Effects of central vasotocin and mesotocin manipulations on social behavior in male and female zebra finches

Male and female zebra finches (Taeniopygia guttata; total n = 40) were fitted with chronic guide cannulae directed at the lateral ventricle and were tested for aggression, affiliation, and partner preference following infusions of mesotocin (MT), vasotocin (VT), their antagonists, and vehicle control. Aggressive behavior was tested in a mate competition paradigm and tests of intersexual affiliation and partner preference were conducted following 1 day of cohabitation with an opposite-sex individual. These tests also provided data on male courtship singing. The results demonstrate a modest dose-dependent facilitation of aggression by VT, but not MT, in both male and female finches. However, only males were sensitive to infusions of a vasopressin antagonist, suggesting that endogenous VT is more important for behavioral modulation in males. Peptide effects were specific to aggression, as no treatments influenced intersexual affiliation, partner preference, or male courtship singing. Thus, in contrast to rodents, partner preference is not readily induced by VT or MT in this species. However, the potential necessity of endogenous VT and MT for natural pair-bond formation remains to be tested.     

Sex-specific effects of handling time on an index of immune activity in zebra finches

Recently, there has been considerable interest in the role of the immune system in shaping life-history evolution, sexual selection strategies, and indexes of individual quality. The most frequently used assay of immune function, particularly in avian field studies, is the phytohemagglunitin (PHA) skin test. PHA is injected subcutaneously into the wing web, and the magnitude of the resultant swelling has traditionally been interpreted as an index of an individual's cell-mediated immunocompetence. The test follows one of two protocols: the traditional two-wing injection protocol, with one wing web injected with PHA and the other with phosphate-buffered saline (PBS), or the simplified one-wing protocol that omits the PBS injection. In this technical comment, we alert researchers to the importance of considering handling time when performing the PHA test. We show that zebra finches (Taeniopygia guttata) subjected to the two-wing protocol had a lower wing-web swelling than individuals injected in one wing. In males, handling time explained over 50% of the variation in an individual's skin swelling response; females were relatively unaffected by handling time. We suggest that caution should be exercised when comparing the magnitude of wing-web swelling across studies in which the alternate protocol was followed. In addition, the recording of handling time, and its inclusion in subsequent statistical analyses, may aid in the detection of subtle differences across treatments.     

Sex-specific effects of yolk testosterone on survival, begging and growth of zebra finches

Yolk androgens affect offspring hatching, begging, growth and survival in many bird species. If these effects are sex-specific, yolk androgen deposition may constitute a mechanism for differential investment in male and female offspring. We tested this hypothesis in zebra finches. In this species, females increase yolk-testosterone levels and produce male-biased sex ratios when paired to more attractive males. We therefore predicted that especially sons benefit from elevated yolk androgens. Eggs were injected with testosterone or sesame oil (controls) after 2 days of incubation. Testosterone had no clear effect on sex-specific embryonic mortality and changed the pattern of early nestling mortality independent of offspring sex. Testosterone-treated eggs took longer to hatch than control eggs. Control males begged significantly longer than females during the first days after hatching and grew significantly faster. These sex differences were reduced in offspring from testosterone-treated eggs due to prolonged begging durations of daughters, enhanced growth of daughters and reduced growth of sons. The results show that variation in maternal testosterone can play an important role in avian sex allocation due to its sex-specific effects on offspring begging and growth.     

Sex-specific effects of maternal immunization on yolk antibody transfer and offspring performance in zebra finches

Trans-generational antibody transfer constitutes an important mechanism by which mothers may enhance offspring resistance to pathogens. Thus, differential antibody deposition may potentially allow a female to differentiate offspring performance. Here, we examined whether maternal immunization with sheep red blood cells (SRBC) prior to egg laying affects sex-specific yolk antibody transfer and sex-specific offspring performance in zebra finches (Taeniopygia guttata). We showed that immunized mothers deposit anti-SRBC antibodies into the eggs depending on embryo sex and laying order, and that maternal exposure to SRBC positively affects the body size of female, but not male offspring. This is the first study reporting sex-specific consequences of maternal immunization on offspring performance, and suggests that antibody transfer may constitute an adaptive mechanism of maternal favouritism.     

