A simple systems approach to neural-immune communication

Camp. B&hem. Physiol. Vol. 115A, No. 1. pp. l-10, Copyright 0 1996 Elsevier Sarnce Inc.

1996

ISSN 0300-9629/96/$15.00 SSL>I 0300-9629(95)02130-2

ELSEVIER

REVIEW

A Simple Systems Approach to Neural-Immune Communicati.on Andrea L. Clatworthy DEPARTMENT OF INTEGRATIVE BIOLOGY, UNIVERSITYOF TEXAS MEDICAL SCHOOL AT HOUSTON, HOUSTON, TEXAS 77225, U.S.A. Communicated by M. Thorndyke,

ABSTRACT.

The marine mollusc Aplysia californica

immune communication accessible

of defense

induces a cellular cantly increased.

cells (haemocytes)

as a useful model system to study neural-

of nociceptive

Haemocytes

IL-l/3 can enhance

nervous system that is easily

the expression

sensory function.

of numerous haemocytes

The feasibility

of injury-induced

raises the interesting

i.e. the

in Aplysia

around the liga-

haemocytes

is signifi-

sensory hyperexcitability possibility

coupled

that cytokines

with the detection

released from activated

nerves may modulate

of

haemo-

nociceptive

of using results from simple system such as Aplysia to formulate testable hypothe-

systems is also discussed.

Invertebrate,

nerves

at regions of axonal injury. The finding that human recombinant

to an injured nerve or to a foreign body close to peripheral

ses in more complex WORDS.

by the accumulation

of peripheral

sensory neurons having axons close to the responding

also accumulate

in Aplysia haemolymph

cytes attracted

at the target site. Loose ligation

defense response as evidenced

ture. The excitability

KEY

is emerging

level hecause it has a well characterized

and it shares with mammals similar basic cellular defensive responses to wounded or non-self,

accumulation

(ir)IL-1

at the mechanistic

Editorinl Board

neuron,

immune,

COMP BIOCHEM PHYSIOL 115A;l:l- 10, 1996. injury, neuropathic

INTRODUCTION

is overwhelming evidence in vertebrates that the immune system can modulate neuronal activity through the release of paracrine factors from immunocompetent cells (47,52). Cytokines, such as interleukin-1 (IL-l) and tumor

There

necrosis factor (TNF), can influence neuronal signaling properties, neuronal survival during development, and regeneration of injured neurons (18,34). Cytokines may also play a role in neuropathological processes. For example, IL1 and TNF have been found in the central nervous system of patients with diseases such as Alzheimer’s, AIDS, multiple sclerosis and bacterial meningitis (22,42,48). The co-localization of activated macrophages and damaged neurons in brain injury and in some degenerative brain diseases coupled with the finding that procedures that reduce the recruitment or activation of macrophages reduce cell death in some experimentally induced brain pathologies implicate the involvement of the immune system in the pathology of some neurodegenerative disorders (16,21). Unfortunately, the complexity of mammalian nervous and immune systems make detailed cellular analyses of neural-immune interactions a formidable prospect. A useful apAddress reprint requests to: A. L. Clatworthy, Department of Integrative Biology, University of Texas Medical School at Houston, P.O. Box 20708, Houston, Texas 77225 U.S.A. Tel. (713) 794-1186; Fax (713) 794-1349; e-mail [email protected]. Recaved 10 April 1995; revwd 24 April 1995; accepted 19 Cktober 1995.

pain

preach is to develop simpler models that share fundamental mechanisms with mammalian systems. In this regard, mollusts represent unique model systems because they have relatively simple nervous systems and furthermore, they share with mammals basic cellular defensive responses to non-self or wounded-self, i.e. the directed migration and accumulation of defense cells around foreign agents or at injured sites. One mollusc in particular, the marine mollusc A@ysia californica, has emerged as a useful system to study neuralimmune

communication.

