Prior exposure to a single stress session facilitates subsequent contextual fear conditioning in rats. Evidence for a role of corticosterone

Previous studies showed that exposure of rats to chronic restraint stress for 21 days enhances subsequent contextual fear conditioning. Since recent evidence suggest that this effect is not dependent on stress-induced neurodegenerative processes, but to elevated training-elicited glucocorticoid release in chronically stressed animals, we aimed to explore here whether a single exposure to restraint stress, which is not expected to induce neuronal damage, would also affect contextual fear conditioning. We also questioned whether post-training corticosterone levels might be associated with any potential effect of stress on fear conditioning. Adult male Wistar rats were exposed to acute restraint stress for 2 h and, two days later, trained in the contextual fear conditioning task, under training conditions involving either moderate (0.4 mA shock) or high (1 mA shock) stress levels. The results showed that acute stress enhanced conditioned freezing at both training conditions, although data from the 1 mA shock intensity experiment only approached significance. Stressed animals were shown to display higher post-training corticosterone levels. Furthermore, the facilitating effect of prior stress was not evident when animals were trained in the hippocampal-independent auditory-cued conditioning task. Therefore, these findings support the idea that stress experiences preceding exposure to new types of stressors facilitate the development of contextual fear conditioning. They also indicate that not only repeated, but also a single exposure to aversive stimulation is sufficient to facilitate context-dependent fear conditioning, and suggest that increased glucocorticoid release at training might be implicated in the mechanisms mediating the memory facilitating effects induced by prior stress experiences.     

Behavioral and cardiac responses after intracerebroventricular corticotropin-releasing hormone (CRH) administration: role of adrenal cortical hormones.

Intracerebroventricularly (icv) administered corticotropin-releasing hormone (CRH) produces a dose-dependent increase in heart rate in association with behavioral activation. The present study was designed to investigate whether these CRH-induced responses are dependent on adrenal function. The effects of adrenalectomy (ADX) and subsequent corticosterone replacement were studied. Administration icv of 300 ng of CRH failed to produce behavioral activation and tachycardia in ADX rats. Corticosterone replacement restored the CRH-induced behavioral response to preoperative levels, whereas the CRH-induced tachycardia was partially restored. This latter result may be related to the fact that the baseline heart rate of ADX animals appeared to be significantly higher than that of corticosterone-treated ADX animals. It is concluded that circulating adrenal corticosterone in ADX rats is involved in the expression of the behavioral and cardiac effect of central CRH.     

Dehydration-induced drinking decreases Fos expression in hypothalamic paraventricular neurons expressing vasopressin but not corticotropin-releasing hormone

Water-restricted (WR) rats exhibit a rapid suppression of plasma corticosterone following drinking. The present study monitored Fos-like immunoreactivity (Fos) to assess the effect of WR-induced drinking on the activity of vasopressin (VP)-positive magnocellular and parvocellular neurons and corticotropin-releasing hormone (CRH)-positive parvocellular neurons in the paraventricular nucleus of the hypothalamus. Adult male rats received water for 30 min (WR) in the post meridiem (PM) each day for 6 days and were killed without receiving water or at 1 h after receiving water for 15 min. In WR rats, Fos increased in VP magnocellular and parvocellular neurons but not CRH neurons. After drinking, Fos was reduced in VP magnocellular and parvocellular neurons but did not change in CRH neurons. To assess the severity of osmotic stress, rats were sampled throughout the final day of WR. Plasma osmolality, hematocrit and plasma VP were increased throughout the day before PM rehydration, and plasma ACTH and corticosterone were elevated at 1230 and 1430, respectively, showing that WR activates hypothalamic-pituitary-adrenal activity during the early PM before the time of rehydration. To determine the effects of WR-induced drinking on CRH neurons activated by acute stress, WR rats underwent restraint. Restraint increased plasma ACTH and corticosterone and Fos in CRH neurons; although rehydration reduced plasma ACTH and Fos expression in VP neurons, Fos in CRH neurons was not affected. These results suggest that inhibition of VP magnocellular and parvocellular neurons, but not CRH parvocellular neurons, contributes to the suppression of corticosterone after WR-induced drinking.     

