Nonshocked Rats Research Articles

The effects of enhancing endocannabinoid signaling and blocking corticotrophin releasing factor receptor in the amygdala and hippocampus on the consolidation of a stressful event

Current clinical and pre-clinical data suggest that both cannabinoid agents and blockage of CRF through corticotrophin releasing factor receptor type 1 (CRFr1) may offer therapeutic benefits for post-traumatic stress disorder (PTSD). Here we aim to determine whether they are more effective when combined when microinjected into the basolateral amygdala (BLA) or CA1 area of the hippocampus after exposure to a stressful event in the shock/reminders rat model for PTSD.Injection of the fatty acid amide hydrolase (FAAH) inhibitor URB597 after the shock into either the BLA or CA1 facilitated extinction, and attenuated startle response and anxiety-like behavior. These preventive effects of URB597 were found to be mediated by the CB1 receptor.Intra-BLA and intra-CA1 microinjection of the CRFr1 antagonist, CP-154,526 attenuated startle response. When microinjected into the BLA, CP-154,526 also attenuated freezing behavior during exposure to the first reminder and decreased anxiety-like behavior. The combined treatment of URB597 and CP-154,526 was not more effective than the separate treatments.Finally, mRNA levels of CRF, CRFr1 and CB1r were significantly higher in the BLA of rats exposed to shock and reminders compared to non-shocked rats almost one month after the shock.Taken together, the results show that enhancing endocannabinoid signaling in the amygdala and hippocampus produced a more favorable spectrum of effects than those caused by the CRFr1 antagonist. The findings suggest that FAAH inhibitors may be used as a novel treatment for stress-related anxiety disorders.

Effects of footshocks on anxiety-like behavior and mRNA levels of precursor peptides for corticotropin releasing factor and opioids in the forebrain of the rat

Corticotropin releasing factor (CRF) and dynorphin are neuropeptides that are associated with the negative emotional states. Experimental evidence indicates that dynorphin neurons located in the nucleus accumbens and CRF neurons in the bed nucleus of the stria terminalis (BST) and the central nucleus of the amygdala (CeA) mediate anxiety-like behaviors immediately after the stressful experience (24–48h). The present study was done to evaluate if changes in the levels of the mRNA for these peptides in the striatum, BST, and CeA were associated with the long-lasting avoidance of novelty, a measure of an anxiety-like state, in a subset of rats exposed to unpredictable and moderately intense footshocks (5×2s of 1.5mA). Shocked rats with enhanced fear to a novel tone 24h after the footshocks (high responders; HR) displayed long-lasting avoidance in the elevated T-maze whereas shocked rats with low levels of acute fear (low responders; LR) had low levels of avoidance similar to nonshocked rats. An increase in the level of proCRF mRNA was detected in the CeA of the HR compared to LR and nonshocked rats but not in other areas of the brain sampled. In contrast, prodynorphin and proenkephalin mRNA levels in the striatum, BST and CeA were not different between HR, LR and nonshocked rats. This study provides evidence that CRF neurons in the CeA may play a role in the anxiety-like state produced in a subset of rats exposed to footshocks.

The long-term impact of footshock stress on addiction-related behaviors in rats

We investigated the impact of electric shocks – frequently used to model post-traumatic stress disorder in rodents – on behaviors relevant to drug abuse in rats. Rats exposed to 10 shocks of 3mA over 5min showed a robust conditioned fear 28 days later, which confirms the traumatic nature of shock exposure. A different set of rats was studied in the conditioned place preference paradigm beginning with the 27th post-shock day. 10mg/kg morphine induced a marked place preference in both shocked and non-shocked rats. Although the magnitude of place preference was not affected, extinction was markedly delayed in shocked rats. We also investigated tolerance to the hyperthermic effects of morphine. A low dose (5mg/kg) that was administered 4 weeks after shock exposure robustly increased body temperature in both shocked and non-shocked rats. Repeated injections resulted in a mild tolerance in non-shocked controls; yet, morphine readily increased body temperature in these rats on the 5th day of injections. In contrast, the temperature-heightening effect of morphine was abolished in shocked rats after 2 days. Thus, shock exposure considerably delayed the extinction of place preference induced by, and dramatically accelerated the tolerance to the effects of, morphine. Our study shows that electric shocks durably affect behavior in tests relevant to drug abuse in conjunction with the development of post-traumatic stress disorder-like behavioral dysfunctions.

