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The study of the brain by magnetic resonance imaging (MRI) allows to obtain detailed anatomical images, useful to describe specific encephalic structures and to analyze possible variabilities. It is widely used in clinical practice and is becoming increasingly used in veterinary medicine, even in exotic animals; however, despite its potential, its use in comparative neuroanatomy studies is still incipient. It is a technology that in recent years has significantly improved anatomical resolution, together with the fact that it is non-invasive and allows for systematic comparative analysis. All this makes it particularly interesting and useful in evolutionary neuroscience studies, since it allows for the analysis and comparison of brains of rare or otherwise inaccessible species. In the present study, we have analyzed the prosencephalon of three representative sauropsid species, the turtle Trachemys scripta (order Testudine), the lizard Pogona vitticeps (order Squamata) and the snake Python regius (order Squamata) by MRI. In addition, we used MRI sections to analyze the total brain volume and ventricular system of these species, employing volumetric and chemometric analyses together. The raw MRI data of the sauropsida models analyzed in the present study are available for viewing and downloading and have allowed us to produce an atlas of the forebrain of each of the species analyzed, with the main brain regions. In addition, our volumetric data showed that the three groups presented clear differences in terms of total and ventricular brain volumes, particularly the turtles, which in all cases presented distinctive characteristics compared to the lizards and snakes., (© 2024. The Author(s).)

Double-pulse laser-induced breakdown spectroscopy (DP-LIBS) was optimized for microspatial analyses of fossil and recent snake vertebrae. As complimentary techniques, solution analysis by inductively coupled plasma mass spectrometry and synchrotron radiation X-ray microtomography was utilized in order to determine the overall concentration of the selected elements in the samples and to visualize nondestructively the fossil sample microstructure, respectively. Elemental mapping of pathological bony tissue by DP-LIBS has been proven as a powerful tool for considering the osteitis deformans phases in fossil vertebrae.

This is the first description of haematopoiesis in snakes. Studies were carried out on the following species belonging to Ophidia: Bothrops jararaca, Bothrops jararacusu, Waglerophis merremii, Elaphe taeniura taeniura, Boa constrictor,and Python reticulatus. Smears of the peripheral blood and histological preparations from the vertebrae, ribs, liver, and spleen were studied under a light and electron microscope. Myeloid cells were present in the following locations in the vertebrae: the neural spine, zygoapophysial processes, floor of the neural canal, lacunae in the bodies of vertebrae and also inside the ribs. Although the vascular system was well developed, especially around the ribs, vessels inside the marrow cavities were scarce, both in the ribs and elsewhere where haematopoiesis was found. Venous sinuses were well developed in the vertebrae and in the rib regions from their costal head towards the middle area. They consisted of one layer of fine endothelial cells. Mature cells in the process of migration into the general circulation were only sporadically encountered when venous sinuses were studied on perfusion-fixed specimens. In contrast, almost every sinus venosus contained protrusions directed towards the lumen, filled mostly with mature and immature blood cells. Various stages of their formation were seen in the cross sections of venous sinuses ranging from small, newly formed to large, elongated ones, filled with many fully developed and some maturing blood cells. In many cases the apices of the protrusions were ruptured, and mature blood cells, as well as a few immature ones, were seen in their vicinity. This observation led us to a new hypothesis that blood cells are released from the extravascular space into the lumen of venous sinuses. In snakes, these cells are released into the systemic circulation mainly via the rupture of protrusions filled with mature blood cells and, to a lesser degree, by transcytosis as known in mammals. In the spleens from young specimens, 1-2 foci of haematopoiesis were encountered where lymphopoiesis predominated. Haematopoiesis was not detected in the liver.

The anatomical distribution of neurons and nerve fibers containing corticotropin-releasing factor (CRF) has been studied in the brain of the snake, Natrix maura, by means of immunocytochemistry using an antiserum against rat CRF. To test the possible coexistence of CRF with the neurohypophysial peptides arginine vasotocin (AVT) and mesotocin (MST) adjacent sections were stained with antisera against the two latter peptides. CRF-immunoreactive (CRF-IR) neurons exist in the paraventricular nucleus (PVN). In some neurons of the PVN, coexistence of CRF with MST or of CRF with AVT has been shown. Numerous CRF-IR fibers run along the hypothalamo-hypophysial tract and end in the outer layer of the median eminence. In addition, some fibers reach the neural lobe of the hypophysis. CRF-IR perikarya have also been identified in the following locations: dorsal cortex, nucleus accumbens, amygdala, subfornical organ, lamina terminalis, nucleus of the paraventricular organ, nucleus of the oculomotor nerve, nucleus of the trigeminal nerve, and reticular formation. In addition to all these locations CRF-IR fibers were also observed in the lateral septum, supraoptic nucleus, habenula, lateral forebrain bundle, paraventricular organ, hypothalamic ventromedial nucleus, raphe and interpeduncular nuclei.