Acquisition and extinction of conditioned suppression of a graft-vs-host response in the rat

Injection of rats with cyclophosphamide (CY) after their consumption of a novel saccharin-flavored drinking solution results in a conditioned aversion to saccharin and a conditioned suppression of immune responses. In this study, female Lewis X Brown Norwegian F1 rats were conditioned by pairing saccharin with 50 mg/kg CY. Seven weeks later (day 0), a graft-vs-host response (GvHR) was induced in these animals by injecting splenic leukocytes from Lewis donors into a rear footpad. At this time, some conditioned animals were reexposed to saccharin, the conditioned stimulus. During the 7-wk interval between conditioning and immunization, subgroups of conditioned rats were given 0, 4, 9, or 18 extinction trials (saccharin followed by saline injections). Animals receiving 4, 9, or 18 extinction trials showed a greater preference for saccharin on day 0 than did animals receiving no extinction trials, but these groups did not differ among themselves; all conditioned groups showed a lower preference for saccharin than placebo-treated animals. There was a clear effect of number of extinction trials on the GvHR. Animals receiving 9 or 18 extinction trials did not differ from controls, whereas animals receiving 0 or 4 trials had a milder GvHR than did conditioned rats that were not reexposed to saccharin at the time of immunization. These results confirm a previous report of conditioned suppression of a GvHR, demonstrate that conditioned immunopharmacologic responses are subject to experimental extinction, and indicate that conditioned immunosuppression can be dissociated from conditioned taste aversion.     

Conditioned suppression of contact sensitivity is independent of sympathetic splenic innervation

The present study investigated the role of sympathetic innervation of the spleen in conditioned suppression of a contact hypersensitivity (CHS) reaction. Behavioral conditioning was achieved by pairing saccharin drinking solution (conditioned stimulus, CS) with injection of cyclosporin A (CsA, 20 mg/kg; unconditioned stimulus, UCS). Four days after sensitization of the animals by application of a 5% 2,4-dinitrochlorobenzene (DNCB) to abdominal skin, the animals were challenged by applying a 1% DNCB solution to the ear. The CHS response was monitored by measuring the degree of ear swelling. Saccharin re-presentation reduced ear swelling to a magnitude that approached that achieved by CsA treatment. Histological examination demonstrated that the conditioned reduction of ear swelling was produced by a reduced leukocyte infiltration of the ear. Prior sympathetic denervation of the spleen did not alter the conditioned suppression of the CHS response. These data indicate that behavioral conditioning using CsA produces alterations of CHS that, unlike conditioned prolongation of heart allograft survival, are independent of sympathetically regulated conditioned alterations in the spleen.     

Neonatal handling enhances contextual fear conditioning and alters corticosterone stress responses in young rats

Previous studies have indicated that neonatal handling influences development of hypothalamic-pituitary-adrenal (HPA) control of corticosterone. In addition, corticosterone influences memory consolidation processes in contextual fear conditioning. Therefore, neonatal handling may affect hippocampal-dependent memory processes present in contextual fear conditioning by influencing the development of HPA control of corticosterone. To investigate the effects of neonatal handling on early learning, rat pups were either handled (15-min removal from home cage) on the first 15 days after birth or left undisturbed in their home cage. Handled rats and nonhandled rats were fear conditioned at 18, 21, or 30 days of age and then tested at two time points--24 h following conditioning and at postnatal day 45. Subsequently, at approximately postnatal day 60, rats were exposed to restraint stress and corticosterone levels were assessed during restraint and recovery. Handled and nonhandled rats did not differ significantly in their freezing response immediately following footshock on the conditioning day. However, when tested for contextual fear conditioning at 24 h following conditioning and at postnatal day 45, handled rats showed more freezing behavior than nonhandled rats. When exposed to restraint stress, handled rats had a more rapid return of corticosterone to basal levels than nonhandled rats. These results indicate that neonatal handling enhances developmentally early memory processes involved in contextual fear conditioning and confirms previously reported effects of neonatal handling on HPA control of corticosterone.     

