I. Introduction

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Sensory systems have the role of informing the brain about the state of the external environment and the internal milieu of the organism. Pain is a perception, and as such, it is one of the outputs of a system in more highly evolved animalsthe nociceptive systemwhich itself is a component of the overall set of controls responsible for homeostasis. In this context, pain constitutes an alarm that ultimately has the role of helping to protect the organism: it both triggers reactions and induces learned avoidance behaviors, which may decrease whatever is causing the pain and, as a result, may limit the (potentially) damaging consequences. At the beginning of the twentieth century, Sherrington (1910) developed this concept and introduced the term nociception (from the Latin nocere, "to harm"). It seems appropriate to take the view of Dennis and Melzack (1983) that pain/nociception has at least three functions: 1) to warn the individual of the existence of real tissue damage; 2) to warn the individual of the probability that tissue damage is about to occur by realizing that a stimulus has the potential to cause such damage; and 3) to warn a social group of danger as soon as it exists for any one its members. Behaviors resulting from pain can facilitate other fundamental biological functions, such as the maintenance of tissue "trophicity" and regeneration (notably in the processes of inflammation and healing). The importance of these behaviors is well illustrated in humans through pathological cases of congenital insensitivity to painful stimuli, in which truly natural experiences can have catastrophic consequences.

The complexity of nociceptive systems, which ultimately produce pain, has increased during evolution as a result of the pressure to avoid organic lesions or their aggravation (Walters, 1994). One can easily see the evolutionary advantage of cutaneous, muscular, and articular pains. However, the Sherringtonian concept of a nociceptor alarm system is more debatable in the context of visceral pain, given that serious lesions can develop painlessly in noninflammatory conditions and that viscera may contain only a few fibers that respond preferentially to nociceptive stimuli (Cervero, 1991, 1994; McMahon et al., 1995).

Therefore, like other body functions, the physiological system that generates pain can also be affected by pathological processes. In the context of chronic pain, which can last months or even years, the physiological protective effect gives way to a pathological state that is not only useless but also highly distressing. There are models of chronic pain in animals such as the rat with induced arthritis and rats that have had various lesions to the central or peripheral nervous systems (Colpaert, 1987; Butler, 1989; Dong, 1989; Rossitch, 1991; Zeltser and Selzer, 1994; Seltzer, 1995; Tjølsen and Hole, 1997; Kauppila, 1998). However, these fall outside the purview of this review, which is restricted to models of acute pain, i.e., pain evoked by a brief noxious stimulus and generated by a nociceptive system functioning normally within its physiological limits.

The absence of verbal communication in animals is undoubtedly an obstacle to the evaluation of pain. There are circumstances during which there can be little doubt that an animal is feeling painnotably when it is responding to stimuli through vocal responses such as squealing or groaning. On the other hand, it is far more difficult to certify that at a given moment, an animal feels no pain because it is presenting no typical physical signs or overt behaviors. This is particularly so given that we know that immobility and/or prostration are sometimes the only responses accompanying pain. The question of pain in animals can be approached only with anthropomorphic references, although differences probably do exist by comparison with humans, notably in respect of certain cerebral structures (Bateson, 1991). In this regard, the degree of cortical development has to be considered (Vierck, 1976), and it is reasonable to conclude that differences do exist between humans and animals, at least, but perhaps not only, with respect to the psychological repercussions. Neurological observations of human patients allow us to make some comparisons. Thus, like Lineberry (1981), one might question whether it is appropriate to consider any pain in patients who have undergone frontal lobotomies as being similar to the pains we feel: although the pain that they experience is unaltered at a sensory level, it has lost its emotional and motivational dimensions (Freemann and Watts, 1946; Foltz and White, 1962; Sweet, 1973). Furthermore, the International Association for the Study of Pain (IASP) defines pain as "an unpleasant sensory and emotional experience associated with actual or potential tissue damage or described in terms of such damage" (Merskey et al., 1979).

