Localization of prolactin receptor in the newt brain

In the male newt Cynops pyrrhogaster, prolactin (PRL) acts directly on the central nervous system and induces courtship behavior. As a step to elucidate the localization of neurons on which PRL acts, we developed a polyclonal antibody against an oligopeptide having a sequence completely identical with a part of the sequence of PRL receptors (PRLRs) of two species of newts, C. pyrrhogaster and C. ensicauda, and performed an immunohistochemical study with this antibody. PRLR-immunoreactive cells were observed in the medial amygdala, anterior preoptic area, magnocellular preoptic nucleus, suprachiasmatic nucleus, nucleus of the periventricular organ, ventral hypothalamic nucleus, and choroid plexus. We also performed in situ hybridization with a ³⁵S-labeled newt PRLR antisense RNA probe and detected signals in the preoptic area and choroid plexus. Colocalization of both PRLR-like immunoreactivity and arginine vasotocin-like or mesotocin-like immunoreactivity was demonstrated in the magnocellular preoptic nucleus. This is the first report of PRLR localization in the amphibian brain.This study was supported by a research grant from Waseda University to S.K. and by a grant-in-aid from the Ministry of Education, Science, and Culture of Japan to I.H., F.T., and S.K.     

Entrainment of the circadian locomotor activity rhythm in the Japanese newt by melatonin injections

We examined whether melatonin can act as a synchronizing agent within the circadian system of amphibians by testing the ability of melatonin injections to entrain the circadian locomotor activity rhythm of a newt (Cynops pyrrhogaster). Under constant darkness, all newts (13 cases) showing the free-running rhythms were subcutaneously injected with 10 g melatonin at the same time every other day for at least 30 days. Subsequently, they were injected with vehicle (1% ethanolic saline) instead of melatonin for at least another 30 days. In 10 of the 13 newts, the locomotor activity rhythms could be entrained to a period of 24 h by melatonin injections but not by vehicle injections. During the entrained steady-state, the active phase of an activity-rest cycle preceded the time of melatonin injections as previously reported in other diurnal species. These results suggest that the endogenous circadian rhythm of melatonin concentration may be involved in synchronizing circadian oscillator(s) within the newt's circadian system.     

Genetic structure of natural populations of the red-spotted newt,Notophthalmus viridescens

Genetic variation is described at 15 loci in 2 neotenic and 12 nonneotenic populations of red-spotted newts. Though high levels of genetic similarity (I=0.990) were found among all populations, allele frequencies at six of the eight most polymorphic loci show significant heterogeneity across populations. Change in allele frequencies at two of these loci (Pep-2 and Ldh-1) is significantly correlated with latitude. Interspecific homologies are established for newt peptidases based on substrate specificities and lactate dehydrogenases based on tissue distribution, thermal stability, and kinetic properties. Nonneotenic populations are highly variable (H=0.157) and neotenic populations are only slightly, but significantly, less variable (H=0.120). The high levels of heterozygosity detected in nonneotenic populations may result from large effective population size and/or environmental heterogeneity. The unexpectedly high heterozygosity values obtained for the neotenic populations may indicate adult dispersal or the presence of some previously undetected red efts at these localities. In any case, a major change in life history has apparently had little effect on the genetic structure of these populations.This research was supported by grants from the Blakeslee Fund of Smith College.     

Micro-habitat use within a guild of newt larvae (Trituridae) in an Alpine lake

Population and ecological parameters such as numbers of larvae, microhabitat use, niche breadth and niche overlap of three species of syntopic larval newts (Alpine newt Triturus alpestris, Italian crested newt T. carnifex, and common newt T. vulgaris) were studied for two years in a small pond at 1160 m a.s.l. in NE Slovenia. Differences in microhabitat partitioning among larval newts were small. The largest niche breadth was estimated for larval T. alpestris, and the narrowest estimate was for larval T. carnifex in both years. Ecological differences seem to be very small and quite variable among sites and years. It appears that the developmental stage and size of newt larvae are more important in explaining resource partitioning than the characteristics of each species. Because of the absence of potential invertebrate predators and adult newts in the second half of the breeding season, the injuries could only be caused by intra-and interspecific predation attempts.     

