Oxytocin actions within the supraoptic and paraventricular nuclei: differential effects on peripheral and intranuclear vasopressin release

In response to forced swimming (FS), AVP is released somato-dendritically within the supraoptic nucleus (SON) and paraventricular nucleus (PVN), but not from neurohypophyseal terminals into blood. Together with AVP, oxytocin (OXT) is released within the SON and PVN. Here, we studied the role of intra-SON and intra-PVN OXT in the regulation of local AVP release and into the blood in male rats. Within the SON, bilateral retrodialysis of an OXT receptor antagonist (OXT-A) increased local AVP release in response to FS [60 s, 21 degrees C, vehicle twofold, not significant (ns); OXT-A: 15-fold increase, P < 0.05] without significantly affecting basal AVP release. In addition, local OXT-A elevated plasma AVP secretion under basal conditions (twofold increase, P < 0.05) without further elevation after FS. Within the PVN, exposure to FS elevated local AVP release, reaching significance only in the OXT-A group (vehicle: 1.4-fold, ns; OXT-A: 1.6-fold increase, P = 0.050). Bilateral OXT-A into the PVN did not affect peripheral AVP secretion either under basal or stress conditions. Basal ACTH concentrations tended to be elevated by local OXT-A within the PVN (1.7-fold increase, P = 0.076). In contrast, the swim-induced ACTH secretion was attenuated after retrodialysis of OXT-A within both the SON (at 5 min) and PVN (at 15 min) (P < 0.05 both) compared with vehicle. The results demonstrate a receptor-mediated effect of OXT within the SON and PVN on local and neurohypophyseal AVP release, which depends upon the activity conditions. Further, while exerting an inhibitory effect on hypothalamo-pituitary-adrenal axis activity under basal conditions, hypothalamic OXT is essential for an adequate acute ACTH response.     

Chronic enhancement of brain oxytocin levels causes enduring anti-aggressive and pro-social explorative behavioral effects in male rats

Oxytocin (OXT) has been implicated in the regulation of social behaviors, including intermale offensive aggression. Recently, we showed that acute enhancement of brain OXT levels markedly suppressed offensive aggression and increased social exploration in resident rats confronted with an intruder in their home territory. Moreover, a different responsivity to the exogenous OXTergic manipulation was observed among individuals based on their baseline aggression. In this study we aimed at evaluating the behavioral response to chronically enhancing or attenuating central OXT levels, and at scrutinizing whether the trait-aggression moderates the treatment-induced behavioral changes. To this end, resident male wild-type Groningen rats were continuously (via osmotic minipumps) intracerebroventricularly infused with synthetic OXT or a selective OXT receptor (OXTR) antagonist for 7 days. Changes in behavior were assessed performing a resident–intruder test before and at the end of the treatment period, as well as after 7 days of withdrawal. Chronic infusion of OXT was found to selectively suppress aggression and enhance social exploration. Chronic blockage of OXTRs instead increased introductory aggressive behavior (i.e. lateral threat), yet without affecting the total duration of the aggression. The magnitude of the anti-aggressive changes correlated positively with the level of baseline aggression. Interestingly, OXT-induced behavioral changes persisted 7 days after cessation of the treatment. In conclusion, these findings provide further evidence that enhanced functional activity of the central OXTergic system decreases social offensive aggression while it increases social explorative behavior. The data also indicate that chronically enhancing brain OXT levels may cause enduring anti-aggressive and pro-social explorative behavioral effects.     

