Neuro-immune associative learning

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6 Neuro-Immune AssociativeLearning

M.-B. Niemi . G. Pacheco‐Lopez . H. Engler . C. Riether . R. Doenlen . M. Schedlowski

1

# 200

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124

1.1

Cross Talk between Central Nervous System and Immune System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124

1.2

Classical Conditioning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

1.3

Neuroimmune Associative Learning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

1.4

Historical Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

2

Evoking Neuroimmune Associative Learning Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

3

Conceptual Framework for Neuroimmune Associative Learning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

3.1

Association Phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

3.2

Recall Phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

4

Principles of Neuroimmune Associative Learning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131

4.1

General Learning Rules of Neuroimmune Associative Learning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131

4.2

Features of Neuroimmune Associative Learning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132

5

Mechanisms of Neuroimmune Associative Learning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

5.1

Neurobiology of Association and Recall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

5.2

Peripheral Mediation of the Conditioned Effects on Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

6

Clinical Implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

6.1

Animal Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

6.2

Human Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

7

Methodological Considerations for Neuroimmune Associative Learning Experiments . . . . . . . . . 141

8

Closing Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142

8 Springer ScienceþBusiness Media, LLC.

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124 6 Neuro-immune associative learning

Abstract: Neuroimmune associative learning constitutes the concomitant presentation of a neutral stimu-

lus (conditioned stimulus, CS) and an immunomodulating agent (unconditioned stimulus, US). Repre-

sentation of the CS alone is able to activate the centrally stored engram often resulting in changes in

immune parameters that mimic those formed by the US (conditioned immune response). This experimen-

tal model enables to study and dissect the afferent (at association) as well as efferent (at recall) pathway of

brain–immune communication. In this chapter, we will introduce this behavioral conditioning paradigm,

review the available literature focusing on features of the conditioned and unconditioned stimuli, and the

conditioned responses, the so far knownmechanisms steering the conditioned response, and the underlying

learning principles. Moreover, a theoretical framework modeling the pathways as well as the guidelines how

to design neuroimmune associative learning experiments will be provided. Finally, the clinical feasibility of

this behavioral approach will be discussed as a supplement to standard therapies with the aim of optimizing

individual healing conditions.

List of Abbreviations: CRs, conditioned responses; CTA, conditioned taste avoidance; SRBCs, sheep red

blood cells; 6‐OHDA, 6‐hydroxydopamine

1 Introduction

Clinical observations and experimental evidence demonstrate the intensive bidirectional communication

between the central nervous system (CNS) and the immune system (for review, see Elmquist et al., 1997;

Straub and Schedlowski, 2002; Tracey, 2002; Dantzer, 2004; Glaser and Kiecolt‐Glaser, 2005). We termed the

process of sensing and encoding immune inputs to the CNS, and further associating themwith the memory

traces of exteroceptive clues as neuroimmune associative learning. Retrieving such engrams may result in a

complex repertory of physiological responses affecting neurobehavioral, endocrine, as well as immune

parameters. Furthermore, neuroimmune associative learning paradigms can be employed to experimentally

study the principles by which the immune and the nervous system exchange information.

1.1 Cross Talk between Central Nervous System and Immune System

One put a lymphocyte into a culture dish, added an antigen, and out came an antibody. So who needed a

nervous system? (Spector, 1996).

The paradigm of total independence of the immune system has been dismissed by experimental

evidence accumulating mainly during the last three decades. Here are only some relevant issues related to

the present topic enlisted.

A whole series of neuropeptides, neurotransmitters, and neuroendocrine hormones are endogenously

produced by immune cells (for review, see Blalock, 1994; Tayebati et al., 2002; Warthan et al., 2002) and

many cytokines are found to be produced and have significant biological activity in the central and

peripheral nervous system (Blalock, 2005).

1. Stimulation or silencing of distinct brain areas affects immune functioning by different mechanism (for

a detailed review, see Meisel et al., 2005; Wrona, 2006; Ziemssen and Kern, 2007).

2. There is evidence for rich neural connections with lymphoid tissue (Steinman, 2004; Straub, 2004), and

receptors for neurotransmitters are also present on lymphocytes.

Immune‐to‐brain pathway: On the afferent pathway, peripheral immunological changes are signaled to the

CNS, and different ascending pathways (neural and humoral) have been identified. The vagus nerve

provides the major neural pathway identified to date. The initial chemosensory transduction events

occur in immune cells, which respond to specific chemical components expressed by dangerous microorgan-

isms. These immune chemosensory cells release mediators, such as cytokines, to activate neural elements,

including primary afferent neurons of the vagal sensory ganglia. Primary afferent activation initiates

local reflexes (e.g., cardiovascular and gastrointestinal) that support host defense (Goehler et al., 2000).

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Neuro-immune associative learning 6 125

This neural afferent pathway is complemented by a humoral afferent pathway that involves cytokines or

other immunotransmitters transported in the blood or produced at the level of the circumventricular

organs. It is possible that such immunotransmitters cross the blood–brain barrier or originate a secondwave of

cytokines produced in the brain parenchyma (Banks, 2006). Depending on their source, these locally produced

cytokines can either activate neurons that project to specific brain areas or diffuse by volume transmission into

the brain parenchyma to reach their targets. Activation of neurons by cytokines can be direct or indirect. The

way the neural pathway of transmission interacts with the humoral pathway remains to be elucidated;

however, it has been proposed that each pathway may engage in different conditions (e.g., localized vs.

systemic infections), and thus codify different information (Dantzer et al., 2000).

Brain‐to‐immune pathway: There are three main pathways: the hypothalamo–pituitary–adrenal (HPA)

axis (humoral), the sympathetic–adrenal–medullary axis and the parasympathetic nervous system includ-

ing the vagus nerve (both neural). Activation of the HPA axis results in the production of glucocorticoid

hormones and catecholamines (Meisel et al., 2005), regulating cytokine balance (for review, see Ziemssen

and Kern, 2007) and vice versa. The sympathetic nervous system regulates immunity by innervation of

lymphoid organs and the release of noradrenaline, and a hormonal component that regulates immunity

systemically through the release of adrenaline from the medulla of the adrenal glands (Sternberg, 2006).

Coupling of the sympathetic nervous system and the HPA axis leads in the spleen to stronger effects

through activation of b‐adrenoceptors and glucocorticoid receptors (Straub, 2004).

In summary, the CNS and immune system intensively and extensively interact, sharing pathways,

messengers, and their receptors.

1.2 Classical Conditioning

Since the CNS has the capability to alter the activity of the immune system as well as of many other organs/

systems, it can be hypothesized that behavioral approaches such as classical conditioning may be able to

modulate immune functions. Any paradigm of associative learning or classical conditioning comprises two

basic phases: association and recall. During association phase, a neutral stimulus (to become a conditioned

stimulus: CS) is paired with an unconditioned stimulus (US), which elicits vigorous responding (uncondi-

tioned response, UR) (Domjan, 2005). Contingent pairing(s) of the CS and the US leads to the establish-

ment of a temporal/causal association of both stimuli, stored within the CNS. This learned association can

then be identified during recall by the emergence of new responses to the CS, which can be now presented in

the absence of the US. These new responses are termed conditioned responses (CRs) and they usually

mimic the URs.

1.3 Neuroimmune Associative Learning

During an association trial, a gustatory/olfactory stimulus as a CS (e.g., saccharin solution; taste, chocolate

milk; flavor, or camphor; odor) precedes the administration of an immunomodulating agent, that is, US

(e.g., cyclosporine A, cyclophosphamide, polyinosinic: polycytidylic acid or antigens). Paired administra-

tion of these stimuli leads to a CS–US association. During recall, the CS is able to produce some effects,

formerly ascribed just to the immunomodulating compound (US) (> Figure 6-1). This phenomenon

implies that the total effects of a drug are composed of pharmacological effects per se plus potential CRs

(Pacheco‐Lopez et al., 2006). Neuroimmune associative learning has been reported employing visual and/or

auditory stimuli such as a light or a tone as CS (MacQueen et al., 1989; Palermo‐Neto and Guimaraes, 2000;

Irie et al., 2002; Costa‐Pinto et al., 2005), or touch stimuli such as scratching or heating of the skin as CS

(Metal’nikov and Chorine, 1926; Nicolau and Antinescu‐Dimitriu, 1929). However, the naturalistic relation

of postprandial immunotoxicological consequences facilitates neuroimmune associative learning that

employs gustatory/olfactory clues as CS. It contributes to the feasibility, duration, and magnitude of the

association. Therefore, it is not surprising that stimuli causing gastrointestinal irritation get more easily

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. Figure 6-1

Neuroimmune associative learning. During association, animals are exposed to a conditioned stimulus (CS)

paired with an immunomodulating unconditioned stimulus (US). During recall, the CS is presented without the

US, evoking the formerly learned association. This is often accompanied by avoidance behavior and a complex

physiological response that may also affect peripheral immune functions

126 6 Neuro-immune associative learning

associated with the postprandial taste/odor memories (e.g., after saccharin intake) than with an acoustic or

visual stimulus, since under natural circumstances the former are more likely to become associated

(Domjan et al., 2004).

