Localization and Partial Characterization of Melatonin Receptors in Amphioxus, Hagfish, Lamprey, and Skate

General and Comparative Endocrinology 110, 67–78 (1998) Article No. GC977042

Localization and Partial Characterization of Melatonin Receptors in Amphioxus, Hagfish, Lamprey, and Skate Adam J. Vernadakis,1 William E. Bemis, and Eric L. Bittman2 Department of Biology and Programs in Organismic and Evolutionary Biology and Neuroscience and Behavior, University of Massachusetts, Amherst, Massachusetts 01003 Accepted December 2, 1997

Through its secretion of melatonin, the pineal complex of vertebrates exerts a range of physiological effects including regulation of circadian rhythms, seasonal reproduction, metamorphosis, and body color change. Little is known about phylogenetic differences in the distribution and characteristics of melatonin binding sites in fishes. We used in vitro autoradiography to examine binding of [2-125I]iodomelatonin (IMEL) in 20-mm frozen sections of amphioxus (Branchiostoma lanceolatum), Atlantic hagfish (Myxine glutinosa), larval and adult lamprey (Petromyzon marinus), little skate (Raja erinacea), and rainbow trout (Oncorhynchus mykiss). Tissue was incubated with IMEL in the presence or absence of unlabeled melatonin (1 mM, in order to assess nonspecific binding). A concentration of 32 pM IMEL was used for single point assays and competition studies. No specific binding was found in hagfish or amphioxus, which lack a pineal complex. In the optic tecta of lamprey, skate, and trout, IMEL binding is highly specific (melatonin : N-acetylserotonin G 5- methoxytryptophol : serotonin). Scatchard analysis revealed that the tectal binding sites are of high affinity (Kd 5 36, 38, and 50 pM) and low capacity (Bmax 5 8.1, 19.8, and 21.8 fmol/mg protein) in lamprey, skate, and trout, respectively. In adult lampreys, intense specific IMEL binding is found in the optic tectum (layer I G II G III) and preoptic nucleus (pars parvocellularis G magnocellularis).

1 Present address: University of Massachusetts Medical School, 55 Lake Avenue N., Worcester, MA 01655. 2 To whom correspondence should be addressed at Department of Biology, University of Massachusetts, Amherst, MA 01003. Fax: (413) 545-3243. E-mail: [email protected].

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Binding was less intense and consistent in the same areas of ammocoete brain. In skates and trout, intense specific binding is found in optic tectum, lateral geniculate body, diencephalic preoptic and suprachiasmatic nuclei, basal hypothalamus, and the medial pallium. These results indicate that specific melatonin binding sites are present in all craniate taxa examined except in hagfish. Although we cannot rule out the possibility that melatonin receptors are secondarily lost in hagfish, their absence in amphioxus makes this unlikely. We speculate that melatonin actions in early vertebrates may have included regulation of visual and endocrine responses to light. r 1998 Academic Press Key Words: melatonin; Branchiostoma; Myxine; Petromyzon; Raja. Melatonin (N-acetyl, 5-methoxytryptamine) is synthesized in the pineal gland and photoreceptors of all vertebrates studied to date. The functions of this compound include regulation of disk shedding, pigment aggregation, and calcium-activated dopamine release in the retina, suggesting that it may originally have played a paracrine role (Gern and Karn, 1983). Melatonin also has endocrine effects in a variety of vertebrates: removal of the pineal gland or administration of exogenous melatonin affects several physiological functions including metamorphosis in lampreys and anurans (Eddy, 1969; Delgado et al., 1987), blanching in dermal melanophores of amphibians (McCord and Allen, 1917; Lerner et al., 1958; Bagnara, 1964), sun compass orientation in salamanders (Adler and Taylor,

