Opioid antinociception in amphibians

0361-9230/88 $3.00 + .OO

Brain Research Bulletin, Vol. 21, pp. 959-962.0 Pergamon Press plc, 1988.Printed in the U.S.A.

Opioid Antinociception in Amphibians CRAIG Department

of Cell Biology

W. STEVENS’

and Neuroanatomy,

University

of Minnesota,

Minneapolis,

MN 55455

STEVENS, C. W. Opioid antinociception in amphibians. BRAIN RES BULL 21(6) 959-%2, 1988.-Systemic and spinal administration of opioids produces a behaviorally defined antinociception in a variety of mammalian models. Although endogenous opioid peptides and opioid binding sites are ubiquitous throughout phylogeny, little attention has been paid to

the function of endogenous opioid systems(s) or development of nociceptive models in nonmammalian species. Recent work has shown that the amphibian, Rana pipiens, provides an appropriate model for assessment of opioid antinociception and that endogenous opioid systems may likewise modulate the central processing of noxious information in amphibians as well as mammals. Opioid

Amphibian

Morphine

Antinociception

Acetic-acid test

Intraspinal

Analgesia

did not attempt to identify the opioid ligands present. They found that the brains of all vertebrates examined contained substantial amounts of endogenous opioids, with higher concentrations in nonmammalian species than in mammals. Although they note that increasing presence of cerebral cortex, which contains very low levels of opioid content, may dilute the subcortical concentrations of opioids in mammals, it is interesting that the highest concentrations were found in the amphibian, Bufi, marinus (37). More recent radioimmunoassay and immunohistological studies have shown that high concentrations of Metenkephalin exist in the pituitary, hypothalamus, extrahypothalamic brain, retina, skin and blood of the grass frog, Rana pipirns (19). A detailed anatomical examination of the diencephalon revealed Met-enkephalin fibers in infundibular nuclei, median eminence, and neurohypophysis in Rana trmporaria (11) and Rana catesbiana (55). Met-enkephalin immunoreactivity has been detected in spinal cord of Rana esculenta with stained fibers in dorsal horn, and intermediate and central gray area; immunoreactive perikarya were also noted (22). The processing of proopiomelanocortin in the frog pituitary produces beta-endorphin (31) and RIA methods show higher concentrations of beta-endorphin in the whole pituitary and hypothalamic brain extracts in frogs than in corresponding tissues in the rat (20). The third major opioid peptide, dynorphin, is similarly distributed in the toad, Bufo marinus, and in the rat (8). Immunoreactive dynorphin is highest in the pars intermedia, with substantial amounts in brain and spinal cord; these authors also note that dynorphin concentrations are higher in the toad than in the rat. A fourth class of endogenous opioids unique to amphibians are the dermorphins. Discovered by Erspamer and colleagues in the skin extracts of South American Phyllomedusa frogs (6,23), these opioids are incredibly potent; from 2,000-5,000 times more potent than morphine in smooth

THE discovery of stereospecific opioid binding sites (29,38, 42) and endogenous opioid peptides (17, 49, SO) in mammalian CNS ushered in a new era of opioid pharmacology and the intense research that followed resulted in the establishment of a major new neurotransmitter system-the endogenous opioid system (EOS). Although the activity of the EOS in mammals has been shown to modulate a number of behaviors, the inhibition of nociceptive behavior is the single most accessible indicator of EOS function, and in light of potential clinical value, remains the focus of opioid research. In contrast to the vast literature on EOS and modulation of nociception in mammalian vertebrates, little attention has been focused on the possible function(s) of EOS or the development of behavioral models for antinociception in nonmammalian vertebrates. This brief review presents the evidence for EOS in amphibians and suggests that, as mammals, a role for opioid antinociception exists in amphibians. BIOSYNTHESIS AND LOCALIZATION OF ENDOGENOUS OPIOID PEnIDES It is well accepted that most biologically-active peptides are derived from posttranslational processing of larger polypeptide precursors (42). At present, all the major endogenous opioid peptides characterized in mammals can be linked to three separate precursors; proopiomelanocortin, proenkephalin, and prodynorphin (35). The human genomic sequences to all three endogenous opioid precursors has also been deduced using recombinant techniques (21,24,27). The evidence for the existence of these families of endogenous opioids in lower vertebrates, and especially amphibians, follows below. Soon after the isolation of Met- and Leu-enkephalin from porcine brain extract (17), a survey of endogenous opioid factors in CNS extracts from representative vertebrates from cyclostomes to primates was undertaken (37). These authors assayed endogenous opioid content by their ability to displace [3Hl-naloxone binding from rat brain homogenates but

‘Requests for reprints should be addressed to Dr. Craig W. Stevens, Department of CBN, 4-135 Jackson Hall, University of Minnesota, Minneapolis,

MN 55455.