1.4 Historical Overview

The first documented cases of behavioral conditioned immune effects are of anecdotal character and date

back to the late nineteenth century. Mackenzie (1886) reported a patient who was suffering from a severe

coryza at the sight of an artificial rose, which the presence of natural roses invariably produced in her case.

Others reported similar findings in that environmental stimuli that have been associated with an allergen in

the past provoke allergic symptoms in sensitive patients by a picture of a hay field (Hill, 1930, cited in Ader

and Cohen, 1992).

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Neuro-immune associative learning 6 127

The first scientifical reports of classically conditioned immune functions arose from Russian scientific

contemporaries of I. P. Pavlov. Lukyanenko (1961) cites an observation of Makukahin (1911) and a report

by Voronov and Riskin (1925) as possibly the first ones to demonstrate ‘‘conditioned leukocytic reactions.’’

However, according to Luk’yanenko they could not be interpreted properly and were not carried forward.

In the 1920s, Metal’nikov and his colleagues at the Pasteur Institute in Paris, France systematically

documented that an immune response could be elicited without the presence of an antigen but just by

evoking a previous experience, that is, CS exposure. After scratching or heating the skin of guinea pigs with

a warmed metallic plate as CS, the animals received intraperitoneal (i.p.) injections of various bacteria‐derived compounds such as small doses of either Bacillus anthrax, a Staphylococcus filtrate, tapioca

emulsion, or Vibrio cholera as US. After association phase and a delay to allow the return to baseline levels

was completed, the CS alone yielded significant and rapid influx of polymorphonuclear leukocytes into the

peritoneal cavity (Metal’nikov and Chorine, 1926, 1928). As a proof of the biological relevance of the

elicited conditioned response on the immune system, it was reported that conditioned animals survived to

lethal doses of V. cholera or Streptococcus if they were previously behaviorally evoked. Follow‐up experi-

ments replicated and extended these findings (Nicolau and Antinescu‐Dimitriu, 1929; Ostrovskaya, 1929).

Additional evidence occurred largely provided by A. O. Dolin in the Soviet Union and his fellows Krylov,

Flerov, and Luk’yanenko. Both specific and nonspecific immune reactions and both immunosuppression

and immunoenhancement due to conditioning were achieved in mice, rats, guinea pigs, rabbits, dogs, oxen,

monkeys and also in humans (Dolin and Krylov, 1952; Doroshkevich, 1954; Vygodchikov, 1955). At the

same time, but less recognized, reports from Romania showed a conditioned increase in phagocytic activity

of blood polymorphonuclear cells in dogs (Benetato, 1955; Baciu et al., 1965).

In addition, a series of reports published in Switzerland documented conditioned asthma‐likeresponse by using an auditory CS in guinea pigs (Noelpp and Noelpp‐Eschenhagen, 1951a–c, 1952a–c).Such findings were replicated by Ottenberg et al. (1958), and extended to humans (Dekker et al., 1957;

Turnbull, 1962).

In 1975, Ader and Cohen published their seminal work in which they constructed the term behaviorally

conditioned immunosuppression, which retrospectively set the stage for the field of psychoneuroimmunology.

During that time, R. Ader was working on extinction of conditioned taste aversion employing saccharin

taste as CS paired with a drug that induced significant visceral malaise. He realized that some rats of the

conditioned group died during the course of a series of extinctions trials (i.e., CS representation). Moreover,

those animals had received the largest of three different amounts of saccharin and displayed the most

pronounced conditioned taste avoidance (CTA) behavior (Ader, 1974). Knowing the additional immuno-

suppressive properties of the drug employed as US, cyclophosphamide: CY, Ader and his colleague,

immunologist N. Cohen, tested the hypothesis that the increased mortality on the conditioned animals

during extinction phase was related to a compromised immune status resulted from a conditioned

immunosuppressive response. This hypothesis was systematically assessed and confirmed showing that

rats receiving paired administration of saccharin and CY not only displayed a strong CTA but also a reduced

production of antibody titers on challenge with sheep red blood cells (SRBC) (Ader and Cohen, 1975). This

initial finding was verified by independent laboratories basically under the same conditions (i.e., Rogers

et al., 1976; Wayner et al., 1978), and further extended and elaborated (Ader et al., 1982).

2 Evoking Neuroimmune Associative Learning Responses

As a synopsis, >Table 6-1 is provided summarizing several neuroimmune associative learning protocols in

which immune functions are evaluated after recall phase.

Different antigens have been applied as US eliciting conditioned immunoenhancement effects, for

example, T‐dependent antigens such as ovalbumin (Chen et al., 2004; Huang et al., 2004), keyhole limpet

hemocyanin (Ader et al., 1993), T‐independent antigens such as lipopolysaccharides (Bull et al., 1991), or

superantigens such as staphylococcal enterotoxin (Pacheco‐Lopez et al., 2004), as well as viral synthetic

patterns such as poly I:C (Solvason et al., 1993; Coussons‐Read et al., 1994; Demissie et al., 1995; Hsueh

et al., 1995, 1999, 2002; Kuo et al., 2001; Chao et al., 2005). On the other hand, immunosuppressive drugs

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. Table 6-1

Overview of conditioning protocols affecting immunity

Conditioned response

Unconditioned

stimulus

Conditioned

stimulus Species References

NK‐cell activity ↑ Poly I:C Camphor odor Mouse Hsueh et al.

(1995, 1999,

2002); Kuo et al.

(2001)

NK‐cell activity ↑ Interferon‐b Camphor odor Mouse Solvason et al.

(1993)

NK‐cell activity ↑ Arecoline Camphor odor Mouse Demissie et al.

(1995)

Neutrophil activity ↑ Poly I:C Camphor odor Mouse Chao et al. (2005)

Antibody response ↓ Cyclophosphamide SAC Rat Ader et al. (1975,

1979); Cohen

et al. (1979)

Antibody response of IgM and

IgG isotypes ↑

Hen egg‐whitelysozyme

SAC Rat Alvarez‐Bordaet al. (1995);

Madden et al.

(2001)

Antibody response and

T‐lymphocyte proliferation ↓

Cyclophosphamide SAC Mouse Neveu et al.

(1986)

Anti‐SRBC antibody titers ↑ Sheep red blood

cells

SAC/LiCl Rat Jenkins et al.

(1983)

Anti‐OVA antibody titers ↑ Ovalbumin SAC Rat Chen et al. (2004)

Anti‐OVA antibody titers ↑ Ovalbumin Electro‐stimulation

(2 ms/2 Hz/2 or 4 V)

Rat Huang et al.

(2004)

Anti‐OVA antibody titers ↑ Ovalbumin electroacupuncture Rat Huang et al.

(2004)

Antibody response ↓ Keyhole limpet

hyacinine

Chocolate milk Mouse Ader et al. (1993)

Secretion of mast‐cellprotease II ↑

Egg albumin

antigen

Flashing light and

noise by ventilation

fans

Rat MacQueen et al.

(1989)

Primary mixed lymphocyte

response ↓

Antilymphocyte

serum

SAC Rat Kusnecov et al.

(1983)

Splenocyte proliferation, IL‐2and IFN‐g production, and mRNA

expression and synthesis ↓

Cyclosporine A SAC Rat Exton et al.

(1998, 2000);

Pacheco‐Lopezet al. (2005)

T helper:T suppressor subset

ratio ↑

Levamisole SAC Rat Husband et al.

(1987)

NK‐cell activity, IL‐2, lymphocyte

proliferation ↓

Morphine sulfate Distinctive

environment

Rat Coussons‐Readet al. (1994)

IL‐2, IFN‐g, and corticosterone

plasma levels ↑

Staphylococcal

enterotoxin B

SAC Rat Pacheco‐Lopezet al. (2005)

Nitric oxide ↓ Heroin Distinctive

environment

Rat Szczytkowski and

Lysle (2007)

Corticosterone production ↑ Interleukin‐1 b SAC/LiCl or

peppermint odor

Mouse Dyck et al.

(1990)

128 6 Neuro-immune associative learning

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Neuro-immune associative learning 6 129

like cyclophosphamide (CY) and cyclosporine A (CsA) have been successfully employed as US, resulting in

conditioned immunosuppressive effects when associated with an appropriate CS such as taste/flavor (Ader

and Cohen, 1975; Exton et al., 2001).

