Behavioral reactions of fishes to pain stimuli

L.S.Chervova.

The sensation of pain as a factor for protection has been formed during natural salection from the fundamental property of animals, i.e., the ability to identify unfavorable external stimuli and respond to them. This property is inherent, to a certain degree, in representatives of all phylogenetic levels (Kavaliers, 1988). Pain is caused by stimuli that lead to the damage of the orgsnism or which threaten to do so. The functional essence of pain lies not in the discrimination of the quality of the external stimulus, but in the sensation in the pains impact that is simultaneously a factor that induces th animal to leave its source. Pain as a type of sensory modality is difficult for study and analysis since "pain" is interpreted as several complex physiological processes: information, sensation, emotion, or neurophysiological phenomenon; therefore, neigher the nature of pain, nor the range of sensations involved in this concept has a definite determination, even for people. In humans, the formation of pain as an act of consciousness is a complicated process and is determined by biological and social factors (Orbeli, 1966).

Since the anatomical and functional characteristics of the structures of the brain of representatives of different phylogenetic levels do not suggest that all animals can have realized sensations that are similar to thouse in man, the term "pain" cannot be considered precise with respect to animals. When considering pain in animals, it would be more proper to use a more correct term "nociception" (from Lat. noceo, to harm, damage, spoil) that means the reception of the aversive stimulus and the process of its conduction along the structures of the nervous system up to the brain where the pain experience is mediated neuronally (Sherrington, 1906). One of the definition of "pain" in animals may be the following "an aversive sensory experience caused by an actual or potential injury that elicits protective motor and vegetative reactions, results in learning avoidance and may modify specific species behavior" (Zimmerman, 1991). Probably, it must be considered that when authors use the term "pain" in the scientific literature for convenience, speaking about animals, they mean namely nociception rather than a complex subjective and emotional sensation that is characteristic of man.

In fish, pain sensation and behavior induced by them have not be purposefully studied as yet. The information available is fragmentary and scarce. The main anatomical, physiological, and biohemical components of nociception in fish are similar to those in mammals. Pain receptors in fish, i.e., nociceptors, as with other vertebrates, represent free nerve endings of spinal (on the trunk) and trigeminal (on head) nerve fibers. In fish, free nerve endings are localized over the entire body surface including fins. They have been recently discovered in the olfactory epithelium in Brachydanio rerio (Hansen and Zeiske, 1998). In some areas, free nerve endings approach the epidermis surface at a distance of 0,5 ?m (Whitear, 1971). Studies performed on Cyprinus carpio, Parasalmo mykiss, and Gadus morhua indicated that in these fishes, nociceptive sensitivity is on the entire body surface. The most sensitive to nociceptive stimuli in these species were the lobe of the caudal fin, dorsal and pectoral fins, skin around eyes, and epithelium of the olfactory sac; the skin of the head and body surface was less sensitive (Chervova et al., 1994; Chervova, 1977). The high density of nociceptors on fins is likely to be related to the fact, in particular, that fins are damaged in fish during their nest-building activity or agressive interactions. The information from nociceptors comes to the neurons of the posterior horn of the spinal cord or the trigeminal caudal nucleus of the brain stem; then, it is distributed in the central nervous system though many pathways. First, to motoneurons that send impulses to motor muscles. This provides for the formation of a rapid behavioral response, i.e., a motor protective response (shuddering, drawing back of extremity, avoidance). In parallel, the nociceptive information is delivered to the ascending tracts that conduct it to higher brain centers where other components of the pain responce of becoming alert and vegetative and emotional responses are formed (Breazile and Kitchel, 1969).

Processes that occur in the central nervous system in response to nociceptive stimulation have not been studied in fish. However, substance P, a neuropeptide that is a mediator of nociception in mammals, was found in the peripheral nerve endings of Gadus morhua and Parasalmo mykiss (Johnsson et al., 1998), in the spinal cord of the Dasyatis sabina (Ritchie and Leonard, 1983) and in different regions of the brain of Carassius auratus (Sharma et al., 1989) and Salmo salar (Vecino and Ecstrom,1991). In the spinal cord and trigeminal ganglion of Petromizon marinus, neurons were found that respond by pulsed discharges to a strong squeezing of skin that leaves traces of pinching, punctures with an acute needle, and cauterization up to appearance of white spots (Martin and Wickelgren, 1971, Matthews and Wickelgren, 1978).

