Decreased maternal behavior and anxiety in ephrin-A5-/- mice.

During development of the nervous system, molecular signals mediating cell-cell interactions play critical roles in the guidance of axonal growth and establishment of synaptic functions. The Eph family of tyrosine kinase receptors and their ephrin ligands has been shown to mediate neuronal interactions in the development of topographic axon projection maps in several brain regions, and the loss of Eph activities result in defects in select axonal pathways. However, effects of deficiencies of the Eph signals on animal behavior have not been well documented. In this study, we showed that inactivation of a ligand of the Eph receptors, ephrin-A5, resulted in defects in maternal behavior and alterations in anxiety. Female ephrin-A5 -/- mice show significant defects in nest building and pup retrieval. In addition, lower levels of anxiety were observed in both male and female null mice. These changes were not due to deficiencies in estradiol, progesterone or corticosterone levels. Our observations suggest that ephrin-A5 plays a key role in the development and/or function of neural pathways mediating mouse maternal care and anxiety.

Methylmercuric chloride induces activation of neuronal stress circuitry and alters exploratory behavior in the mouse.

Methylmercury (MeHg) is a well-known neurotoxicant, responsible for neurological and cognitive alterations. However, there is very little information available on the effects of MeHg administration on activation of murine neuronal pathways involved in the stress response, and whether this is altered as a function of repeated exposure to MeHg. Moreover, interactions between MeHg and other psychogenic and inflammatory stressors have yet to be fully determined. Acute i.p. exposure of male C57BL/6J mice to MeHg (2-8 mg/kg) dose-dependently attenuated exploratory behavior in the open field in the presence and absence of a novel object. In addition, increased numbers of c-Fos immunoreactive cells appeared in response to acute i.p. and i.c.v. MeHg within thalamic (anterior paraventricular nucleus of the thalamus (PVA)/posterior paraventricular nucleus of the thalamus (PV)), hypothalamic (paraventricular nucleus of the hypothalamus (PVN)), central amygdaloid nucleus (CeC), septal and hippocampal (dentate gyrus) nuclei, medial bed nucleus (BSTm) and the locus coeruleus (Lc). The increase in c-Fos positive cells in response to acute i.p. and i.c.v. MeHg did not appear to be influenced further by open field exposure. Repeated administration of MeHg led to an attenuation of most parameters of open field behavior altered by acute MeHg. However, increased c-Fos was significant in the CeC, Dg, supracapsular bed nucleus (BSTs), and Lc. Moreover, open field exposure after repeated treatments resulted in significant c-Fos responses in similar areas. Interestingly, 3 days after the final repeated MeHg dose (2 or 4 mg/kg) c-Fos increases to an immunogenic stressor (LPS) were not affected by MeHg pretreatment. These results demonstrate that systemic exposure to acute and repeated MeHg serves to activate the brain's stress circuitry, and furthermore appears to engage normal neuronal habituation processes.

Potentiation of interleukin-1beta adjuvant effects on the humoral immune response to antigen in adrenalectomized mice.

OBJECTIVES: Activation of the hypothalamic-pituitary-adrenal (HPA) axis is a primary effect of interleukin-1 (IL-1), with elevated glucocorticoids considered a mechanism for negative feedback immunoregulation. However, there is little direct evidence of such a functional relationship between IL-1-mediated immunoregulation and neuroendocrine influences elicited by IL-1. Therefore, the goal of this study was to examine whether the known adjuvant effects of IL-1 are altered in the absence of neuroendocrine feedback due to adrenalectomy. METHODS: Male BALB/c mice subjected to adrenalectomy (ADX) or sham surgery were administered with saline or recombinant human IL-1beta (rhIL-1beta) and at the same time immunized with 100 microg ovalbumin (OVA). In vivo and in vitro measures of antigen-specific IgG antibody production, IL-6 production and spleen cell proliferation were taken 6 and 12 days later. RESULTS: It was demonstrated that administration of rhIL-1beta at a dose that activates the HPA axis resulted in a significant augmentation of serum anti- OVA IgG antibody levels. Interestingly, this augmentation was potentiated in ADX animals. In addition, the in vitro spleen cell memory IgG antibody response to OVA was significantly augmented in rhIL-1beta-treated animals, and again, further potentiated in ADX animals. Interestingly, while hrIL-1beta treatment augmented antigen-stimulated IL-6 production - suggesting an effect of IL-1 on antigen-specific T helper 2 cell memory formation - potentiation was not evident in ADX animals. CONCLUSIONS: These results are consistent with the concept of HPA axis-mediated neuroendocrine feedback on excessive immune responsiveness due to IL-1. Such feedback may prevent disturbances to the self-limiting functions of the immune system, which are important to the prevention of autoimmune diseases, some of which involve elevated IL-1 production.

