Neuropeptide binding reflects convergent and divergent evolution in species-typical group sizes

Neuroendocrine factors that produce species differences in aggregation behavior ("sociality") are largely unknown, although relevant studies should yield important insights into mechanisms of affiliation and social evolution. We here focused on five species in the avian family Estrildidae that differ selectively in their species-typical group sizes (all species are monogamous and occupy similar habitats). These include two highly gregarious species that independently evolved coloniality; two territorial species that independently evolved territoriality; and an intermediate, modestly gregarious species that is a sympatric congener of one of the territorial species. Using males and females of each species, we examined binding sites for (125)I-vasoactive intestinal polypeptide (VIP), (125)I-sauvagine (SG; a ligand for corticotropin releasing factor, CRF, receptors) and a linear (125)I-V(1a) vasopressin antagonist (to localize receptors for vasotocin, VT). VIP, CRF and VT are neuropeptides that influence stress, anxiety and/or various social behaviors. For numerous areas (particularly within the septal complex), binding densities in the territorial species differed significantly from binding in the more gregarious species, and in most of these cases, binding densities for the moderately gregarious species were either comparable to the two colonial species or were intermediate to the territorial and colonial species. Such patterns were observed for (125)I-VIP binding in the medial bed nucleus of the stria terminalis, medial septum, septohippocampal septum, and subpallial zones of the lateral septum; for (125)I-SG binding in the infundibular hypothalamus, and lateral and medial divisions of the ventromedial hypothalamus; and for the linear (125)I-V(1a) antagonist in the medial septum, and the pallial and subpallial zones of the caudal lateral septum. With the exception of (125)I-SG binding in the infundibular hypothalamus, binding densitites are positively related to sociality.     

Endogenous vasotocin exerts context-dependent behavioral effects in a semi-naturalistic colony environment

Arginine vasotocin (VT), and its mammalian homologue arginine vasopressin (VP), are neuropeptides involved in the regulation of social behaviors and stress responsiveness. Previous research has demonstrated opposing effects of VT/VP on aggression in different species. However, these divergent effects were obtained in different social contexts, leading to the hypothesis that different populations of VT/VP neurons regulate behaviors in a context-dependent manner. We here use VP antagonists to block endogenous VT function in male zebra finches (Taeniopygia guttata) within a semi-natural, mixed-sex colony setting. We examine the role of VT in the regulation of aggression and courtship, and in pair bond formation and maintenance, over the course of three days. Although our results confirm previous findings, in that antagonist treatment reduces aggressive mate competition during an initial behavioral session during which males encounter novel females, we find that the treatment effects are completely reversed within hours of colony establishment, and the antagonist treatment instead facilitates aggression in later sessions. This reversal occurs as aggression shifts from mate competition to nest defense, but is not causally associated with pairing status per se. Instead, we hypothesize that these divergent effects reflect context-specific activation of hypothalamic and amygdalar VT neurons that exert opposing influences on aggression. Across contexts, effects were highly specific to aggression and the antagonist treatment clearly failed to alter latency to pair bond formation, pair bond stability, and courtship. However, VT may still potentially influence these behaviors via promiscuous oxytocin-like receptors, which are widely distributed in the zebra finch brain.     

Vasotocin neurons in the bed nucleus of the stria terminalis preferentially process social information and exhibit properties that dichotomize courting and non-courting phenotypes

Neurons within the medial bed nucleus of the stria terminalis (BSTm) that produce arginine vasotocin (VT; in non-mammals) or arginine vasopressin (VP; in mammals) have been intensively studied with respect to their anatomy and neuroendocrine regulation. However, almost no studies have examined how these neurons process stimuli in the animals' immediate environment. We recently showed that in five estrildid finch species, VT-immunoreactive (-ir) neurons in the BSTm increase their Fos expression selectively in response to positively-valenced social stimuli (i.e., stimuli that should elicit affiliation). Using male zebra finches, a highly gregarious estrildid, we now extend those findings to show that VT-Fos coexpression is induced by a positive social stimulus (a female), but not by a positive non-social stimulus (a water bath in bath-deprived birds), although the female and bath stimuli induced Fos equally within a nearby control region, the medial preoptic nucleus. In concurrent experiments, we also show that the properties of BSTm VT-ir neurons strongly differentiate males that diverge in social phenotype. Males who reliably fail to court females (“non-courters”) have dramatically fewer VT-ir neurons in the BSTm than do reliable courters, and the VT-ir neurons of non-courters fail to exhibit Fos induction in response to a female stimulus.     

