T. V. Chudinova

Core-and-belt organisation of the mesencephalic and forebrain auditory centres in turtles: Expression of calcium-binding proteins and metabolic activity

The distribution of immunoreactivity to the calcium-binding proteins parvalbumin, calbindin and calretinin and of cytochrome oxidase activity was studied in the mesencephalic (torus semicircularis), thalamic (nucleus reuniens) and telencephalic (ventromedial part of the anterior dorsal ventricular ridge) auditory centres of two chelonian species Emys orbicularis and Testudo horsfieldi. In the torus semicircularis, the central nucleus (core) showed intense parvalbumin immunoreactivity and high cytochrome oxidase activity, whereas the laminar nucleus (belt) showed low cytochrome oxidase activity and dense calbindin/calretinin immunoreactivity. Within the central nucleus, the central and peripheral areas could be distinguished by a higher density of parvalbumin immunoreactivity and cytochrome oxidase activity in the core than in the peripheral area. In the nucleus reuniens, the dorsal and ventromedial (core) regions showed high cytochrome oxidase activity and immunoreactivity to all three calcium-binding proteins, while its ventrolateral part (belt) was weakly immunoreactive and showed lower cytochrome oxidase activity. In the telencephalic auditory centre, on the other hand, no particular region differed in either immunoreactivity or cytochrome oxidase activity. Our findings provide additional arguments in favour of the hypothesis of a core-and-belt organisation of the auditory sensory centres in non-mammalian amniotes though this organisation is less evident in higher order centres. The data are discussed in terms of the evolution of the auditory system in amniotes.

Differences in parvalbumin and calbindin chemospecificity in the centers of the turtle ascending auditory pathway revealed by double immunofluorescence labeling.

Using double immunofluorescence labeling, quantitative ratio between parvalbumin- and calbindin-containing neurons, neurons that co-localize both peptides, as well as the intensity of their immunoreactivities were studied in the brainstem, midbrain and forebrain auditory centers of two chelonian species, Testudo horsfieldi and Emys orbicularis. In the spiral ganglion and first-order cochlear nuclei, highly immunoreactive parvalbumin-containing neurons predominated, and almost all neurons in these nuclei also exhibited weak immunoreactivity to calbindin. The number of strongly calbindin-immunoreactive (-ir) cells increased in the second-order brainstem auditory centers (the laminar cochlear nucleus, superior olivary complex, lateral lemniscal nucleus), and co-localization with parvalbumin in some of them was observed. In the midbrain, a complementary distribution of parvalbumin and calbindin immunoreactivity was found: the central (core) region of the torus semicircularis showed strong parvalbumin immunoreactivity, while the laminar (belt) nucleus was strongly calbindin-ir. In the thalamic nucleus reuniens, almost complete topographic overlapping of the parvalbumin-ir and calbindin-ir neurons was shown in its dorsomedial region (core), with the intensity of immunoreactivity to calbindin being much higher than that to parvalbumin. The predominance of calbindin immunoreactivity in neurons of the dorsomedial region of the nucleus reuniens is correlated with the existence of the dense calbindin-ir terminal field in its projection area in the telencephalon. We conclude that the turtle auditory pathway is chemically heterogeneous with respect to calcium-binding proteins, the predominance of parvalbumin in the brainstem and midbrain centers giving way to that of calbindin in the forebrain centers; the portion of neurons co-localizing both peptides nonlinearly decreases from lower to higher order centers.

Distribution of calcium-binding proteins in the pigeon visual thalamic centers and related pretectal and mesencephalic nuclei. Phylogenetic and functional determinants