T-lymphocyte activation increases hypothalamic and amygdaloid expression of CRH mRNA and emotional reactivity to novelty.

Stimulation of T-cells with staphylococcal enterotoxin B (SEB) significantly elevates interleukin-2 (IL-2) and contemporaneous activation of the hypothalamic-pituitary-adrenal (HPA) axis and c-fos in the paraventricular nucleus (PVN) of BALB/cByJ mice. Such neural signaling may promote cognitive and emotional adaptation before or during infectious illness. Because corticotropin-releasing hormone (CRH) is an anxiogenic neuropeptide that may mediate the stressor-like effects of immunological stimuli, we measured neuronal CRH mRNA alterations in mice challenged with SEB. Increased CRH mRNA levels were observed in the PVN and central nucleus of the amygdala (ceA) 4-6 hr after SEB administration. This was associated with plasma ACTH increases, which could be abrogated by the systemic administration of anti-CRH antiserum. Additional experiments did not support a role for IL-2 or prostaglandin synthesis in activating the HPA axis. Behavioral experiments testing for conditioned taste aversion did not confirm that SEB challenge promotes malaise. However, consistent with the notion that central CRH alterations induced by SEB may affect emotionality (e.g., fear), SEB challenge augmented appetitive neophobia in a context-dependent manner, being marked in a novel and stressful environment. It is hypothesized that immunological stimuli generate a cascade of events that solicit integrative neural processes involved in emotional behavior. As such, these data support the contention that affective illness may be influenced by immunological processes and the production of cytokines and are consistent with other evidence demonstrating that autoimmune reactivity is associated with enhanced emotionality.

Differential immune reactivity to stress in BALB/cByJ and C57BL/6J mice: in vivo dependence on macrophages.

Inbred BALB/cByJ and C57BL/6J mice not only differ in their neuroendocrine and behavioral reactivity to stress, but also their ability to mount appropriate immune responses to various pathogens. Because evidence suggests that stress may bias humoral or cell-mediated immune responses in these mouse strains, we assessed the effects of acute (1 h) physical restraint on the humoral immune response to keyhole limpet hemocyanin (KLH). Restraint exposure in close proximity to immunization with KLH enhanced the number of primary antigen-specific IgM and IgG producing splenic B cells in BALB/cByJ mice, but not in C57BL/6J mice. These effects might be determined at the level of macrophage antigen presenting cells, because BALB/cByJ mice immunized with KLH as a particulate antigen (i.e., encapsulated in liposomes) displayed the same stressor enhanced antibody response as they did to free, unencapsulated KLH. In addition, these mice showed enhanced production of the IgG1 subtype of IgG, but not the IgG2a subtype. Conversely, stressed C57BL/6J mice revealed an enhanced IgG2a response, although this was observed only under conditions of immunization with liposome-encapsulated KLH. In a final experiment involving only the BALB/cByJ strain, the depletion of macrophages in the spleen by administration of liposomes containing dichloromethylene biphosphonate (DMDP) 2 days before immunizing the mice with free KLH and restraint exposure, blocked the restraint-induced enhancement of humoral immune responses. These data suggest a possible intermediary role for macrophages in stressor-induced immunomodulation in vivo, which may be a potential point of divergence that explains the differential immune reactivity to KLH of BALB/cByJ and C57BL/6J mice exposed to an acute stressor.

Hypothalamic-pituitary-adrenal activation by the bacterial superantigen staphylococcal enterotoxin B: role of macrophages and T cells.