Allopregnanolone induces state-dependent fear via the bed nucleus of the stria terminalis

Gonadal steroids and their metabolites have been shown to be important modulators of emotional behavior. Allopregnanolone (ALLO), for example, is a metabolite of progesterone that has been linked to anxiety-related disorders such as posttraumatic stress disorder. In rodents, it has been shown to reduce anxiety in a number of behavioral paradigms including Pavlovian fear conditioning. We have recently found that expression of conditioned contextual (but not auditory) freezing in rats can be suppressed by infusion of ALLO into the bed nucleus of the stria terminalis (BNST). To further explore the nature of this effect, we infused ALLO into the BNST of male rats prior to both conditioning and testing. We found that suppression of contextual fear occurred when the hormone was present during either conditioning or testing but not during both procedures, suggesting that ALLO acts in a state-dependent manner within the BNST. A shift in interoceptive context during testing for animals conditioned under ALLO provided further support for this mechanism of hormonal action on contextual fear. Interestingly, infusions of ALLO into the basolateral amygdala produced a state-independent suppression of both conditioned contextual and auditory freezing. Altogether, these results suggest that ALLO can influence the acquisition and expression of fear memories by both state-dependent and state-independent mechanisms.     

Molecular correlates of emotional learning using genetically selected rat lines

The genetic contributions to active avoidance learning in rodents have been well established, yet the molecular basis for genetically selected line differences remains poorly understood. To identify candidate genes influencing this active avoidance paradigm, we utilized the bidirectionally selected Syracuse high- and low-avoidance (SHA and SLA) rat lines that markedly differ in their two-way active avoidance behavior. Rats were phenotyped, rested to allow recovery from testing stress and then hippocampi were dissected for gene expression profiling (Affymetrix U34A chips; approximately 7000 known genes), comparing SLA to SHA. Next, a subset of differentially expressed genes was confirmed by real-time PCR (RT-PCR) in hippocampi. Additional studies at the protein level were performed for some genes. Using triplicate arrays on pooled hippocampal samples, differentially expressed genes were identified by microarray suite 5.0 and robust multi-array average analyses. By RT-PCR analysis in hippocampi, eight genes were nominated as potential candidate genes consistent with the differential expression from the microarray data. Four genes, Veli1 (mlin-7B), SLC3a1, Ptpro and Ykt6p, showed higher expression in SHA hippocampi than SLA. Four genes, SLC6A4, Aldh1a4, Id3a and Cd74, showed higher expression in SLA hippocampi than SHA. The active avoidance behavioral difference between lines probably emerges from 'many small things'. These potential candidate genes generate hypotheses for future testing in human association and rodent studies. Differences in levels of a pleiotropic gene like Ptpro and SLC6A4 suggest that small differences over a lifespan may contribute to large behavioral differences.     

用条件性逃避反应建立简易的大鼠耳鸣检测方法

目的：用较简单的行为实验建立检测动物耳鸣的方法。方法：按照巴甫洛夫条件反射原理,参照Jastreboff的方法,建立“中断背景声音-大鼠逃跑”的条件反射;不再给电击,观察不同组别大鼠条件反射消退过程的行为表现,用公认的水杨酸耳鸣造模法来验证本方法的可靠性。结果：水杨酸组动物停止背景声音时不出现或较少出现逃跑反应,因为大鼠耳内仍有声音存在(耳鸣声),即条件刺激时有耳鸣的动物出现的逃跑反应次数小于无耳鸣的对照动物。结论：本实验设计可检测动物是否有耳鸣。     

Urocortin 1 administered into the hypothalamic supraoptic nucleus inhibits food intake in freely fed and food-deprived rats

Peptides of the corticotropin-releasing hormone/Urocortin (CRH/Ucn) family are known to suppress appetite primarily via CRH₂ receptors. In the rat hypothalamic supraoptic nucleus (SON), synthesis of both Ucn1 and CRH₂ receptors has been reported, yet little is known about the effects of Ucn1 in the SON on feeding behaviour. We first established the dose-related effects of Ucn1 injected into the SON on the feeding response in both freely fed and 24-h food-deprived rats. A conditioned taste avoidance paradigm was performed to investigate possible generalised effects of local Ucn1 treatment. Administration of Ucn1 into the SON at doses equal to or higher than 0.5 μg significantly decreased food intake in both freely fed and food-deprived rats. The Ucn1-mediated suppression of food intake was delayed in freely fed as compared to food-deprived animals. Conditioning for taste aversion to saccharine appeared at 0.5 and 1 μg of Ucn1. Both the early and the delayed onset of anorexia observed after intra-SON injection of Ucn1 under fasting and fed conditions, respectively, suggest the possible involvement of different CRH receptor subtypes in the two conditions, while the conditioned taste aversion seems to be responsible for the initial latency to eat the first meal in these animals.     