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1980), circadian rhythms in lampreys, teleosts, reptiles, birds, and mammals (Kavaliers, 1981; Armstrong, 1989; McArthur et al., 1991; Morita et al., 1992; Chabot and Menaker, 1992), and photoperiodically controlled seasonal breeding in teleosts and tetrapods [see de Vlaming and Olcese (1981) and Bittman (1993) for review]. Evidence for the synthesis of melatonin in invertebrates has been presented (Vivien-Roels and Pevet, 1993), but it remains to be determined whether this compound plays a physiological role in invertebrates and whether enzymes which participate in the synthesis of melatonin are a plesiomorphic feature of chordates. Melatonin’s actions are most likely mediated by high-affinity, G-coupled cell membrane receptors. Binding studies employing [2-125I]melatonin (IMEL) in amphibians, birds, and mammals have characterized a receptor whose affinity is approximately 50 pM and which displays little affinity for N-acetylserotonin, serotonin, or other physiologically occuring indoleamines (Rivkees et al., 1989; Reppert et al., 1994; Reppert et al., 1995; Dubocovich, 1995). Autoradiographic studies using this ligand have revealed the existence of melatonin receptors in the central nervous system, although the distribution of these binding sites varies taxonomically among the tetrapods. The concentration of melatonin receptors varies across the course of development in birds and mammals (Carlson et al., 1991; Vanecek and Kosari, 1994), but little evidence for such maturational changes has been gathered in other taxa. Reports of the occurrence of specific melatonin binding sites in teleost fishes (Martinoli et al., 1991; Eckstrom and Vanacek, 1992; Davies et al., 1994) suggest that this pineal hormone began to play a physiological role early in chordate evolution. Teleosts are highly derived, however, and little attention has been devoted to the possible existence and distribution of melatonin receptors in cartilaginous fishes, lampreys, hagfishes, and protochordates. To our knowledge, the only report of melatonin binding in lampreys predated the availability of IMEL. The low specific activity of [3H]melatonin precluded an accurate and exhaustive determination of receptor localization (Joss, 1977, 1981). In order to address these issues, we utilized IMEL to characterize and map melatonin binding sites in the brains and pituitaries of amphi-

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Vernadakis, Bemis, and Bittman

oxus, Atlantic hagfish, sea lamprey, little skates, and rainbow trout. To assess possible changes in melatonin receptor concentration and distribution during lamprey metamorphosis, we also examined IMEL binding in ammocoete larvae.

MATERIALS AND METHODS Amphioxus (Branchiostoma floridae) were purchased from the Gulf Specimen Co. (Panacea, FL). Animals were maintained in sea water during shipment and were sacrificed by placement on dry ice during the evening on the day following their arrival in the laboratory. Atlantic hagfish (Myxine glutinosa) were caught in traps baited with fish at an approximate depth of 100 m near Shoals Marine Station (Portsmouth, NH) during July of 1990. Animals were main-

FIG. 1. IMEL binding in amphioxus (Branchiostoma). Saggital sections through the anterior end of the animal were incubated with IMEL (A) in the absence (total binding) or (B) presence (nonspecific binding) of unlabeled melatonin. The tissue section which generated the total binding autoradiogram was stained with cresyl violet (C). Note that IMEL binding is not displaceable by unlabeled melatonin. The discrepancy between (A) and (B) reflects the small size of the animal; the adjacent sections contain different amounts of the nervous system. Abbreviation: ns, nervous system. Scale bars in this and subsequent figures indicate 1 mm.

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FIG. 2. IMEL binding in Atlantic hagfish (Myxine glutinosa). Midsaggital section showing (A) total binding, (B) nonspecific binding, (C) cresyl-stained section which generated autoradiogram shown in (A). Abbreviations: c, cartilage; ha, habenula; ot, optic tectum; te, telencephalon; vt, ventral thalamus; pr, hippocampal primordium. No specific binding of IMEL was evident.

tained in sea water until dissection of the brain and pituitary the following day. Adult lampreys (Petromyzon marinus) of both sexes were caught either at the Holyoke fish lift (Holyoke, MA) or by electroshock in the Connecticut River and its tributaries during their upstream migration in June of 1990. Ammocoete larvae, ranging in length from 6 to 14 cm, were collected at the same time and brought to the laboratory in river water. Little skates (Raja erinacea) were purchased from the Marine Biological Laboratory (Woods Hole, MA) and transported to the laboratory in sea water. Adult rainbow trout (Oncorhyncus mykiss) were obtained from domestic stock by the Conte Anadromous Fish Research Laboratory (Turners Falls, MA) and kept in

tanks of river water overnight until sacrifice. Ammocoetes and skates were anesthetized in MS222 prior to dissection of the brain and pituitary, while lampreys and rainbow trout were decapitated without anesthesia. Tissues were rapidly frozen on powdered dry ice and stored at 280°C until sectioning. In adults, we dissected the head to remove the brain with the hypophyseal complex attached. Because it was not possible to dissect out the central nervous system of either amphioxus or ammocoete larvae, entire animals were sectioned in the saggital plane. This report is based upon results from 6 to 15 individuals of each species. Tissues were sectioned on a cryostat at 20 µm.

