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Microvascularization of the cerebellum in the turtle, Pseudemys scripta elegans (Reptilia). A scanning electron microscope study of microvascular corrosion casts, including stereological measurements.

Cerebellar blood supply and microvascular patterns were studied in 12 freshwater turtles Pseudemys scripta elegans by scanning electron microscopy (SEM) of microvascular corrosion casts and histology. Vascular densities were estimated by point counting methods from casts and thin sections (7 microns). Short A2-arterioles and recurrent branches from A3-arterioles supply the capillary bed of the molecular layer, while V2 and V3 venules drain it. The Purkinje cell layer is supplied by horizontal branches ("parallel arteries") of A4 and A3 arterioles, which capillarize toward the granular and molecular layers. V2, V3 and V4 venules drain the areas supplied by A3 arterioles. The deep granular and subependymal layers are supplied by A4 arterioles, those adjacent to the Purkinje cell layer also by A3 arterioles. The areas supplied by A4 arterioles drain via V4 venules. The granular and Purkinje cell layers taken together have an estimated vascular density of 4.1%, while in the molecular layer this value is 3.3%. Our findings largely confirm published data from Testudo geometrica and Pseudemys scripta elegans with respect to gross supply and drainage. The microvascular patterns are similar to those of the human cerebellar cortex, particularly the basic patterns of intracortical arterioles and venules. The molecular layer, like that in the human brain, has a circulatory bed largely independent of that of the Purkinje cell and granular layers. In contrast to the human brain, a cerebellar pial capillary network is present in the brain of the turtle.

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Connections of the basal telencephalic areas c and d in the turtle brain.

Tracer substances were injected into the basal telencephalic areas c and d of the turtle brain. These areas (Acd) have recently been shown to be connected reciprocally with the dorsal spino-medullary region, though the particular subregions involved in these projections remained unclear. We demonstrated that the efferent projections of area d terminate predominantly within or immediately adjacent to the trigeminal nuclear complex and in the high cervical spinal gray. The dendritic domain of the vagus-solitarius complex and the dorsal column nuclear complex might also receive some basal telencephalic efferents. The afferent projections to Acd, on the other hand, arise predominantly in the dorsal column nuclei as defined according to cytoarchitectural and hodological criteria. A few retrogradely labeled cells were found in the vagus-solitarius complex, the principal trigeminal nucleus and the high cervical spinal cord. Numerous labeled cells were found in the dorsolateral isthmo-rhombencephalic tegmentum, especially the n. visceralis secundarius, the n. vestibularis superior and parts of the lateral lemniscal complex. Aminergic cell populations projecting to Acd were the n. raphes inferior and superior, the locus coeruleus, the substantia nigra, pars compacta and the ventral tegmental area. Other meso-diencephalic cell groups were the griseum centrale (including the n. laminaris of the torus semicircularis), the n. interpeduncularis dorsalis, the nucleus of the fasciculus longitudinalis medialis, the nucleus and the nucleus interstitialis of flm, the n. interstitialis commissuralis posterior and then n. caudalis. Several hypothalamic regions, the reuniens complex and the perirotundal region of the thalamus also appeared to project heavily to Acd. Telencephalic areas retrogradely labeled after injection of tracer into Acd and its immediate surroundings were the rostral part of the lateral (olfactory) cortex, adjacent regions of the basal dorsal ventricular ridge and the n. centralis amygdalae, the n. tractus olfactorius lateralis as well as the areas g and h. The data suggest that areas c and d may correlate best with the 'extended' amygdala in mammals; further correlation with structures similar to the ventral striopallidum, however, cannot be excluded. Homostrategies are discussed with regard to the processing of higher-order somatovisceral information in turtles, birds and mammals.

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A correlative light-, fluorescence- and electron-microscopic study of neuroepithelial bodies in the lung of the red-eared turtle, Pseudemys scripta elegans.

