Tecto-thalamo-telencephalic interrelationships following visual deafferentation

21-23 months after uni- and bilateral enucleation of the eyes, the turtles Emys orbicularis and Testudo horsfieldi exhibited atrophy, gliosis and residual degenerative changes in the visual nerves and tracts and in the two upper layers of the optic tectum, as well as a reduced density of fibres in the tecto-thalamic tract on the deafferentated side. Electrophysiological experiments on the turtles have shown that tectal impulses en route to the general cortex of the forebrain are relayed in the n. rotundus. Conduction of impulses along the tecto-geniculo-cortical path, found in intact animals, ceases, which is apparently due to transsynaptic changes in the surface layers of the optic tectum.     

Comparative study of dendritic spines in three principal cortical areas in the forebrain ofTestudo horsfieldi andEmys orbicularis

Methods of light and electron microscopy were used to compare dendritic spines of cortical neurons in three principal areas (dorsal, medial, and lateral cortex) of the forebrain inTestudo horsfieldi andEmys orbicularis. Differences between the spines in each of the three areas were found to be common to both species. Differences in the distribution of dendrites in proximal, middle, and distal segments of the dendrites and the density, shape, length, and fine structure of these formations were all taken into account. Besides the undoubted similarity between the species, differences were found in the fine structure of the spines and in their quantitative distribution in homonymous cortical zones and are connected with the character of processing of incoming information, which is largely dependent on the ecological features of the species.A. N. Severtsov Institute of Evolutionary Morphology and Ecology of Animals, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 9, No. 2, pp. 142–148, March–April, 1977.     

Neurochemical characteristics of the turtle optic tectum: comparison with other reptilian species and birds

Data on distribution of biologically active substances in the turtle optic tectum are compared with results of similar experiments on other reptilian as well as on avian species. In two turtle species (Testudo horsfield and Emys orbicularis), immunoreactivity to monoamines (5-HT and TH), NPY, as well as NADPH-d activity were similarly distributed in neuropil of the SGFS retinorecipient part and in that of the SGP/SAP periventricular layers. Immunoreactivity to neuropeptides SP and m-Enk was maximal in neuropil of the SGFS non-retinorecipient part. The periventricular layers were characterized by the abundant radial SP- and mENK-ir as well as the NADPH-d-positive neurons. Diffusely dispersed ChAT-ir elements and many ir fibers perpenducilar to the tectal surface were observed in the SGFS retinorecipient part; the SGFS non-retinorecipient part contained a dense plexus of thick ir fibers and diffusely distributed ir terminals. The GABA ir cells were the most numerous in the tectum; they were spread in all tectal layers. Thus, various biologically active substance located in superficial retinorecipient tectal sublayers could affect processing and transmission of information via ascending dendrites of neurons in deeper layers. The cells containing SP, m-Enk, and NADPH-d had laminar organization in SGP; via the system of ascending and descending axons, they are able to affect other structures within and outside of the optic tectum. Putative sources of tectal modulatory innervation are discussed. In all studied reptilian and avian species, the principal similarity is revealed in the neurochemical organization. Some differences might be explained by the level of tectal differentiation due to factors of phylogenetic evolution and/or adaptive specialization.     

Dark and light neurons in the cortical plate of the forebrain of the swamp turtle]

Electron microscopic study of the neuronal composition of Emys orbicularis cortical zones as compared with the light microscopy data has revealed that the distinctions in the cell cytoplasm ultrastructure with respect to the dendrite branching pattern and the peculiarities of their localization in the cortical plate are suggestive of a possible metabolic heterogeneity and can serve as a foundation of the morpho-functional characteristics of the neurons. A few neuronal types of neurons have been revealed: 1. light neurons distinguished by the cytoplasm matrix devoid of any electron density, a poorly developed endoplasmic reticulum and a few cytoplasmatic organells and inclusions; 2. light neurons having slight osmophility of the cytoplasm matrix, a more developed endoplasmic reticulum with elongated cisternae, a great number of organells including neurofilaments; 3. dark neurons having a high degree of cytoplasm matrix and the nucleus, a large number of the nuclei, cytoplasm, dendrites and axons in contrast to the above described patterns.     