In contrast with the polymorphic nature of the pain that is described as a sensation in humans, pain in animals can be estimated only by examining their reactions. This is essentially the same difficulty that is faced by the pediatrician, the geriatrician, or the psychiatrist dealing with patients incapable of expressing themselves verbally. In those cases as well, the symptomatology is not unequivocalit has to be taken in context and placed in an inventory, because its meaning will differ depending on the degree of maturation (or degradation) of the nervous system. In addition, one must never forget that the existence of a reaction is not necessarily evidence of a concomitant sensation (Hardy et al., 1943). Indeed, anesthetists see a dissociation between these phenomena every working day. Consequently, we must consider reactions within a more global context, which includes other considerations such as the homeostatic mechanisms of the animal (see Section XII.E.).

We will restrict this review to mammals, particularly rodents, since they are used in almost all animal models of pain. Reviews of nociception and/or pain in other species can be found in the articles by Kavaliers (1988), Bateson (1991), and Walters (1994). The essential mechanisms that make it possible for an organism to react to a stimulus, which might endanger its existence (including sensory perception), exist throughout the animal kingdom, except perhaps in arthropods and particularly in insects (Eisemann et al., 1984; Walters, 1994).

Generally speaking, the most reliable signs of pain are physical ones. Research in humans and in animals has focused on various biochemical indicators (catecholamines, corticoids, opioids, etc.), but these all seem to be without specificity. One can say the same for other methods such as electrophysiological parameterselectroencephalograms, evoked potentials, etc. (Molony, 1986; Ichinose et al., 1999). For the time being, the study of behavioral reactions provides the only indicator of the perceived disagreeable sensation resulting from a stimulus that would be algogenic in humans, but it must never be forgotten that these responses are often not very specific; for example, escape can result from any disagreeable stimulus whether or not it is noxious.

Descriptions of the "signs" of pain have been published on several occasions in a veterinary or an animal-welfare context (Gibson and Paterson, 1985; Morton and Griffith, 1985; Flecknell, 1986; Sanford et al., 1986; American Veterinary Medical Association, 1987; Sanford, 1992, 1994; Baumans et al., 1994). Above all else, it must be emphasized that these signs have no unequivocal value and that each species expresses pain in a manner related to its own behavioral repertoire. It is in that context that the description of each becomes interesting and allows the inventory of the principal clinical signs to be refined (Gibson and Paterson, 1985; Morton and Griffith, 1985).

For example, one can distinguish the following reactions produced by a (presumably) painful focus: 1) responses organized by centers that are relatively "low" within the hierarchy of the central nervous system; and 2) more complex responses organized by higher centers in the central nervous system. The former can be elicited in decerebrate animals and have been termed "pseudoaffective reflexes" (Woodworth and Sherrington, 1904; Sherrington, 1906b). They include basic motor responses (withdrawal, jumping, contractures, etc.), neurovegetative reactionsgenerally in the context of Selye's "alarm reaction", with an increase in sympathetic tone (tachycardia, arterial hypertension, hyperpnea, mydriasis, etc.), and vocalization.

The more complex reactions include conditioned motor responses, which result from a period of learning and sometimes can be very rapid, e.g., cattle avoiding an electrical enclosure. In general, the significance of these has as much to do with preventing new damage as with avoiding aggravating existing lesions. Behavioral reactions (escape, distrust of objects responsible for painful experiences, avoidance, aggressiveness, etc.) or modifications of behavior (social, food, sexual, sleep, etc.) are often observed. By comparison with the responses discussed above, these behaviors provide evidence of far more integrated reactions within the hierarchical organization of the central nervous system. If the stimulus is sufficiently intense, the reaction will be escape or attack. However, it must be noted that even if active motor reactions are frequent, passive motor responses are observed just as often in animalsimmobility allowing the animal to preserve a painless posture. Whereas brief and sharp pains are associated with phasic motor responses (withdrawal, startle reactions), lasting pains may be associated with contractures, the consequence of which is to immobilize the painful region. This explains, for example, the frequent hunching of the back in dogs suffering from a slipped disc or abdominal pain. It reminds one of reflexes involving abdominopelvic muscles in humansthe classic "board-like" abdomen seen during peritonitis, hyperalgesic sciatica, etc. Furthermore, in animals, motor atonia is a general response to sickness, whether or not they are in pain. However, there can be spectacular exceptions, such as colic in horses or pancreatitis in dogs.