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STEVENS

muscle bioassav (10) and algesiometric measures in rodents (45). This extraordinary opioid contains a D-alanine in its heptapeptide sequence and is the first D-amino acid containing peptide to be isolated in vertebrates (23). Recent genetic studies have shown that the D- form of alanine is racemized posttranslationally from the genetically encoded L-alanine (34). Dermorphin-like immunoreactivity has also recently been detected in brain extracts from mammals (4). LOCALIZATION

OF OPtOfD RECEPTORS

While the presynaptic side of EOS is highlighted by neurons and fibers staining for endogenous opioid peptides, the postsynaptic side of EOS is characterized by opioid receptors to which the released endogenous opioids bind and produce an effect. Opioid receptors have been characterized into four main subtypes by various biochemical and pharmacological criteria: mu, delta, kappa, and sigma opioid receptors [see (14,54)]. The first comparative study of opioid binding sites in a wide variety of vertebrates and invertebrates found significant stereospecific binding tissues of all species examined, including the amphibian, B&t tnurinlts (30). A regional analysis of opioid binding sites in goldfish brain revealed high densities in the forebrain. ventral midb~in, tectum, and medulla (37). These authors note that as the limbic system of mammals contains the highest densities of opioid receptors and is thought to have evolved from the olfactory forebrain areas of lower vertebrates, it is interesting to note that the highest levels of binding are seen in olfactory forebrain areas of shark, goldfish, and chick (37). A phylogenetic comparison of opioid receptor subtypes found that delta to mu ratio was lower in primitive amphibians (the salamanders Axolotl and Pkurodelrs) than in the rat (2). A comparison of mu and delta binding sites in brain homogenates from goldfish, frogs (X~~opus hwis), turtles, and rats revealed that the mu to delta ratio is greatest in rats, intermediate in turtles and frogs, and very small in goldfish (3). As other work in mammals suggests that mu and delta opioid receptors mediate the antinociceptive and respiratory effect of opioids, respectively (28), these authors note that lack of mu opioid receptors in goldfish suggests that opioid-mediated antinociceptive systems first arose in amphibians (3). They further note that the ontogeny of opiate binding sites in the developing rat reflects this phylogenetic trend towards higher mu-delta ratio. This phylogenetic trend was confirmed by measurement of mudelta binding ratios in brain homogenates from mammalian striata or striatal homologous in various nonmammalian species; mu binding exceeding delta binding only in warm-blooded species and a significant negative correlation was seen between percentage of delta binding and human relatedness as determined by cytochrome C sequence homology (12). Their data on Xf~}z~~~~.~ luc+s confirm earlier observations of about equal ratios of mu and delta binding sites in amphibians. Kappa and sigma opioid receptors have been reported in membrane-bound and solubilized opiate binding sites from toad brain (36,39). The toad brain was found to contain 60-7@% kappa or sigma sites, 20-3% mu sites and few or no delta sites. These results were confirmed by similar studies in an unspecified frog species (9). Although both groups do not address the conflict with earlier reports suggesting equal mu/delta binding, it is possible that earlier studies employed delta ligands with some binding activity at mu or kappa opioid sites.

NEURAL

PATHWAYS

SUBSERVING

NOClCEYTfON

The neural substrates conveying nociceptive information in mammals has been aptly elucidated, particularly at the spinal level, and the reader is referred elsewhere for an exhaustive review (53). Nociceptive pathways in amphibians are much less characterized, however, cutaneous nociceptors in frog skin are similar to those found in mammals; i.e.. free-nerve endings arising as the Deripheral terminations of thinly myelinated or unmyelinated primary afferents (41). These cutaneous receptors have been extensively studied since the classic experiments of Adrian (If and are characterized as nociceptors and nociceptive fibers primarily by electrophysiolog~cal parameters (conduction velocity, size, and duration of action potentials) evoked by presentations of noxious stimuli. These determinations are valid due to comparisons of nociceptive afferent activity in higher vertebrates and man (53), however, behavioral studies of nociceptive models in intact amphibians have only recently been made (see below). Central terminations of small-diameter dorsal root afferents are primarily found in the dorsal field of the spinal cord, although some investigators show afferent fibers penetrating to the ventral horn (25). Second-order neurons within the dorsal horn which receive direct dorsal root input have not been identified, nor having ascending nociceptive pathways been well-demonstrated (40). However, electrophysioIogica1 studies do show that electrical stimulation of the frog sciatic nerve produces evoked potentials in posterior thalamic nuclei and hypothalamus (5 I). OPIOID ANTINOCICEF’TION

