Betty Zimmerberg

Sex differences in anxiety behavior in rats: Role of gonadal hormones

These experiments examined the role of gonadal hormones at both the organizational and activational time periods on sex differences in plus-maze behavior. In the first experiment, adult female Long-Evans rats were found to spend more time on the open arms of the plus maze than adult males, indicating less anxious behavior. In the second experiment, male and female subjects received a neonatal treatment (chemical castration with flutamide or tamoxifen, vehicle injection, or no injection) and a prepubertal treatment (gonadectomy, sham surgery, or no surgery). Adult females receiving either neonatal tamoxifen or prepubertal ovariectomy spent less time on the open arms than control females, but females who received both treatments were the most defeminized subjects. Males were not affected by the absence of gonadal hormones at either time period. These experiment indicate that female gonadal hormones play an important role both organizationally and activationally in plus-maze behavior. The role of the GABA receptor complex in mediating this effect is discussed. Knowledge of sex differences in plus-maze behavior may help to make this maze a more useful tool in investigating anxiety behavior in rats.

Reduction of rat pup ultrasonic vocalizations by the neuroactive steroid allopregnanolone

Several of the recently characterized neuroactive steroids have been proposed to have anxiolytic effects in behavioral models when subjects were tested as adults. In this experiment, the effects on infant subjects were examined using the isolation distress model of anxiety. The production of ultrasonic vocalizations in week-old rat pups after maternal separation was assessed after ICV injections of vehicle or allopregnanolone (1.25-5 micrograms), or sham injections. Subjects were also observed for activity and behavioral responses and tested on three measures of sedation. Allopregnanolone caused a dose-dependent decrease in ultrasonic vocalizations, with increasing motor incoordination, ataxia, and turning at the higher doses. Sex differences were not observed for any measure. These results suggest the GABAA receptor binding site for neuroactive steroids is behaviorally active in neonates as well as in adults, and that the anxiolytic effects of the neuroactive steroids at this site may be dissociable from their sedative effects at low doses.

Social isolation stress during the third week of life has age-dependent effects on spatial learning in rats.

Despite extensive research on the relationship between acute stress and hippocampal function in adults, little is known about the short- and long-term effects of prolonged juvenile stress on learning, memory, and other hippocampal functions. This experiment investigated whether spatial learning would be altered in juvenile and adult rats previously exposed to a chronic stressor: 6 h of social isolation (SI) daily at 15-21 days of age. SI was found to increase circulating plasma levels of corticosterone (CORT) and allopregnanolone (3-alpha,5-alpha-pregnan-20-one; 3,5-THP) at 1 h after separation on the fourth day, indicating that the isolation was an effective stressor. When tested as juveniles (post-natal (PN) 22-24), spatial learning was impaired on the Morris water maze in the previously isolated subjects compared to non-isolated controls. However, when tested as adults (PN 92-94), subjects previously exposed to SI during the third week of life demonstrated more rapid learning of the task than controls. These results are discussed in light of research on the effects of CORT on the developing hippocampus.

Spatial learning of adult rats with fetal alcohol exposure: deficits are sex-dependent

The effects of prenatal alcohol exposure on learning in adult offspring were studied in a spatial task in a T-maze. Male and female Long-Evans rats were selected from litters whose dams had received one of three treatments: alcohol in a liquid diet (35% ethanol-derived calories, 35% EDC), pair-fed nutritional control (0% ethanol-derived calories, 0% EDC) or standard control (lab chow, LC). The task included trial-independent (reference memory) and trial-dependent (working memory) components: subjects were required to make a fixed left-right discrimination, and then to alternate left and right choices to escape water. Prenatal alcohol exposure was associated with a greater number of reference errors for both sexes; only males from the alcohol prenatal treatment group, however, were impaired on the working memory component. These results suggest that prenatal exposure to alcohol can cause behavioral dysfunctions that persist into adulthood. Secondly, the pattern of memory impairments suggests that both sexes may be equivalently damaged in neural areas subserving reference memory, but that males are selectively more vulnerable in neural areas subserving working memory.