Zimmermann (1986) re-interpreted the IASP definition of pain so that it could be applied to animals: "an aversive sensory experience caused by actual or potential injury that elicits progressive motor and vegetative reactions, results in learned avoidance behavior, and may modify species specific behavior, including social behavior".

The term nociceptive refers to the potential of a stimulus to produce a tissue lesion and a reaction from the organism. The "algogenic" character of a stimulus is defined by its capacity to produce painin an affective and motivational as well as a sensory context. None of these can be observed directly in animals. Cervero and Merskey (1996) recently discussed these terms and gave some examples related to acute pain. In the same way that menthol excites cold receptors without being a thermal stimulus, capsaicin evokes a sensation of burning without producing tissue damage. Thus, it is a nociceptive stimulus (it activates nociceptors) and an algogenic stimulus (it produces pain), but it is not harmful (and as such cannot be called a "noxious" stimulus). Similarly, a thermal stimulus of 45°C may or may not be harmful depending on the duration of its application (Stoll and Greene, 1959).

In this review, we describe and critically analyze the most commonly used behavioral tests of nociception in animals (Fig. 1). However, we do not claim that the review will be exhaustive. For example, some often complex tests that depend on a period of learning by the animals have been omitted deliberately (Hill et al., 1957; Weiss and Laties, 1958; Weitzman et al., 1961; Evans, 1964; Lineberry, 1981; Chapman et al., 1985; Hammond, 1989; Vierck et al., 1989). We will simply illustrate this type of method with an example (Fig. 2). In addition, complementary information can be found in other reviews (Jacob, 1966; Domer, 1971; Vyklicky, 1979; Vierck and Cooper, 1984; Wood, 1984; Chau, 1989; Watkins, 1989; Dubner, 1994; Tjølsen and Hole, 1997; Dubner and Ren, 1999).

View larger version (27K): [in this window] [in a new window]   Fig. 1.   A, evolution of the number of original articles published during each of the years between 1970 and 1999 (ordinate) in which the authors used one of the five most common tests of nociception (based on Medline). B, relative proportions of these categories of articles appearing during 1999. Of all of these tests, the tail-flick and the hot plate tests remain the most commonly used. Note that the rate of publications regarding the tail-flick, hot plate, and writhing tests stabilized in the 1990s, whereas there was a progressive increase in the number of articles describing the use of the formalin test and the various different tests involving withdrawal of the paws from mechanical stimuli.

View larger version (37K): [in this window] [in a new window]   Fig. 2.   Results from a test involving a substantial amount of learning by the experimental animal (rhesus monkey). Short bursts of electrical stimuli were applied through electrodes implanted in the trigeminal ganglion of the animal. The stimulus intensity (ordinate) was automatically increased in preset "steps" (approximately 0.75 V) every 5 s. However, the animals had been trained over a period of several weeks to press a lever to reduce this stimulus intensity and the intensity at which they did this was taken as the pain threshold. Thus, upward deflections on the records were produced by subthreshold, automatic increases in the stimulus intensity, whereas downward deflections indicate that the pain threshold had been reached (and that the animal had pressed the lever). The graphs show the effects with time (abscissa) on this pain threshold of three doses of morphine administered intravenously. Records traced from original chart recordings in Weitzman et al. (1961) and modified for clarity of presentation with permission.

Before describing the most commonly used tests, we will consider successively the ethical problems posed by the study of acute pain in animals, the choice of stimulus, and which type of response can or should be monitored. However, the main part of this review will be devoted to a critical analysis of these tests. Most notably, we will comment on relationships between tests of acute pain and motor responses and then consider successively the sensitivity, specificity, and predictiveness of the tests. The analysis of their sensitivities will lead us to pose the question of which fibers underlie the observed responses and what meaning can be ascribed to measurements of reaction time when a stimulus is gradually increasing in intensity. We will also consider some methods of analyzing the results of the tests. Finally, we will examine factors that can disturb the measurement of behavioral responses in animals; in this respect, we pay particular attention to intercurrent physiological functions.