Aplysia have been used exten-

sively over the past 30 years to study cellular and subcellular mechanisms underlying some forms of associative and nonassociative learning (28) and more recently, injury-related neuronal plasticity (12,74). During the course of these studies, the nervous system has been well characterized at functional, cellular and molecular levels. Moreover, evidence from recent studies demonstrates that the induction of a cellular defense response close to peripheral nerves in Aplysia can modulate the excitability of a population of well defined mechanosensory neurons having axons close to responding “inflammatory cells” or haemocytes ( 13) (also see below). This finding highlights the feasibility of using this mollusc as a model system to understand fundamental mechanisms underlying neural-immune interactions. Below I present a general overview of the molluscan cellular defense system emphasizing similarities between basic cellular defense responses in molluscs and mammals. This is accompanied by evidence illustrating why the nervous

A. L. Clatworthy

2

system of Aplysia is well suited for the detailed celullar analyses required to understand basic mechanisms underlying neural-immune interactions. Last, I discuss how results from

Although lacking an antibody-based immune system, some degree of specificity is conferred on the molluscan immune system by the presence of lectin-protein complexes

this simple model system can be used to formulate testable hypotheses regarding neural-immune communication in more phylogenetically complex animals.

with carbohydrate-specific binding properties that have the ability to opsonize and agglutinate non-self material. Natu-

The Molluscan

Molluscs comprise one of the most successful animal phyla, being second only to arthropods in number of species. They have existed since the Precambrian era, which reflects a remarkable ability to adapt to environmental stresses. This implies adaptation to traumatic stresses that may be presented externally by predators and internally by micro and macro parasites. The principal line of cellular defense and internally

of a variety of

molluscs, including Aplysia, are capable of agglutinating a variety of bacteria and vertebrate red blood cells (29,43,5 1,

Cellular Defense System

against both externally

rally occurring substances in the haemolymph

generated

injury is

phagocytosis or encapsulation involving phagocytic cells (68). The principal phagocytic cell is the haemocyte (also referred to in the literature as the immunocyte, leucocyte

77). These agglutinins appear to play an important role in the defense against potential pathogens in some molluscs by acting as opsonins. reported that the serum that renders cells more cytes. In a more recent

For example, Prowse and Tait (49) of Helix aspersa contains a substance suitable for phagocytosis by haemostudy, Renwrantz and Stahmer (51)

isolated agglutinating material from the haemolymph of Mytilus that promotes phagocytosis of yeast cells by Mytilus haemocytes.

Phagocytosis of foreign agents in some inverte-

brate species can occur in the absence of humoral factors suggesting that there may be constitutive or cytophilic com-

or amebocyte), which wanders freely in haemolymph and through loose connective tissue. Sub-populations of haemo-

ponents on phagocyte surface membranes that are capable of interacting with foreign materials. In support of this, Renwrantz and Stahmer (51) reported that Ca*+ ions alone

cytes have been identified in molluscs on the basis of functional and staining characteristics (10,37,44). Haemocytes are multifunctional, having roles in such diverse functions

(in the absence of serum factors) increase the phagocytic activity of Mytilus haemocytes. This latter observation was interpreted to indicate that the activation of divalent cation-

as digestion, excretion, wound healing, glycogen storage and transport (2,9,61,71). Because molluscs are prostostome animals, not on the lineage leading directly to the vertebrates, any structural and functional similarities between

dependent recognition molecules occurs at the haemocyte surface. Mucin, an inhibitor of the Mytilus humoral agglutinin, also inhibits the attachment of yeast cells to the haemocyte surface, suggesting that these membrane recognition com-

their blood cells and vertebrate blood cells may be analogous (products of convergent evolution). However, phago-

ponents

cytosis represents such a primitive mechanism (3), it is likely that both molluscs and mammals retained this capa-

are structurally similar to circulating

opsonins. In

contrast, membrane-bound and serum opsonins in the mollusc Biomphalatia glabrnta differ in their carbohydrate specificities,

bility, which evolved phylogenetically through common ancestors. Therefore, some structural and functional aspects of

suggesting that the opsonins in plasma are not simply a free form of the membrane-bound recognition molecule (20). Interestingly, vertebrates have retained a family of non-immuno-

vertebrate and molluscan blood cells may be homologous. Indeed, there appear to be striking similarities between molluscan haemocytes and vertebrate phagocytic cells of the myeloid lineage (granulocytes and monocytes). For exam-

globulin, lectin-like molecules (the pentraxins) of which Creactive protein is a well characterized example (36). Severing or crushing of a nerve in a variety of molluscs (including Helisoma trivoluis, Lymnaea stagnalis, Mytilus

ple, one of the defense strategies of molluscan haemocytes, like mammalian macrophages, involves phagocytosis, incorporating the release of highly reactive oxygen metabolites

edulis and Aolysia californica) evokes a cellular defense reaction as characterized by the directed migration and adher-