Lymphoid aggregates in gonads of embryos, hatchlings, and young of turtles with temperature-dependent sex determination

Cellular infiltrations forming lymphoid-like aggregates were previously observed in gonads of two turtle species exhibiting temperature-dependent sex determination (TSD): at hatching in Chelydra serpentina; at and after hatching in Emys orbicularis. We show here that such aggregates are also present in gonads of Testudo graeca by the end of embryonic development, suggesting that their occurrence is general in turtles. Since in C. serpentina, infiltrations were observed mainly in testes exhibiting remnants of the germinal epithelium, it was assumed that their occurrence was an expression of maleness leading to rejection of this epithelium. The generality of this hypothesis was tested in E. orbicularis by looking for lymphoid-like aggregates in three types of gonads (testes, ovotestes, and ovaries) and for the stages at which they occur. Gonads were from embryos, hatchlings, and young incubated at various temperatures. Ovotestes obtained by treatment with an aromatase inhibitor of eggs incubated at female-producing temperature were also examined. In these gonads, the differentiation of Sertoli cells in testicular cords/tubes was ascertained by expression of SOX9. Moreover, the cell composition of aggregates was determined on electron micrographs. Aggregates appear in ovaries and ovotestes by the end of embryonic development and are present in the majority of these gonads at hatching, and at least up to one year after hatching. They are composed mainly of lymphocytes and fibroblasts. Aggregates are not present in typical testes. Since they occur in most ovaries, they cannot be seen as an expression of maleness. Rather, lymphocytic infiltration and formation of lymphoid aggregates in turtle gonads can be seen as components of the immune system, and can be under the control of gonadal endogenous sex steroids.     

Demonstration of afferent connections of the forebrain in Emys orbicularis turtles by the peroxidase method

The transport of horseradish peroxidase (HRP) out of the injection site in the dorsal ventricular ridge was studied in turtles Emys orbicularis. Labeled cells in the forebrain were observed in the paleostriatum among fibers of the lateral forebrain bundle. In the thalamus most of cells containing the granular HRP reaction product were located in the n. rotundus, n. reuniens and perirotundal nuclei (n. dorso-medialis anterior, n. magnocellularis thalami, n (centralis) lateralis, n. dorso-medialis). Fewer labeled cells were revealed in the n. anterior and n. ventralis. The density of labeled cells in the majority of all thalamic nuclei increased if the HRP was extended from the dorsal ventricular ridge into the neostriatum and the pallial thickening with adjacent general cortex. HRP positive cells in the pretectal area, nuclei of the posterior commissura and mesencephalic ventro-lateral tegmentum were observed only in cases when the enzyme was diffused from the injection site into the neostriatum, while the HRP retrograde transport to n. geniculatus lateralis, pars dorsalis was revealed only when HRP was extended into the pallial thickening and adjacent general cortex. Ascending connections of the paleostriatum, thalamic nuclei and mesencephalic tegmentum with telencephalic structures, mainly with the dorsal ventricular ridge, were discussed.     

Sources of the innervation of the carotid reflexogenic zone in representative reptiles

The investigation is dedicated to study sources of the carotid reflexogenic zone innervation in 43 tortoises (Testudo horsfieldi and Emys orbicularis). In 7 tortoises fine preparation of the vessels and nerves of the cervical area after V. P. Vorob'ev has been performed. In 13 animals descending branch of the glossopharyngeal nerve has been resected. In 4--the caudal ganglion of this nerve and in 9 tortoises the caudal ganglion of the vagus nerve have been resected. In 10 tortoises adrenergic nervous plexuses are studied after Falck-Govyrin method, and cholinergic ones--after Karnovsky-Roots. As demonstrate anatomical investigations, to the carotid reflexogenic zone of the tortoises, situating in the area of the common carotid artery base, the branches of the glossopharyngeal and of the vagus nerve approach. The experiments with resection of these nervous conductors demonstrate that by the end of 3 days after the operation myelin nervous fibers of various thickness are at the stage of granular decay. Cholinergic and adrenergic nervous fibers and plexuses are revealed histochemically in the carotid zone.     