Neuroepithelial bodies (NEB) were identified for the first time in the respiratory tract of a reptile by the use of combined morphological and histochemical methods. In the red-eared turtle, Pseudemys scripta elegans, NEB were found within the trabecular epithelium of the respiratory tract, mainly in the branching regions of the trabeculae. An intracellular formaldehyde-induced fluorescent compound was identified as 5-hydroxytryptamine (5-HT) by means of microspectrofluorometry. Subsequent histochemical staining of the same fluorescent sections showed the 5-HT-containing cells to be argentaffin. In electron micrographs cell clusters characterized by the presence of distinctive cytoplasmic, membrane-bounded dense granules (+/- 100 nm) were observed, correlating with the distribution of the yellow-fluorescent epithelial cells. The granules of the NEB are positive when the argentaffin technique is performed directly on ultrathin sections. Cells of the NEB extend into the lumen of the airway via apical microvilli and a single modified cilium displaying a 9 X 2 + 0 or 8 X 2 + 2 microtubular pattern. Unmyelinated axons containing mostly small, clear vesicles were seen in close association with NEB cells, often forming synaptic junctions. Occasionally, axons containing a few small dense-cored vesicles were found. The relationship between NEB cells and capillaries, the images of emiocytotic granule release and the occurrence of synaptic contacts between axons and granule-containing cells are indicative of endocrine secretion. These features and the presence of intracytoplasmic granules containing 5-HT may justify the inclusion of NEB-cells of the turtle lung into the diffuse neuroendocrine system. Furthermore, structurally these cells appear to represent sensory elements capable of an intrapulmonary receptor-secretory function.

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Freeze-fracture study of the turtle lung. 2. Rod-shaped particles in the plasma membrane of a mitochondria-rich pneumocyte in Pseudemys (Chrysemys) scripta.

In thin sections of the lung of the fresh-water turtle Pseudemys (Chrysemys) scripta some pneumocytes can be distinguished from the remaining pulmonary epithelial cells by a larger amount of mitochondria. In these cells the typical features of type-I and type-II cells are absent. Freeze-fracture replicas reveal rod-shaped particles in the apical plasma membrane of a small population of pneumocytes, which by cytological criteria seem to be identical with the mitochondria-rich cells observed in thin sections. It is assumed that these cells represent a distinct type of pneumocytes in the turtle lung and that they are a member of the group of mitochondria-rich cells present in some ion-transporting epithelia. The function of these cells in this location remains to be determined.

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Freeze-fracture study of the turtle lung. 1. Intercellular junctions in the air-blood barrier of Pseudemys scripta.

The air-blood barrier in the lung of the turtle Pseudemys scripta was studied by means of freeze-fracture replicas in an attempt to give a detailed account on the structural organization of the intercellular junctions. Between the pneumocytes, zonulae occludentes containing 4-19 strands in the apico-basal direction are present; they are considered to be physiologically very tight. In the endothelium, fasciae occludentes, i.e., discontinuous occluding junctions can be found, composed of up to 4 strands. These junctions are regarded to be very leaky. The findings are discussed in relation to recent physiological results, suggesting that in comparison with 'dry' mammalian lungs, the turtle lung is a rather wet lung based on its much larger transcapillary fluid filtration into the interstitium. In addition, small maculae communicantes are demonstrated between the pneumocytes; they possibly serve for metabolic coupling.

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Immunocytochemical study of the hypothalamic magnocellular neurosecretory nuclei of the snake Natrix maura and the turtle Mauremys caspica.

An immunocytochemical study of the magnocellular neurosecretory nuclei was performed in the snake Natrix maura and the turtle Mauremys caspica by use of antisera against: (1) a mixture of both bovine neurophysins, (2) bovine oxytocin-neurophysin, (3) arginine vasotocin, and (4) mesotocin. Arginine vasotocin- and mesotocin-immunoreactivities were localized in individual neurons of the supraoptic and paraventricular nuclei, with a distinct pattern of distribution in both species. The same cells appeared to be stained by the anti-oxytocin-neurophysin and antimesotocin sera. The supraoptic nucleus can be subdivided into rostral medial and caudal portions. In N. maura, but not in M. caspica, neurophysin-immunoreactive neurons were found in the retrochiasmatic nucleus. No immunoreactive elements were seen in the suprachiasmatic nucleus of both species after the use of any of the antisera. A dorsolateral aggregation of neurophysin-containing cells, localized over the lateral forebrain bundle, was present in both species. Magnocellular and parvocellular neurophysin-immunoreactive neurons were present in the paraventricular nucleus of both species. In the turtle, the paraventricular neurons were arranged into four distinct layers parallel to the ependyma; these neurons were bipolar with the major axis perpendicular to the ventricle, and many of them projected processes toward the cerebrospinal-fluid compartment. In N. maura a group of large neurons of the paraventricular nucleus was found in a very lateral position. The posterior lobe of the hypophysis and the external zone of the median eminence contained arginine vasotocin- and mesotocin-immunoreactive nerve fibers. The lamina termialis of both species was supplied with a dense bundle of fibers containing immunoreactive neurophysin. Neurophysin-immunoreactive fibers were also present in the septum, some telencephalic regions, including the cortex and the olfactory tubercule, in the paraventricular organ, and the periventricular and periaqueductal gray of the brainstem.

