Paramyxine Atami Research Articles

Characteristics of the brown hagfish Paramyxine atami transthyretin: Metal ion-dependent thyroid hormone binding

Transthyretin (TTR) is a vertebrate-specific protein involved in thyroid hormone distribution in plasma, and its gene is thought to have emerged by gene duplication from the gene for the ancient TTR-related protein, 5-hydroxyisourate hydrolase, at some early stage of chordate evolution. We investigated the molecular and hormone-binding properties of the brown hagfish Paramyxine atami TTR. The amino acid sequence deduced from the cloned hagfish TTR cDNA shared 33–50% identities with those of other vertebrate TTRs but less than 24% identities with those of vertebrate and deuterostome invertebrate 5-hydroxyisourate hydrolases. Hagfish TTR, as well as lamprey and little skate TTRs, had an N-terminal histidine-rich segment, allowing purification by metal-affinity chromatography. The affinity of hagfish TTR for 3,3′,5-triiodo-L-thyronine (T3) was 190 times higher than that for L-thyroxine, with a dissociation constant of 1.5–3.9nM at 4°C. The high-affinity binding sites were strongly sensitive to metal ions. Zn2+ and Cu2+ decreased the dissociation constant to one-order of magnitude, whereas a chelator, o-phenanthroline, increased it four times. The number of metal ions (mainly Zn2+ and Cu2+) was approximately 12/TTR (mol/mol). TTR was also a major T3-binding protein in adult hagfish sera and its serum concentration was approximately 8μM. These results suggest that metal ions and the acquisition of N-terminal histidine-rich segment may cooperatively contribute to the evolution toward an ancient TTR with high T3 binding activity from either 5-hydroxyisourate hydrolase after gene duplication.

RFamide peptides in agnathans and basal chordates

Since a peptide with a C-terminal Arg-Phe-NH2 (RFamide peptide) was first identified in the ganglia of the venus clam in 1977, RFamide peptides have been found in the nervous system of both invertebrates and vertebrates. In vertebrates, the RFamide peptide family includes gonadotropin-inhibitory hormone (GnIH), neuropeptide FF (NPFF), prolactin-releasing peptide (PrRP), pyroglutamylated RFamide peptide/26RFamide peptide (QRFP/26RFa), and kisspeptins (kiss1 and kiss2). They are involved in important functions such as the release of hormones, regulation of sexual or social behavior, pain transmission, reproduction, and feeding. In contrast to tetrapods and jawed fish, the information available on RFamide peptides in agnathans and basal chordates is limited, thus preventing further insights into the evolution of RFamide peptides in vertebrates. In this review, we focus on the previous research and recent advances in the studies on RFamide peptides in agnathans and basal chordates. In agnathans, the genes encoding GnIH, NPFF, and PrRP precursors and the mature peptides have been identified in lamprey (Petromyzon marinus) and hagfish (Paramyxine atami). Putative kiss1 and kiss2 genes have also been found in the genome database of lamprey. In basal chordates, namely, in amphioxus (Branchiostoma japonicum), a common ancestral form of GnIH and NPFF genes and their mature peptides, as well as the ortholog of the QRFP gene have been identified. The studies revealed that the number of orthologs of vertebrate RFamide peptides present in agnathans and basal chordates is greater than expected, suggesting that the vertebrate RFamide peptides might have emerged and expanded at an early stage of chordate evolution.