Electron-microscopic investigations of vasoactive intestinal peptide (VIP)-like immunoreactive terminal formations in the lateral septum of the pigeon

Vasoactive intestinal peptide (VIP)-like immunoreactive terminal fields were examined in the lateral septum of the pigeon by means of immunocytochemistry. According to light-microscopic observations, these projections originated from VIP-like immunoreactive cerebrospinal fluid (CSF)-contacting neurons, which are located in the ependymal layer of the lateral septum and form a part of the lateral septal organ. The processes of these cells gave rise to dense terminal-like structures in the lateral septum. Pre-embedding immuno-electron microscopy revealed that VIP-like immunoreactive axon terminals had synaptoid contacts with perikarya of small VIP-immunonegative neurons of the lateral septum, which were characterized by an invaginated nucleus, numerous mitochondria, a well-developed Golgi apparatus, endoplasmic reticulum and a small number of dense-core vesicles (about 100 nm in diameter). VIP-like immunoreactive axons were also seen in contact with immunonegative dendrites in the lateral septum. In both axosomatic and axodendritic connections, VIP-like immunoreactive presynaptic terminals contained large dense-core vesicles, clusters of small vesicles and mitochondria. These findings suggest that VIP-immunoreactive neurons of the lateral septal organ project to small, presumably peptidergic nerve cells of the lateral septum and that the VIP-like neuropeptide serves as a neuromodulator (-transmitter) in this area.     

Activational effects of estradiol and dihydrotestosterone on social recognition and the arginine-vasopressin immunoreactive system in male mice lacking a functional aromatase gene

In rodents, parts of the arginine-vasopressin (AVP) neuronal system are sexually dimorphic with males having more AVP-immunoreactive cells/fibers than females. This neuropeptide neuronal system is highly sensitive to steroids and has been proposed to play an important role in the processing of olfactory cues critical to the establishment of a social memory. We demonstrate here that gonadally intact male aromatase knockout (ArKO) mice, which cannot aromatize androgens into estrogens due to a targeted mutation in the aromatase gene, showed severe deficits in social recognition as well as a reduced AVP-immunoreactivity in several brain regions. To determine whether this reduction is due to a lack of organizational or activational effects of estrogens, we assessed social recognition abilities and AVP-immunoreactivity in male ArKO and wild-type (WT) mice when treated with estradiol benzoate (EB) in association with dihydrotestosterone propionate (DHTP) in adulthood. Adult treatment with EB and DHTP restored social recognition abilities in castrated ArKO males since they showed normal female-oriented ultrasonic vocalizations and were able to recognize an unfamiliar female using a habituation-dishabituation paradigm. Furthermore, adult treatment also restored AVP-immunoreactivity in the lateral septum of ArKO males to levels observed in intact WT males. These results suggest that social recognition in adulthood and stimulation of AVP expression in the adult mouse forebrain depend predominantly on the estrogenic metabolite of testosterone. Furthermore, our results are in line with the idea that the organization of the AVP system may depend on androgen or sex chromosomes rather than estrogens.     

Catecholaminergic innervation of the suprachiasmatic nucleus in the adult rat: ultrastructural relationships with neurons containing vasoactive intestinal peptide or vasopressin