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Ultrastructural changes in the thymus of the turtle Mauremys caspica in relation to the seasonal cycle.

Changes in the ultrastructure of the thymus of the turtle Mauremys caspica, with special reference to its non-lymphoid components, were studied in relation to the seasonal cycle. The thymic cortex contains framework-forming epithelial-reticular cells and free macrophages, while the medulla includes, in addition, mature and presumptive pro-interdigitating cells. The ultrastructural features of these cells are generally similar to those described for non-lymphoid components of the mammalian thymus. The turtle thymus undergoes cortical involution in spring, with recovery periods in May-June and during autumn. A moderate involution occurs in winter. At the beginning of spring, cortical (but not medullary) epithelial-reticular cells show degenerative changes, probably related to high levels of circulating testosterone. In spring and autumn, mature interdigitating cells are absent, but macrophages, monocytes, and pro-interdigitating cells are found. During May-June, the cortical epithelial-reticular population recovers and macrophages, monocytes, and interdigitating cells are actively phagocytic. In summer, the epithelial-reticular cells in both cortex and medulla display normal ultrastructural features; mature and immature interdigitating cells are absent and some macrophages are detected occasionally. The results suggest that non-lymphoid components of the reptilian thymus can play a role in governing T-lymphocyte differentiation, and that the thymic cortex and medulla exhibit different cycles of seasonal activity.

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Inhibitory effects of excitatory amino acids on pyramidal cells of the in vitro turtle medial cortex.

The electroencephalogram of the in vitro brain of the turtle Chrysemys d' orbigny shows spontaneous random large sharp waves (LSWs) which may be compared to interictal spikes. In order to evaluate the role of excitatory amino acids (EAAs)--in particular through the N-methyl-D-aspartate (NMDA) receptor--in the generation of LSWs, the bath application of NMDA and its antagonists 3-((+/-)-2-carboxypiperazin-4y)-propyl-1-phosphonic acid (CPP) and DL-2-amino-5-phosphonovaleric acid (APV), was performed in the whole open hemisphere (WOH) in vitro. Field recordings in WOH showed that both CPP and APV unexpectedly increased LSW amplitude. Consistently, NMDA in the bath suppressed the LSWs. Iontophoretically applied glutamate, kainate and NMDA produced a hyperpolarization of intracellularly recorded medial cortex pyramidal cells both in WOH and in slices. The EAA-induced hyperpolarization was tetrodotoxin (TTX) and bicuculline sensitive and reversed close to -70 mV. It would therefore seem to be due to the activation of gamma-aminobutyric acid (GABA) interneurons. The NMDA could also produce an excitation of pyramidal cells--always following a previous inhibitory phase. In some cases rhythmic bursting discharges or plateau potentials were observed. These NMDA effects were mainly elicited by a direct effect on pyramidal cells. A long-lasting hyperpolarizing response following the NMDA excitatory phase was also observed. This long-lasting response was an intrinsic property of pyramidal cells since it was TTX resistant. This study demonstrates that GABAergic interneurons from the turtle medial cortex can be activated by EAAs, a mechanism that can account for the effects of NMDA antagonists on LSWs.

2-Amino-5-phosphonovalerate↗

Spinocerebellar projections in the turtle. Observations on their origin and terminal organization.

