Modulation of immune status by a conditioned aversive stimulus: evidence for the involvement of endogenous opioids.

Recent research has shown that presentations of an unconditioned aversive stimulus, such as electric shock, can induce alterations of immune function in rats. Furthermore, it has been demonstrated that an innocuous stimulus paired with an unconditioned aversive stimulus can acquire immunomodulatory properties. Research has suggested that endogenous opioid activity is responsible for the alterations of immune function by unconditioned aversive stimulation. The present study evaluated the effect of administration of opiate receptor antagonists, naltrexone and N-methylnaltrexone, on the immunomodulatory effect of a conditioned stimulus (CS) that had been paired with electric footshock. Naltrexone dose-dependently attenuated the CS-induced suppression of the in vitro proliferative response of splenic lymphocytes to concanavalin A, lipopolysaccharide, and a combination of ionomycin and phorbol myristate acetate. Naltrexone also attenuated the CS-induced reduction in natural-killer cell activity. In contrast, the quaternary form of naltrexone, N-methylnaltrexone, did not significantly attenuate the CS-induced immunomodulatory effects. Collectively, these findings indicate that endogenous opioid activity is involved in CS-induced alterations of immune function. Moreover, the lack of effectiveness of N-methylnaltrexone in attenuating the CS-induced immunomodulatory effect suggests that the opioid receptors involved in the effect are located in the central nervous system.     

Effect of a conditioned aversive stimulus on the immune response in three strains of rats

In the present study we investigated the effect of a brief exposure (15 s) to a conditioned aversive stimulus (CS) on the proliferative response of spleen and peripheral blood lymphocytes (PBL) in Lewis, Fischer 344 and Sprague-Dawley rats. Plasma levels of ACTH and corticosterone were also measured. For conditioning, rats were exposed to 10 presentations of a 5 s duration foot-shock (1.6 mA) preceded by a 15 s tone. Seven days later, animals were exposed to the auditory signal without electric shock. Significant differences were found in both the kinetics and the magnitude of altered mitogenic responsiveness of PBL between the different strains of rats. Enhancement of PBL responsiveness to mitogens was observed in Fischer and Sprague-Dawley rats immediately after exposure to the CS. A significant decrease in the response of PBL to mitogens was found in Lewis and Sprague-Dawley rats 10 min after exposure to the CS. The PBL response of Sprague-Dawley and Fischer rats returned to baseline at 30 min, but not in Lewis rats.

Proliferative activity of spleen lymphocytes in response to the CS was suppressed from baseline in all rat strains, but the timing and degree of suppression differed. Fischer rats had the largest percentage of suppression. The earliest suppression of spleen mitogenic function after exposure to the CS was in Fischer rats, while the Lewis rats had the latest onset of suppression, with the Sprague-Dawley rats being intermediate. Plasma levels of ACTH and corticosterone peaked at 10 min in all strains of rats. The magnitude of hormonal elevation differed in the different rat strains, suggesting that corticosterone may not have a variable immunomodulatory role in each strain. These data suggest that a brief psychological stressor results in activation of the HPA axis and is associated with strain-dependent alterations of lymphocyte responsiveness to non-specific mitogens. The short-term exposure to a CS which produces different parameters of lymphocyte functional modulation, provides a useful tool to study the mechanisms of stressor-induced immune alteration.     

Evidence for the involvement of macrophage-derived nitric oxide in the modulation of immune status by a conditioned aversive stimulus

Prior work in our laboratory has demonstrated that exposure to a conditioned aversive stimulus developed through pairings with electric shock results in pronounced alterations of immune status. These conditioned alterations of immune status.include a decrease in natural killer cell activity, decreased production of interleukin-2 and γ-interferon by concanavalin A (ConA)-stimulated splenocytes and a profound suppression of the mitogenic responsiveness of T and B lymphocytes to mitogens. The present study examines the role of macrophage-derived nitric oxide in the conditioned stimulus-induced suppression of lymphocyte proliferation by measuring the level of nitrite accumulation in culture, determining the effect of macrophage depletion, and assessing the effect of N^G-monomethyl-l-arginine (L-NMMA), a specific inhibitor of the l-arginine-dependent nitric-oxide synthesizing pathway, alone and in combination with l- or d-arginine. The results show that the conditioned suppression of the mitogenic responsiveness of splenocytes to ConA is accompanied by a marked increase in nitrie accumulation. Both the depletion of macrophages and the addition of L-NMMA attenuates the conditioned suppression of ConA-stimulated lymphocyte proliferation. Furthermore, the addition of excess, l-arginine, but not d-arginine, counteracts the effect of L-NMMA. The present findings show that the neuroendocrine alterations induced by a conditioned aversive stimulus suppress lymphocyte proliferation through alteration of the production of nitric oxide by macrophages.     