Organization of histamine-containing neurons in the brain of the crested newt,Triturus carnifex

The distribution of immunoreactivity for histamine was studied in the brain of the urodele Triturus carnifex using the indirect immunofluorescence method. Histamine-immunoreactive cell bodies were localized in the caudal hypothalamus within the dorsolateral walls of the infundibular recesses. These immunoreactive cell bodies were pear-shaped, bipolar and frequently of the cerebrospinal-fluid-contacting type. Histaminergic nerve fibers were detected in almost all parts of the brain. Dense innervation was seen in the telencephalic medial pallium and ventral striatum, the neuropil of the preoptic area, the septum, the paraventricular organ, the posterior commissure, the caudal hypothalamus, the ventral and lateral mesencephalic tegmentum. Medium density innervation was observed in the lateral mesencephalic tegmentum and optic tectum. Poor innervation was present in the telencephalic dorsal pallium and in the central gray of the medulla oblongata. Few fibers occurred in the olfactory bulbs and in the telencephalic lateral pallium. Double immunofluorescence staining, using an antibody against tyrosine hydroxylase, showed that histamine-immunostained somata and those containing tyrosine-hydroxylase-like immunoreactivity were co-distributed in the tuberal hypothalamus. No co-occurrence of histamine-like and tyrosine hydroxylase-like immunostaining was seen in the same neuron. The pattern of histamine-immunoreactive neurons in the newt was similar to that described in other vertebrates. Our observations, carried out on the apparently simplified brain of the newt confirm that the basic histaminergic system is well conserved throughout vertebrates.     

Biogenesis of myeloid bodies in regenerating newt (Notophthalmus viridescens) retinal pigment epithelium

Summary Myeloid bodies are believed to be differentiated areas of smooth endoplasmic reticulum membranes, and they are found within the retinal pigment epithelium in a number of lower vertebrates. Previous studies demonstrated a correlation between phagocytosis of outer segment disc membranes and myeloid body numbers in the retinal pigment epithelium of the newt. To test the hypothesis that myeloid bodies are directly involved in outer segment lipid metabolism and to further characterize the origin and functional significance of these organelles, we examined the effects on myeloid bodies of eliminating the source of outer segment membrane lipids (neural retina removal) and of the subsequent return of outer segments (retinal regeneration) in the newt Notophthalmus viridescens. Light- and electron-microscopic analysis demonstrated that myeloid bodies disappeared from the pigment epithelium within six days of neural retina removal. By week 6 of regeneration, rudimentary photoreceptor outer segments were present but myeloid bodies were still absent. However, at this time, the smooth endoplasmic reticulum in some areas of the retinal pigment epithelial cells had become flattened, giving rise to small (0.5 m long), two-to-four layer-thick lamellar units, which are myeloid body precursors. Small myeloid bodies were first observed one week later at week 7 of retinal regeneration. This study revealed that newt myeloid bodies are specialized areas of smooth endoplasmic reticulum. It also showed that a contact between functional photoreceptors and the retinal pigment epithelium is essential to the presence of myeloid bodies in the epithelial cells.     

Limb regeneration of the adult newt (Notophthalmus viridescens) in the absence of the spleen

Summary It has been suggested that the immune system might figure prominently in the regulation of forelimb regeneration. However, neither the nature of this influence nor the aspect(s) of regeneration influenced are clearly known. The determination of which components of the immune system are indispensable for regeneration would be a logical first step in attempting to address such questions. This investigation, therefore, examined the effects of removing the spleen, a major lymphoid organ in the newt, upon the progress of regeneration. Splenectomies performed concomitantly with or after forelimb amputation failed to alter the time course of regeneration. Splenectomies, but not sham-splenectomies, performed prior to amputation reduced the time required to achieve successive stages of regeneration under some, but not all conditions, i.e., when performed 10–20 days before amputation, during the late fall and winter. Up until 35 days after amputation, no gross morphological distortions were observed as a result of splenectomy. It was concluded that the spleen is not required for regeneration to occur.Portions of this work constitute part of the thesis submitted by M.E. Fini in partial fulfillment of the requirements for the M.S. degree in Biology at Boston College     

