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.     

Levels of arginine-vasopressin in cerebrospinal fluid during passive avoidance behavior in rats

The concentration of immunoreactive arginine-vasopressin (IR-AVP) was measured in the cerebrospinal fluid (CSF) during acquisition and retention of passive avoidance behavior. IR-AVP level in CSF of male Wistar rats immediately after the learning trial was increased; the rate of which was related to the intensity of the electric footshock during the learning trial and the avoidance latency as measured 1 day after the learning trial. Immediately after the 24 h retention test IR-AVP levels were significantly increased in rats subjected to the low (0.25 mA) shock intensity during the learning trial, but IR-AVP levels of rats exposed to the high shock (1.0 mA) were under the limit of detection. If the retention test was postponed till 5 days after the learning trial, the increase of IR-AVP level in the CSF was related to avoidance latencies which reflect the intensity of aversive stimulation (electric footshock). The results suggest an association between central AVP release and passive avoidance behavior and may be indicative of the role of this peptide in neuronal mechanisms underlying learning and memory processes.     

Neurohypophyseal peptide levels in CSF and plasma during passive avoidance behavior in rats

Levels of vasopressin (AVP), oxytocin (OXT), and neurophysin (NP) in CSF and plasma of rats were determined during acquisition and retention of passive avoidance behavior. None of the levels of neurohypophyseal peptides in CSF were changed either during the adaptation period, or during acquisition or the retention of this behavior. Moreover, no differences were found in hormone levels in CSF of the various groups of rats subjected to different shock intensities during the acquisition trial. The marked differences in individual latencies of nonavoiding rats, and the differences in latencies due to a different shock intensity applied during the learning trial were not reflected by changes in CSF hormone levels. Neither AVP nor NP levels in plasma were affected by the different shock intensities applied, when measured at 20 min after the learning trial. In contrast, a decrease in plasma OXT levels was observed after application of a shock intensity of 0.25 mA during the learning trial. During retention of the passive avoidance response plasma levels of AVP, OXT and NP were not different from the levels found in the nonshocked groups. It is suggested that under the conditions used in this study the CSF is apparently not involved in the distribution of neurohypophyseal peptides to their possible sites of behavioral action in the brain.     

Graded footshock stress elevates pituitary cyclic AMP and plasma beta-endorphin, beta-LPH corticosterone and prolactin

Male rats were subjected to 15 min of various intensities of footshock current (0.0, 0.2, 0.4, 0.8, 1.6, 2.4, 3.2m A) on a variable interval schedule with an average intershock interval of 30 sec (30 shocks/15 min session). Each shock lasted 5 sec. Animals were sacrificed immediately after being removed from the shock box. Two similar studies were conducted. In the first experiment, rats were sacrificed by microwave irradiation and pituitary cyclic AMP levels were determined. In the second study, rats were decapitated and plasma hormones (prolactin, corticosterone, beta-endorphin, beta-LPH) were measured by radioimmunoassay. Although all biochemical indices of stress measured increased as shock intensity increased, some differences among the substrates measured were observed with respect to threshold intensity, range of proportional response and maximal response.     

Tyrosine prevents behavioral and neurochemical correlates of an acute stress in rats

Exposure of rats to an acute, uncontrollable stressor can increase brain norepinephrine (NE) turnover and decrease locomotor and exploratory behavior. We examined the ability of exogenous tyrosine, NE's amino acid precursor, to protect rats from developing these neurochemical and behavioral changes when stressed.Animals pretreated with saline or tyrosine (200 mg/kg, i.p.) were subjected to tail shock (15 v, 2 mA, 5 sec/30 sec) or to no shock during a 60-min period. Exposure to shock depleted NE and increased its turnover [as indicated by altered NE and 3-methoxy-4-hydroxy-phenylethylene-glycol sulfate levels (MHPG-SO₄)] within the locus coeruleus, the hippocampus and the hypothalamus. Behavioral deficits were observed using measures of locomotion, standing on hind legs, and hole-poking in an open-field apparatus. Animals given tyrosine before shock displayed neither shock-induced NE depletion nor the deficits in locomotion and hole-poking; brain MHPG-SO₄ levels tended to be greater than after shock alone. These observations suggest that the stress caused NE to be released from some neurons more rapidly than it could be restored by synthesis or reuptake, thereby impairing noradrenergic transmission and NE-dependent exploratory behaviors. Tyrosine administration presumably enhanced the transmitter's synthesis in stressed animals, thereby preventing both the neurochemical and the behavioral deficits.     