Morphine sulfate as US paired with a distinctive environment as CS served to achieve a condi-

tioned suppression of NK‐cell activity, IL‐2 production, and lymphocyte proliferation (Coussons‐Readet al., 1994).

3 Conceptual Framework for Neuroimmune Associative Learning

The following section aims to provide a framework which takes basic assumptions about stimuli and sites of

action within neuroimmune associative learning into account. Within classical conditioning theory, a

prerequisite for its occurrence is that the CNS must sense both the CS (changes in the external environ-

ment) and the US (changes in the internal environment), where the signals are then processed, associated,

consolidated and recalled at evocation time (Eikelboom and Stewart, 1982; Bovbjerg, 2003; Pacheco‐Lopezet al., 2006, 2007b). Accordingly, the CNS must initiate both the UR and the CR. Translated to neuroim-

mune associative learning, this implies that only immune changes that are detected by the brain can serve as

US, in turn, only immune changes that are executed by the brain should then be called CR. However, the

nature of the US and the CR in most of the neuroimmune associative learning protocols so far reported is

not evident (> Figure 6-2).

3.1 Association Phase

During association phase, there are two possible USs that may be detected by the brain. A directly perceived

US, which is defined as a stimulus that itself is recognized by the CNS. The second kind is an indirectly

perceived US, which is defined to be signaled by intermediary molecules that are then indeed detected by the

CNS. In that case those molecules are the genuine US and the applied drug is a sham US, or indirectly

perceived US. This has important implications, for example, pairing of a CS with a compound that uncondi-

tionally decreases body temperature as US could result in a conditioned hypothermic response; however,

conditioned hyperthermia is also possible, that is, ‘‘paradoxically conditioned effects’’ or ‘‘counterconditioning

effects’’ (e.g., Bull et al., 1991). The proposed framework predicts such possibilities also for conditioned immune

responses and provides a tentative explanation. As Eikelboom and Stewart (1982) proposed, it is necessary to

correctly determine the nature of the US and UR to predict the direction of the CR.

There are two possible afferent pathways for any US, regardless whether directly or indirectly perceived –

a humoral and a neural pathway (see > Section 1.1). Within the neural pathway, the US information, on

detection, may be translated into neural activity. This sensing process requires immunoceptive capacities of

the CNS (Goehler et al., 2000; Blalock, 2005).

The humoral afferent pathway is required more often for indirectly perceived USs that induce molecules

that reach the brain via the blood stream as well as for USs that are not detected locally by the immune

system. In addition, if an indirectly perceived US affects several cell types, all of the involved molecules

become candidates for serving as a genuine US to be detected by the CNS. This implies a more complex,

longer, and therefore maybe slower signaling process for indirectly perceived US compared with directly

perceived US. Therefore, it can be assumed that the CNSmay take longer to respond to an indirectly perceived

US than to a directly perceived US.

3.2 Recall Phase

The CR represents the ultimate proof that an association has formerly taken place. There are two possible

pathways by which immune functions can be modulated by the CNS: the humoral efferent pathway and the

neural efferent pathway (see > Section 1.1). During recall phase, humoral efferent pathway may affect

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. Figure 6-2

Theoretical framework for neuroimmune associative learning. At association, the conditioned stimulus (CS) can

potentially be associated with two possible unconditioned stimuli (US). The US directly detected by the CNS is

defined as a directly perceived US. The stimulus that needs one or more intermediary molecules released

by another system to be detected by the CNS is termed indirectly perceived US. Any US, indirectly or directly

perceived, has two possible afferent pathways to the CNS: a neural afferent pathway and a humoral efferent

pathway. At recall, the CNS canmodulate immune function via these twopathways. Thehumoral efferent pathway

may imply changes in neuroendocrine mediators that directly or indirectly modify the immune response. The

neural efferent pathway is supported by direct innervations of primary and secondary lymphoid organs

130 6 Neuro-immune associative learning

neurohormones that in turn affect immune responses. These peripheral effects are diffuse and long‐lastingas any neuroendocrine responses. Direct innervation of primary and secondary lymphoid organs (Elenkov

et al., 2000; Mignini et al., 2003; Tracey, 2007) may be part of the neural efferent pathway. Since several

immune parameters such as T‐cell differentiation (Sanders and Kohm, 2002a; Sanders and Straub, 2002b),

hematopoiesis (Miyan et al., 1998; Artico et al., 2002), T‐ and B‐cell activity (Downing and Miyan,

2000), NK‐cell activity (Katafuchi et al., 1993; Hori et al., 1995), and inflammatory responses (Czura and

Tracey, 2005; Pavlov and Tracey, 2005) are affected by neural activity, and they may also be subjected to be

affected by neuroimmune associative learning protocols. It may also be the case that besides the analyzed

and reported parameters there are still others not yet identified that are controlled or initiated by the

conditioning procedure. The picture becomes more complex considering that the immune system under-

takes sensitization (memory) and habituation (tolerance) processes, basically independent from the CNS.

In addition, it is known that several immune functions underlie circadian rhythms (Buijs et al., 2006).

These conditions complicate the prediction of the final nature and magnitude of the CR, often requesting

experimental trials. In this regard, some immune parameters modulated at recall may be the bizarre

reflection of neural activity that cannot be explained by orthodox learning and memory rules. For instance,

one such scenario would be that the delay between two recall trials is not long enough for certain immune

functions to return to baseline levels. Such successive recall trials may yield an additive effect in these and

related immune parameters rather than an extinction, which may occur in the neural correlates that elicited

these conditioned immune effects. In summary, immune responses can be affected recalling neuroimmune

engrams, but this does not necessarily imply that such immune responses were behaviorally conditioned.

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Neuro-immune associative learning 6 131

4 Principles of Neuroimmune Associative Learning

The following section introduces findings of neuroimmune associative learning experiments and discusses

them with regard to rules of orthodox learning and memory theory that are commonly accepted to apply

when nonimmune USs are employed. This is insofar of interest as the conditioned taste aversion paradigm,

which is employed in the majority of neuroimmune associative learning experiments, has certain unique

features. It has been reported that odor–immune association can be established under long interstimulus

intervals (e.g., up to 24 h) (Hsueh et al., 1992). Taste–visceral associations are weak with interstimulus

delays longer than 4 h (Hiramoto et al., 1992; Solvason et al., 1992). Taste/odor–immune association could

occur after only a single CS–US association, whereas visual/auditory/touch–immune association requires

further reinforcement trials (Domjan, 2005). Therefore, given the CNS–immune communication complex-

ity, it seems worth to take a closer look at rules applying to a given neuroimmune associative learning.

4.1 General Learning Rules of Neuroimmune Associative Learning

Intensive training by increasing the number of CS–US trials during association strengthens the conditioned

response. This is a commonly agreed principle in diverse conditioning paradigms and was first described by

Pavlov (1927) and also applying to neuroimmune associative learning (Espinosa et al., 2004). Although one

learning trial conditioning (e.g., pairing antigenic challenge with saccharin, Alvarez‐Borda et al., 1995;

Madden et al., 2001) has been reported many times, it is not a phenomenon general to all USs employed in

neuroimmune associative learning protocols. Therefore, a paradigm including several association trials may

produce more reliable CRs (Espinosa et al., 2004). In this regard, it has been reported that the magnitude of

the conditioned effects on the immunity is larger after intensive learning (Niemi et al., 2007).

Extinction is defined as the reduction in magnitude and duration of the conditioned response as a

consequence of unreinforced trials (i.e., CS alone) (Szczytkowski and Lysle, 2007). The extinction rate was

directly related to the volume of CS presented on the association trial (Ader, 1974). This principle has been

proven to apply to neuroimmune associative learning in many other studies (e.g., Bovbjerg et al., 1984;

Lysle et al., 1988). In contrast, a recent finding indicates that evoking a consolidated taste–CsA engram

several times resulted in a stronger conditioned immunosuppression (Niemi et al., 2007). Similar heterodox

findings have been reported on an analog taste–immunosuppression engram resulting from pairing

saccharin and cyclophosphamide. Here, the immunosuppression was also more pronounced after several

CS unreinforced exposures (Ader and Cohen, 1975; Rogers et al., 1976; Wayner et al., 1978). Possible

explanations for such peculiar results are due to an insufficient delay between recall trials (too‐near trials),in that immune functions do not have enough time to return to baseline levels, an additive effect may result

in a cumulative immunosuppression. An alternative hypothesis is that the conditioned immunosuppressive

state occurring at first recall is sensed by the CNS and acts as a reinforcement to induce reconsolidation

(Berman and Dudai, 2001; Eisenberg et al., 2003; Dudai, 2006) of the taste–immunosuppression engram

and hereby working as an additional association trial.