The effects of ghrelin/GHSs on AVP mRNA expression and release in cultured hypothalamic cells in rats

Ghrelin, the endogenous ligand for growth hormone secretagogues (GHSs) receptor (GHS-R), increases adrenocorticotropin (ACTH) and cortisol (corticosterone) as well as GH secretion in humans and animals. However, the site of GHSs action to induce ACTH secretion is not fully understood. To clarify the mechanisms of the action of ghrelin/GHSs on ACTH secretion, we analyzed the effects of KP-102 and ghrelin on the mRNA expression and release of corticotropin releasing factor (CRF) and arginine vasopressin (AVP), ACTH secretagogues, in monolayer-cultured hypothalamic cells of rats. Incubation of cells with KP-102 for 4 h and 8 h and with ghrelin for 4 h significantly increased AVP mRNA expression and release without changing CRF mRNA expression. CRF levels in culture media were undetectable. Suppression of GHS-R expression by siRNA blocked ghrelin- and KP-102-induced AVP mRNA expression and release. NPY significantly increased AVP mRNA expression and release. Furthermore, treatment of cells with anti-NPY IgG blocked KP-102-induced AVP mRNA expression and release. We previously reported that KP-102 significantly increases NPY mRNA expression in cultured hypothalamic cells. Taken together, these results suggest that ACTH secretion by ghrelin/GHSs is induced mainly through hypothalamic AVP, and that NPY mediates the action of ghrelin/GHSs.     

Angiotensin (1-7) contributes to nitric oxide tonic inhibition of vasopressin release during hemorrhagic shock in acute ethanol intoxicated rodents

Aims

Acute ethanol intoxication (AEI) attenuates the arginine vasopressin (AVP) response to hemorrhage leading to impaired hemodynamic counter-regulation and accentuated hemodynamic stability. Previously we identified that the ethanol-induced impairment of circulating AVP concentrations in response to hemorrhage was the result of augmented central nitric oxide (NO) inhibition. The aim of the current study was to examine the mechanisms underlying ethanol-induced up-regulation of paraventricular nucleus (PVN) NO concentration. Angiotensin (ANG) (1-7) is an important mediator of NO production through activation of the Mas receptor. We hypothesized that Mas receptor inhibition would decrease central NO concentration and thus restore the rise in circulating AVP levels during hemorrhagic shock in AEI rats.

Main methods

Conscious male Sprague–Dawley rats (300–325 g) received a 15 h intra-gastric infusion of ethanol (2.5 g/kg + 300 mg/kg/h) or dextrose prior to a fixed-pressure (~ 40 mm Hg) 60 min hemorrhage. The Mas receptor antagonist A-779 was injected through an intracerebroventricular (ICV) cannula 15 min prior to hemorrhage.

Key findings

PVN NOS activity and NO were significantly higher in AEI compared to DEX-treated controls at the completion of hemorrhage. ICV A-779 administration decreased NOS activity and NO concentration, partially restoring the rise in circulating AVP level at completion of hemorrhage in AEI rats.

Significance

These results suggest that Mas receptor activation contributes to the NO-mediated inhibitory tone of AVP release in the ethanol-intoxicated hemorrhaged host.     

Low inborn anxiety correlates with high intermale aggression: link to ACTH response and neuronal activation of the hypothalamic paraventricular nucleus

Aggression constitutes a central problem in several psychopathologies, including anxiety and depression disorders and antisocial behaviors. In particular, the activity of the hypothalamic-pituitary-adrenocortical (HPA) axis has been associated with aggression-related disorders. The present study assessed whether genetically determined levels of anxiety-related behavior influence the level of intermale aggression and whether this is associated with differences in neuroendocrine responsiveness and neuronal activation in the brain. Adult male Wistar rats bred for high (HAB) or low (LAB) anxiety-related behavior were used, as well as non-selected rats (NAB) with an intermediate anxiety level. LAB residents displayed more aggressive behavior than HAB and NAB residents during the resident-intruder (RI) test. Moreover, an inverse correlation was found between the level of anxiety and the level of aggression. The plasma corticotropin (ACTH) response to RI-test exposure was significantly higher in LABs than in HABs and NABs, indicating that a higher level of aggression was linked to an elevated hormonal stress response. Furthermore, LAB residents showed more neuronal activation in the parvocellular part of the hypothalamic paraventricular nucleus (PVN) than HAB residents 1 h after the RI-test. In addition, a tendency toward a higher number of c-Fos-positive cells in LABs compared with HABs was observed in the medial amygdala, hypothalamic attack area and central amygdala, areas relevant for the regulation of aggression. These data demonstrate that low trait anxiety is correlated with high intermale aggression. Furthermore, the increased neuronal activation of the PVN along with the higher ACTH responsiveness might underlie the display of high aggression.     