Catecholaminergic fibers in the suprachiasmatic nucleus of adult rats were investigated by use of light- and electron-microscopic immunocytochemistry. The suprachiasmatic nucleus receives a modest density of tyrosine hydroxylase-containing axons, homogeneously distributed in the nucleus and forming varicosities throughout its entire rostro-caudal extension. Immunolabeling with antibodies against dopamine showed that this catecholamine input comprises a dopaminergic component. Many tyrosine hydroxylase-positive cells were localized at the immediate periphery of the suprachiasmatic nucleus. With electron-microscopic examination, dendrites of these neurons were found within the limits of the nucleus as well as at a border zone between the suprachiasmatic nucleus proper and the optic tract where they received unlabeled synapses, providing a morphological support for a possible role of dopaminergic neurons in the integration and/or transfer of light-related signals. More than 91% of catecholaminergic axonal varicosities were found to establish morphologically defined synapses with dendrites. To investigate whether these synapses might be shared with neurons of one or both of the two main peptidergic populations of the nucleus, namely vasoactive intestinal peptide- and vasopressin-containing neurons, we carried out doublelabeling experments combining immunoperoxidase and immunogold-silver labeling. Results showed only a few cases of direct association of the catecholaminergic terminals with these peptidergic categories. In both types of dually stained sections, catecholaminergic synapses were preferentially made with unlabeled dendrites. The homogeneous distribution of tyrosine hydroxylase-immunoreactive fibers in the suprachiasmatic nucleus could therefore reflect a lack of significant catecholaminergic innervation of both vasoactive intestinal peptide- and vasopressin-synthesizing neurons.     

Vasoactive intestinal polypeptide (VIP) immunoreactivity of endocrine-like cells in the feline pyloric mucosa

Summary Immunoreactivity to VIP by endocrine-like cells in the feline pyloric mucosa was examined by using three kinds of region-specific anti-porcine VIP sera. VIP-immunoreactive endocrine-like cells were detected clearly with all of the VIP antisera used. They were located mainly around the neck of the pyloric glands. Some of these endocrine-like cells showed dilution-dependent immunoreactivity against VIP antisera. The immunostaining intensity of VIP-immunoreactive endocrine-like cells showing dilution-independence could not be distinguished from those of nerve elements. The present results suggest that the immunoreactivity with properties very similar to those of authentic VIP may be present in the endocrine-like cells of the feline pyloric glands.     

Localization of vasopressin-, vasoactive intestinal polypeptide-, peptide histidine isoleucine-and somatostatin-mRNA in rat suprachiasmatic nucleus

Summary Messenger RNAs (mRNA) coding for vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), somatostatin and vasopressin were localized in the suprachiasmatic nucleus (SCN) of the rat hypothalamus using in situ hybridization histochemistry. Specific mRNA coding for each of these peptides was distributed in areas coextensive with the immunohistochemical localization of the appropriate peptide. The autoradiographic signal produced with probes to VIP and PHI created dense concentrations of silver grains over neuronal perikarya in the ventrolateral SCN, and the coextensive distribution of both VIP-and PHI-mRNAs suggests that both peptides are synthesized within the same neurons. The distribution of somatostatin-mRNA was distinct from that of VIP and PHI. Labeled neurons are observed at the interface of the two SCN subdivisions and the distribution of these neurons is identical to those shown to contain somatostatin immunoreactivity. Vasopressin-mRNA is also differentially concentrated within neurons in the dorsomedial subdivision of the SCN in an area that is coextensive with vasopressin-immunoreactive perikarya. The discrete pattern of hybridization for each of these mRNAs indicates that each of these peptides are synthesized in SCN neurons and reaffirms the differential distribution of each of these chemically defined cell populations within cytoarchitecturally distinct subdivisions of the nucleus.     

Breeding-context-dependent relationships between song and cFOS labeling within social behavior brain regions in male European starlings (Sturnus vulgaris)

Male European starlings (Sturnus vulgaris) sing throughout the year, but the social factors that motivate singing behavior differ depending upon the context in which song is produced. In a non-breeding context (when testosterone concentrations are low), starlings form large, mixed-sex flocks and song is involved in flock cohesion and perhaps maintenance of social hierarchies. In contrast, in a breeding context (when testosterone concentrations are high), male song plays a direct role in mate attraction. How the nervous system ensures that song production occurs in an appropriate context in response to appropriate stimuli is not well understood. The song control system regulates song production, learning, and, to some extent, perception; however, these nuclei do not appear to regulate the social context in which song is produced. A network of steroid hormone sensitive nuclei of the basal forebrain and midbrain regulates social behavior. The present study used the immediate early gene cFOS to explore possible involvement of these regions in context-dependent song production. Numbers of cFOS-labeled cells in the medial bed nucleus of the stria terminalis, anterior hypothalamus, and ventromedial nucleus of the hypothalamus related positively only to song produced in a breeding context. In contrast, numbers of cFOS-labeled cells in three zones of the lateral septum related positively only to song produced in a non-breeding context. Taken together, these data suggest differential regulation of male starling song by social behavior nuclei depending upon the breeding context in which it is produced.     