Priority effects of the early breeding fire salamander on the late breeding banded newt

Early breeding intraguild predators may have advantages over late breeding predators via priority effects; early breeding predators may reduce shared prey resources before late breeders appear and may also prey upon the late breeders. Here we show that predatory larvae of the late-breeding predatory banded newt, Triturus vittatus vittatus, occupy the same temporary pond toward the end of the developmental period of the early-breeding predatory fire salamander, Salamandra salamandra, resulting in a large size disparity between larvae of these two species while they co-occur. We conducted outdoor artificial pool experiments to assess priority effects of large larval Salamandra at the end of their larval development period, on recently hatched larval Triturus. We also assessed how artificial vegetation may influence larval Triturus performance in the presence or absence of Salamandra Salamandra, introduced into the experimental pools two weeks prior to the newt larvae, strongly reduced invertebrate prey abundance shared by these two predatory urodeles and with only a one week period of overlap, strongly reduced abundance of Triturus larvae. The artificial vegetation had only a small ameliorating effect on Triturus survival when Salamandra was present. Triturus size at metamorphosis (snout-tail length) was significantly larger in the Salamandra pools, presumably due to a combination of a strong “thinning effect” and greater vulnerability of smaller Triturus individuals to predation by Salamandra. Time to metamorphosis was not significantly affected by Salamandra. These results have conservation implications as T. v. vittatus is listed as highly endangered and may also explain the largely negative spatial association of the two species. Handling editor: K. Martens     

Axon regeneration across the site of injury in the optic nerve of the newt Triturus pyrrhogaster

Summary The process by which axons regenerate following a freeze injury to the optic nerve of the newt was analyzed by light and electron microscopy. Freezing destroys cellular constituents in a one millimeter segment of the nerve, leaving intact the basal lamina and the blood supply to the eye. No axons are seen at the site of injury one to seven days post lesion. This contrasts with the persistence of normal-appearing but severed unmyelinated axons within the cranial stump which thus give a false appearance of early regeneration. The first axon sprouts traverse the lesion and enter the cranial stump by ten days. The number of regenerating axons increases rapidly thereafter with no signs of random growth at the site of injury. These axon sprouts tend to be somewhat larger than normal unmyelinated axons and contain dense core vesicles and abnormal organelles similar to those in growing axons in tissue culture. The persisting basal lamina inside the optic sheath appears to provide continuity across the site of injury, to orient axon sprouts, and to favor an orderly process of axon regeneration without neuroma formation.The authors wish to express their gratitude to Barbara Heindel and Jill Jones for extremely helpful technical assistance. This work was supported by grants NS 10864 and NS 05666 from the U.S. Public Health Service and by the Medical Research Service of the Veterans Administration     

Vesicle and granule content of sympathetic ganglion cells during limb regeneration of the newt Triturus

Summary Fine structural observations were made on the vesicle and granule content of ganglion cells in the posterior subclavian ganglion and peripheral nerve fibers of the upper forelimb of the newt Triturus. The populations of vesicles and granules in normal ganglion cells and nerve fibers were compared with those observed after limb transection. In normal neurons, clear vesicles range in size from 250 to 1000 Å in diameter, but are most frequently 400–500 Å. Vesicles with dense contents (granules) also vary greatly in size, but most are 450–550 Å in diameter and correspond to dense-core vesicles. Large granules that contain acid phosphatase activity are thought to be lysosomes. During limb regeneration, in both the ganglion cells and peripheral nerves, the ratio of dense vesicles to clear vesicles increases. There is a large increase in number of dense granules with a diameter over 800 Å, particularly in the peripheral regenerating fibers. This study shows that regenerating neurons differ from normal in their content of vesicular structures, especially large, membrane-bounded granules.This work was supported by grants from the National Science Foundation (GB 7912) and from the National Cancer Institute (TICA-5055), National Institutes of Health, United States Public Health Service.     

