual.

Delay of behavioral estrus in hamsters and phenobarbital.

The onset of behavioral estrus was used as a phase marker of the hamster timing system in SLD 16:8 (dark 20:00-04:00). TZ was injected between 11:00 of cycle day 3 and noon of cycle day 4 when onset of estrus was determined. At no time did injection of TZ cause a phase advance in SLD 16:8. Small delays of estrus resulted from 11:00-16:00 injections but marked delays began with the 17:00 injection. Phenobarbital was injected between noon and 19:30 on cycle day 3. Injections between noon and 16:00 had no effect but all later injections beginning at 17:00 delayed estrus, the 17:30 injection causing the greatest delay. Diazepam also markedly delayed estrus when tested at 17:30. These results with three drugs support results with light pulses that 18:00 in SLD 16:8 marks the same phase of the 24-h hamster timing system as the onset of wheel running does in DD, LL, and WLD. These findings with three GABA potentiators extend to SLD previous evidence based on the onset of wheel running in DD, LL and WLD that GABA may be involved in hamster timekeeping and its responses to light and drugs.

How hamsters keep time: the 6 pm to 6 am light-sensitive period.

A recent review of the pineal literature revealed that when hamsters are exposed to 24-hour light:dark (LD) cycles with less than 12 hours of darkness (summerlike, SLD), the nightly period of pineal melatonin synthesis (PPMS) begins close to the midpoint of the dark period ("midnight") and ends at lights-on irrespective of the length of the dark period or time of day presented. New evidence based on the onset of behavioral estrus every 4 days indicated that the 24-hour hamster clock controlling timing of estrus (4:30 PM) and the PPMS has a 12-hour light-sensitive period (LSP) set to 6 PM-6 AM in LD 16:8 (dark 8 PM-4 AM, SLD) by a balance in opposing actions of evening and morning light [Alleva: Pineal Research Reviews, Volume 5, Alan R. Liss, Inc., New York, 1987]. Present experiments focus on how this balance is maintained. When lights-off was advanced to 6 PM in SLDs ranging from LD 12.5:11.5 (dark 6:15 PM-5:45 AM) to 18:6 (dark 9 PM-3 AM), the onset of estrus later that day was advanced in every SLD. However, when lights-on was delayed to or beyond 6 AM, the onset of estrus was unaffected. Thus, the balance is maintained by a resistive force (blocking without a delaying action) of evening light and an advancing action of morning light. In this balancing process all evening light from 6 PM to lights-off but only the first 5 minutes or less of morning light were involved. The advancing action of morning light was characterized in LD 13:11 and 18:6 by imposing on the night before estrus a 5:30 PM-6:30 AM dark period scanned with a 15-minute light pulse. Shifts in onset of estrus later that day were plotted vs. time of the light pulse. The resulting phase response curves (PRCs) were similar and comprised only an advancing curve, which rose about 10 PM, peaked at 2 AM, and returned gradually to normal at 6 AM. In contrast, a PRC obtained from LD 12:12 (dark 6 PM-6 AM) was sinelike, comprising a 6 PM-9 PM delaying curve followed by an advancing curve similar to those from SLD. An hypothesis based on these findings is presented to explain how hamsters would keep constant AM-PM time throughout summer.

Characterization of uterine growth response to cholera toxin in hamsters and test of heat-labile enterotoxin from Escherichia coli.

Cholera toxin (CT) and the heat-labile enterotoxin from Escherichia coli, when injected intraperitoneally into cycling hamsters but not rats or mice, induced a massive uterine growth similar to that normally induced by the implanting blastocyst during pregnancy. CT and heat-labile enterotoxin are the only known agents that have this action in any species. Uterine weight reached a maximal sixfold increase 48 h after injection of CT. Concurrent injection of estrogen, progesterone, and CT increased the maximal response to eightfold and eliminated differences in the response to CT injected on different days of the 4-day hamster estrous cycle. The dose response for CT, heat-labile enterotoxin, and CT plus estrogen plus progesterone was most linear (r greater than 0.93) when the logarithm of uterine weight was plotted against the dose of toxin. The hamster uterine weight response can serve as a simple, highly precise, and highly specific bioassay for CT and heat-labile enterotoxin.

Induction of estrogen and progesterone receptors and decidualization in the hamster uterus by cholera toxin.

Cholera toxin (CT) injected ip on day 1 (day of ovulation) of the 4-day hamster estrous cycle, when circulatory progesterone is high and estrogen low, induced a massive uterine decidual reaction, a progesterone-dependent growth normally triggered by the implanting blastocyst. However, CT injected ip on day 3, when circulatory estrogen is high and progesterone low, did not induce a decidual reaction but, instead, intensified the effects of estrogen (stromal edema and stimulation of the mucosa). These cycle day effects were reproduced in one uterine horn injected intraluminally with CT, but not in the other horn of the same animal given solvent alone as a control. The intrauterine injection of CT had no effect on the concentration of serum estrogen or progesterone. The decidual reaction resulting from intrauterine injection of CT on day 1 was accompanied by increases in estrogen receptor (femtomoles per mg DNA) in both cytoplasm and nucleus. In long term ovariectomized hamsters, an ip or intrauterine injection of CT induced only histological effects of estrogen (stromal edema and mucosal mitosis) without affecting circulatory estrogen. These estrogenic effects were accompanied by increases in receptors for estrogen and progesterone in both cytoplasm and nucleus. CT injected ip into ovariectomized hamsters primed with estrogen intensified the stromal edema and mucosal mitosis and resulted in progesterone and estrogen receptor levels equal to or greater than those after the administration of CT or estrogen alone. When progesterone was included in the priming (estrogen + progesterone + CT), all receptor levels were decreased, and a massive decidual reaction resulted. Thus, the induction of estrogen receptor by CT may have been the primary event that triggered the decidual reaction. Whether CT-induced estrogen receptor is mediated by cAMP, a known mediator of CT, remains to be determined.

Cholera toxin: mitogen for Harderian glands of golden hamsters.

1. Within 2 days, a single injection of 10 micrograms cholera toxin induces a 6-20-fold increase in mitotic activity in the Harderian glands of male and female golden hamsters. 2. Neither hypophysectomy nor ovariectomy had any influence on this response. 3. The cellular proliferation does not appear to involve cAMP nor is it the result of stress, nor a release from an early mitotic block. 4. Nuclear polyploidy increases within a few hours after treatment. 5. DNA density per unit area increases within 24 hr and is maintained for at least 3 days.

Use of vaginal concretions of calcium carbonate to detect ovulation in hamsters.

Concretions of calcium carbonate normally appear every third and fourth da of the 4-da hamster cycle da 1 = day of ovulation). We presently found that the 2-da relation between ovulation and concretion formation was maintained even after ovulation was delayed 1 or more da by centrally acting drugs or by exposing the hamsters to constant light. It is therefore possible to identify days of ovulation in vaginal washing records of both normal and treated hamsters by counting back 2 da from each sequence of concretions.

Calcium carbonate concretions: cyclic occurrence in the hamster vagina.

Three crystalline forms of calcium carbonate were identified in washings of the hamster vagina. Spherical concretions of vaterite and hexagonal concretions of calcite predominate on days 3 and 4 of the 4-day estrous cycle. Dumbbell-like concretions of aragonite predominate during pregnancy and pseudopregnancy. Each polymorph is associated with an acid-insoluble matrix. Concretions disappear after ovariectomy and reappear during daily injections of estrogen and progesterone.