Spinocerebellar projections were studied in the turtle, Pseudemys scripta elegans, by both anterograde (injections of 35S-methionine into various spinal segments) and retrograde (injections of horseradish peroxidase into the cerebellar cortex) tracing techniques. After unilateral HRP injections, labeled neurons were found on both sides of the spinal cord. The total number of retrogradely labeled spinocerebellar tract neurons was relatively small and neuronal aggregations were not observed. Most spinocerebellar tract neurons were found in the ventral horn, dorsal to the motoneuronal cell group. Some cells lay in the spinal intermediate zone and in the dorsal horn. The fibers mainly ascended in the lateral funiculus, some in the ventral and possibly some in the dorsal funiculi. Spinal projections terminated within the cerebellar granular layer. The spinocerebellar target area extended along the whole medio-lateral extent and covered about the rostral four fifths of the cerebellar plate. Individual target areas, separated from each other, as are the anterior and posterior spinocerebellar target areas of higher vertebrates, could not be identified. Similar to mammals, however, the terminal field in the turtle was not a homogenous one but consisted of zones of mossy fiber terminations of varying sizes and intensities.

Afferent Pathways↗

Somatosensory and movement-related properties of red nucleus: a single unit study in the turtle.

Extracellular recordings were performed from turtle red nucleus neurons to examine their responsiveness to peripheral somatic stimulation and to study differences between rubral sensory and movement-related responses. In pentobarbital sodium-anesthetized or decerebrate turtles, red nucleus neurons could be divided into two categories based on their response characteristics. The first group, which included 87% of neurons studied, had low spontaneous rates of activity and responded with excitation to electrical stimulation of the spinal cord or the cerebellum, or during active movement of the contralateral limbs. Neurons in this category were likely to be rubrospinal cells. The remaining 13% of cells studied had higher rates of spontaneous discharge and were inhibited by electrical stimulation or during active movement. These cells might be rubral GABAergic interneurons. Single red nucleus neurons responded with excitation and/or inhibition to somatosensory stimulation. Unlike the motor fields, which were restricted to a single contralateral limb, red nucleus sensory receptive fields were wide and often bilaterally distributed. Rubral responsiveness to sensory stimulation was found to be significantly diminished during active limb movements, thereby suggesting that sensory inputs to the red nucleus are not used for the on-line modification of motor commands. Inactivation of the cerebellar cortex enhanced the sensory responsiveness of rubral neurons and expanded the size of red nucleus receptive fields. These results suggest that the red nucleus receives substantial sensory input, and that the cerebellar cortex can modify the flow of sensory information to the red nucleus.

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Chromosome banding pattern conservatism in birds and nonhomology of chromosome banding patterns between birds, turtles, snakes and amphibians.

The G-banded karyotypes of 4 species of birds representing the orders Galliformes, Columbiformes and Musophagiformes were compared. Banding pattern homology between orders was limited t 5o 5 major chromosome arms and the Z chromosome. Even in these major chromosome arms pericentric and paracentric inversions produced alteration of the banding pattern sequences. Addition of constitutive heterochromatin was responsible for changes in banding patterns in the Z chromosome. The chromosome banding patterns of an emydid turtle, Terrepene carolina, 5 species of boid snakes of the genera Liasis, Acrantophis, and Sanzinia and the African clawed-frog. Xenopus muelleri, were also compared to the bird chromosome banding patterns. No homology was observed between any of these major groups: bird, snake, turtle, amphibian. However, intergroup homology was apparent. - The data obtained do not support reports of broad interordinal direct homology of the macrochromosomes of birds and refutes the idea of a primitive bird karyotype with 3 pairs of "Agroup' chromosomes and 3 pairs of "B group' chromosomes. - The major mechanisms responsible for chromosome evolution in birds appear to be centric and tandem fusions, paracentric and pericentric inversions, and addition or deletion of heterochromatin.

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Immunohistochemical demonstration of serotonin neurons in the central nervous system of the turtle (Clemmys japonica).

