Immunoreactive vasotocin in the zebra finch brain (Taeniopygia guttata).

UNLABELLED: The distribution of vasotocin (VT)-immunoreactive cells and fibers in the zebra finch brain was studied with immunocytochemical techniques. A large number of VT-immunoreactive cells was found in the nucleus preopticus anterior and nucleus paraventricularis. In the lateral parts of the hypothalamus diffusely organized VT-immunoreactive cells were present. Some of these cells were found close to and embedded in the optic tract. An extra-hypothalamic parvocellular cell group was found at the level of the anterior commissure in the dorsal diencephalon. VT-immunoreactive fibers and varicosities were present in a number of extrahypothalamic regions i.e. the septum, the area ventralis of Tsai, the lateral habenula, the optic tectum, the substantia grisea centralis, the nucleus tractus solitarii, the lateral medulla, the nucleus intercollicularis and in the archistriatum surrounding the nucleus robustus archistriatalis. The VT-immunostaining in the lateral septum and dorsal diencephalon showed no sex differences and can not be influenced by testosterone administration. IN CONCLUSION: the topography of the central VT-immunoreactive network is similar to that in another song bird i.e. the canary; but seasonal and testosterone-dependent changes in VT-immunostaining in the lateral septum and dorsal diencephalon are only observed in the canary.

Effect of a vasotocin analog on singing behavior in the canary.

Groups of juvenile and 1-year-old male canaries were treated briefly with the vasotocin (VT) analog desGly(NH2)9d(CH2)5-[Tyr(Me)2,Thr4, Orn8]VT (dGVTA) during four time intervals between September and February. The canaries received subcutaneously testosterone-containing silastic implants at the start of the VT analog treatment to assure that despite age and season differences the birds would all have comparable plasma levels of testosterone. The VT analog was administered subcutaneously (0.7 micrograms/100 microliters) during the first 3 days (3 injections daily) of chronic testosterone treatment. Observations on the singing behavior were carried out between Day 8 and Day 30 after implantation of the testosterone-filled silastic tubing. The short-term administration of the VT analog influenced the amount of singing behavior during a 30-min observation interval measured 1 to 4 weeks later. Despite age differences the effect of dGVTA held and seemed more related to season than to age. The song duration (seconds of song/30 min) was affected in a dual mode. In early autumn the VT analog enhanced song duration of testosterone-primed canaries, but the same VT analog decreased song duration in the period November/January. These results suggest that the neuropeptide VT is implicated in control of seasonal changes in singing behavior.

Ontogenetic and seasonal changes in immunoreactive vasotocin in the canary brain.

Using immunocytochemistry the central VT system of the canary was examined during ontogeny and throughout the season. At 4 weeks of age no VT-immunoreactive staining in the bed nucleus of the stria terminalis and lateral septum was present, but VT-containing cells and fibers were clearly visible in the nucleus paraventricularis of the hypothalamus. At 13 weeks of age a weak VT immunoreactivity appeared in the bed nucleus of the stria terminalis and lateral septum. This VT immunostaining increased progressively till at the age of 28 weeks adult levels were reached. In adulthood, cells and fibers in the lateral septum and the bed nucleus of the stria terminalis contained the highest amounts of immunoreactive VT during the breeding season, whereas during the molt, in august, a decreased VT immunostaining was noted. These seasonal changes in VT immunostaining coincided with variations in circulating testosterone level, which is high during the breeding season and low during the molt. In the ventral capsular region surrounding the nucleus robustus archistriatalis a clear VT-immunoreactive fiber pattern was demonstrated at the age of 8 and 13 weeks in both female and male canaries. Although the size of this nucleus varies during the season no seasonal changes in the VT-immunoreactive pattern surrounding the RA were detected. The data suggest that the activity of the VT system slowly increases during development. At adulthood seasonal and gonadal hormone-dependent changes occur in the limbic VT system only. The VT immunostaining associated with the nucleus robustus archistriatalis however, is resistant to seasonal and gonadal influences.

Vasotocin target sites in the capsular region surrounding the nucleus robustus archistriatalis of the canary brain.