Distribution of osmoregulatory peptides and neuronal-glial configuration in the hypothalamic magnocellular nuclei of desert rodents

The desert rodents Psammomys obesus and Gerbillus tarabuli live under extreme conditions and overcome food and water shortage by modes of food and fluid intake specific to each species. Using immunohistochemistry and electron microscopy, we found that the hypothalamic magnocellular nuclei, and in particular, their vasopressinergic component, is highly and similarly developed in Psammomys and Gerbillus. In comparison to other rodents, the hypothalamus in both species contains more magnocellular VP neurons that, together with oxytocin neurons, accumulate in distinct and extensive nuclei. As in dehydrated rodents, many magnocellular neurons contained both neuropeptides. A striking feature of the hypothalamic magnocellular system of Psammomys and Gerbillus was its display of ultrastructural properties related to heightened neurosecretion, namely, a significant reduction in glial coverage of neuronal somata and dendrites in the hypothalamic nuclei. There were many neuronal elements whose surfaces were directly juxtaposed and shared the same synapses. Their magnocellular nuclei also showed a high level of sialylated isoform of the Neural Cell Adhesion Molecule (PSA-NCAM) that underlies their capacity for neuronal and glial plasticity. These species thus offer striking models of structural neuronal and glial plasticity linked to natural conditions of heightened neurosecretion.     

Testosterone decreases the potential for song plasticity in adult male zebra finches

Zebra finches are age-limited learners; males crystallize their songs at 90 days and do not subsequently alter those songs. However, a variety of interventions, including deafening and syringeal denervation, result in long-term changes to the crystallized song. These changes can be prevented by lesioning nucleus LMAN. As different social contexts for song production result in differential activation of LMAN, we asked whether the social context experienced by adult males would affect their ability to alter their songs in response to syringeal denervation. Males able to see and direct their songs to females made fewer changes to their songs than did males that could hear but not see females, but this trend was not significant. The volume of a male's HVc, a forebrain song control nucleus, also failed to predict the degree to which a male would change his song. However, testis mass was significantly correlated with the number of changes made to the song, indicating that variations in testosterone modulate adult song plasticity. We directly tested the effect of circulating testosterone on adult song plasticity by implanting adult males with either testosterone or flutamide, a testosterone receptor blocker, and tracking song changes triggered by ts nerve injury. As predicted, males implanted with testosterone changed their songs less than did males that received flutamide implants. These results suggest that the high testosterone concentrations associated with sexual maturity and song crystallization in zebra finches continue to act in adult males to reduce the potential for vocal plasticity.     

The interaction of testosterone and breeding phase on the reproductive behavior and use of space of male zebra finches

It is well known that androgens play a critical role in mediating the reproductive behavior of males. However, many laboratory experiments that examined the effects of testosterone in male songbirds typically limited their investigations to the early phase of breeding. We sought to determine the influence of testosterone on social behavior, pair bonding, nesting, and use of space in captive zebra finch (Taeniopygia guttata) males as a function of breeding stage (pre-laying, incubation, and nestling phases). Fourteen males were released into an aviary with 14 females and allowed to breed for 7 weeks. Half of the males were given testosterone implants and half were given control implants. During the pre-laying phase, testosterone-implanted males spent significantly more time in nesting activities than control birds and more time elapsed from starting to build a nest to when their mates initiated egg-laying. During the incubation phase, testosterone-implanted subjects spent significantly more time in female-directed and undirected singing. Use of space varied between hormone conditions depending upon breeding phase: there was no difference during pre-laying, but during the incubation and nestling phases, testosterone-implanted subjects used significantly more space. This significant increase in "home range" during the latter phase of the breeding cycle coincides with results from field studies on other species. These results underscore the importance of considering breeding phase in assessing the behavioral sensitivity to hormones.     