Distribution of vasoactive intestinal peptide-like and neurophysin-like immunoreactive neurons and acetylcholinesterase staining in the ring dove hypothalamus with emphasis on the question of an avian suprachiasmatic nucleus

Summary Two nuclei, termed here the medial hypothalamic nucleus and the lateral hypothalamic retinorecipient nucleus, are possible homologs of the mammalian suprachiasmatic nucleus. As the mammalian suprachiasmatic nucleus is characterized by a dense concentration of vasoactive intestinal peptide (VIP)-and neurophysin (NP)-immunoreactive neurons and an absence of acetylcholinesterase (AChE) staining, we decided to examine these factors in the ring dove hypothalamus. Neither the medial hypothalamic nucleus nor the lateral hypothalamic retinorecipient nucleus contained either VIP-or NP-like immunoreactive neurons. The lateral hypothalamic retinorecipient nucleus stained darkly for AChE. Although there was some overlap in the distribution of VIP-and NP-like immunoreactive neurons, a clustering of both types into a well defined nucleus was not observed. Therefore, an avian homolog to the mammalian suprachiasmatic nucleus must differ in its chemoarchitecture from that of mammalian species described to date.     

Synaptology of three peptidergic neuron types in the central nucleus of the rat amygdala

The synaptology of neurotensin (NT)-, somatostatin (SS)- and vasoactive intestinal polypeptide (VIP)-immunoreactive neurons was studied in the central nucleus of the rat amygdala (CNA). Three types of axon terminals formed synaptic contacts with peptide-immunoreactive neurons in the CNA: Type A terminals containing many round or oval vesicles; Type B terminals containing many pleomorphic vesicles; and Type C terminals containing fewer, pleomorphic vesicles. Peptide-immunoreactive terminals were type A. All three types of terminals formed symmetrical axosomatic and asymmetrical axodendritic contacts. However, type B and peptide-immunoreactive terminals frequently formed symmetrical axodendritic synaptic contacts. VIP-immunoreactive terminals also formed asymmetrical axodendritic contacts. SS- and NT-immunoreactive terminals commonly formed symmetrical contacts on SS- and NT-immunoreactive cell bodies, respectively. VIP-immunoreactive axon terminals were postsynaptic to nonreactive terminals. Type B terminals appeared more frequently on VIP neurons than on NT or SS neurons.     

Anorexigenic effects of pituitary adenylate cyclase-activating polypeptide and vasoactive intestinal peptide in the chick brain are mediated by corticotrophin-releasing factor

Intracerebroventricular (ICV) injection of pituitary adenylate cyclase-activating polypeptide-38 (PACAP) or vasoactive intestinal peptide (VIP) inhibits feeding in chicks. However, the underlying anorexigenic mechanism(s) has not yet been investigated. The present study investigated whether these peptides influence the activity of corticotrophin-releasing factor (CRF) neural pathways in the brain of chicks. Firstly, we found that ICV injections of PACAP and VIP increased plasma corticosterone concentrations. The corticosterone-releasing effect of PACAP was completely attenuated by co-injection of astressin, a CRF receptor antagonist, but this effect was only partial for VIP. These results demonstrated that CRF neurons mediate the actions of PACAP and, to a lesser extent, VIP, and suggest that the signaling mechanisms differ between the two peptides. This difference may arise from the two peptides interacting with different receptors because the corticosterone-releasing effect of PACAP, but not VIP, was completely attenuated by co-injection of PACAP (6–38), a PACAP receptor antagonist. Finally, we examined the effect of ICV co-injection of astressin on the anorexigenic effects of PACAP and VIP and found that the effects of both peptides were attenuated by astressin. Overall, the present study suggests that the anorexigenic effects of PACAP and VIP are mediated by the activation of CRF neurons.     

