Richard J. Servatius

Delayed startle sensitization distinguishes rats exposed to one or three stress sessions: Further evidence toward an animal model of PTSD

Posttraumatic stress disorder (PTSD) may occur in humans exposed chronically to stressors or after a single exposure to a traumatic event. A distinguishing feature of patients with PTSD is an exaggerated startle response, evident long after the traumatic event. We have observed similar abnormalities in our animal model of a chronic stress state. Rats exposed to 3 days (3DS) of our stress regimen (2-hr sessions of 40, 2 mA tailshocks) have exhibited a consistent pattern of persistent physiological and behavioral abnormalities including an exaggerated startle response several days after stressor cessation. In contrast, rats exposed to a single stress session (1DS) have exhibited many, but not all, of the persistent abnormalities displayed by 3DS rats. The present experiment compared the startle responding of 3DS and 1DS rats 4, 7, and 10 days after stressor cessation. Consistent with previous work, stressed rats exhibited elevated basal plasma corticosterone (CORT) levels the first day poststressor. These CORT levels were sensitive to the number of stressor exposures with higher CORT levels in 3DS rats than in 1DS rats. As for startle responding, the 1DS rats exhibited an exaggerated startle response 7 days poststressor, whereas startle sensitization was apparent 10 days poststressor in 3DS rats. Thus, the appearance of an exaggerated startle response after stressor cessation appears to be related to the number of stress session exposures. These animal models, the 3DS and 1DS rats, may be useful to gain insight into the neurobehavioral changes associated with PTSD.

A chronic stress state in rats: Effects of repeated stress on basal corticosterone and behavior ☆

The chronic stress state has previously been defined as persistent visceral arousal coupled with behavioral abnormalities. To determine the number of stressor exposures necessary to induce a chronic stress state, male rats were given 2 hours of inescapable shock on 10, 7, 4, or 3 consecutive days. The 3-day stress group had the most pervasive changes in the variables measured: persistently elevated basal plasma corticosterone (CORT), continued weight loss in the post-stressor period, and abnormal behavior. More exposures to the stress regimen did not produce higher CORT levels or greater behavioral changes. Acutely stressed rats, exposed to 1 day of inescapable shock, had persistent CORT elevations without the other changes seen in the 3-day stress group. The data suggest that 3 days of our stress regimen are sufficient to produce a state of chronic stress and that some signs of this state begin to appear as early as the first exposure to our inescapable stress regimen.

Exposure to inescapable stress persistently facilitates associative and nonassociative learning in rats

The present study examined the proactive effects of inescapable stress on aversive Pavlovian conditioning. Stressed rats were restrained and exposed to 90 1-mA tailshocks. Twenty-four hours later, all rats were exposed to 10 conditioned stimuli (CS; 350 ms of white noise at 85 dB). Rats then received either paired training in which the CS coterminated with a 100-ms, 0.7-mA periorbital shock or the same stimuli presented in an explicitly unpaired fashion. After the unpaired exposures, these rats were also exposed to paired training. Previously stressed rats exhibited persistent sensitization to the white-noise stimulus. Stressed rats exposed to unpaired stimuli, and no longer exhibiting a sensitized response, acquired the eyeblink conditioned response at a facilitated rate when these stimuli were presented in a paired fashion. These results also demonstrate that the effect of stress on classical conditioning is long-lasting, in excess of 48 hr.

The Contribution of Stressor Intensity, Duration, and Context to the Stress-Induced Facilitation of Associative Learning

Exposure to an acute stressor of restraint and intermittent tailshock facilitates acquisition of the classically conditioned eye-blink response 24 h after stressor cessation. The contribution of stressor intensity, duration, and context was determined. Male rats exposed to 90 or 30 1-mA tailshocks exhibited sensitization to an auditory cue of 86 dB and facilitated acquisition of the conditioned response, whereas rats exposed to 90 0.5-mA tailshocks or restraint alone only exhibited sensitization. Rats exposed to the 5 1.0-mA tailshocks (and 5 min of restraint) exhibited neither sensitization nor facilitated acquisition. Rats stressed in the same context 48 and 96 h earlier exhibited sensitization and facilitated acquisition relative to those stressed in a different context. Neither stressed group exposed to unpaired stimuli responded to the CS, and thus there was no pseudodconditioning. Together, these results dissociate the stress-induced sensitization from the facilitated learning. They also indicate that the facilitated learning is particularly sensitive to stressor intensity and contextual cues.