Multichannel processing of environmental information constitutes a fundamental basis of functioning of sensory systems in the vertebrate brain. Two distinct parallel visual systems - the tectofugal and thalamofugal exist in all amniotes. The vertebrate central nervous system contains high concentrations of intracellular calcium-binding proteins (CaBPrs) and each of them has a restricted expression pattern in different brain regions and specific neuronal subpopulations. This study aimed at describing the patterns of distribution of parvalbumin (PV) and calbindin (CB) in the visual thalamic and mesencephalic centers of the pigeon (Columba livia). We used a combination of immunohistochemistry and double labeling immunofluorescent technique. Structures studied included the thalamic relay centers involved in the tectofugal (nucleus rotundus, Rot) and thalamofugal (nucleus geniculatus lateralis, pars dorsalis, GLd) visual pathways as well as pretectal, mesencephalic, isthmic and thalamic structures inducing the driver and/or modulatory action to the visual processing. We showed that neither of these proteins was unique to the Rot or GLd. The Rot contained i) numerous PV-immunoreactive (ir) neurons and a dense neuropil, and ii) a few CB-ir neurons mostly located in the anterior dorsal part and associated with a light neuropil. These latter neurons partially overlapped with the former and some of them colocalized both proteins. The distinct subnuclei of the GLd were also characterized by different patterns of distribution of CaBPrs. Some (nucleus dorsolateralis anterior, pars magnocellularis, DLAmc; pars lateralis, DLL; pars rostrolateralis, DLAlr; nucleus lateralis anterior thalami, LA) contained both CB- and PV-ir neurons in different proportions with a predominance of the former in the DLAmc and DLL. The nucleus lateralis dorsalis of nuclei optici principalis thalami only contained PV-ir neurons and a neuropil similar to the interstitial pretectal/thalamic nuclei of the tectothalamic tract, nucleus pretectalis and thalamic reticular nucleus. The overlapping distribution of PV and CB immunoreactivity was typical for the pretectal nucleus lentiformis mesencephali and the nucleus ectomamillaris as well as for the visual isthmic nuclei. The findings are discussed in the light of the contributive role of the phylogenetic and functional factors determining the circuits׳ specificity of the different CaBPr types.

Components of the pigeon tectothalamic visual pathway, revealed with aid of study of cytochrome oxidase activity and immunoreactivity to calcium-binding proteins

Distribution of activity of cytochrome oxidase (CO) and immunoreactivity to parvalbumin (Pv) and calbindin (Cb) was studied in the optic tectum of the pigeon (Columba livia). In the first link of the tectofugal pathway in the central gray layer (SGC = layer 13), small amounts of the CO-active and Pv-immunoreactive (Pv-ir) cellular bodies were revealed in its internal part (sublayer 13b). Some of these neurons located along the SGC lower boundary had long dendritic processes ascending into the superficial tectum layer (SGF). In the retinorecipient SGF sublayers and particularly in neuropil of the sublayers 4 and 7, the high CO activity correlating with Pv-immunoreactivity was found. It is suggested that a great contribution to metabolic activity of these sublayers is made by the largely branching dendritic processes of Pv-ir neurons of sublayer 13b. The projectional neurons SGC located in its external part (sublayer 13a) were CO-inactive and contained Cb. They sent long dendrites into sublayer 5b; in its neuropil, the high density of Cb-immunoreactivity and a moderate CO activity were detected. Thus, the tectal link of the pigeon tectofugal visual channel consists of two components—the Pv-specific, highly metabolically active and the Cb-specific, metabolically less active ones that transduce visual information from different retino-recipient SGF sublayers. The absence of the significant amount of CO-positive bodies of projectional neurons in SGC can be due to that metabolically more active are their dendritic arborizations in the SGF sublayers.

Metabolic activity of the thalamic and telencephalic auditory centers of reptiles.

329 The lemniscal (classical projection) and extralemniscal parts of mammalian sensory systems, including the auditory system have different levels of metabolic activity. The lemniscal parts have a higher activity of cytochrome oxidase (CO) and an increased ability to accumulate 2-deoxyglucose (2-DG), whereas the extralemniscal parts are characterized by a lower level of metabolic activity [1]. A similar distribution of metabolic activity was observed in the auditory system of birds (in the mesencephalic, thalamic, and telencephalic auditory centers) [2‐4]. Earlier, we showed that the mesencephalic auditory center, torus semicircularis (TS), of some reptiles, such as turtles and lizards, has a higher CO activity in the central nucleus of the lemniscal pathway than in the peripheral extralemniscal part [5]. However, it is not clear whether the metabolic activity in the rostral centers of reptilian auditory system, viz. the auditory relay thalamic nucleus (n. reuniens, Re) and its projection zone in the telencephalon (the ventromedial part of anterior dorsal ventricular ridge, Advr), have selective distribution. In this work, we studied the distribution of the activity of the mitochondrial oxidative enzyme CO in the rostral auditory centers of two turtle species, Testudo horsfieldi and Emys orbicularis. The experiments were performed with four T. horsfieldi and four E. orbicularis. The CO activity was measured by the standard histochemical method [6] with the use of bovine heart cytochrome Σ (Sigma). The CO activity was estimated by the density of histochemical staining of frozen frontal brain slices with a thickness of 50 µ m. Thalamus. The distribution of the CO activity in the Re in both turtle species was similar and heterogeneous. In the rostral level of the thalamus, we found a CO-active band-shaped structure that continuously spread from the ventricle in the lateral direction below the ventral border of the thalamic center of the tectofugal visual system (Rot) and coincided with the location of the n. anterior (nA). The medial part of this structure is a rostral pole of Re (Figs. 1a and 2a). The middle Re level had two areas with a high CO activity. The larger dorsocentral area occupied almost the entire nucleus except for a small ventrolateral part and extended to the somatic nucleus n. ventralis (nV) below the Rot. This area was continuously connected with the rostral bandshaped structure (Figs. 1b and 2b). Like the rostral part of the Re, this area was filled with stained punctuate terminals with sparse CO-positive cells located in the lat