Molecular cloning of cytochrome P450 side-chain cleavage and changes in its mRNA expression during gonadal development of brown hagfish, Paramyxine atami

Since hagfishes are considered the most primitive vertebrate known, extant or extinct, studies on their reproduction are indispensable for understanding phylogenetic aspects of vertebrate reproduction. However, little information is available on the endocrine regulation of the gonadal function in the hagfish. Based on EST analysis of the testis of the brown hagfish (Paramyxine atami), P450 side chain cleavage (CYP11A), which is the first and essential enzyme for steroidogenesis in jawed vertebrates, was cloned. The deduced amino acid sequence of hagfish CYP11A shows high identity to other animal forms especially in two functional domains, adrenodoxin binding domain and heme-binding domain. In the phylogenetic analysis, hagfish CYP11A forms a clade with the vertebrate CYP11A. Following the real-time PCR analysis, CYP11A mRNA expression levels were clearly correlated to the developmental stages of gonads in both sexes of the brown hagfish. By in situ hybridization, CYP11A mRNA signals were found in the theca cells of the ovarian follicles and Leydig cells and the tubule-boundary cells of the testis. These molecular and histological evidences are suggesting that CYP11A plays functional roles as a steroidogenic enzyme in gonadal development. Moreover, native GTH purified from hagfish pituitary stimulated the transcriptional levels of CYP11A in the organ-cultured testis in vitro, clearly suggesting that the steroidogenic activity of the hagfish is under the control of the pituitary GTH. It is suggested that vertebrates, during their early evolution, have established the pituitary–gonadal reproductive system.

Relationships between Plasma Concentrations of Sex Steroid Hormones and Gonadal Development in the Brown Hagfish,Paramyxine atami

The relationship between sex steroid hormone profiles in plasma and gonadal function in hagfish is poorly understood. In the present study, plasma concentrations of estradiol, testosterone, and progesterone were examined with respect to gonadal development, sexual differences, and possible function of atretic follicles in the brown hagfish, Paramyxine atami, using a time-resolved fluoroimmunoassay. Plasma concentrations of these three hormones were low in juveniles of both sexes. In females, plasma estradiol showed a significant correlation with ovarian development, with the highest concentrations in late vitellogenic adults. Plasma testosterone and progesterone also increased significantly in non-vitellogenic adult females; however, plasma testosterone showed no significant differences among adult females at different ovarian developments, while plasma progesterone was significantly lower in late vitellogenic adults than it was in non-vitellogenic adults. Vitellogenic females that possessed atretic follicles showed significantly lower concentrations of all three hormones than females that only possessed normal follicles. In males, no significant differences were found in plasma estradiol or testosterone levels among groups of different developmental stages of the testis, while plasma progesterone showed a clear inverse relationship with testicular development. Thus, differences were found in plasma sex steroid hormone profiles between male and female P. atami. Moreover, plasma estradiol showed a significant correlation with ovarian development, which suggests that estradiol is involved in the regulation of ovarian development. The present study also revealed that steroid hormone production was strongly suppressed in females that possessed atretic follicles in their ovaries.

Effects of estradiol or testosterone treatment on expression of gonadotropin subunit mRNAs and proteins in the pituitary of juvenile brown hagfish, Paramyxine atami

A single functional gonadotropin (GTH) comprising two subunits, α and β, was recently identified in the pituitary of brown hagfish (Paramyxine atami). Little is known about the feedback mechanisms that regulate these GTH subunits by sex steroids in the hagfish. The present study was designed to examine feedback effects of estradiol and testosterone on mRNA expression and protein expression of both GTHα and GTHβ subunits in the pituitary of the juvenile P. atami. Intraperitoneal administration of estradiol over the course of 27days resulted in substantial accumulation of immunoreactive (ir)-GTHα and ir-GTHβ in the adenohypophysis, but testosterone treatments over 27days had no effects on ir-GTHα or ir-GTHβ. Estradiol treatment for 1, 2, 4 or 14days had no effect on GTHα mRNA levels. In contrast, after 2days of estradiol treatment, GTHβ mRNA levels had increased significantly from baseline, while these levels were not affected after treatment over 1, 4, or 14days. After 14days of testosterone treatment, both GTHα and GTHβ mRNA levels had decreased significantly from baseline levels. These results indicate that estradiol acted primarily to suppress the secretion of GTH, and hence resulted in the accumulations of ir-GTHα and ir-GTHβ in the pituitary. On the other hand, testosterone appeared to suppress both the synthesis and the secretion of GTH. Thus, estradiol and testosterone probably differ in their effects on the regulation of pituitary GTH synthesis and secretion in juvenile hagfish.

