Identification of specific pathways of communication between the CNS and NK cell system

Life Sciences, Vol. 53, pp. 527-540 Printed in the USA

Pergamon Press

MINIREVIEW IDENTIFICATION OF SPECIFIC PATHWAYS OF COMMUNICATION BETWEEN THE CNS AND NK CI:~.|J+ SYSTEM Raymond Hiramoto, Vithal Ghanta, Brent Solvason, Joan Lorden, Chi-Mei Hsueh, Carolyn Rogers, Sossiena Demissie, Nancy Hiramoto Departments of Microbiology, Biology and Psychology, University of Alabama at Birmingham, University Station, Birmingham, Alabama 35294, USA (Received in final form June 2, 1993) Summarv The specific signals and pathways utilized by the natural killer (NK) cell system and the central nervous system (CNS) that results in the conditioned response (CR) is not clearly understood. Single trial conditioning of the NK cell activity provides us with a model to probe the mechanisms of communication between two major systems (Immune and CNS) which are involved in the health and disease of the individual. The studies show that the IFN-~ molecules possess the properties attributed to the unconditioned stimulus (US). IFN-I3 can penetrate the CNS and evoke the elevation of NK cell activity in the spleen. This unconditioned response (UR) can be linked to a specific conditioned stimulus (CS). Specific odors such as camphor provide a neural pathway for the CS to associate with the US. Evidence is presented that in conditioning there are two locations where memory develops. The CS/US association is made centrally and its memory is stored at a central location, but the memory for the specificity of the odor is presumably stored in the olfactory bulbs. The CS recalls the CR by triggering the olfactory neural pathway which, in turn, signals the hypothalamic-pituitary axis to release mediators that modulate the activity of NK cells in the spleen. These results imply that through conditioning one has direct input into the regulatory hypothalamus that controls the internal environment of the organism and the health and disease of the individual. Consequently, it is not inconceivable that through this approach we might be able to alter the course of a disease process. The evidence for the immune svstem-CNS interactions It has long been known that the immune, the nervous, and the endocrine systems are functionally interconnected, but the detailed mechanisms of this interconnection in health and disease have not been elucidated. In order to understand how "messages" between the immune system (IS) and CNS are received and acted upon, it is essential that models be developed which can identify specific pathways of communication between these systems. We have used Pavlovian conditioning to probe the interactions and pathways of communication between the CNS and IS. The conditioning of the natural killer cell response allows us to test if interferon 13(IFN-B) generated by the IS acts as a signal to the CNS and whether such signals are able to induce lasting effects or associative memories in the CNS. The conditioning model provides evidence of functional, bidirectional communication between the IS and CNS. Several other lines of investigation support the idea that such communication is both feasible and probable. Since lymphoid cells have receptors for products of the CNS it is clear that neurotransmitters, hormones and neuropeptides can affect the cells of the Correspondence: R.N. Hiramoto, Ph.D., Department of Microbiology, University of Alabama at Birmingham, University Station, Birmingham, AL 35294-0007 0024-3205/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd All rights reserved.

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immune system. A variety of IS generated signals have also been known to affect the CNS. Besedovsky et al., (1) have shown the existence of a glucocorticoid increasing factor (GIF) of lymphoid cell origin and its integrated action at the level of the pituitary-hypothalamic axis. Factors from lipopolysacchadde (LPS)-treated peritoneal exudate macrophages that inhibit ACTHinduced steroidogenesis (2) or factors from ConA supernatants that have the property of decreasing the activity of brain noradrenergic neurons (3) have been reported. The cytokine interleukin-1 (IL-1) which is also produced by glial cells (4) can produce fever and can increase slow wave sleep when injected into the lateral ventricle of the brain (5). These results indicate that IL-1 might directly or indirectly alter brain functions. Substances such as histamine and serotonin, classic brain neurotransmitters, are released by immune responses. They could act as messengers, provided they can penetrate the brain. Smith & Blalock (6) have shown that in response to Newcastles' disease infection, lymphoid ceils can produce both ACTH and endorphin-like substances. These agents could, therefore, act as immune messengers and affect the feedback mechanisms that control these polypeptide hormone levels. There is also evidence that thymosin is a physiologic modulator of the pituitary-adrenal cortical axis. Removal of the thymus gland, a major source of thymosin, decreases the circulating levels of ACTH, I~-endorphin and cortisol. Though injection of thymosin ctl into rats intracerebrally results in significant elevation of corticosterone, it had no effect upon serum LH levels. However, thymosin IM is found to stimulate LH but not corticosterone. It appears that different thymosin peptides might exert differential effects upon the neuroendocrine circuits (7). A third example of IS-CNS interaction is the regulation of interleukin production by a feedback loop involving the CNS. Interleukin- 1 is produced by stimulated macrophages involved in activation of lymphocytes and stimulation of lymphokine production (8). IL-1 causes hepatocytes to produce acute phase proteins and alters prostaglandin production (9). In human and animals the production and action of IL-1, and mediators of inflammation are inhibited by glucocorticoids (10, 11). A relationship between adrenocortical and immune cell function is evidenced by the fact that animals producing an immune response to various antigens show at the same time, increased glucocorticoid blood levels in proportion to the magnitude of the immune response (12). Factors derived from activated lymphoid cells can also produce this increase in glucocorticoids (13). Here it appears that IL-1 produced in response to the foreign antigen has signaled the CNS to release neuroendocrine regulatory factors ACTH and corticosterone (14). What is important is that what was initiated by the immune system through its cytokines is detected by the CNS. The hypothalarnic pituitary-adrenal axis shuts down the immune response by increasing the production of anti-inflammatory glueocorticoids. These, in turn, block the production and action of lymphokines such as IL-2 and IFN-'t, and the production of prostaglandins (15). Glucocorticoids can also block IL-1 production by macrophages and inhibit class II MHC expression which is necessary for antigen presentation (11). Therefore, the increased levels of glucocorticoids can interfere with several critical steps in the immune response. Thus the evidence for signals that originate within the immune compartment that could result in modulation of neurologic events is quite extensive. These studies support the concept that the CNS and the immune system are in constant communication and communicate in part through mediators which are released into the circulation. The lwoblem Conditioning of the immune system provides specific proof that the CNS can regulate immune functions. Despite the fact that numerous examples of conditioned alteration of immunologic activities have been described and documented for nearly 100 yrs (16), the underlying mechanisms resulting in conditioned elevation or suppression of the immune activity are largely unknown and conjectural. Recently it was suggested by Grossman et al., (17) that "neural/endocrine signals act on the immune system in conjunction with immunological stimuli in a way that leads to "storage" of the association (memory) of those two kinds of stimuli in the immune system rather than in the brain." Without a complete understanding of how the central nervous system-immune system interaction occurs, it has been difficult to put together a coherent, orderly, and logical picture of how this is taking place. Consequently, the study of conditioning of immune activities has remained largely at a phenomenological level.

