Electrophysiological study of the functional connections of the hypothalamus with the forebrain in the tortoise Emys orbicularis

During stimulation of the posterior hypothalamus, the evoked potentials with short latent periods, high amplitude and poor exhaustion by rhythmic stimulation were recorded in the hippocampal cortex. In the piriform cortex, the evoked potentials exhibited longer latent periods and complex configuration. Less readily the evoked potentials appeared in the neocortex, their latency being very large. During stimulation of the anterior hypothalamus, maximum activity was also localized in the hippocampal cortex. The data obtained indicate close connection between hypothalamic structures and the hippocampal cortex. The latter is presumably the main projectional area for the ascending afferentation from the hypothalamus.     

Features of the dynamics of the forebrain EEG of Emys orbicularis during the solving of an extrapolation problem

Sharp EEG changes are recorded in bioelectrical activity of the dorsal cortex and dorsal ventricular edge in marsh tortoises in conditions of free movement during solving of an extrapolation task (a test of elementary reasoning ability). These changes of a pathological character, accompanied by neurotic states, were observed in some animals having correctly solved the task several times in succession (2-5), beginning with the first presentation. Such changes of EEG and behaviour were not found in tortoises that committed errors at first presentations of the task and only gradually learned correct solving. Formation of the adequate behaviour can proceed by two means: on the basis of elementary reasoning ability and learning. Disturbance of adequate behaviour in the experiment with characteristic changes of EEG testifies to a difficult state of the animal during solving of the extrapolation task.     

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.     

Absence of purinoceptors in the heart of the turtle (Emys orbicularis)

The effects of adenosine, adenosine 5'-triphosphate (ATP), a slowly degradable ATP analogue beta,gamma-methylene ATP (APPCP) and a degradation resistant ATP analogue alpha,beta-methylene ATP (APCPP) were examined on the turtle heart. Adenosine, ATP, APPCP and APCPP had no effect on the rate or force of contraction of either the atrium or ventricle. The effects of acetylcholine and noradrenaline were also examined on the turtle heart. Acetylcholine decreased the force and rate of contraction of turtle atria in a concentration-dependent manner. Noradrenaline increased the rate of contraction but caused a slight decrease in the force of contraction of the atrium. Neither acetylcholine nor noradrenaline produced an inotropic effect on the ventricle.     

H-Y Antigen Expression in Temperature Sex-Reversed Turtles (Emys orbicularis)

H-Y antigen has been used as a marker for the heterogametic sex and is assumed to be an organizing factor for the heterogametic gonad. In the turtle Emys orbicularis , H-Y antigen is restricted to the female cells, indicating a female heterogamety (ZZ/ZW) sex-determining mechanism. Moreover, the sexual differentiation of the gonads is temperature sensitive, and complete sex reversal can be obtained at will. In this framework the relationships between H-Y antigen, temperature, and gonadal phenotype were studied. Mouse H-Y antiserum was absorbed with blood and gonadal cells of control wild male and female adults, and with blood and gonadal cells from three lots of young turtles from eggs incubated at 25–26°C (100% phenotypic males), at 30–30.5°C (100% phenotypic females), or at 28.5–29°C (majority of females with some males and intersexes). The residual activity of H-Y antiserum was then estimated using an immunobacterial rosette technique. In adults, both blood cells and gonadal cells were typed as H-Y negative in males and as H-Y positive in females. In each of the three lots of young, blood cells were H-Y negative in some individuals and H-Y positive in others. The proposed interpretation is that the H-Y negative individuals were genotypic males (ZZ) and the H-Y positive were genotypic females (ZW). The gonads of these animals were then pooled in different sets according to their sexual phenotype and to the presumed genotypic sex (i.e., blood H-Y phenotype). Testicular cells were typed as H-Y negative in genotypic males as well as in the presumed sex-reversed genotypic females; likewise, ovarian cells were typed as H-Y positive in genotypic females as well as in the presumed sex-reversed genotypic males. These results provide additional evidence that H-Y antigen expression is closely associated with ovarian structure in vertebrates displaying a ZZ/ZW sex-determining mechanism.     

Study of afferent connections of the visual cortex in the ventral bank of the cruciate sulcus in cats by the method of retrograde axonal transport of horseradish peroxidase

To study the possible pathways along which visual signals reach a visual zone discovered by the writers in the cat frontal cortex in the ventral bank of the cruciate sulcus, horseradish peroxidase was injected into a site previously identified by physiological experiments. Stained neurons were discovered in visual areas of the cortex (lateral suprasylvian and ectosylvian), in the parietal cortex (areas 5 and 7), and also in small numbers in the prefrontal and limbic cortex. Stained neurons were found in the following nuclei in the thalamus: n. medialis dorsalis, intralaminar nuclei (nn. centralis lateralis, paracentralis, centralis medialis), nn. ventralis medialis, anteromedialis, and reuniens. Many stained neurons were found in the claustrum and a few in the substantia grisea centralis in the midbrain. The principal sources of inputs to the cortical area investigated are thus centers of the visual system or parts of the brain directly connected with it.Institute for Problems in Information Transmission, Academy of Sciences of the USSR, Moscow. Brain Institute, All-Union Mental Health Research Center, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 17, No. 1, pp. 43–49, January–February, 1985.     

Thalamic neuronal reactions of the tortoise, Emys orbicularis, to visual stimuli

Reactions of 300 neurons and characteristics of their receptive fields (RF) in response to moving stimuli and diffuse illumination have been investigated in three hypothalamic regions of the tortoise Emys orbicularis. With respect to their reactions to different stimuli, the neurons investigated were divided into two groups. Neurons of the 1st type (85%) were activated by the stimuli of any size exhibiting large, small and mean RF. Adaptive changes were usually weak and mean (63%); strong changes were less frequent (37%). Some of the neurons (33) did not react to diffuse illumination, whereas other ones produced reactions of different types: 57% of the neurons exhibited a spontaneous activity, 9%--directional sensitivity. Neurons of the 2nd type (15%) reacted only to large stimuli and were characterized by large RF, strong adaptation (89%), low reactivity to diffuse illumination (46%); 35% of the neurons revealed the spontaneous activity, 20%-directional sensitivity.     

Afferent and efferent connections of the area postrema demonstrated by the horseradish peroxidase method

The previous autoradiographic study of the efferent connections of the arca postrema have been completed by using horseradish peroxidase (H.R.P.) as a marking agent. These two labelling techniques have made it possible to determine two categories of structures connected with A.P.: those with afferent connections (Nucleus ambiguus), or efferent connections (mesencephalic nucleus of V, Locus coeruleus, griseum centrale and inferior and superior colliculi) and those having both types of connections (Nucleus tractus solitarii (N.F.S.), Dorsal vagal nucleus, nucleus intercalatus, hypoglossal nucleus). The afferent and efferent pathways of the A.P. identified with H.R.P. coincide with the catecholaminergic pathways demonstrated in the rat by Lindvall and Bj?rklund (1974).     

Isolation of microsatellite markers in the Emys orbicularis complex and development of multiplex PCR amplification

We report the development of 15 new microsatellite markers for Emys orbicularis and Emys trinacris. A survey of 20 individuals showed that all loci are highly polymorphic with 3–14 alleles per locus. Additionally, 22 Glyptemys muhlenbergii primers were checked for cross-species amplification, with 14 being amplified successfully and polymorphic (2–14 alleles). A set of eight markers was selected and combined into two multiplex PCRs. Levels of genetic diversity were assessed in 648 individuals covering the complete distribution area. The number of alleles ranged from 13 to 24 and observed heterozygosity varied between 0.515 and 0.852.     

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.     

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.