Staphylococcal enterotoxin B (SEB) is a bacterial superantigen which stimulates T cells bearing the V beta 8 motif on the T-cell receptor. This stimulation is MHC class II dependent, and in vivo results in a rapid and pronounced T-cell cytokine response. Based on previous evidence that SEB stimulates corticosterone production in BALB/c mice, which possess a high percentage of V beta 8+ T cells, we explored the effects of SEB on the hypothalamic-pituitary-adrenal (HPA) axis and identified the peripheral immunologic cellular requirements for these effects. Administration of SEB stimulates corticosterone in a dose-dependent manner, with peak production of corticosterone occurring by 2 h after intraperitoneal challenge with 50 micrograms SEB. Challenge with staphylococcal enterotoxin A, which activates V beta 3+ and V beta 11+ T cells (deleted during ontogenesis in BALB/c mice), did not increase ACTH or corticosterone production. Furthermore, SEB challenge increased plasma ACTH, which accounted for the increased plasma corticosterone, and increased the expression of c-fos in the PVN region of the hypothalamus. In vivo elimination of macrophages did not prevent the corticosterone response to SEB, suggesting that pituitary-adrenal activation does not require macrophages. However, when mice were pretreated with the T-cell immunosuppressant cyclosporin A, the significantly increased ACTH and corticosterone production in response to SEB was dramatically attenuated. These results demonstrate that bacterial superantigens can stimulate the HPA axis, and that functional T cells may play an obligatory role in this effect.

Interleukin 6 modulates interleukin-1-and stress-induced activation of the hypothalamic-pituitary-adrenal axis in male rats.

This study examined the interaction between interleukin 6 (IL-6) and IL-1 and between IL-6 and stress on the activation of the hypothalamic-pituitary-adrenal (HPA) axis. Coadministration of IL-6 (100 ng/rat) with IL-1 (20 or 100 mg/ rat) resulted in synergistic stimulation of the HPA axis, as determined by increased plasma levels of ACTH and corticosterone (CORT) which were greater in rats that received both cytokines than in rats receiving either cytokine alone. Concomitant administration of IL-6 (100 ng/rat) with exposure to a novelty stressor also synergistically stimulated the activation of the HPA axis, as IL-6-treated rats subjected to novelty stress had greater increases in plasma levels of ACTH and CORT than vehicle-treated rats exposed to novelty stress or rats receiving IL-6 alone. However, concomitant administration of IL-6 (100 ng/rat) did not significantly affect restraint stress induced elevation of plasma levels of ACTH and CORT, although IL-6 tended to prolong restraint stress induced elevation of plasma levels of CORT. These findings indicate a modulatory role for IL-6 stimulated HPA axis activity in response to IL-1 or a novelty psychological stressor, but not for restraint stress.

Suppression of lymphocyte mitogenesis in different rat strains exposed to footshock during early diurnal and nocturnal time periods.

The present study examined stressor interactions with genotype and light/dark cycle. Male Brown Norway (BN), Fischer 344 (F344), Lewis (from two different vendors: Lew/CR and Lew/H) and Sprague Dawley (SD) rats were exposed to footshock either in the early light or early dark circadian phase. Immediately after footshock, the spleen and whole blood proliferation to PHA and Con A was assessed. To provide endocrine indices of stress, serum was measured for corticosterone and interleukin-6 (IL-6). All rats showed significant increases in serum corticosterone and IL-6 following footshock either in the light or the dark. Rat strain differences were noted in the IL-6 response, while the corticosterone response was strong for all strains. The criterion for 'suppression' of lymphocyte proliferation was p < .05 (as determined by ANOVA) compared to non-shocked controls. Spleen: with the exception of BN rats, the other strains showed suppressed spleen cell proliferation to PHA and Con A both in the light and the dark. BN rats failed to show suppression of mitogenic activity to PHA when footshock was given in the light. Peripheral blood lymphocytes: suppression in Lew rats from either vendor, and in F344 and BN rats, did not vary with time of day nor with the type of mitogen tested. SD rats did not show suppression to PHA if shocked in the light. These results highlight the generality of stressor-induced mitogenic lymphocyte proliferation during the early diurnal and nocturnal periods of the day.