The location of the somata, course of the main tracts, and fiber distribution of the serotonin neurons in the turtle brain were studied using the peroxidase-antiperoxidase (PAP) immunohistochemical method with antibodies against serotonin (5-hydroxytryptamine). The somata of the serotonin neurons were distributed in the reticular formation of the brainstem from the mesencephalon to the lower medulla level and in a resticted region of the hypothalamus, viz. the paraventricular organ (PVO). In the PVO the serotonin neurons were seen to have the appearance of cerebrospinal fluid-contacting neurons. Analysis of serial sections cut in the frontal and sagittal planes revealed a widespread distribution of the serotonin immunoreactive fibers in the turtle brain. Prominent concentrations of the serotonin immunoreactive fibers were found in the lateral portion of the striatum, the ventral portion of the septum, the nucleus corporis geniculati lateralis, the nucleus pretectalis, the nucleus isthmi parvocellularis, the optic tectum, and the lateral edge of the reticular formation of the brainstem. Ascending and descending serotonin pathways could be defined: the ascending pathway originated mainly from the nucleus profundus mesencephali caudalis, nucleus lemnisci lateralis, nucleus reticularis isthmi and, less prominently, from the nucleus raphe superior pars lateralis, and the descending pathway arose predominantly from the nucleus raphe inferior. The fibers of the ascending pathway projected widely in the prosencephalon and mesencephalon, via the medial forebrain bundle. The descending pathway ran through the ventral and lateral portion of the medulla oblongata and spinal cord.

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Morphology of geniculocortical axons in turtles of the genera Pseudemys and Chrysemys.

An analysis has been made of the morphology of axons in the geniculocortical pathway of turtles using the anterograde transport of horseradish peroxidase in both in vivo and in vitro preparations. Following injections of HRP into the dorsolateral thalamus, labeled axons could be traced from the dorsal lateral geniculate complex to the telencephalon. They are unbranched and free of varicosities within the diencephalon. They travel in the dorsal peduncle of the lateral forebrain bundle, through the basal telencephalon and dorsally into the pallial thickening. Many axons are situated deep in the pallial thickening and bear numerous varicosities that often appear apposed to the proximal dendrites or somata of neurons retrogradely labeled by thalamic injections of horseradish peroxidase. Individual axons continue from the pallial thickening into the dorsal cortex where they shift dorsally and bear varicosities as they course from lateral to medial in the superficial third of layer 1. These data indicate that the terminal zone of the dorsal lateral geniculate complex within the telencephalon of turtles is more extensive in the mediolateral direction than previously believed. Geniculate axons bear varicosities both within the pallial thickening as well as the dorsal cortex, but have different relationships to potential postsynaptic elements in the two areas. Geniculocortical axons overlie somata and proximal dendrites of neurons in the pallial thickening, but intersect the distal dendrites of neurons in the dorsal cortex.

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Morphology of primary afferents to the spinal cord of the turtle Pseudemys scripta elegans.

The morphology of primary afferents to the spinal cord of the turtle Pseudemys scripta elegans was studied by means of intra-axonal injections of horseradish peroxidase. A total of 74 collaterals arising from 34 different afferents in 22 animals was investigated. Within this sample, a division into three morphologically distinct collateral types appeared possible. Collaterals of the same parent axon could always be classified to the same type. Type A collateral arborizations could be found within area I-II and III of the spinal grey matter. The number of presynaptic boutons per collateral varied considerably. However, collaterals of the same parent axon usually possessed a similar general appearance. Type B collaterals terminated within area IV and V-VI. The general shape and number of boutons could differ considerably between collaterals of different parent fibers but also between collaterals of the same axon. Type C collaterals formed terminal arborizations in the lateral parts of areas IV, V, VI and VII-VIII and demonstrated a fair constancy in general appearance and number of presynaptic boutons. Type A collaterals are thought to be derived from fibers innervating various cutaneous receptors. Terminal arborizations of type C collaterals are fully overlapping with the dorsal dendritic trees of turtle lumbar motoneurons. It is suggested that type C collaterals form contacts with these motoneurons and arise from muscle spindle innervating afferents. The origin of type B collaterals is less clear, attractive possibilities may be found in joint and/or tendon organs.

Afferent Pathways↗

Immunocytochemical and biochemical studies of histamine in the retina of the turtle Pseudemys scripta.

A combination of immunocytochemical and biochemical methods was used to study histamine in the turtle retina. Histamine-like immunoreactivity was localized within paraboloids of certain cone photoreceptors by use of two different antisera directed against histamine. Preincubation of eyecups in Ringer's containing 10 microM histamine selectively increased the immunoreactivity of these photoreceptor paraboloids. The present localization of histamine in paraboloids indicated that, although histamine is in photoreceptors of the turtle retina, it may play some metabolic or neuromodulatory role, and not function as a neurotransmitter.

