Carol K. Kellogg

Anxiolytic effects of 3α-hydroxy-5α[β]-pregnan-20-one: endogenous metabolites of progesterone that are active at the GABAA receptor

Abstract The effects of intracerebroventricular administration of reduced metabolites of progesterone on locomotor activity and on exploration in the elevated plus-maze were assessed in adult female rats. Allopregnanolone (3α-hydroxy-5α-pregnan-20-one; 1.25, 5.0, and 10 μm) and pregnanolone (3α-hydroxy-5β-pregnan-20-one; 2.5, 5.0, and 10 μg) elicited anxiolytic effects and, at the highest dose tested, allopregnanolone resulted in sedation. In contrast, the 3β-hydroxy-epimer of allopregnanolone was without effect in either behavioral paradigm. The anxiolytic response to pregnanolone was blocked by picrotoxin (0.75 mg/kg, i.p.), a dose that by itself did not affect behavior in the plus-maze. These data suggest that the anxiolytic effect of 3α-hydroxy metabolites of progesterone is mediated by brain GABAA receptors in a stereospecific manner, and are in good agreement with the well-documented in vitro effects of these steroids as potent modulators of the GABAA receptor.

Treatment with an Anabolic-Androgenic Steroid Affects Anxiety-Related Behavior and Alters the Sensitivity of Cortical GABAA Receptors in the Rat

The putative psychotropic effect of the anabolic-androgenic steroid, testosterone propionate (TP), was determined in intact adult male rats after 1 or 2 weeks of continued exposure via subcutaneously implanted capsules. Behavior was assessed in a novel open-field arena and in the elevated plus-maze. In addition, gamma-aminobutyric acid (GABA)-stimulated 36chloride (Cl-) influx was determined in cerebral cortical synaptoneurosomes as a function of TP exposure. The weight of the prostate gland was taken and blood serum level of total testosterone (T) was assayed. One week of TP exposure (approximately 3.5-5.0 mg/kg per day) resulted in anxiolytic behavior, as evidenced by an increase in the exploration of the open arms of the elevated plus-maze. The behavioral effect in the elevated plus-maze was not observed in animals exposed to TP for a 2-week period. Ambulation scores in the novel open field did not change as a function of TP exposure. Blood T levels were increased 7-fold by 1 week of exposure, and increased 10-fold in animals with implants for a 2-week period. After 1 week of TP exposure, the concentration of GABA that elicited 50% of the maximal Cl- influx in cortical synaptoneurosomes (i.e., EC50) was significantly decreased; this effect was not seen in animals exposed to TP for 2 weeks. The maximal efficacy of the GABAA receptor-gated Cl- influx was not affected after 1 or 2 weeks of TP treatment. Thus, 1 week of treatment with TP resulted in anxiolytic behavior that was accompanied by an increase in the sensitivity of cortical GABAA receptors. However, the behavioral and neurochemical changes were no longer present after 2 weeks of TP exposure. These results are discussed in terms of the agonist effects of reduced androgen metabolites at the GABAA receptor and the possible development of tolerance to these effects.

Ovarian endocrine status modulates the anxiolytic potency of diazepam and the efficacy of !g-aminobutyric acid-benzodiazepine receptor-mediated chloride ion transport.

The effect of ovarian steroid hormones on the behavioral and neurochemical sensitivity of the gamma-aminobutyric acid (GABA)-benzodiazepine (BZD) receptor chloride ion channel complex was studied. Locomotor activity and behavior in the elevated plus maze were examined in female rats of various ovarian states as was the efficacy and potency of GABA-stimulated chloride uptake in cortical synaptoneurosomes from proestrous and ovariectomized rats. A significant increase in the exploration of the open arms of the plus maze was observed in lactating females, in relation to diestrous, proestrous, ovariectomized, and pregnant females. The anxiolytic effect of diazepam (DZ) was decreased in ovariectomized females, in relation to proestrus females. Although 1.0 mg/kg DZ in proestrous females resulted in significant anxiolytic activity, this dose was ineffective in ovariectomized females but was reinstated by injection of estradiol benzoate and progesterone. A reduced efficacy of GABA-stimulated chloride ion transport in cortical synaptoneurosomes from ovariectomized females, in relation to that from proestrous females, was observed. Furthermore, the facilitative effect of DZ on the potency of GABA-stimulated chloride ion influx that was observed in cortical synaptoneurosomes from proestrous females was absent in synaptoneurosomes from ovariectomized females. These results are discussed in terms of the effect of ovarian steroids and reduced metabolites on GABA-BZD receptor-mediated functions.