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.

Stressor-induced alterations of immune function: mechanisms and issues.

This paper reviews the role of catecholamines and the hypothalamic-pituitary-adrenal system in the mediation of stress-induced immune changes in both human and animal subjects. There is evidence to support the importance of these factors in mediating stressor effects on certain immune parameters, but further research is needed to define the specific circumstances in which they are relevant. Therefore, discussion of such issues as sex, genotype, stress history, environment, and stressor characteristics is provided to suggest possible ways to increase our understanding of stressor effects on immune function. Since the imposition of a stressor disrupts physiological homeostasis, understanding the capacity of the immune system to function under such conditions is of prime importance in predicting disease onset and outcome.

Exposure to physical and psychological stressors elevates plasma interleukin 6: relationship to the activation of hypothalamic-pituitary-adrenal axis.

Interleukin 6 (IL-6) is a pleiotropic cytokine produced by the cells of immune and nonimmune origin. Increased production of IL-6 is associated with disturbances of homeostasis, such as trauma, sepsis, or inflammatory diseases. Endotoxemia, tissue injury, or immune inflammatory reactions as well as physical or psychological stress are known to cause increased production of IL-6. We have confirmed this by showing that rats exposed to electric footshock, physical restraint, or a conditioned aversive stimulus have increased levels of plasma IL-6. Interestingly, the kinetics of the increase in plasma IL-6 resembled that of increase in plasma corticosterone. As no detectable endotoxin was found in the plasma samples from stressed and nonstressed rats and there is no evidence of tissue damage and inflammation in situations of restraint or conditioned aversive stimulus, a nonimmune origin of IL-6 is possible. Thus, the releasing of IL-6 into plasma may be under the regulation of neural and endocrine responses to stress. This hypothesis is supported by the decreased production of IL-6 in cultures of splenic cells and peripheral blood mononuclear cells from stressed animals. Furthermore, substantial attenuation of increased plasma IL-6 was achieved by adrenalectomy but not by pretreatment with the beta-receptor antagonist propranolol. The important role of the adrenal gland in the IL-6 response to stress suggests that increased plasma IL-6 may be part of the hormonal responses to stress. As IL-6 induces acute-phase proteins along with glucocorticoids from the adrenal, and regulates the secretion of various hormones from neuroendocrine and endocrine tissues, it is possible that stress-induced increase in plasma IL-6 contributes to the maintenance of homeostasis.

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 micrograms KLH and 14 days later were intradermally injected with 25 micrograms 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.

Inescapable footshock exposure differentially alters antigen- and mitogen-stimulated spleen cell proliferation in rats.

A variety of stressors have been shown to influence specific and non-specific measures of immune function in laboratory animals. One of the most common tools used to evaluate lymphocyte function is the non-specific mitogen proliferation assay. Assessment of this function in the rat spleen has revealed profound suppression following restraint, electric shock, and re-exposure of animals to a fearful context. However, there have been no studies that have compared the effects of stressor exposure on mitogen- and specific antigen-stimulated spleen cell proliferation. Therefore, the present study addressed this issue through experiments in which rats were immunized intraperitoneally with 1 microgram cholera toxin and exposed to acute (one session) or repeated (three consecutive daily sessions) footshock. The results showed that footshock exposure prior to immunization inhibited cholera toxin stimulated spleen cell proliferation 7 days after immunization. Acute or repeated footshock exposure 5-7 days after cholera toxin immunization depressed non-specific spleen cell proliferation, while augmenting the proliferative response to specific antigen. From these observations it can be hypothesized that footshock exposure either differentially regulates lymphocyte activation by clonal and polyclonal signals, and/or naive and memory cells react differently to stressor exposure.

Corticosterone-independent alteration of lymphocyte mitogenic function by amphetamine.