Activation of vasopressinergic and oxytocinergic neurons during aging in the Wistar rat

The activity of the hypothalamo-neurohypophyseal system (HNS) was determined in male Wistar rats from 3 to 32 months of age. Plasma levels of vasopressin (AVP) and oxytocin (OXT) were measured by means of a radioimmunoassay. In addition, the distribution of the Golgi apparatus marker enzyme thiamine-pyrophosphatase (TPP-ase) was measured as a parameter for neurosecretory activity in the hypothalamic supraoptic and paraventricular nuclei (SON and PVN). Plasma levels of radioimmunoassayable AVP were increased in the 32-month-old animals. Plasma levels of radioimmunoassayable OXT in 32-month-old animals did not differe from the levels found in the youngest group, but were higher than in 11-month-old animals. Neurosecretory activity in the SON was similar in 3- and 32-month-old animals, whereas in the PVN neurosecretory activity was increased in the 32-month-old animals. Urine excretion decreased between 6 and 11 months of age and remained on the same level until 32 months of age. In other words, instead of a loss of HNS function as has been suggested in the literature, an increased neurosecretory activity was observed in aged rats.     

Catecholamine distribution and relationship to magnocellular neurons in the paraventricular nucleus of the rat

Summary The distribution of catecholamine synthesizing enzymes within the paraventricular nucleus of the rat hypothalamus is elucidated immunocytochemically by use of antibodies to tyrosine hydroxylase, dopamine -hydroxylase, and phenylethanolamine-N-methyltransferase. Tyrosine hydroxylase-immunostained cell bodies are localized in the periventricular stratum and adjacent parvocellular regions, but rarely in magnocellular subnuclei of the paraventricular nucleus. Tyrosine hydroxylase-immunostained fibers are present in greatest density in the periventricular zone, and moderate density in the parvocellular and magnocellular subnuclei. Dopamine -hydroxylase-immunostained fibers are remarkably dense in the posterior magnocellular division of the paraventricular nucleus, especially in the dorso-lateral portion where vasopressin-containing cells predominate. Noradrenergic fiber input to these magnocellular neurons is likely since phenylethanolamine-N-methyltransferase-immunostained fibers are sparse in magnocellular subnuclei of the paraventricular nucleus. Dual immunocytochemical staining of thick and thin tissue sections demonstrates with clarity an anatomical association of dopamine -hydroxylase-immunostained fibers and magnocellular neurons. Dopamine -hydroxylase-immunostained and phenylethanolamine-N-methyltransferase-immunostained fibers are dense in the medial parvocellular component of the paraventricular nucleus; distinct features of both antisera are presented.     

Coexistence of NADPH-diaphorase with vasopressin and oxytocin in the hypothalamic magnocellular neurosecretory nuclei of the rat

Coexistence of NADPH-diaphorase with vasopressin and oxytocin was studied in the magnocellular neurosecretory nuclei of the rat hypothalamus by use of sequential histochemical and immunocytochemical techniques in the same sections. Coexistence was found in all the nuclei examined (supraoptic, paraventricular, circular, fornical, and in some isolated neurons located in the hypothalamic area between the paraventricular and supraoptic nuclei). The ratios of neurons expressing both markers (NADPH-diaphorase and vasopressin, NADPH-diaphorase and oxytocin) in each of the nuclei were very similar. Although further studies must be carried out, the partial coexistence found in all nuclei suggests that NADPH-diaphorase is probably not related to general mechanisms involving vasopressin and oxytocin, but rather in specific functions shared by certain hypothalamic neuronal cell populations.     

