Distribution of somatostatin immunoreactivity in the brain of the snake Bothrops jararaca.

The distribution of perikarya and fibers containing somatostatin was studied in the brain of the snake Bothrops jararaca by means of immunohistochemistry using an antiserum against synthetic somatostatin. Immunoreactive perikarya and fibers were localized in telencephalic, diencephalic and mesencephalic areas. In the telencephalon, numerous immunoreactive perikarya were found in the medial, dorsomedial, dorsal and lateral cortex, mainly in the deep plexiform layer, less so in the cellular layer, but not in the superficial plexiform layer. Immunoreactive perikarya were also observed in the dorsal ventricular ridge, the nucleus of the diagonal band of Broca, amygdaloid complex, septum and lamina terminalis. In the diencephalon, labelled cells were observed in the paraventricular, periventricular hypothalamic and in the recessus infundibular nuclei. In the mesencephalon, immunoreactive perikarya were seen in the mesencephalic reticular formation, reticular nucleus of the isthmus and torus semicircularis. Labelled fibers ran along the diencephalic floor and the inner zone of the median eminence, and ended in the neural lobe of the hypophysis. Other fibers were observed in the outer zone of the median eminence close to the portal vessels and in the septum, lamina terminalis, retrochiasmatic nucleus, deep layers of the tectum, periventricular gray and granular layer of the cerebellum. Our data suggest that somatostatin may function as a mediator of adenohypophysial secretion as well as neurotransmitter and/or neuromodulator which can regulate the neurohypophysial peptides in the snake B. jararaca.

Reproductive cycle of the Neotropical Crotalus durissus terrificus: I. Seasonal levels and interplay between steroid hormones and vasotocinase.

Crotaline snakes present delayed fertilization and sperm storage because secondary vitellogenesis is not completed by the time of mating. The release of vitellogenesis and synchrony between ovulation and fertilization suggest a steroidal modulation. We investigated changes of sexual steroid levels during reproduction in the Neotropical rattlesnake Crotalus durissus terrificus, analyzing macroscopical variations of reproductive condition (vitellogenesis, pregnancy, and post-partum) and plasma levels of estradiol, progesterone, and vasotocinase cystine aminopeptidase (CAP) activity over 2 years. Data showed 44.4% non-reproductive snakes (40.1% primary vitellogenesis and 4.3% post-partum) and 55.6% reproductive (36.8% secondary vitellogenesis and 18.8% pregnant). Estradiol was low in spring and summer, increasing in autumn till it peaked in winter. Estradiol in secondary vitellogenesis was significantly higher than in primary vitellogenesis, or in pregnant and post-partum females, Progesterone dropped significantly in autumn compared to summer, winter, and spring. Pregnant females showed the highest levels of progesterone compared to primary or secondary vitellogenesis, or post-partum females. CAP activity showed lowest values in reproductive females in autumn and greatest levels in post-partum females. A significant negative linear relationship was obtained between CAP activity and estradiol. The combination of morphological observations, levels of steroids and CAP activity allowed us to suggest a similar morphological reproductive pattern between temperate and tropical rattlesnakes, and to infer the role of estradiol, progesterone and CAP activity on vitellogenesis, gestation and sperm storage, respectively.

Reproductive cycle of the Neotropical Crotalus durissus terrificus: II. Establishment and maintenance of the uterine muscular twisting, a strategy for long-term sperm storage.

Crotaline snakes store sperm by means of a uterine musculature twisting (UMT). We investigated the influence of plasma levels of estradiol and progesterone and vasotocinase cystine aminopeptidase (CAP) activity on UMT formation and maintenance, and the in vitro uterine reactivity for AVT in Crotalus durissus terrificus in primary or secondary vitellogenesis with or without UMT. Frequency of females in secondary vitellogenesis with UMT is significantly higher than in primary one. Estradiol levels did not vary in all conditions studied, however, significantly low levels of progesterone were found in snakes in secondary vitellogenesis with UMT compared to those without it. UMT is always observed when high levels of estradiol and low levels of progesterone are detected. CAP activity did not change in the presence of UMT. AVT produced concentration-response contractions of the isolated uterus of snakes in all stages analysed and the pD2 value and maximum contractile response were significantly higher in primary vitellogenesis without UMT than in other reproductive conditions, indicating that uterus of those snakes presents a higher contractile capacity which may favour UMT establishment. In conclusion, we show a relationship of UMT and estradiol/progesterone balance and a possible participation of AVT in UMT formation and maintenance in the Neotropical rattlesnake.

The distribution of vasotocin and mesotocin immunoreactivity in the brain of the snake, Bothrops jararaca.