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FIG. 3. IMEL binding in ammocoete larva of the sea lamprey (Petromyzon marinus). (A, B, and C) Coronal sections through the entire head of ammocoete larva; (D, E, and F) coronal sections of adult brain. (A, D) Total IMEL binding; (B, E) nonspecific binding; (C, F) cresyl-stained tissue sections. Specific melatonin binding was detected in both larval and adult lampreys, but was more extensive in adults. Abbreviations: ce, cerebellum; cp, choroid plexus; e, eye; IV, fourth ventricle; mt, midbrain tegmentum; n, notochord; p, pharynx.

Sections were thaw-mounted onto gelatin/chrome alum-subbed slides and stored dessicated at 280°C until binding studies were performed. Slides were removed and allowed to air dry for 10 min at this time. Sections were then preincubated for 1 h at 22°C in 0.01 M phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (BSA; pH 7.4). Slides used for

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single point binding studies were then transferred to buffer containing IMEL (25–80 pM) for 1 h. Matched slides were incubated in IMEL to which additional unlabeled melatonin had been added to produce a final concentration of 1 µM in order to permit subsequent assessment of nonspecific binding. In order to perform saturation analyses, additional slides contain-

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ing optic tectum of lamprey, skate, or trout were incubated at one of seven concentrations of IMEL (5–400 pM), in the presence or absence of unlabeled melatonin. In order to assess specificity of binding, an additional set of sections from these three species was incubated in buffer containing IMEL plus various concentrations of unlabeled melatonin, N-acetylserotonin, serotonin, or 5-methoxytryptamine. Slides were next incubated in ice-cold PBS/BSA for 15 min, transferred to PBS for 15 min, rapidly air dried, and apposed to film (Hyperfilm, Amersham, Arlington Hts, IL), for 10–24 days. Each casette contained commercial 125I microscale standards (Amersham) for quantification of binding. Film was developed in D-19 (Kodak, Rochester, NY) and fixed. Slides were stained in cresyl violet for anatomical reference. Autoradiograms were analyzed using the NIH Image program (version 1.44) on a Macintosh IIX computer. For anatomical reference to the hagfish and lamprey brains, we used the atlases of Jansen (1930) and Johnston (1902), respectively. An atlas of Raja clavata (Smeets et al., 1983) was consulted for analysis of skate brain. The procedure of Nazarali et al. (1990) was used to calculate the concentration of protein in brain tissue so that Bmax could be estimated.

RESULTS Although IMEL binding was restricted to the nervous system of amphioxus, corresponding to the line of pigmented dorsal ocelli, addition of unlabeled melatonin did not interfere with labeling (Fig. 1). Specific IMEL binding was absent in Atlantic hagfish, although in this species total binding was essentially absent (Fig. 2). In contrast, specific IMEL binding was evident in the nervous system of both larval and adult lamprey. In ammocoete larvae, IMEL binding sites were most concentrated in the tectum, preoptic nucleus, and lateral geniculate body (Figs. 3A–3C; Table 1; Kennedy and Rubinson, 1977). In adult lamprey, specific IMEL binding was found in these same areas, but in much higher concentrations (Figs. 3D–3F and 4; Table 2). The larger size of the brain permitted resolution of the tectal binding, and revealed a higher concentration of specific binding sites in the surperficial layers. In addition, IMEL binding sites were

TABLE 1 Specific IMEL Binding (fmol/mg Protein) in Brain Structures of Ammocoete Larvae of the River Lamprey (Petromyzon marinus) fmol bound/mg protein

Area Telencephalon Olfactory bulb Pallium (rostral) Pallium (caudal) Diencephalon Preoptic nucleus Pars magnocellularis Pars parvocellularis Pineal Dorsal hypothalamic/ventral thalamic nucleus Nucleus corporis geniculati lateralis Inferior lobe of hypothalamus Mesencephalon Tectum Tegmentum Commisura ansulata Hindbrain Medulla oblongota