(including superoxide, hydrogen peroxide) and degradative enzymes (e.g. lysozyme, aglucuronidase), and the secretion of agglutinating and cytotoxic molecules (1,17,29,45,46, 51). The haemocytes of the bivalve mollusc My&s edulis contain a range of antioxidant enzymes (e.g. catalase, superoxide dismutase), which serve to minimize potential damage to adjacent tissues and cells caused by reactive oxygen metabolites (46). The immunocytochemical location of these enzymes in Mytilus is in general agreement to that reported for a wide range ofvertebrate species. In vertebrates, nitric oxide (NO) has emerged as a macrophage bacteriocidal agent (24). Molluscan haemocytes also produce a bacteriocidal factor that has been identified pharmacologically

as NO (39).

ence of haemocytes to the lesioned area (14,60,62) (see also Fig. 2). In Mytilus, naloxone injections into the area of nerve severance counteract the migration of haemocytes, suggesting the involvement of opioid peptides in this response. In support of this, injection of DAMA (a synthetic analog of Met-enkephalin) into Mytilus induces the directed movement of haemocytes to the site of the injection (62). The finding that the effect can be blocked by naloxone, coupled with the &lure of injections of other neuroactive factors (including dopamine, serotonin, substance I’) to induce directed migration, suggest the involvement of specific opioid receptors in this response. Specific immune cells in mammalian systems are also capable of responding to and synthesizing opioid peptides (50,59). In rats, injec-

Neural-Immune Communication in a Simple System

tions or chronic infusion of beta-endorphin alin into cerebrospinal

or Met-enkeph-

fluid causes a similar directed migra-

3

induces the formation of an endogenous

IL- 1 -like molecule

tion of neutrophils, macrophages and lymphocytes (53,69). In vitro studies have revealed that DAMA induces similar conformational changes in sub-populations of Mytilus hae-

in Mytilus that has a stimulatory action on a sub-population of haemocytes (65). The stimulatory effect of (ir)IL-1 extracted from DAMA-stimulated Mytiius haemocytes is reversed by administration of a specific IL-1 antibody. A simi-

mocytes increase

lar opioid-induced formation of IL-1 occurs mononuclear peripheral blood leukocytes (65).

and human granulocytes, i.e. flattening and an in cellular area and perimeter (63). Incubating

Mytilus haemolymph with DAMA reversible increase in the adherence

produces a naloxoneof haemocytes to glass

(62). Opioid peptides induce a similar enhancement in the adherence properties of human neutrophils in a naloxonereversible manner (70). Mammalian mononuclear phagocytes produce IL-1 and TNF. The haemocytes of a variety of invertebrates, including molluscs, express IL-1 and TNF alpha immunoreactivity (14,25,58). Beck and Habicht (5,6) characterized IL-1 from various invertebrate species and found basic similarities in structure and biological properties of vertebrate and inverte-

in human

Mytilus haemocytes also respond to the endotoxin LPS in a similar manner to human macrophages, i.e. with the production of immunoreactive (ir)TNF and (ir)IL-1 (26). (ir)IL-6 (another product of monocytes/macrophages) has recently been detected in Mytilus haemolymph (27). Recombinant human IL-6 fails to activate Mytilus haemocytes directly, but it does potentiate IL-l-induced haemocyte activation. This effect can be blocked by IL-6 antibodies, suggesting that it is mediated specifically via IL-6 receptors. A small population of glial cells that are immunopositive for IL-1 have been found in the pedal ganglion of Mytilus (41,66). Exogenous IL-6 enhances the amount of endoge-

brate IL-l. For example, human IL-l has a M, of 17,500 and two major charged forms (35). IL-1 isolated from both deuterostome and prostostome invertebrates has a molecu-

nous IL-1 derived from DAMA-stimulated pedal ganglia, an effect that is reversed by the addition of anti-IL-6 (27).

lar weight of 18,000-22,000 and at least two major charged forms (4,5,6). IL-1 isolated from the starfish Asterias forbesi stimulated murine thymocyte and fibroblast proliferation

