Calcium transients in the garter snake vomeronasal organ

The signaling cascade involved in chemosensory transduction in the VN organ is incompletely understood. In snakes, the response to nonvolatile prey chemicals is mediated by the vomeronasal (VN) system. Using optical techniques and fluorescent Ca(2+) indicators, we found that prey-derived chemoattractants produce initially a transient cytosolic accumulation of [Ca(2+)](i) in the dendritic regions of VN neurons via two pathways: Ca(2+) release from IP(3)-sensitive intracellular stores and, to a lesser extent, Ca(2+) influx through the plasma membrane. Both components seem to be dependent on IP(3) production. Chemoattractants evoke a short-latency Ca(2+) elevation even in the absence of extracellular Ca(2+), suggesting that in snake VN neurons, Ca(2+) release from intracellular stores is independent of a preceding Ca(2+) influx, and both components are activated in parallel during early stages of chemosensory transduction. Once the response develops in apical dendritic segments, other mechanisms can also contribute to the amplification and modulation of these chemoattractant-mediated cytosolic Ca(2+) transients. In regions close to the cell bodies of the VN neurons, the activation of voltage-sensitive Ca(2+) channels and a Ca(2+)-induced Ca(2+) release from intracellular ryanodine-sensitive stores secondarily boost initial cytosolic Ca(2+) elevations increasing their magnitude and durations. Return of intracellular Ca(2+) to prestimulation levels appears to involve a Ca(2+) extrusion mediated by a Na(+)/Ca(2+) exchanger mechanism that probably plays an important role in limiting the magnitude and duration of the stimulation-induced Ca(2+) transients.     

Immune response in the garter snake (Thamnophis ordinoides).

Garter snakes (Thamnophis ordinoides) were immunized with hen egg albumin, human gamma-globulin and Keyhole limpet haemocyanin in Freund's adjuvant. Antibody was consistently detected by radioimmunoelectrophoresis and in three different gamma- and beta-globulin precipitin lines called IgM (approximately or equal to 20S), Ig-1 (approximately or equal to 9S) and Ig-2 (approximately or equal to 8-5S). Early antibody (day 31 after immunization) was frequently Ig-M whereas Ig-2 and especially Ig-1 were detectable for the longest duration (992 days). After immunization with antigen in Freund's adjuvant, Ig-1 serum concentration showed the greatest increase, from almost undetectable levels to the most prominent immunoglobulin in immune serum.     

Ultrastructure of the tubular nephron of the garter snake Thamnophis sirtalis

Ultrastructural features of the tubular nephron of the garter snake, with special reference to modifications for conservation of water, were studied using transmission electron microscopy, freeze-fracture and tracer experiments. Although a nephric loop (loop of Henle) is lacking, the tubules appear to be structurally well adapted for efficient ion and water reabsorption. The most prominent features are well developed microvilli in the proximal tubule and elaborate lateral folds, particularly in the distal tubule and collecting ducts. The latter structures are highly interdigitated, creating complex intercellular channels, perhaps facilitating transepithelial fluid transport. Only the proximal tubule actively absorbs and degrades protein tracers from the lumen. The cells of the collecting duct secrete mucus which may precipitate and bind urate salts in the lumen. This may be significant in the excretion of these salts, a process which combines maximal removal of salts and nitrogenous wastes with minimal loss of water.     

Ultrastructure of the renal corpuscle of the garter snake Thamnophis sirtalis

The renal corpuscle of the garter snake was examined using scanning and transmission electron microscopy, freeze-fracture and tracer experiments. The renal corpuscle was small and glomerular architecture simple. The visceral epithelium was composed of irregular, highly branched podocytes with broad, interdigitating foot processes. Numerous cell junctions, including fasciae occludentes and adhaerentes, occurred between podocytes. The podocytes bore numerous microvilli which projected into the urinary space and contained large bundles of microfilaments. The podocytes lay on a basal lamina which appeared to act as a primary filtration barrier.     

The effect of shedding on skin homografts in the garter snake,thamnophis sirtalis

All skin homografts shed a layer of keratin or an outer epidermal generation when shedding appeared in the recipient. Skin homografts did not express the timing of the shedding cycle found in the donor animal. It was concluded that the timing of shedding is determined by the recipient but not by the skin. The response of skin homografts was not affected by age, sex or the stage between sheddings when grafts were taken from donors. A review of mammalian work shows that skin homografts in the rat show the timing of the hair growth cycle found in the donor although eventually these homografts also become synchronized with the recipient. Less complex mechanisms seem to be involved in the shedding cycle in garter snakes than in the hair-growth cycle which evolved in mammals.     