Studies on the formation and state of determination of the trunk organizer in the newt,Cynops pyrrhogaster

Summary The exact localization of the presumptive trunk organizer was determined by means of vital staining at the initiation of gastrulation (0 h embryo) and subsequently in 6, 9, 12 and 24 h embryos.The progressive changes in the self-differentiation and inductive capacity of the trunk organizer were studied in isolation cultures (sitting drop) and in sandwich cultures with competent gastrula ectoderm. In the 0 and 6 h embryo cultures the excised trunk organizer predominantly formed atypical ectoderm. A dramatic change in differentiation and inductive capacity occurred in the 9 h embryo. The positive cases — 83% of the isolation and 50% of the sandwich cultures — mainly formed notochord and somites, accompanied by spinal cord and hindbrain in the sandwich cultures. Although no further change in self-differentiation occurred from that time onwards, a gradual increase in inductive capacity was recognized.     

Topographical relationships between catecholamine-and neuropeptide-containing fibers in the median eminence of the newt,Triturus alpestris

Summary Dopaminergic and peptidergic nerve fibers were simultaneously demonstrated with a double-labeling technique at the ultrastructural level. The first antibody, raised against tyrosine hydroxylase, was applied during the preembedding phase and visualized with the peroxidase method. The second antibody, raised against one of the peptides met-enkephalin, somatostatin or gonadotropin-releasing hormone (GnRH), was applied to the ultrathin sections and visualized with gold-labeled goat anti-rabbit IgG. The fibers of both categories were present in the zona externa of the median eminence, frequently contacting the basal lamina of the portal vessels. In addition, topographical relationships between different types of nerve fibers were observed in the perivascular areas, although there were no morphological signs of synaptic specializations. Using serial sections, it could be established that one GnRH-fiber contacted both a dopaminergic fiber and a fiber immunoreactive for met-enkephalin. The observations support earlier physiological data concerning the regulation of the hypothalamo-hypophyseal axis, with special emphasis on the release of neurohormones in the median eminence of the newt.     

True enamel matrix of the newt,Triturus pyrrhogaster,contains no sulfated glycoconjugates

Summary The ultrastructural distibution and histochemical properties of sulfated glycoconjugates were investigated in the developing enamel of the adult newt, Triturus pyrrhogaster, by use of the high-iron diamine thiocarbohydrazide silver proteinate (HID-TCH-SP) staining and enzymatic digestion methods. Development and ultrastructure of the enamel were also studied. After deposition of the mantle dentin matrix to a certain thickness, the first enamel matrix, globular in shape, appeared in juxtaposition to the dental basement membrane and tended to be intermixed with the previously deposited dentin matrix. Subsequently, enamel matrix was deposited outside (ameloblastic side) of the dental basal lamina and formed a true enamel layer. Thus, developing enamel of the newt consists of two layers: (1) an inner layer made up of a dentin-enamel mixed matrix and (2) an outer layer composed of only true enamel matrix. HID-TCH-SP precipitates resulting from the abovementioned studies were found in the mixed matrix and were identified as chondroitin sulfates; in contrast, the true enamel matrix contained no sulfated glycoconjugates.     