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Pumose of Conditionin~ the NK Cell Activity In an effort to shed light on mechanisms and pathways involved in conditioning of immune activity, we developed a paradigm for conditioning the NK cell activity. Our studies have been aimed at attempting to understand three aspects of the conditioning model: 1) during the CS/US association how is the NK cell system (or IS) signaling the CNS? 2) where are the essential circuits for the association? and 3) when the conditioned animal is reexposed to the CS, how is the CNS "signaling" to the NK cell system to elevate the NK cell activity? During the CS/US association how is the NK cell svstem communicating with the CNS? In our general conditioning paradigm, we postulated that when the poly I:C is given after the CS (camphor odor) exposure, the relevant information conferred by the US (poly I:C) must reach the CNS. Because of its position in the autonomic and neuroendocrine systems, the hypothalamus (HT) is likely to be a critical CNS site for the receipt of IS information. The hypothalamus in turn must activate neuronal and/or neuroendocrine pathways that signal back to the NK cell system. This signaling to and from the CNS is an unconditional and reflexive property essential to the US. In other words, when the appropriate US information reaches the HT, it immediately returns a signal back to the NK cell system. This complete loop of communication is shown in Fig. 1A. Through an as yet unknown associative process, the signal which is initiated by the US becomes linked to the CS within the CNS (Fig. 1B). Subsequent exposure of the conditioned animal to the CS (Fig. 1C) now allows the CNS to communicate with the NK cell system, through a pathway that was previously utilized by the US. Our studies show that poly I:C, a double stranded synthetic RNA, can serve as an effective US in conditioning NK cell activity (18). Poly I:C mimics a viral infection and induces the synthesis of IFN-ct and -8 and raises NK cell activity in the spleen. Injection of poly I:C caused IFN-ct and -13to peak in the circulation at 6 hr, followed by NK cell activity which peaked at 24 hr (19). IFN-~ but not IFN-ct can replace poly I:C as the US; this implies that the activity of the US resides in IFN-~. Based on our initial studies, we surmised that IFN-13 must reach the CNS for CS/US association to take place. Our subsequent studies have shown that the cistema magna (CM) route can be used to transfer signals directly into the CNS. IFN-13 injected at 102 IU into the CM produced a statistically significant increase in the NK cell activity in the spleen. This indicates that IFN-~ can act within the CNS to trigger an up-regulatory signal from the CNS to the NK cell system. The dose used (102 IU of IFN-13) is too low to produce an NK cell response in the spleen when injected peripherally (sc or iv), but it can be used as the US in a conditioning experiment if injected directly into the CM in mice preexposed to camphor (CS). Through conditioning, we showed that the US message can be placed directly into the brain and the information resulting from the CS/US association was stored in memory for subsequent recall. Other studies from our laboratory also support the view that IFN-~ must penetrate the CNS for CS/US linkage to take place. If rabbit anti-IFN (100 neutralizing units) was injected into the CM 24 hr before CS/US linkage was made, we found that learning did not take place and the conditioned response (CR) was not made when the animals were reexposed to the CS. Injection of normal rabbit serum into the CM did not prevent the CS/US association and subsequent recall of the CR was evoked (20). Therefore, the IFN message produced by the injection of poly I:C can be effectively neutralized centrally by specific antiserum to IFN but not by normal rabbit serum injected into the CM. Interestingly, introducing neutralizing antibody of a known specificity into the CNS provides us for the first time with a means to identify specific mediators to the CNS which are responsible for the conditioning of a specific behavioral activity. The Irue US is what the CNS perceives We have treated the administration of poly I:C as a US. This is not altogether accurate. In taste aversion learning, animals learn to avoid saccharin on the basis of their association with drug injections that cause some discriminable internal state usually assumed to be nausea. In taste aversion learning, it is not the drug injection per se that causes the unconditioned stimulus. It is

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A. Propertyof the US

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B. CS/USAssociation

CNS .

Loop~

IFN-B

C N S , ~ - I ) CS

Loop~

US

e) us . ~

C. Recallof the CR Half Loop/ Is4 Fig. 1 In (A), the property of the US is depicted, poly I:C injection induces IFN-a,-~l which signals the CNS (hypothalamus) which in turn responds by signaling back to produce the unconditioned response in the IS and thus establishing the communication loop. In (B), CS/US conditioning is effected by 1) exposing the animal to the CS (camphor odor), followed by 2) injection of the US (poly I:C) ip. Here the CS signal which is presented f'mst is stored in memory. The US signal which follows establishes the communication loop. The CS "ties in" to the US loop somewhere within the CNS, presumably in the hypothalamus. In (C), the recall of the conditioned response (CR) is initiated by the CS which activates the "memory site" and triggers the "half loop" pathway of communication which was initially established by the US (A). the effect of the nausea perceived centrally. Because IFN is produced in response to poly I:C injection and because we have shown it is effective ff injected into the CNS, it is possible that this molecule serves as the US. Whether the CS/US learning takes place or not would also depend on the signals from the US reaching the I-IT. In this regard, the question of using higher or lower concentrations of the US (poly I:C or antigens) might be critical. We can condition NK cell response with different doses of poly I:C (Table 1). Suboptimal doses of poly I:C do not lead to CS/US linkage. The suboptimal dose of poly I:C can raise the NK cell activity in the peripbery, but apparently does not induce sufficient amounts of IFN in the plasma to reach the CNS. While NK cells are elevated in the spleen IFN mRNA appears not to be made in sufficient quantities by spleen cells to be detectable in mice given suboptimal doses of poly I:C. Injection of 20~tg poly I:C induces mRNA for IFN-~ and -a in the spleen (unpublished observations) and IFN levels (10,000 - 20,000 units/ml) are readily detectable in the plasma (19). The amount of IFN-~ produced in the