Persistent stress-induced sensitization of adrenocortical and startle responses

We assessed the functional adrenocortical and behavioral state of rats previously exposed to repeated stressor presentations. In Experiment 1, the whole-body startle response to threshold (91 dB) and suprathreshold (96 dB) stimuli was assessed in rats given 3 daily sessions (3DS) of 40, 2-mA tailshocks. The 3DS rats showed an exaggerated startle response to the threshold auditory stimulus 4 days poststressor compared to nonshocked controls (CON). An exaggerated startle response in stressed rats was not evident either 1 day or 10 days poststressor. In Experiment 2, adrenocortical sensitization and behavioral reactivity were assessed in rats exposed to 1 day (1DS) or 3 days of our stress regimen. Stressed rats exhibited elevated basal plasma corticosterone (CORT) levels 1 day poststressor which recovered by 9 days poststressor. Stressed rats also exhibited suppressed open-field activity 4 days poststressor. On the 10th day poststressor, rats were exposed to a single tailshock. The 1DS and 3DS rats showed both a sensitized and prolonged CORT response to stressor reexposure compared to control rats which received only the single tailshock. In addition, on the 11th day poststressor 3DS rats exhibited a moderate recapitulation of the elevated basal CORT levels seen after the initial stressor exposures. Thus, exposure to our stress regimen produces a chronic stress state in rats characterized by persistent behavioral and adrenocortical sensitization, as well as suppressed open-field activity and elevated basal CORT levels. Rats exhibiting a chronic stress state may be appropriate as a model for the study of stress-related psychophysiological illnesses, such as posttraumatic stress disorder.

Repeated stress persistently elevates morning, but not evening, plasma corticosterone levels in male rats

Repeated exposures to a stressor in our rat model of a chronic stress state cause elevated plasma corticosterone levels in the morning for several days after the last stressor. However, plasma corticosterone levels are normally characterized by a circadian rhythm with low levels for much of the morning and higher levels near the onset of darkness. The current experiment examined the question of whether the elevated morning levels after stressor exposures were accompanied by other changes in this circadian rhythm. Male rats were given restraint-shock stressor sessions for 0, 1, or 3 days, after which plasma samples were collected for 3 days at 0900 h and at three other times around the circadian peak (1400, 1800, and 2200 h). Plasma corticosterone levels at 0900 h were elevated for the first 2 days after three stressor exposures and for 1 day after a single stressor exposure compared to those in nonstressed controls. However, levels at 1400, 1800, and 2200 h were not different in stressed and control rats on the first 2 days after stressor exposures. In addition, the amplitude of the corticosterone rhythm was suppressed after three stressor exposures, but not after one. This decrease in amplitude was mostly due to increased morning levels, inasmuch as the evening levels were similar in stressed rats and controls. Because the hypothalamic-pituitary-adrenal axis is more sensitive to glucocorticoid feedback in the morning, our data suggest that the mechanisms mediating feedback at this time of day may be disrupted by repeated stressor exposures.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytokine Levels during Pregnancy Influence Immunological Profiles and Neurobehavioral Patterns of the Offspring

Abstract:u2002 The underlying causes of autism spectrum disorders (ASD) are unknown, but clinical and experimental studies indicate immune mechanisms, in general, and cytokine dysregulation, in particular, as contributing factors in their etiology. We developed a prenatal mouse model of autism to demonstrate that circulating levels of defined cytokines in pregnant dams could influence fetal development and behavioral characteristics in their offspring. We administered daily injections of murine IL‐2 (0.4 μg in phosphate‐buffered saline [PBS]) to pregnant mice during mid‐gestation, and analyzed their offspring (IL‐2 pups) in comparison to offspring of pregnant mice injected with vehicle only (PBS pups). Significant levels of IL‐2 were present in amniotic fluid and tissues from embryos of dams given radiolabeled IL‐2, indicating that the injected IL‐2 crossed the placenta and entered the fetuses. Lymphocytes from IL‐2 pups demonstrated accelerated T cell development, with a skewing toward TH1 cell differentiation. IL‐2 pups also showed in vitro proliferative and cytotoxicity responses that were significantly higher than control PBS pups when stimulated with syngeneic B lymphoma cells or allogeneic spleen cells. In addition to their previously shown increases in open‐field activity, grooming and rearing behavior, offspring of IL‐2‐injected (vs. PBS‐injected) dams also displayed abnormal new motor learning as assessed through acquisition of the classically conditioned eyeblink response. These results suggest that increases in maternal levels of IL‐2 during pregnancy induce in their offspring long‐lasting increased vulnerability to neurobehavioral abnormalities associated with autism, and provide a valid animal model to determine the underlying immunological mechanisms.