The Aplysia Nervous System

and protein synthesis suggesting there is conservation of the three dimensional structure required for interaction with cytokine receptors (4). Preliminary results from T. K. Hughes (personal communication) suggest that there is sequence similarity between human IL-1 and (ir)IL-1 isolated from the haemocytes of Mytilus. Complementary DNA was synthesized from human peripheral blood leukocyte (PBL) and Mytilus haemocyte RNA via reverse transcriptase with random heximer primers. PCR was performed using human and Mytilus IL- 1 a and /?, TNFa and IL-6 primers on the reverse transcriptase products. A Southern analysis was run utilizing a cDNA probe for human IL-l/Z Although PCR products

The central nervous system of Aplysia is distributed across 10 major ganglia, each comprising 1,000 to 10,000 nerve cell bodies and many more glial support cells. Aplysia neurons are typically monopolar, that is, the cell body gives rise to only one axon and the dendritic branches emanate from this one axon. Nerve cell bodies are large, ranging in diameter from approximately 30 pm to 500 pm. The location of the cell bodies on the outer surface of the ganglion together with their pigmentation and large size allows visualization and identification of individual cells under a dissecting microscope. Thus, it is relatively simple to record intracellu-

were seen with other primers, only human and Mytilus products amplified with IL-lpprimers hybridized to the IL-

larly from single, identified neurons using microelectrodes. One group of neurons in Aplysia that has received particular attention is a large, homogeneous cluster of sensory

lp probe. In addition, preliminary results from a Northern analysis of human RNA isolated from PBLs and Mytilus

cells located on the ventrocaudal (VC) surface of each pleural ganglion (Fig. 1). Each VC sensory neuron sends a single

RNA isolated from human cDNA encoding for human

axon out through an identified ipsilateral pedal nerve, e.g. p8, p9. Each cluster innervates virtually all the ipsilateral

haemocytes, probed with a TNFa, suggest that Mytilus

respond to human monokines IL-1 similar to human granulocytes-the their area and perimeter. These efcan be blocked by prior treatment

body surface (73). The VC sensory cells are activated by mechanical stimulation within their receptive field and respond most vigorously to noxious stimulation (11). Consequently, they have been characterized as wide dynamic range nociceptors. Functionally, they play an important role in triggering defensive withdrawal reflexes following nox-

with anti-IL-l or TNF, respectively, suggesting they are mediated through specific surface receptors (25). Some of the effects of IL-1 in vertebrates are attributed to its induction of TNF. A similar mechanism appears to exist in Mytilus evidenced by a partial blockade of the IL-1 response by administration of antibodies to TNF. The remainder of the IL1 response can be abolished by anti-IL-l. Opioid challenge

ious stimulation. Of particular interest are the recent findings that this population of nociceptive mechanosensory neurons show long-term changes in their electrophysiological properties following axonal injury (12,74) and following the induction of a foreign body response in close proximity to peripheral nerves (13). Both these manipulations result in the accumulation of numerous haemocytes close to sen-

haemocytes express a human TNFa mRNA. Mytilus haemocytes and TNF in a manner cells flatten, increasing fects of IL-1 and TNF

mRNA

for TNFa

that is similar to

A. L. Clatworthy

vc

PLEURAL

SENSORY CLUSTER

GANGLION

/

the distance of the crush site from the sensory neuron soma. For example, hyperexcitability produced by crushing a sensory axon at a distance of 1 cm from the soma is expressed after a latency of approximately 24 hours. This delay suggests that some of the signals initiating the sensory plasticity

p2

1\

the number of action potentials elicited by a l-second intracellular depolarizing pulse. There is a delay in the expression of crush-induced sensory hyperexcitability that is related to

{PEDAL-PLEURAL CONNECTIVE

are conveyed from the crush site to the soma by slow retrograde axonal transport (23). Potential induction signals include interruption of trophic signals coming from the periphery, release of substances from damaged sensory axons or support cells, and release of factors from cells of the cellular defense system that are attracted to the injury and possi-

_I(3N

the injury site. A similar cellular defense response is induced by nerve

p9 FIG. 1. Schematic diagram of the left pleural and pedal ganglion of Aplysia. Symmetrical clusters of neurons are located in the right pleural and pedal ganglia. VC pleural sensory cell bodies are large (30-40 pm) and easily accessible for intracellular recording. Each sensory cell sends a single axon out to the periphery through a specific pedal nerve, e.g. sensory neurons innervating the tail send an axon out to nerve p9. Sensory cells innervate only ipsilateral body regions. Modified after Walters et al. (73).