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circulation is important as this mediator may penetrate the CNS during the CS/US learning. Conditioning can be achieved with 10,000 IU of IFN-13 but not with 1000 IU injected iv. However, if the US signal is introduced directly into the CNS, IFN-13 at 100 units injected into the CM can condition the mice. Anti-IFN-I~ (100 neutralizing units) injected into the CM 24 hr before conditioning can block conditioning with high doses of peripherally administered poly I:C (20gg). Because the antiserum is introduced 24 hr before the CS/US association, it is likely that <100 neutralizing units are present 24 hr later. Therefore, the effective dose of US administered centrally which could allow conditioning to take place would probably be < 100 IU of IFN-13. TABLE 1 The effect of dose of the US needed for conditioning Poly I:C (gg/mouse) a

Conditioned response observed

40.0

+

36.0

+

20.0

+

10.0

___

5.0 b 2.5 b 1.0b apoly I:C was given intraperitoneally. bSuboptimal doses. + = conditioned association was observed. - = no conditioned association. Our studies show that the events taking place in the periphery (spleen, lymph nodes, etc.) from injection of poly I:C are independent of what is being detected in the CNS (21). When poly I:C (20~tg) is given, the IFN-13 produced penetrates the blood brain barrier and signals a rise in the NK cell response. This complete loop of communication initiated by IFN-13 is a property of the US (Fig. 1A). We have dissociated what is detected by the CNS from the effects produced peripherally. The unconditioned stimulus (US) must be the stimulus detected by the brain and not the exogenous substance injected to activate the NK cells in the lymphoid organs. In light of this argument, it becomes apparent that the US mediators must be produced in sufficient amounts to activate the CNS and become a part of the circuit, or no linkage with the CS will occur. An important point we wish to emphasize is that IFN-13 is involved as a signal to the neural circuitry and is not a memory molecule. The memory is a property of the circuit underlying the association of the CS and US. The CS/US association that has been integrated within the memory circuit can be altered by changes in environmental conditions. One cannot rule out completely the role of the experirnental environment. Uncontrolled variables introduced during the experiment such as sounds, odors of other animals, pheromones, lighting, handling, and strange personnel can not only alter the direction of the conditioned response but negate it altogether (22). Where is the memory of the CS/US conversation stored? The learned NK cell response is specific in that animals conditioned with camphor or

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citronella odor produce the CR only in response to the odor to which they were conditioned (23). This stimulus selectivity suggests that the association is stored centrally and not in the immune system as proposed by Grossman et al. (17). Within the CNS, there are several candidate storage sites. It has been shown by others that memory for pheromone odor specificity resides in a neural circuit in the olfactory pathway close to the receptors (24). It seems likely that the memory for the odor used as a CS in NK cell conditioning would also be stored in the sensory pathway. We have shown that conditioning occurs over interstimulus intervals (ISis) as long as 1-2 days (25). This implies that a memory of the odor CS is retained at least until the US is administered 1-2 days later. Since conditioning and recall also take place under anesthesia (26) and over long ISis the olfactory pathway appears to be a plausible site for retention of CS information. NK cell activity has been conditioned to tastes (saccharin-LiC1) and light cues (27) as well as odor cues. Memory for cues other than odors may be located in different circuitry near their organs of reception. Although the memory of the CS may be stored in the sensory pathway, it also seems probable that information about CSs converges along a pathway to or in the hypothalamus close to the efferent mechanism. Memory for the CS/US association may be stored at such a site. During conditioning, multiple responses may be associated with the CS. We have used the same 3-day conditioning paradigm to condition both fever and NK cell activity (28). This means that the CS must have access to multiple response pathways and suggests that the CS/US link is made central to the primary sensory pathway. In the case of NK cell activity, a conditioned elevation was observed whether the CS/US presentations were overlapped or were separated by ISis of 1 - 2 days. With the same conditioning paradigm, we found that when the CS and US presentations overlap, conditioned core body temperature is elevated (fever), but when the ISI is increased to 1 day, the conditioned core body temperature is decreased (unpublished observations). Since the memory for the CS is introduced one day before exposure to the US in both NK and temperature conditioning, the CNS processes the US (IFN-~) differently for NK cell activity and fever production. It is not clear why these divergent responses occug, however, these results imply differences in efferent mechanisms and circuits. NK cell activity appears to be activated by endocrine pathways (29-31). Thermoregulation is effected by multiple tissues and systems under autonomic control, including sweat glands, brown adipose tissue, the cardiovascular system and the respiratory system (32). For example, sympathetic and parasympathetic innervadon of skin capillaries are involved in controlling heat gain and loss (33). Thermogenesis in brown adipose tissue is stimulated by catecholamine activation of adenylate cyclase through sympathetically innervated ~adrenergic receptors (34). Storage of the CS/US link at a central site such as the hypothalamus would give the the CS access to both endocrine and autonomic effector mechanisms. Ease of retention of memory mieh~ glcpend on the nature of the interacting systems It is noteworthy that conditioning the NK response to an olfactory cue can be accomplished with relative ease. A one trial conditioning paradigm has been adequate. The reasons for this are unknown. It may be a property of the olfactory system, the subcortical circuitry involved in the association, or the similarities between the olfactory and immune systems. The ease of association seen in conditioning of immune responses is similar to that seen in other responses that have immediate importance for the survival of the organism such as taste aversion learning and imprinting. The rapidity with which such associations are formed is clearly adaptive. The olfactory system and the immune system have evolved a number of shared characteristics that contribute to their roles in species and individual survival. Both systems possess specificity, memory, self-recognition, and clonal expansion (olfactory receptors can multiply). Another interesting attribute of conditioned immunobiologic responses to olfactory cues is the fact that long delays between CS and US exposure are tolerated. Delayed associative learning has been demonstrated with interstimulus intervals of 2 days. Although chemical stimuli may be relatively enduring, it is unlikely that the stimuli employed in the conditioning experiments are physically present after 2 days (25). The fact that conditioned responses can still be obtained may