Localization and osmotic regulation of vesicular glutamate transporter-2 in magnocellular neurons of the rat hypothalamus

In this report we present immunocytochemical and in situ hybridization evidence that magnocellular vasopressin and oxytocin neurons in the hypothalamic supraoptic and paraventricular nuclei express type-2 vesicular glutamate transporter, a marker for their glutamatergic neuronal phenotype. To address the issue of whether an increase in magnocellular neuron activity coincides with the altered synthesis of the endogenous glutamate marker, we have introduced a new dual-label in situ hybridization method which combines fluorescent and autoradiographic signal detection components for vasopressin and vesicular glutamate transporter-2 mRNAs, respectively. Application of this technique provided evidence that 2% sodium chloride in the drinking water for 7 days produced a robust and significant increase of vesicular glutamate transporter-2 mRNA in vasopressin neurons of the supraoptic nucleus. The immunocytochemical labeling of pituitary sections, followed by the densitometric analysis of vesicular glutamate transporter-2 immunoreactivity in the posterior pituitary, revealed a concomitant increase in vesicular glutamate transporter-2 protein levels at the major termination site of the magnocellular axons. These data demonstrate that magnocellular oxytocin as well as vasopressin cells contain the glutamatergic marker vesicular glutamate transporter-2, similarly to most of the parvicellular neurosecretory neurons examined so far. The robust increase in vesicular glutamate transporter-2 mRNA and immunoreactivity after salt loading suggests that the cellular levels of vesicular glutamate transporter-2 in vasopressin neurons are regulated by alterations in water–electrolyte balance. In addition to the known synaptic actions of excitatory amino acids in magnocellular nuclei, the new observations suggest novel mechanisms whereby glutamate of endogenous sources can regulate magnocellular neuronal functions.     

Changes in catecholamine levels and turnover rates in hypothalamic,vocal control,and auditory nuclei in male zebra finches during development

The catecholamines norepinephrine (NE) and dopamine (DA) have been implicated in the sexual differentiation of brain and behavior and in species-specific learning in several species. To determine if these neurotransmitters might be involved in sexual differentiation of the vocal control system and song learning in male zebra finches, NE and DA levels and turnover rates were quantified in 10 behaviorally relevant brain nuclei [6 vocal control (VCN), 2 auditory (AN), and 2 hypothalamic (HN)] at four critical points during sexual differentiation of the VCN and the period of song learning, 25, 35, 55, and 90 days of age. Some birds were pretreated with α-methyl-para-tyrosine (αMPT) to allow estimation of NE and DA turnover rates. NE and DA levels in microdissected nuclei were quantified using high-performance liquid chromatography with electrochemical detection. αMPT treatment suppressed catecholamine synthesis just as effectively in juveniles as it does in adults and proved an effective method for estimating NE and DA turnover rates. Patterns of NE and DA function in most VCN and AN over development were quite different from those in HN in which NE and DA function changed gradually and showed no striking peaks. NE turnover rates changed significantly over development in all six VCN [nucleus interfacialis (Nlf), high vocal center (HVC), nucleus robustus of the archistriatum (RA), dorsomedial portion of the intercollicular nucleus (DM), Area X of the parolfactory lobe, and lateral portion of the magnocellular nucleus of the anterior neostriatum (IMAN)]; one AN [nucleus mesencephalicus lateralis pars dorsalis (MLd)], and one HN [preopticus anterior (POA)]. NE levels changed significantly in two VCN (Nlf and Area X). In Nlf, RA, Area X, IMAN, and MLd, NE levels and/or turnover rates showed a striking peak at day 25, which was not seen in HN. Both DA levels and turnover rates changed profoundly over development in 5 of 6 VCN (Nlf, RA, DM, Area X, and IMAN) and both AN (MLd and Field L). These nuclei showed striking peaks in DA levels and turnover rates, primarily on day 35 and/or 55, which then declined profoundly by day 90. This contrasted with the minimal change in DA turnover rates seen in one HN (POA) and the sixth VCN, HVC. In several VCN and AN, NE and DA levels and turnover rates during development reached levels never seen in adult males. Previous research has shown that catecholamine function is heightened in VCN during development compared to surrounding tissues. Our data demonstrate that NE and DA function during development shows pronounced peaks in most VCN not seen in HN. This is interesting because both VCN and HN are hormone sensitive, and both show hormone-modulated NE and DA function in adult males. The timing of these peaks suggests that increased catecholaminergic function may be involved in sexual differentiation of the VCN and song learning in finches. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 329–346, 1998     

