Calcium-Binding Proteins in the Turtle Thalamus. Analysis in the Light of Hypothesis of the “Core-Matrix” Thalamic Organization in Relation to the Problem of Homology of Thalamic Nuclei among Amniotes

Distribution of immunoreactivity (IR) to Ca-binding proteins (CaBPr) (calbindin, Calb, parvalbumin, Parv., and calretinin, Calr) was studied in the thalamus of the Central Asian terrestrial turtles (Testudo horsfieldi) and fresh water turtles (Emys orbicularis). There has been established a wide spread of these proteins, which combines overlapping and a relative alternation of distribution of different CaBPr in individual nuclei. A comparison of IR was made in two relay nuclei of the visual system, GLd and Rot. Both nuclei had IR to all CaBPr, but with different degree of intensity. In the terrestrial turtles, the amounts of Calb-, Parv-, and Calr-IR neurons in the cellular plate of the GLd were close. In this plate and in the neuropil part of this nucleus there was observed CaBPr-innervation of various density. Calr-IR neurons in the GLd of the fresh water turtles dominated over Parv- and Calb-IR neurons, whose detection varied significantly. In Rot, a clear predominance of Calb-IR neurons was shown over Parv- and Calr-IR cells by constancy of their detection, the number (1.5–2-fold higher), and intensity of the immune label, as well as the highest density of Calb and Parv innervation. The character of IR in the Rot was similar in the both turtle species. In the auditory and somatic relay thalamic nuclei and in the non-sensory anterior thalamic nuclei (Dma, Dla) there were present neurons and terminals with IR to all CaBPr without any predominance of Parv-IR in the relay nuclei and Calb-IR in the anterior thalamic nuclei. The constant and characteristic feature of Enta in the turtles of both species is a dense population of Parv-IR neurons, whose topography and cellular composition coincide with those of population of GABA-IR neurons in this nucleus. The data obtained have shown that the alternative presence of different CaBPr in the relay sensory and non-sensory thalamic nuclei, which has been established as a characteristic feature of the mammalian thalamus, is not characteristic at all of turtles. It seems that in the course of evolution there occurred a reorganization of distribution of different CaBPr in thalamic nuclei of amniotes due to changes of their functional loading. The reptilian thalamic sensory relay nuclei are likely to be represented mainly by less specific parts comparable with Calb-IR matrix of specific nuclei in the higher amniotes (mammals), while their more specialized (core) Parv-IR regions are formed later in evolution. Therefore, the distribution of Parv- and Calb-IR neurons in the turtle thalamic nuclei cannot be a criterion at evaluation of homology of thalamic nuclei in amniotes, but permits judging about the degree of their specialization.     

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 cytochrome oxidase activity in the turtle optic tectum with special reference to the tectofugal visual pathway

Using immunohistochemistry and a tracer technique we investigated the distribution in the optic tectum of turtles (Emys orbicularis and Testudo horsfieldi) of the calcium-binding proteins (CaBPr) parvalbumin (PV), calbindin (CB) and calretinin (CR) before and after labeling of the nucleus rotundus (Rot) with horseradish peroxidase. The optic tectum activity of the cytochrome oxidase (CO) was studied in parallel. In the principal link of the tectofugal visual pathway (central gray layer, SGC) in both chelonian species, the sparse PV-ir as well as CB- and CR-ir neurons were found significantly varying both in number and the intensity of immunoreactivity of their bodies and dendrites. In contrast, the superficial (SGFS) and deeper periventricular (SGP) tectal layers comprised numerous cells immunoreactive to all three CaBPr in different proportions. Only few retrogradely labeled tectorotundal SGC neurons expressed PV, CB or CR. The very large PV-ir neurons in SGC and SAC were not retrogradely labeled; morphologically they matched the efferent neurons with descending projections. SGC neurons of two chelonian species differed in the level of CO activity. Intense immunoreactivity to all three CaBPr and high CO activity were detected in both species in SGFS neuropil with some differences in sublaminar distribution patterns. The peculiarities of the CaBPr and CO activity distribution patterns in different segments of SGC neurons are discussed as related to the laminar organization of the turtle tectum and its retinal innervation. It is suggested that in the projection tectorotundal SGC neurons the CaBPr are concentrated mainly in their distal dendrites that contact retinal afferents in the superficial retinorecipient tectal layer.     