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be due to the absence of other interfering novel stimuli in the laboratory environment or may represent an important characteristic of olfactory memory. When the conditioned animal is reexDosed to the CS. how does the CNS signal the NK cell system and cause/tn elevation of the NK cell activity? Here we postulate that during the CS/US association, the IS communicates with the CNS by specific mediators for the different USs employed (IFN-~ for NK, perhaps IFN-'t or IL-2 for cytotoxic T-lymphocytes (CTL), IL- 1 for LPS, etc) but the CNS "talks back" to the IS by using essentially the same set of common neuroendocrines released by the pituitary gland (ACTH, endorphins, prolactin, TSH, GH, LH, FSH, MSH, etc). Fig. 2 provides the hypothetical pathway of CS/US learning, memory, and recall of the NK cell activity. A brief description of where drugs are presumed to block the pathway is shown. In order to understand possible pathways and mechanisms that might be involved in learning and recall of the conditioned response, we have used different drugs to either prevent the CS/US association or block the recall of the learned response. Table 2 summarizes the drugs used and their effects on acquisition or recall. Additional information is provided on the mechanisms of drug action to identify whether certain neuronal structures, neuronal pathways, or receptors are or are not involved in the response. Studies were initiated to determine if opioid receptors, central catecholamines, peripheral sympathetic system, or endocrine pathways influence the triggering of the CR. Our studies with naltrexone (NTX) and quaternary NTX (QNTX) show that NTX which blocks central and peripheral opioid receptors when injected prior to CS/US conditioning did not interfere with CS/US learning, indicating that opioid pathways are not involved in the acquisition of the conditioned response. However, in animals which have been conditioned, naltrexone injected prior to exposure to the CS, blocked the CR. QNTX, which does not penetrate the CNS, did not block the CR. This implies that central opioid receptors (or pathways) are involved in the recall of the CR, i.e. the effector pathway from the CNS to the IS to trigger the NK cell activity requires activation of central opioid receptors. The fact that QNTX did not block the CR implies that opioids may not be the only signal mediating the enhancement of NK cell activity in the spleen (29). These studies opened up two areas for consideration: 1) the possibility that both the aminoterminal (opioid end) and the carboxyl-terminal end of endorphin and ACTH (35) might be involved in signaling the activation of the NK cell activity. If both terminals were involved, it would explain why QNTX block of the opioid receptors on NK cell would not prevent their ability to respond. This possibility was supported by our studies showing glycyl-glutamine, the carboxyl terminal end of 13-endorphin could also stimulate NK cell activity. These studies imply that the endocrine pathways might be involved in the CR (31), and 2) the second important possibility was that the opioid pathway might be stimulated directly with opioids injected into the CNS to activate the effector arm. This meant that by injection of opioids (met-enkephalin) into the cistera magna, we could begin to study the effector pathway leading from within the CNS out to the IS without the need for conditioning the animals. We have tested this possibility. The injection of metenkephalin into the CM of mice whose NK cell activity is preactivated show signals emanating from the CNS which can augment the NK cell activity in the spleen (36). However, CM injection of met-enkephalin did not mimic every aspect of the CR because both NTX and QNTX were able to block the response. The roles of the adrenergic innervation of the spleen and central catecholamine pathway were tested. We have shown that depletion of central and peripheral catecholamine by treatment of animals prior to CS/US learning with amantadine or diethyldithiocarbamate or with reserpine after CSKIS association leads to blocking of the CR. Depletion of peripheral catecholamines alone with systemic 6-hydroxydopamine (6-OHDA), throughout conditioning and recall did not prevent learning or recall of the NK cell response. Therefore central catecholamines are required for learning and recall of the CR, but peripheral catecholamines are not essential to the bidirectional communication between the CNS and IS (30). We showed met-enkephalin, glycyl-glutamine, and naltrexone, when injected into mice, had no substantial effect on augmenting the resting NK cell activity. However, met-enkephalin (amino terminal end) and glycyl-glutamine (carboxyl-terminal

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PATHWAYS OF CS/US LEARNING, MEMORY, AND RECALLOF NK CELL ACTIVITY

CS { MOB

(I) { AOB

US (IFN-B) (2) I~ (3) NALTREXONE 1 CENTRAL ~-- HYPOTHALAMUS COMPARTMENT (5b) PITUITARY (4) PERIPHERAL ACTH (6) ENDORPHIN COMPARTMENT +