Gonadal hormones during puberty organize environment-related social interaction in the male rat

This study examined the role of gonadal androgens during puberty on the development of environment-related social interaction (SI) in male rats. SI in an unfamiliar environment versus SI in a familiar environment was evaluated in young adult rats as a function of sex and gonadal status. Intact male rats at 60 days of age exhibited a differential response to the two environments, whereas SI in intact female rats at 60 days was equivalent in the two environments. Furthermore, male rats castrated as juveniles and tested for SI at 60 days displayed a pattern of environment-related SI similar to SI in intact adult female rats. This effect of juvenile castration on SI in male rats was prevented by chronic exposure to testosterone propionate (TP) over Days 30 through 60. SI in male rats castrated in adulthood, on the other hand, was not altered either 2 or 4 weeks postcastration. The results from this study indicate that pubertal secretions of gonadal androgen(s) are necessary for the development of environment-related SI in male rats. In contrast, secretions of gonadal androgens in adulthood do not appear to be critical for the continued expression of environment-related SI, as suggested by the observation that environment-related SI in male rats remains unchanged by castration in adulthood.

Benzodiazepines: influence on the developing brain

Publisher Summary This chapter discusses the influence of benzodiazepine (BDZ) on the developing brain. In recent years, neural receptors mediating the pharmacological effects of many neuroactive drugs are identified. Of particular pertinence to this discussion, specific neural binding sites for the anxiolytic BDZ compounds are identified and the interaction of these drugs with their binding sites correlates well with their anxiolytic, anticonvulsant, sedative and muscle-relaxant properties. The full expression of drug interaction with neural receptors during development can also depend upon the neural interactions that receptive cells make with other neurons and upon the maturational state of specific neural circuitry. Developmental exposure to BDZs is associated with neural, physiological and behavioral alterations in both clinical and experimental studies. The scope of the discussion is limited to the presentation of results of research on the effects of in utero exposure to BDZs in rats. To understand the implications that binding of drug to specific sites in fetal brain, it is necessary to demonstrate a response in the fetal brain to the presence of the drug.

Audiogenic seizures: Relation to age and mechanisms of monoamine neurotransmission

Mice with a genetically determined susceptibility to audiogenic seizures were utilized to analyze the ontogeny of central monoamine neurotransmission in relation to a behavior with age-specific properties. Levels of noradrenaline (NA), dopamine (DA), and 5-hydroxytryptamine (5-HT) were measured in forebrain and hindbrain regions at 14, 21, 28, and 42 days postnatal age in genetically sensitive or resistant strains of mice. An in vivo estimate of tyrosine and tryptophan hydroxylase activity was obtained at the same ages by following the accumulation of 3,4-dihydroxyphenylalanine (DOPA) and 5-hydroxytryptophan (5-HTP) respectively, after the administration of a centrally effective L-amino acid decarboxylase inhibitor (R04-4602, 800 mg/kg). At 14 days, there was a faster rate of accumulation of DOPA in both the forebrain and hindbrain of the sensitive mice compared to mice of the nonsensitive strain. At 21 days, the age of maximal sensitivity in the sensitive mice, the levels of NA were significantly lower in both regions of the sensitive mice, but the accumulation of DOPA was similar between strains at this age. There was also a slightly lower level of 5-HT in the forebrain of sensitive mice at 21 days accompanied by a slower rate of accumulation of 5-HTP in this region. In the hindbrain of the sensitive animals however, the rate of accumulation of 5-HTP was faster than in the sensitive strain. At 28 days, some impairment in mechanisms within NA-containing neurons in the sensitive mice was still apparent (including lower NA levels). At 42 days, there were no differences in amine levels, however, the levels of accumulated DOPA and 5-HTP were significantly lower in the sensitive strain. The results suggest that in the sensitive mice, developmental differences in mechanisms of monoamine storage and/or synthesis may exist which could contribute to deficient amounts of physiologically releaseable transmitter.

Adolescent development influences functional responsiveness of noradrenergic projections to the hypothalamus in male rats

Hypothalamic noradrenergic utilization in vivo and the in vitro depolarized release of norepinephrine (NE) were measured at 28, 42, and 70 days of age in male rats to determine the impact of adolescent development on the functional responsiveness of this transmitter system. At each age, function was determined in control rats and rats challenged by restraint. NE utilization in vivo was estimated by measuring the decrease in NE levels following administration of a synthesis inhibitor, alpha-methyl-p-tyrosine methyl ester (alpha-MT, 250 mg/kg). The half-life of approach to a new steady-state was determined. To measure depolarized release of NE, the hypothalamus was incubated in vitro in a high potassium (50 mM) medium and the percent of endogenous NE released into the medium was determined. The in vivo results indicated that hypothalamic NE utilization in control animals decreases as animals mature. Additionally, the in vitro results indicated that the percent NE released upon depolarization also decreased with maturation in control animals. Restraint shifted the NE decay curve measured in vivo to the right at all ages. Overall, however, restraint tended to increase NE utilization at 70 days, have little effect at 42 days, and retard utilization at 28 days. Furthermore, restraint markedly reduced the depolarized release of NE at 28 days, had no effect at 42 days and slightly, but significantly, increased release at 70 days. Restraint significantly increased plasma corticosterone at all ages. Hypothalamic NE projections are important to an organisms regulatory responses, and changes that take place over adolescence in this system may be important for the emergence of adult-typical responses as well as render adolescents vulnerable to specific dysfunctions.