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Dynamics of the cone-horizontal cell circuit in the turtle retina.

The model of the catfish retina (Siminoff, in press) has been extended to the turtle retina with incorporation of color-coding. The turtle retina contains 6 types of cones of which 4 are red-sensitive and the other 2 are green- and blue-sensitive, respectively. The cone-horizontal circuit incorporates negative feedback from the L-HC to all the cones having input to the L-HC. By use of systems analysis, Laplace transforms and the convolution theorem, impulse responses, that give information as to gain and phase, for the cone-types and L-HC were simulated. As with the catfish retina, negative feedback gain was proportional to the dc level of the L-HC and therefore, the mean illuminance level. It was shown that this mechanism can be an important factor in chromatic adaptation, since the gains of the various cone-types are preferentially altered dependent on mean illuminance level and wavelength of the background light.

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Synaptic organization of the outer plexiform layer of the turtle retina: an electron microscope study of serial sections.

Neural connections in the outer plexiform layer of the Pseudemys turtle retina have been studied by electron microscopy of serial ultrathin sections. While the distinguishing features of the photoreceptors have been described elsewhere, in this paper we describe the patterns of connectivity between identified second order neurons and identified photoreceptors or amongst second order neurons themselves. Basal telodendria emitted from double cone pedicles interconnect the two members of the double cone. Three morphologically different types of junction are made between bipolar cells and cone pedicles. H1 horizontal cells can be distinguished from H2 horizontal cells and synapses occur between them. Axon terminals of H1 cells are presynaptic to H1 cell bodies. Photoreceptors, H1 cell bodies and H1 axon terminals engage in electrical junctions while chemical synapses occur from both types of horizontal cell to bipolar cells. On rare occasions, bipolar cell dendrites were seen to be presynaptic to other bipolar cell dendrites. The significance of some of these contacts for the electrophysiological findings on the OPL of the turtle retina is discussed.

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Synaptic analysis of amacrine cells in the turtle retina which contain tyrosine hydroxylase-like immunoreactivity.

This study examined amacrine cells in the retina of the turtle Pseudemys scripta elegans, which were labelled using an antiserum directed against tyrosine hydroxylase (an enzyme participating in catecholamine synthesis). These cells were investigated using both light and electron microscopy. Labelled somata were located in the inner nuclear layer near the border of the inner plexiform layer. The dendritic arborizations of these neurons were tristratified and arborized in strata 1 and 3 and near the border between strata 4 and 5. Serial tangential sections taken through the entire inner plexiform layer of a 1 mm-2 region in mid-peripheral retina were examined. All of the synapses associated with labelled profiles were counted and classified. The majority (84%) of the synapses involving labelled processes represented output, while the remaining 16% represented synaptic input. The synaptic output of the labelled processes was as follows: 87% onto unlabelled amacrine cells, 4% onto ganglion cells, 9% onto unidentified cell processes. None of the synaptic output from labelled processes was onto bipolar cells. The synaptic input to these labelled cells was from bipolar cells (29%) and from unlabelled amacrine cells (71%). A well labelled amacrine cell was serially sectioned and examined at the ultrastructural level to analyze its synaptic connectivity. Immunoreaction product was located diffusely throughout the cytoplasm and in large vesicles. The synaptic organization of the cell was directed primarily toward output. The labelled processes were postsynaptic and presynaptic to unlabelled amacrine cell processes in strata 1 and 3 and at the border between strata 4 and 5. Synaptic input from bipolar cells was seen exclusively near the border between strata 4 and 5. Labelled processes were presynaptic to ganglion cell processes in stratum 1 and at the border between strata 4 and 5, but not in stratum 3. Quantitative studies suggested that amacrine cell inputs and outputs were evenly distributed across the dendritic arborization, while bipolar cell inputs and outputs to ganglion cells were concentrated on the distal parts of the dendritic arborization. No labelled processes were seen in the outer plexiform layer, indicating that the cells with tyrosine hydroxylase-like immunoreactivity in the turtle retina were true amacrine cells and not interplexiform cells.

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