The ependymal cells of the subcommissural organ (SCO) of the snake Natrix maura display long basal processes which terminate either on blood vessels or on the leptomeninges. The cell body and the basal processes contain a secretory material detectable immunocytochemically at the light-microscopic level using an antibody raised against bovine Reissner's fiber. The present investigation deals with the ultrastructural location in these cells of the (i) immunoreactive material; (ii) concanavalin A (Con A)- and wheat-germ agglutinin (WGA)-binding sites. In the subnuclear region the immunoreactive material was located within dilated cisternae of the rough endoplasmic reticulum and had affinity for Con A but not for WGA. In the supranuclear region the secretory material was exclusively located within numerous granules. Since all these granules showed affinity for WGA, they can be regarded as "post-Golgi" elements. Thus, at variance with the situation in the mammalian SCO, in the ophidian SCO most of the secretion is stored in secretory granules rather than in dilated cisternae of the rough endoplasmic reticulum. In the perivascular and leptomeningeal endings the immunoreactive material was located within granules which, because of their affinity for WGA, should also be regarded as true secretory granules derived from the Golgi apparatus. It is concluded that these granules are transported along the basal processes and accumulated in the perivascular and leptomeningeal endfeet. This observation favours the view of a local release of the content of these granules, since there is no evidence for a reverse transport of these granules all the way back from the distal termination to the apical pole, to be finally released into the ventricle.

The distribution and cytoarchitectonic pattern of the magno- and parvocellular hypothalamic nuclei of the cobra. Naja naja, are described at the light-microscopic level. With respect to their tinctorial affinity to paraldehyde fuchsin (AF) as a representative of the Gomori-type of stains, the magnocellular neurons belong to the "AF-positive" and the parvocellular neurons to the "AF-negative" elements. In addition to the supraoptic and paraventricular nuclei proper, two accessory aggregations of magnocellular neurons, the nucleus retrochiasmaticus and nucleus circularis, can be identified. Although in a peculiar location, they may be regarded as subunits of the supraoptico-paraventricular neurosecretory complex. As many as 22 "AF-negative" nuclear areas are identified in the hypothalamus of the cobra. The nucleus periventricularis hypothalami of earlier authors is subdivided into several circumscribed neuronal complexes. The nucleus arcuatus, nucleus hypothalamicus lateralis and nucleus lateralis recessus infundibuli are well developed. An attempt is made to interpret the significance of these nuclei on a comparative and phylogenetic basis.

Dual innervation of snake cerebral blood vessels by adrenergic and cholinergic fibres was demonstrated with the use of histochemical methods. Although the nerve plexuses are somewhat less dense, the essential features of innervation of the blood vessels are similar to those of mammals with the exception that the adrenergic plexuses are more prominent than the cholinergic plexuses. The major arteries of the cerebral carotid system have a rich nerve supply. However, the innervation is less rich in the basilar and poor in the spinal (vertebral) arteries. Although the arteries supplying the right side of head are poorly developed, three pairs of arteries, cerebral carotids, ophthalmics and spinals, supply the snake brain. The carotids and ophthalmics are densely innervated and are accompanied by thick nerve bundles, suggesting that the nerves preferentially enter the skull along those arteries. Some parenchymal arterioles are also dually innervated. Connection between the brain parenchyma and intracerebral capillaries via both cholinergic and adrenergic fibres was observed. In addition cholinergic nerve fibers, connecting capillaries and the intramedullary nerve fibre bundles, were noticed. Capillary blood flow may be influenced by both adrenergic and cholinergic central neurons. The walls of capillaries also exhibit heavy acetylcholinesterase activity. This may indicate an important role for the capillary in the regulation of intracerebral blood flow.