Enhancement of DMBA induced mammary tumours by intermittent whole body hyperthermia (42 degrees C)

Wistar Female rats bearing DMBA induced mammary tumours were subjected to whole body hyperthermia 42 degrees C dry heat exposure for 15 minutes daily for 6 weeks. The control group was maintained at a room temperature of 25 degrees C. Hyperthermia induced significant growth stimulation of breast tumour compared to the controls. Plasma estradiol was slightly decreased while total T4 and TSH values remained unchanged in heat stressed rats. Plasma prolactin was significantly increased together with enhanced synthetic activity of pituitary prolactin cells. It is concluded that heat acting as stressor accelerates breast tumor growth, probably by influencing synthesis of prolactin. Therefore the hormone dependency of tumours should be considered before hyperthermia is used as an anticancer modality.     

Startle responses to electric shocks: measurement of shock sensitivity and effects of morphine, buspirone and brain lesions

The present study developed a new protocol to assess shock sensitivity in rats. Male Wistar rats were subjected to footshock stimuli ranging from 0 to 1.6 mA (0.1 s) in a startle apparatus and startle responses elicited by shocks were measured. Acoustic stimuli (95, 105, or 115 dB) were dispersed within the shock series serving as a control measurement of motor performance. Results indicated that the magnitude of shock startle responses significantly increased with the shock intensity in a linear trend. Morphine (8.0 mg/kg) and buspirone (1.0, 2.5, or 5.0 mg/kg), both of which possessing analgesic effects, depressed shock startle but had no such effect on acoustic startle. The effect of morphine was readily reversed by pretreatment of naloxone (1.0 mg/kg). To investigate the neural basis underlying this response, radio-frequency lesions of various structures implicated in processing of nociceptive or aversive information were undertaken. Lesions of the ventroposterior thalamic nucleus, insular cortex, or amygdala decreased startle reactivity to electric shocks but not to acoustic stimuli. Lesions of the anterior cingulate gyrus or medial prefrontal cortex, while altered the reactivity to acoustic stimuli, had no effect on the shock-elicited startle. These results suggested that the amplitude of startle in response to electric shocks provide a quantitative measurement of shock sensitivity within an extended range of stimulus intensities. Performing this response may engage the the central nociceptive pathway.     

A conspecific attenuates prolactin responses to open-field exposure in rats

Acute exposure to a novel environment, such as an open field, generally results in a prolactin surge, while several days of exposure to the open field is often characterized by a decline in prolactin. As exposure to the open field is a psychological stressor, altering the animal's interpretation of the event should alter prolactin levels. In the present study, juvenile male and female rats were habituated to the open field for 1 or 5 days prior to testing in the chamber alone or with a same-sex conspecific. Levels of prolactin were measured across all rats, and play (pins) was recorded for animals tested with a conspecific. Five days of habituation to the chamber resulted in lower levels of prolactin and more play than 1 day of habituation. Across both conditions of habituation, testing with a conspecific caused lower levels of prolactin than testing alone. In addition, play and prolactin were negatively correlated. The presence of a conspecific in a stressful situation may have reduced stress by altering the animal's negative interpretation of the open field. Further, as the intensity of the social interaction increased (more play), prolactin levels decreased.     

Effect of illness on hormonal response to footshock stress

The corticosterone (CORT) and prolactin (PRL) responses to 1.0 mA of footshock were measured in healthy rats and rats with pneumonia. No differences in basal PRL levels were seen, but basal CORT levels were significantly increased in the sick animals. Healthy rats showed a significant increase in both PRL and CORT after receiving footshock whereas the sick rats showed no changes. The adapative value of the current findings are unclear at this time.     

Sensitivity of anterior pituitary hormones to graded levels of psychological stress

The effect of graded levels of stressor intensity on anterior pituitary hormones was studied in adult male rats. Corticosterone, considered as a reflection of ACTH release, and prolactin responses showed a good correlation with the intensity of the stressors. On the contrary, neither LH, GH nor TSH release showed a parallelism with the intensity of the stressors in spite of the fact that they clearly responded to all the stimuli. It appears that the hormones of the anterior pituitary might be divided into two groups: those whose response is sensitive to the levels of emotional arousal elicited by stress, and those displaying a clear but stereotyped response during stress. However, other alternative explanations might exist to justify the present results. The neural mechanisms underlying the two types of response are at present unknown. These data indicate that only the pituitary-adrenal axis and prolactin have some potential utilities as quantitative indices of emotional arousal elicited by currently applied stressors in the rat.     