Neurochemical Organization of the Turtle Pretectum: An Immunohistochemical Study. Comparative Analysis

A comparative analysis of neurochemical organization of pretectal nuclei was performed on the basis of results of our studies on immunoreactivity to monoamines (tyrosine hydroxylase—TH and serotonin—5-HT), neuropeptides (substance P—SP, met-enkephalin—mEnk, and neuropeptide Y, NPY), and gamma-aminobutyric acid (GABA) in pretectum of two turtle species, as well as of the corresponding literature data obtained in other reptile species, birds, and mammals. Their presumed homologous pretectal nuclei were shown to have both similar and different immunohistochemical features. A conclusion is made that species of divergent lines of amniotes have not only the evolutionary conservatism of the neurochemical organization of pretectal structures and their projection pathways but also its certain plasticity due to rearrangements in the course of phylogenetic development.     

Distribution of calcium-binding proteins parvalbumin and calbindin in the thalamic auditory center in pigeons

Distribution of calcium-binding proteins (CaBPr) parvalbumin (PV) and calbindin (CB) in the thalamic auditory center (nucleus ovoidalis, Ov) was studied in the pigeon (Columba livia). Two parts of Ov were distinguished on the basis of their cytoarchitectonics and distribution of PV and CB immunoreactivity. The central lemniscal region (core, nCe) contains both dense PV-ir neuropil and PV-ir neurons overlapped with scant CB-ir neuropil and weaker stained CB-ir neurons. The peripheral extralemniscal region (belt), consisting of peri/paraovoidal nuclei (Ovl, Ovm, SPO), contains only CB-ir neuropil and strongly stained CB-ir neurons morphologically differing from CB-ir neurons in the nCe. A comparative analysis of our data on the distribution of PV and CB immunoreactivity in the thalamic auditory relay center in pigeons and related literature data obtained on other avian, reptilian and mammalian species indicates high evolutionary conservatism of its extralemniscal region across all sauropside amniotеs and mammals in contrast to plasticity of its central lemniscal region due to adaptive, ecologically dependent transformations during the evolution.     

Neurons of visual thalamic nuclei projecting to telencephalon express different types of calcium-binding proteins: A combined immunocytochemical and tracer study

In turtles (Testudo horsfieldi, Emys orbicularis), immunoreactivity to calbindin (CB), parvalbumin (PV), calretinin (CR) and co-localization of CB and PV were studied in neurons of the visual thalamic nuclei (Rot, GLd) projecting to the telencephalon using a combination of immunohistochemical and tracer methods. The prevalence of CB-immunoreactive (-ir) neurons in Rot, CB-ir and CR-ir neurons in GLd, and a smaller number of PV-ir neurons in both nuclei was shown. Double immunofluorescent labeling revealed that within both nuclei PV and CB are colocalized in most PV-ir and fewer CB-ir neurons. After injection of horseradish peroxidase into the Rot and GLs telencephalic projection fields, retrograde labeling was found in corresponding thalamic projection neurons immunoreactive to all the three proteins. After introduction of the fluorescent tracer Fluo-gold into the same telencephalic regions, retrograde labeling was detected in Rot and GLd neurons immunoreactive only to PV and CB as well as in neurons with colocalization of both proteins. These findings provide further evidence that in turtles the CB component prevails in the rotundo-telencephalic pathway while the CB/CR component is dominant in the geniculotelencephalic pathway. The role of functional specialization in segregation of neurons expressing distinct types of calcium-binding proteins is postulated.     

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.     