Molecular phylogenetics of emydine turtles: taxonomic revision and the evolution of shell kinesis

The 10 extant species of emydine turtles represent an array of morphological and ecological forms recognizable and popular among scientists and hobbyists. Nevertheless, the phylogenetic affinities of most emydines remain contentious. Here, we examine the evolutionary relationships of emydine turtles using 2092 bp of DNA encoding the mitochondrial genes cyt b, ND4, and adjacent tRNAs. These data contain 339 parsimony informative characters that we use to erect hypotheses of relationships for the Emydinae. Both maximum parsimony and maximum likelihood methods yield a monophyletic Emydinae in which all but three nodes are well resolved. Emys orbicularis, Emydoidea blandingii, and Clemmys marmorata form a monophyletic clade, as do the species of Terrapene. Clemmys muhlenbergii and Clemmys insculpta form a third monophyletic group that may be sister to all other emydines. Clemmys guttata is problematic and probably related to Terrapene. Based on this phylogeny, and previous molecular work on the group, we suggest the following taxonomic revisions: (1) Clemmys should be restricted to a single species, C. guttata. (2) Calemys should be resurrected for C. muhlenbergii and C. insculpta. (3) Emys should be expanded to include three species: E. orbicularis, E. blandingii, and E. marmorata. Furthermore, our analyses show that neither kinetic-shelled nor akinetic-shelled emydines form monophyletic groups. Therefore, shell kinesis was either independently gained in Emys and Terrapene or secondarily lost in E. marmorata and C. guttata. Parsimony, paleontological evidence, and the multiple origins of shell kinesis in related turtle lineages (especially geoemydines) support the independent origin of plastral kinesis.     

Extrinsic cells, immunoreactive to Ca-binding protein, as sources of thalamic visual centres in tortoises

Extrinsic sources of calcium-binding proteins involved in immunoreactive innervation of the visual thalamic nuclei Rot and GLd in turtles (Testudo horsfieldi and Emys orbicularis) were studied using HRP tracing method and immunohistochemistry. In 1.5-4.5 months after monocular enucleation calbindin (Calb)-, parvalbumin (Parv)- and calretinin (Calr)-labeling was found in fragments of degenerated retinal fibers in the contralateral optic tract and in some retinorecipient structures (optic tectum, GLd and GLv). Changes in GLd were detected in its neuropil part. in 2.0-3.5 months after unilateral ablation of tectum and pretectum, the densities of Parv-, Calb- and Aclr-immunoreactivity terminals and fibers were diminisched in the ipsilateral n. Rot, with the maximum effect seen in Parv. Following HRP injection into the visual thalamus (Rot and GLd), retrogradely labeled cells with Parv label only, were revealed in the ventrothalamic nucleus Enta, pretectal nucleus Ptv, and in all types of Ca-binding proteins (CaBPr) in separately labeled cells of the optic tectum. Thus, it has been shown that thalamic visual centers in turtles have multiple extrinsic cells, which serve as sources of CaBPr projections. The present data suggest that organization of CaBPr inputs to visual thalamus in reptiles (turtle) and higher amniotes are fundamentally similar.     

Calcium-binding proteins and metabolic activity in thalamotelencephalic parts of the turtle visual system

Distribution of three calcium-binding proteins (CaBPr) calbindin (CB), calretinin (CR) and parvalbumin (PV) in parallel with metabolic activity (cytochrome oxidase, CO) was studied in telencephalic projection zones of the tecto- and thalamofugal visual pathways in experiments on the Horsfield's terrapin Testudo horsfieldi and the pond turtle Emys orbicularis. It was shown that the nucleus rotundus (Rot) and dorsal lateral geniculate nucleus (GLd) terminal fields in both zones (dorsolateral region of the anterior ventricular ridge, Advrdl and dorsolateral cortex, Cxdl, respectively) were CB-immunoreactive (-ir) in the both studied turtle species. The highest density of CB-ir terminals and the focus of rotundal projections in the Advrdl core coincided precisely. The GLd terminal field in Cxdl also was CR-ir. The PV contribution to innervation of both projectional zones was much lower, especially to innervation of Cxdl from GLd. In spite of similar CB-ir innervation, the projectional field of the tectofugal pathway of Advrdl had the much higher CO activity than of that of the thalamofugal pathway in Cxdl. The neurons immunoreactive to all three CaBPr types were distributed in Cxdl in different ratios in each of layers. In the visual Advrdl area the overwhelming majority were PV-ir neurons, whereas CB-ir neurons were absent. The conclusion is made that in spite of the CB- or CB/CR-immunoreactivity predominates over the PV-immunoreactivity in both thalamotelencephalic pathways of the visual system, the tectofugal (rotundo-Advrdl) pathway having the higher metabolic activity.     

Die Bedeutung der Haupt- und Nebenaugen für die photomenotaktische Orientierung der Trichterspinne Agelena labyrinthica (Cl.)1

Yawning in reptiles was investigated in field observations of various lizard and tortoise species and in laboratory experiments with the tortoises Testudo h. hermanni and Emys orbicularis. In the experiments the animals' reactions to various conditions of temperature, air O₂ and CO₂ content, fatigue and hunger, were tested. Yawning and related or similar motor patterns are described and discussed.     