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 tortoise 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 that of the thalamofugal pathway in Cxdl. The neurons immunoreactive to all three CaBPr types were distributed in Cxdl in different ratios in each layer. 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- and 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 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.

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.

Homologous thalamic nuclei of the tectofugal visual system of reptiles and birds have different immunoreactivities towards calcium-binding proteins

210 Determination of homology is the main tool in the research of evolution of living organisms from their ancestral forms [1, 2]. However, there is hardly any other problem of comparative neurobilology that is that far from its complete solution. A large number of various traits of brain structures have been used to determine homology: from morphological and funcc tional to molecular and genetic. The wider the range of these traits, the more probable their homology is. However, there are a number of conditions limiting the value of the comparison of chosen traits at different hierarchical levels of the body [1, 2]. The introduction of histochemical and particularly immunohistochemm ical studies has created one more criterion for deterr mination of homology of brain structures and the whole functional systems that is based on similar content of biologically active substances, neurotranss mitters/neuromodulators, including calciummbinding proteins (CaBPr) [1, 3, 4]. The role of the latter as effective functional markers is important both at the level of single neurons and at the level of cell aggree gates within nuclei [1]. Apparently, neurochemical parameters of both individual brain structures and the whole functional systems are highly conserved in the phylogeny of vertebrates. These systems include, e.g., the dopaminergic mesencephaloostriatal system, seroo toninergic system of the raphe nuclei, and mesenn cephalofugal cholinergic system. On the contrary, some structures that are considered homologous according to some criteria have different neurochemii cal parameters, so their plastic reorganization could have taken place during evolution. Apparently, a neuu rochemical marker alone is not sufficient for making a conclusion about homology, though the similarity of chemical organization is typical for many homologous structures. At present, there is no criterion that could be a touchstone in determination of homology [1, 2]. In this study, the results of comparative analysis of immunoreactivity towards two calciummbinding proo teins (CaBPr), parvalbumin (PV) and calbindin (CB), in the thalamic nucleus of the n. rotundus (Rot) tectofugal visual system of reptiles (tortoises and tur tles) and birds (pigeons) are presented. The homology of the tectofugal visual pathway and its thalamic relay the Rot in two related taxons (reptiles and birds) is generally acknowledged [5]. It is interesting to investii gate if the calcium balance in neurons of homologous thalamic nuclei is regulated by the same or different CaBPr with different buffer and nonnbuffer properties. The results were obtained from Middle Asian terr restrial turtle (tortoises) (Testudo …

Metabolic activity of pigeon thalamic and telencephalic auditory centers

Distribution of activity of mitochondrial oxidative enzyme cytochrome oxidase (CO) was studied in the thalamic (Ov) and telencephalic (field L) auditory centers of the pigeon Columbia livia. Different levels of CO activity are found in the core and belt of the centers: the high CO activity in the core of Ov (nCe) and telencephalic field L2 and the much lower or absent in the peripheral regions (Ovl, Ovm, SPO and L1 and L3). Comparison of our data with those of various avian and reptile species confirms the concept of the common plan of rostral auditory centers in sauropsid amniotes by the principle of the center-periphery (core-belt), which is characteristic of the corresponding mammalian centers. The separation of the central and peripheral parts of these centers is better pronounced in birds than in reptiles.