MOLECULAR SIGNATURES OF TRAUMA-HEMORRHAGIC SHOCK-INDUCED LUNG INJURY

The etiology of trauma-hemorrhagic shock (T/HS)-induced acute lung injury has been difficult to elucidate because of, at least in part, the inability of in vivo studies to separate the noninjurious pulmonary effects of trauma-hemorrhage from the tissue-injurious ones. To circumvent this in vivo limitation, we used a model of T/HS in which T/HS lung injury was abrogated by dividing the mesenteric lymph duct. In this way, it was possible to separate the pulmonary injurious response from the noninjurious systemic response to T/HS by comparing the pulmonary molecular responses of rats subjected to T/HS, which did and did not develop lung injury, with those of nonshocked rats. Using high-density oligonucleotide arrays and treatment group comparisons of whole lung tissue collected at 3 h after the end of the shock or sham-shock period, 139 of 8,799 assessed genes were identified by significant analysis of microarrays. Hemorrhage without the secondary effects of lung injury modulated the expression of 21 genes such as interleukin 1beta, metallothionein-2, and myeloctomatosis oncogene (c-myc). In response to injury, 42 genes were identified to be differentially expressed. Upregulated genes included the L1 retroposon and guanine deaminase, whereas downregulated genes included catalase and superoxide dismutase 1. Real-time polymerase chain reaction confirmed the differential expression for selected genes. PathwayAssist analysis identified interleukin 1beta as a central regulator of two subpathways of stress response-related genes (c-myc and superoxide dismutase 1/catalase) as well as several unrelated genes such as lipoprotein lipase. Our model system provided a unique opportunity to distinguish the molecular changes associated with T/HS-induced acute lung injury from the systemic molecular response to T/HS.

Endocannabinoids at the spinal level regulate, but do not mediate, nonopioid stress-induced analgesia

Recent work in our laboratories has demonstrated that an opioid-independent form of stress-induced analgesia (SIA) is mediated by endogenous cannabinoids [Hohmann et al., 2005. Nature 435, 1108]. Non-opioid SIA, induced by a 3-min continuous foot shock, is characterized by the mobilization of two endocannabinoid lipids—2-arachidonoylglycerol (2-AG) and anandamide—in the midbrain periaqueductal gray (PAG). The present studies were conducted to examine the contributions of spinal endocannabinoids to nonopioid SIA. Time-dependent increases in levels of 2-AG, but not anandamide, were observed in lumbar spinal cord extracts derived from shocked relative to non-shocked rats. Notably, 2-AG accumulation was of smaller magnitude than that observed previously in the dorsal midbrain following foot shock. 2-AG is preferentially degraded by monoacylglycerol lipase (MGL), whereas anandamide is hydrolyzed primarily by fatty-acid amide hydrolase (FAAH). This metabolic segregation enabled us to manipulate endocannabinoid tone at the spinal level to further evaluate the roles of 2-AG and anandamide in nonopioid SIA. Intrathecal administration of the competitive CB 1 antagonist SR141716A (rimonabant) failed to suppress nonopioid SIA, suggesting that supraspinal rather than spinal CB 1 receptor activation plays a pivotal role in endocannabinoid-mediated SIA. By contrast, spinal inhibition of MGL using URB602, which selectively inhibits 2-AG hydrolysis in the PAG, enhanced SIA through a CB 1-selective mechanism. Spinal inhibition of FAAH, with either URB597 or arachidonoyl serotonin (AA-5-HT), also enhanced SIA through a CB 1-mediated mechanism, presumably by increasing accumulation of tonically released anandamide. Our results suggest that endocannabinoids in the spinal cord regulate, but do not mediate, nonopioid SIA.

Radix Angelica Sinensis extracts ameliorate scopolamine- and cycloheximide-induced amnesia, but not p-chloroamphetamine-induced amnesia in rats.

The effects of the methanolic extract of Radix Angelica Sinensis (Umbellifera) (abbreviated as RAS extract) and n-hexane fraction of RAS extract (RAS(H) fraction) on the various drugs-induced amnesia in rats were studied by using passive avoidance task. RAS extract (1 g/kg) significantly prolonged the shortened step-through latency induced by SCOP and CXM, but not PCA. Furthermore, RAS(H) fraction (1 g/kg) also significantly prolonged the shortened step-through latency induced by SCOP and CXM but not PCA. RAS extract at any dose alone did not influence the step-through latency in the training trial produced by non-shocked rats, but it plus PCA prolonged the latency compared with PCA alone. However, RAS(H) fraction (1 g/kg) prolonged the latency in the training trial produced by non-shocked rats, but it plus any induced drugs did not differ from any induced drugs alone. These results suggest that the attenuating effects of RAS extract on the various drugs-induced amnesia were related to the memory processes. n-Hexane fraction of RAS extract might be one of the active fractions of RAS extract in the treatment of amnesia.