Behavioral responses that in mammals are considered to be markers of primary pain sensitivity that is on the verge of the tactile sensation and are controlled, as is supposed, by the limbic system are primary motor stsrt responses, a simple nonspecific avoidance (Wall, 1992). These responses are observed in fish in response to pain stimuli (Rekubratskii, 1967; Jansen and Green, 1970; Sickarulidze and Kadagishvili, 1974; Ehrensing et al., 1982). In mammals, nociceptor stimulation is accompanied by an affective response, i.e., vocalization. Special sounds that are emitted at wounding were also recorded in fish. In Misgurnus fossilis, these sounds were generated by the swimming bladder and were characterized by a frequency spectrum of 0 to 4000 Hz with maxima of 500, 1500, and 3000 Hz and continued for 490 ms, on average. According to these parameters, the pain "shout" of Misgurnus fossilis differed from sounds that accompanied food seizure, feeding, or spawning behavior (Nikol'skii et al., 1968).

To assess coordinated responses to a pain stimulus (that in mammals are likely to be controlled by the cortex of the brain, for instance, an attack of the stimulus source, rubbing slightly of the stimulation site), it was suggested to use a method of conditional avoiding for other than mammals and voiseless species (Charpentier, 1968). In bony fish (that have no cortex of the brain) the conditioned reflex of avoiding the aversive stimulus is easily formed if rapid impetuous swimming is peculiar to these fish in nature. Thus, Trachurus mediterraneus ponticus was taught to avoid a light signal that was followed by a pain electric stimulus after 12 to 13 combinations. In slow-moving fish, "ambuscaders", as with Neogobius melanostomus and Symphodus roissali, a reflex of avoidance failed to form in response to an conditional stimulus. In response to an electric stimulus, these fish shuddered, performed a short dart aside, and came to a standstill. They responded to the subsequent pain stimuli by an increase in respiration rate (Rekubratskii, 1967).

Under social interactions, the animal is permanently confronted with situations when it is compelled, to a certain degree, to show no response to nociceptive impacts for the sake of more important biological motivations, such as the search for food, reproduction, maintenance of social contacts, and maintenance of progeny. Therefore, in animals with a developed central nervous system, there are different neuronal mechanisms that perform a control and limiting function with respect to nociceptive afferent information on the segmentary and brain levels. Similar biological systems of control have an adaptive significance since they provide for a steady state of the organism in such situations when, for instance, the nociceptive impulsion from injured site reaches the tolerance level. The regulating impact in this case is made by the endogenous antinociceptive system that maintance and regulates the threshold of pain sensitivity on the needed functional level. This system is represented maimly by endogenous opioid peptides (enkephalins and endorphins) and opioid receptors (targets for endogenous opioids) that are subdivided into three main classes mu, delta and kappa (Dhawan et al., 1996).

Opioid peptides, or endogenous morphines, act similarly to exogenous opiates, i.e., morphine and codeine that are contained in the opium poppy (Papaver somniferum). Opioid peptides are linked with opiate receptors and, similarly to morphine, suppress pain responses. In addition, they exert significant effects on learning and memory, sexual and reproductive behavior, feeding, social behavior, locomotory patterns, temperature control, etc (Krieger, 1983).

In fish, as with land animals, components of the opioid antinociceptive system, i.e., endogenous opioid peptides and opioid receptors were also found. Opioid peptide beta-endorphin was found in pituitaries of Squalus acantias, Merluccius productus, Oncorhynchus kisutch (Pezalla et al., 1978), in the basal hypothalamus of the small spotted dogfish Scyliorhynus canicula (Vallarino et al., 1989b), in hypothalamic nuclei of n.tuberis and n. preopticus of Boops boops ( Vallarino, 1985), and in the mesencephalon and medulla oblongata of Parasalmo mykiss; beta-endorphin distribution in the central nervous system corresponds to the pattern of the peptide containing neurons in the basal hypothalamus of the tetrapods (Vallarino et al., 1989a). Enkephalins were described in the preoptic nucleus, neuroendocrine cells of hypothalamus, olfactory lobe, visceral nuclei and cerebellum of Carassius auratus (Reaves and Hayward, 1979; Finger, 1981; Schulman et al., 1981), in different structures of the forebrain, interbrain, mesencephalon, medulla oblongata, and cerebellum of Parasalmo mykiss and Salmo salar (Vecino and Ekstrom, 1991; Vecino et al., 1992), in nuclei of the suture of the reticular formation of truncus cerebri in Hydrolagus colliei, Heterodontus francisci, and Squalis acantias (Stuesse et al., 1995).