Mitochondrial phylogeography of the European pond turtle, Emys orbicularis (Linnaeus 1758)

The phylogeny and phylogeography of Emys orbicularis was inferred from mitochondrial nucleotide sequences of the cytochrome b gene analysed by DNA sequencing and RNA heteroduplex analysis. Within the family Emydidae the monotypic genus Emys is affiliated with the nearctic taxa Emydoidea blandingii and Clemmys marmorata. The analysis of 423 individuals of E. orbicularis, originating throughout its distribution range, revealed a remarkable intraspecific differentiation in 20 different haplotypes with distinct geographical ranges. Maximum parsimony analysis produced a star-like phylogeny with seven main lineages which may reflect separations in the late Pliocene. The haplotype distribution examined by partial Mantel tests and analysis of molecular variance revealed a substantial effect of glacial periods. This historical perspective suggests the existence of multiple glacial refugia and considerable Holocene range expansion which was modulated by climatic traits. Further support is gained for the occurrence of long-term parapatry in glacial refugia.     

Reptile brain cerebrosides and cerebroside sulfates

Studies have been made on the content of cerebrosides and cerebroside sulfates, as well as on their fatty acid composition in the brain of reptiles, subclass Anapsida (tortoises Emys orbicularis and Testudo horsfieldi) and subclass Lepidosauria (lizards Agama caucasica, A. sanguinolenta, Phrynocephalus mystaceus and snake Natrix tesselata). Total content of cerebrosides and cerebroside sulfates is higher in the brain of Lepidosaurians than in that of Anapsids. In the brain of tortoises, the content of cerebroside fraction with hydroxy fatty acids is significantly higher than of the fraction with normal fatty acids, which is also typical of the brain of homoiothermic mammals and birds. In the brain of Lepidosaurians, concentration of hydroxycerebrosides is considerably lower than of cerebrosides with normal fatty acids, which is similar to lower vertebrates -- amphibians and fishes. Low content of hydroxycerebrosides was found in all the Lepidosaurians investigated, irrespectively of their ecological conditions, being therefore dependent on their phylogenetic position. The composition of fatty acids, both normal and hydroxyderivates, as well as that of glycolipids from the brain of Anapsids and Lepidosaurians is essentially similar. However, some interspecific differences were noted in the pattern of fatty acids of cerebrosides and cerebroside sulfates of the brain, which concern the content of saturated and long chain fatty acids.     

Characterization of microsatellite loci in the European pond turtle Emys orbicularis

A set of eight highly polymorphic microsatellite markers was isolated and characterized from a genomic library enriched for dinucleotide repeats in the European pond turtle, Emys orbicularis. The markers were tested for polymorphism in a total of 33 turtles sampled in two natural ponds in the nature reserve of Kerkini, northern Greece. Number of alleles varied from 10 to 18, and expected heterozygosity ranged between 0.738 and 0.921. This novel set of loci will be particularly useful to assess fine-scale population structure and for parentage analysis in E. orbicularis.     

Pretectal and Tectal Connections Revealed by Tracing Technique in theTurtle Intergeniculate Leaflet

Using antero- and retrograde tracer techniques, it has been shown that the intergeniculate leaflet nucleus (IGL) in turtles (Testudo horsfieldi, Emys orbicularis) has reciprocal connections with pretectum and tectum. Neurons projecting to this nucleus are located in the retino- and non-retinorecipient nuclei of pretectum and, mostly, in the deep periventricular layer of tectum. Neurons containing neuropeptide Y (NPY) and GABA are a possible source of projections from IGL to the same structures of pretectum and tectum. The data obtained allow concluding the presence of a morphological substrate for interaction of the circadian and non-circadian (visual-motor, perceptive) visual systems in reptiles as well as in other amniotes—birds and mammals.     