Polyclonal antibodies against vasotocin (AVT) and mesotocin (MST) were used to explore the distribution of these peptides in the brain of the snake Bothrops jararaca. Magnocellular AVT- and MST-immunoreactive (ir) perikarya were observed in the supraoptic nucleus (SON), being AVT-ir neurons more numerous. A portion of the SON, in the lateroventral margin of the diencephalon ventrally to optic tract, showed only AVT-ir perikarya and fibers. However, the caudal most portion displayed only mesotocinergic perikarya. Parvocellular and magnocellular AVT- and MST-ir perikarya were present in the paraventricular nucleus (PVN) being AVT-ir fibers more abundant than MST-ir. Vasotocinergic perikarya were also found in a dorsolateral aggregation (DLA) far from the PVN. Mesotocinergic perikarya were also present in the recessus infundibular nucleus and ependyma near to paraventricular organ. Nerve fibers emerging from supraoptic and paraventricular nuclei run along the diencephalic floor, internal zone of the median eminence (ME) to end in the neural lobe. Also a dense network of AVT- and MST-ir fibers was present in the external zone of the ME, close to the vessels of the hypophysial portal system. As a rule, all regions having vasotocinergic and mesotocinergic perikarya also showed immunoreactive fibers. Vasotocinergic and mesotocinergic fibers but not perikarya were found in the lamina terminalis (LT). Moreover AVT-ir fibers were present in the nucleus accumbens and MST-ir fibers in the septum. In mesencephalon and rhombencephalon MST-ir fibers were more numerous than AVT-ir fibers. Vasotocinergic and mesotocinergic fibers in extrahypothalamic areas suggest that these peptides could function as neurotransmitters and/or neuromodulators in the snake B. jararaca.

Angiotensin receptor in the heart of Bothrops jararaca snake.

Angiotensin II interacts with specific cell surface angiotensin AT1 and AT2 receptors and, in some vertebrates, with an atypical angiotensin AT receptor. This study was designed to characterize the angiotensin receptor in the heart of Bothrops jararaca snake. A specific and saturable angiotensin II binding site was detected in cardiac membranes and yielded Kd=7.34+/-1.41 nM and B(max)=72.49+/-18 fmol/mg protein. Competition-binding studies showed an angiotensin receptor with low affinity to both angiotensin receptor antagonists, losartan (2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]imidazole) and PD123319 ((s)-1-(4-[dimethylamino]-3-methylphenyl)methyl-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylate). Studies on the intracellular signaling pathways showed that phospholipase C/inositol phosphate breakdown and adenylylcyclase/cyclic AMP generation were not coupled with this angiotensin receptor. An adenylylcyclase enzyme sensitive to forskolin was detected. The results indicate the presence of an angiotensin receptor in the heart of B. jararaca snake pharmacologically distinct from angiotensin AT1 and AT2 receptors. It seems to belong to a new class of angiotensin receptors, like some other atypical angiotensin AT receptors that have already been described.

Corticotropin-releasing hormone-like immunoreactivity in the brain of the snake Bothrops jararaca.

The distribution of corticotropin-releasing hormone in the brain of the snake Bothrops jararaca was studied immunohistochemically. Immunoreactive neurons were detected in telencephalic, diencephalic and mesencephalic areas such as dorsal cortex, subfornical organ, paraventricular nucleus, recessus infundibular nucleus, nucleus of the oculomotor nerve and nucleus of the trigeminal nerve. Immunoreactive fibres ran along the hypothalamo-hypophysial tract to end in the outer layer of the median eminence and the neural lobe of the hypophysis. In general, immunoreactive fibres occurred in the same places of immunoreactive neurons. In addition, immunoreactive fibres were observed in the septum, amygdala, lamina terminalis, supraoptic nucleus, nucleus of the paraventricular organ, ventromedial hypothalamic nucleus and interpeduncular nucleus. These results indicate that, as for other vertebrates, corticotropin-releasing hormone in B. jararaca brain, besides being a releasing hormone, may also act as a central neurotransmitter and/or neuromodulator.

The vasopressor action of angiotensin in the snake Bothrops jararaca.

1. Carotid blood pressure from anesthetized B. jararaca snakes was recorded in order to study angiotensin action in this reptile. 2. Whereas [Asn1,Val5] AII and AIII were less potent than [Asp1,Ile5] AII and [Asp1,Val5] AII, [Sar1,Ile5] AII was slightly more potent. 3. Captopril abolished the responses to AI (0.01-3 micrograms/kg). 4. [Sar1,Ala8] AII was uneffective but [Sar1,Leu8] AII or phenoxybenzamine were able to reduce AII vasopressor responses. 5. These results led to the conclusion that the vasopressor response of AII in B. jararaca is due to an interaction with its own receptor but, part of the AII receptor population seems to be coupled to the sympatho-adrenal system. Moreover, structural requirements seem to be necessary for the AII response in B. jararaca.