Immunocytochemical analysis of calcitonin gene-related peptide and vasoactive intestinal polypeptide in Merkel cells and cutaneous free nerve endings of cats

Summary Calcitonin gene-related peptide (CGRP)-and vasoactive intestinal polypeptide (VIP)-immunoreactivity were observed to coexist in Merkel cells of cats. No differences in peptide content were found between Merkel cells located in epithelia of the hard palate, in hairy and glabrous skin of the upper lip, and in vibrissae follicles. CGRP-and VIP-immunoreactive nerve fibres were also found near CGRP/VIP-immunoreactive Merkel cells. In the vibrissae follicles some CGRP-and VIP-immunoreactive nerve terminals end abutting on the glassy membrane. Other CGRP immunoreactive nerve fibres penetrate the epithelium of the skin and end within it. Electron microscopy of vibrissae follicles revealed that Merkel cell neuntes are not immunostained and that immunostained nerve fibres form unmyelinated bundles before ending freely. Thus, CGRP-and VIP immunoreactive nerve fibres in cat skin do not end as Merkel cell neuntes but as different kinds of free nerve endings.     

Organization and interrelationship of neuropeptides in the central amygdaloid nucleus of the rat

The organization and interactions of neuropeptides in the central nucleus of the amygdala (Ce) were studied using single and double label immunocytochemical techniques. Immunocytochemical localization of substance P (SP), neurotensin (NT), met-enkephalin (m-ENK), somatostatin (SS) and vasoactive intestinal polypeptide (VIP) revealed all of these peptides within discrete regions of the Ce. The regions differed from the classical medial and lateral anatomical divisions reported for the Ce. Instead, three easily recognizable neuropeptidergic subdivisions were evident: a medial zone, a central zone and a lateral capsular zone. Two types of interrelationships between peptides were noted. The first involved a peptidergic fiber in apposition to a peptidergic perikarya. The most prevalent peptidergic interaction of this type occurred between SP and NT. The second interrelationship involved two different peptidergic fibers in apposition to an immunonegative cell. Two interactions of this type were commonly observed. The first involved NT and m-ENK fibers simultaneously apposed to an unstained cell. The second involved SP and m-ENK fibers adjacent to the same immunonegative cell. The interactions between peptidergic systems may suggest a role of these substances in the regulation of autonomic functions in the Ce.     

Innervation of lower airways and neuropeptide effects on bronchial and vascular tone in the pig

Summary The occurrence and distribution of peptide-containing nerve fibres [substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), neuropeptide Y (NPY)] and noradrenergic nerve fibres [tyrosine hydroxylase (TH)- and dopamine beta hydroxylase (DBH)-positive] in the airways of the pig were studied by means of immunohistochemistry. SP- and CGRP-immunoreactive (-IR) nerve fibres were present close to and within the lining respiratory epithelium, around blood vessels, within the tracheobronchial smooth muscle layer and around local tracheobronchial ganglion cells. The content of CGRP- and neurokinin A (NKA)-like immunoreactivity (-LI) measured by radioimmunoassay (RIA) was twice as high in the trachea compared to that in the peripheral bronchi. SP was a more potent constrictor agent than NKA on pig bronchi in vitro. CGRP had a relaxant effect on precontracted pig bronchi. On blood vessels CGRP exerted a relaxant effect that was more pronounced on pulmonary arteries than on bronchial arteries. VIP/PHI-IR fibres were seen in association with exocrine glands and in the tracheobronchial smooth muscle layer. VIP-positive nerve fibres were abundant around blood vessels in the trachea but sparse or absent around blood vessels in the peripheral bronchi. This histological finding was supported by RIA; it was shown that the content of peptides displaying VIP-like immunoreactivity (-LI) was 18 times higher in the trachea compared to peripheral bronchi. VIP was equally potent as CGRP in relaxing precontracted pig bronchi in vitro. Both bronchial and pulmonary arteries were relaxed by VIP. NPY was colocalized with VIP in tracheal periglandular nerve fibres and in nerve fibres within the tracheobronchial smooth muscle layer. NPY was also present in noradrenergic (DBH-positive) vascular nerve fibres. The content of NPY was much higher (15-fold) in the trachea compared to small bronchi. NPY caused a contraction of both pulmonary and bronchial arteries. The bronchial smooth muscle contraction to field stimulation in vitro was purely cholinergic. A non-cholinergic relaxatory effect following field stimulation was observed after bronchial precontraction. Capsaicin had no effect on pig bronchi in vitro.     

