A.K. Salm

Effects of prenatal stress on defensive withdrawal behavior and corticotropin releasing factor systems in rat brain

Exposure of pregnant rats to stress results in offspring that exhibit abnormally fearful behavior and have elevated neuroendocrine responses to novelty and aversive stimuli. This study examined the effects of prenatal stress on plasma corticosterone, adrenal weight, defensive withdrawal behavior, and the density of receptors for corticotropin releasing factor (CRF) in the amygdala. Pregnant Sprague-Dawley rats were stressed by daily handling and saline injection (s.c., 0.9%, 0.1 mL) during the last week of gestation. Male offspring were studied at adulthood (60-120 days of age). Adrenal hypertrophy and increased plasma corticosterone were observed in the prenatally stressed offspring. Defensive withdrawal, an ethological measure of the conflict between exploratory behavior and retreat, was quantified in naive offspring, and in offspring exposed to restraint stress (2 h). Restraint stress increased defensive withdrawal in both control and prenatally stressed offspring. Both naive and restraint-stressed prenatally stressed offspring exhibited increased defensive withdrawal compared to control offspring. There was a significant interaction between prenatal stress and restraint stress, suggesting increased vulnerability of prenatally stressed offspring. The effects of restraint in the defensive withdrawal test were reduced by intracerebroventricular administration of the CRF antagonists, alpha-helical CRF9-41 (20 microg every hour) or D-phe(12), Nle(21, 38), C(alpha)-MeLeu(37)]-CRF((12-41)) (5 microg every hour) during the restraint period. The difference between control and prenatally stressed offspring was abolished by the CRF antagonists, suggesting that increased activation of CRF receptors may be a factor in the behavioral abnormalities of prenatally stressed rats. Measurement of CRF receptors in amygdala revealed a 2.5-fold increase in binding in prenatally stressed offspring. In light of previous work from this laboratory demonstrating increased content and release of CRF in amygdala from prenatally stressed offspring, the present study suggests that the increased fearfulness of prenatally stressed rats may be a consequence of increased activity of CRFergic systems in the amygdala.

Prenatal stress affects the developmental trajectory of the rat amygdala.

The amygdala plays a critical role in generating the emotion of fear, and alterations in amygdala fear processing are thought to underlie the acquisition and maintenance of anxiety disorders. The prenatally stressed (PS) rat displays hormonal, behavioral and brain anatomical similarities to anxious humans and is useful to study the neurobiological underpinnings of pathological anxiety. We studied PS and control male rats at postnatal days 7 (P7), P25, P45 and P60. Using unbiased stereological analyses we examined the volumes, anterior–posterior lengths and total numbers of neurons and glia of the basolateral (BL), central (Ce) and lateral (La) amygdalar nuclei. We found prenatal stress-associated differences in the developmental trajectories of each nucleus. These were apparent in some measures as early as P7, most extensive at P25 and resolved by P45, at least as seen by Nissl staining. These changes were not a result of differential brain growth. This early divergence in developmental trajectories seen here may be the harbinger of PS rat amygdalas that ultimately function very differently in adulthood.

Mild prenatal stress in rats is associated with enhanced conditioned fear

We tested the hypothesis that prenatal stress would enhance conditioned fear in adult rats. Pregnant Sprague-Dawley rats were stressed by exposure to a novel environment and subcutaneous injection of saline (0.1 ml 0.9% NaCl) at random times daily from Days 14 to 21 of pregnancy. When compared to adult control (CON) male rats from unmanipulated pregnancies, adult prenatally stressed (PS) male rats showed increased freezing behavior in response to acute footshock as well as increased freezing behavior the next day in the same context, without shock delivery. In another experiment, the gestational stressor was examined for elevations in corticosterone and ACTH. At gestational days (G)15, G17, G19 and G21, maternal and fetal plasma was collected. Analysis showed elevations in corticosterone and ACTH in the PS dams when compared to the CON dams. Additionally, increased corticosterone was found in the PS fetuses when compared to the CON fetuses. Finally, some CON and PS litters were examined for alterations in length of gestation, number of pups born, bodyweight on postnatal day (P)1 and anogenital distance on P1 and differences were not found. In conclusion, our data demonstrate that a mild stressor during gestation, sufficient to raise plasma corticosterone and ACTH, is associated with enhanced conditioned fear during adulthood.

Early emergence of increased fearful behavior in prenatally stressed rats

We have been studying the mildly prenatally stressed (PS) rat as a potentially useful animal model of anxiety disorders. Previously we have demonstrated that there are anatomical and biochemical alterations in the amygdalas of adult PS offspring and that these offspring show increased fearful behaviors. However, human data indicate that anxiety disorders often present first in early childhood and then persist throughout adolescence and adulthood. To determine if PS rats also model this characteristic of human anxiety disorders, here we asked whether behavioral indices of increased fear would be detectable at an early age. We tested the hypotheses that young PS rats would show increased behavioral fearfulness in response to an acute stressor and that this would increase with age. A mild prenatal stressor, consisting of removal of the dam from the home cage and administration of a subcutaneous injection of 0.1 ml of 0.9% saline daily, was administered during the last week of pregnancy. Offspring were tested in the defensive-withdrawal apparatus before and after exposure to restraint stress at 25, 45 and 60 days of age. PS animals showed increased defensive-withdrawal behavior following the stressor and were more fearful following restraint when compared to controls (CON). This was significant at P45 and increased to P60. Hence, fearful behaviors in PS rats emerge prior to sexual maturation and increase in magnitude thereafter, further validating our model as a means to investigate the underpinnings of anxiety disorders.