0.000 0.015 0.000

0.110 0.191 0.000 0.037 0.103 0.040 0.136 0.033 0.073 0.048

evident in the medial pallium, rostral and dorsal hypothalamus and ventral thalamus, inferior hypothalamus, and medulla oblongata. Saturation studies performed to characterize IMEL binding sites in adult lamprey tectum indicated a Kd of 36 pM and an apparent Bmax of 8.1 fmol/mg protein (Fig. 5). The anatomical distribution of specific IMEL binding sites in the little skate generally resembled that of the adult lamprey (Fig. 6). The highest concentration of binding sites was in the superficial layers of the tectum (Table 3). Within the forebrain, the lateral geniculate body, rostral hypothalamus, and preoptic nucleus also contained high concentrations of binding sites. Unlike the lamprey, however, the skate brain had high concentrations of binding sites in interpeduncular nucleus and torus semicircularis. High concentrations of IMEL binding sites were also detected in telencephalic, diencephalic, and mesencephalic structures. Saturation studies of tectal binding indicated a Kd of 38 pM and an apparent Bmax of 19.8 fmol/mg protein (Fig. 7). Competition studies confirmed specificity of binding (Fig. 8). Among the compounds studied, only melatonin was able to compete with IMEL for the tectal binding site. N-Acetylserotonin and 5-methxytryptophol were 4–6 orders of magnitude less effective as competitors, and serotonin failed to inhibit IMEL binding at any concen-

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FIG. 4. Pseudocolor image of autoradiogram generated by sagittal section of adult lamprey brain. Most intense total IMEL binding is represented in red, and least intense in blue. Abbreviations: hab, habenula; gen, geniculate body; hyp, hypothalamus; med, medulla; mt, midbrain tegmentum; olf, olfactory lobe; ot, optic tectum; po, preoptic area; thal, thalamus. Nonspecific binding was negligible in these structures.

tration (Ki values calculated at 3 3 1026, 5 3 1024, and .1022 M, respectively; Fig. 8). Specific IMEL binding in rainbow trout was similar to that described previously in salmonids (Eckstrom and Vanecek, 1992; Davies et al., 1994). Specific binding is most prominent in sensory structures including the tectum, semicircular torus, and accessory optic nucleus (data not shown). Within the diencephalon, high concentrations of IMEL binding sites were found in inferior, ventral, and lateral hypothalamus, and preoptic nucleus. The molecular and granular layers of the cerebellum also contained concentrations of IMEL binding sites. Our saturation studies in trout tectum

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indicated a Kd of 50 pM. In competition studies, only melatonin effectively displaced IMEL from its binding sites (Ki values for melatonin, NAS, 5-methoxytryptophol, and serotonin were 3 3 10210, 3 3 1026, 5 3 1024, and .1022 M, respectively; data not shown).

DISCUSSION The present experiments confirm and extend our understanding of melatonin binding sites among craniates and suggest that melatonin binding is a primitive

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feature of a group including lampreys plus gnathosomes. The affinity and specificity of IMEL binding sites in lampreys and skates is comparable to that previously described in earlier studies of salmonids and tetrapods (Martinolli et al., 1991; Eckstrom and Vanacek, 1992; Weichmann and Wirsig-Weichman, 1993). However, specific IMEL binding was not evident in the nervous system of either amphioxus or hagfish. Although other, still more sensitive techniques may reveal melatonin receptors or related proteins in these species, the present data are most consistent with the hypothesis that melatonin receptors originated in the common ancestor of lampreys and gnathostomes. Alternatively, but less likely, amphioxus and hagfish may have independently ceased to express a melatonin receptor common to vertebrates and invertebrates. We might detect melatonin binding sites in hagfish at different developmental stages or at different times of year, although this seems unlikely to us. Our interpretation is that the gene(s) encoding melatonin receptor protein(s) may have evolved and become

TABLE 2 Specific IMEL Binding (fmol/mg Protein) in Brain Structures of Adult Lampreys Area Telencephalon Olfactory bulb Pallium (rostral) Pallium (caudal) Corpus striatum Diencephalon Preoptic nucleus Pars magnocellularis Pars parvocellularis Dorsal hypothalamic/ventral thalamic nucleus Nucleus corporis geniculati lateralis Inferior lobe of hypothalamus Infundibulum Neurohypophyseal lobe Mesencephalon Tectum Layer 1 Layer 2 Layer 3 Tegmentum Commisura ansulata Hindbrain Medulla oblongota Tuber trigeminum Substantia griasea periventricularis

fmol bound/mg protein 0.231 0.191 0.503 0.532

1.321 2.690 2.270 3.696 1.428 0.576 0.209

3.582 2.536 1.674 1.648 1.707 0.367 0.005 0.009

FIG. 5. (A) Saturation study characterizing binding of IMEL to the tectum of the sea lamprey. Open squares indicate total binding, closed squares represent nonspecific binding (determined by incubation in the presence of 1 µM unlabeled melatonin), and filled diamonds represent specific binding. (B) Scatchard transformation of binding data.