Change in the signal properties of a conditioned stimulus during two-way avoidance conditioning in rats

Acquisition of two components of conditioned active avoidance behavior by rats was studied. First presentations of electroshock evoked a number of different behavioral reactions. However, after five trials many rats learned to escape punishment running away to another part of a shuttle-box. The efficiency of the avoidance reaction conditioning significantly depended on the ability of an animal to learn the correct escape reaction to the unconditioned stimulus. However, some animals were incapable for acquisition of the conditioned reaction despite their high level of successful escapes. Increase in the number of negative reactions to the conditioned stimulus (light) at the next stage of learning suggests that the conditioned stimulus becomes the signal of forthcoming punishment. The ability of an animal to identify the conditioned stimulus as a signal significantly affected the efficiency of conditioned avoidance acquisition.     

The reactivity of the hypophyseal-adrenal system in rats with different forms of adaptive behavioral pathology]

The dexamethasone suppression test (DST) was applied to male Wistar rats with different models of depression: group with the learned helplessness, group with informational neurosis provided by time-deficit conditioned avoidance training, as well as groups of rats of two strains selected for low (KLA)--and high (KHA) avoidance learning. The pre-dexamethasone basal and stress-induced corticosterone levels were similar in intact rats and those exposed to inescapable shock. The dexamethasone administration (5 mkg/kg) failed to decrease the serum corticosterone level in rats with learned helplessness. The informational neurosis increased significantly the basal corticosterone level and decreased the stress response. Serum corticosterone levels were similar in KLA and KHA rats. These results give evidence that two stress-induced rat models of depression with similar behavioural disturbances (reduction of escape/avoidance reactions) exhibit marked differences in the activity of hypothalamo-pituitary-adrenal (HPA) axis.     

Fear conditioning and shock intensity: the choice between minimizing the stress induced and reducing the number of animals used

Many fear conditioning studies use electric shock as the aversive stimulus. The intensity of shocks varies throughout the literature. In this study, shock intensities ranging from 0 to 1.5 mA were used, and the effects on the rats assessed by both behavioural and biochemical stress parameters. Results indicated a significant difference with respect to defaecation and freezing behaviour between controls and those animals that received a shock. Significant differences in corticosterone levels were also noted between controls and those groups that received a shock. No significant differences were found between the shock groups with regards to the stress parameters measured in our fear conditioning paradigm, indicating that the two shock groups were similarly stressed. Increased significance levels were noted for freezing behaviour as well as a lower standard error of means was found in the highest shock intensity group. We therefore recommend using the higher shock intensity (1.5 mA) as the rats in the higher shock intensity group were more homogeneously fear-conditioned and therefore the results should be more reproducible and robust than in the lower shock intensity group. This would allow for fewer rats to be used in order to gain an accurate impression of the conditioning paradigm employed.     

Corticotropin-releasing hormone and pituitary-adrenocortical responses in chronically stressed rats

Brain corticotropin-releasing hormone (CRH) concentration and pituitary adreno-cortical responses were examined in chronically stressed rats: body restraint stress (6 h/day) for 4 or 5 weeks. Stressed rats showed a reduction in weight gain. CRH concentration in the median eminence and the rest of the hypothalamus were not different between control and chronically immobilized rats. The anterior pituitary adenocorticotropic hormone (ACTH) concentration was elevated in chronically stressed rats, whereas plasma ACTH and corticosterone levels did not differ from the control values. The median eminence CRH concentration was reduced to the same extent at 5 min after onset of ether exposure (1 min) in chronically immobilized rats and controls. However, plasma ACTH and corticosterone showed greater responses to ether stress in chronically immobilized rats than in control rats. Plasma ACTH and corticosterone responses to exogenous CRH were not different between control and chronically immobilized rats, while the response to arginine vasopressin (AVP) was significantly greater in chronically immobilized rats. These results suggest that chronic stress caused an increase in the ACTH-secreting mechanism and that pituitary hypersensitivity to vasopressin might at least be partly responsible for this.     