大凉螈繁殖生态

大凉螈是我国特有的珍稀有尾两栖动物,其种群数量目前呈现明显下降趋势,然而涉及该物种保护的繁殖生态学研究仍十分匮乏。通过融合围栏陷阱及标志重捕的样方调查法,对大凉螈石棉栗子坪种群繁殖个体和变态登陆幼体的迁徙、繁殖群体种群大小、繁殖场内雌雄有效性比变化等进行了研究。运用Jolly-Seber法估测了繁殖种群大小,运用单因素方差分析或Kruskal-Wallis秩和检验比较了不同时期进入繁殖场的雄性大凉螈头体长及体重,运用t检验或者Wilcoxon秩和检验比较了雌雄性间形态上的差异,运用t检验、t′检验或Wilcoxon秩和检验比较了野外抱对雄性与非抱对雄性间的体征差别,运用Pearson相关分析探讨了雌性产卵量与其身体形态的关系,同时观察了卵的孵化情况。研究结果表明:大凉螈的繁殖季为每年的4月下旬到7月下旬,幼体最早于8月上旬变态登陆。估测调查地繁殖场内雄性大凉螈繁殖种群大小约为391尾,雄螈较雌螈更早进入繁殖场且在场内停留时间更长,体重较轻的雄螈较晚迁入繁殖场。有效性比明显偏雄(雌/雄:0.03—0.10)。雌雄间具明显性二型性,雌性个体的头体长、体重及肥满度均大于雄性,而雄性的尾高和尾长占全长的比例则大于雌性。对比自然抱对雄性和非抱对雄性个体发现,抱对个体在头体长、体重和尾高等体征方面显著大于非抱对个体,暗示这些形态特征可能在雄性竞争配偶的过程中起到关键作用。雌螈在室内条件下平均产卵数为176枚,产卵历时2—4 d,产卵量与雌性肥满度正相关,卵的平均孵化期为15.7 d,孵出幼体平均全长为9.74 mm。     

The response of the atrium to direct mechanical wounding in the adult heart of the newt,Notophthalmus viridescens

Summary Wound repair and proliferation were examined in the injured newt atrium with light- and electron-microscopic techniques including autoradiography. Hearts were injured by removing a piece approximately 0.5 mm² of the atrial wall. The five-day wound was an endothelial and mesothelial-lined blood clot bordered by a 150-m necrotic zone. Repair progressed from the periphery inward with areas of macrophage activity replaced by fibroblasts and connective tissue. The wound at 25 days consisted of a scar with few myocytes. There was no difference in the proliferative behavior between the right and left atria. Proliferative cells were localized to a 500-m reactive zone surrounding the wound. The maximum mesothelial cell thymidine-labeling index of 20.5% and mitotic index of 1.4% were seen 5 days after injury. The peak connective tissue cell thymidine-labeling index of 10.2% and mitotic index of 0.4% were seen 10 days after wounding. The peak thymidine-labeling index of 9.8% for myocardial cells was recorded 10 days after injury with a mitotic index of 0.2%. Proliferation returned to control levels by 25 days post-injury. Electron microscopy demonstrated that myocytes engaged in DNA synthesis were indistinguishable from control myocytes. Z-band material was not observed in mitotic myocytes, but myofilaments and junctions were present.     

Distribution of catecholaminergic and serotoninergic systems in forebrain and midbrain of the newt,Triturus alpestris (Urodela)