Inactivation of the interpositus nucleus blocks the conditioned response acquired by a somatosensory conditioned stimulus in rabbit eyeblink conditioning.

1. Earlier studies suggest that the memory trace for the conditioned eyeblink reflex is formed and maintained in the interpositus nucleus (IPN) in the deep cerebellar nuclei when either an auditory or visual stimulus is used as a conditioned stimulus (CS). 2. In the present study, the eyeblink reflex of the rabbit was conditioned to a somatosensory CS (an airpuff onto the back). 3. In well-trained animals, the IPN was reversibly inactivated by local cooling and the existence of the learned responses to the CS was then tested. 4. The reversible IPN inactivation blocked the memory trace the somatosensory CS. The finding further supports the view that IPN-mediated memory trace formation is not dependent on the modality of the CS.     

Induction of c-Fos immunoreactivity in the rat forebrain by conditioned and unconditioned aversive stimuli.

The protein product of the c-fos proto-oncogene was immunocytochemically localized in forebrain regions of adult male Lewis rats subjected to a physically aversive footshock stimulus or a Pavlovian-conditioned, non-aversive, auditory stimulus. Animals receiving the conditioned stimulus were first conditioned by repeatedly pairing electric footshock, the unconditioned stimulus (US), with an auditory cue, the conditioned stimulus (CS). These animals were later tested with the CS in the absence of the US, a procedure which, like footshock itself, suppresses immune function. In animals exposed to the conditioned or unconditioned stressor, c-Fos was strongly expressed in cells of the paraventricular nuclei (PVN) of the hypothalamus, some of which contain corticotropin-releasing hormone (CRH), and other forebrain areas directly associated with autonomic function, the ventral lateral septal nuclei (LSV), the medial amygdaloid nuclei (AME), the sensorimotor cortex, the basal ganglia and thalamic nuclei. Control animals exhibited very little or no c-Fos in the above areas. The identified forebrain nuclei can now be targeted for further study aimed at elucidating their role in stress-induced immune alteration.     

Suppression of conditioned avoidance by 8-OH-DPAT in the rat

Summary Rats were trained to perform an aversely motivated discriminative task in a shuttle-box. The conditioned avoidance response was selectively suppressed by 8-OH-DPAT in a dose-dependent manner (25–100 g·kg^–1). There were no statistically significant deficits in discriminative performance. The present results suggest antipsychotic-like properties of 8-OH-DPAT.     

A conditioned stimulus decreases extracellular dopamine in the nucleus accumbens after the development of a learned taste aversion

The conditioned taste aversion (CTA) paradigm and microdialysis were used to determine if extracellular dopamine in the nucleus accumbens is related to the reward value of a stimulus. Intraorally applied saccharin caused a 37% increase in DA in naive rats and a 40% decrease in subjects with a CTA to this taste. These results suggest that accumbens DA is not just a function of arousal but is related to stimulus reward.     

Alleviation of response suppression to conditioned aversive stimuli by lesions of the dorsal noradrenergic bundle

Rats with neurotoxic lesions of the dorsal ascending noradrenergic bundle (DB) were compared with sham-operated (SH) controls on the acquisition, steady state and extinction of response suppression maintained by a classical (conditioned suppression) or an instrumental (discriminated punishment) contingency. DB lesions interfered neither with the acquisition of the reference response of sucrose-rewarded barpressing nor with unconditioned responding to the overhead flashing light subsequently used as a signal of shock. The acquisition of discriminated response suppression was also unaffected by the lesion under both types of contingency. However, once discriminated suppression had stabilized, both the conditioned and the discriminative stimulus used were significantly less effective in maintaining suppression in DB animals than in SH controls provided that low intensity footshock (0.2 mA) was used as the unconditioned stimulus (UCS). Upon increase of UCS intensity (to 0.5 mA) normal suppression was observed in the DB group under both contingencies. Extinction of the classical contingency reinstated the difference between DB and SH performance: DB lesion resulted in significantly faster extinction of fear. In contrast, extinction of the discriminated punishment contingency was unaffected by the lesion, although generalized response suppression dissipated faster in the DB than in the SH animals trained under this condition. Our results offer no support for the reinforcement hypothesis of DB function (normal acquisition of barpressing and of discriminated suppression of barpressing); mixed support (greater initial generalization of suppression in DB animals) and contradiction (more rapid extinction of conditioned suppression in DB animals) for the attentional hypothesis; and weak support (reduced suppression and more rapid extinction of suppression in DB animals, but only within limited experimental parameters) for the anxiety hypothesis of DB function. Hence none of the extant theories of DB function offer a ready explanation of the pattern of results presented here. A simple interpretation which conforms with the sparsity of positive behavioural findings in the literature on DB lesions is that forebrain noradrenaline contributes to the detection and utilization of conditioned stimuli; but that this contribution is critical only for the detection of stimuli with low associative strength.     