Somato-dendritic mechanisms underlying the electrophysiological properties of hypothalamic magnocellular neuroendocrine cells: A multicompartmental model study

Magnocellular neuroendocrine cells (MNCs) of the hypothalamus synthesize the neurohormones vasopressin and oxytocin, which are released into the blood and exert a wide spectrum of actions, including the regulation of cardiovascular and reproductive functions. Vasopressin- and oxytocin-secreting neurons have similar morphological structure and electrophysiological characteristics. A realistic multicompartmental model of a MNC with a bipolar branching structure was developed and calibrated based on morphological and in vitro electrophysiological data in order to explore the roles of ion currents and intracellular calcium dynamics in the intrinsic electrical MNC properties. The model was used to determine the likely distributions of ion conductances in morphologically distinct parts of the MNCs: soma, primary dendrites and secondary dendrites. While reproducing the general electrophysiological features of MNCs, the model demonstrates that the differential spatial distributions of ion channels influence the functional expression of MNC properties, and reveals the potential importance of dendritic conductances in these properties. Action Editor: Eric De Schutter     

Relationship of CRF-immunostained cells and magnocellular neurons in the paraventricular nucleus of rat hypothalamus

The distribution of corticotropin-releasing factor (CRF), vasopressin (VP) and oxytocin (OXY) containing neurons within the magnocellular and parvocellular divisions in the paraventricular nucleus (PVN) of rat hypothalamus is described in brains from normal untreated, colchicine treated and adrenalectomized animals. Double immunostained preparations using glucose oxidase-antiglucose oxidase (GAG) complex combined with PAP complex to visualize two antigens with contrasting colors in the same tissue sections were employed. Separate and distinct populations of cells containing the immunoreactive (ir) elements were seen. Immunostained CRF neurons present in the ventral medial portion of the posterior magnocellular division were juxtaposed to oxytocin-ir perikarya in colchicine treated and adrenalectomized animals. CRF-ir cells were for the most part concentrated in the medial parvocellular component of PVN. An intimate anatomical proximity between CRF-ir and VP-ir perikarya was evident in this medial parvocellular division in brains of adrenalectomized animals; this area is normally VP-ir poor except in the adrenalectomized rats. This extension of VP-ir cells into this CRF rich region and the very close approximation between the two cell bodies suggests potential cell to cell communication following perturbation of the brain-pituitary-adrenal axis. No evidence for the co-existence of two peptidergic systems in the same neuron was apparent in the present study.     

Relationship of ACTH1-39-immunostained fibers and magnocellular neurons in the paraventricular nucleus of rat hypothalamus

Dual antigen immunocytochemical staining procedures were used in the same tissue section to determine the distribution of ACTH immunostained fibers and varicosities within the magnocellular and parvocellular divisions in the paraventricular nucleus (PVN) of rat hypothalamus and elucidate its anatomical relationship to vasopressin (VP) and oxytocin (OXY)-containing neurons. Double immunostained preparations using glucose oxidase-antiglucose oxidase complex combined with PAP complex to visualize two antigens with contrasting colors in the same tissue section were employed. ACTH-immunoreactive (ir) fibers were distributed throughout the periventricular stratum and the parvocellular component of the PVN; in the latter area fibers were particularly dense in the ventral medial portion of the medial parvocellular division. Dual immunostained sections revealed a close anatomical association between opiocortin fibers and oxytocin and vasopressin parvocellular neurons. ACTH immunostained fibers were present in the anterior and medial magnocellular component of PVN and in the ventral medial portion of the posterior magnocellular division; these immunoreactive fibers were in intimate proximity to oxytocin-ir perikarya. The very close approximation between the ACTH-ir fibers and oxytocin-containing cell bodies suggests potential cell to cell communication between the two peptidergic systems in PVN. Few ACTH immunostained fibers were seen in the dorsal lateral portion of the posterior magnocellular division in which vasopressinergic neurons predominate. The present anatomical study supports pharmacological and physiological studies which indicate that opioids can influence the activity of magnocellular PV neurons. This study also elucidates an anatomical relationship between opiocortins (ACTH1-39) and parvocellular PV neurons which suggests that the opiocortin system may play a role in the regulation of both the neuroendocrine and autonomic activities of specific PV neurons.     