Distribution of metabolic activity (cytochrome oxidase) and immunoreactivity to calcium-binding proteins in turtle brainstem auditory nuclei

Using histochemical and immunohistochemical techniques, distribution of activity of oxidative mitochondrial enzyme cytochrome oxidase (CO) and of immunoreactivity to calcium-binding proteins has been studied in spiral ganglion and auditory nuclei of brainstem in two turtle species. It has been shown that immunoreactivity to calbindin, parvalbumin, and calretinin in neurons and neuropil of nuclei of cochlear and superior olivary complexes, in nucleus of lateral lemniscus, and in spiral ganglion neurons coincides topographically with the high CO activity. The similarity of the studied metabolic and neurochemical characteristics of these auditory centers in reptiles, birds, and mammals indicates the existence of some common principles of their organization in amniotes in spite of phylogenetic differences and peculiarities of specialization of the auditory system in different species.     

LACK OF CALBINDIN-D28K ALTERS RESPONSE OF THE MURINE CIRCADIAN CLOCK TO LIGHT

A strong stimulus adjusting the circadian clock to the prevailing light-dark cycle is light. However, the circadian clock is reset by light only at specific times of the day. The mechanisms mediating such gating of light input to the CNS are not well understood. There is evidence that Ca²⁺ ions play an important role in intracellular signaling mechanisms, including signaling cascades stimulated by light. Therefore, Ca²⁺ is hypothesized to play a role in the light-mediated resetting of the circadian clock. Calbindin-D28k (CB; gene symbol: Calb1) is a Ca²⁺ binding protein implicated in Ca²⁺ homeostasis and sensing. The absence of this protein influences Ca²⁺ buffering capacity of a cell, alters spatio-temporal aspects of intracellular Ca²⁺ signaling, and hence might alter transmission of light information to the circadian clock in neurons of the suprachiasmatic nuclei (SCN). We tested mice lacking a functional Calb1 gene (Calb1^?/?) and found an increased phase-delay response to light applied at circadian time (CT) 14 in these animals. This is accompanied by elevated induction of Per2 gene expression in the SCN. Period length and circadian rhythmicity were comparable between Calb1^?/? and wild-type animals. Our findings indicate an involvement of CB in the signaling pathway that modulates the behavioral and molecular response to light. (Author correspondence: urs.albrecht@unifr.ch)     

A fluorescence microscopic study of the distribution of monoamines in the hypothalamus of the cat

Three distinct groups of monoamine (MA)-containing nerve cell bodies have been visualized in the hypothalamus and preoptic area of the cat by means of the Falck-Hillarp fluorescence histochemical technique. First, numerous small-sized catecholamine (CA) type neurons were disclosed within the ventral half of the periventricular area in the supraoptic and middle hypothalamic regions. The round to oval neurons of this medio-ventral group were more especially abundant around the base of the third ventricle, within the arcuate and supraopticus diffusus nuclei. Numerous medium-sized CA perikarya identified as the dorsal group, were also mapped out in the dorsal and posterior hypothalamic areas. Finally, a small population of both CA and serotonin (5-hydroxytryptamine, 5-HT)-containing neurons was disclosed within the lateral area of the middle and mammillary hypothalamic regions. These multipolar or elongated neurons which compose the lateral group were lying either along the ventrolateral surface of the hypothalamus or around the ventrolateral aspect of the fornix. In addition to these three MA cell groups, a few cells displaying a fluorescence of the CA type were also visualized in the so-called “dorsal chiasmatic nucleus” after α-methyl-dopa treatment. High density of CA axon terminals were found, on the other hand, in the external layer of the median eminence, in the dorsomedial, paraventricular, supraoptic and suprachiasmatic nuclei, and also within nucleus interstitialis of stria terminalis. In the present study, however, it was not possible to identify with certainty any concentration of 5-HT axon terminals in the cat hypothalamus. Therefore, except for the lateral cell group which could be peculiar to the cat, the topographical distribution of MA nerve cell bodies and axon terminals in the hypothalamus of the cat appears similar to the morphological organization of the MA neuronal elements in the hypothalamus of the rat.     

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.     