Brain stem neurons that fire selectively to a conditioned stimulus for shock.

In the brain stem reticular formation of rats, 10 neurons (1 in each of 10 animals) were found that responded by an increase in activity to a CS paired with foot shock but not to a neutral CS nor to a CS signalling the delivery of water to a thirsty rat. Nine of these neurons showing a conditioned response to shock (CR-S cells) were clustered in the region of nucleus pontis caudalis; the other CR-S cell was localized to nucleus gigantocellularis. The possibility that the increase in firing to the CS for shock was an unconditioned response to the specific stimulus used as this CS was controlled for. No correlation was observed between the activity of CR-S cells and gross movements of the rat. The data suggest that neurons in the pontine reticular region of the brain stem may participate in the reaction of a rat to an aversive situation such as fear.     

A trigeminal conditioned stimulus yields fast acquisition of cerebellum-dependent conditioned eyeblinks

Classical conditioning of the eyeblink response in the rabbit is a form of motor learning whereby the animal learns to respond to an initially irrelevant conditioned stimulus (CS). It is thought that acquired conditioned responses (CRs) are adaptive because they protect the eye in anticipation of potentially harmful events. This protective mechanism is surprisingly inefficient because the acquisition of CRs requires extensive training - a condition that is unlikely to occur in nature. We hypothesized that the rate of conditioning in rabbits could depend on CS modality and that stimulating mystacial vibrissae as the CS could produce CR acquisition faster than the traditional auditory or visual stimulation. We tested this hypothesis by conditioning naïve rabbits in the delay paradigm using a weak airpuff CS (vCS) directed to the ipsilateral mystacial vibrissae. We found that the trigeminal vCS yields significantly faster CR acquisition. We next examined if vCS-evoked CRs are dependent on the intermediate cerebellum in the same fashion as CRs evoked by the traditional auditory CS. We found that vibrissal CRs could be abolished by inactivating the cerebellar interposed nuclei (IN) with muscimol. In addition, injections of picrotoxin in the IN shortened the onset latency of vibrissal CRs. These findings suggest that the tone and vCS-evoked CRs share similar cerebellar dependency.     

Contribution of the ventral tegmental area to cocaine-seeking maintained by a drug-paired conditioned stimulus in rats

Stimuli paired with cocaine can maintain cocaine-seeking as conditioned reinforcers. Although converging evidence implicates a role for the ventral tegmental area (VTA) in the primary reinforcing effects of drugs of abuse, the role of the VTA in mediating associative influences over drug-seeking remains to be determined. The present study therefore investigated the contribution to CS-maintained cocaine-seeking under a second-order schedule of reinforcement of the VTA, and its terminal regions, the nucleus accumbens (NAcc) core and basolateral amygdala (BLA), previously shown to be critical for the acquisition of this behaviour. Inactivation of the VTA or NAcc core by direct infusion of a mixture of the GABA(A) and GABA(B) receptor agonists, baclofen and muscimol, decreased cocaine-seeking when rats were drug-free, while drug-seeking after self-administered cocaine was increased only when they were infused into the NAcc core. Inactivation of the BLA had no effect on drug-seeking. These results are discussed with reference to critical and distinct contributions of the VTA, NAcc core and BLA to drug-seeking maintained by conditioned reinforcers     

Suppression of conditioned fear by administration of CCKB receptor antisense oligodeoxynucleotide into the lateral ventricle.

We investigated the role of CCK in the development of anxiety by determining whether CCKB receptor antisense suppressed intracellular Ca(2+) concentration in vitro or suppressed conditioned fear stress in vivo. First, for the in vitro studies, we used rat pituitary tumor GH3 cells since these cells have CCKB receptors. GH3 cells were stimulated by 10 microM CCK-4; intracellular Ca(2+) concentration was measured. The CCKB receptor antisense at 1 or 10 microM reduced the subsequent response to 10 microM CCK-4 in a time-dependent manner. Second, for the in vivo studies, the CCKB receptor antisense, sense, random sense, or saline was infused at a constant rate for 6 days into rat lateral ventricles via mini-osmotic pumps. Individual rats were then subjected to 30 min of inescapable electric footshock in a chamber with a grid floor. Twenty-four hours later, the rat was again placed in the chamber and observed for 5 min without shocks. This study showed that CCKB receptor antisense significantly suppressed intracellular Ca(2+) concentration in GH3 cells and significantly reduced freezing behavior in rats, indicating that the CCKB receptor plays an important role in anxiety.     