Characterization of Novel RFamide Peptides in the Central Nervous System of the Brown Hagfish: Isolation, Localization, and Functional Analysis

RFamide (RFa) peptides play various important roles in the central nervous system in both invertebrates and vertebrates. However, there is no evidence of the existence of any RFamide peptide in the brain of hagfish, one of the oldest lineages of vertebrates. In this study, we sought to identify novel RFamide peptides from the brains of hagfish (Paramyxine atami). We identified four novel RFamide peptides, which had the C-terminal Pro-Gln-Arg-Phe-NH2 structure. cDNA cloning revealed that the identified RFamide peptides are encoded in two types of cDNA. Molecular phylogenetic analysis of the two precursors indicated that the hagfish RFamide peptides belong to the PQRFamide peptide group that includes mammalian neuropeptide FF and AF. Based on immunohistochemistry and in situ hybridization, hagfish PQRFamide peptide precursor mRNA and its translated peptides were localized in the infundibular nucleus of the hypothalamus. Immunoreactive fibers were terminated on blood vessels in the infundibular nucleus. Dense immunoreactive fibers were also observed in other brain regions. We further showed that one of the hagfish PQRFamide peptides significantly stimulated the expression of gonadotropin-β mRNA in the cultured hagfish pituitary. These results indicate that the control mechanism of gonadotropin expression by a hypothalamic neuropeptide evolved in the agnathan brain. This is the first evidence describing the identification of RFamide peptides in the hagfish brain. This is also the first report showing the regulation of gonadotropin expression by a homolog of neuropeptide FF that belongs to the PQRFamide peptide group in any vertebrate.

Evolutionary Implications of Lactate Dehydrogenases (LDHs) of Hagfishes Compared to Lampreys: LDH cDNA Sequences from Eptatretus burgeri, Paramyxine atami and Eptatretus okinoseanus

Heart muscles of hagfishes Paramyxine atami and Eptatretus okinoseanus express the B4 isozyme of lactate dehydrogenase [L-LDH: NAD oxidoreductase, EC 1.1.1.27] (LDH-B4) whereas their skeletal muscles express LDH-A4. To examine the relationship of hagfish LDHs to lamprey and other vertebrate LDHs, we determined the cDNA sequences of LDH-A from three hagfishes and compared them with previously published sequences. A phylogenic tree shows that hagfishes diverged just after lampreys. The deduced amino acid sequences showed ten regions common to all vertebrate LDHs examined, i.e., the active site, the pocket recognizing the substrate-coenzyme complex, part of a loop at the surface, and the substrate binding site. The cyclostomate-specific regions (S1, S2) were located in the neighborhood of the active site loop. Three regions, IGS1, IGS2 and IGS3, seem to have altered their structures during the differentiation of LDH isozymes, and the regions remain in LDH-B of vertebrates hitherto examined. IGS2 and IGS3, which are in the neighborhood of the active site, may regulate catalytic activity. There were differences in six amino acid residues (6, 10, 20, 156, 269, and 341) in LDHs of hagfishes. These differences might reflect the tolerance to high pressure and low temperature of LDHs from hagfishes at different habitat depths.

Pressure-adaptive differences in lactate dehydrogenases of three hagfishes: Eptatretus burgeri, Paramyxine atami and Eptatretus okinoseanus

The tolerance of abyssal pressures likely depends on adaptive modifications of fish proteins. However, structural modifications of proteins which allow functioning at high pressure remain unclear. We compared the activities of lactate dehydrogenase (LDH), an important enzyme in glycolytic reaction, in three hagfishes inhabiting different depths under increased pressure. LDH in Eptatretus okinoseanus, found at a depth of 1,000 m, was highly active at high pressure of 100 MPa maintaining the activity at 70% of that at 0.1 MPa. In contrast, LDH activity in Paramyxine atami, found at 250-400 m, decreased to 55% at 15 MPa, and that in Eptatretus burgeri, found at 45-60 m, was completely absent at 5 MPa. The result suggests that subunit interaction of the LDH-tetramer is more stable in E. okinoseanus than that in P. atami and E. burgeri under high-pressure conditions. We found six amino acid substitutions between the three LDH primary structures. Accordingly, these amino acid residues are likely to contribute to the stability of the E. okinoseanus LDH under high-pressure conditions.