Regarding the phenomenon of passive forgetting (the retention rate after a delay between association

and recall phases), Markovic et al. (1988) found excellent retention of CS–US association 8 weeks after

association. Here, rats were sensitized with ovalbumin injections; afterward, conditioning comprised

saccharin as CS and ovalbumin as US. No extinction occurred during a 6‐day test period in terms of the

CTA behavior.

Contingency is defined as the occurrence of CS and US and is important for achieving associative

learning. Ader and Cohen (1982) demonstrated this in showing that a partial reinforcement protocol

significantly reduced the conditioned immunosuppressive effect. Apparently, contingency in neuroimmune

associative learning adheres also to common learning principles.

Backward association would be the case if the US precedes the occurrence of the CS. Odor–poly I:C

backward conditioning resulted in conditioned increased NK‐cell activity (Solvason et al., 1992). This

should not be possible for directly (fast) perceived US (see > Section 3), whereas an indirectly perceived

US, which takes longer to be sensed by the CNS, is more likely to occur in parallel with sensory and

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132 6 Neuro-immune associative learning

encoding steps of the CS, and therefore may become associated. In this case, a backward conditioned group

may help to delineate the nature of a given US or to discover the genuine US, respectively. An alternative

approach would be to systematically vary the CS–US interstimulus interval. In addition, if the UR kinetic is

known, the determination of the time point of the most pronounced CR enables extrapolation to the

genuine US that mediates the relevant information.

Latent inhibition, the CS preexposure may retard or diminish CS–US engram, thus reducing the

strength of the CR at recall. Such learning interference phenomena have been documented also in

neuroimmune associative learning (e.g., saccharin–ovalbumin Chen et al., 2004). However, the naturalistic

relation among stimuli should be considered to estimate the relevance of such a phenomenon in a given

conditioning protocol.

4.2 Features of Neuroimmune Associative Learning

Neuroimmune associative learning across life span: The strength of neuroimmune associative learning seems

to be age‐dependent. For instance Spector et al. (1994) report that old mice (24 months old) were able to

display a conditioned enhancement of NK activity in an odor–poly I:C conditioning protocol, but young

(3 months old) mice showed a stronger conditioned response. This reflects very much the unconditioned

response, which is detectable, although weaker in aged mice. This assumption is in line with reports

demonstrating a diminished conditioned immunosuppression in aged animals (Gorczynski, 1987b, 1991).

Two main factors could be the basis of aging effects on neuroimmune associative learning. Learning and

memory deficits are a common characteristic of CNS senescence (Nomura and Hori, 1996). However it

should also be considered that innervations to the immune organs change across life span, reducing

significantly during elderly stages. As has been reviewed (> Section 3.2), such neuroimmune efferent

neural pathways may be essential to induce the conditioned effect on immunity. Thus, it cannot be assumed

that a given conditioning protocol could be extrapolated to any stage of life.

Gender effects: Few systematic attempts have been undertaken to elucidate the role of gender or estruous

cycle on neuroimmune associative learning. Spector et al. (1994) reported conditioned increases in NK‐cellactivity in both male and female mice. However, it should be taken into account that a significant effect of

gonadal hormones on the neural (Kritzer et al., 2007) as well as on immune processes (Cutolo et al., 2006)

has been documented. Thus, it may not be surprising to find gender effects in a given neuroimmune

associative learning protocol.

Compensatory or paradoxical conditioned effects: These effects on immune responses have been reported

after evocation of a given neuroimmune associative learning. For example, Gorczynski and Kennedy (1984)

varied the time of day at which the initial association trial began and found that conditioned suppression

was developed by taste cues paired with CYduring the light portion of the diurnal cycle, whereas association

that began during the dark portion of the diurnal cycle resulted in either no CR or a conditioned

immunoenhancement. The authors hypothesized that the background level of neuroendocrine hormones

is critical to the direction of the CR to the taste cue paired with CY. However, Siegel et al. (1987) posited an

alternative hypothesis, indicating that the observation that most conditioning studies of regulatory

responses use stimuli other than taste cues suggest that the immunosuppression results in evoking a

taste–CY association may be unique to the use of taste cues. Other cues, inadvertently present in the

association regimen may control CRs different than those CRs elicited by taste cues (e.g., cue to conse-

quence specificity, Garcia and Koelling, 1967b).

Consciousness: An interesting finding is that association and recall phases can occur in anesthetized

subjects. Hsueh et al. (1992) reported that mice associate CS (camphor odor) and US (poly I:C) under

anesthesia. Second, if conditioned consciously, they could recall under anesthesia, even when CS/US

interstimulus interval was separated by 1–2 days. Since this unusual long interval differed from conscious

learning where the organism seeks information using a logical perceptual relation among events, the

authors reasoned that CS/US learning must be taken place unconsciously by different rules.

Immune history: Different immune histories among subjects may result in a divergent response to the

same immune stimulus. This yields a different immune reaction signaled to the CNS that may become

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Neuro-immune associative learning 6 133

associated with a CS. Therefore, at recall phase, the conditioned response may vary in magnitude or even in

direction depending on the initial immune reaction. Evidence supporting this hypothesis comes from a

study showing that in an anaphylaxis model, physiological responses to first and second antigen exposure

differs drastically: CTA response (Djuric et al., 1988; Markovic et al., 1988), immunological, neural, and

behavioral responses (Costa‐Pinto et al., 2005). For instance, a recent study documented a highly significant

difference in lipopolysaccharide‐induced CTA behavior depending on the individual immune history (i.e.,

tolerant vs. naıve) (Pacheco‐Lopez et al., 2007a). Drug tolerance is another phenomenon that may affect

neuroimmune associative learning. Repeated treatment with a drug may reduce its specific effect on the

organism, a phenomenon termed drug tolerance. Therefore, if used as US with several CS–US pairings, the

signaling of this drug may alter with each administration. If so, this yields to different information possibly

becoming associated with the CS (e.g., Dyck et al., 1987). The first pairing may involve a different US signal

than the second, where it may be less pronounced.

Another key finding comes from experiments of odor–poly I:C conditioning in which several immune

responses (e.g., NK cell, neutrophil, and cytotoxic T cell) are affected on recall. However, a systematic

analysis led to the conclusion that the enhanced immune responses after recall depended on the immuno-

logical history of the particular subject (Demissie et al., 1997, 2000).

Species differences: Differences among species in a given conditioning paradigm do also exist. Although

conditioned immunosuppression in a saccharin–CsA conditioning paradigm is apparent in all three

species, it is reported that mice did not develop CTA behavior (Niemi et al., 2006), but rats do whereas

humans have reported a reduced palatability of the conditioned taste (Goebel et al., 2002). Dissociation of

CTA behavior and conditioned immunosuppression was found here in mice (e.g., Bovbjerg et al., 1984).

CS/US administration route: The delivery route of CS and US may affect the conditioned response. For

instance, it was found that the mode of administration of saccharin (CS) and ovalbumin (US) significantly

affects the conditioned behavioral response. The most effective mode to induce a conditioned taste aversion

behavior was CS periorally and US intraperitoneally. Strikingly, this mode yielded the mildest symptoms of

anaphylactic shock (UR) compared with CS intravenously/US intravenously mode and CS periorally/US

intravenously (weakest CTA response) (Markovic et al., 1988). Such results are in concordance with the

naturalistic relation of postprandial immunotoxicological consequences. Although according to established

associative learning rules, the extent of the UR determines the extent of the CR (Domjan, 2005); the

magnitude of the CR is often smaller than the UR. However, in the earlier case changing the route of

administration led to violation of this law.

Neuroimmune engram specificity: Solvason et al. (1991) demonstrated that a given CS creates specific

and independent neuroimmune engrams. Two odors (camphor and citronella oil) were paired with poly I:C

as a US. Mice were able to discriminate between the two CS when reexposed to either one of them in the

sense that they showed a CR to that CS that had been paired with the US during association phase, but not

to the other one.

Different CS served successfully in different conditioning protocols. Among the reported data are environ-

ments (Coussons‐Read et al., 1994; Szczytkowski and Lysle, 2007); visual (MacQueen et al., 1989) and auditory

(Harris and Fitzgerald, 1989;MacQueen et al., 1989); stimuli or such complex procedures like surgical sham skin

grafts (Gorczynski et al., 1982) (see also >Table 6‐1 and >Table 6‐2). In general, it can be assumed that

novelty, intensity, distinctiveness, and uniqueness are relevant characteristics of an effective CS.

