Beth E.F. Wee

Characteristics of the spinal nucleus of the bulbocavernosus are influenced by genotype in the house mouse

Sex differences and differences related to genotype have been observed in the nervous system. These observations provide the opportunity to relate differences in neural structure to functional differences. The spinal nucleus of the bulbocavernosus (SNB) was examined in castrated and gonadally intact male house mice from the following 3 genotypes: C57B1/6J, DBA/2J, and the B6D2F1 (F1) hybrid (resulting from a cross between a C57B1/6J female and a DBA/2J male). The number and size of SNB neurons were determined from thionin-stained thick sections from spinal cords of these mice. The gonadally intact DBA males had significantly fewer SNB motoneurons than either the C57 or F1 males, but no strain differences were observed for the size of SNB cells. Castration of adults significantly reduced SNB neuronal number, but not somatic area, in the C57s (the maternal strain) and reduced somatic area, but not neuronal number, in the DBAs (the paternal strain). Both characteristics of the SNB were reduced by castration in the F1 hybrid. Thus, the size and number of SNB neurons appear to be inherited independently and the influence of gonadal hormones on these traits varies with genotype.

Retention of masculine sexual behavior following castration in male B6D2F1 mice

The reduction of masculine sexual behavior following castration varies widely among genotypes. In contrast to the loss of sexual behavior by castrated males of other strains, males of the B6D2F1 genotype retain the ejaculatory reflex for many weeks after castration. The present study examined this retention phenomenon. Masculine sexual behaviors were measured before and after castration or sham operation in male C57BL/6J, DBA/2J, and B6D2F1 mice. Castrated C57BL/6J and DBA/2J males showed a rapid decline in copulatory behavior. In contrast, 30% of the B6D2F1 males continued to ejaculate 25 weeks after castration. Regardless of whether or not sexual behaviors were retained, levels of plasma testosterone and hypothalamic nuclear estrogen receptors were reduced by castration. These results suggest that the intra- and inter-strain differences in the retention of sexual behavior following castration are not due to differences in levels of steroid hormones. Further, some B6D2F1 males retain the ability to copulate in the absence of gonadal hormone levels required for the maintenance of sexual behavior in other genotypes.

Midazolam, a short-acting benzodiazepine, resets the circadian clock of the hamster

Treatment with the short-acting benzodiazepine, triazolam, has been found to induce changes in both behavioral and endocrine circadian rhythms in hamsters. The objective of this study was to determine if these effects of triazolam could be generalized to other short-acting benzodiazepines. Therefore, the effects of midazolam on the biological clock of the hamster were examined in detail. A phase-response curve and a dose-response curve were measured to determine the effects of a single intraperitoneal injection of midazolam on the circadian clock of hamsters free-running in constant light. Midazolam injections produced maximal phase advances at circadian time (CT) 6 and 9 and maximal phase delays at CT 15 and 21. Doses of 2.5 mg or larger produced phase shifts that were significantly different from those produced by the vehicle controls. In addition, the phase-shifting effects of midazolam were completely blocked by administration of the benzodiazepine receptor antagonist, RO 15-1788, indicating that the phase-shifting actions of midazolam are mediated via benzodiazepine receptors. These results indicate that the previously reported effects of triazolam on the circadian clock can be generalized to other short-acting benzodiazepines.

Administration of carbachol into the lateral ventricle and suprachiasmatic nucleus (SCN) produces dose-dependent phase shifts in the circadian rhythm of locomotor activity

The cholinergic agonist, carbachol, induces phase-dependent shifts in the timing of the circadian rhythm of locomotor activity (CRLA). The effects of carbachol injections into the lateral ventricles of hamsters were compared between circadian times that produce phase delays vs. phase advances in the CRLA. The shape of the dose-response curves and the ED50 for carbachol injections were similar for the two injection times. The second experiment demonstrated the dose-dependence of phase advances produced by carbachol injections into the area of the suprachiasmatic nucleus. These results indicate that carbachol exerts similar dose-dependent actions for both phases advances and phase delays.

Hormonal restoration of masculine sexual behavior in long-term castrated B6D2F1 mice

In contrast to the facilitative effects reported for other rodents, testosterone treatment at the time of castration previously was reported to inhibit masculine sexual behavior in male B6D2F1 mice. Males of this genotype vary in their behavioral response to castration. Some castrates retain sexual behaviors for many weeks after surgery, whereas others do not. In the present study, we sought to determine the effects of exogenous steroid hormone treatment on castrated B6D2F1 mice that had ceased to show copulatory behavior. Testosterone propionate and estradiol benzoate restored copulatory behavior to precastration levels in B6D2F1 males that did not retain sexual behaviors after castration.