Gonadotropin-like and adrenocorticotropin-like cells in the pituitary gland of hagfish, Paramyxine atami; immunohistochemistry in combination with lectin histochemistry

The pituitary system of the hagfish remains an enigma. The present study has aimed to detect possible adenohypophysial hormones in the pituitary gland of the brown hagfish, Paramyxine atami, by means of immunohistochemistry in combination with lectin histochemistry. Rabbit antisera raised against ovine luteinizing hormone (LH)beta, proopiomelanocortin (POMC)-related peptides, and the growth hormone/prolactin family of tetrapod and fish species were used, and 25 kinds of lectins were tested. Three different types of adenohypophysial cells were revealed in the pituitary of brown hagfish. The first was stained with both anti-ovine LH beta and several D-mannose-binding lectins, such as Lens culinaris agglutinin and Pisum sativum agglutinin. This cell type predominated in the adenohypophysis in adults with developing gonads and thus appeared to be involved in the regulation of gonadal functions. The second was negative for anti-ovine LH beta but was stained with several N-acetylglucosamine-binding lectins, such as wheat germ agglutinin and Lycopersicon esculentum lectin. This cell type exhibited a weak positive reaction with anti-lamprey adrenocorticotropin (ACTH) and thus appeared to be related to POMC-like cells. The second cell type was found in the adenohypophysis regardless of the developmental state of the gonads. The third cell type was negative for both antisera and lectins. Since this cell type was numerous in juveniles and adults without developing gonads, most cells of this type were probably undifferentiated. These findings suggest that GTH and ACTH are major adenohypophysial hormones in the hagfish.

Identification of a natriuretic peptide (NP) in cyclostomes (lamprey and hagfish): CNP-4 is the ancestral gene of the NP family

In bony fishes, natriuretic peptides (NPs) comprise a hormone family that is composed of seven subtypes; ANP, BNP, VNP that have an intramolecular ring and N- and C-terminal extensions, and four CNPs (CNP-1 to -4) that lack the C-terminal extension. To assess the ancestral molecule of the NP family, we determined the NP sequences in several species of two extant cyclosotome groups, lampreys and hagfishes. A cDNA encoding CNP was cloned from the heart and brain of three phylogenetically distant species of lampreys, Geotria australis, Lampetra japonica, and Petromyzon marinus. In the deduced prohormone sequence of each species, two potential processing signals, lysine-lysine (KK) that is commonly present in CNP precursors, and arginine-X-X-arginine (RXXR) for furin-like proprotein convertase (PC) that is typical for CNP-4 were present. The deduced mature peptides that are released at each signal were highly conserved among three species; 100% cleaved at KK and >92% processed at RXXR. In L. japonica, the CNP gene was expressed almost exclusively in the heart and brain. Meanwhile, a cDNA encoding NP with a C-terminal tail sequence was cloned from the heart and brain of three hagfish species in different genera, Myxine glutinosa, Eptatretus cirrhatus, and Paramyxine atami. The precursor sequences including the prosegment had >80% identity among the three hagfish species. A processing signal, RXXR, is also conserved in the prosegment of all hagfish NPs. The molecular phylogenetic analyses inferred that the lamprey CNP and hagfish NP belong to the CNP-4 group, even though the hagfish NP has a C-terminal sequence extended from the intramolecular ring. The presence of a processing signal, RXXR, in the prosegment of cyclostome NPs supports the above classification. Based on the current findings, we suggest that the ancestral gene of the NP family is CNP-4.