Because the goal of these animal models is the understanding of acute pain in humans, we make reference throughout this review to analogous experimental situations in humans. Notwithstanding the normal caution one must have with any anthropomorphic approach, we believe this is a necessary counterbalance to the reductionism of the so-called basic sciences.

	II. Ethical Problems

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As with all biomedical research involving animals, pain research presents ethical problems at two levels (Morton and Griffith, 1985). From a general point of view, investigators have to follow the recommendations of ethics committees and, notably, those of international scientific review boards so as to ensure a given level of physiological well being in the animal. Indeed, if the animal is miserable or in a state of stress in which neurovegetative reactions are exacerbated, it is clear that scientific observations will not be valid from a physiological point of view. Thus, it is not only for moral reasons but also for scientific reasons that some rules have to be observed.

The second point is more specific to studies on pain (Wall, 1975; Sternbach, 1976; Zimmermann, 1983; Casey and Handwerker, 1989; Roberts, 1989). The ethics committee of the IASP has formulated a certain number of recommendations on this subject. The practical consequences of these are summarized below (Covino et al., 1980; Zimmermann, 1983). In a preamble, the committee stated that experiments are indispensable if we are to gain a better understanding of the mechanisms of pain. As in other areas of biomedical research, the attitudes of scientists is conditioned by how they regard their subject of study: they have to consider the animal not as an object but as a living being gifted with sensations. The committee, while acknowledging that some experiments have the aim of trying to reproduce chronic syndromes in animals, stated clearly that experimental protocols have to minimize or avoid pain (this notion could, a priori, seem paradoxical; however, as we shall see, one can study nociception without producing pain).

The committee's other recommendations with respect to studies of acute pain may be summarized as stating that 1) experiments involving the study of pain on conscious animals must be reviewed beforehand by scientists and lay persons, and the potential benefit of these experiments must be shown; 2) as far as possible, the scientist must test the painful stimuli on himself or herself, and this should apply to most noninvasive stimuli; 3) the scientist should carefully assess all behavioral and physiological changes in the animal and report them in resulting manuscripts; 4) as in other areas of neuroscience, there must be no question of using animals paralyzed with a neuromuscular blocking agent without a general anesthetic or an appropriate surgical procedure that eliminates sensory awareness; and 5) the duration of experiments must be as short as possible, and the number of animals involved must be kept to the minimum.

Studies in conscious animals most commonly involve monitoring the threshold for obtaining a response to a stimulus that would produce pain if applied to humans. Such responses include flexion reflexes and vocalization. In such experimental scenarios, the stimulus is stopped once the response has been obtained. Sometimes algogenic substances are applied brieflygenerally under conditions similar to when they are used in studies of experimental pain in humans.

	III. Input and Output: the Stimulus and the Response

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There are numerous tests of nociception, and in this review, we do not give an exhaustive list. In his magnificent review published in 1957, Beecher cited 60 original publications related to the description, development, and application of experimental tests of pain in animals. Twenty-seven of these publications were based on the use of thermal stimuli, whereas 10 involved electrical and 23 mechanical stimulation. None was based on the use of chemical stimulation. By comparison, at that time the corresponding numbers of studies in humans were 167 publications: 63 based on thermal, 49 on electrical, 46 on mechanical, and 9 on chemical stimulation.

Experimental studies on conscious animals are often designated "behavioral studies". Sometimes, this may seem to be stretching the meaning of the word "behavioral", but what it means is simply and implicitly that all responsesincluding simple withdrawal reflexesare part of an animal's behavioral repertoire. The behavioral tests that are used to study nociceptionnociceptive testsconstitute "input-output" systems that function via "black boxes", which the neurobiologist wishes to decode. As a result, when describing these tests, one must specify the characteristics of the input (the stimulus applied by the scientist) and the output (the reaction of the animal).

Thus, describing these tests should be a simple matter of first accounting for the nature of the stimulus (electrical, thermal, mechanical, or chemical) and then describing the behavioral parameters that are measured. This latter task may involve defining the responses as a function of their increasing complexity (e.g., one might distinguish reflexes from more integrated reactions). In fact, the inputs and outputs of these systems are very intimately linked by the physical characteristics (notably the temporal nature) of the stimulus.