Opioid receptors were also found in the brain of fish. In Carassius auratus, many opiate receptor binding sites were found in olfactory bulbs and in tegmentum mesencephali (Pert et al., 1974), as well as in the region of the forebrain that is homologous to the striatum of land animals (Edley et al., 1982) as well as in the forebrain of spiny dogfish, smooth dogfish, and in the brain of Mixine (Pert et al., 1974). mu-Opioid receptors that mediate mainly antinociceptive effects have not been found in fish for a long time; this fact suggested that mu-opioid antinociception appears during evolution first in amphibians. Later, mu- and kappa-opioid receptors were found in the brain of Carassius auratus (Brooks et al., 1993) and in different structures of the brain of coho salmo parr (Ebbesson et al., 1993). In addition, in Catostomus commersoni and Brachydanio rerio were recently isolated complementary DNA and cloned mu-, delta-, and kappa-opioid receptors that are coded by them (G-proteins); the latter displayed high similarity with the respective opioid receptors in mammals according to their amino-acid sequences (Darlison et al., 1997; Barrallo et al., 1998).

It was shown that opiates and opiod peptides modulate the nociceptive behavior of fish. In Carassius auratus, the effects of morphine on the agitation swimming response caused by the pain electric stimulus applied to the dorsal fin was studied. Morphine that was added in stages (by 140 mkg) in the aquarium (1 l), was consumed by fish through gills and had an analgetic effect. These effects were displayed by an increase of the electric stimulus needed for the appearance of an active swimming response. As with higher vertebrates, morphine tolerance was formed: its analgetic effect decreased following repeated injection (Jansen and Green, 1970). In another experiment, in Carassius auratus, at an electric stimulation that was gradually increased and applied just caudally to the fish's dorsal fin, the response of shuddering was recorded. The threshold of this response increased under the effects of morphine added to water (Ehrensing et al., 1982). The analog of beta-endorphin injected intracranially and intraperitoneally significantly lengthened the latent period of the response of avoiding a strong acoustic stimulus (108 dB) in freely swimming Carassius auratus. The behavioral effect was stronger at the intracranial injection that proves the effects of beta-endorphin on the central mechanisms of behavioral control (Olson et al., 1978). In addition morphine that was added to the aquarium water affected the aggressive behavior of convict cichlids, Cichlasoma nigrofasciatum, significantly decreasing the number of attacks of the fish-resident on the fish-intruder (Avis and Peeke, 1975). The intranasally injected mu-opioid analgetics dermorphin and beta-casomorphin decreased the magnitude of behavioral responses of Gadus morhua and Parasalmo mykiss to nociceptive electric stimuli (Chervova et al., 1992, 1994). Using a new behavioral test, it was found that nociceptive thresholds in Cyprinus carpio significantly increasd following the intramuscular injection of agonists mu-, delta-, and kappa-opioid receptors, tramadol, DADLE, and U-50488, respectively (Chervova and Lapshin, 1999).

Thus, on the basis of the data presented, it is reasonably safe to suggest that fish, similar to higher vertebrates, have nociceptive and opioid antinociceptive systems that on the level of central mechanisms take part in the control of fish behavior.

ACNOWLEDGEMENTS

Author is thankful to Prof. A.O.Kasumyan for his help in preparing this paper.

REFERENCES

Avis, H.H., and Peeke, H.V.S., Differentation by Morphine of Two Types of Aggressive Behavior in the Convict Cichlid (Cichlasoma nigrofasciatum), Psychopharmacologia (Berl), 1975, vol. 43, p. 287-288.

Barrallo, A., Gonzalez-Sarmiento, R., Porteros, A., et al., Cloning, Molecular Characterization, and Distribution of a Gene Homologous to delta-Opioid

Receptor from Zebrafish (Danio rerio), Biochemical and Biophysical Research

Breasile, J.E., and Kitchel, R.L., Pain Perception in Animals, Fed. Proceed., 1969, vol. 28, no. 4, pp. 1379-1382.

Brooks, A.I., Standifer, K.M., Ciszewska, G.R., et al., Expression of kappa-3 and mu Binding Sites in Bufo marinus (Giant Toad) and Carassius auratus (Goldfish) Brain, Soc. Neurosci. Abstr., Opioids: Recep., III, 1993, vol. 19, no. 5, p. 1156.

Charpentier, J., Analisis and Measurement of Pain in Animals, a New Conception of Pain, Pain. Soulairac, A., Cahn, J. and Charpentier, J., Eds., London: Academic Press. 1968.

Chervova, L.S., Pain Sensitivity and Behavior of Fishes, J. of Ichthyol., 1997, vol. 37, no. 1, pp. 98-102.

Chervova, L.S., Kamenskii, A.A., Malyukina, G.A., et al., Investigation of the Mechanism of Intranasal Effect of Dermorphin in Representatives of Two Classes of Vertebrates, Zh. Evol. Biokhim. Fiziol., 1992, vol. 28, no. 1, pp. 45-48.