Carbachol phase shifts the circadian rhythm of locomotor activity in the Djungarian hamster

The effects of light on the circadian system may involve the neurotransmitter acetylcholine (ACh). The purpose of the present study was to determine whether carbachol, a cholinergic agonist, mimics the phase-shifting effects of light on the circadian rhythm of locomotor activity (CRLA) in the Djungarian hamster. Phase response curves (PRCs) were measured for blind animals injected intracerebroventricularly with either vehicle or carbachol at different circadian times. Carbachol produced significant phase delays in the CRLA when administered at circadian time (CT) 12-16 and small, but significant phase advances when given at CT 8. No consistent phase shifts were observed in response to carbachol injections at other circadian times. Phase shifts produced by carbachol injections at CT 8 and 12 were similar for both sighted and blind animals. Light pulses to sighted hamsters produced phase advances at CT 20 and phase delays at CT 12. Both the carbachol PRC and the PRC for light pulses in the Djungarian hamster have phase delay regions in the early subjective night. However, these two PRCs have different phase advance regions. These results indicate that carbachol mimics some of the effects of light on the circadian system in the Djungarian hamster and support the hypothesis that cholinergic mechanisms may play a role in mediating some of the phase-shifting effects of light on the circadian clock of mammals.

Environmental influences on masculine sexual behavior in mice

Retention of masculine copulatory behaviors following castration varies among B6D2F1 male mice. In the present study, we examined the effect of test environment and the amount of behavioral testing after castration, on retention of copulatory responses in castrated B6D2F1 male mice. Results showed that weekly behavioral testing after castration was not necessary for the retention of ejaculatory reflexes. However, the test environment had a major effect. Following castration, 26% of the males completely stopped showing ejaculatory responses when tested in the test arenas. When these males were tested in their home cages, 75% achieved at least one ejaculatory response in four home cage tests. Castrated males that continued to copulate in the test arena situation also achieved ejaculatory reflexes in their home cages. These results indicate that for some B6D2F1 males, the retention of sexual behaviors after castration is influenced by environmental factors.

Increased Hypothalamic Somatostatin Expression in Mice Transgenic for Bovine or Human GH

Acute studies of GH removal by hypophysectomy or GH replacement in adult rats have shown that GH has a positive influence on its hypothalamic inhibitory hormone somatostatin (SRIH). The present study was undertaken to assess the effect of lifelong exposure to elevated GH on the development and differentiation of SRIH‐producing hypothalamic neurons, including comparison of differing GH levels and heterologous species of GH. Expression of somatostatin peptide and mRNA was evaluated using respective immunocytochemistry and in situ hybridization in brains of transgenic mice bearing constructs of either human (hGH) or bovine (bGH) linked to metallothionein (MT) promoter or bGH linked to phosphoenolpyruvate carboxykinase (PEPCK) promoter. Nontransgenic littermates served as controls. All transgenic constructs resulted in high levels of circulating heterologous GH and significantly elevated body weights. Both bGH levels and body weights were higher in PEPCK‐bGH than in MT‐bGH mice; mean weights were not different between MT‐bGH and MT‐hGH mice. Numbers of SRIH‐immunoreactive neurons in the hypophysiotropic periventricular nucleus (PeN) of transgenic mice showed a two‐fold increase (P<0.01) relative to control animals; the number of SRIH‐positive cells in the medial basal hypothalamus (MBH) was comparable for transgenic and control mice. Total SRIH mRNA in situ hybridization intensity also showed a two‐fold increase (P<0.05) in the PeN of all transgenic mice compared with controls, and was not elevated in the MBH. The higher levels of GH produced in PEPCK‐bGH transgenic mice led to greater weight gain, but not to greater SRIH expression than in other GH‐transgenic mice, suggesting that the increased SRIH cell number and mRNA in the PeN of MT‐GH‐transgenic mice may represent a plateau of maximal feedback stimulation. The results indicate that lifelong elevated heterologous GH in mice stimulates hypothalamic SRIH expression markedly. It is not known whether this mechanism is direct or indirect via a mediator of GH such as IGF, but the heterologous GH appears to be specific to these hypophysiotropic neurons.