Shedding facilitates exposure learning in the garter snake (Thamnophis sirtalis)

Young garter snakes were exposed to a specific chemical odor for five consecutive days. Following training, the snakes were individually tested for their preferences for the training odor vs. an unfamiliar odor. Snakes that were trained and tested prior to — or following — their initial shedding displayed no selective responsiveness towards the odors. However, nine snakes which shed for the first time during the training period spent significantly more time near the training odor as compared to the novel odor during subsequent testing. It was concluded that endocrine changes which initiate and accompany shedding may account for the preference for the familiar odor shown by the snakes which shed during training. The possible adaptive value of such exposure learning was also discussed.     

Histochemical and electron microscopic observations on the sperm receptacles in the garter snake oviduct

The sperm storage receptacles in the oviduct of the garter snake, Thamnophis sirtalis, were studied using histological, histochemical, and electron microscopic techniques. The receptacles are located in the posterior infundibulum and are readily distinguished from other structures within the oviduct. The luminal epithelium consists of secretory cells and ciliated cells. The latter cell type also lines the ducts leading to the receptacles. The distribution of lipids, carbohydrates, and several enzymes (succinic dehydrogenase, acid and alkaline phosphatases, nonspecific esterase, lipase, sorbitol dehydrogenase) in the infundibulum is described. At the electron microscopic level sperm heads are seen to be closely associated with the surface of receptacle cells, often indenting the luminal plasma membrane. The possibility of a “Sertoli cell-like” relationship is discussed. Formation of cytoplasmic vacuoles in receptacle cells is temporally associated with ovulation and may be part of a mechanism for sperm release from receptacle cells. No contractile elements were observed immediately around the receptacles. Evidence is presented which suggests that free fructose and glucose are produced by the oviduct, possibly to be utilized for sperm metabolism at the time of ovulation and sperm release.     

Immunocytochemical localization of four hormones in the pancreas of the garter snake, Thamnophis sirtalis

Insulin, glucagon, somatostatin, and pancreatic polypeptide (PP) were localized in the pancreas of the common garter snake, Thamnophis sirtalis, by light and transmission electron microscopic (TEM) immunocytochemistry. Colloidal gold-protein A was used for TEM localization and the peroxidase--antiperoxidase complex technique was used for light microscopy. The glucagon-containing A cells and the insulin-positive B cells were the most numerous cell types. The somatostatin-containing D cells made up about 15% of the endocrine cells. PP-positive F cells were a minor cell type. The only topographic arrangement of the cells within the endocrine-rich areas that was apparent was the peripheral localization of the D and F cells. Cells of a specific cell type were sometimes grouped together. At the electron microscopic (EM) level, the gold particles (indicating the presence of hormone) were localized nearly exclusively over the secretory granules of the reactive cells. The alpha-granules were the largest found and were predominantly electron dense with a moderately electron-dense periphery. PP-containing granules were the smallest. The somatostatin-reactive delta-granules were round and moderately electron opaque. The beta-granules were heterogeneous in appearance. The morphognomy of the secretory granules of the major endocrine cell types is qualitatively similar to that of mammals. Whether or not the quantitative and/or associative differences contribute to the marked metabolic differences between reptiles and mammals, remains to be determined.     

Behavioral and hormonal responses to corticosterone in the male red-sided garter snake, Thamnophis sirtalis parietalis

Stress and glucocorticoids are generally thought to suppress reproductive function at multiple levels. We tested the hypotheses that exogenous corticosterone would suppress sexual behavior in a dose-dependent manner, as well as drive a decrease in plasma testosterone levels in the male red-sided garter snake, Thamnophis sirtalis parietalis. We examined this by challenging individual males with intraperitoneal injections of exogenous corticosterone, and subsequently exposing them to sexually attractive females or taking a blood sample. Previous work has demonstrated a hormonal but no behavioral response to stress in this species. In this study, increasing concentrations of exogenous corticosterone rapidly suppressed mating behavior in a threshold manner. However, exogenous corticosterone had no effect on plasma levels of testosterone. Thus, these data suggest that the mechanism is in place for corticosterone to suppress mating behavior in this species and that these effects do not occur because of an indirect effect on plasma levels of testosterone but rather are the direct effect of the hormone itself. In addition, the negative relationship observed previously between plasma levels of corticosterone and testosterone in this species was probably not the direct result of corticosterone acting on the hypothalamic-pituitary-gonadal (HPG) axis. Rather, our results seem to indicate that the negative associations between the hypothalamic-pituitary-adrenal axis (HPA) and the HPG axis occur at other levels of these neuroendocrine pathways.     