Amphetamine, a neural stimulatory agent with acute effects mimicking those of stress, is shown here to elevate plasma corticosterone levels and suppress spleen and peripheral blood lymphocyte (PBL) mitogenic responses to concanavalin A (Con A) and phytohemagglutinin (PHA) when administered to rats. Pretreatment of the rats with propranolol, a nonselective beta-adrenergic receptor antagonist, totally prevented the amphetamine-induced suppression of lymphocyte mitogenic reactivity to Con A and PHA in the spleen and to PHA in the peripheral blood; however, the PBL mitogenic response to Con A was only partially restored. Although the amphetamine-induced alterations in immune function were prevented by propranolol pretreatment, the elevated plasma corticosterone response was not. This suggests that corticosterone is not modulating the mitogenic activity of splenic lymphocytes or PHA-reactive PBLs. On the other hand, Con A-reactive PBLs may be affected by corticosterone and/or other mechanisms, which may include the catecholamines.

Adjuvant effects of freely ingested cholera toxin on systemic antibody and DTH responses to protein antigen.

Recent evidence suggests that immunological handling of antigen in the gut may be different when animals voluntarily ingest antigen in their drinking fluid. Therefore, we investigated whether the adjuvant effects of CT were evident in mice that voluntarily ingested CT together with keyhole limpet hemocyanin (KLH) in 1.0 ml chocolate milk. Initially, it was found that ingestion of 2 mg KLH for up to four times (one ingestion per week) did not induce serum IgG anti-KLH antibody; however, KLH-specific IgG was detected if KLH was ingested together with 10 micrograms CT. Furthermore, 50 days after only one ingestion of CT+KLH, the serum IgG response to intraperitoneal challenge with 100 micrograms KLH was significantly elevated with respect to the response of mice who previously had ingested either KLH alone, CT alone, or drinking fluid only. This observation was repeated in further experiments showing an enhanced IgG response to 100 micrograms KLH ip given 7 or 14 days after a single ingestion of 2 or 5 mg KLH mixed with different doses (0.5-20 micrograms) CT. The lowest effective CT dose for serum IgG anti-KLH adjuvanticity was found to be 1 microgram; the highest effective dose was 10 micrograms, there being no additive effect with 20 micrograms CT. It was also found that simultaneous ingestion of KLH (2 or 5 mg) with CT (0.5-20 micrograms) followed 7 or 14 days later by 100 micrograms KLH ip, increased the 24 hr footpad DTH response to 50 micrograms KLH administered 43 days after the ip challenge.(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of dexamethasone on behaviorally conditioned immunomodulation and plasma corticosterone.

Utilizing a conditioned taste aversion paradigm we have previously shown that rats re-exposed to a saccharin solution previously paired with cyclophosphamide (CY) demonstrate significantly reduced in vitro mitogen-induced spleen cell proliferation and IgM secretion assessed 24 h after saccharin re-exposure. In this report treatment of similarly conditioned rats with dexamethasone (DEX) either before conditioning or before re-exposure abrogated the conditioned modulation of pokeweed mitogen (PWM)-induced spleen cell proliferation. The finding that DEX pretreatment on the conditioning day was as effective in abrogating the conditioned response as DEX treatment prior to the test day does not support pituitary-adrenal mediation of the conditioned immusuppressive effect following re-exposure of conditioned animals to the CS. There was no significant conditioned immunosuppression observed with respect to PHA- and Con A-induced proliferation and the influence of DEX on these parameters could not be assessed. The effect on PWM-induced IgM production was inconclusive since the reduced IgM response among conditioned animals was of only borderline significance.

Modulation of mitogen-induced spleen cell proliferation and the antibody-forming cell response by beta-endorphin in vivo.