CsR

ADRENAL

Fig. 2 Pathways are identified by number. During CS/US association (1 & 2), the CS (camphor odor) is detected by the main olfactory bulb (MOB) and/or accessory olfactory bulb (AOB). IFN-[~ which serves as the US is perceived directly by the CNS. The CS/US association (1 & 2) is not blocked by naltrexone or dexamethasone. Therefore, opioids and the HPA axis appear not be directly involved in the CS/US learning process. Depletion of catecholamine (CA) prevented both the CS/US learning and recall of the CR. The general depletion (3 & 4) caused by reserpine, amantadine, diethyldithiocarbamate can be occuring both centrally and peripherally. Chemical sympathectomy with 6-OHDA of peripheral (compartment) ~-adrenergic nerves (4) did not prevent CS/US learning or recall. Therefore information to and from the CNS during CS/US learning or CS recall does not appear to utilize this neuronal pathway and supports the view that depletion of central (3) CA levels are involved in both CS/US learning and CS recall. The recall of the conditioned response is interrupted by naltrexone (5b) and dexamethasone (5a). Therefore it is likely that CS (recall) triggers an opioid mediated pathway (5b) in the CNS. This pathway leading to enhancement of NK cell activity in the spleen can be directly activated by injection of met-enkephalin (opioid) into the cisterna magna. Dexamethasone (glucocorticoid agonist) (5a) on the other hand inhibits the HPA axis by feedback inhibition, consequently, the release of ACTH and endorphins are prevented from being secreted by the pituitary gland. This supports the view that in recall of the conditioned response the HPA axis is utilized. The fact that the inhibition produced by dexamethasone (5a) does not interfere with the enhancement of NK cell activity which can be produced by a direct injection of metenkephalin into the CM indicates that the block by dexamethasone (5a) occurs ahead of the block by naltrexone (Sb). Quaternary naltrexone (QNTX) does not penetrate the blood brain barrier and blocks opioid receptors in the periphery only (4). QNTX did not prevent the recall of the conditioned response. Therefore, while opioid receptors on NK cells might be involved in activating the NK cell response, opioids are not the only signals that can enhance NK cell activity. It has been shown that the carboxyl terminal end of endorphin and ACTH can also activate NK cell activity. Because multiple signals can activate NK cell activity, QNTX would not be able to completely establish a block of NK cell activation.

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TABLE 2 Drugs disrupting memory site or neuronal track

Drugs

Dose a

Effect on: CS/US memory

Effect on: CS recall track

Mechanisms of action

Reserpine

2.5mg/kg

+

+

Depletes catecholamines (CA)

Naltrexone

10mg/kg

-

+

Block central and peripheral opioid receptors

Dexamethasone

1001~g/kg

-

+

Feedback inhibitor of the HPA axis

6-OHDA

150mg/kg

Quaternary Naltrexone

10mg/kg

Indomethacin

10mg/kg

Blocks PGE 2 synthesis and fever production

Ketamine

85mg/kg

Blocks the inhibition of mitral cells by granular cells (ketamine is an NMDA receptor blocker)

Lithium Chloride

125mg/kg

Sodium Carbonate Rompun (Xylazine)

0.2ml (1% soln) 13mg/kg

Chemical sympathectomy of peripheral adrenergic system -

Peripheral opioid receptor blocker

-

Blocks the enzymatic hydrolysis of inositol monophosphate

+

Dissociates H+-ATPase into subunits

-

-

tx2-adrenoreceptor agonist

aAll compounds were given intraperitoneaUy. Dosages used were obtained from the literature. no effect, + response is disrupted. *Can change the perception within the CNS of the incoming CSKIS signals. -

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end) of 13-endorphin appear to enhance the NK cell response in animals whose NK cell activity has been preactivated with l~tg poly I:C. In contrast, naltrexone has no effect. In vitro studies with those drugs at various concentrations (10.4 to t0-SM) on preactivated NK cells were unable to enhance NK cell activity. This suggests that in animals making a conditioned response, endorphin and AC'I~ (35) released by the pituitary might not be directly involved in the activation of NK cells, but that an accessory cell might be involved in triggering the NK cell activity through production of possibly IFN-ct and -13or other mediators (31). In some of our initial studies we used saccharin and lithium chloride (Sac-LiC1) as a combined CS (18,37). We found that treatment with Sac-LiCI produced better conditioning than with saccharin alone as the CS. Therefore, LiCI (125mg/kg) did not interfere with CS/US learning or recall of the conditioned response. The phosphoinositide (PI) system is an important second messenger for neurotransmitters. The blockade by lithium of the enzymatic hydrolysis of inositol monophosphate (38) can alter PI signal transduction by limiting the regeneration of inositol, an essential precursor of PI synthesis, and block the PI mediated response to carbachol in hippocampal slices (39). Lithium has been shown to impair muscarinic cholinergic responses in the hippocampus. The cholinergic blockade by lithium appears to involve the PI cycle in that it elevates concentrations of inositol-l-phosphate in the presence of muscarinic agonists (40,41). Lithium-treatment of rats in vivo reduces muscarinic stimulation of PI turn over in brain slices which have been measured biochemically (42). Although these changes can take place in the CNS under the influence of lithium, the dose of lithium chloride used in our studies of 125mg/kg may be too low to affect CNS functions directly. Nevertheless, changes can be perceived by the CNS due to the effect of lithium (elicitor of gastrointestinal upset) on the gastrointestinal system. Role of the hvoothalamus in conditionine The hypothalamus receives both exteroceptive and interoceptive information and is a critical component of systems that protect the organism by maintaining the constancy of the internal environment. Everything that the brain "knows" about the external environment sensed by touch, taste, smell, sight, sound, pressure, balance, or temperature is conveyed to the hypothalamus through its connections with the limbic forebrain and lower brainstem. It is also the principle regulator of autonomic functions and, therefore, controls all aspects of the internal environment by way of descending projections to brainstem autonomic regions and the pituitary. If the cerebral cortex is removed from a rat, the reproductive functions can still be realized, i.e. fertilization, normal labor, and feeding of the offspring. Many of these reproductive activities are organized at the level of the HT and brain stem. If however, a rat is subjected to emotional stress induced by unpleasant, shrill sounds, the reproductive function is depressed. Therefore under the proper circumstances, automatic hypothalamic control of the reproductive function can be interrupted if the activity of the organism must be diverted to the demands of the external environment. The HT functions to a large degree automatically, responding continuously to signals from the body. Regulation of the internal environment is carried out through the autonomic nervous system (sympathetic and parasympathetic systems which exert opposite effects on the tissues and organs) and the endocrine glands. The HT regulates directly or indirectly growth and development, the thyroid gland, the adrenal gland, lactation (prolactin), sleep, emotion, appetite (hunger and satiation), heat regulation, energy metabolism, cardiac activity, vascular tension, water and saline balance, functions of the gastrointestinal (GI) tract, urination, development and senescence of the female reproductive system, and possibly senescence of the organism as a whole (43,44). In view of the influence of the HT on virtually all aspects of the visceral function, it would be surprising if the immune system was not also under its regulation. The HT is the guard of the organism's capacity to react because it always switches on automatically without involvement of consciousness. For example, under the influence of any unconditioned stimulus, such as pain, or any change in composition of the internal environment caused by hemorrhage, decrease or increase in blood sugar, infectious or inflammatory activity, information relating to the alteration will be conveyed to the HT and it, in turn, would make an appropriate response to bring the change under control. This argument suggests that the HT is a site at which one would expect to find cells responsive to the IS signals that mediate USs in conditioning.