Enhancement of Antigen-Specific Humoral and Cell-Mediated Immunity by Electric Footshock Stress in Rats

The effect of footshock stress on the induction phase of sensitization to keyhole limpet hemocyanin (KLH) introduced intraperitoneally was studied in adult male Sprague– Dawley rats. Rats were shocked at Days −1, 0, 1, and 3 relative to sensitization with 50 μg KLH and 14 days later were intradermally injected with 25 μg KLH or were noninjected. Anti-KLH IgG levels were measured in serum by ELISA and were enhanced in stressed versus control rats shocked on Days 0 or 1; splenocyte proliferation to KLH in vitro was also found to be enhanced in shocked rats compared to that in nonshocked rats. Skin at the challenge sites was removed and histologically examined for infiltrate density. There was an increased infiltrate in animals shocked on Days 0 or 1 in comparison to nonshocked controls. The increased humoral and cell-mediated anti-KLH immunity in stressed rats is evidence for enhanced immune function by exposure to footshock proximal to the induction phase of the immune response. The possibility of a generalized increase in immune function in stressed rats is doubtful since splenocyte proliferation to the T-cell mitogens concanavalin A (Con A) and phytohemagglutinin and the B-cell mitogen lipopolysaccharide showed no alteration between control and stressed rats at the time of sacrifice.

Comparative effects of electroconvulsive shock and haloperidol on in vivo tyrosine hydroxylation and tetrahydrobiopterin in the brain of rats with 6-hydroxydopamine lesions

We have evaluated the effects of electroconvulsive shock (ECS) and haloperidol treatment on the in vivo tyrosine hydroxylation rate and the tetrahydrobiopterin levels in the nigrostriatal system of 6-OHDA-lesioned rats. The rate of DOPA accumulation was significantly decreased by 96% in the ipsilateral striatum and by 50% in substantia nigra of the 6-OHDA-lesioned rats compared to the control activity of contralateral non-lesioned striatum and substantia nigra. The loss of total biopterin was found to be 75% and 50% in the ipsilateral striatum and substantia nigra, respectively. Following administration of haloperidol, the rate of DOPA accumulation increased significantly in the striatum and substantia nigra on the lesioned side compared to that in the vehicle treatment group. Application of ECS also significantly increased the rate of DOPA accumulation in the ipsilateral striatum and substantia nigra compared to that obtained in the non-shocked rats. The biopterin levels in the nigrostriatal system of 6-OHDA-lesioned rats were elevated significantly in the striatum after haloperidol treatment; in contrast the biopterin levels were unchanged in response to ECS. Our results show that both haloperidol and ECS significantly enhanced the rate of in vivo tyrosine hydroxylation in the striatum and substantia nigra of rats with greater than 90% lesions. These results suggest that the nigrostriatal system, although up-regulated following 6-OHDA lesions, still maintains the potential for further up-regulation of dopaminergic function in response to haloperidol and ECS treatment.

5‐Hydroxytryptamine1A Receptor Agonists in Animal Models of Depression and Anxiety

The effects of different doses of buspirone, 3-dipropyl-amino-5-hydrochromar (NDO 008) and 8-hydroxydipropyl-aminotetralin (8-OH-DPAT) (administered intraperitoneally) were studied in tests of anxiolytic and antidepressant action in rats. These tests included the elavated plus maze test, the forced swim test, stress-induced suppression of open-field behavior, and the differential-reinforcement-of-low-rates-of-behaviour-72 sec (DRL 72 s) test. Buspirone (0.125 mg/kg) and NDO 008 (1.0 to 2.0 mg/kg) produced anxiolytic activity in the elevated plus maze, whereas 8-OH-DPAT did not in the doses employed. All three compounds increased activity in the forced swim test, although buspirone did so at a lower dose than NDO 008 and 8-OH-DPAT. In the stress-induced suppression test of open field activity all three compounds induced an antidepressant-like effect at different doses dependent on whether footshock (stressor) was presented 24 hr before or just prior to the open-field test. All three compounds even caused some reduction of activity in the non-shocked rats. 8-OH-DPAT (1.0 mg/kg) produced a significant and reliable increase in the Reinforcement/Response rate quotient in the DRL 72s test. These diverse results may provide an indication of potential clinical efficacy of the 5-HT1A agonists in the treatment of anxiety and depression.