Memorization of contextual and CS conditioned fear response (freezing) in a one-trial acquisition paradigm.

In fear-conditioned Wistar rats freezing was induced by the delivery of a series of footshocks paired to tones (CS) in a specific conditioning chamber (context). CS and contextual fear were acquired in the same single conditioning session without preexposition to the conditioning chamber (day 1). Different groups of animals were conditioned employing three increasing US (footshock) intensities (0.25, 0.5, 0.75 mA). During the retention sessions context and CS conditioned freezing (fear response) were measured using a paradigm that fulfilled the following conditions: i) CS freezing retention was measured in a context different from the conditioning one; ii) CS and context freezing were measured at increased delays after the training session (days 3 and 4, 14 and 15, 28 and 29). The results show that there are significant differences between CS and context freezing retention, which are clearly related to delay after the initial session and to US intensity. In particular: 1) conditioned freezing to a discrete tone is better retained than conditioned freezing to context (irrespective of US intensity); 2) context freezing is directly related to US intensity much more than to tone freezing; 3) context freezing is easier to extinguish than tone freezing. The results are discussed in relation to previous ones and to their relevance to freezing genesis neural correlates.     

Corticosterone responses, hurdle-jump acquisition, and the effects of dexamethasone using classical conditioning of fear

Male hooded rats were habituated, classically conditioned with 30 CS-UCS (light-footshock) pairings, and subsequently tested for corticosterone response or instrumental hurdle-jump acquisition. In Experiment 1, corticosterone levels were lowest during chamber placement alone (during habituation), higher during presentations of the CS after conditioning with a low shock intensity, even higher during classical conditioning with the low shock intensity, and highest during classical conditioning or CS presentations involving a high shock intensity. Injections of the synthetic glucocorticoid dexamethasone before both conditioning and hurdle-jump acquisition sessions (Experiment 2) did not affect acquisition occurring during early trials, but produced slow hurdle-jump speeds late in the session. This agrees with previous findings of glucocorticoid facilitation of fear extinction, but does not indicate a simple suppression of fear by dexamethasone. When dexamethasone was given only prior to the classical conditioning session (Experiment 3) hurdle-jump acquisition was poor only on the early trials, and corticosterone levels after 60 min of CS presentations were higher than control values. These results agree with the proposal of a state-dependent effect of dexamethasone on memory retrieval.     

Does estradiol treatment normalize the hypothalamic-pituitary-adrenal axis in streptozotocin-induced ovariectomized diabetic female rats?

We recently found circulating corticosterone (CS) levels to be significantly lower in diabetic female rats as compared with proestrous control animals. This reduction in CS was correlated with the hypoestrogenic state of the diabetic female. It was the purpose of this study to evaluate basal and corticotropin releasing hormone (CRH)-stimulated CS secretion in ovariectomized (OVX) control (C) and streptozotocin-induced diabetic (D) rats given blank, 5 mcg and 20 mcg estradiol (E2) implants to determine if adrenal CS secretion in the diabetic is normalized by E2 treatment. After 3 weeks of diabetes, pituitary-adrenal function was assessed in rats from each group with a CRH stimulation test. The remaining rats were sacrificed for determination of CS, E2, testosterone and fructosamine in serum. Suppressed CS secretion in OVX female diabetic rats was partially restored with E2 therapy. Basal CS levels were significantly higher in 20 mcg E2 treated C and D rats compared with OVX rats. However, C rats had significantly higher basal CS compared with D rats in similarly E2 treated groups. The CS response to CRH stimulation was not different between OVX female diabetic and control rats. Estrogen enhanced the CS response to CRH stimulation in control animals but not in diabetic animals suggesting altered estrogen action at the pituitary level in diabetic animals.     

Effect of L-NAME, a specific nitric oxide synthase inhibitor, on corticotropin-releasing hormone-elicited ACTH and corticosterone secretion.