A comparison of plasma prolactin levels in young female Long-Evans and Holtzman rats as measured by Nb2 lymphoma bioassay and radioimmunoassay

This study was conducted to determine the plasma levels of prolactin in prepubertal and young, postpubertal, proestrus rats of mammary tumor-susceptible (Sprague-Dawley) and tumor-resistant (Long-Evans) strains using a sensitive bioassay-Nb2 lymphoma cell replication. Prepubertal Long-Evans rats had significantly higher levels of prolactin than did Holtzman Sprague-Dawley rats of the same age. Likewise, Long-Evans rats secreted significantly more prolactin into the blood on the afternoon and evening of proestrus than did Holtzman rats. Finally, ovariectomized Long-Evans rats released more prolactin into the blood at 1 day, but not at 8 or 15 days, of treatment with diethylstilbestrol. Prolactin levels determined by conventional radioimmunoassay and by bioassay were similar except on the afternoon of proestrus, when, in both strains of rats, the bioassay to radioimmunoassay ratio increased significantly above 1.0 during the late evening. In addition, the ratio was significantly less than 1.0 in the early and late afternoon in the Holtzman rats, but not Long-Evans rats. These data indicate that a strain of rats that is resistant to experimentally induced mammary cancer has higher prolactin levels in the blood than does a strain that is susceptible to mammary cancer at a time when mammary gland growth is rapid. Furthermore, there are times during the proestrus prolactin surge when the bioassay yielded higher and lower values of prolactin than radioimmunoassay of the same samples, suggesting functional heterogeneity of prolactin that may impact on mammary gland or other target tissue function.     

Stressors produce a concurrent decrease and increase in reflex amplitude in rats treated with naloxone

The effect of inescapable foot-shock on the tail-flick response and on the startle response to brief shocks and brief tones was studied in rats. In the first experiment, 25 minutes of inescapable foot shock (stressor) produced a significant increase in tail-flick latency which was antagonized by the opioid antagonist naloxone (2.0 mg/kg). In the second experiment, the startle response to an electric shock to the tail was significantly diminished by the stressor, and this effect was not significantly reduced by naloxone. However, the size of the startle response to a brief tone was significantly increased in rats treated with naloxone. Thus, rats injected with naloxone had a decreased startle to shock but an increased startle to tone following inescapable foot shock. Finally, tones which preceded shocks by one second produced a facilitation of the startle response to the shocks in tests that followed exposure to the stressor. This facilitation was not affected significantly by naloxone. These results indicate that the changes in the startle response following the stressor were not mediated exclusively by endogenous opioids.     

Differential Behavioral and Endocrinological Effects of Corticotropin-Releasing Hormone (CRH) in the Syracuse High- and Low-Avoidance Rats

The Syracuse high- and low-avoidance rats, which have been selectively bred for good (SHA/Bru) or poor (SLA/Bru) avoidance learning in a two-way shuttle box, differ in emotionality. This experiment investigated the effect of corticotropin-releasing hormone (CRH), administered centrally (0, 0.1, 0.5, and 1.0 μg), on conditioned suppression and on the hypothalamic–pituitary–adrenocortical system. Three groups of animals were used: SHA/Bru rats conditioned at 0.21 or 0.43 mA and SLA/Bru rats conditioned at 0.21 mA. The results confirm those of previous studies which found that SLA/Bru rats show greater conditioned suppression than the SHA/Bru rats at the low shock intensity and that at 0.43 mA, the SHA/Bru animals acquire a level of conditioning comparable to that of the SLA/Bru animals at 0.21 mA. The results show that the nonlinear behavioral effect of CRH is independent of strain and produces comparable effects in animals of both strains, but only when level of conditioning is equated. Adrenal and plasma concentrations of corticosterone increased in all three groups of animals as a direct linear function of dose of CRH. Both greater levels of conditioning and larger amounts of CRH increase the synthesis of corticosterone more in SHA/Bru animals than in the SLA/Bru animals. Thus, genetic variation, which differentiates the behavioral and endocrinological characteristics of these animals, shows that these effects of CRH can be independent of each other and suggests that some minimal level of conditioned fear is necessary for CRH to exert its anxiogenic effect.     