Early studies employing massive doses of systemic morphine (600-1000 mgikg) in frogs noted proconvulsive effects exclusively (7, 15, 52) and it was not until much later that an attempt to measure antinociceptive activity in the intact frog was made (26). Subcutaneously administered morphine (20,60, or 100 mgikg) showed no significant antinociceptive effects as assayed in the electric grid test or hot plate measures (26). More recent work has shown that systemic morphine (10 or 100 mgikg) induces a significant increase in nociceptive threshold in Rum pipiens, as measured using the acetic acid test, which may be a more approprtate measure ot nociceptive threshold in frogs (32). This test is described fully elsewhere (32,43); briefly, eleven dilutions of acetic acid, equally spaced on a logarithmic scale from 0.26 to 15 M, are numbered in order of increasing concentration from 0 to 10. The nociceptive threshold is defined as the number of lowest concentration of acetic acid which causes a wiping response when applied to the dorsum of a frog’s thigh. The dilute acid drop is washed off with a stream of distilled water if no response occurs within 5 seconds or immediately if the wiping response occurs. Thus, this test is “response controlled” by the animal, i.e., the elicitation of nociceptive behavior immediately terminates the noxious stimulus, and as no tissue damage occurs, repeated measurements may be made to determine the time course of antinociceptive action. The wiping response remains intact after high spinal transection in the frog (13); in this respect it may be likened to the tail-flick nociceptive test in rodents. The results obtained by Pezalla (32) were the first example of opioid antinociceptive activity, specifically blocked by the opioid antagonist naloxone, in an intact nonmammalian vertebrate model. The apparent insensitivity of frogs to systemic morphine (as attested by the astronomical doses ad-

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OPIOIDS AND FROGS ministered in the early studies cited above) is apparently due to poor distribution of morphine into the amphibian CNS. In support of this hypothesis, further investigations by Pezalla and the present author demonstrated a potent antinociceptive action of morphine and other alkaloids injected directly into the lumbar spinal cord of unanesthetized frogs (43,44). This antinociceptive activity was measured on the acetic acid test, and was dose-dependent and blocked by concurrent intraspinal injection of naloxone. The exquisite sensitivity of the intact frog spinal cord to submicrogram quantities of morphine is similar to that obtained following intraspinal injection in mice; however, in the latter model the predominant motor effect (as evidenced by Straub tail occurring at same dosages at tail-flick antinociception) confounds the interpretation of the antinociceptive effects (18). In the frog, motor effects are also seen following systemic and intraspinal opiates, however the dosages to obtain these effects are higher than those needed to produce antinociception (33). Intraspinal injection of the three representative endogenous opioid peptides; Met-enkephalin, beta-endorphin, and dynorphin also produces a dose-dependent increase in nociceptive thresholds after nanogram quantities in Ram pipirns (47). Although the scant evidence for opioid antinociception in amphibians is obscured by the wealth of information obtained describing the powerful influence of opioid antinociception in mammalian models, a few comparisons may already be drawn. The antinociceptive activity after systemic administration of opioids in amphibians requires a greater dose; this may be due to differences in distribution from site of administration (subcutaneous or intraperitoneal in rodents vs. SC into dorsal lymph sac in amphibians) to target neurons in the CNS; in fact, it has been shown that the kappa opioid, ethylketocyclazocine, penetrates the blood-brain barrier less so in the toad than in the rat (5). The differential ratio of opioid receptor subtypes might also account for the apparent behavioral insensitivity of systemic opioids in amphibians. Direct intraspinal administration of opioids in amphibians and mammals appear comparable after morphine and other mu-preferring opioids; the recent evidence that Met-

enkephalin and dynorphin in frog spinal cord also produce potent. dose-dependent antinociception suggests that additional involvement of delta-opioid and kappa-opioid, respectively, systems as well. In contrast, spinal administration of the opioid-inactive enantiomer, dextrorphan, produces a dose-dependent antinociception in frogs (44); this has not been reported in other mammalian models. As dextrorphan demonstrates highest affinity for sigma opioid/phencyclidine (PCP) receptors (56), it is possible that such receptors, as well as a putative endogenous ligand for these receptors, exists in amphibian spinal cord. Secondly, the spinal administration of dynorphin in rodents produces a marked dysfunction which is not separated from antinociceptive actions (46), however, intraspinal dynorphin in amphibians produces a potent antinociception without any discernible motor effects (47). CONCLUSION

The basic CNS structures common to all vertebrates is well-established in amphibians (16) and the localization of endogenous opioid system(s) across vertebrate classes is found in phylogenetically old structures. Nociceptive pathways documented in mammals have been anatomically and histochemically identifed in amphibians, although much further work in this area is needed. Basic research into the mechanisms and regulation of endogenous opioid systems is the foundation for potential clinical benefit. The amphibian model may be the most parsimonious behavioral unit available; and, in view of decreased neural complexity, the importance of a comparative approach, and contemporary distaste for employing infraclass experimental subjects, may be a fruitfull approach to further investigation of opioid action and the EOS.

ACKNOWLEDGEMENTS

Sincere appreciation to V. Mancini Stevens for manuscript preparation, to Dr. Paul Pezalla for helpful discussions, and to Dr. Tony L. Yaksh for encouragement and support.

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