Stressor-induced changes in mitogenic activity are not associated with decreased interleukin 2 production or changes in lymphocyte subsets

Splenic lymphocytes from Lewis rats that received presentations of physically aversive electric shock demonstrated a marked reduction in responsiveness to T-cell mitogens such as concanavalin A. This study examined cellular mechanisms which may be responsible for this functional alteration. There was no difference in distribution of T-cell subsets from shocked and nonshocked rats. There was no difference in the production of interleukin 2 (IL-2) nor was there a difference in the percentage of IL-2 receptor positive T cells or T-cell subsets after culture for 24 hr. However, there was a marked lack of mitogenic stimulation in splenocytes from shocked rats when stimulated with the calcium ionophore A23187. This indicates a defect in the biochemical pathways necessary to activate T-cell mitogenesis.

Conditional hypoalgesia is attenuated by Naltrexone applied to the periaqueductal gray

The ventrolateral periaqueductal gray (vPAG) is an important component in a brainstem system involves in the endogenous modulation of nociception and defensive behavior. The present study was conducted to determine if opioid receptors within the vPAG contribute to the hypoalgesia seen in rats during presentation of a Pavlovian CS for footshock. Independent groups of animals received microinjections of either 5.0 μg naltrexone HCl or vehicle into the vPAG prior to being placed in an observation chamber in which shock had been delivered 24 h earlier. Nociceptive reactivity was measured with the formalin test. Naltrezone treatment attenuated conditional hypoalgesia but did not affect formalin-induced behavior in non-shocked rats. Naltrexone had no effect on the amount of defensive freezing behavior observed during the test session. These results indicate that conditional hypoalgesia as measured by the formalin test involved the activation of documented brainstem antinociceptive systems.

Individual differences in taste, body weight, and depression in the “helplessness” rat model and in humans

The helplessness paradigm is used extensively in basic stress research and is an experimental model of clinical depression. In Experiment 1, exposure to unsignaled, inescapable shock resulted in finickiness about drinking a weak quinine solution, as previously reported. In contrast, exposure to escapable shock resulted in marked individual differences in finickiness that were predicted by prestress body weight. A more sensitive index of finickiness was used in Experiment 2, and a correlation between body weight and finickiness was observed in nonshocked rats. In Experiment 3, measures of quinine reactivity and body weight predicted depressive symptomatology in a nonclinical human sample. Although research in the helplessness paradigm usually focuses on environmental determinants of distress, the paradigm may help identify and explain individual differences in, or intrinsic modulation of, stress and depression.

Anxiolytics and stress-induced behavioural and cardiac responses: a study of diazepam and ipsapirone (TVX Q 7821)

The present study has been designed to investigate the effects of the 5-HT 1A receptor agonist, ipsapirone (TVX Q 7821), a representative of a novel class of anxiolytics, and the classical benzodiazepine anxiolytic, diazepam, on cardiac and behavioural responses in an emotional stress situation. The emotional stress of fear of punishment, induced by training male Wistar rats in an inhibitory avoidance situation, was followed by a bradycardiac response relative to similarly trained, but non-punished, freely moving rats. The behavioural response of stressed rats was immobility in the dark compartment in which an electric footshock (0.6 mA a.c. for 3 s) had been administered as punishment a day earlier. Diazepam administered i.p. in doses of 2.5 mg/kg and 7.5 mg/kg caused a decrease in the interbeat interval (IBI) in shocked and non-shocked rats whereas ipsapirone administered i.p. in doses of 2.5 and 12.5 mg/kg decreased the IBI in shocked rats only. Ipsapirone diminished the duration of immobility in both shocked and non-shocked animals whereas diazepam decreased immobility in shocked rats only. These results suggest a differential effect of the two anxiolytics on the behavioural and cardiac responses to an emotional stress situation. It is suggested that ipsapirone has an anxiolytic-like effect and ‘anti-stress’ action that is clearly reflected in the cardiac physiology in an anxiety-inducing situation.