Summary Mapping of monoaminergic systems in the brain of the newt Triturus alpestris was achieved with antisera against (1) thyrosine hydroxylase (TH), (2) formaldehyde-conjugated dopamine (DA), and (3) formaldehyde-conjugated serotonin (5-HT). In the telencephalon, the striatum was densely innervated by a large number of 5-HT-, DA-and TH-immunoreactive (IR) fibers; IR fibers were more scattered in the amygdala, the medial and lateral forebrain bundles, and the anterior commissure. In the anterior and medial diencephalon, TH-IR perikarya contacting the cerebrospinal fluid (CSF-C perikarya) were located in the preoptic recess organ (PRO), the organum vasculosum laminae terminalis and the suprachiasmatic nucleus. Numerous TH-IR perikarya, not contacting the CSF, were present in the posterior preoptic nucleus and the ventral thalamus. At this level, DA-IR CSF-C neurons were only located in the PRO. In the posterior diencephalon, large populations of 5-HT-IR and DA-IR CSF-C perikarya were found in the paraventricular organ (PVO) and the nucleus infundibularis dorsalis (NID); the dorsal part of the NID additionally presented TH-IR CSF-C perikarya. Most regions of the diencephalon showed an intense monoaminergic innervation. In addition, numerous TH-IR, DA-IR and 5-HT-IR fibers, orginating from the anterior and posterior hypothalamic nuclei, extended ventrally and reached the median eminence and the pars intermedia of the pituitary gland. In the midbrain, TH-IR perikarya were located dorsally in the pretectal area. Ventrally, a large group of TH-IR cell bodies and some weakly stained DA-IR and 5-HT-IR neurons were observed in the posterior tuberculum. No dopaminergic system equivalent to the substantia nigra was revealed. The possible significance of the differences in the distribution of TH-IR and DA-IR neurons is discussed, with special reference to the CSF-C neurons.Abbreviations AM amygdala - CAnt commissura anterior - CH commissura hippocampi - CP commissura posterior - Ctm commissura tecti mesencephali - DH dorsal hypothalamus - DTh dorsal thalamus - FLM fasciculus longitudinalis medialis - Fsol fasciculus solitarius - H habenula - LFB lateral forebrain bundle - ME median eminence - MFB medial forebrain bundle - NID nucleus infundibularis dorsalis - nIP neuropil of nucleus interpeduncularis - NPOP nucleus preopticus posterior - NS nucleus septi - OVLT organum vasculosum laminae terminalis - PD pars distalis - Pdo dorsal pallium - PHi primordium hippocampi - PI pars intermedia - Pl lateral pallium - PN pars nervosa - PRO preoptic recess organ - Ptec pretectal area - PVO paraventricular organ - Ra nucleus raphe - Rm nucleus reticularis medius - SCO subcommisural organ - ST striatum; strm stria medullaris thalami - strt stria terminalis thalami - TM tegmentum mesencephali - TO tectum opticum - TP tuberculum posterius - trch tractus cortico-habenularis - trmp tractus mamillopeduncularis - VH ventral hypothalamus - Vm nucleus motorius nervi trigemini - VTh ventral thalamus - II optic nerve     

Lactate sensitive cells in newt kidneys

Summary The blood supply of duck muscle spindles from the extensor pollicis and the extensor digitorum comminis wing muscles has been studied by light and electron microscopy. Capillaries usually accompany the nerve bundle that innervates the spindle, approaching at an oblique angle around the midequatorial region. Capillaries may run for some distance along the surface of the outer capsule, or penetrate partially into the outer capsule and lie between layers of the capsule cell processes. Some capillaries also penetrate the outer capsule, running into the periaxial space and continuing further towards the polar region. They have been shown to be in close contact with intrafusal muscle fibres, from the juxta-equatorial to the polar region, but have not been encountered among sensory terminals in the mid-equatorial region.     

The distribution of GABA-like-immunoreactive neurons in the brain of the newt,Triturus cristatus carnifex,and the green frog,Rana esculenta

Summary The distribution of -aminobutyric acid (GABA) immunoreactivity was studied in the brain of two amphibian species (Triturus cristatus carnifex, Urodela; Rana esculenta, Anura) by employing a specific GABA antiserum. A noteworthy immunoreactive neuronal system was found in the telencephalic dorsal and medial pallium (primordium pallii dorsalis and primordium hippocampi) and in the olfactory bulbs. In the diencephalic habenular nuclei there was a rich GABAergic innervation, and immunoreactive neurons were observed in the dorsal thalamus. In the hypothalamus the GABA immunoreactivity was found in the preoptic area, the paraventricular organ and in the hypothalamo-hypophysial complex. In the preoptic area of the frog some GABA-immunoreactive CSF-contacting cells were shown. In the optic tectum immunolabeled neurons were present in all the cellular layers. A rich GABAergic innervation characterized both the fibrous layers of the tectum and the neuropil of the tegmentum and interpeduncular nucleus. In the cerebellum, in addition to the Purkinje cells showing a variable immunopositivity, some immunoreactive cell bodies appeared in the central grey. Abundant immunolabeled nerve fibers in the acoustico-lateral area and some immunopositive neurons in the region of the raphe nucleus were observed. In conclusion, the GABAergic central systems, well-developed in the amphibian species studied, were generally characterized by close similarities to the pattern described in mammals.Dedicated to Professor Valdo Mazzi (Dipartimento di Biologia Animale, Università di Torino), in honor of his 70th birthday