Prenatal stress effects are partially ameliorated by prenatal administration of the neurosteroid allopregnanolone

This study examined the effects of exposure to prenatal stress on young and adult rats, and whether the concomitant administration of an anxiolytic neurosteroid, allopregnanolone (3-alpha-hydroxy-5 alpha-pregnan-20-one), could ameliorate some of the behavioral dysfunctions associated with prenatal stress. Pregnant dams were assigned to one of five treatment groups on gestational day 14. These groups were exposed to either 1) restraint for 45 min three times daily; 2) a vehicle injection twice daily; 3) 5 mg/kg allopregnanolone twice daily; 4) restraint with allopregnanolone injections; or 5) nonhandled controls. Assays for plasma allopregnanolone concentrations indicated that exogenous allopregnanolone injections significantly raised circulating levels to a comparable degree in gestational day 20 dams and their fetuses. At 7 days of age, however, subjects prenatally exposed to allopregnanolone either alone or with restraint now had lower circulating levels compared to the other groups, suggesting some negative compensatory change. Behavioral results suggested that the effects of prenatal stress on affective behaviors (ultrasonic vocalizations emitted after a brief maternal separation at 7 days of age, and plus-maze behavior at 70 days of age) could be reversed by coadministration of allopregnanolone. When locomotor activity was assessed at 16 and 60 days of age, no comparable reversal effect was observed. In fact, the allopregnanolone groups had results similar to those of the restraint alone group. Thus, for some neuronal systems, allopregnanolone may exert either a direct teratogenic effect or an indirect effect due to neurosteroid-induced behavioral changes in the pregnant dam.

Sex differences in corpus callosum: Influence of prenatal alcohol exposure and maternal undernutrition

The functional significance of sex differences in the size of the corpus callosum was investigated using a prenatal alcohol exposure paradigm that influences the fetal hormonal milieu. Adult male and female Long-Evans rats were selected from one of 3 prenatal treatment histories: prenatal alcohol-exposed (35% ethanol-derived calories), nutritional control (0% ethanol-derived calories) or standard control (lab chow). Subjects were assessed for open field activity at 85 days of age. At 100 days of age, midline sagittal areas of the corpus callosum and the anterior commissure were determined for these subjects. Male control subjects had significantly larger callosal areas than female controls. Prenatal alcohol exposure significantly decreased the total callosal area, and abolished this sexual dimorphism. When the callosal measurements were analyzed using Denenbergs regions, differential effects of prenatal alcohol exposure, undernutrition and sex were dissociable by subarea. There were no significant sex differences or effects of prenatal alcohol exposure in the midline sagittal area of the anterior commissure. Callosal size was negatively correlated to open field activity, suggesting a possible role in normal exploratory behavior and to the overactivity observed after prenatal alcohol exposure.

PRENATAL EXPERIENCE AND POSTNATAL STRESS MODULATE THE ADULT NEUROSTEROID AND CATECHOLAMINERGIC STRESS RESPONSES

Allopregnanolone (3α‐hydroxy‐5α‐regnan‐20‐one) is a neuroactive steroid recently shown to be involved in the neurochemical stress response via its positive modulation of the GABAA receptor complex. This experiment investigated the effects of postnatal stress (daily maternal separation during the first week of life) on the subsequent adult response to a stressor (10 min forced swim) in Long–Evans rats from one of three prenatal treatment groups (alcohol, pair‐fed and control). Indices of stress response were allopregnanolone concentrations in plasma, cortex and hippocampus, and dopamine and norepinephrine concentrations in prefrontal cortex, nucleus accumbens and striatum. Females had higher levels of allopregnanolone than males in both plasma and brain. Prenatal alcohol exposure combined with early maternal separation stress resulted in an increase in the endogenous levels of allopregnanolone in the prefrontal cortex and hippocampus of adult offspring in response to a stressor compared to subjects without a prior history of postnatal stress ; this effect was greater in females. This increased allopregnanolone was also associated with decreased dopamine and norepinephrine levels in the prefrontal cortex. In the prenatal alcohol‐exposed offspring, postnatal maternal separation blunted the increase in dopamine levels in the striatum seen in both control groups. Postnatal maternal separation increased norepinephrine levels in the nucleus accumbens regardless of prenatal experience, while in the prefrontal cortex only prenatal diet condition (pair‐feeding and alcohol) resulted in lower norepinephrine levels. The results of this experiment suggest that experience, both pre‐ and postnatal, can have long‐term consequences for the developing neurochemical responses to stressors.