Neurochemical characteristics of the turtle optic tectum: Comparison with other reptilian species and birds

Data on distribution of biologically active substances in the turtle optic tectum are compared with results of similar experiments on other reptilian as well as on avian species. In two turtle species (Testudo horsfield and Emys orbicularis), immunoreactivity to monoamines (5-HT and TH), NPY, as well as NADPH-d activity were similarly distributed in neuropil of the SGFS retinorecipient part and in that of the SGP/SAP periventricular layers. Immunoreactivity to neuropeptides SP and m-Enk was maximal in neuropil of the SGFS non-retinorecipient part. The periventricular layers were characterized by the abundant radial SP- and mENK-ir as well as the NADPH-d-positive neurons. Diffusely dispersed ChAT-ir elements and many ir fibers perpenducilar to the tectal surface were observed in the SGFS retinorecipient part; the SGFS non-retinorecipient part contained a dense plexus of thick ir fibers and diffusely distributed ir terminals. The GABA ir cells were the most numerous in the tectum; they were spread in all tectal layers. Thus, various biologically active substances located in superficial retinorecipient tectal sublayers could affect processing and transmission of information via ascending dendrites of neurons in deeper layers. The cells containing SP, m-Enk, and NADPH-d had laminar organization in SGP; via the system of ascending and descending axons, they are able to affect other structures within and outside of the optic tectum. Putative sources of tectal modulatory innervation are discussed. In all studied reptilian and avian species, the principal similarity is revealed in the neurochemical organization. Some differences might be explained by the level of tectal differentiation due to factors of phylogenetic evolution and/or adaptive specialization.     

Ultrastructure of the joint receptors of the tortoise (Testudo graeca. Emys orbicularis).

The ultrastructure of the spray-like ramified encapsulated corpuscles with the primitive inner core from the joint capsules of the large limb joints of the tortoise (Testudo graeca and Emys orbicularis) was examined. Each of the branches of the receptor consists of three components. Through the middle of the receptor branche runs the nerve terminal, containing in the receptor matrix numerous mitochondria, tiny light vesicles and neurofilaments and neurotubules running in the axial way. The nerve terminal gives off on some places among the inner core cells tiny finger-like processes. The axon is surrounded by the inner core cells and their irregular plasmatic processes. Among the inner core cells and their irregular plasmatic processes there is a labyrinth of spaces, connected centrally with the periaxonal space and with the boundary space on the periphery. The inner core cells are covered on the surface, turning to the boundary space by the basal membrane. The inner core has a very primitive structure, it still lacks the typical lamellar structure. The capsule of the receptor is formed by flat cells, which surround the inner core in 1--3 layers. Between the capsule of the receptor and the inner core is the boundary space, containihg sporadical collagenous fibrils. The structure of the spray-like ramified encapsulated corpuscles with the primitive inner core from the joint capsules of the tortoise is analogous to the simple lamellar receptors from the skin of some reptiles (Von Düring 1973, 1974). The primitive structure of the inner core of the joint receptors in the tortoise reminds of the structure of the inner core of the developing simple (paciniform) corpuscles (Polá?ek and Halata 1970) and Pacinian corpuscles (Malinovsky 1974). The observed nerve endings represent a primitive, early stage in phylogeny development of the lamellar mechanoreceptors.     

Dating cryptodiran nodes: origin and diversification of the turtle superfamily Testudinoidea

The superfamily Testudinoidea is the most diverse and widely distributed clade of extant turtles. Surprisingly, despite an extensive fossil record, and increasing amount of molecular data available, the temporal origin of this group is still largely unknown. To address this issue, we used a comprehensive molecular dataset to perform phylogenetic and molecular dating analyses, as well as seven fossil constraints to calibrate the ages of the nodes in the phylogeny. The molecular dataset includes the complete mitochondrial genomes of 37 turtle species, including newly sequenced mitochondrial genomes of Phrynops hilarii, Emys orbicularis, Rhinoclemmys punctularia, and Chelonoidis nigra, and four nuclear markers. Our results revealed that the earliest divergences within crown testudinoids occurred around 95.0 Mya, in the early Late Cretaceous, earlier than previously reported, raising new questions about the historical biogeography of this group.     

Selective specificity of calcium-binding proteins calbindin and calretinin expression in the magnocellular neurosecretory hypothalamic nuclei of tortoises and turtles

We have studied the distribution of calcium-binding proteins in the magnocellular neurosecretory nuclei of nonapeptidergic neurosecretory nuclei of the preoptic–hypothalamic complex in a tortoise (Testudo horsfieldi) and a pond turtle (Emys orbicularis) using immunohistochemistry. We have found that different types of cells in the paraventricular and supraoptic nuclei predominantly express calbindin and, to a lesser extent, calretinin, but not parvalbumin. The selective calbindin/calretinin control of the neurohormone secretion in these hypothalamic nuclei is an evolutionary conservative feature typical of reptiles and mammals.