Ascending projections of the dorsal column in a garter snake (thamnophis siritalis): A degeneration study

Ascending axons in the dorsal column of garter snakes were examined following hemisection of the spinal cord at segment levels 2, 3, 4, 11, 13, and 31. After postoperative survival periods of 11 to 28 days, sections of the spinal cord and brain were processed with a silver method to demonstrate degenerated axons and preterminals. The study demonstrated that most ascending degenerated axons are located in the outer half of the dorsal column. The somatotopic pattern of ascending fibers is evident, whereby dorsomedial fibers are primarily of caudal origin and the more dorsolateral axons are from rostral cord segments. Rostral to segment 31, all spinal segments appear to project to a strip of dorsal column adjacent to the dorsal median septum. From the septum, axons descend to terminate somatotopically on cells of the nucleus of Bischoff located caudal to the obex of the medulla. Dorsal column degeneration ascends to the level of the dorsal column nuclei, where most fibers terminate. Degeneration from caudal cord segments terminates on caudo-medial cells of the dorsal column nuclei, while rostral cord segments project to rostro-lateral cells. The dorsal column nuclei consist of an expanded lateral part between tractus descendens trigemini and the vago-solitary complex, and a contiguous, thin medial lamina of cells dorsal and medial to the vagal nuclei. The somatotopic pattern of degeneration in the dorsal column nuclei, probably of dorsal root origin, follows the mammalian organization, which suggests that the garter snake has primitive nuclei gracilis and cuneatus. Other terminal sites of degenerating fibers, although probably of spinal gray origin, are nucleus commissura infima, nucleus descendens vestibuli, and area postrema.     

The muscle-tendon junctions of fast and slow fibres in the garter snake: ultrastructural and stereological analysis and comparison with other species

Summary The muscle-tendon junctions of fast and slow muscle fibres from the costocutaneous muscles of garter snakes have been analyzed by scanning and transmission electron microscopy and by a stereological protocol. The junctions of the two fibre types are morphologically similar, and consist of anastamosing processes which increase the surface area of the muscle-tendon interface and significantly reduce the stress at the junction when the fibres contract. The muscle-tendon junctions of these fibres are analogous to adhesive joints between stiff adherends and fibre-reinforced composite materials. Stereological estimates of the area of surface membrane devoted to force transmission, relative to the cross-sectional area of myofilaments (the interfacial ratio), have produced values of about 14 for the fast fibres and about 16 for the slow fibres. This difference is small but statistically significant. Interfacial ratios of about 14 and 10 were previously determined for fast and slow fibres, respectively, of both chickens and mice. Thus the slower fibres of the snake differ from those of the mouse and chicken, in that their interfacial ratio is greater than that of fast fibres.     

The immunological synapse

The induction of a proper adaptive immune response is dependent on the correct transfer of information between antigen-presenting cells (APCs) and antigen-specific T cells. Defects in information transfer may result in the development of diseases, e.g. immunodeficiencies and autoimmunity. A distinct 3-dimensional supramolecular structure at the T cell/APC interface has been suggested to be involved in the information transfer. Due to its functional analogy to the neuronal synapse, the structure has been termed the "immunological synapse" (IS). Here, we review molecular aspects concerning IS formation, appearance, and cessation. In addition, proposed functions of the IS are discussed. The process of IS formation occurs in a sequential manner, initially causing a remarkable large-scale redistribution of a number of integral membrane and cytosolic proteins. At the T cell/APC interface the structure comprises in its nascent stage a non-random pattern of protein distribution. The protein pattern is regulated during development of the mature IS and is finally organized into concentric rings of co-receptors and adhesive molecules surrounding the T cell antigen receptor (TCR). The relocations of proteins are influenced by passive as well as active mechanisms. Considering the IS as a device enabling cell-cell communication, clarification of its exact function is of huge general as well as therapeutic interest.     