Differences in affective behaviors and hippocampal allopregnanolone levels in adult rats of lines selectively bred for infantile vocalizations.

Allopregnanolone, 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha,5 alpha-THP), a progesterone metabolite, is an endogenous neurosteroid mediating affective behaviors via its positive modulation of GABA(A) receptors. In order to better understand the role of this neurosteroid in individual differences in affective behavior, we used an animal model based on selective breeding for an infantile affective trait, ultrasonic vocalizations (USV). Adult male and female (in either proestrus or diestrus) rats that had been bred for low (low line) or high (high line) rates of USV after maternal separation were tested in a series of affective behavioral tests: open field, emergence, social interaction, defensive freezing, and the Porsolt forced swim task. Concentrations of allopregnanolone in combined hippocampus and amygdala tissue were then measured. low line subjects showed significantly lower anxiety and depression responses in the emergence, open field, and Porsolt forced swim tasks than did high line subjects. Proestrus females exhibited less affective behaviors than diestrus females or males. Allopregnanolone levels in hippocampus/amygdala were significantly higher in low line subjects compared to high line subjects, and in proestrus females compared to diestrus females and males. These data indicate that: (1) affective behaviors in lines selectively bred for an infantile anxiety trait exhibit selection persistence into adulthood; and (2) levels of allopregnanolone in the limbic system parallel selected disparities in affective behavior, suggesting a selection for alterations in the neurosteroid/GABA(A) receptor system in these lines.

The effects of cocaine on maternal behaviors in the rat

Although recent research has focused on the behavioral teratology of cocaine, there has been little attention centered on the effects of cocaine use on parenting behaviors after birth. This experiment investigated the effects of cocaine on parental (generally termed maternal) behavior in the rat. Four types of adult rats were tested for these effects over a 10-day testing period: lactating primiparous female dams, primiparous females, nulliparous females, and males. Subjects were injected daily with cocaine (5 mg/kg) or saline. Ten minutes postinjection each subject was placed in a cage with three stimulus pups and nesting material, and observed for several measures of maternal behavior for 5 minutes, including sniffing, licking, and retrieving pups. Subjects were also rated on a maternal behavior scale at 60 minutes and 24 hours postinjection, and on nest quality. Cocaine reduced both the total number and duration of sniffing and licking maternal behaviors, and resulted in a lower maternal behavior rating at 60 minutes compared to saline-treated subjects. Among those subjects that retrieved pups within the first 5 minutes, cocaine-injected subjects retrieved for a longer duration. These results also suggest impaired maternal behavior, since rapid retrievals would have been more efficient in maintaining a nest huddle. Thus, cocaine impaired the parenting ability of both male and female rats, with and without previous parenting experience.

Prenatal alcohol exposure influences the effects of neuroactive steroids on separation-induced ultrasonic vocalizations in rat pups

Fetal alcohol exposure has been reported to be associated with hyper-responsiveness to stress. Using a maternal separation paradigm, this study examined whether prenatal alcohol exposure affected sensitivity to neurosteroid modulation of stress. We have shown that the neuroactive steroid allopregnanolone reduces ultrasonic vocalizations (USVs) after brief maternal separation in week-old rat pups. Prenatal alcohol exposure, however, resulted in reduced sensitivity to this neurosteroid. In this studys first experiment, the behavioral effects of pregnenolone sulfate, a neurosteroid with reportedly opposite modulatory effects on the GABAA receptor, were characterized. Pregnenolone sulfate had a triphasic effect on the production of ultrasonic vocalizations and on open field activity. Blockade of conversion of pregnenolone sulfate to allopregnanolone via the 5 alpha-reductase inhibitor 4-MA also blocked the drug-related reduction in USVs, but not the higher-dose augmentation. The enzyme inhibitor alone had no significant effects on USV production, nor did progesterone. These results suggest that the neuroactive steroid pregnenolone sulfate may play an independent role in the stress response after maternal separation as well as being a precursor for the anxiolytic neurosteroid allopregnanolone. In the second experiment, prenatal alcohol exposure was found to eliminate both the low dose USV-reducing effect and the higher dose USV-increasing effect. These results support previous results demonstrating that prenatal alcohol exposure may cause an altered sensitivity to the neuromodulatory effects of neurosteroids.