sory axons, providing the framework for potential immune interactions to occur. Plasticity in Aplysia Mechanosensory

neural-

VC

Neurons

bly to the presence of non-self, e.g., pathogens that could enter through a wound. Figure 2 illustrates that in Aplysia, the potential for interactions between haemcjcyte-derived factors and injured sensory axons certainly exists. Within 1 day of axonal crush, numerous haemocytes accumulate at

Induced by Injury

Body wall injury in Aplysia was recently shown to be associated with profound changes in the properties of the injured sensory neurons that innervate that area (72). These changes show remarkable similarities to inj my-related sensory plasticity in mammals (76). Common features include enhanced sensitivity of peripheral sensory elements, enlargement of receptive fields and long-term enhancement of central excitability. The plasticity was initially assumed to be mediated by a combination of spike activity in the sensory neuron produced by the noxious stimulus coupled with the release of neuromodulators from nearby facilitatory interneurons. However, long-term enhancement of sensory excitability can also be produced by crushing peripheral nerves containing sensory axons under anesthetic conditions where spike activity and neuromodulator release are blocked at the time of crush (12,74). Sensory hyperexcitability is expressed in the sensory neuron soma as a decrease in action potential threshold and after-hyperpolarization, an increase in action potential duration and an increase in

damage in mammalian preparations. It has been reported that macrophages that invade a site of injury on rat peripheral nerve release IL-l, TNF and lL-6 (75). Although the full extent of their actions on nervous system function is as yet unclear, one of the functions of IL-1 appears to stimulate Schwann cells directly to produce nerve growth factor in the distal stump to promote regeneration of the injured nerve (30,3 1). Indeed, exogenous application of IL-1 to injured nerves can increase the regeneration of axons frotn sensory ganglia (18). The induction of an inflammatory response in rat dorsal root ganglia enhances previously crushed axons in the associated viding further evidence for a regenerative mune cells following nerve injury (32). It

regeneration of dorsal root profunction of imis possible that

one of the roles of the haemocytes attracted to the crush site in Aplysia is to initiate or modulate the regenerative process that occurs following crush of peripheral nerves (19,67). It is of interest in this regard that molluscan haemocytes extend fine processes around the neurites of isolated neurons in culture. The haemocytes leave “tracks” on the substratum suggesting they inay have a guidance function (60). The effects of haemolymph on neurite outgrowth of Aplysia neurons in dissociated cell culture have also been examined (57). The presence of haemolymph in the culture-medium enhanced cell survival and the initiation and extension of neurite outgrowth, suggesting that factors present in the haemolymph intluence regenerative processes. It is interesting to speculate that these “factors” may be derived from haemocytes.

Neural-lmmune Interactions in Aplysia Strong nerve stimulation (in the absence of injury) activates the haemocytes of Mytilus in a naloxone reversible

Neural-Immune Communication in a Simple System

FIG. 2. Light (A,B) and electron (C) microscopic views of cross sections through control (A), and crushed (B,C) pedal nerves 1 day after unilateral pedal nerve crush. There are few cells (black dots) associated with the control, uncrushed nerve section. In contrast, note the large accumulation of cells associated with the crushed nerve section. The higher power electron micrograph (C) of a section through the crushed nerve illustrated in (B) shows that the cells associated with the crushed nerve are haemocytes. Vesicles located in the cytoplasm contain dense material that resembles phagocytic vesicles characteristic of haemocytes.

manner (65). This suggests that the nervous system can in-

nerves. Contralateral

fluence haemocyte function either directly or indirectly via endogenous opioid substances. Indeed, there is considerable overlap between the extracellular signals utilized by the cel-

ligation of peripheral nerves induced a foreign body reaction as evidenced by the accumulation of numerous haemocytes at the ligation site. The cotton thread was too large to be

lular defense and nervous systems in molluscs. For example, it has been demonstrated that the nervous system, the haemocytes and the cell-free haemolymph of a variety of molluscs contain the neuroactive factors, dopamine and noradrenaline (38,40). A novel high-affinity dopamine receptor has

phagocytosed so was instead encapsulated-that is surrounded-by haemocytes. Five to 30 days after ligation, the excitability of the soma of VC sensory neurons having axons in ligated nerves was significantly increased compared