Chervova, L.S., and Lapshin, D.N., Opioid modulation of nociceptive thresholds in fish, Abstr. 30th International Narcotics Research Conference. Saratoga Springs, NY, USA, July 10-15, 1999, p. 81.

Chervova, L.S., Lapshin, D.N., and Kamenskii, A.A., Pain Sensitivity of Trout and Analgesia Caused by Dermorphin Administrated Intranasally, Dokl. Biol. Scienc., 1994, vol. 338, pp. 424-425.

Darlison, M.G., Greten, F.R., Harvey, R.J., et al., Opioid Receptors from a Lower Vertebrate (Catostomus commersoni): Sequence, Pharmacology, Coupling to a G- Protein-Gated Inward-Rectifying Potassium Channel (GIRK1) and Evolution, Proc. Natl. Acad. Sci. USA. Neurobiology, 1997, vol. 94, pp. 8214-8219.

Dhawan, B.N., Cesselin, F., Raghubir, R., et al., International Union of Pharmacology. X11. Classification of Opioid Receptors, Pharmacological Reviews, 1996, vol. 48, no 4, pp. 567-592.

Ebbesson, L.O.E., Daviche, P., and Ebbesson, S.O.E. Distribution and Changes in mu- and kappa-Opiate Receptors in the Midlife Critical Period of Natural Development in Coho salmon, Oncorhynchus kisutch, Abstr. Amer. Assoc. Anat., 106th Ann. Meet. Joint. Jpn. Assoc. Anat., San Diego, Calif., March 27-31, 1993, Anat. Rec., 1993, Suppl., p. 48.

Edley, S.M., Hall, L., Herkenham, M., and Pert, C.B., Evolution of Striatal Opiate Receptors, Brain Research, 1982, vol. 249, pp. 184-188.

Ehrensing, R.H., Michell, G.F., and Kastin, F.J., Similar Antagonism of Morphine Analgesia by MIF-1 and Naloxone in Carassius auratus, Pharmacol. Biochem. and Behav., 1982, vol. 17, pp. 757-761.

Finger, T.E., Enkephalin-like Immunoreactivity in the Gustatory Lobes and Visceral Nuclei in the Brains of Goldfish and Catfish, Neuroscienc., 1981, vol. 12, pp. 2747- 2758.

Hansen, A., and Zeiske, E., The Periferal Olfactory Organ of the Zebrafish, Danio rerio: an Ultrastructural Study, Chem. Senses, 1998, vol. 23, pp.39-48.

Jansen, G.A., and Greene, N.M., Morphine Metabolism and Morphine Tolerance in Goldfish, Anesthesiology, 1970, vol. 32, pp. 231-235.

Johnsson, M., Axelsson, M., and Holmgren, S., Localization of Neuropeptides in Perivascular Nerves Innervating Veins in the Atlantic Cod (Gadus morhua) and the Rainbow Trout (Oncorhynchus mykiss), Abstr. of Vlll Intern. Symp. on Fish Physiol., 15-18 August, 1998, Uppsala, Sweden, p. 34.

Kavaliers, M., Evolutionary and Comparative Aspects of Nociception, Brain Research Bull., 1988, vol. 21(6), pp. 923-931.

Krieger, D.T., Brain Peptides: What, Where, and Why?, Science, 1983, vol. 222, pp. 975-985.

Matthews G., and Wickelgren, W.O., Trigeminal Sensory Neurons of the Sea Lamprey, J. Comp. Physiol., 1978, vol. 123, pp. 329-333.

Martin, A.R., and Wickelgren, W.O., Sensory Cells in the Spinal Cord of the Sea Lamprey, J. Physiol., 1971, vol. 212, pp. 65-83.

Nikolskii, I.D., Protasov, V.R., Romanenko, E.V., and Shishkova, E.V., Zvuki ryb. Atlas (Sounds of fishes. Atlas), Moscow: Nauka, 1968.

Olson, R.D., Kastin, A.J., Michell, G.F., et al., Effects of Endorphin and Enkephalin Analogs on Fear Habituation in Goldfish, Pharmacol. Biochem. and Behav., 1978, vol. 9, pp. 111-114.

Orbeli, L.A., Problema boli (Problem of pain), Izbrannie trudy, Moscow-Leningrad: Nauka, 1966, vol. 4, pp. 129-190.

Pert, C.B., Aposhian, D., and Snyder, S.H., Phylogenetic Distribution of Opiate Receptor Binding, Brain Research., 1974, vol. 75, pp. 356-361.