Both oxytocin and vasopressin may influence alloparental behavior in male prairie voles

Neuropeptides, especially oxytocin (OT) and arginine vasopressin (AVP), have been implicated in several features of monogamy including alloparenting. The purpose of the present study was to examine the role of OT and AVP in alloparental behavior in reproductively na?ve male prairie voles. Males received intracerebroventricular (ICV) injections of artificial cerebrospinal fluid (aCSF), OT, an OT receptor antagonist (OTA), AVP, an AVP receptor antagonist (AVPA), or combinations of OTA and AVPA and were subsequently tested for parental behavior. Approximately 45 min after treatment, animals were tested for behavioral responses to stimulus pups. In a 10-min test, spontaneous alloparental behavior was high in control animals. OT and AVP did not significantly increase the number of males that showed parental behavior, although more subtle behavioral changes were observed. Combined treatment with AVPA and OTA (10 ng each) significantly reduced male parental behavior and increased attacks; following a lower dose (1 ng OTA/1 ng AVPA), males were less likely to display kyphosis and tended to be slower to approach pups than controls. Since treatment with only one antagonist did not interfere with the expression of alloparenting, these results suggest that access to either OT or AVP receptors may be sufficient for the expression of alloparenting.     

The distribution of vasopressin-, oxytocin-, and neurophysin-producing neurons in the guinea pig brain

Summary The location, cytology and projections of vasopressin-, oxytocin-, and neurophysin-producing neurons in the guinea pig were investigated using specific antisera against vasopressin, oxytocin or neurophysin in the unlabeled antibody enzyme immunoperoxidase method. Light microscopic examination of the neurons of the supraoptic and paraventricular nuclei shows that hormone is transported not only in axons, but also in processes having the characteristics of dendrites. Neurons were found to contain only vasopressin or oxytocin; all neurons containing neurophysin appear to contain either vasopressin or oxytocin. In the neural lobe, vasopressin and oxytocin terminals are intermingled. In the median eminence, vasopressin and oxytocin fibers are intermingled in the internal zone. In a caudal portion of the median eminence, a number of vasopressin and neurophysin (but few oxytocin) axons enter the external zone from the internal zone, and surround portal capillaries. In the supraoptic nucleus, vasopressin neurons outnumber oxytocin neurons with a ratio of at least 5:1. The paraventricular nucleus is separated into two distinct groups of neurons, a lateral group consisting of only vasopressin neurons, and a medial group consisting of only oxytocin neurons. In addition to axons passing to the neurohypophysis, a number of axons appear to interconnect the supraoptic and paraventricular nuclei.Supported by the Deutsche Forschungsgemeinschaft (SFB 51, C/21 and C/27), (We 608/3)Acknowledgements. The authors are greatly indebted to Mmes. R. Köpp-Eckmann, B. Reijerman, A. Scheiber, I. Wild and Mr. U. Schrell for technical assistance, to Mmes. P. Campbell and U. Wolf for editorial assistance, and to Dr. R.R. Dries and Ferring Pharmaceuticals, Kiel, for the generous provision of high quality peptides