Experiments were conducted which compared the in vivo effects of beta-endorphin (BEP), gamma-endorphin (gamma EP), methionine-enkephalin (Met-ENK), and acetylated BEP(1-27) on the in vitro proliferative response of rat spleen cells to concanavalin A (ConA). In addition, the influence of BEP administration on the primary and secondary antibody-forming cell (AFC) response to the soluble antigen keyhole-limpet hemocyanin (KLH) was examined. Intravenous administration of BEP enhanced the spleen cell proliferative response to ConA assessed 3 hr after a single bolus infusion. Conversely, infusion with AcBEP(1-27) suppressed the proliferative response, whereas no effects of intravenous gamma EP or Met-ENK treatment were observed. The enhancing effect of BEP administration was not detectable 24 hr after a single infusion, but could be maintained over a 44 hr period by multiple infusions. The primary AFC response to KLH was suppressed by a dose of 1 nmole BEP only. On the other hand, the secondary IgG AFC response to KLH was enhanced by 10 pmoles BEP, while the IgM and IgA AFC responses remained unaltered by BEP treatment. The anamnestic in vitro proliferative response of spleen cells cultured with KLH was not altered if BEP was administered at the time of secondary KLH immunization. These results extend previous observations of BEP-induced modulation of in vitro immune function by demonstrating that opioid and nonopioid forms of BEP administered in vivo alter the capacity of spleen cells to proliferate and develop antibody responses to antigen.

Behaviorally conditioned suppression of mitogen-induced proliferation and immunoglobulin production: effect of time span between conditioning and reexposure to the conditioning stimulus.

Rats were subjected to taste aversion conditioning using the immunosuppressive drug cyclophosphamide (CY) as the unconditioned stimulus (UCS) paired with saccharin, the conditioned stimulus (CS), and were reexposed to the CS at 2, 5, or 10 days after a single conditioning trial. Twenty-four hours after reexposure the rats were sacrificed and spleen cells assayed for mitogen-induced proliferation and immunoglobulin production. A robust conditioned taste aversion (CTA) was observed irrespective of the day of CS reexposure. However, only conditioned rats reexposed to the CS 2 days after training displayed a conditioned reduction in proliferative responses to PHA and PWM. These rats also exhibited a reduction in the synthesis of IgM, but not IgG or IgA, by spleen cells cultured with PWM. These effects were not observed in conditioned rats reexposed 5 or 10 days after conditioning. In another experiment, rats were subjected to a backward conditioning (UCS prior to CS) training trial, tested 2 days later for the presence of CTA, and sacrificed 24 h later for assessment of immune function as described above. The results of this experiment demonstrated that rats do not develop an aversion to saccharin when it is first presented 4 h after CY, and no alterations in spleen cell proliferation and immunoglobulin production were noted. The data show that the CTA response established by explicit association between CY and saccharin depresses in vitro spleen cell proliferation and IgM production only when elicited shortly after the conditioning trial.

In vivo effects of beta-endorphin on lymphocyte proliferation and interleukin 2 production.

Experiments were undertaken in rats to investigate the effects of in vivo infusion of beta-endorphin (BEP) on subsequent Con A-induced proliferation and interleukin 2 (IL-2) production by spleen cells in vitro. BEP administration induced a dose-dependent enhancement of the proliferative response to Con A. Infusion of the opiate antagonist naloxone (NAL) inhibited the Con A response and infusion of NAL prior to BEP resulted in even further inhibition. None of these treatments resulted in detectable alterations in IL-2 production after 48 h in culture. To demonstrate a direct interaction between BEP and lymphocytes, spleen cells were incubated in vitro with varying concentrations of BEP and/or NAL. Enhanced Con A-induced proliferation was observed following incubation with BEP in the range 10(-12) to 10(-9) M (levels comparable to the effective in vivo doses) and this effect was abrogated by NAL pretreatment (10(-6) M). These data indicate a role for BEP in enhancing lymphocyte reactivity which is to some extent dependent on opiate receptors on the cell surface. This report extends the evidence obtained from in vitro experiments implicating endogenous opioids in modulation of host immunity by demonstrating that these effects can be obtained in vivo.

Synergism of a compound unconditioned stimulus in taste aversion conditioning.

Anti-lymphocyte serum (ALS) and lithium chloride have both previously been used successfully as unconditioned stimuli in taste aversion conditioning paradigms in rats. This report substantiates those findings but shows that when the two stimuli are given as a compound unconditioned stimulus in association with saccharin flavoured drinking solution, the conditioned taste aversion response following a second exposure to saccharin alone is more profound than that following conditioning with either ALS or LiCl alone. These results demonstrate synergism between the two stimuli when given together.