widely expressed only after epiphyseal structures were present to supply high circulating titers of melatonin (Gern and Karn, 1983). Although amphioxus and hagfish lack pineal glands, Lacalli et al. (1994) have proposed on ultrastructural grounds that the dorsal lamellar body in the dorsal roof of the posterior cerebral vesicle of amphioxus is a homologue of the vertebrate pineal body. Nevertheless, we are aware of no evidence of melatonin synthesis or secretion from

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FIG. 6. IMEL binding in the brain of the little skate (Raja erinacea). Coronal sections showing arranged from rostral (left) to caudal (right) illustrating (A, D, G) total IMEL binding, (B, E, H) nonspecific binding, (C, F, I), cresyl-stained sections. Abbreviations: h, hypothalamus; th, thalamus; oc, optic chiasm; ot, optic tectum; r, reticular nucleus.

this or any other structure in either amphioxus or hagfish. In lampreys, the pineal develops as larvae approach metamorphosis. We found that the concentration of IMEL binding sites in adults greatly exceeded that in ammocoetes. Possibly, expression of melatonin binding proteins reflects maturational events, including the development of the pineal gland and the secretion of

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melatonin, which may be under endocrine and/or photoperiodic control. Pinealectomy of larval lampreys delays or prevents metamorphosis (Eddy, 1969; Cole and Youson, 1981), indicating that melatonin receptors such as those described here may have an important physiological role. In anurans, melatonin’s integumentary effects occur only in larval stages (Bagnara, 1964); thus restriction of melatonin receptor

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expression to a particular phase of development is a likely possibility. Melatonin binding in the central nervous system serves as yet unknown function(s), but it may influence neuroendocrine structures that regulate the pituitary–thyroid axis. Although effects of melatonin on dermal melanophores for which this

TABLE 3 Specific IMEL Binding (fmol/mg Protein) in Brain Structures of Little Skate (Raja erinacea) Area Telencephalon Subpallium Pallium Superficial basal area Basal forebrain bundle Diencephalon Preoptic nucleus Postoptic commissure Suprachiasmatic nucleus Medial hypothalamic nucleus Inferior hypothalamic lobe Lateral lobe nucleus Ventral thalamus Nucleus corporis geniculati lateralis Nucleus of the saccus vasculosis Habenula Thalamic eminence Mesencephalon Nucleus of the longitudinal medial fasicle Nucleus ruber Interpeduncular nucleus Dorsal Ventral Lateral lemniscus Commisure ansulata Tegmentum Tectum Layer 1 Layer 2 Layer 3 Layer 4 Layer 5 Layer 6 Semicircular torus Nucleus of the lateral torus Hindbrain Cerebellum Granular layer Molecular layer Raphe nucleus Reticular nucleus Other structures Optic nerve Optic chiasm

fmol bound/mg protein 1.285 0.228 0.253 1.270 3.057 2.764 3.798 2.786 0.385 0.701 1.846 3.699 1.116 1.262 1.200 1.901 0.488 2.345 6.290 4.404 1.251 0.936 12.768 10.749 10.415 5.270 4.749 5.788 4.966 1.057

0.341 0.528 1.123 2.510 1.277 1.270

FIG. 7. (A) Saturation experiment characterizing binding of IMEL in the tectum of the little skate. Symbols are as in Fig. 4. (B) Scatchard transformation of binding data.

compound was named were the first to be described (McCord and Allen, 1917), our study does not support speculation that integumentary actions represent the primitive function of melatonin. In addition to IMEL binding sites in diencephalic sites which might be predicted to regulate limbic and endocrine function, sensory structures were found to have high concentrations of melatonin receptors in lampreys, skates, and trout. The in vitro binding method which we used to identify IMEL binding sites requires use of unfixed tissue and is thus incompatible with techniques employed to identify a possible homologue

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FIG. 8. Competition study indicating specificity of IMEL binding in skate tectum. Open squares represent melatonin; 3, N-acetylserotonin; open diamonds, 5-methoxytryptophol; X, serotonin.