Pezalla, P.D., Clarke, W.C., Lis, M., et al., Immunological Characterization of b- Lipotropin Fragments (Endorphin, b-MSH, and N-Fragment) from Fish Pituitaries, General and Comparative Endocrinology, 1978, vol. 34, pp. 163-168.

Reaves, T.A.J., and Hayward, J.N., Immunohistochemical Identification of Enkephalinergic Neurons in the Magnocellular Preoptic Nucleus of the Goldfish, Carassius auratus, Cell and Tissue Research, 1979, vol. 299, pp. 147-151.

Rekubratskii, V.A., Ekologicheskie stereotipy pischedobyvatelnogo i zaschitnogo povedenija ryb (Ecological stereotypes of foraging and defensive behavior of fishes), Povedenie i recepcii ryb (Behavior and receptions of fishes), Moscow: Nauka, 1967, pp. 121-125.

Ritchie, T.C., and Leonard, R.B., Immunohistochemical Studies on the Distribution and Origin of Candidate Peptidergic Primary Afferent Neurotransmitters in the Spinal Cord of an Elasmobranch Fish, the Atlantic Stingray (Dasyatis sabina), J. of Comp. Neurol., 1983, vol. 213, pp. 414-425.

Schulman, J.A., Finger, T.E., Brecha, N.C., and Karten, H.J., Enkephalin Immunoreactivity in Golgi Cells and Mossy fibers of Mammalian, Avian, Amphibian and Teleost Cerebellum, Neurosci., 1981, vol. 6, pp. 2407-2416.

Sharma, S.C., Berthoud, V.M., and Breckwoldt, R., Distribution of Substance P-Like Immunoreactivity in the Goldfish Brain, J.Comp. Neurol., 1989. V. 279. P. 104-116.

Sherrington C.S., Integrative Action of the Nervous System, Cambridge: Cambridge University Press, 1906.

Sikharulidze, N.I., and Kadagishvili, A.Ja., K izucheniju emocional'noj pamjati u ryb (Study of the emotional memory of different force stimuli in fish), Soobschenija AN Gruz.SSR, 1974, no 73, p. 181.

Stuesse, S.L., Stuesse, D.C., and Cruce, W.L.R., Raphe Nuclei in Three Cartilaginous Fishes, Hydrolagus colliei, Heterodontus francisci, and Squalus acantias, J. of Comp. Neurol., 1995, vol. 358, pp. 414-427.

Vallarino, M., Occurance of b-Endorphin-Like Immunoreactivity in the Brain of the Teleost, Boopa boops, Gen. Comp. Endocrinol., 1985, vol. 60, pp. 63-69.

Vallarino, M., Delbende, C., Tranchand Bunel, D., et al., Proopiomelanocortin (POMC)-Related Peptides in the Brain of the Rainbow Trout, Salmo gairdneri, Peptides, 1989 (a), vol. 10, pp. 1223-1230.

Vallarino, M., Tranchand Bunel, D., Delbende, C., et al., Distribution of the Pro- Opiomelanocortin-Derived Peptides, Alpha-Melanocyte-Stimulating Hormone (a- MSH), Adrenocopticotropic Hormone (ACTH), and Beta-Endorphin in the Brain of the Dogfish Scyliorhynus canicula: An Immunocytochemical Study, J. of Exper. Zool. Suppl., 1989 (b), vol. 2, pp. 112-121.

Vecino, E., and Ekstrom, P., Distribution of met-Enkephalin, leu-Enkephalin, Substsnce-P, neuropeptide Y, FMRFamide and Serotonin Immunoreactivities in the Optic Tectum of the Atlantic Salmon (Salmo salar L.), J. of Comp. Neurol., 1991, vol. 299, pp.229-241.

Vecino, E., Pinuela, C., Arevalo, R., et al., Distribution of Enkephalin-Like Immunoreactivities in the Central Nervous System of the Rainbow Trout: an Immunocytochemical Study, J. of Anatomy, 1992, vol. 180, pp. 435-453.

Whitear, M., The Free Nerve Endings in Fish Epidermis, J.Zool.(Lond.), 1971, vol. 163, pp. 231-236.

Wall, P.D., Defining 'Pain in Animals', Animal Pain, Short, C.E. and Van Poznak, A., Eds., New-York: Churchill Livingstone, 1992, pp. 63-79.

Zimmerman, M., Behavioural Investigations of Pain in Animals, Assessing Pain in Farm Animals, Duncan, I.J.H. and Molony, V., Eds. Luxemburg: Commission of the European Communities, 1986, pp. 30-35.