Calcium binding protein expression in the optic tectum of Alligator during development

The onset and distribution of the calcium binding proteins, calretinin, calbindin, and parvalbumin, were examined in the optic tectum of Alligator mississipiensis embryos between Stages 18 and 26–28. The immunoreactivity of each calcium binding protein correlated well with the results from the Western blot experiments. In terms of onset and distribution, calretinin expressison was the most widespread of the three calcium binding proteins that were examined, and was also the earliest to be visualized. Calbindin expression occurred next, whereas parvalbumin expression was the most limited and appeared last. For small calretinin (+) neurons, the pattern of immunoreactivity during development was from inside to outside, whereas for the larger cells, it was from outside to inside. For calbindin immunoreactive cells in the superficial zone, the pattern was from outside to inside. The distribution of the parvalbumin immunopositive neurons did not change significantly over the time period examined. Similar data on other amniotes is limited. However, the pattern in Alligator shares some similarities with kittens in regards to the distribution of calbindin and parvalbumin in the developing superior colliculus. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 899–910, 2013     

Phylogenetic position of turtles among amniotes: evidence from mitochondrial and nuclear genes

Maximum likelihood analysis, accounting for site-heterogeneity in evolutionary rate with the Gamma-distribution model, was carried out with amino acid sequences of 12 mitochondrial proteins and nucleotide sequences of mitochondrial 12S and 16S rRNAs from three turtles, one squamate, one crocodile, and eight birds. The analysis strongly suggests that turtles are closely related to archosaurs (birds+crocodilians), and it supports both Tree-2: (((birds, crocodilians), turtles), squamates) and Tree-3: ((birds, (crocodilians, turtles)), squamates). A more traditional Tree-1: (((birds, crocodilians), squamates), turtles) and a tree in which turtles are basal to other amniotes were rejected with high statistical significance. Tree-3 has recently been proposed by Hedges and Poling [Science 283 (1999) 998-1001] based mainly on nuclear genes. Therefore, we re-analyzed their data using the maximum likelihood method, and evaluated the total evidence of the analyses of mitochondrial and nuclear data sets. Tree-1 was again rejected strongly. The most likely hypothesis was Tree-3, though Tree-2 remained a plausible candidate.     

Historical Burden In Systematics And The Interrelationships Of 'Parareptiles'

The interrelationships within the clade comprised of turtles, pareiasaurs, and procolophonid-like taxa are investigated via a cladistic analysis incorporating 56 characters. A single most parsimonious tree was found (80 steps, c. i. = 0·8) in which the successive outgroups to turtles are: pareiasaurs, Sclerosaurus, lanthanosuchids, procolophonoids (=Owenetta, Barasaurus and procolophonids), and nyctiphruretians (= nycteroleterids). Thus, as suggested recently by other workers (Reisz, in Fischman, 1993) turtles are the highly modified survivors of a radiation of poorly-known reptiles commonly called ‘parareptiles’. Pareiasaurs are united with turtles on the basis of twenty unambiguous derived features which are absent in other basal amniotes (=‘primitive reptiles’) and reptiliomorph amphibians: for example, the medially located choana, enlarged foramina palatinum posterius, blunt cultriform process, fully ossified medial wall of the prootic, opisthotic-squamosal suture, lateral flange of exoccipital, loss of ventral cranial fissure, thickened braincase floor, ‘pleurosphenoid’ ossification, reduced presacral count, acromion process, trochanter major, reduced fifth pedal digit, and presence of transverse processes on most caudals. Recent phylogenetic proposals linking turtles with captorhinids, with dicynodonts, and with procolophonoids are evaluated. None of the proposed traits supporting the first two hypotheses is compelling. The procolophonoid hypotheses is supported by only one synapomorphy (the slender stapes). All other synapomorphies proposed in favour of the above groupings either occur in many other primitive amniotes, or are not primitive for turtles, or are not primitive for the proposed chelonian sister-group. Nyctiphruretus and Lanthanosuchids and nycteroleterids, often considered to be seymouriamorph amphibians, are demonstrated unequivocally to be amniotes. The ‘rhipaeosaurs’, currently considered to be pareiasaur relatives, are shown to be a heterogenous assemblage of seymouriamorphs, therapsids and nycteroleterids. The phylogeny proposed here indicates that many of the traits of the earliest known turtle, Proganochelys, previously interpreted as unique specialisations, also occur in pareiasaurs and other near outgroups of turtles, and must instead represent the primitive chelonian condition: for example, the wide parietals and the short quadrate flange of the pterygoid. The sequence of acquisition of chelonian traits is discussed: many features once thought to be diagnostic of turtles actually characterize larger groupings of procolophonomorphs, and must have evolved long before the chelonian shell appeared. These traits include most of the chelonian-pareiasaur synapomorphies listed above, and many others which characterize more inclusive groupings found in this analysis. In putting Proganochelys much closer to the main line of chelonian evolution, in elucidating the sequence of acquisition of chelonian traits, and in reducing greatly the number of differences between turtles and their nearest relatives, this study helps bridge one of the major gaps in the fossil record. The failure of previous cladistic analyses to identify correctly the nearest relatives of turtles is attributed to biased character selection, caused by an over-reliance on cranial characters deemed ‘important’ by earlier workers, and by a tendency to shoehorn ‘parareptile’ taxa into phylogenies derived from analyses restricted to ‘mainstream’ groups such as synapsids, diapsids, turtles, and ‘captorhinomorphs’. Many of the synapomorphies that resolve turtle origins are postcranial, and the three nearest outgroups to turtles are all highly bizarre groups which were dismissed as ‘too specialized’ by early workers and continued to be inadequately assessed even by workers using a cladistic framework.     