In humans and in animals, experimental studies of the mechanisms underlying acute pain necessitate the use of appropriate stimuli to provoke the sensation. To be adequate, these stimuli have to be quantifiable, reproducible, and noninvasive (Beecher, 1957; Lineberry, 1981). Although thermal and electrical stimuli can meet these requirements, they also have serious drawbacks.

1. Electrical Stimulation. The application of electrical stimuli has the advantages of being quantifiable, reproducible, and noninvasive and of producing synchronized afferent signals. However, it also has serious disadvantages. First, electrical stimuli are not a natural type of stimulus like those encountered by an animal in its normal environment. More importantly, intense electrical stimuli excite in a nondifferential fashion all peripheral fibers, including large diameter fibers, which are not directly implicated in nociception, as well as fine A and C fibers, which mediate sensations of cold and hot as well as nociceptive information. Furthermore, this type of stimulation completely short-circuits peripheral receptors, thus preventing any study of peripheral transduction mechanisms with these methods. On the other hand, this last disadvantage becomes an advantage when one wants to study the actions of a systemically administered substance in the central nervous system: as long as the substance has no action on peripheral fibers, any effect will be of central origin because the transduction processes have been bypassed. Finally, there are difficulties introduced by variations in the impedances of the tissues being stimulated, although these can be minimized by the use of a constant current stimulator and the monitoring of the voltage as well as the current of the applied stimulus. However, this precaution is not always respected, which may explain the difficulties that are sometimes encountered, notably, the variability in evoked vocal responses (Fennessy and Lee, 1975). It must be emphasized that the use of a constant current stimulator does not solve all the problems because it allows one only to verify that a given current has been delivered, not that it has gone in a consistent fashion to the intended target (e.g., the paws of an animal). The possibility always exists that a variable amount of current may be shunted through other conducting media (e.g., the urine of the animal). To assess whether this is happening, it is essential to monitor the voltage required to generate the current, because this will vary with the impedance of the tissues and thus will indicate whether this impedance is changing during an experiment.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   A, when radiant heat is used, the percentage of the energy reflected by the skin (ordinate) depends on the wavelength emitted by the source of radiation (generally an incandescent lamp). In the visible and adjacent infrared spectra, it also depends on the degree of pigmentation of the skin (white skin: solid line; black skin: broken line). From data from Hardy (1980). B, the percentage of the energy transmitted through the skin (ordinate) also depends on the wavelength emitted by the source of radiation and decreases within depth. Modified from Hardy et al. (1956) with permission. C, the electromagnetic emission spectrum of a lamp varies with the intensity of the electrical current that is being applied to it. In this example, using a lamp in a commercially available apparatus that is marketed for the tail-flick test, it can be seen that at full power, the spectrum is centered in the infrared range (around 0.9 µm). When the power is reduced, the center shifts gradually to the right. (Graph courtesy of Michel Morel, Philips, 54700 Pont à Mousson, France.)

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Relationship between the thermal energy necessary to increase the temperature to the threshold for "stinging" pain and the initial temperature of the skin. In four subjects, radiant heat from a preheated lamp controlled by an obturator was applied for 3 s to a 3.5-cm2 area of skin on the face that had been blackened by India Ink. The threshold fell in a linear fashion with the increase in temperature and tended toward a value of approximately 45°C. Adapted with permission from Hardy et al. (1951). Copyright 1951 American Association for the Advancement of Science.