Two different types of ependymal cells were found in the subcommissural organ (SCO) of Natrix maura. Most secretory cells showed morphological features resembling the general structure and ultrastructure of cells in the SCO of other vertebrates. This report describes a second population of cells lining a portion of the dorsal groove of the SCO. These cells were not selectively stained by chromalum-hematoxylin and, under the electron microscope, they were characterized by scarce surface differentiations, sparse apical cytoplasm and short basal processes. Flat, parallel cisternae of the rough endoplasmic reticulum produced vesicles that appeared to be transported to the well-developed Golgi apparatus. Dense secretory granules about 200 nm in diameter were found in the Golgi region. Similar granules were seen in the vicinity of the apical plasma membrane; some of them opened toward the ventricle. All these characteristics clearly differentiate this cell group from the other secretory cells lining the SCO laterally and ventrally.

The adrenergic innervation of the pulmonary vasculature of the file snake Acrochordus granulatus was examined by use of glyoxylic acid-induced fluorescence. Perivascular plexuses of blue-green fluorescent nerves are observed around the common pulmonary artery, the anterior and posterior pulmonary arteries, the arterioles leading to the gas exchange capillaries of the lung, the venules draining the lung, and the anterior and posterior pulmonary veins. Adrenergic nerves are also associated with the visceral smooth muscle of the lung septa and other tissues. Thus, adrenergic control of pulmonary blood flow may occur either at the common pulmonary artery or more regionally within the lung. Regional control of blood flow in the elongate lung of this snake may be important in matching pulmonary perfusion with the distribution of respiratory gas. Glyoxylic acid-histochemistry and immunohistochemistry revealed that populations of cells located in the common pulmonary artery contain the indoleamine 5-hydroxytryptamine. Many of the cells are intimately associated with varicose blue-green fluorescent nerves. It is proposed that the 5-hydroxytryptamine-containing cells may be involved in intravascular chemoreception.

The pancreas from eleven species of snakes representing both advanced and primitive families has been investigated for the presence of eleven regulatory peptides reported to occur in the mammalian endocrine pancreas. Of the eleven peptides studied, insulin, pancreatic glucagon and somatostatin were present in endocrine cells within the islets of all the species investigated. The neuropeptide, vasoactive intestinal polypeptide, was located within nerve terminals innervating the islets in the Boidinae, Colubrinae, Elaphidae and Crotalidae but absent from the Natricinae investigated. No immunoreactivity was demonstrable with the antisera to substance P, met-enkephalin, C-terminal gastrin, bombesin, glicentin and gastric inhibitory polypeptide. Pancreatic polypeptide-like immunoreactivity was demonstrable only in the boid snakes and exclusively stained by a C-terminal specific antiserum.

Ascending spinal projections in the caiman (Caiman crocodilus) were demonstrated with Nauta and Fink-Heimer methods following hemisections of the third spinal segment in a series of twelve animals. These results were compared with earlier data in the literature obtained from a turtle, a snake, and a lizard using the same experimental and histological procedures. The results show remarkable similarities considering that each species represents a different reptilian order with different evolutionary history and habitat. However, the caiman displays several important peculiarities. Although the dorsal funiculus of the caiman contains the largest number of ascending spinal projections of the four species examined, this funiculus has not differentiated into cuneate and gracile fasciculi as is the case in the tegu lizard. The ventro-lateral ascending spinal projections follow a fundamentally similar general morphologic pattern in the four species with only minor variations. The anatomical arrangement in the caiman and tegu lizard appears most similar in the high cervical and the medullary regions; however, this is not the case in midbrain and thalamic regions where considerably more extensive projections are seen in the caiman. In the caiman an extensive spinal connection to the ventro-lateral nucleus of the dorsal thalamus is present; this connection is reminiscent of the mammalian spinal projection to the ventro-basal complex. The caiman has in common with the other three reptilian species a small projection to another dorsal thalamic region that is apparently homologous to the mammalian intralaminar nuclei, which are the destination of the mammalian paleospinothalamic tract.

Zonulae occludentes are shown by freeze-fracture to be pleomorphic along the garter snake nephron. In the neck and proximal segments the occluding junctions are moderately complex with frequent discontinuities in their junctional fibrils. Junctional depth and complexity are maximal in the distal and collecting segments and discontinuities in fibrils are absent. Comparison of these results with similar observations on other tissues indicates that the zonulae occludentes in the neck and proximal segments are "intermediate" to "leaky" and that they may be "very light" in the distal and collecting segments. The findings suggest that in the garter snake nephron transepithelial flow of fluid may occur primarily by passive diffusion through the zonulae occludentes in the neck and proximal segments and by cell-mediated osmotic flow in the distal and collecting segments. Gap junctions occur only in the proximal tubule and areprobably involved in low resistance, intercellular movement of ions.