Neuropeptide Y localization in telencephalic and diencephalic structures of the ground squirrel brain

The distribution of neuropeptide Y-immunoreactive (NPY-IR) perikarya, fibers, and terminals was investigated in the brain of two species of hibernatory ground squirrels, Spermophilus tridecemlineatus and S. richardsonii, by means of immunohistochemistry. In the telencephalic and diencephalic structures studied, distinct patterns of NPY-IR were observed which were essentially identical in male and female animals of both species. No differences in amount or distribution of NPY-IR structures were observed between animals which had been in induced hibernation for several months before sacrifice in March/April and those sacrificed one week after their capture in May. In some brain structures (e.g., the hypothalamic arcuate nucleus), IR cell bodies were observed only after pretreatment with colchicine. NPY-IR perikarya and fibers were found in the cerebral cortex, caudate nucleus-putamen, and dorsal part of the lateral septal nucleus. Dense fiber plexuses were seen in the lateral and medial parts of the bed nucleus of the stria terminalis. The numbers of IR perikarya observed in the medial part of the nucleus increased following intraventricular colchicine injections. The accumbens nucleus exhibited few IR cells and many fibers. Claustrum and endopiriform nuclei showed a considerable number of stained cells and fibers that increased in number and staining intensity in colchicine-treated ground squirrels. The induseum griseum showed a small band of IR cell bodies and varicose fibers. Bipolar of multipolar IR cells and varicose fibers were found in the basal nucleus of the amygdala. Dense fiber plexuses as well as IR terminals were seen in the median, medial, and lateral preoptic areas of the hypothalamus. Terminals and relatively few fibers were located in the periventricular, paraventricular, and supraoptic nuclei. The anterior, lateral, dorsomedial, and ventromedial hypothalamic nuclei contained relatively large numbers of terminals and fibers. In the suprachiasmatic nuclei, dense terminals were distributed mainly in the ventromedial subdivision. In the median eminence, immunoreactive terminals were concentrated in the external layer, with fibers predominant in the internal layer. NPY-IR perikarya were observed only in the arcuate nucleus of the hypothalamus and only following colchicine treatment. In the epithalamus (superficial part of the pineal gland and habenular nuclei), varicose fibers appeared mainly in perivascular locations (pineal) or as a dense plexus (habenular nuclei). These results from ground squirrels are discussed in comparison to those obtained in other species and with regard to considerations of the physiological role of NPY.     

Retinofugal and retinopetal projections in the teleost Channa micropeltes (Channiformes)

Summary Retinofugal and retinopetal projections were investigated in the teleost fish Channa micropeltes (Channiformes) by means of the cobaltous lysine and horseradish peroxidase (HRP) tracing techniques. Retinofugal fibers cross completely in the optic chiasma. A conspicious lamination is present in those parts of the optic tract that give rise to the marginal branches of the optic tract. This layering of optic fibers continues in the marginal branches to mesencephalic levels. Retinal projections to the preoptic and hypothalamic regions are sparse; they are more pronounced in the area of pretectal nuclei. The medial pretectal complex and the cortical pretectal nucleus are more fully differentiated than in other teleostean species. Further targets include the thalamus and the optic tectum. The course of major optic sub-tracts and smaller fascicles is described. Retinopetal neurons are located contralaterally in a rostral and a caudal part of the nucleus olfactoretinalis, and in a circumscribed nucleus thalamoretinalis. The present findings are compared with reports on other teleost species.     