View larger version (27K): [in this window] [in a new window]   Fig. 5.   Relationships between the thermal energy necessary to increase the temperature to a pain or nociceptive threshold and the stimulus duration. A, variations in the threshold for "stinging" pain in six healthy volunteers (thin solid curve). This graph also shows a curve obtained in a paraplegic patient by measuring the threshold for producing a reflex withdrawal movement of the foot when its dorsal surface was stimulated (broken curve). Very similar curves were obtained with similar protocols in guinea pigs by measuring the threshold for producing a reflex in back muscles by stimulation of the previously shaven back (thick solid curve) and in rats by measuring the threshold for producing a reflex movement of the tail (tail-flick, thick dotted curve). Adapted from Hardy et al. (1953) with permission. Each of these curves tends toward a limit (the rheobase) below which one can never evoke a sensation or a response. The four rheobases are very close together. B, in this experiment, 11 healthy volunteer subjects were asked to differentiate stinging from burning pain. With stimulus durations of less than 3 s (left gray zone), the subjects were unable to distinguish the two pains. It was with durations between 5 and 10 s (right gray zone) that they could most easily determine the threshold for "burning" pain. Moreover, this pain was produced by weaker stimulus intensities than was stinging pain. For example, at an intensity of 120 mcal/s/cm2, it was necessary to double the duration to pass from the threshold for burning pain to that for stinging pain (arrows). Adapted from Bigelow et al. (1945) with permission.

View larger version (78K): [in this window] [in a new window]   Fig. 6.   Schematic representation of heat transfer through the skin at rest (A) and when subjected to heat by a radiant lamp (B), a contact thermode (C), or a CO2 laser (D). For clarity of presentation, we have indicated a diaphragm close to the cutaneous surface during the exposure to radiant heat (B). It should be noted 1) that radiation from an incandescent lamp easily passes through the thickness of the skin (B) in a fashion dependent on the wavelength emitted by the source of radiation (Fig. 3B); 2) that there is a finite speed of conduction of heat within the skin (C); and 3) that radiation from a CO2 laser penetrates little more than 100 µm (D).

View larger version (19K): [in this window] [in a new window]   Fig. 7.   A, when subjected to a constant-power heat source, the temperature of the skin surface increases in proportion to the square root of time. This increase is more gradual deep within the skin (broken white line). Moreover, an additional inaccuracy is introduced at the beginning of stimulation by the thermal inertia of the lamp when it is not preheated. The combination of these factors can give an illusion of linearity in the increase in temperature. B, increase in temperature can be proportional to time when contact thermodes are used. The more gradual increase in temperature within the skin (broken white line) is highly dependent on the rate of heating of the thermode. C, when part of the body is immersed in a hot, soaking fluid such as water, the temperature of the skin interface almost immediately achieves the temperature of the bath if the bath has an adequate thermostatic control and the water is agitated. In this case, one must bear in mind that the increase in temperature within the skin (broken white line) will be smoother and more gradual. D, diagrammatic representation of the activity evoked in cutaneous receptors by different temperatures applied to the skin (Hensel, 1973; Duclaux and Kenshalo, 1980; Meyer et al., 1994; Treede et al., 1995). When the temperature is gradually increased from the normal value for the skin (around 30°C with an ambient temperature of 20°C) to within the noxious range, there is a successive activation of thermoreceptors and then C- and A-polymodal nociceptors. At the highest temperatures, high-threshold mechanoreceptors and cold receptors are also activated. On the basis of the recruitment of these different receptors, four successive periods shown in A, B, and C can be defined: 1) thermoreceptors; 2) thermoreceptors and C-polymodal nociceptors; 3) C- and A-polymodal nociceptors; and 4) polymodal nociceptors, high-threshold mechanoreceptors, and cold receptors.

3. Mechanical Stimulation. The application of a noxious mechanical stimulus can be progressive or coarse. Responses produced by noxious mechanical stimuli are graded in relation to the intensity and/or duration of the stimulus, from reflexes up through vocalizations ultimately to complex motor behaviors. The stimulus is stopped as soon as a response is obtained. The type of mechanical stimulus used by von Frey in the last century (Handwerker and Brune, 1987) is often almost revered by neurologists, but it has the disadvantage of activating low-threshold mechanoreceptors as well as nociceptors. Consequently, the stimulus is not specific. There are also technical difficulties in applying mechanical stimuli, especially in freely moving animals. In addition, when mechanical stimuli are truly nociceptive, they are likely to produce changes in the tissues (sensitization or actual lesions). Furthermore, conventional techniques do not allow noxious mechanical stimuli to be delivered rapidly and briefly enough to produce synchronous excitation of the nerve fiberswith disadvantages identical to those discussed above for thermal stimuli. Finally, especially in small animals such as rodents, the parts of the body that are stimulated are themselves small, which can produce problems for the scientist in separating cause (stimulus) and effect (reaction). This problem is so great that the most common mechanical stimuli (pinches) are really double stimuli. There are also animal models of visceral pain triggered by mechanical stimuli, in this case involving the dilatation of hollow organs (see Section VI.C.).