Abstract The aim of this study was to characterize neurohypophyseal hormone receptors in the brain of the canary (Serinus canarius) by using autoradiographical and biochemical procedures with a radioiodinated vasotocin analogue, [(125) l]d(CH(2))(5)[Tyr(Me)(2), Thr(4), Orn(8), Tyr-NH(2) (9)]vasotocin ([(125) I]-OTA). This synthetic analogue was used previously to identify a population of oxytocin-like receptors in the rat brain that have high affinity for vasotocin. In vitro autoradiography revealed high affinity binding sites for [(125) I]-OTA in the canary brain in the archistriatum surrounding the nucleus robustus archistriatalis. Scatchard analysis of [(125) I]-OTA binding to a synaptic membrane fraction prepared from the archistriatal region including the nucleus robustus archistriatalis indicated the presence of a single population of binding sites (K(d)= 0.05 nM; B(max)= 4 fmol/mg protein). Displacement studies revealed that the order of potency of certain peptides to inhibit [(125) I]-OTA binding was as follows: vasotocin (K(i)= 0.4 nM) > oxytocin = vasopressin > mesotocin (K(i)= 8.0 nM). The administration of testosterone to female canaries did not affect [(125) I]-OTA labelling in the archistriatum detected by autoradiography and did not influence [(125) I]-OTA binding constants in the membrane binding assay. In conclusion, this study provides morphological and biochemical evidence of a vasotocin-target site in the archistriatum in close association with the central vocal control circuit in the canary brain.

Diurnal variation in vasopressin and oxytocin messenger RNAs in hypothalamic nuclei of the rat.

Diurnal changes in the expression of the vasopressin (VP) and oxytocin (OT) genes in the supraoptic (SON), paraventricular (PVN) and suprachiasmatic nuclei (SCN) of the rat were investigated by dot-blot and in situ hybridization of the VP and OT mRNAs. A significant diurnal variation in VP mRNA level was measured in the SCN, with highest levels around 17.00 h and lowest levels around midnight. No variations in levels of VP mRNA and OT mRNA were detected in SON and PVN. The data indicate that the regulation of the VP gene in the SCN is independent of that in the magnocellular nuclei.

The distribution and plasticity of [3H]vasopressin-labelled specific binding sites in the canary brain.

[3H]Vasopressin was used to detect and to quantitate specific binding sites with in vitro receptor autoradiography in the canary brain. A discrete regional distribution of [3H]vasopressin-labelled binding sites was observed. A high density of specific binding sites was present in the medial posterior hypothalamic nucleus, the superficial layer of the optic tectum, the area ventralis of Tsai (AVT), the nucleus pretectalis, the habenula, the nucleus of Darkschewitch and the nucleus interstitialis. A low density occurred in the nucleus robustus archistriatalis of female birds. However, after testosterone treatment the density of the sites in this area as well as in the AVT increased 2-fold. [3H]Vasopressin binding was displaced to the same extent by excess vasopressin as well as by vasotocin. Moreover, the [3H]vasopressin-labelled brain regions are innervated by immunoreactive vasotocin fibers and terminals. It seems likely therefore that the labelled sites represent putative vasotocin receptors.

Testosterone-sensitive vasotocin-immunoreactive cells and fibers in the canary brain.

A sexual difference in vasotocin (VT) immunoreactivity was observed in the canary brain. The male canary displays denser VT innervation of the lateral septum and more VT-immunoreactive cells are visible in the dorsal diencephalon than in the female canary. This sex difference is the consequence of gonadal influences. Castration of male canaries resulted in a substantial decrease of VT immunoreactivity in these septal and diencephalic cells and fibers. Testosterone administration to castrated males restored the original intensity of fiber and cell staining. Testosterone treatment of female canaries induced strong VT immunostaining in the above regions similar to that in the male canary.

Organization of vasotocin-immunoreactive cells and fibers in the canary brain.

The distribution of vasotocin (VT)-immunoreactive neuronal perikarya and fibers in the canary (Serinus canaria) was investigated with immunohistological techniques. The results suggest that most VT-stained cell bodies are located in three diencephalic regions. First, a large number of densely packed neurons are found in the paraventricular nucleus (PVN) and the anterior preoptic nucleus. Neurons here vary widely in size and shape. Small-size rounded neurons and large-size multipolar neurons appear to concentrate in separate subdivisions. Second, a series of loosely organized cell groups of medium- to large-size cells occurs in the lateral parts of the hypothalamus. These aggregates of neurons apparently correspond to subdivisions of the supraoptic nucleus (SON). Third, diffusely distributed, lightly stained cells are found dorsal to the paraventricular nucleus in the dorsal diencephalon. A number of cells of this group seem to be located in the basal septal area and bed nucleus of the stria terminalis. Immunoreactive fibers and varicosities concentrate in brain regions that are associated with neuroendocrine, autonomic, and limbic functions. Axons from the PVN and SON form compact bundles of the hypothalamohypophysial tract in the lateral hypothalamus and then funnel into the internal zone of the medium eminence (ME). Furthermore, a heavy innervation seems to be present in the palisadal, external zone of the ME. A substantial number of fibers appear to leave the PVN toward extrahypothalamic areas. Most extrahypothalamic VT fibers innervate telencephalic and brainstem regions that are thought to be involved in mediation of limbic and autonomic functions. These areas include the lateral and medial septum, the lateral habenula, the substantia grisea centralis, the area ventralis (Tsai), the locus coeruleus, raphe nuclei, the nucleus tractus solitarii, and lateral medulla. In addition, fibers with immunoreactivity for VT innervate structures such as the optic tectum and the nucleus ovoidalis that have been implicated in sensory processing of visual and auditory information. Finally, VT fibers and varicosities occur in centers including the nucleus robustus archistriatalis and nucleus intercollicularis that have been implicated in vocal control.