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Neural basis for olfactorv memory In discussing memory for odor conditioning, it might be instructive to review mechanisms of olfactory recognition in reproductive behavior. It has been reported that mice have an olfactory memory for pheromones located at the ftrst relay (accessory olfactory bulb) in the sensory system. This recognition is acquired with one-trial learning and depends on activation of norepinephrine at the time of mating. This memory lasts for several weeks (24). In the mouse mating normally results in 90-100% of the female mice becoming pregnant (24). It was noticed that if the mated female is removed from the stud male immediately after the mating and returned to the stud male 6 hr later, pregnancy failed. If the female is put with a strange male immediately after the mating, pregnancy failed. But, if the female mouse is left in the cage with the odor of the urine of the stud male for 6 hr and then removed, pregnancy is sustained. The mated female requires the odor of the male stud's pheromones for 4-6 hr in order for recognition to occur in the brain to allow the pregnancy to take place. The duration of this 4-6 hr recognition period indicates that memory is being laid down in the CNS and this laying down of memory can be blocked by the odor of a strange male or if the mated female is removed from the presence of the original stud's pheromones which are present in the urine. Recognition of odors such as pheromones might be thought to require the cortical system to which the main olfactory bulb (MOB) projects. The main olfactory bulb neurons project primarily to the pyriform cortex but direct projection to the insular cortex has been described as well (45-48). MOB is also connected to lateral and tuberal HT by way of the anterior olfactory nucleus and amygdala (49). Studies by Lloyd-Thomas & Keverne (50) and Keverne et al., (51) showed that memory to odor was not impaired in pregnancy blocking experiments by lesioning of the MOB. They conclude that the process of recognition (memory) that occurs is a function of the vomeronasal system and its central projections. The central projections of the vomeronasal system are distinct from those of the MOB (52,53). The vomeronasal projections are subcortical to the neuroendocrine hypothalamus via the accessory olfactory bulb (AOB) and amygdala (54). Connections with the hippocampus were not involved in storage of olfactory (pheromone) memory. Selway & Keverne (55) showed hippocampal lesions did not disrupt memory for familiar odors. It is not certain that the smaller molecules used in the conditioning studies are also processed by the AOB system; however, the work of Keverne et al. provides an interesting model for conditioned immune responses. Based on their studies, the accessory olfactory system and its projections to the hypothalamus appear to be a likely candidate for storage of memory, not only to produce the neuroendocrine changes needed to recall the CR but perhaps odor induced structural alterations in the AOB sustains the process of recognition of familiar odor. The circuitry of the AOB is relatively simple. Mitral ceils receive afferents from the vomeronasal nerve and project to the medial amygdala, forming the excitatory pathway to the hypothalamus for odor signals received by the vomeronasal receptors. The mitral cells form reciprocal dendrodendfitic synapses with granule cells, the main class of interneurons in the AOB. Granule cells are depolarized by an excitatory input from the mitral cells and in turn provide a feedback inhibition via -t-aminobutyric acid (GABA) release. This interaction between mitral and granule cells regulate mitral cell activity by feedback inhibition. It has been shown that norepinephrine (NE) reduced the inhibition exerted by granule cells on mitral cells (56). If NE reduces the feedback inhibition of granule to mitral ceils, then sustained excitation of mitral cells will occur. It has been reported for rat olfactory bulbs that phencyclidine-like compounds, ketamine and MK801 which block N-methyl-Daspartate (NMDA) receptors blocked the inhibition of mitml cells by blocking intrinsic excitatory feedback circuits in the olfactory bulb. Since GABA inhibitory pathways were not inhibited, ketamine effect was not due to a direct block of the dendrodendritic inhibition initiated by granule cells, and the mechanism has not been clearly delineated (57). Nevertheless, it is of interest to note that mice conditioned under ketamine/rompun (K/R) anesthesia or exposed to the CS to recall the conditioned response under anesthesia produced a better CR than animals trained and tested, which were not anesthetized (26). If ketamine treatment is responsible for preventing the feedback inhibition of mitral cells at the CS/US learning or the recall stage, the effect of the CS signal to the HT would be maintained longer and this might explain why the memory for the CR is observed to be stronger in ketamine treated vs non-ketamine treated animals. The role of rompun (xylazine) an o.2-adrenoceptor agonist on the response is unclear at this time.