Pentobarbital anesthesia: interference in the effects of other pharmacological agents and of electrical stimulation on prolactin secretion

In adult male rats, a pretreatment regimen of serial injections of dexamethasone (1 mg/kg), morphine (20 mg/kg) and pentobarbital (40 mg/kg) was evaluated for use in conjunction with studies on the effects of hypothalamic electrical stimulation on prolactin secretion. Serum prolactin levels were measured before and 15 min after electrical and sham stimulation of the hypothalamic arcuate nucleus in rats subjected to either the pharmacological regimen or to pentobarbital anesthesia alone. Pentobarbital alone caused a transient rise in serum prolactin levels, which obscured any effect of electrical or sham stimulation; this interference was not overcome by the addition of dexamethasone and morphine treatment. Thus, the result indicate that the acute stimulatory affect of pentobarbital anesthesia on prolactin release may interfere with further manipulation of prolactin-controlling mechanisms, by either pharmacological or surgical means. Furthermore, the dexamethasone-morphine-pentobarbital pretreated rat does not provide an adequate preparation for studing the effects of electrical stimulation on prolactin secretion.     

Effects of a Post-Shock Injection of the Kappa Opioid Receptor Antagonist Norbinaltorphimine (norBNI) on Fear and Anxiety in Rats

Exposure of rats to footshocks leads to an enduring behavioral state involving generalized fear responses and avoidance. Recent evidence suggests that the expression of negative emotional behaviors produced by a stressor is in part mediated by dynorphin and its main receptor, the kappa opioid receptor (KOR). The purpose of this study was to determine if a subcutaneous injection of the long-acting KOR antagonist norbinaltorphimine (norBNI; 15.0 and 30.0 mg/kg) given 2 days after an acute exposure of rats to footshooks (5×2 s episodes of 1.5 mA delivered over 5 min) attenuates the expression of lasting fear and anxiety. We report that exposure of rats to acute footshock produced long-lasting (>4 weeks) fear (freezing) and anxiety (avoidance of an open area in the defensive withdrawal test). The 30 mg dose of norBNI attenuated the fear expressed when shock rats were placed in the shock context at Day 9 but not Day 27 post-shock. The same dose of norBNI had no effect on the expression of generalized fear produced when shock rats were placed in a novel chamber at Days 8 and 24. In contrast, the 30 mg dose of norBNI produced consistent anxiolytic effects in shock and nonshock rats. First, the 30 mg dose was found to decrease the latency to enter the open field in the defensive withdrawal test done 30 days after the shock exposure. Second, the same high dose also had anxiolytic effects in both nonshock and shock rats as evidence by a decrease in the mean time spent in the withdrawal box. The present study shows that systemic injection of the KOR antagonist norBNI had mixed effect on fear. In contrast, norBNI had an anxiolytic effect which included the attenuation of the enhanced avoidance of a novel area produced by a prior shock experience.     

Monoamine Receptors in an Animal Model of Affective Disorder

After a relatively mild course of uncontrollable shocks, two distinct groups of rats can be defined in terms of their performance in learning to escape from a controllable stressor. Response-deficient (RD) rats do not learn to terminate the controllable stressor, whereas nondeficient (ND) rats learn this response as readily as do untreated control rats. The current studies were designed to determine the neurochemical correlates of the behavioral differences between these groups of rats. The major findings concerned postsynaptic beta-adrenergic effects in the hippocampus of RD rats. These included an up-regulation of beta-adrenergic receptors and, in parallel experiments, an increase in the sensitivity of adenylyl cyclase to stimulation by norepinephrine. There was no difference in brain levels of catecholamines between the three groups of rats. A statistically significant increase in levels of 5-hydroxytryptamine was noted in the hippocampus and hypothalamus of RD rats as compared to levels in ND rats, but no significant differences were measured between groups of rats in terms of S1 or S2 serotonergic receptor binding. These results implicate both beta-adrenergic and serotonergic mechanisms in the behavioral deficit caused by uncontrollable shock.     