5 Mechanisms of Neuroimmune Associative Learning

5.1 Neurobiology of Association and Recall

The neural network involved in taste–visceral associative learning includes mainly sensory and hedonic

pathways (Sewards and Sewards, 2002; Sewards, 2004). Among the involved brain structures are consistently

the nucleus tractus solitary, parabracchial nucleus, medial thalamus, amygdala, and insular cortex (Yamamoto

et al., 1994). In particular, the insular cortex subserves the association, retrieval, retention, and extinction of

taste–visceral memories (Nerad et al., 1996; Bermudez‐Rattoni et al., 1997, 2004; Pacheco‐Lopez et al., 2007b),

Page 12

. Table 6-2

Conditioned immune responses on animal disease models

Disease

model

Conditioned

response

Unconditioned

stimulus

Conditioned

stimulus Species References

Allergy Delayed‐typehypersensitivity

response ↓

Cyclophosphamide SAC Mouse Roudebush and

Bryant (1991)

Delayed‐typehypersensitivity

response ↑

Cyclophosphamide SAC Mouse Bovbjerg et al.

(1987)

Tumor

model

Survival time of

tumor‐bearinganimals ↑

Poly I:C Camphor odor Mouse Ghanta et al.

(1987, 1988)

Tumor growth ↓ Allogenic DBA/

2 spleen cells

Camphor odor Mouse Ghanta et al.

(1990)

Myeloma growth ↓

cytotoxic

T‐lymphocyte

response to

YC8 tumor ↑

Poly I:C Camphor odor Mouse Ghanta et al.

(1995)

Increased and

decreased

development of

DMBA‐inducedtumors ↑↓

Cyclophosphamide

cimedine (histamine

type II‐receptorantagonist)

SAC Mouse Gorczynski et al.

(1985)

Autoimmune

disease

Proteinuria and

mortality ↓

Cyclophosphamide SAC Mouse Ader and Cohen

(1982)

Grafting Cytotoxic

T‐lymphocyte

precursor cells ↑

C57BL/6 lymphoid

cells inoculated i.p.

Sham skin

grafts

Mouse Gorczynski et al.

(1982)

Heart graft survival ↑ Cyclosporine A SAC Rat Grochowicz

et al. (1991)

Arthritis Arthritic

inflammation

SAC‐vanilladrink

Rat Klosterhalfen and

Klosterhalfen

(1983)

Asthma Histamine release ↑ Bovine serum

albumin (with prior

sensitization)

Methylsulfide

and

triethylamine

odor

Guinea

pig

Russell et al.

(1984); Dark

et al. (1987);

Peeke et al.

(1987)

Histamine ralease ↑ Ovalbumin (with

prior sensitization)

Dimethylsulfide

odor

Guinea

pig

Irie et al. (2001,

2002, 2004)

134 6 Neuro-immune associative learning

and it has been postulated that the insular cortex may integrate gustatory and visceral stimuli (Sewards and

Sewards, 2001). More recently, using the neuronal activity marker c‐Fos, it was possible to confirm the

preponderant role of the insular cortex in conditioned increase of antibody production (taste–ovalbumin)

(Chen et al., 2004). In particular, reexposure to the CS yielded significant increase in c‐Fos expression in all

insular areas (granular, dysgranular, and agranular) 120 min postrecall, confirming previous observations

employing an excitotoxic lesioning approach (Ramırez‐Amaya and Bermudez‐Rattoni, 1999).Regarding other forebrain structures, the amygdala seems to play an important role during the

formation of aversive ingestive associations (Reilly and Bornovalova, 2005), and is also relevant for

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Neuro-immune associative learning 6 135

limbic–autonomic interaction (Swanson and Petrovich, 1998). A series of reports has indicated that the

insular cortex and the amygdala are key structures in conditioned immunosuppression after evoking taste–

CY and odor–CY associations (Ramırez‐Amaya et al., 1996, 1998).

It has also been proposed that the ventromedial hypothalamic nucleus, widely recognized as a satiety

center (Vettor et al., 2002), is intimately associated with sympathetic facilitation in peripheral tissues

(Saito et al., 1989), including modulation of peripheral immune reactivity (Okamoto et al., 1996). In

agreement with previous reports employing a taste–CY engram (Ramırez‐Amaya et al., 1996, 1998, 1999),

the neural substrates involved in the immunosuppression resulting in evoking a taste–CsA association in

rats have been identified (Pacheco‐Lopez et al., 2005). The conditioned effect on the immune system that

reduced splenocyte responsiveness and cytokine production (IL‐2 and IFN‐g) was affected by brain

excitotoxic lesions. These data show that the insular cortex is essential for acquiring and evoking this

conditioned response. In contrast, the amygdala seems to mediate the input of visceral information

necessary at association time, whereas the ventromedial hypothalamic nucleus appears to participate in

the output pathway to the immune system, needed

(> Figure 21-3 ).

to evoke the behaviorally conditioned immune response

. Figure 6‐3At evocation time, conditioned animals are reexposed to the conditioned stimulus (CS). This gustatory

information is centrally processed through brain stem relays (nucleus tractus solitarius: NTS, parabracchial

nucleus: PBN), reaching the insular cortex (IC). This neocortex, together with the amygdala (Am), is indispens-

able during the association phase, and is also necessary in evoking conditioned ingestive behavior (aversion/

avoidance). The ventromedial nucleus of the hypothalamus (VMH) is essential for evoking the immunosup-

pressive conditioned response in the periphery, also recruiting other hypothalamic nuclei such as the lateral

hypothalamus (LH)

Several attempts have been undertaken to elucidate central processing in conditioning protocols

applying antigen as US. Brain lesions of specific brain areas revealed that, again, the insular cortex and

the amygdala are indispensable for associating saccharin with an immune response induced by a protein

antigen (Ramırez‐Amaya and Bermudez‐Rattoni, 1999). Although the hippocampal plasticity is modulated

by neuroimmune interactions (Avital et al., 2003; Ikegaya et al., 2003; Balschun et al., 2004), this limbic

structure seems not to be involved in the taste–immune association process (Ramırez‐Amaya and

Bermudez‐Rattoni, 1999). In the same report, the authors successfully conditioned an almond odor with

hen egg lysozyme antigen, and insular cortex lesion blocked this association. Interestingly, this neocortex is

not essential for odor‐conditioned aversive behavior (Kiefer et al., 1982).

In a modified classical passive avoidance paradigm, ovalbumin‐immunized mice avoid a context that

has been paired with the allergen (Costa‐Pinto et al., 2005). Ovalbumin aerosol was employed as an aversive

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136 6 Neuro-immune associative learning

US, the CS was a usually preferred dark compartment of a box. After association phase, animals avoided

this compartment and spent more time in the bright (usually aversive) compartment of the box. The

expression of c‐Fos expression was tracked after airway ovalbumin challenge and it was found to be

enhanced in the hypothalamic paraventricular nucleus and the central nucleus of the amygdala in

sensitized mice. Therefore, it is likely that the immediate hypersensitivity reaction in allergic asthma,

which is characterized by IgE‐mediated mast‐cell deregulation with concomitant histamine release, plays

a major role in the reported brain activity (Costa‐Pinto et al., 2005). In addition, these structures are

commonly linked to emotional behavioral patterns that are also involved in conditioned taste aversion

(Bermudez‐Rattoni, 2004).Regarding neural mechanisms behind the odor–poly I:C conditioning paradigm, it has been reported

that IFN‐b is the major input to the CNS during association phase. It can replace poly I:C as US to acquire a

conditioned increase in NK‐cell activity; interestingly, this is not the case for IFN‐a (Solvason et al., 1988).

This association can be achieved by intravenous (i.v.) administration of IFN‐b at a dose of 10,000 IU, but

not at 1,000 IU. Further, if IFN‐b was directly delivered into the CNS via the cisterna magna, a smaller dose

(100 IU) sufficed to be associated with the CS. In turn, the CR was blocked by injecting anti‐IFN‐b(100 neutralizing units) into the cisterna magna 24 h before association (Solvason et al., 1993). The afferent

pathway for association yielding conditioned increased NK‐cell activity seems to be a different one to that of

the conditioned fever in the same associative learning conditioning protocol (Rogers et al., 1992). The i.p.

injection of sodium carbonate blocked CS–US association for enhancement of NK‐cell activity, but leftconditioned fever response unaffected. Conversely, indomethacin treatment which is known to prevent

prostaglandin synthesis blocked the conditioned fever response but not the conditioned NK‐cell response.Using a pharmacological approach, the neurochemical features of the conditioned effect enhancing NK‐cellactivity in rodents (odor–poly I:C association) have been described in detail by one group. Administering

lidocaine centrally blocked both association and recall of the CR (Rogers et al., 1994a) and the CNS sensory

processes of the CS, but not of the US. Similarly, peripheral and central treatment with monosodium

glutamate (Ghanta et al., 1994) or sodium carbonate (Rogers et al., 1994b) apparently blocks association,

but not CS perception. Moreover, the hypothalamic arcuate nucleus is necessary for association, but not for

recall of the CR (Ghanta et al., 1994). Recall in this paradigm seems to be mediated by central opioid

pathways; peripheral injection of the opioid receptor antagonist quaternary naltrexone (not penetrating the

CNS) does not affect recall of the CR. Naltrexone given before association did not prevent the conditioned

effect (Solvason et al., 1989), indicating independency of central opioid pathways.