been localized to Mytilis haemocytes (64). A similar receptor was found on mouse thymocytes. Various neuroactive peptides including

substance

P, somatostatin

and neurotensin

have been found in the haemocytes of the mollusc Viviparus ater (38). In addition, a sub-population ofMytilis haemocytes

pedal nerves were not ligated. Loose

to contralateral, control sensory neurons with axons in non-ligated nerves. Figures 4A and 4B illustrate that spike threshold

and after-hyperpolarization

were reduced,

and

spike amplitude and duration were increased. Sensory neurons on the ligated side also fired more spikes in response

contain a Met-enkephalin-like factor (62). To begin to determine whether the cellular defense system can influence nervous system function in molluscs, Clatworthy et al. (13) examined whether an accumulation

to a standard 1 -set intracellular depolarizing pulse compared with control sensory neurons (Fig. 4C). The finding that the induction of a foreign body response close to sensory axons influences sensory excitability is particularly interesting because the sensory axon is not a locus traditionally

of haemocytes close to peripheral nerves in Aplysia could influence the excitability of sensory cells with axons in those nerves. Figure 3 illustrates the experimental set-up used in these experiments. A strip of cotton wrapped loosely around pedal nerves containing VC mechanosensory axons served as the stimulus to activate the cellular defense system and produce an accumulation of haemocytes close to the

associated with neuromodulation. The recorded increase in sensory excitability is qualitatively similar to that seen following axonal injury (12,74). However, the effects are unlikely to be accounted for by ligation-induced injury of sensory axons because both morphological and electrophysiological evidence indicate that axons in ligated nerves were undamaged and able to con-

A. L. Clatworthy

bathed overnight

Cerebral Ganglia

jury-induced

in vehicle.

Both sides showed typical in-

sensory plasticity.

However,

sensory cells re-

corded from IL-1 treated ganglia fired significantly more spikes in response to a 1-set depolarizing pulse than sensory cells recorded from ganglia that were bathed in vehicle. Sensory spikes recorded from the experimental side were also significantly broader than those recorded from control sensory neurons.

Comrnonalities Invertebrate

Control Nerves

Between

and Vertebrate

Systems

These results from a simple model system raise the possibil-

Loosely Ligated Nerves

ity that cytokines released from activated components of the cellular defense system may modulate sensory function.

FIG. 3. In vitro preparation used to examine immune modud lation of sensory excitability in Aplysia. A unilateral foreign body was induced by loosely ligating pedal nerves with a strip of cotton at a distance of approximately 1 cm from the pedal ganglion. The excitability of VC sensory neuron cell bodies (indicated by dots) having axons in these nerves was tested between 1 and 30 days later and compared to the excitability of contralateral VC sensory neurons having axons in non-ligated nerves. The appearance of evoked spikes in sensory neuron somata following electrical stimulation of nerve stumps containing axons of recorded sensory neurons distal to the ligation at the end of each experiment was taken as evidence of healthy axons ( 13).

An important

duct action potentials. Also, a significant increase in action potential amplitude was recorded following loose ligation,

vertebrate and vertebrate sensory systems (76). Findings indicative of an immune-mediated increase in excitability of Aplysia nociceptive sensory neurons (13,14) suggest that in

an effect not associated with injury-induced sensory plasticity. Furthermore, the latency to onset of sensory hyperexcitability following ligation was approximately 5 days. This contrasts with the shorter latency (2-3 days) of sensory plasticity induced by crushing pedal nerves at a similar distance from the sensory cell body. The increased latency might reflect the time required for a sufficient haemocytes

number of

to aggregate close to sensory axons to exert an

effect. Thus, an intriguing possibility is that the sensory changes associated with implantation of a foreign body close to peripheral nerves are mediated by factors released from haemocytes that are activated by the presence of a foreign agent. Potential factors include IL-1 and TNF. Detectable levels of (ir)IL-1 and TNF are present in the haemolymph of both Aplysia and Mytilus (14,25). Also, these cytokines have neuromodulatory effects in Aplysia. For example, IL-1 and TNF modulate ionic channels in the soma of identified neurons of Aplysia (54-56). Furthermore, preliminary studies suggest that IL-1 can significantly enhance the expression of injury-induced sensory plasticity in Aplysia (14). Briefly, one set of pleural-pedal ganglia and associated pedal nerves, that had been cut at a distance of approximately 1 cm from the plural ganglion, were bathed in IL-lp (2050 units/ml) overnight. The contralateral, control pleuralpedal ganglia were treated identically except they were