This study was designed to determine the role of endogenous nitric oxide (NO) in the corticotropin-releasing hormone (CRH)-induced ACTH and corticosterone secretion, as well as possible involvement of hypothalamic dopamine and noradrenaline in that secretion in conscious rats. CRH given i.p. stimulated dose-dependently the pituitary-adrenocortical activity measured 1 h later. Dexamethasone (0.2 mg/kg i.p.) injected 1 h before CRH (1 microg/kg i.p.) totally abolished the CRH-elicited ACTH and corticosterone secretion, indicating a predominantly pituitary site of CRH-evoked stimulation. L-arginine (120 mg/kg i.p.) and N(omega)-nitro-L-arginine methyl ester (L-NAME 5-10 mg/kg i.p.) did not markedly affect the basal plasma ACTH and corticosterone levels. L-NAME given 15 min before CRH markedly, but not significantly, augmented the CRH-induced ACTH response, and enhanced more potently and significantly the corticosterone response. Pretreatment with L-arginine, a substrate for NOS, slightly diminished the CRH-induced ACTH response and considerably reduced the corticosterone response. L-arginine also significantly reversed the L-NAME-evoked increase in the CRH-induced ACTH and corticosterone secretion. L-NAME did not markedly alter the CRH-induced hypothalamic dopamine and noradrenaline levels, while L-arginine significantly increased noradrenaline level. However, those alterations were not directly correlated with the observed changes in ACTH and corticosterone secretion. These results indicate that in conscious rats NO plays a marked inhibitory role in the CRH-induced ACTH secretion and inhibits more potently corticosterone secretion. Hypothalamic dopamine and noradrenaline do not seem to be directly involved in the observed alterations in ACTH and corticosterone secretion.     

Effects of corticotropin releasing hormone on appetitive behaviors.

Corticotropin releasing hormone (CRH) is a 41-residue hypothalamic neuropeptide that has been shown to have potent behavioral effects in animals and has been implicated in clinical disorders in man. This review focuses on those aspects of the behavioral effects of CRH related to food-associated behaviors. The effects of CRH on food intake are compared with its effects on performances maintained by food presentation, and contrasted with the effects of CRH on performances maintained by other events. The effects of CRH antagonists and drugs that interact with the behavioral effects of CRH are also reviewed, particularly with respect to their direct effects on food intake. Lastly, data assessing the effects of CRH administration on central neurotransmitter levels are presented and compared with levels seen in clinical populations. The effect of CRH on food intake seen in animals is consistent with a putative role for CRH in clinical syndromes where appetite suppression is apparent. Since some of the effects of CRH on food intake are subject to pharmacological intervention, strategies directed at peptidergic mechanisms of psychiatric disorders should be explored.     

Ontogenetic differences in sensitivity to LiCl- and amphetamine-induced taste avoidance in preweanling rats

When amphetamine is associated with a tastant conditioned stimulus, rats learn to avoid the taste even when employing doses that promote conditioned place preference. One hypothesis raised to account for this effect proposes that taste avoidance induced by amphetamine may be motivated by fear. A sensitive period has been identified in the rat (until postnatal day 10) in which infants learn conditioned appetitive effects to stimuli to which aversions are conditioned after this period. Exogenous administration of corticosterone within this period reverses this effect, generating aversive conditioning. In the present study, we tested conditioning of aversions to amphetamine or LiCl, within and after the sensitive period (Experiments 1 and 2). A third experiment evaluated unconditioned rejection of an aversive quinine solution within the sensitive period. Finally, we tested whether corticosterone administration before conditioning modulates amphetamine-induced taste avoidance. After the sensitive period, infant rats rejected the solution paired with amphetamine or LiCl after 2 conditioning trials, but within the sensitive period, aversions were conditioned only by LiCl and after 4 conditioning trials. Amphetamine-induced taste avoidance was not observed even when corticosterone was administered before conditioning. Additionally, during the sensitive period, a low LiCl dose promoted conditioned taste preference. According to Experiment 3, parameters employed in this study were suitable to yield rejection of aversive solutions within the sensitive period. These results suggest that during the sensitive period, there is a notable resistance to the acquisition of taste avoidance induced by amphetamine. The present experimental framework may represent a useful tool for studying mechanisms underlying taste avoidance and aversion effects.