Central catecholamines seem to be essential, and glutamate – but not GABA – is also required at recall

stage (Hsueh et al., 1999; Kuo et al., 2001). In particular, reserpine treatment before recall, which

unspecifically depletes central and peripheral catecholamine contents, blocked the CR (Hiramoto et al.,

1990). More recently, it has been shown that a‐ and b‐adrenoceptor antagonists or dopamine (DA)‐1 and

DA‐2 receptor antagonists given shortly before recall also blocked the CR (Hsueh et al., 1999).

Furthermore, it has been demonstrated that cholinergic, as well as serotonergic, central systems are

required in triggering conditioned NK‐cell response (Hsueh et al., 2002). At association, acetylcholine is

believed to act through nicotinic, M2‐, and M3‐muscarinic receptors, whereas at recall M1‐, M2‐ and M3‐muscarinic receptors have been identified to be crucially involved. In both association and recall phase,

serotonin acts through 5‐HT1 and 5‐HT2 receptors to affect the CR.

Neurotransmitter contents in certain brain areas during recall stage have been analyzed, revealing a

significantly higher norepinephrine content in the cerebellum and dopamine content in the striatum and

hippocampus in conditioned animals compared with controls (Hsueh et al., 1999). Interestingly, glutamate

contents at recall in this paradigm in the same brain areas did not differ between groups (Kuo et al., 2001).

In an identical conditioning protocol (camphor odor, poly I:C) with enhanced neutrophil activity as CR

instead of NK‐cell activity (Chao et al., 2005) measured levels of tyrosine hydroxylase in several brain areas

24 h after recall to localize action sites of catecholamines. Conditioned animals displayed significantly more

tyrosine hydroxylase expressing neurons in the hypothalamus, cortex, and locus coeruleus compared with

control animals. But this is unlikely to be related to a neural memory process since a 24 h memory trace is

rather unusual.

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Neuro-immune associative learning 6 137

In addition to classical neurotransmitters, cytokines have been demonstrated to play an important role

within the CNS, modulating neuronal and glial function in nonpathological settings such as learning and

memory processes (Balschun et al., 2004; Dantzer, 2004; Tonelli et al., 2005). Specifically, proinflammatory

cytokines, such as IL‐1, IL‐6, and TNF‐a, have been shown to modulate spatial learning tasks, as well as

long‐term potentiation phenomena (Gibertini, 1996; Schneider et al., 1998; Fiore et al., 2000; Banks et al.,

2001; Matsumoto et al., 2001, 2002; Rachal Pugh et al., 2001; Lynch, 2002). In this sense, it can be assumed

that cytokines may be a significant factor in the associative processes occurring during behavioral condi-

tioning of immune functions. Apart from these neuromodulatory properties, proinflammatory cytokines

seem to play an important part in the afferent pathway between the immune system and the CNS

(Besedovsky and del Rey, 1996; Turnbull and Rivier, 1999; Dantzer, 2004). Therefore, it can be hypothesized

that central cytokines may act as mediators in the brain during an ‘‘immune‐sensing’’ phase in the

association phase. These hypotheses are supported by observations that (1) receptors for these proinflam-

matory cytokines are expressed in the CNS (Szelenyi, 2001; Sredni‐Kenigsbuch, 2002), (2) peripheral

immune changes affect central cytokine production and cytokine receptor expression in the brain (Pitossi

et al., 1997; Del Rey et al., 2000), and (3) cytokines can act as unconditioned stimuli to induce conditioned

taste aversion/avoidance (Tazi et al., 1988; Dyck et al., 1990; Janz et al., 1991; Hiramoto et al., 1993).

The underlying mechanisms when using SEB as US are completely unknown. However, systemic IL‐2administration has been found to modify central monoamine activity (Lacosta et al., 2000). Similarly,

striatum catecholamine concentrations followed a dose–response curve in reaction to increased peripheral

SEB immunization (Pacheco‐Lopez et al., 2004). Therefore, association may involve T‐cell‐derived cyto-

kines like IL‐2 signaling to the CNS.

5.2 Peripheral Mediation of the Conditioned Effects on Immunity

The available data suggest that the effects of conditioning could be mediated by a preferential effect on

T cells. Conditioned suppression of lymphoproliferative responses in rats and mice, for example, has been

observed in response to T‐cell mitogens but not (or less reliable) in response to B‐cell mitogens (Neveu

et al., 1986; Kusnecov et al., 1988; Lysle et al., 1990, 1991). Immune adoptive transfer experiments also

suggest that conditioning may be mediated by T‐cell changes (Gorczynski, 1987a). Splenocytes from

conditioned or experimentally naıve animals were transferred into irradiated naıve or conditioned animals

that were or were not subsequently reexposed to the CS. The observed increases or decreases in the

antibody‐forming cell response to SRBC depended on the donor cells and the conditioning treatment

experienced by the recipient. The separate transfer of enriched Tand B cells into naıve or conditioned animals

suggested that conditioning effects were attributable to the adoptively transferred T cells (Gorczynski, 1991).

However, the specificity of whether conditioning can modulate the antibody response to different types of

antigens has not been resolved.

With respect to adrenocortical influences, it is reasonable to hypothesize that conditioned alterations in

immunologic reactivity could be mediated by conditioned neuroendocrine changes. In experiments in

which humoral immune responses were assessed in conditioned animals, the recall of a taste–LiCl

association did not affect antibody responses to SRBC, in contrast to the immunosuppression that was

observed after evoking the taste–CY engram (Ader et al., 1979). This finding indicates that an immuno-

suppressive drug was needed to cause immunosuppression during association phase, thus supporting a

taste–immunosuppression engram interpretation.

In follow‐up experiments, circulating levels of adrenocortical steroids were artificially elevated by

exogenous administration of corticosterone at the time of antigen injection to mimic the stress response

occurring in animals reexposed to the taste paired with sickness (Ader et al., 1979). Elevated glucocorticoid

levels did not significantly lower antibody titers. These data would appear, a priori, to exclude the possibility

of a stress‐mediated phenomenon in the genesis of conditioned immunosuppression.

However, experimental data, in which the delayed‐type hypersensitivity (DTH) response was used as an

index of T‐cell function, support the hypothesis that changes in the immune function of animals subjected

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138 6 Neuro-immune associative learning

to conditioned taste aversion might be better viewed as a secondary consequence of the psychological

conflict affecting thirsty animals that are exposed to a taste solution previously associated with sickness

(Kelley and Dantzer, 1988). According to this hypothesis, the immunosuppressive status after evocation

phase is a consequence of the conflict of the strong motivation to drink in fluid‐deprived animals against

the aversive memories associated to the CS. Accordingly, it should be possible to induce immunosuppres-

sion in conditioned animals even when no immunosuppressive drug is used during association phase (as

US). After a taste–LiCl association, conditioned animals showed a strong conditioned taste aversion, but

also an immunosuppressive status in the T‐cell function only when a forced choice test was implemented,

but not in a two‐bottle preference procedure which eliminates the psychological conflict of thirst versus

aversive memories (Kelley et al., 1984, 1985). In contrast to this stress hypothesis, a serum corticosterone

time–course study performed to examine the possible involvement of glucocorticoids in conditioned

immunosuppression of the DTH response has been published (Roudebush and Bryant, 1991). Animals

were sacrificed 30, 60, 90, 120 min and 24 h after evoking a taste–CY engram. No significant differences in

serum corticosterone levels were detected between nonconditioned controls and the conditioned groups at

any time point. Supporting this line of thinking, several other research groups have consistently reported that

the suppression of splenic T‐cell proliferation was independent of the stressor‐induced increase in adrenocor-

tical activity (Mormede et al., 1988; Lysle et al., 1990; Exton et al., 1998).