Neuropeptide Y modulates the action of vasodilator agents in guinea-pig epicardial coronary arteries

Recently, we have demonstrated that guinea-pig epicardial coronary arteries are supplied by numerous nerve fibres containing neuropeptide Y (NPY) immunoreactivity. However, examination of vasomotor responses revealed that NPY did not elicit a contractile response in these arteries. In contrast, acetylcholine (ACh), calcitonin gene-related peptide (CGRP), substance P and vasoactive intestinal polypeptide (VIP) all relaxed precontracted arteries. In the present study, we have used histochemical, immunohistochemical and in vitro pharmacological techniques, in order to further investigate the possible role of NPY in guinea-pig epicardial coronary arteries. A double-immunofluorescence staining technique revealed that CGRP and substance P were co-localized in nerve fibres distinct from those displaying NPY immunoreactivity. Furthermore, using a method combining immunofluorescence and histochemical techniques, we observed that putative cholinergic nerve fibres (identified by their acetylcholinesterase content) and NPY-immunoreactive nerve fibres are two different nerve populations. An in vitro pharmacological method demonstrated that NPY markedly inhibited the relaxant responses mediated by ACh, VIP, substance P and isoprenaline but had no effect on CGRP. These results suggest that NPY-containing nerves associated with guinea-pig epicardial coronary arteries may be predominantly involved in modulating the action of vasodilator agents.     

Role of vasoactive intestinal polypeptide (VIP) in regulating the pituitary function in man

Intramuscular injection of synthetic VIP (200 micrograms) resulted in a rapid increase in plasma prolactin (PRL) concentrations in normal women, which was accompanied by the 4- to 7-fold increase in plasma VIP levels. Mean (+/- SE) peak values of plasma PRL obtained 15 min after the injection of VIP were higher than those of saline control (28.1 +/- 6.7 ng/ml vs. 11.4 +/- 1.6 ng/ml, p less than 0.05). Plasma growth hormone (GH) and cortisol levels were not affected by VIP in normal subjects. VIP injection raised plasma PRL levels (greater than 120% of the basal value) in all of 5 patients with prolactinoma. In 3 of 8 acromegalic patients, plasma GH was increased (greater than 150% of the basal value) by VIP injection. In the in vitro experiments, VIP (10(-8), 10(-7) and 10(-6) M) stimulated PRL release in a dose-related manner from the superfused pituitary adenoma cells obtained from two patients with prolactinoma. VIP-induced GH release from the superfused pituitary adenoma cells was also shown in 5 out of 6 acromegalic patients. VIP concentrations in the CSF were increased in most patients with hyperprolactinemia and a few cases with acromegaly. These findings indicate that VIP may play a role in regulating PRL secretion in man and may affect GH secretion from pituitary adenoma in acromegaly.     

The central nervous system of Bulla gouldiana: peptide localization

In the present study, we describe the structure of the central nervous system (CNS) of the marine gastropod Bulla gouldiana, and compare it with the structure of the CNS of the related mollusc, Aplysia californica. In addition, we performed an immunohistochemical analysis of a series of peptides, and the synaptic vesicle protein, synapsin I, in the central nervous system of B. gouldiana. The most common peptide in the B. gouldiana nervous system is the molluscan cardioexcitatory peptide (FMRFamide), which is present in a significant proportion of B. gouldiana neurons. A smaller number of neurons exhibit immunoreactivity to antisera raised against the calcitonin gene related peptide, vasopressin, vasoactive intestinal peptide, cholecystokinin, galanin and enkephalin. In some instances there is colocalization of two or more peptides. Very few neurons or axons exhibit synapsin I-like immunoreactivity. The patterns of immunoreactivity to these antisera is quite similar to the patterns that have been described in other gastropods, including Lymnaea stagnalis and Aplysia californica. These observations emphasize the importance of FMRFamide-like compounds in phylogenetically old nervous systems and indicate that compounds similar to mammalian peptides are present in the gastropod. Thus, the production of a wide variety of peptide molecules and their use in neuronal function appears to be a highly conserved phylogenetic process.