Distribution of GABA-immunoreactive neurons in the brain of adult and developing salamanders (Pleurodeles waltli,Triturus alpestris)

The distribution of GABAergic neurons in brains of the family Salamandridae (Pleurodeles waltli, Triturus alpestris) has been investigated immunohistochemically with an antibody against gamma-aminobutyric acid (GABA). In adult animals, immunoreactive neurons, fibers, and terminals are abundantly labeled. In the telencephalon, pallial areas contain fewer GABAergic neurons and fibers than basal forebrain areas. The amygdalar complex and the habenulae have a complex pattern of GABA-immunoreactivity that is especially pronounced within the neuropil. The pretectal and basal optic systems are provided with GABAergic neurons, corroborating electrophysiological results. The dorsal thalamus and parts of the torus semicircularis are almost completely devoid of GABA-immunoreactive neurons. In the torus, magnocellular neurons known to project to the contralateral counterpart are distinctly GABA-immunoreactive. During ontogeny, GABAergic neurons arise early when the first reflexive movements occur after mechanical stimulation. At stage 28, cells are labeled initially near the nucleus of the medial longitudinal fasciculus, which is the first supraspinal tract to appear in ontogeny. At stage 30 (still before hatching), GABAergic neurons are found in the pretectum, immunoreactive neurons arising in the dorsal tegmentum slightly later. Both systems are known to mediate basic reflexes in gaze stabilization. The commissura posterior is GABAergic at early stages suggesting an important functional role in homonymous inhibition between both sides. Thus in salamanders, the neurotransmitter GABA displays a complex distribution, similar to that in other vertrebrates. This pattern emerges early in ontogeny.     

Level of metabolic activity (cytochrome oxidase) as an index of functional significance of tectofugal and thalamofugal channels of the reptilian visual system

Using histochemical determination of activity of the mitochondrial oxidative enzyme cytochrome oxidase (CO) the metabolic activity in structures of brain of turtles and lizards was shown to be higher in centers of the tectofugal channel (tectal SGC, pretectal Ptv, thalamic Rot, telencephalic visual area of Advr of) than in the centers of the thalamofugal channel (thalamic GLd, Cxdl and Path of telencephalon) of the visual system. Some interspecies differences in distribution of CO activity in the tectal, thalamic, and telencephalic visual centers were revealed between terrestrial and pond turtles and lizards. The obtained data confirm the idea of the dominating role of the tectofugal visual pathway in information processing and organization of the usual behavior of reptiles.     

Central projections of the lagena (the third otolith endorgan of the inner ear) in the pigeon

Central projections of the lagena were studied in the pigeon using transport of biotinylated dextran amine (BDA) that was locally applied to the lagenar epithelium through the opened cochlear canal. Descending (dorsocaudal part) and superior (middle part) vestibular nuclei were the main rhombencephalon structures with the maximum density of labeled fibers and terminals. Lesser numbers of labeled fibers were observed in the ventral part of the lateral vestibular nucleus and also in the medial vestibular nucleus; single labeled fibers were found in the cochlear nuclei. In the cases where BDA diffused not only in the lagena but also on the basilar papilla after application of the marker to the cochlear canal, considerable numbers of labeled fibers were observed in the cochlear nuclei; apart from this, the pattern of distribution of labeled fibers in the vestibular nuclei did not differ in general from that described above (in the case of a sufficiently local application of BDA only to the lagena). Efferent lagenar neurons were localized ventrally with respect to the vestibular nuclei, in particular in the nucl. reticularis pontis caudalis. Neirofiziologiya/Neurophysiology, Vol. 40, No. 3, pp. 199–210, May–June, 2008.