Prenatal diazepam exposure: Effects on auditory temporal resolution in rats

The maturation of temporal acuity in the auditory system of the rat was evaluated following prenatal exposure to diazepam (DZ; 0, 2.5, 10 mg/kg to the dam on each of the last 8 days of gestation). Acuity was defined by the effectiveness of brief silent periods in white noise (gaps) as inhibitors of an immediately subsequent acoustic startle reflex. In uninjected control rats the gaps became effective at 25–28 days of age, and onset was delayed in prenatally exposed animals according to dose. No DZ was present in the rat fffspring at this time of testing. Injections of DZ were given to these same animals in adulthood (0, 0.5, 1.5, 4.5 mg/kg). These injections reduced the inhibitory effect of the gap on startle behavior in all animals, but less so in those previously exposed to DZ in utero. The threshold of temporal acuity in the rat approximates that found in humans and the function matures at the same relative rate in the two species. Further, administration of DZ in the adult human has the same effect on temporal acuity in the auditory system as it does in the adult rat. There are presently no data available on the effects on human auditory development of exposure to DZ in utero. The data we present have considerable relevance for clinical investigation given the importance of temporal acuity in the perception of speech and, thus, in the maturation of language ability.

Endogenous levels of 5 alpha-reduced progestins and androgens in fetal vs. adult rat brains.

5 Alpha-reduced metabolites of certain steroids have been shown to have important functions in adult brains and may play a role in brain development. To assess which 5 alpha-reduced steroid metabolites may have an impact during development, endogenous levels of 5 alpha-reduced androgens and progestins and their parent hormones were measured in male and female fetal brains over the last 5 days of gestation. These levels were compared to levels measured in adult male and female brains (evaluated at different stages of the estrous cycle). Neither the brain levels of parent hormones nor of their 5 alpha-reduced metabolites varied as a function of fetal sex or of gestational age. Therefore, the data from the two sexes were combined. In fetal brains, the levels of the progesterone reduced metabolites were 20-fold higher than levels of progesterone itself whereas levels of testosterone reduced metabolites were 10-fold lower than testosterone levels. In contrast to fetal brain, conversion of progesterone to reduced metabolites was much lower in adult brain, but the level of 5 alpha-reduced androgens was 3-10-fold higher than the level of testosterone in all adult tissue, indicating more conversion of androgen to 5 alpha-reduced metabolites in adult than in fetal brains. These results imply that the reduction of progesterone to reduced metabolites may play a critical role in brain development.

Prenatal diazepam exposure in rats: long-lasting, receptor-mediated effects on hypothalamic norepinephrine-containing neurons

The concentration and turnover of catecholamines (CAs) were measured in the cortex, hippocampus, and hypothalamus of rats exposed in utero to diazepam (DZ, 1.0, 2.5, or 10.0 mg/kg/day) over gestational days 13-20. Prenatal DZ induced a regionally specific, dose-related decrease in the level of norepinephrine (NE) (maximum decrease, 65%) and turnover rate of NE (maximum decrease, 85%) in the hypothalamus of 90-day-old adult rats. Dopamine levels were not altered in this region and neither of the CAs were altered in the other regions. Dividing the prenatal exposure period into two shorter periods revealed that late gestation (days 17-20) was the period when factors influencing NE function in the hypothalamus were most sensitive to DZ. Analysis of the development of the CA in the hypothalamus demonstrated that the effect of the prenatal exposure on NE levels did not become apparent until after 28 days of age. However, at 28 days, there was a dose-related increase in turnover rate of NE (maximum increase, 52%). Concurrent administration of the specific benzodiazepine (BZ) antagonist RO15-1788 with DZ (2.5 mg/kg) to pregnant rats effectively reversed the effects of DZ in the hypothalamus of the adult offspring, suggesting that the effects were mediated via the BZ receptor. These data have provided insight into the intricate processes of neuronal development; in particular the importance of target cell-nerve terminal interactions and the role of early developing receptors.