Predatory aggression, but not maternal or intermale aggression, is associated with high voluntary wheel-running behavior in mice

Predatory (towards crickets), intermale, and maternal aggression were examined in four replicate lines of mice that had been selectively bred for high wheel-running (S) and in four random-bred control lines (C). In generation 18, individual differences in both predatory and intermale aggression were significantly consistent across four trial days, but predatory and intermale aggression were uncorrelated both at the individual level and among the eight line means. Latencies to attack crickets were significantly lower in S lines as a group. Intermale aggression, however, did not differ between S and C lines. S lines were significantly smaller in body mass, but did not differ in either testes mass or plasma testosterone. In generations 28 and 30, respectively, S and C lines did not differ in either maternal or intermale aggression. However, significant differences among the individual lines were found for maternal aggression, and one S line exhibited an extremely high mean time of aggression (>120 sec for a 5-min test). Maternal and intermale aggression were not correlated among the eight line means or at the level of individual variation. Overall, our results suggest: (1) predatory aggression and voluntary wheel-running are positively related at the genetic level; (2) predatory and intermale aggression are unrelated at a genetic level; and (3) maternal and intermale aggression are not tightly related at the genetic level. Possible relationships between predatory aggression, dopamine, and wheel-running behavior are discussed.     

Thyroid gland responses to intermale aggression in an inherently aggressive wild rat

The aim of the present investigation was to ascertain whether intermale aggression (by fighting) affects thyroid gland activity in wild rat. Following intermale aggression for three hours, thyro-follicular epithelial height was significantly decreased and colloidal content in follicular lumen increased only in the subordinate rats whereas the thyroid gland of the dominant rats was not perceptibly changed. Thus, aggression inhibits thyroid gland activity only in the subordinate rats. It is suggested that this effect may be due to psychosomatic rather than physical stress during aggression.     

Relationships between rapid changes in local aromatase activity and estradiol concentrations in male and female quail brain

Estradiol-17β (E₂) synthesized in the brain plays a critical role in the activation of sexual behavior in many vertebrate species. Because E₂ concentrations depend on aromatization of testosterone, changes in aromatase enzymatic activity (AA) are often utilized as a proxy to describe E₂ concentrations. Utilizing two types of stimuli (sexual interactions and acute restraint stress) that have been demonstrated to reliably alter AA within minutes in opposite directions (sexual interactions = decrease, stress = increase), we tested in Japanese quail whether rapid changes in AA are paralleled by changes in E₂ concentrations in discrete brain areas. In males, E₂ in the pooled medial preoptic nucleus/medial portion of the bed nucleus of the stria terminalis (POM/BST) positively correlated with AA following sexual interactions. However, following acute stress, E₂ decreased significantly (approximately 2-fold) in the male POM/BST despite a significant increase in AA. In females, AA positively correlated with E₂ in both the POM/BST and mediobasal hypothalamus supporting a role for local, as opposed to ovarian, production regulating brain E₂ concentrations. In addition, correlations of individual E₂ in POM/BST and measurements of female sexual behavior suggested a role for local E₂ synthesis in female receptivity. These data demonstrate that local E₂ in the male brain changes in response to stimuli on a time course suggestive of potential non-genomic effects on brain and behavior. Overall, this study highlights the complex mechanisms regulating local E₂ concentrations including rapid stimulus-driven changes in production and stress-induced changes in catabolism.     