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The striking feature of olfactory recognition for pheromones is its specificity, and the fact that the olfactory memory resides in a neural circuitry located close to the olfactory receptors which can distinguish the stud male from other males. The site where memory is stored may be the AOB and its subcortical pathway to the hypothalamus. In conditioning of the NK cell activity, there axe certain features that parallel these observations. Odor recognition by way of the vomeronasal system can occur subcortically. If memory for camphor odor is being established through this subcortical pathway, it should be possible to link an odor readily to a US that activates the neuralendocrine reflex circuitry. For example, stimulation of nerve endings by an infant nursing a breast transmits afferent nerve impulses via the spinal cord and brain stem to the HT. Release of hypothalamic prolactin releasing factor activates the pituitary to secrete prolactin (PRL) which circulates back to the breast to stimulate milk production and secretion. This is an example of afferent limb stimulation of a neural pathway and the efferent limb of hormonal secretion (58). In a conditioned animal, the CS (camphor odor) signal is an afferent limb stimulation of a neural pathway to the HT which activates the efferent limb of a hormonal secretion, corticotropin releasing hormone (CRH) which causes the pituitary to secrete ACTH and endorphin. Both ACTH and endorphin circulate back to the IS and either enhance or suppress its activity. However, one should note that the afferent signal to the HT from the US was a soluble mediator, IFN-13 which induces a hormonal efferent limb which circulates back to activate the IS. This response is initiated by the US and after conditioning, the CS triggers this response reflexively. Conclo~i0n~ Studies of conditioned NK cell activity were undertaken to provide some basis for understanding how the IS and CNS communicate. Conditioning of the NK cell activity was employed as a model to learn how the IS-CNS interaction can lead to expression of a specific response initiated by cues to the CNS. When a foreign antigen (virus, bacteria) or substance such as poly I:C is injected into the body, the immune system detects the sudden change in the internal milieu of the organism either by the neuronal route or by mediators that signal the CNS. Poly I:C which simulates such an infection induces the production of IFN-a, -[~ and raises NK cell activity in the spleen. We have shown that the mediator that signals the CNS is IFN-~. In response to the IFN-I3 afferent signal from the periphery, the HT responds by activating either neuronal or neuroendocrine effector pathways or both in an effort to re-establish homeostatic balance. In this regard, a true US signal must be one that the CNS detects and immediately responds to by activation of an efferent pathway. Such US (afferen0 signals to the CNS can be readily linked to a CS signal (odor of camphor). Our results suggest that during the CS/US association, two memories develop. The memory for the CS is stored in the olfactory system, and the memory for the CS/US association is stored outside the sensory system, possibly in a site such as the HT. When the conditioned animal is reexposed to the CS, we postulate that activation of specific olfactory receptors triggers the neural circuits (where the CS memory is stored) and sends nerve impulses to the HT. These signals activate the hypothalamic-pituitary axis to release neuroendocrine hormones (ACTH, ~lendorphin, prolactin, etc.,) which can stimulate the up-regulation of preactivated NK cells in the spleen. The fact that the specific cytotoxic lymphocyte (CTL) activity to an allogeneic antigen can be similarly conditioned suggests that parallel pathways are employed by the IS to communicate with the CNS and that the capacity of the CNS to trigger the generation of a specific CTL or NK cell activity is not limited to these two systems alone (59). We postulate that for these immune activities, specific mediators are used by the immune system to interact with the CNS. We also believe that the CNS, in turn, employs the same neuroendocrine signals from the pituitary to trigger the specific response in the immune system. The responses that are up-regulated are critically dependent on the activation state of the immune cells in question. Acknowledm'nents The studies reported were supported by grants CA37570 and AG10263 from the National Institutes of Health.

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References 1. H.O. BESEDOVSKY, A. DEL REY, E. SORKIN, W. LOTZ and U. SCHWULERA, Clin. Exp. Immunol. 59 622-628 (1985). 2. J.C. MATHISON, R.D. SCHREIBER, A.C. LA FOREST and R.J. ULEVITCH, J. Immunol. l~O 2757-2762 (1983). 3. H.O. BESEDOVSKY, A. DEL REY, E. SORKIN, M. DAPRADA and M. KELLER. Cell. Immunol. 48 346-355 (1979). 4. A. FONTANA, E. WEBER and J-M. DOYER, J. Immunol. 133 1696-1698 (1984). 5. J. M. KRUEGER, J. WALTER, C. DINARELLO, M.L. WOLFF and L. CHEDID, Am. J. Physiol. 246 994-999 (1984). 6. E.M. SMITH and J.E. BLALOCK, Proc. Natl. Acad. Sci. USA 78 7530-7534 (1981). 7. N.R. HALL, J.P. MCGILLIS, B.L. SPANGELO and A.L. GOLDSTEIN, J. Immunol. 135 806s-81 Is (1985). 8. S.B. MIZF~I,, Immunol. Rev. 63 51-72 (1982). 9. C.A. DINARELLO, J. Clin. Immanol. 5 287-297 (1985). 10. A. KEIJSO and A. MUNCK, J. Immunol. 133 784-791 (1984). 11. D.S. SNYDER and E.R. UNANUE, J. Immunol. 129 1803-1805 (1982). 12. H.O. BESEDOVSKY, E. SORKIN, M. KELLER and J. MULLER, Proc. Soc. Exp. Biol. Med. 1~0 466-470 (1975). 13. H.O. BESEDOVSKY, A. DEL REY and E. SORKIN, J. Immunol. 126 385-387 (1981). 14. H.O. BESEDOVSKY, A. DEL REY and E. SORKIN, J. Immunol. 13~ 750s-754s (1985). 15. HIRATA, S. TOYASHIMA, J. AXELROD and M.J. WAXDAL, Proc. Natl. Acad. Sci. USA 77 862-865 (1980). 16. R. ADER and N. COHEN, Psvchoncuroimmunolo~v II, R. Ader, D.L. Felton and N. Cohen, (Eds), 611-646, Academic Press, Inc. (1991). 17. Z. GROSSMAN, R.B. HERBERMAN and S. LIVNAT, Int. J. Neurosci. 64 275-290 (1992). 18. B. SOLVASON, V.K. GHANTA and R.N. HIRAMOTO, J Immunol 140 661-665 (1988). 19. V.K. GHANTA, N.S. HIRAMOTO, H.B. SOLVASON, S.K. TYRING, N. H. SPECTOR and R.N. HIRAMOTO, J. Neurosci. Res. 18 10-15 (1987). 20. H.B. SOLVASON, V.K. GHANTA, S-J. SOONG and R.N. HIRAMOTO, Prog. NeuroEndocrinlmmunol. 4258-264 (1991). 21. B. SOLVASON, V.K. GHANTA, and R.N. HIRAMOTO, J. Neuroimmunology, In press. 22. B. SOLVASON, V.K. GHANTA, S-J. SOONG, C.F. ROGERS, C-M HSUEH, N. HIRAMOTO, and R. HIRAMOTO, Proc. Soc. Exp. Biol. Med. 199 199-203 (1992). 23. H.B. SOLVASON, V.K. GHANTA, J.F. LORDEN, S-J. SOONG and R.N. HIRAMOTO, Int. J. of Neurosci. 59 101-117 (1991). 24. P. BRENNAN, H. KABA, and E.B. KEVERNE, Science 250 1223-1226 (1990). 25. R.N. HIRAMOTO, V.K. GHANTA, J.F. LORDEN, H.B. SOLVASON, S-J. SOONG, C.F. ROGERS, C-M. HSUEH and N.S. HIRAMOTO, Prog. NeuroEndocrinlmmunol. 5 13-20 (1992). 26. C-M HSUEH, J.F. LORDEN, R.N. HIRAMOTO and V.K. GHANTA, Life Sci. 50 20672074 (1992). 27. A.L. GEE and D.R. JOHNSON, FASEB J. 6A1995 (1992). 28. R.N. HIRAMOTO, V.K. GHANTA, C. ROGERS and N. HIRAMOTO, Life Sci. 49 93-99 (1991). 29. H.B. SOLVASON, R.N. HIRAMOTO and V.K. GHANTA, Brain. Behav. Immun. 3 247262 (1989). 30. R.N. HIRAMOTO, H.B. SOLVASON, V.K. GHANTA, J. LORDEN and N. HIRAMOTO, Pharmacol. Biochem. Behav. 36 51-56 (1990). 31. V.K. GHANTA, C.F. ROGERS, C-M. HSUEH, N. HIRAMOTO, S-J. SOONG and R.N. HIRAMOTO, Int. J. Neurosci. 61 135-143 (1991). 32. C.J. GORDON and J.E. HEATH, Ann. Rev. Physiol. 48 595-612 (1986). 33. C.A DINARELLO, Fever. Boniford & Henderson (Eds.) (1989). 34. D.G. NICHOLS and R.M. LOCKE, Physiol. Rev. 641 1-64 (1984). 35. J.J. MCGLONE, E. LUMPKIN and R.L. NORMAN, Endocrinology 129 1653-1658 (1991).