Importantly, both conditioned and stressor‐induced alterations in immune and nonspecific defense

responses have been attributed to the action of central and peripheral catecholamines. For instance, it has

been reported that both chlorpromazine and amitriptyline, both centrally acting, abolish the immunosup-

pressive status elicited by recalling a taste–CYengram (Gorczynski and Holmes, 1989). Furthermore, it has

also been reported that the b‐adrenergic antagonist, propranolol, blocked the immunosuppressive effects of

a conditioning stress paradigm (Lysle et al., 1992). Supporting the involvement of peripheral catechola-

mines, nadolol (a/b‐adrenergic antagonist that does not cross the blood–brain barrier) blocked the electric

shock‐induced suppression of splenic, but not peripheral, blood lymphocyte proliferation following

mitogenic stimulation ex vivo (Cunnick et al., 1990). In this regard, we have previously revealed that the

immunosuppressed status after evoking a taste–CsA engram is not related to the activation of the HPA axis

and is merely mediated by the neural innervation of the spleen, via noradrenaline–b‐adrenoceptors‐dependent mechanisms (Exton et al., 1999, 2002; Xie et al., 2002). It has been demonstrated that splenocyte

reactivity is modulated in part by tonic inhibition from the splenic nerve (Okamoto et al., 1996) and

sympathetic splenic innervation seems to be under the central control of the ventromedial hypothalamus

(Katafuchi et al., 1993, 1994). While electrical stimulation of the ventromedial hypothalamus has been

found to arouse sympathetic activity (Saito et al., 1989), the lateral hypothalamus seemed to do the

opposite (Bernardis and Bellinger, 1993). Thus, the lateral hypothalamus may exert immunoenhancing

properties (Wrona and Trojniar, 2003) in part by antagonizing the ventromedial hypothalamus, and thereby

reducing sympathetic tone inhibition. Importantly, such hypothalamic regulation of sympathetic activity

seems to be modulated by the insular cortex (Allen et al., 1991; Cechetto and Chen, 1992; Oppenheimer

et al., 1992; Butcher and Cechetto, 1998).

Concerning the conditioned enhancement of NK‐cell activity, several mechanisms have already been

elucidated. On the neural efferent pathway, sympathetic innervation of the spleen seems not to be responsi-

ble for mediating the conditioned response. Despite peripheral sympathectomy using 6‐hydroxydopamine

(6‐OHDA) between association and recall, the CR still occurred (Hiramoto et al., 1990). However, splenic

denervation should give final evidence. Interestingly, applying the same conditioning protocol, but measur-

ing neutrophil activity as UR and CR, respectively, peripheral sympathectomy before recall completely

abrogated the CR (Chao et al., 2005). On the humoral efferent pathway, elevated plasma adrenocorticotropic

hormone (ACTH) levels and splenic IFN‐a expression were measured in conditioned animals at recall time

(Hsueh et al., 1994b); no effect was found for b‐endorphin concentrations. Peripheral administration of the

synthetic glucocorticoid dexamethasone blocked recall, but not association of the CR, assumingly by

negative feedback inhibition of HPA axis activity (Hsueh et al., 1994a). Dexamethasone treatment before

recall resulted in elevated neutrophil activity in conditioned animals compared with controls, indicating that

this specific CR is independent of HPA activity (Chao et al., 2005).

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Neuro-immune associative learning 6 139

6 Clinical Implications

6.1 Animal Models

>Table 6‐2 summarizes conditioned effects affecting critical immune responses in animal disease models

with potential clinical application.

The DTH response refers to an overreaction produced by the immune system given a presensitized

immune state of the host. A conditioned decrease (Roudebush and Bryant, 1991) and increase (Bovbjerg

et al., 1987) of this response has been observed by pairing saccharin with CY. Similarly, Exton et al. (2000)

reported a conditioned suppression of the contact hypersensitivity reaction by pairing saccharin with CsA.

Conditioning protocols attested a significant impact on tumor‐related models. For instance, tumor

growth was demonstrated to be enhanced as well as delayed by applying different US (CY and cimedine, a

histamine type II receptor antagonist) (Gorczynski et al., 1985). Moreover, the survival time of tumor‐bearing mice has been prolonged in conditioned animals (Ghanta et al., 1988).

Evoking a taste–CY engram, Ader and Cohen (1982) demonstrated a conditioned retardation in

proteinuria and mortality in New Zealand hybrid mice which are prone to develop an autoimmune disease

resembling human lupus erythematosus. Most interestingly, in a follow‐up study they could show that

the lupus‐prone MRL‐lpr/lpr mice displayed a weaker CTA compared with congenic controls. The

authors interpreted that the lupus‐prone animals seek to regain homeostasis by consuming the tasting

solution to achieve the therapeutic immunosuppressive status elicited by evoking the taste–CY engram

(Grota et al., 1987).

Another example of conditioning effects on immunity is documented by grafting experiments. Gorczynski

et al. (1982) performed skin grafting as CS in combination with i.p. injected lymphoid cells of another

mouse strain as US. Reexposing the conditioned animals to the sham‐grafting procedure, they showed

an increase in cytotoxic T‐lymphocyte precursor cells specific for alloantigens on the grafted tissue.

In addition, Grochowicz et al. (1991) reported a conditioned prolongation of the survival time of heart

allografts in rats evoking a saccharin–CsA engram. In follow‐up experiments, Exton and colleagues

(1999) demonstrated a significant prolongation of the survival of transplanted hearts, including long‐term survival (>100 days) of transplants in 20% of the animals that were conditioned and additionally

subtherapeutically treated with CsA. In addition, employing taste–CsA association Klosterhalfen and

Klosterhalfen (1983, 1990) extended the conditioned immunosuppression to an arthritis model. Before

induction of adjuvant arthritis, rats were dosed with cyclophosphamide (US) after presenting a distinctive

saccharin/vanilla solution (CS). Conditioned rats showed no external signs of a proliferation of inflamma-

tion, whereas approximately half of the animals in the control groups developed small lesions.

A series of studies dealt with conditioning of asthma‐like symptoms, anaphylactic shock (Noelpp

and Noelpp‐Eschenhagen, 1951c; Djuric et al., 1988; Palermo‐Neto and Guimaraes, 2000), or histamine

release, respectively (Dark et al., 1987; Irie et al., 2001, 2002, 2004) that have been already reviewed

(> Sections 1.3, 1.4, and 4.2).

6.2 Human Studies

This section summarizes the few, but promising research reports of conditioned effects modulating immune

responses in human subjects, indicating potential therapeutic outcomes of this behavioral approach.

A Japanese study from the early 1960s reported a conditioned dermatitis response in adult male subjects

elicited by evoking a specific association (CS: blue solution topically applied also contains 2% raw extract

Rhus vernicifera: US) (Ikemi et al., 1962). A case report of asthmatic patients suffering from skin sensitivities

to house‐dust extract and grass pollen shows a conditioned effect. The subjects were exposed to these

allergens by inhalation (Dekker et al., 1957). After a series of conditioning trials, they experienced allergic

attacks after inhalation of the neutral solvent used to deliver the allergens. This work showed not only fast

conditioning of the asthmatic attack (CR), but also tenacious retention, that is, lack of extinction. Similarly,

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140 6 Neuro-immune associative learning

presenting a novel‐tasting and novel‐appearing drink as CS with house‐dust mite allergen as US to patients

with allergic rhinitis yielded an associative learning (Gauci et al., 1994). In addition, pairing an olfactory cue

as CS with nasal challenge with seasonal grass allergens to investigate conditioning of the immediate

hypersensitivity reaction in hay fever (seasonal allergic rhinitis) produced conditioned increase of histamine

release and decrease nasal airflow at recall (Barrett et al., 2000). Furthermore, there was evidence of

extinction and a follow‐up experiment showed a more pronounced conditioned effect after three associa-

tion trials compared with one, indicating a reinforcement effect. An elevated mast‐cell tryptase in mucosa

was observed when an intranasal saline application was given simultaneously with the CS at recall. Another

type of allergic reaction, the DTH response, was tested in healthy volunteers who received five monthly

tuberculin skin tests (Smith and McDaniel, 1983). In this conditioning protocol, both tuberculin (US) and

saline were injected; the latter was taken from a green vial (CS�) and the former was drawn from a red vial

(CSþ). On the test day, the color labeling of the substances was reversed. Although the saline injections did

not induce a skin reaction (erythema and induration), the severity of the symptoms was significantly

blunted in all the subjects tested when the tuberculin was drawn from the green vial (i.e., conditioned

compensatory effect), where subjects expected their reactions to be negative. However, a similar protocol

using various allergens (e.g., mite dust, fur) taken from colored vials did not result in conditioned

modulation of skin reactions in the subjects tested (Booth et al., 1995).

Another series of experiments investigated the role of conditioning in the context of cancer treatment

and chemotherapy (Bovbjerg, 2003). Some chemotherapeutic agents such as CY have immunosuppressive

effects. For the patient, often the treatment visits to the hospital become aversive and act as CS–US

association trials pairing whereby a variety of features such as white coat, distinct smell when entering

the building, the clinic itself, its smell, the clinician’s voice, and so on may act as distinctive salient stimuli

(CS) that are contingently paired with the chemotherapy (US) (e.g., CY). Immune function was assessed in

cancer patients in the hospital before chemotherapy and compared with assessments conducted at home.