Growth Hormone Releasing Hormone Expression During Postnatal Development in Growth Hormone-Deficient Ames Dwarf Mice:mRNA in situ Hybridization

Several genetic mutations in mice and rats that produce lifelong growth hormone (GH) deficiency result in overexpression of GH-releasing hormone (GHRH) mRNA in hypothalamic arcuate nucleus neurons. In order to examine the development of this condition, GHRH mRNA expression was quantified in Ames dwarf (df/df) and normal (DF/?) mice at 1 (day of birth), 3, 7, 14, 21 and 60 postnatal days (d) following in situ hybridization. Total mRNA was assessed using computer-assisted densitometry after X-ray film autoradiography, and mRNA expression per neuron was quantified by counts of grains per cell after emulsion autoradiography. Total GHRH mRNA was the same in dwarf and normal mice at 1, 3 and 7d. GHRH mRNA in dwarfs increased at 14d to 240% of that in DF/? (p < 0.005); the percentage overexpression in dwarf mice remained ≧200% through 60d, although total GHRH mRNA increased in both dwarfs and normals during this period. GHRH mRNA per neuron was the same in normal and dwarf mice at 1d, then increased in dwarfs to 190% of that in normals at 3d (p < 0.05), and rose to 300% of normal levels by 7d and beyond (p < 0.005). There was no sexual dimorphism in expression by either measure in normal or dwarf mice. These results indicate that an increase in GHRH mRNA in Ames dwarf mice is first detectable at 3d, a period of approximately 7d after the failure to initiate GH production, which occurs normally at embryonic day 17.5. The onset of GHRH overexpression occurs earlier than the decline of either hypophysiotropic somatostatin or dopamine in Ames dwarf mice. This difference may be due to the stimulatory action of GHRH, as opposed to the inhibitory effects of factors examined previously.

HYPOPHYSIOTROPIC SOMATOSTATIN EXPRESSION DURING POSTNATAL DEVELOPMENT IN GROWTH HORMONE-DEFICIENT AMES DWARF MICE : MRNA IN SITU HYBRIDIZATION

Lifelong deficiency of growth hormone (GH) in spontaneous or transgenic dwarf mice has been shown to be accompanied by reduced hypophysiotropic somatostatin (somatotropin release-inhibiting hormone, SRIH) expression in hypothalamic anterior periventricular nucleus (PeN). However, the postnatal developmental pattern of SRIH expression in the absence of GH is unknown. Therefore, SRIH mRNA levels in GH-deficient Ames dwarf (df/df) mice and normal (DF/?) littermates were determined both in adults, to compare with other GH-deficient models, and at selected days of postnatal development, to determine the effects of GH deficiency on SRIH neuron development. DF/? and df/df mice of both sexes at postnatal ages 1, 3, 7, 14, 21 and 60 days (adult) were examined. In situ hybridization and image analysis were used to quantify the relative abundance of total SRIH mRNA in the PeN, and SRIH mRNA per cell was determined in PeN and medial basal hypothalamus (MBH). In adult df/df mice, total PeN SRIH mRNA was 45% (p < 0.05) of that in DF/? littermates, which is consistent with studies of other GH-deficient dwarf mice. In developing animals, SRIH expression in the PeN of DF/? mice began at 3 days of age and increased at subsequent ages to reach adult levels. In df/df mice, PeN SRIH mRNA levels at 60 days were significantly greater than at 1-21 days of age (p < 0.05). However, levels were not different over 1-21 days of age, and were consistently lower in df/df than DF/? mice. The difference in total PeN SRIH mRNA between df/df and DF/? mice was statistically significant at 7 days, and at each subsequent age. There were no differences between DF/? and df/df mice in the number of grains per cell in either PeN or MBH at any age. Thus, the reduced total hypophysiotropic SRIH mRNA in GH-deficient Ames dwarf mice appears developmentally shortly after initial detectability of SRIH in the PeN. Because SRIH mRNA per cell was the same for DF/? and df/df mice, the decreased total mRNA in dwarfs suggested reduced SRIH cell numbers in PeN, which was corroborated by immunocytochemical findings. The reduction of SRIH in df/df mice at 7 days of age suggests that GH production during embryonic or very early postnatal development is important to activation of PeN SRIH transcription.