Glutamate release in the ventromedial hypothalamus of the female rat during copulation: Modulation by estradiol

Binding of glutamate or its ionotropic receptor agonists in the ventromedial hypothalamus (VMH) of female rats inhibits both appetitive and consummatory aspects of sexual behavior. Because vaginocervical stimulation activates glutamate neurons in the VMH, and administration of estradiol benzoate (EB) and progesterone (P) delays this effect, the present study examined the effects of hormonal priming on glutamate release within the VMH of female rats paired with sexually vigorous males. Ovariectomized, sexually experienced rats were implanted with guide cannula aimed at the ventrolateral VMH, through which microdialysis probes were inserted prior to testing. Females were assigned randomly to one of three hormone treatment conditions: EB + P, EB alone, or the oil vehicle. Testing was conducted over 5 h, including a 120-min period of habituation to the testing chamber, a 60-min period of baseline sample collection, and a 120-min period during which a sexually vigorous male was introduced into the testing chamber. Dialysates were collected every 20 min during the test and were analyzed for glutamate using HPLC. Females primed with oil had large and significant increases in glutamate release from baseline once the male was introduced to the chamber. Treatment with EB alone decreased glutamate release in response to male cues. Although treatment with EB + P did not differ significantly from EB alone, the degree of reduced glutamate release was less than with EB alone. These results indicate that priming with EB reduces glutamate transmission in the VMH in response to male cues. Taken together with our previous findings, estradiol blunts the activation of glutamate neurons in the VMH thus allowing female rats to copulate.     

Circulating obestatin is increased in patients with cardiorenal syndrome and positively correlated with vasopressin

Obestatin regulates fluid and electrolyte homeostasis mainly by opposing the action of vasopressin (AVP). We measured plasma concentration of obestatin and AVP in patients with cardiorenal syndrome (CRS). Plasma AVP and obestatin concentration were measured in 34 patients with type II CRS. The data were compared to that in 31 patients with chronic kidney disease (CKD), 41 patients with chronic heart failure (CHF) and 30 healthy subjects. Obestatin was significantly higher in the patients with CRS (355.8 ± 85.1 pg/ml) than that in the healthy controls (212.3 ± 37.9 pg/ml, P < 0.01), the patients with CKD (246.7 ± 34.3 pg/ml, P < 0.01) and the patients with CHF (258.4 ± 112.1 pg/ml, P < 0.01). AVP was also significantly higher in the patients with CRS (65.1 ± 36.0 pg/ml) than that in the healthy controls (38.5 ± 20.1 pg/ml, P < 0.01), the patients with CKD (50.4 ± 24.8 pg/ml, P < 0.01) and the patients with CHF (54.6 ± 16.3 pg/ml, P < 0.01). Plasma concentration of obestatin was positively correlated with AVP plasma concentration in the overall analysis that included subjects from all disease categories (r = 0.219, P < 0.05), but not within the CRS group. Plasma obestatin and vasopressin were elevated in patients with CRS. Plasma obestatin concentration seemed to be positively correlated with plasma AVP.     

Neurogenesis of galanin cells in the bed nucleus of the stria terminalis and centromedial amygdala in rats: a model for sexual differentiation of neuronal phenotype

Male rats possess twice as many cells that express arginine-vasopressin (AVP) in the bed nucleus of the stria terminalis (BST) and centromedial amygdala (CMA) as do females. This sex difference may arise from sex differences in the induction of AVP expression in galanin (GAL)-expressing cells, which themselves do not differ in number between males and females. To test whether AVP expression could arise from a single pool of galaninergic cells, we determined whether the cell birth profile of GAL-immunoreactive (ir) cells was similar to that of AVP-ir cells. Dams were injected with the cell birth marker bromodeoxyuridine (BrdU) on one of seven gestational dates, ranging from embryonic day 11 (E11) to E17. The resulting offspring were sacrificed at 3 months of age. Processing their brains for the presence of either GAL and BrdU, or AVP and BrdU immunoreactivity revealed that in both the BST and CMA, the majority of GAL-ir and AVP-ir cells were labeled with BrdU on E12 and E13. In contrast, most other cells in the same region were labeled on E14 and E15. The similarity in the timing of cell birth of the GAL-ir and AVP-ir cells is consistent with the idea that GAL-ir cells in the BST/CMA constitute a single pool of cells that may be induced to express AVP during development.     