of the suprachiasmatic nucleus in lampreys, including anterograde tracing of retinal projections and immunocytochemistry (Weigle et al., 1996). Nevertheless, rostral diencephalic and telencephalic IMEL binding is apparent in this general region of the brain of adult lamprey (Fig. 4). Our finding is also consistent with evidence from nonmammalian tetrapods, in which high concentrations of binding sites have been identified in the optic tectum, the thalamus, and their projection areas (Weichmann and Wirsig-Weichman, 1993; Cassone et al., 1995; Rivkees et al., 1989; Dubocovich, 1995). It is unknown whether melatonin acts at any of these sites to modulate sensitivity to sensory input. Although pinealectomy disrupts entrainment of circadian rhythms of locomotor activity in lampreys (Morita et al., 1992), it is not established whether systemic replacement of melatonin can reinstate rhythmicity or regulate phase in lampreys as has been established in some other vertebrates (Armstrong, 1989; Chabot and Menaker, 1992; Chiba et al., 1995; Heigle and Gwinner, 1995). Teleosts fishes have extensive pinealofugal projections to regions including tectum, diencephalon, and brainstem, so that melatonin might act as a transmitter in many areas of the brain (Eckstrom, 1984; Eckstrom and van Veen, 1984; Puzdrowski and Northcutt, 1989; Yanez et al., 1993). Thus some of the binding sites found in the present study may subserve local rather than endocrine influences of melatonin. The

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Vernadakis, Bemis, and Bittman

extensive distribution of IMEL binding sites suggests that melatonin’s influence over CNS function may be widespread, and it is difficult to identify any principal candidates for sites at which melatonin might regulate circadian rhythms. Although a crude localization of a circadian pacemaker to the preoptic nucleus has been accomplished in the hagfish Eptatretus stouti (OokaSouda et al., 1993), the absence of central melatonin receptors in Atlantic hagfish (present results) makes it unlikely that this site is a target of melatonin action. Anatomical definition of circadian pacemakers in the CNS of lampreys and skates would help to identify which of the many structures which bind melatonin are likely to respond to this hormone by changing circadian period and phase. We are not aware of previous studies of melatonin action or IMEL binding sites in elasmobranchs. Given the evidence for melatonin receptors in lampreys (present study) and teleosts (Martinolli et al., 1981; Eckstrom and Vanecek, 1992; Davies et al., 1994), the presence of IMEL binding sites in skates is to be expected. Our study also extends the anatomical description of the distribution of melatonin binding sites in the trout beyond those reported in Salmo salar and O. mykiss by Eckstrom and Vanecek (1992) and Davies et al. (1994). The recent cloning of melatonin receptors in amphibians, birds, and mammals (Ebisawa et al., 1994; Reppert et al., 1994) may make it possible to identify and sequence the gene(s) responsible for the binding shown in the present study of fishes and basal craniates. The putative production of melatonin in insects (VivienRoels and Pevet, 1993; Tilden et al., 1994) and dinoflagellates (Poggeler et al., 1992) suggests that receptormediated functions of this indoleamine may occur in other taxa as well. Nevertheless, the absence of specific binding in amphioxus and hagfish reported here raises the possibility of independent evolution of melatonin receptors, if they exist, in non-chordates. It will be of great interest to clone and sequence genes responsible for melatonin receptors in lampreys as well as cartilaginous and bony fishes in order to compare their sequences and intronic organization with those which encode melatonin receptors in tetrapods. Such comparative study may enable us to determine whether a single primitive receptor subtype exists, and whether

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homologous genes are present in amphioxus and hagfish.

ACKNOWLEDGMENTS These studies were supported by NSF BNS86-16935, NIMH RO1-44132, and KO2 MH00914 to E.L.B., and NSF BSR 88-06539 and BSR92-20938 to W.E.B. Hagfish were collected with the expert help of Drs. John B. Heiser and Rick Martini of the Shoals Marine Lab. We thank the Holyoke fish lift (Massachusetts Cooperative Fish and Wildlife Unit) and Dr. Steven McCormick of the Conte Anadromous Fish Laboratory (Turners Falls, MA) for assistance in collection of lampreys. A preliminary report of this research was presented at the Annual Meeting of the Society for Neuroscience (Neurosci. Abstr. 17,129.10).

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