Functional imaging of conditioned aversive emotional responses in antisocial personality disorder

Individuals with antisocial personality disorder (n = 12) and healthy controls (n = 12) were examined for cerebral regional activation involved in the processing of negative affect. A differential aversive classical conditioning paradigm was applied with odors as unconditioned stimuli and faces as conditioned stimuli. Functional magnetic resonance imaging (fMRI) based on echo-planar imaging was used while cerebral activity was studied during habituation, acquisition, and extinction. Individually defined cerebral regions were analyzed. Both groups indicated behavioral conditioning following subjective ratings of emotional valence to conditioned stimuli. Differential effects were found during acquisition in the amygdala and dorsolateral prefrontal cortex. Controls showed signal decreases, patients signal increases. These preliminary results revealed unexpected signal increases in cortical/subcortical areas of patients. The increases may result from an additional effort put in by these individuals to form negative emotional associations, a pattern of processing that may correspond to their characteristic deviant emotional behavior.     

A single application of an aversive stimulus to eliminate chronic cough

There is increasing evidence that punishment can modify certain types of respiratory malfunctioning. In the present case, a single application of a mild electric shock totally suppressed the chronic coughing exhibited by a 14-yr-old boy. Follow-up after 2$\text{1}\text{2}$ yr indicates no further appearance of this behavior.     

Cerebral glucose utilization after aversive conditioning and during conditioned fear in the rat

Regional cerebral glucose utilization (rCMRglu) was studied in rats with and without previous aversive conditioning. Four groups of rats were studied. Two groups of rats were aversely conditioned by placing them in a shock chamber (conditioned stimulus) where they received random footshocks. The two remaining groups were placed in the shock chamber but not conditioned. Regional CMRglu and systemic parameters (heart rate, mean arterial blood pressure (MABP), blood gases and pH, plasma catecholamines, and plasma glucose) were measured in unconditioned and conditioned rats in the presence and in the absence of the conditioned stimulus. The changes in rCMRglu described below appeared to be global and not limited to specific regions. Results are as follows: (1) transferring unconditioned rats to the shock chamber had no significant effect on rCMRglu even though the systemic parameters indicated a stress response. It appears that stress capable of inducing changes in heart rate, MABP, and plasma catecholamines is not necessarily accompanied by increases in cerebral glucose utilization. (2) Conditioned rats not exposed to the shock chamber at the time rCMRglu was measured had decreased rates of rCMRglu compared to rats that were not conditioned. Except for plasma epinephrine, which increased after conditioning, systemic parameters were not affected. (3) Conditioned fear, elicited by transferring conditioned rats to the shock chamber, increased rCMRglu when compared to a control group that was conditioned to footshock using the same paradigm but not exposed to the shock chamber at the time rCMRglu was measured. The systemic parameters indicated a stress response in conditioned rats transferred to the shock chamber.(ABSTRACT TRUNCATED AT 250 WORDS)     

Suppression of conditioned fear by administration of CCKB receptor antagonist PD135158

In order to examine the involvement of CCK in the formation of anxiety, we have investigated whether CCKB receptor antagonist PD135158 suppressed conditioned fear stress. Rats were individually subjected to 30 min of inescapable electric footshock in a chamber with a grid floor. First, the rats were individually subjected to 30 min of footshock. Twenty-four h after the footshock, the rats were again placed in the chamber and observed for 5 min without shocks. PD135158 was administered 30 min before placing the rats in the chamber again. Secondly, PD135158 was administered 30 min before footshock. Thirdly, PD135158 was administered 5 min after footshock. Administration of PD135158 30 min before conditioned fear stress significantly reduced freezing behavior. Administration of PD135158 30 min before footshock also significantly reduced freezing behavior. But, administration of PD135158 5 min after footshock did not significantly reduce freezing behavior. PD135158 blocked not only the acquisition but also the expression of conditioned fear. These results suggest that the CCKB receptor might play an important role in conditioned fear stress and that it might be related to anxiety.     

Suppression of conditioned fear by administration of CRF receptor antagonist CP-154,526

In order to examine the involvement of corticotropin-releasing hormone (CRF) receptor in the formation of anxiety, we investigated whether CRF receptor antagonist CP-154,526 suppressed conditioned fear stress. First, rats were individually subjected to 30 min of footshock. Twenty-four hours after footshock, the rats were again placed in the chamber and observed for 5 min without shock. CP-154,526 was administered 30 min before placing the rats in the chamber again. After that, CP-154,526 was once more administered 30 min before applying footshock. Administration of CP-154,526 30 min both before conditioned fear stress (placing the rats inside the cage but not applying footshock) and before actual footshock significantly reduced freezing behavior. These results show that CP-154,526 blocked both the acquisition and expression of conditioned fear, thus suggesting that the CRF receptor might be related to anxiety.