In situ hybridization of oxytocin messenger RNA: macroscopic distribution and quantitation in rat hypothalamic cell groups.

Oxytocin mRNA was detected in the rat hypothalamus by in situ hybridization to a single stranded 35S-labelled DNA probe and the distribution of oxytocin mRNA-containing cell groups was studied at the macroscopic level. Specificity of hybridization was confirmed by comparison to vasopressin mRNA hybridization in parallel tissue sections. Cell groups containing oxytocin mRNA were confined to a set of hypothalamic cell groups, i.c. the supraoptic, paraventricular, anterior commissural nuclei, nucleus circularis and scattered hypothalamic islets. These cell groups displayed similar densities of autoradiographic signals indicating that the oxytocin gene is expressed at approximately the same average level at these various sites.

Vasopressin gene expression is attenuated in the fetal Brattleboro rat.

Due to a genetic defect the homozygous Brattleboro rat is unable to synthesize vasopressin gene products but still transcribes a mutant vasopressin mRNA from the gene. To study the influence of vasopressin gene products on the development of vasopressin gene expression, vasopressin mRNA levels of the supraoptic and paraventricular nucleus were measured at fetal day 20, postnatal day 1, 15 and 30 in the Wistar rat and in the heterozygous and homozygous Brattleboro rat by Northern blot analysis and in situ hybridization. In the homozygous Brattleboro rat of fetal day 20 and postnatal day 1, no or minute amounts of vasopressin mRNA were detectable but vasopressin mRNA was readily detectable at postnatal day 15 and 30. The Wistar rat and heterozygous Brattleboro rat had abundant vasopressin mRNA at fetal day 20 with increasing amounts towards postnatal day 30. The results indicate that vasopressin gene expression in the development of the homozygous Brattleboro rat is attenuated, possibly due to the absence of vasopressin gene products.

Autoradiographic localization of binding sites for the arginine-vasopressin (VP) metabolite, VP4-9, in rat brain.

A discrete neuro-anatomical pattern of binding sites was observed for a principal metabolite of arginine-vasopressin (VP4-9) after incubation of tissue sections with [35S]VP4-9 and autoradiography. [35S]VP4-9-labeled binding sites were highly concentrated in the pineal gland, the nucleus tractus solitarii (nts), the arcuate nucleus region (an) and the organum vasculosum lamina terminalis (ovlt). The distribution of these sites is distinctly different from the putative VP and oxytocin receptor systems in rat brain. It is possible that the VP4-9 binding sites are also involved in memory processes and/or that the VP metabolite exerts additional effects on the brain.

Topography of binding sites for neurohypophyseal hormones in rat brain.

Binding sites for arginine-vasopressin (AVP) and oxytocin (OT) were detected in neuroanatomically defined regions of rat brain by using in vitro incubation of 32 micron brain sections with the tritium-labelled ligands and autoradiography with LKB Ultrofilm. Sites were quantified by computerized densitometry of the film images. The highest amount of [3H]AVP-labelled binding sites were observed in the ventral subiculum of the hippocampus, central amygdala nucleus, lateral septum, olfactory nucleus and nucleus tractus solitarii, while the dentate gyrus contained a large amount of low affinity binding. The binding pattern of [3H]OT was more discrete. Relatively high concentrations of OT binding sites were observed in the central amygdala, ventral subiculum and olfactory nucleus. The lateral septum and the nucleus tractus solitarii were virtually devoid of displaceable OT binding while no significant association of OT with the dentate gyrus was found. The topography of binding sites for neurohypophyseal hormones corresponds to the localization of responsive sites in behavioural, biochemical and electrophysiological studies.

Intracellular CBG-like molecules in the rat pituitary.

The localization of transcortin (CBG) in pituitary cells of the rat was investigated using the peroxydase-antiperoxydase (PAP) technique. A rabbit antiserum against purified rat plasma transcortin was used as the primary antiserum. Transcortin-like (CBG-like) immunoreactive products were found in the cytoplasma of certain cells in the anterior pituitary, but not in the intermediate lobe and weakly in the posterior pituitary. It is postulated that the CBG-like molecules participate in the cellular uptake process of corticosterone, thereby modulating the feedback signal of this steroid on pituitary function.