Immunohistochemical detection of gonadotropin-like material in the pituitary of brown hagfish ( Paramyxine atami) correlated with their gonadal functions and effect of estrogen treatment

Since hagfish are members of the most primitive group of living vertebrates, studies on their reproduction are indispensable for understanding phylogenetic aspects of vertebrate reproductive system. Nevertheless, our knowledge of the reproductive physiology of the hagfish, especially of the pituitary–gonadal axis, is almost completely lacking. In the present study, the relationship between the amount of immunoreactive gonadotropin (GTH)-like material in the pituitary gland and gonadal conditions was examined in the brown hagfish, Paramyxine atami. First, pituitary sections were stained immunohistochemically with anti-ovine LHβ, and the degrees of the accumulation of GTH-like material were compared among three different groups of gonadal conditions; juveniles and adults with and without developing gonads. Immunoreactive GTH-like material was heavily accumulated in adults with developing gonads, whereas it was not or only weakly accumulated in juveniles or adults without developing gonads. Thus, there was a strong positive correlation between the amount of GTH-like material and gonadal conditions. Second, effect of estradiol benzoate on GTH-like material was examined using three groups of juvenile hagfish: initial control, sham control, and experimental animals. Experimental animals received estradiol benzoate resolved in sesame oil intraperitoneally every third day for 1 month, whereas sham control animals received the same doses of sesame oil. GTH-like material was heavily or moderately accumulated in most estrogen-treated animals, whereas it was not or weakly accumulated in initial or sham control animals. Thus, estrogen treatment in juvenile hagfish resulted in the large increase in the amount of GTH-like material. From these results, it is suggested the presence of not only GTH but also the hypophysial–gonadal feedback system in the hagfish.

Immunohistochemical detection of gonadotropin-like material in the hagfish pituitary, correlated with their gonadal conditions

In the present study, immunoreactive gonadotropin (GTH)-like material was examined in the hagfish pituitary, in correlation with their gonadal conditions. For this purpose, three species of hagfish, Myxine glutinosa, Eptatretus burgeri and Paramyxine atami, were used. In all species, GTH-like cells were observed in the adenohypophysis. They were stained with several antibodies to LH-related GTHs, such as ovine LHβ, human LHβ, bullfrog LH, and salmon GTHIIβ. In the subsequent study, the relationship between the amount of GTH-like material and gonadal conditions was examined in Paramyxine atami. The adenohypophysis of adult animals exhibited well-developing features, and about half of the adenohypophysial cells were stained intensely with anti-ovine LHβ. On the other hand, the adenohypophysis of juveniles exhibited less developed features with few or no cells stained with anti-ovine LHβ. These results suggest that a GTH-gonadal system is truly present in the hagfish.

A germline restricted, highly repetitive DNA sequence in Paramyxine atami: an interspecifically conserved, but somatically eliminated, element.

In some species of hagfish, the phenomenon of chromosome elimination occurs during embryogenesis. However, only two repetitive DNA families are known to be represented in chromosomes that are eliminated from somatic cells of the Japanese hagfish Eptatretus okinoseanus. Using molecular analyses, another germ line-restricted, highly repetitive DNA family has been detected in another Japanese hagfish, Paramyxine atami. The repeat unit of this family, which is 83 bp long, has been designated "EEPa1", for Eliminated Element of P. atami 1. DNA filter hybridization using EEPa1 as a probe revealed that this family is shared among several species and is conserved in the germline DNA. Although eliminated, repetitive DNA that is shared interspecifically has not been reported in hagfish species, cases of chromatin diminution and chromosome elimination processes have been described previously in other organisms. The patterns and intensities of hybridization signals suggest that members of the repetitive DNA family defined by EEPa1 have undergone concerted molecular evolution.

Structure of portal vein heart wall in the brown hagfish (Paramyxine atami).