4. Chemical Stimulation. Chemical stimulation involving the administration of algogenic agents represents a slow, or even very slow, form of stimulation. In this respect, chemical stimuli are clearly different from other forms of stimulation; they are also progressive, are of longer duration, and have an inescapable character once they have been applied. As a result, typical reflexes, which necessitate a minimum level of synchronization of activity in primary afferent nerves, are not produced by these stimuli (although reflexes can be facilitated by algogenic agents such as capsaicin; Gilchrist et al., 1996; Yeomans et al., 1996b).

It also seems reasonable to classify tests in terms of the biological function being recorded. The observed reactions cover a very wide spectrum ranging from the most elementary reflexes to far more integrated behaviors (escape, avoidance). In almost every case, it is a motor response that is monitored; vegetative responses are considered only occasionally (Ness and Gebhart, 1988; Gebhart and Ness, 1991; Holzer-Petsche, 1992; Sherman and Loomis, 1994; Holzer-Petsche and Rordorf-Nikolic, 1995; Reina and Yezierski, 1995; Roza and Laird, 1995; Culman et al., 1997; Taylor et al., 1998). It is important to bear this in mind when considering all results obtained from such tests. Indeed the analysis of the results should take account of the possibility that nociceptive processes interact with other linked phenomena, particularly motility itself (Chapman et al., 1985; Schomburg, 1997; see Section VIII.). It is equally necessary to consider nociception along with the other phenomena responsible for the homeostatic balance of the organism (see Section XII.). When you consider that even today there are no major analgesics without secondary side effects, you realize how difficult it is to analyze the results of tests of nociception.

	IV. Behavioral Models of Nociception

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Ideally, a behavioral model for nociception in an animal should possess the characteristics detailed below (Goetzl et al., 1943; Taber, 1974; Lineberry, 1981; Vierck and Cooper, 1984; Ramabadran and Bansinath, 1986; Hammond, 1989; Watkins, 1989; Dubner, 1994; Tjølsen and Hole, 1997).

Specificity.  1) The stimulus must be nociceptive ("input specificity"). Although this is common sense, it is not always easy to confirm that it is being achieved. For example, the appearance of a flexion reflex does not inevitably mean that the stimulus is nociceptive or that it is a nociceptive flexion reflex. Indeed, flexion reflexes are not triggered exclusively by nociceptive stimuli (see Section VIII.). This can lead to misinterpretations.

2) It must be possible in the behavioral model to differentiate responses to nociceptive stimuli from responses to non-nociceptive stimuli. In other words, the quantified response has to be exclusively or preferentially triggered by nociceptive stimuli ("output specificity"). In this respect, one has to bear in mind that some innate or acquired behaviors can be triggered by aversive stimuli that are not nociceptive/painful.

Sensitivity.  3) It must be possible to quantify the response and to correlate this variable with the stimulus intensity within a reasonable range (from the pain threshold to the pain tolerance threshold). In other words, the quantified response must be appropriate for a given type of stimulus and monotonically related to its intensity.

4) The model must be sensitive to manipulations and notably pharmacological ones, which would reduce the nociceptive behavior in a specific fashion. A sensitive test must be able to show effects for the different classes of antinociceptive agents at doses comparable to those used for analgesics in humans.

Validity.  5) The model must allow the differentiation of nonspecific behavioral changes (e.g., in motility and attention) from those triggered by the nociceptive stimulus itself. In other words, the response being monitored must not be contaminated by simultaneous perturbations related to other functions, notably if they have been introduced by a pharmacological agent. The test validity, i.e., the degree to which the test actually measures what it purports to measure, is undoubtedly one of the most difficult problems to resolve (see Section XII.).

Reliability.  6) Consistency of scores must be obtained when animals are retested with an identical test or equivalent form of the test. In this context, the repeated application of the stimulus must not produce lesions.