Damage of GABAergic neurons in the medial septum impairs spatial working memory and extinction of active avoidance: effects on proactive interference.

The medial septum and diagonal band (MSDB) are important in spatial learning and memory. On the basis of the excitotoxic damage of GABAergic MSDB neurons, we have recently suggested a role for these neurons in controlling proactive interference. Our study sought to test this hypothesis in different behavioral procedures using a new GABAergic immunotoxin. GABA‐transporter‐saporin (GAT1‐SAP) was administered into the MSDB of male Sprague–Dawley rats. Following surgery, rats were trained in a reference memory water maze procedure for 5 days, followed by a working memory (delayed match to position) water maze procedure. Other rats were trained in a lever‐press avoidance procedure after intraseptal GAT1‐SAP or sham surgery. Intraseptal GAT1‐SAP extensively damaged GABAergic neurons while sparing most cholinergic MSDB neurons. Rats treated with GAT1‐SAP were not impaired in acquiring a spatial reference memory, learning the location of the escape platform as rapidly as sham rats. In contrast, GAT1‐SAP rats were slower than sham rats to learn the platform location in a delayed match to position procedure, in which the platform location was changed every day. Moreover, GAT1‐SAP rats returned to previous platform locations more often than sham rats. In the active avoidance procedure, intraseptal GAT1‐SAP impaired extinction but not acquisition of the avoidance response. Using a different neurotoxin and behavioral procedures than previous studies, the results of this study paint a similar picture that GABAergic MSDB neurons are important for controlling proactive interference.

A stress-induced anxious state in male rats: Corticotropin-releasing hormone induces persistent changes in associative learning and startle reactivity

BACKGROUNDnExposure to intense inescapable stressors induces a persistent anxious state in rats. The anxious state is evident as increased sensory reactivity and enhanced associative learning.nnnMETHODSnWe examine whether similar neurobehavioral changes are observed after intracerebroventricular (ICV) administration of corticotropin releasing hormone (CRH). Two behaviors were observed: acoustic startle responses (ASRs) and acquisition of the classically conditioned eyeblink response. Male Sprague-Dawley rats were administered ICV CRH either in a single dose (1.0 microg/rat) or in three doses each separated by 30 min.nnnRESULTSnExaggerated ASRs were evident 2 hours after either CRH treatment; however, only the rats given three injections exhibited a persistently exaggerated ASR apparent 24 hours after CRH treatment. Rats administered three injections of CRH also exhibited faster acquisition of the eyeblink conditioned response beginning 24 hours after treatment. Yet, we did not find evidence for a persistent activation of the HPA-axis response; three CRH injections did not lead to elevated basal plasma corticosterone levels the following morning.nnnCONCLUSIONSnRepeated treatment with CRH over a 1.5-hour period models some of the behavioral changes observed after exposure to intense inescapable stressors.

Early acquisition, but not retention, of the classically conditioned eyeblink response is N-methyl-D-aspartate (NMDA) receptor dependent.

N-methyl-D-aspartate (NMDA) antagonists impair performance in some tasks, but whether they impair learning directly or through effects on sensorimotor performance remains controversial. Rats administered a competitive NMDA antagonist, CGP-39551, 24 hr before training could not acquire a classically conditioned eyeblink response. The associative deficit remained evident during training with a high-intensity conditioned stimulus, even though sensory reactivity was unaffected. The antagonist did not alter retention and thus did not affect motor performance of the task. These results extend and confirm studies that implicate NMDA-receptor activation in the acquisition of classically conditioned associations and specifically in tasks not dependent on the hippocampus for learning itself. Moreover, they substantiate recent claims that NMDA receptor activation (and by association, long-term potentiation) may be involved in early processes of procedural memory formation.