The portal vein heart (PVH) wall of the brown hagfish (Paramyxine atami) was observed by light and transmission electron microscopy. Cardiac musculature is well developed in the PVH wall. The myocytes possess arranged myofibrils in the cytoplasm. We suggest that the PVH may act as a pump in blood circulation from the intestines to the liver, as it does in other myxinoid species. Ultrastructural and immunohistochemical findings of the brown hagfish PVH myocytes also indicate that the PVH has an active contraction and atrial natriuretic polypeptide endocrine function. J Morphol 231:225-230, 1997. © 1997 Wiley-Liss, Inc.

Structure of portal vein heart wall in the brown hagfish (Paramyxine atami)

The portal vein heart (PVH) wall of the brown hagfish (Paramyxine atami) was observed by light and transmission electron microscopy. Cardiac musculature is well developed in the PVH wall. The myocytes possess arranged myofibrils in the cytoplasm. We suggest that the PVH may act as a pump in blood circulation from the intestines to the liver, as it does in other myxinoid species. Ultrastructural and immunohistochemical findings of the brown hagfish PVH myocytes also indicate that the PVH has an active contraction and atrial natriuretic polypeptide endocrine function. J Morphol 231:225–230, 1997. © 1997 Wiley-Liss, Inc.

Chromosome elimination in three Baltic, south Pacific and north-east Pacific hagfish species.

We have confirmed that chromosome elimination occurs in the cells of Myxine glutinosa, collected from the Baltic Sea off Sweden, Eptatretus cirrhatus from the south Pacific Ocean off the east coast of New Zealand, and E. stoutii from the north-east Pacific Ocean off Canada, similar to cells of four Japanese hagfish species. In M. Glutinosa, E. cirrhatus type A, E. cirrhatus type B and E. stoutii, the differences in chromosome number between spermatogonia (44, 72, 80 and 48) and somatic cells (28, 34, 34 and 34) were 16, 38, 46 and 14 respectively. The amount of DNA eliminated from presumptive somatic cells averaged 43.5%, 48.7%, 54.6% and 52.8% respectively. Euchromatic chromosomes and/or parts of chromosomes in addition to heterochromatic chromosomes were clearly eliminated in E. cirrhatus and E. stoutii. Adding our previous observations of four Japanese hagfish species, chromosome elimination occurs in all seven of the hagfish species. These results suggest that this phenomenon, chromosome elimination, generally occurs in the order Myxinida. In addition, B-chromosomes were observed in the germ cells of E. cirrhatus and E. stoutii, similar to the cells of E. okinoseanus, E. burgeri and Paramyxine atami (E. atami). This fact suggests that B-chromosomes might exist generally in the family Eptatretidae.

Light and electron microscope studies of the dermal capillaries in three species of hagfishes and three species of lampreys

A dermal capillary network was found in three species of hagfishes (Myxine glutinosa, Paramyxine atami, Eptatretus stoutii) and in the adults of three species of lampreys (Geotria australis, Lampetra japonica, Petromyzon marinus). However, it was found neither in the ammocoetes of any of the species examined, nor in the adults of the lamprey Lampetra fluviatilis. The complex arrangement of dermal capillaries described for adult L. fluviatilis by Czopek & Sawa (1971) was apparently an artefact. The dermal capillaries of hagfishes and lampreys are lined by a continuous endothelium, comprising flattened cells that possess most of the usual complement of organelles found in the endothelia of higher vertebrates. The pericapillary space contains proteoglycans, glycoproteins and tubular microfibrils (diam. 11–15nm) and, in hagfishes, also irregularly arranged collagen fibrils (diam. 40–120 nm). The thickness of the endothelium varied amongst species, ranging from c. 100nm in G. australis and P. marinus to over 500 nm in L. japonica and all three species of hagfishes. In comparison with the endothelial cells of lampreys, those of hagfishes have a better developed basement membrane, possess small desmosomes in the flange‐like intercellular contact regions, and contain numerous plasmalemmal smooth tubules, rather than the smooth vesicles that are typically present in endothelial cells. In addition, the hagfish dermal capillaries have pericytes. Amongst hagfishes, the dermal capillary network is far better developed in Myxine glutinosa and Paramyxine atami, which burrow and may therefore have to rely to some degree on cutaneous respiration, than in Eptatretus stoutii which does not burrow. However, a dermal capillary network is probably important for all species of hagfishes since it would enable the precursors required for synthesizing mucus to be transported to the epidermis, where mucous cells are very abundant. The particularly well‐developed dermal capillary network in Geotria australis may facilitate the transport of oxygen through the unusually thick dermis of their migrating adults as they move around barriers on their upstream migration, and must thus presumably have to employ cutaneous respiration.