Reproducibility.  7) Results obtained with a test must be reproducible not only within the same laboratory but also between different laboratories.

Because no test of nociception meets all these criteria, the choice of which test to use has to be a compromise. Before describing these tests, it is worth noting that in general, they can be divided into two overall categories depending on whether it is a threshold or a supraliminal response to a given stimulus that is being measured. Note that both these categories permit one to investigate only one point on the stimulus-response curve, be it the threshold or an arbitrary point further up the curve. As a result, they allow only a rough appreciation of the gain of the process (Tjølsen and Hole, 1997). For the main part, the models involve rodents, most often the rat. In this review, when the species is not explicitly mentioned, we are referring to models that are based on the rat.

If we restrict ourselves to acute cutaneous and acute visceral pain, it is useful to classify the animal models used on the basis of the physical characteristics of the stimuli. We therefore successively consider tests based on the use of short-duration stimuli (in the order of seconds) and then those based on the use of longer-duration stimuli (in the order of minutes). The former relate to pains of cutaneous origin, with physical stimuli (thermal, mechanical, electrical) applied to small areas, often at increasing intensities. The latter relate to pains of cutaneous or visceral origin, with chemical stimuli (algogenic substances) being applied usually subcutaneously or intraperitoneally. In addition, one can add to the latter category tests based on the distension of hollow organs (visceral mechanical stimulation); such stimuli last for intermediate periods of time.

	V. Use of Short-Duration Stimuli ("Phasic Pain")

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These tests are the most commonly used. In general they 1) involve a short period of stimulation; 2) have somatic rather than visceral sites of stimulation; 3) involve measuring thresholds with the result that they generate no information whatsoever regarding responses to frankly nociceptive stimuli; 4) usually involve measuring the response time to a stimulus of increasing intensity with the explicit or implicit assumption that this reaction time is related to the threshold; 5) involve stimulation of minimal surface areas, with two important exceptions: the hot plate and the electrified grid, where the four paws and tail of the animal are stimulated simultaneously; and 6) can be classified by the nature of the stimulus, be it thermal, mechanical, or electrical.

In tests involving thermal stimuli, it is always the skin that is stimulated. These tests do not involve visceral or musculoskeletal tissues. However, it is important not to forget that radiant heat also stimulates thermoreceptors and that, consequently, the application of a ramped thermal stimulus will result in an organized and unalterable sequence of activation, namely thermoreceptors, then thermoreceptors plus nociceptors, then nociceptors alone, and finally (possibly) nociceptors plus "paradoxical cold" receptors (Fig. 7D). In practice, the animal withdraws itself quickly from the stimulus, and therefore only the first part of this scenario takes place.

The source of nociceptive stimulation can be distant from its target (e.g., radiant heat from a lamp) or can be in direct contact with the skin. Radiant heat constitutes a relatively selective stimulus for nociceptors and has an advantage over the other modes of thermal stimulation in that it produces no tactile stimulus.

We first mention the work of Ercoli and Lewis (1945), who applied the method used in humans by Hardy et al. (1940) to 2200 rats. The beam was directed at the animal's back, which had been shaven the previous day. By simultaneously opening the obturator and starting timers, it was possible to measure two successive response times: that of a local response, which consisted of a "twitch", and then that of a general response involving an "escape" reaction by the animal. The most interesting observation by these authors related to morphine: although it affected both responses, it was more powerful against the second, to such an extent that increasing the dose resulted primarily in an increase in the difference between the two response times, with the second becoming increasingly longer. Andrews and Workman (1941) and Winder et al. (1946) studied similar muscle responses in the dog and the guinea pig.

1. The Tail-Flick Test. There are two variants of the tail-flick test. One consists of applying radiant heat to a small surface of the tail. The other involves immersing the tail in water at a predetermined temperature. Although apparently similar, these two alternatives are actually quite different at a physical level: the cutaneous temperature varies with the square root of time in the first case and more rapidly in the second (Fig. 7, A and C). In addition, the stimulated surface areas can be very different. Indeed it is surprising that authors generally consider these two tests as though they were equivalent.