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36. C-M HSUEH, R.N. HIRAMOTO and V.K. GHANTA, Brain Res. 578 142-148 (1992). 37. R.N. HIRAMOTO, N.S. HIRAMOTO, H.B. SOLVASON and V.K. GHANTA, Ann. N.Y. Acad. Sci. 49¢~ 545-552 (1987). 38. L.M. HALLACHER and W.R. SHERMAN, J. Biol. Chem. 255 10896-10901 (1980). 39. P.F. WORLEY, W.A. HELLER, S.H. SNYDER, J.M. BARABAN, Science 239 14281429 (1988). 40. S.K. FISHER and R.T. BARTUS, J. Neurochem. 45 1085-1095 (1985). 41. R.A. GONZALES and F.T. CREWS, J. Neurosci. 4 3120-3127 (1984). 42. D.A. KENDALL and S.R. NABORSKI, J. Pharmacol. Exp. Ther. 241 1023-1027 (1987). 43. V.M. DILLMAN, The grand biological clock, 1-24, Mir Publishers Moscow, (1989). 44. J. MEITES, Experimental Gerontology 23 349-358 (1988). 45. M.T. SHIPLEY and G.D. ADAMEK, Brain Res. Bull. 12 669-688 (1984). 46. M.T. SHIPLEY and Y. GEINISMAN, Brain Res. Bull. 12 221-226 (1984). 47. L. HEIMER, Brain Behavior Evolu. 6 484-523 (1972). 48. T. TANABE, M. LINO and S.F. TAKAGI, J. Neurophysiol. 38 1284-1296 (1975). 49. R.C. SWITZER, J. DE OLMOS and L. HEIMER, The rat nervous system, G Paximas (Ed), Vol. 1, 1-36, Sydney Academic Press (1985). 50. A. LLOYD-THOMAS and E.B. KEVERNE, Neurosci. 7 907-913 (1982). 51. E.B. KEVERNE, C.L. MURPHY, W.C. SILVER, C.J. WYSOCKI and M. MEREDITH, Chem. Senses Flavor 11 17-23 (1986). 52. E.B. KEVERNE, Trends Neurosci. 6 381-384 (1983). 53. F. SCALIA and S.S. WINONS, J. Comp. Neurol. l~il 31-56 (1975). 54. C.S. LI, H. KABA, H. SAITO and K. SETO, Neurosci. 29 201-208 (1989). 55. R. SELWAY and E.B. KEVERNE, Physiol. Behav. 47 249-252 (1990). 56. C.E. JAHR and R.A. NICOLL, Nature 297 227-229 (1982). 57. I. JACOBSON, A. HAMBURGER and C.D. RICHARDS, Exp. Brain Res. 80 409-414 (1990). 58. J.B. MARTIN, Harrison's Princioles of Internal Medicine, G.W. Thorn, R.D. Adams, E. Braunwald, K.J. Isselhacher ancl R.G. Petersdorf (Eds.), 471-478, McGraw-Hill Book Company (1977). 59. R.N. HIRAMOTO, C-M HSUEH, C.F. ROGERS, S. DEMISSIE, N.S. HIRAMOTO, S-J SOONG and V.K. GHANTA, Pharmac. Biochem. Behav. ~ 275-280 (1993).