Fear-motivated behavior induced by prior shock experience is mediated by corticotropin-releasing hormone systems

In earlier studies, we established that in rats, antagonism of corticotropin-releasing hormone (CRH) receptors reduced the occurence of fear-motivated behavior displayed immediately after administration of electric foot shock. Because exposure to stress is reported to have long-term behavioral and physiological effects, we examined whether central administration of the synthetic CRH receptor antagonist, α-helical CRH(9–41), would also alter fear-motivated behavioral and hormonal responses occuring 24 h after exposure to electric foot shock. Adult male rats were placed in a shock box and administered three, 1.0 mA foot shocks. Twenty-four hours later, rats received an intracerebroventricular infusion of 20 μg of α-helical CRH(9–41) or vehicle 20 min before re-exposure to the shock box. Antagonism of CRH receptors produced a significant reduction in fear-motivated freezing. However, stress-induced analgesia and plasma adrenocorticotropin concentrations did not differ significantly from vehicle-treated animals. In order to assess whether the reduction in freezing was due to intrinsic actions of the CRH receptor antagonist, non-shocked rats received central infusions of α-helical CRH(9–41) prior to re-exposure to the test box. Under these conditions, behavior exhibited by antagonist- and vehicle-treated rats did not differ significantly. Results suggest that the reduction in fear-motivated behavior is mediated by antagonism of endogenous CRH receptors and occurs independently of analgesic and hormonal reactions induced by prior exposure to stress.

Stressor predictability and rat brain noradrenaline metabolism

This study examined the effects of stressor predictability on regional rat brain noradrenaline (NA) turnover, by measuring levels of a principal metabolite of NA (3-methoxy-4-hydroxyphenylenhyleneglycol sulfate, MHPG-SO 4). Male Wistar rats were exposed to one of three shock conditions for 19 hr: nonshock, signalled, and unsignalled shocks. Rats in the shock conditions received shock (1.2 mA intensity, 2 sec duration) on a 2.5 min variable time (VT) either preceded by a 12-sec, 10-W light signal (signal-shock interval of 10 sec) or not preceded by this signal. The tail electrodes for these rats were in series, so that the shock received by all rats was of exactly the same number and duration. After 19 hr in a VT-2.5 min shock session, the rats exposed to unsignalled shock (unpredictable group) showed significantly greater increases in MHPG-SO 4 levels in the hypothalamus, amygdala, midbrain, cerebral cortex, thalamus and locus coeruleus, as well as in plasma corticosterone levels. Rats exposed to signalled shock (predictable group) showed significant increases in MHPG-SO 4 levels in the first four of these regions, as compared to the nonshocked rats. Moreover, the unpredictably shocked rats exhibited greater elevations in MHPG-SO 4 levels in the hypothalamus, amygdala, and thalamus, as well as in plasma corticosterone levels, when compared to the predictably shocked rats. These results are consistent with previous reports showing that unsignalled shock induced extensive somatic effects in comparison to signalled shock. The present study suggests that the presence of a signal attenuates the extent of NA release in some brain regions resulting from irregular inescapable shock stress.

Enhanced passive avoidance retention following posttrain serotonergic receptor antagonist administration in middle-aged and aged rats

The experiments examined the ability of posttrain administration of serotonergic receptor antagonists to attenuate the age-related deficits in avoidance retention normally exhibited by middle-aged (12 months) and aged (22 months) rats. Ketanserin (0, 1.0, 10.0 mg/kg) produced a significant dose-dependent increase in test step-through latencies in both age groups. The suppression of responding did not appear to be due to generalized learned aversion as nonshocked rats, injected with the highest dose of ketanserin, did not exhibit similar elevations in test latencies. In order to determine whether the ketanserin-induced effect could be generalized to other serotonergic antagonists, middle-aged and aged animals were injected with a single dose of mianserin (10.0 mg/kg). This antagonist also significantly increased step-through latencies in both age groups, while not affecting the performance of nonshocked animals. The results provide additional evidence for a role of the serotonergic nervous system in memory, and may have important implications in the development of effective treatment strategies for geriatric-related cognitive disorders.

Brief Footshock Analgesia: Long-Lasting Enhancement Induced by Cathinone, an Amphetamine-Like Agent

In view of the analgesic effects produced by cathinone (CATH), an amphetamine-like agent, and of the interaction of amphetamines with stressful environmental stimuli, the present study evaluated in rats the influence of CATH on the nonopioid analgesia induced by a brief electric footshock (FSA; 3 min of continuous 2.5 mA current). The influence of this combination on body temperature was also evaluated. CATH (5 mg/kg, i.p.) alone induced a brief and slight increase in latency during the hot plate test (HPT), but enhanced and prolonged the analgesic effect induced by FS. In addition, the presentation of the environment (shock box with unelectrified grid) where other rats received FS, caused CATH to induce a slow-rising analgesic effect for 180 min. A hyperthermic response paralleling the analgesic effect was observed in shocked and nonshocked rats receiving CATH. After 24 h, rats that had received both CATH and FS on the previous day showed prolonged latencies on the HPT before and after a 1-min presentation of unelectrified grid. These animals also showed an increased analgesic response to the subsequent application of a 15-second FS. At the same time no differences in body temperature were observed between treatment groups. These results suggest that CATH can interact with environmental stimuli to induce an analgesic effect, the time-course of which depends upon the intensity of the stimulus applied.