Inhibitory effect of alcohol on the established suckling-induced prolactin surge in lactating rats

The effect of acute alcohol infusion on the established suckling-induced prolactin surge in lactating rats was examined. Dams were implanted with an atrial catheter on Day 6 of lactation and blood sampling was done on Day 10. Following the separation of litters from dams for a 6-hr period, a baseline blood sample was removed via a catheter extension. Pups were weighed and returned to dams. Subsequent blood samples were obtained 10, 30, and 60 min after initiation of suckling. Dams were then infused with alcohol doses of 0, 0.5, 1.0, 2.0, or 2.5 g/kg body wt. Infusion (0.1 ml/min) was completed in approximately 30 min. Additional blood samples were obtained 10 30, 60, and 120 min after the termination of infusion. In a separate group of rats, pups were removed from the dam after the first 60 min of suckling and additional blood samples were obtained 40, 70, 90, and 150 min after removal of pups (corresponding to 10-, 30-, 60-, and 120-min samples for rats infused with various alcohol doses). Alcohol, when administered after the establishement of suckling-induced prolactin surge and resulting in blood alcohol levels equal to or greater than legal human intoxication levels, inhibited prolactin release. However, continued suckling for an extended period (120 min in the present study) overcame this inhibitory effect, even when the blood alcohol level was comparable to (2.0 g/kg group) or greater than (2.5 g/kg group) the human legal intoxication level. Furthermore, in rats with established prolactin surges, the patterns of prolactin decline that followed alcohol administration or pup removal were comparable, indicating that similar mechanism(s) may be involved.     

Activation of the arousal response and impairment of performance increase with anxiety and stressor intensity.

The purpose of this study was to determine the effect of trait anxiety and stressor intensity on arousal and motor performance during a pinch task. We examined the steadiness of a precision task in the presence and absence of an imposed stressor on subjects with moderate and low trait anxiety. Subjects with the 26 highest and 14 lowest anxiety scores were assigned to one of three groups: a control group (5 women, 5 men), a moderate-anxiety group (8 women, 8 men), or a low-anxiety group (7 women, 7 men). Subjects in the anxiety groups received electric shocks and experienced significant increases in cognitive and physiological arousal compared with baseline and control subjects, especially subjects in the moderate-anxiety group. Heart rate, systolic blood pressure, and electrodermal activity were elevated during the stressor, whereas diastolic blood pressure was unchanged. Cognitive and physiological arousal tended to increase with stressor intensity and was accompanied by changes in steadiness. Although steadiness was markedly reduced with the highest intensity of shock, the average electromyogram activity was unaffected by the stressor. These findings indicate that the increase in arousal and the impairment of steadiness increased with trait anxiety and with the intensity of the noxious stimulus.     

Effects of immobilization stress on tuberoinfundibular dopaminergic (TIDA) neuronal activity and prolactin levels in lactating and non-lactating female rats.

The effects of immobilization stress on the prolactin secretory response and on the activity of the tuberoinfundibular dopaminergic (TIDA) neurons were determined in intact, virgin female rats on the morning of diestrus or proestrus and in post-partum, lactating female rats. The virgin females exhibited a significant increase in circulating levels of prolactin which was evident by 1 minute and persisted during the immobilization (5 minutes). In contrast, the prolactin secretory response in lactating females was significantly attenuated compared to non-lactating animals. The activity of the TIDA neurons was not altered by the 5 minutes of stress. Even after 30 minutes of immobilization, TIDA neuronal activity was not affected in either the lactating or cycling females. These data suggest that the cycling female rat is capable of a prolactin secretory response to the stressor without inhibition of TIDA neuronal activity. It seems likely that prolactin releasing factors mediate this response. In contrast, stress did not produce a similar prolactin increase during lactation. It seems likely that, during lactation, the pituitary is not sensitive to releasing factors unless the TIDA neurons are inhibited. There appear to be differences in the sensitivity of the pituitary depending on the physiological state of the model employed.     

Inhibitton of prolactin release by stimulation of presynaptic serotonin autoreceptors

The effects of 5-methoxy-N, N-dimethyltryptamine (5-MeODMT), a serotonin agonist with a preferential action on presynaptic autoreceptors, on prolactin release in male rats was determined. Basal serum prolactin levels were not altered after administration of 1.0, 2.0, 5.0, 10.0 or 20.0 mg/kg of 5-MeODMT.Pretreatment with 5-MeODMT reduced prolactin release by agents that depend on serotonergic neurotransmission for part of their prolactin release stimulation. Prolactin release in response to L-5-hydroxytryptophan (5-HTP) or morphine was significantly reduced by pretreatment of the rats with 5-MeODMT.The results of this experiment indicate that 5-MeODMT act as a presynaptic serotonin autoreceptor stimulant and not as a postsynaptic serotonin agonist on the neuronal systems that control prolactin release.