The immunological synapse

The immunological synapse plays a central role in organising the immune system. Through their synaptic activity both T and B cells usually, but not always, acquire the information that critically determines the level and nature of the responses that they make. For T cells much of that information comes from epicrine and paracrine cell-cell interactions in the cluster that forms around a dendritic cell. These interactions are being dissected by experiments in which two populations of TCR-transgenic T cells are combined in vivo. Another important aspect of synaptic activity is the way in which different levels of expression of MHC class II molecules influence Th1/Th2 balance. In exploring this form of control we are learning something of general importance about cis-regulation.     

Facilitation of courtship behavior in the male red-sided garter snake (Thamnophis sirtalis parietalis) following lesions of the septum or nucleus sphericus

Courtship behavior in male red-sided garter snakes is limited to the period immediately following spring emergence. Lesions of the septum or nucleus sphericus prior to hibernation facilitate courtship behavior upon emergence the following spring. These lesions also had a significant effect on the reproductive physiology of the male snake. Snakes with lesions in the nucleus sphericus had a significantly higher concentration of circulating androgens compared to the control group. While animals with lesions in the septum exhibited elevated levels of circulating androgens, they were not statistically elevated when compared to the control group. The quantity of sexual granules in the epithelium of the renal sexual segment, a secondary sexual characteristic of male squamate reptiles, was significantly greater in lesioned animals. These results are the first to demonstrate a central inhibitory control of sexual behavior in a snake.     

Effects of early sex steroid hormone treatment on courtship behavior and sexual attractivity in the red-sided garter snake, Thamnophis sirtalis parietalis

Previous research has shown that exogenous androgen fails to elicit courtship (chin-rubbing) behavior in adult male or female red-sided garter snakes (Thamnophis sirtalis parietalis). However, gonadectomized and intact newborn male and female red-sided garter snakes given silastic capsules containing testosterone exhibit chin-rubbing behavior; gonadectomized and untreated animals do not show this behavior. Both males and females also exhibit chin-rubbing behavior when treated with testosterone as yearlings. Hibernation stimulates chin-rubbing behavior only in males that have received androgen treatment as a neonate or as a yearling. Previous research has also shown that adult females, but not adult males, are courted if given estrogen treatment. Both newborn males and females will elicit chin-rubbing behavior from adult males if given estrogen treatment, indicating production and release of an attractiveness pheromone characteristic of adult females. Male red-sided garter snake breed for the first time on emergence from their second hibernation, whereas females probably do not breed until their third year of life. These data suggest that in the red-sided garter snake, a species that exhibits a dissociated reproductive tactic, sex steroid hormones act to organize central nervous system mechanisms subserving courtship behavior such that temperature, and not hormonal, fluctuations activate sexual behavior in the adult organism.     

Temperature activation of courtship behavior in the male red-sided garter snake (Thamnophis sirtalis parietalis): role of the anterior hypothalamus-preoptic area

Adult male red-sided garter snakes (Thamnophis sirtalis parietalis) received radio-frequency lesions prior to 17 weeks of low-temperature hibernation. Animals found to have bilateral lesions of the anterior hypothalamus-preoptic area (AH-POA) failed to exhibit courtship behavior on emergence from hibernation. Those individuals in which the major portion of the destruction was centered in the anterior POA also exhibited deficits in thermoregulatory behavior. Animals that received unilateral lesions of the AH-POA initiated courtship behavior after controls and had an abbreviated period of courtship; these animals exhibited normal responses to thermal stimuli. Male snakes with lesions outside the AH-POA courted normally and demonstrated no differences in thermoregulatory behavior compared with the surgical controls. These results indicate that in male red-sided garter snakes, an intact AH-POA is critical for the integration of thermal stimuli that activate seasonal courtship behavior.     

Differential effects of lesions of the vomeronasal and olfactory nerves on garter snake (Thamnophis sirtalis) responses to airborne chemical stimuli

The roles of the main (MOS) and accessory (AOS) olfactory systems of garter snakes in response to airborne chemicals were investigated. Preoperatively, all snakes responded to airborne odors with increased tongue-flick rate and duration. Postoperatively, sham-operated snakes responded to airborne odors with increased tongue-flick rates, but snakes with main olfactory nerve cuts failed to respond to the odors, and snakes with vomeronasal nerve cuts responded to nonprey odors only. Preoperatively, exposure to earthworm odor produced more frequent and shorter duration tongue-flicks during locomotion compared with exposure to water. Postoperatively, only sham-lesioned snakes exhibited differential responding to earthworm odors. This study demonstrates that the MOS is critical for the initiation of tongue-flick behavior in response to airborne odors and that discrimination of odors with biological significance requires a functional AOS.