Effects of stress controllability, immunization, and therapy on the subsequent defeat of colony intruders

Three experiments investigated the influence that various stress-controllability manipulations had on the defensive behaviors of rats when they were subsequently tested as intruders in previously established, aggressive colonies of conspecifics. In Experiment 1, naive subjects that had received a session of 80 shocks in a tube showed an enhanced series of defensive responses and received more bites than did a group of restrained nonshocked rats as colony intruders 24 h later. These two measures were also found to be positively correlated within each group. In Experiment 2, a group that was given 80 yoked inescapable shocks, in contrast to a group that had wheel-turn escape training and a restrained nonshocked control group, displayed more defeat and was bitten more frequently when tested as intruders on the following day. In Experiment 3, 60 trials of wheel-turn escape training were given 4 h prior to (i.e., immunization) or after (i.e., therapy) a session of 60 inescapable tube shocks. During resident-intruder testing 24 h later, both of these groups showed less defeat and received fewer bites than did an inescapably preshocked group but did not differ from a restrained nonshocked control group. These findings clearly indicate that stress controllability alters species-typical defensive responses, and their implications concerning other learned helplessness effects and interpretations are discussed.

Shock controllability and opioid substrates of escape performance and nociception: differential effects of peripherally and centrally acting naltrexone.

Rats exposed to inescapable shock exhibited analgesia and a significant impairment of shock-escape learning in a shuttle box situation 24 hr later. In contrast, rats exposed to escapable shock or to no shock displayed neither effect. Naltrexone (10 mg/kg) significantly reduced the analgesia and completely eliminated the escape deficit in inescapably shocked rats but induced hyperalgesia, coupled with a marked deterioration of escape performance, in escapably shocked and nonshocked rats. The same dose of quaternary naltrexone, which has low ability to cross the blood-brain barrier, had no effect on either the antinociception or the escape deficit produced by inescapable shock, although it also induced escape impairment and hyperalgesia in rats preexposed to escapable shock or to no shock. A second experiment demonstrated that both the escape interference and the antinociceptive consequences of prior inescapable shock could be reduced partially by a much lower dose (1 mg/kg) of naltrexone but 50 times this amount of quaternary naltrexone was still without effect. These results imply that the consequences of exposure to inescapable shock are mediated by activation of central opioid processes whereas naltrexone-induced effects in escapably shocked and nonshocked animals may be peripherally mediated. The relevance of these findings to the possible role of nociception in escape performance is discussed.

Differential changes in noradrenaline turnover in specific regions of rat brain produced by controllable and uncontrollable shocks.

A series of three studies was performed to investigate the effects of the ability to avoid or escape shock (controllability) and the lack of ability to do so (uncontrollability) on noradrenergic neurons in various brain regions of male Wistar rats. The levels of noradrenaline (NA) and its major metabolite, 3-methoxy-4-hydroxyphenylethyleneglycol sulfate (MHPG-SO4), in the hypothalamus, amygdala, thalamus, midbrain, hippocampus, cerebral cortex, pons plus medulla oblongata, and basal ganglia were measured fluorometrically. These studies indicated that after 3 hr and 6 hr in a free operant avoidance-escape stress procedure, the experimental rats able to avoid or escape shock showed greater increases in NA turnover (lower NA levels and higher MHPG-SO4 levels) in specific brain regions (i.e., the hypothalamus, amygdala, and thalamus) than the yoked rats unable to control the same shock. After 21 hr of stress, in contrast to stress of either 3-hr or 6-hr duration, the yoked rats exhibited a more marked enhancement of NA turnover in these brain regions than did experimental rats. Once shock-controlling responses had been acquired and well established by experimental rats, the responses of NA neurons in these rats did not differ markedly from those in the nonshocked control rats. Yoked rats given the same repetitive sessions of uncontrollable shock displayed sustained increases in NA turnover preferentially in the hypothalamus and amygdala, compared with the experimental rats. These results suggest that NA release in specific brain regions in the experimental "coping" rats is increased before the rats have learned the effective coping response. However, once a coping response is firmly established, NA release is reduced.