Proliferative responses to T‐cell mitogens were lower for cells isolated from blood samples taken in the

hospital (i.e., after recall) than for home samples (Bovbjerg et al., 1990). These results were replicated in

ovarian cancer patients (Lekander et al., 1995) and pediatric cancer patients receiving chemotherapy (Stock-

horst et al., 2000). In addition, chemotherapy patients often develop conditioned/anticipatory nausea (Andry-

kowski, 1988; Bovbjerg et al., 1990; Morrow et al., 1991; Matteson et al., 2002), anxiety (Jacobsen et al., 1993;

DiLorenzo et al., 1995), and fatigue (Bovbjerg et al., 2005) responses to reminders of chemotherapy. To

resolve such severe consequence of chemotherapy treatment, one may use some of the learning principles

that have been shown to apply to neuroimmune associative learning (> Section 4), for example, latent

inhibition to prevent such undesirable conditioning effects. Several visits to the clinic where the chemo-

therapy will take place should be recommended before starting the chemotherapy. Another possible

behavioral prophylactic therapy may be accomplished before chemotherapy, a specific novel stimulus

that could be administered under the physician’s control, for example, a novel and distinctive tasting/

flavored/colored/sparkling solution. Such artificial stimulus will be categorized and associated to negative

chemotherapy hedonic values; however, it may prevent the development of possible associations to other

high‐protein/caloric food input (Scalera, 2002).

Regarding conditioning of cellular immune parameters, one group assessed the conditionability of

augmentation of NK‐cell numbers and their lytic activity in healthy subjects. Although a conditioned

response was evoked after pairing a given taste with subcutaneous administered adrenaline (Buske‐Kirschbaum et al., 1992, 1994), these effects could not be replicated (Kirschbaum et al., 1992). In multiple

sclerosis patients, four monthly CY infusions (US) were paired with an anise‐flavored syrup (CS) (Giang

et al., 1996). Long‐term treatment with CY decreases blood leukocyte numbers, which often leads to

leukopenia. After a long period such as 6 months of administering the placebo infusion paired with the

drink, 8 out of 10 patients showed a conditioned reduction in peripheral leukocytes numbers. In addition,

by pairing subcutaneously interferon‐g injections (US) with a distinctively flavored drink (CS), it was

possible to induce an elevation of neopterin and quinolinic acid serum levels after evoking such an

association in healthy volunteers (Longo et al., 1999). However, it has been hypothesized that more than

a single associative learning trial pairing a distinctive taste (CS) with interferon‐b injections (US) is

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Neuro-immune associative learning 6 141

necessary to produce immune‐conditioned effects (Goebel et al., 2005). This view is supported by experi-

mental data for healthy male volunteers where the immunosuppressive drug cyclosporine A (US) was

paired four times with a distinctively flavored/colored solution (CS) (Goebel et al., 2002), inducing taste–

immune associative learning. After association, the drink (CS) alone induced conditioned inhibition of

ex vivo cytokine (IL‐2 and IFN‐g), mRNA expression, and cytokine release, as well as of the proliferative

responsiveness of human peripheral blood lymphocytes, similar to the CsA effect. In addition, a study with

patients suffering from allergic house‐dust mite rhinitis received pairing of a novel‐tasting drink (CS) and

the H1‐receptor antagonist, desloratadine (US) on five consecutive days. On reexposure, conditioned

patients displayed a decreased basophil activation, assessed in a skin prick test, and subjective symptom

score to a degree that was similar to drug treated control group (Goebel et al., 2007).

In summary, the reported results imply that introduction of behavioral conditioning to supplement

standard pharmacotherapeutic treatment may enable the physician to maintain some desired physiological

state or diminish pathological states. The outlook will be of reducing undesired side effects and maximizing

the effects of pharmacological therapies with a lower cumulative amount of active drug than is currently

used (Ader and Cohen, 1985).

7 Methodological Considerations for Neuroimmune AssociativeLearning Experiments

The following methodological considerations may improve neuroimmune associative learning establish-

ment in new settings as well as its further experimental development and clinical translation.

Ader (2003) suggested the following experimental groups to be included in conditioning protocols,

testing its impact on immune parameters:

Conditioned group: It constitutes the primary experimental group. After CS–US pairing during associa-

tion, this group is reexposed to the CS during recall.

Conditioned not reexposed group: This group is identically treated as conditioned group during associa-

tion phase, and it is not reexposed to the CS during recall phase. It serves as a control for any direct or

indirect immunomodulating effects of the association procedure per se, as well as for possible residual

effects of the US at time of recall.

Unconditioned response group: This group is also identically treated to conditioned group during

association, and receives additionally US reexposure during recall without the CS. This is the pharmaco-

logical control that defines the magnitude and direction of the UR, and also controls for immunological and

other assays being performed to gather the data.

Latent inhibition group: This group is preexposed to the CS before submitted to the entire conditioning

procedure identical to conditioned group. Here, a reduced CR is expected since preexposure should weaken

the strength of association.

Noncontingent conditioned group: This group receives the same number and amount of stimuli as

conditioned group, but CS and US are not timely paired. During recall, this group is also reexposed to the

CS. It controls for nonassociative factors, and certifies the neutrality of the CS in terms of immunological

effects.

Placebo group: This group is exposed to the CS at times as conditioned group is, but never receives the

US; instead, an immunologically neutral stimulus such as saline is administered as placebo. It controls for

residual CS effects and possible procedural artifacts like handling, injections, and so on.

The difficulty for the investigator lies not so much in inducing conditioned immune alterations, but in

employing the proper controls, both immunological and psychological, to demonstrate the predictability

and reliability of such effects, and consequently, the controllability. Therefore, the more control groups one

employs, the better the data are to interpret. Although it is a matter of costs and in the case of human

studies, in particular such involving patients, an ethical issue.

Some considerations about the nature of both the US and CS are provided in the following paragraphs,

complementing previous elaborations (Spector, 1987).

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142 6 Neuro-immune associative learning

The unconditioned stimulus should be known in detail in terms of immunologic sequel, and the sequel

must be reproducible. This means that applying a drug with an attenuating effect with subsequent

administration is useless for more than one association trial, since different immunological statuses

would be paired with the CS. In addition, the dose level must be reproducible. For ethical reasons, the

drug should have minimal side effects when experimenting with humans.

The conditioned stimulus should be easily perceived by each subject. This means that it should be novel

and distinct. This is not trivial, since the experimenter needs to find a stimulus that is new to a diverse

population that encountered numerous tastes, odors, colors, and so on in daily routine. Note also

that for instance about 30% of the human population may be anosmic to camphor odor (Spector, 1987).

The CS must also be continuously perceived by each subject without attenuation on repetition, similarly

to the US. It must be immunologically neutral and harmless. The conditioning procedure must be carried

out in a constant environment, since its cues have been proven to be part of the CS. The conditioning

procedures often inadvertently manipulate additional stimulus components that may not be identified

explicitly (e.g., the deodorant or voice of the experimenter, the smell of the room, the sound of a church

bell nearby, etc.). A lack of attention to environmental components of the CS may make conditioned effects

in the immune system appear to be small, although they may just be masked by the presence of environ-

mental cues. In addition, it should be taken into account that the development of the CR depends on

the modality of the CS; aversions produced by association with drug‐induced visceral illness are

often specific to gustatory or olfactory components of the CS (Garcia and Koelling, 1967a). The procedure

should be carried out at the same time of the day, since most immunological parameters underlie circadian

rhythm.

Finally, some issues should be solved to standardize a given conditioning protocol with a clinical

perspective. For instance, it is unknown how long the conditioned response lasts. Is reconditioning

possible (to maintain the conditioned response in a longer perspective, extinction must be avoided)?

When does forgetting set in? Are side effects of the US also conditioned? How controllable is the

conditioned response in a population that in contrast to a laboratory animal comprises differing immune

and psychological history/status? In addition, other factors such as age, gender, and cultural background

must be taken into consideration by health practitioners implementing behavioral conditioning as a

supplement therapy.

8 Closing Remarks

Neuroimmune associative learning is one of the most fascinating examples which uncover the multiple

interactions among the nervous, endocrine, and immune systems. Contributions to this line of research are

coming from various fields of expertise, from the molecular to the behavioral level. This behavioral model is

unique in elucidating the CNS–immune interactions, because it unites the afferent (during association

phase) and efferent (during evocation phase) pathway between these systems in one model, but with the

experimental possibility to dissect such interactions. The potential clinical application to use CRs affecting

immunity as supplementary treatment in a therapeutic setting is a further argument to promote the

understanding of the principles behind neuroimmune associative learning.

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