sequel to this work in Aplysia is a consider-

ation of the possible relevance to more phylogenetically advanced organisms. The last section of this review addresses the issue. The VC mechanosensory neurons in Aplysia that were used in the studies described above function as nociceptors, i.e. they respond maximally to damaging or potentially damaging stimuli (11). The enhancement of responsiveness in these sensory neurons following injury or the induction of a foreign body response is therefore functionally similar to hyperalgesia, i.e. a heightened sensitivity to painful stimuli, in mammalian preparations. Indeed, there are many striking similarities between injury-related plasticity in in-

mammalian systems the “immune agonists” (8) may play a role in the production of hyperalgesia. A mammalian model that has been used extensively to study the hyperalgesia that is associated with peripheral nerve injury involves loosely ligating the sciatic nerve in rats with chromic

gut sutures (7). Hyperalgesia is recorded

on the ligated side within a few days. This rat model bears a striking resemblance to the model that has been used to study neural-immune interactions in Aplysia (13). In both systems an increase in the responsiveness sensory neurons is produced following loose ligation of peripheral nerves.

of

In the rat model of hyperalgesia, activated immune cells attracted to the chromic gut used to ligate the sciatic nerve may play a role in the modulation of nociceptive sensory function. Evidence in support of this possibility is that silk sutures, which produce only a small inflammatory response when used to ligate the sciatic nerve (A. L. Clatworthy and P. A. Illich, unpublished observations), fail to produce hyperalgesia in the loose ligation model (33). Clatworthy et al. (15) studied the effect of manipulating the inflammatory response associated with the suture material used to ligate the sciatic nerve on the expression of hyperalgesia in the Bennett and Xie model of neuropathic pain. Severely reducing the magnitude of the inflammatory

\r

Control

‘I,

Ligated

msec

5 msec

20

20 mV

20 mV

c.

-I

1 set

Ligated Side

Control Side

l-

I

40 mV

FIG. 4. Examples of longterm electrophysiological changes iu sensory neuron somata associated with loose ligation of pedal nerves containing their axons. Nerves were ligated 9 days earlier. A. Response of a sensory neuron haying an axon in a ligated nerve and a control sensory neuron to a 20 msec depolarizing pulse. Note the smaller after-hyperpolarization and larger spike amplitude recorded from the neuron on the ligated side. B. Expanded record to show the prolonged duration of the action potential recorded from the sensory neuron on the ligated side. C. Increase in excitability of a sensory neuron having an axon in a ligated nerve compared to a sensory neuron with its axon in a non-ligated nerve. Each cell was tested with a l-set intracelhtlar depolariziig pulse at 2.5 x the 20 msec threshold. Spikes illustrated in this figure are attenuated because of the limited sampling rate of the data acquisition system. Modified after Clatworthy et al. (13).

B.

A.

A. L. Clatworthy

8

reaction

associated

with chromic

gut ligatures by treating

the animals with the steroid dexamethasone significantly reduced the hyperalgesia recorded on the ligated side. Conversely, enhancing the inflammatory response associated with cotton ligatures by soaking them in Freund’s complete adjuvant (Sigma) prior to implantation significantly potentiated the hyperalgesic response. These results suggest that inflammation in close proximity to the ligated nerve plays an important role in the development of hyperalgesia in this model. Furthermore, it provides evidence for a novel site of inflammation-induced modulation of nociceptive function-the sensory axon. These findings from experiments designed on the basis of results from Aplysia illustrate the feasibility of using simple systems to generate results that can be applied to formulate testable hypotheses in more complex systems, in which inflammation, either nonspecifically

or immunologically

mediated,

host defense or in the pathogenesis

is involved

in

of disease.

I am ,qmteftkl to Drs. G. A. Cusrro and E. T. Walters for their comments on the manuscript, to Mr. J. Pastore and Ms. K. Hen&y for Drebnrinn rhe illustrations, and to M. C Thvrnclyke for his assistance. This e&r is dedicatedto the memory Jim P&tore.

of

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