Peripheral immune challenge with viral mimic during early postnatal period robustly enhances anxiety-like behavior in young adult rats.

Inflammatory factors associated with immune challenge during early brain development are now firmly implicated in the etiologies of schizophrenia, autism and mood disorders later in life. In rodent models, maternal injections of inflammagens have been used to induce behavioral, anatomical and biochemical changes in offspring that are congruent with those found in human diseases. Here, we studied whether inflammatory challenge during the early postnatal period can also elicit behavioral alterations in adults. At postnatal day 14, rats were intraperitoneally injected with a viral mimic, polyinosinic:polycytidylic acid (PIC). Two months later, these rats displayed remarkably robust and consistent anxiety-like behaviors as evaluated by the open field/defensive-withdrawal test. These results demonstrate that the window of vulnerability to inflammatory challenge in rodents extends into the postnatal period and offers a means to study the early sequelae of events surrounding immune challenge to the developing brain.

Low-frequency hearing loss in prenatally stressed rats

We investigated the possibility that hearing thresholds are altered in prenatally stressed rats raised in a normal auditory environment. Pregnant dams were assigned randomly to prenatally stressed and control groups. Half of the dams were subjected to the mild stressors of handling, exposure to a novel cage and saline injection at random times during lights-on daily. The hearing thresholds of young adult male offspring were assessed by recording auditory-evoked brainstem responses to 0.5, 1, 2, 4, 8, 16, 32 and 64 kHz pure tones. The resultant audiograms showed that prenatally stressed offspring had significantly higher hearing thresholds than control animals at 1, 2 and 4 kHz (t-tests, P<0.05). The threshold shifts caused by prenatal stress averaged 7.7 dB across frequencies. We conclude that prenatal stress causes low-frequency hearing loss, possibly due to increased vulnerability to noise-induced hearing loss, accelerated cochlear degeneration and/or disrupted cochlear development.

Analysis of extinction acquisition to attenuated tones in prenatally stressed and non-stressed offspring following auditory fear conditioning☆

Stimulus generalization occurs when stimuli with characteristics similar to a previously conditioned stimulus (CS) become able to evoke a previously conditioned response. Experimental data (Lissek et al., 2005) indicate that patients with post-traumatic stress disorder (PTSD), more often show stimulus generalization following fear conditioning when tested under laboratory conditions. Factors surrounding this observation may contribute to two common features of PTSD: 1) hyper-responsiveness to sensory stimuli reminiscent of those associated with the original trauma, and 2) resistance of PTSD to extinction-based therapies. Adverse early experience is considered a risk factor for the later development of PTSD and in the present experiments we hypothesized that stimulus generalization would occur in an animal model of adverse early experience, the prenatally stressed (PS) rat. Adult PS and control (CON) rats underwent extensive pre-habituation to a conditioning chamber followed by conventional auditory fear conditioning. The next day both groups began an extinction regimen where a series of quieter (attenuated), CSs were administered prior to the full 75 dB training CS. When tested in this manner, PS rats froze at significantly lower tone amplitudes than did CON offspring on the first day of extinction training. This suggests that the PS rats had stimulus-generalized the CS to lower decibel tones. In addition to this finding, we also observed that PS rats froze more often and longer during three ensuing days of extinction training to attenuated tones. Group differences vanished when PS and CON rats were extinguished under conventional conditions. Thus, it appears that the two extinction regimens differed in their aversive cue saliency for the PS vs. CON rats. Follow-up prefrontal cortex transcriptome probing suggests that cholinergic and dopaminergic alterations may be involved.

Glutathione S-transferase π-isoform is not altered in bladder tissue from smokers

Glutathione S-transferase π (GSTπ)-isoform is a cytosolic enzyme involved in the detoxification of reactive metabolites generated from environmental pollutants and cigarette smoke. Although cigarette smoking has been implicated in the etiology of bladder cancer, no information yet exists regarding GSTπ in smokers and nonsmokers. This study was carried out to evaluate the immunohistochemical localization and to measure levels of immunoreactive GSTπ in bladder tissue from smokers and nonsmokers. Tissues from patients diagnosed with transitional cell carcinoma (TCC) were obtained from paraffin embedded blocks fixed in formalin. Bladder tissues from smokers (n = 7) and nonsmokers (n = 8), and histologically confirmed cancerous and noncancerous areas of the same patients (n = 10) were studied. The immunoreactive GSTπ was quantified by calculating its optical density with a computerized Olympus BH-2 microscope connected to a DAGE camera. Immunohistochemical staining for GSTπ appeared to be evenly distributed in the cytosol of the transitional epithelium (TE) of the noncancerous regions. In the TE from patients with advanced TCC, the staining intensity appeared to be stronger in the nuclei relative to cytoplasm, an effect that was even more pronounced in poorly differentiated cancers and in cancers with squamoid features. The immunoreactive levels of GSTπ in the superficial TE cells was approximately 1.7-fold higher compared with the rest of the TE layer (p < 0.05) in smokers and nonsmokers. No significant differences (p > 0.05) were observed between smokers and nonsmokers in either of these regions. The higher concentration of GSTπ in the TE is suggestive of the protective role of this enzyme, serving to prevent any potentially harmful xenobiotics from entering the bladder tissue. The lack of differences in the detoxifying enzymes between smokers and nonsmokers suggests that the increased susceptibility of bladder cancer in smokers is probably mediated by other mechanisms.