Variability in the Presence of Elastic Fibre‐like Structures in the Ventral Aorta of Agnathans (Hagfishes and Lampreys)

AbstractThis paper investigates whether elastic fibre‐like structures are present in the ventral aorta of hagfishes and lampreys. Fibres, which are morphologically similar to the elastic fibres of gnathostomatous (jawed) vertebrates, are shown to be present in the ventral aorta of the hagfish Paramyxine atami, and also, but to a lesser extent, the dorsal aorta of this species and the ventral aorta of another hagfish, Eptatretus stouti.The ‘elastic’ tissue formed irregular sheet‐like aggregates, comprising well‐defined amorphous material surrounded by tubular microfibrils, whose diameters ranged from 10 to 15 nm. While this tissue was most abundant in the same region of the aortae as that occupied by the elastica interna in the blood vessels of gnathostomes, it was also found deeper in the wall of these blood vessels. Although tubular microfibrils were found in the ventral aorta of the hagfish Myxine glutinosaand the lamprey Geotria australis, these were never associated with well–defined, amorphous material. This parallels the results of previous studies on Myxine glutinosaand another species of lamprey, Petromyzon marinus.Thus, the elastic fibre–like tissues found in the ventral aortae of P. atamiand E. stoutiprovide the first examples of such structures in this region in agnathan vertebrates.

Immunohistochemical localization of neuropeptide Y-like substance in the brain and hypophysis of the brown hagfish, Paramyxine atami

The distribution of neuropeptide Y-like immunoreactivity in the brain and hypophysis of the brown hagfish, Paramyxine atami, was examined by use of the peroxidase-antiperoxidase method. Immunoreactive cells were found in two areas of the brain, the nucleus hypothalamicus of the diencephalon and the ventrolateral area of the caudal tegmentum, at the level of the nucleus motorius V–VII. The labeled cells of the nucleus hypothalamicus were loosely grouped and recognized as bipolar neurons. Immunolabeled fibers were widely distributed in the brain, showing the highest density in the diencephalon. They were sparse, or absent, in the olfactory bulb, habenula, primordium hippocampi, neurohypophysis, corpus interpedunculare, and dorsolateral area of the medulla oblongata. The fibers appeared to project exclusively from the ventral hypothalamus to various other portions of the brain: the anterolateral areas of the telencephalon via the basal hypothalamus, the pars dorsalis thalami, the dorsocaudal region of the mesencephalon, and the ventromedial portions of the tegmentum and anterior medulla oblongata. These findings suggest that, in the brown hagfish, NPY-like substance is involved in neuroregulation of various cerebral areas, but it may be of little significance in the control of pituitary function.

The Effect of Light Pulses on the Circadian Rhythm in Locomotor Activity of the Hagfish.

Light pulses of 15min to 6h were given to the hagfish Paramyxine atami, individuals displaying a free-running rhythm in locomotor activity under continuous darkness. The fish sensitively responded to the pulses; the active phase was promptly delayed or advanced when pulses were respectively given at the beginning or at the end of the duration of locomotor activity (α). The responses to light pulses in P. atami were characterized both by an initial phase-shift and by the changing period length (τ) in the ensuing active phase. In contrast, a closely related hagfish species Eptatretus burgeri responded to the light pulses by taking another stable course of the ensuing active phase with relatively constant τThese results suggest that the sensitivity to light pulses in P. atami is higher than that in E. burgeri.