In the snake, Natrix maura, and the turtle, Mauremys caspica, the basal processes of the ependymal cells of the subcommissural organ project toward the local blood vessels and the leptomeninges. These processes and their endings were studied using aldehyde-fuchsin (AF), periodic-acid Schiff (PAS), periodic-acid silver-methenamine (PA-SM), concanavalin A (ConA), wheat germ agglutinin (WGA), immunoperoxidase staining (employing an antiserum against bovine Reissner's fiber; AFRU), and conventional transmission electron microscopy. For the purposes of comparison, the ventricular cell pole was also analyzed. The secretory material located in the ventricular cell pole and that present in ependymal endings had only a few staining properties in common, i.e., affinity for AF, ConA, and AFRU at a dilution of 1:1000. On the other hand, PAS, PA-SM, WGA, and AFRU at a dilution of 1:200,000 stained the apical (ventricular) secretory material but not the secretory material of the ependymal processes. The histochemical features of the secretory material located in the terminals of ependymal processes, as well as the presence at these sites of numerous rough-endoplasmic-reticulum cisternae and secretory granules, suggest that secretory material may be synthesized in these terminals. The probable fate of this material, i.e., release to the perivascular and leptomeningeal spaces or transport to the ventricular cell pole, is discussed.

In the present HRP study extensive reticulospinal projections and more modestly developed vestibulospinal pathways have been demonstrated in the snake Python regius. The funicular trajectories of the main reticulospinal pathways have been shown: via the lateral funiculus pass spinal projections of the nucleus reticularis superior pars lateralis, the nucleus reticularis inferior and nucleus raphes inferior; via the ventral funiculus fibers arising in the nucleus reticularis superior and nucleus reticularis medius. Spinal projections of the locus coeruleus and subcoeruleus area reach their targets via both the lateral and ventral funiculi. Two vestibulospinal pathways have been demonstrated: an ipsilateral tractus vestibulospinalis lateralis arising in the ventrolateral vestibular nucleus, and a contralateral tractus vestibulospinalis medialis from the descending and ventromedial vestibular nuclei. After HRP gel implants into the vestibular nuclear complex direct vestibulocollic projections to motoneurons in the rostral spinal cord were observed. Spinal projections from the ventral part of the nucleus reticularis inferior and the descending and ventromedial vestibular nuclei are mainly aimed at the thin "neck area" (approximately the first 50 spinal segments). This area is extensively used in such acts as orientation and prey-catching, requiring a rather delicate brain stem control.

Chromaffin, small granule-containing (SGC)-cells, neurons and the innervation of these cells was studied in the adrenal gland of three species of reptiles (Testudo graeca, Lacerta dugesi, Natrix natrix). 1. After fixation with glutaraldehyde and osmium-tetroxide adrenaline (A)- and noradrenaline (NA)-storing cells can be distinguished by means of the different electron density of their granules: A-granules are moderately electron-dense, while NA-granules show a core of high electron density. The unusually high electron density of a few A-granules in Testudo occasionally required viewing of unstained sections which facilitated the discrimination of the two cell types in this species. In all species studied NA-granules display a remarkable polymorphism which is most pronounced in the tortoise. In this species A-granules are polymorphic, too. Both types of granules show wide variations in size, which are particularly great in the tortoise. This species also exhibits the largest average sizes for A-granules (285 nm), and NA-granules (354 nm). The corresponding parameters for Lacerta and Natrix, are 255 and 179 nm for A- and 323 and 304 nm for NA-granules, respectively. The rough ER in A-cells of the tortoise regularly occurs in the form of circular dilations ('ergastosomes', Kanerva and Hervonen, 1973). Mitochondria sometimes contain longitudinal cristae with a crystalloid internal pattern. Large dense bodies which incorporate granules are abundant in NA-cells. Smaller dense bodies containing a few dense patches and membranes are present in both A- and NA-cells. Intermediate stages between dense bodies and what appear to be A- or NA-granules (if the latter have lost some of their amine-content) are frequently observed.