刺激室旁核及加压素对大鼠胃缺血-再灌注损伤的保护作用

采用夹闭大鼠腹腔动脉30min,松开动脉夹血流复灌1h的胃缺血-再灌注损伤(gastric ischemia-reper-fusion injury,GI-RI）模型,观察了电或化学刺激室旁核（paraventricular nucleus,PVN）及外源性加压素（arginine-va-sopression,AVP）对GI-RI的影响,并对PVN的调控通路进行了初步分析。结果表明：电或化学刺激PVN后,GI-RI显著减轻;损毁双侧孤束核（nucleus tractus solitarius,NTS）或一侧NTS内注射AVP-V1受体阻断剂,均能取消电刺激PVN对GI-RI的效应;去除脑垂体后不影响PVN的作用;切断膈下迷走神经或切除腹腔交感神经节,则能加强电刺激PVN对GI-RI的影响;PVN内注射不同剂量的AVP同样能减轻大鼠GI-RI损伤。结果提示：PVN及AVP对大鼠GI-RI具有保护作用;PVN的这种作用可能是因电或化学刺激后,激活了其中的加压素能神经元,经其下行投射纤维释放AVP作用于NTS神经元的VAP-V1受体,并通过迷走和交感神经介导,从而影响GI-RI;而似与PVN-垂体通路关系不大。     

Interaction of onconase with the human ribonuclease inhibitor protein

One of the tightest known protein-protein interactions in biology is that between members of the ribonuclease A superfamily and the ribonuclease inhibitor protein (RI). Some members of this superfamily are able to kill cancer cells, and the ability to evade RI is a major determinant of whether a ribonuclease will be cytotoxic. The archetypal cytotoxic ribonuclease, onconase (ONC), is in late-stage clinical trials for the treatment of malignant mesothelioma. We present here the first measurement of the inhibition of the ribonucleolytic activity of ONC by RI. This inhibition occurs with K_i = 0.15 μM in a solution of low salt concentration.     

Structure and antioxidant activity of an extracellular polysaccharide from coral-associated fungus, Aspergillus versicolor LCJ-5-4

An extracellular polysaccharide AVP was isolated from the fermented broth of coral-associated fungus Aspergillus versicolor LCJ-5-4. AVP was a mannoglucan with molecular weight of about 7 kDa, and the molar ratio of glucose and mannose was 1.7:1.0. On the basis of detailed one- and two-dimensional nuclear magnetic resonance (1D and 2D NMR) spectroscopic analyses, the backbone of AVP was characterized to be composed of (1 → 6)-linked α-d-glucopyranose and (1 → 2)-linked α-d-mannopyranose units. The mannopyranose residues in the backbone were substituted mainly at C-6 by the side chain of (1 → 2)-linked α-d-mannopyranose trisaccharides units. The antioxidant activity of AVP was evaluated with the scavenging abilities on 1,1-diphenyl-2-picrylhydrazyl (DPPH), superoxide and hydroxyl radicals in vitro, and the results indicated that AVP had good antioxidant activity, especially scavenging ability on superoxide radicals. AVP was a novel extracellular polysaccharide with different structural characteristics from other extracellular polysaccharides and could be a potential source of antioxidant.     

Contractile responses of human thyroid arteries to vasopressin

Aims

In the present study we investigated the intervention of nitric oxide and prostacyclin in the responses to vasopressin of isolated thyroid arteries obtained from multi-organ donors.

Main methods

Paired artery rings from glandular branches of the superior thyroid artery, one normal and the other deendothelised, were mounted in organ baths for isometric recording of tension. Concentration–response curves to vasopressin were determined in the absence and in the presence of either the vasopressin V₁ receptor antagonist d(CH₂)₅Tyr(Me)AVP (10^− 8 M), the nitric oxide synthase inhibitor N^G-monomethyl-l-arginine (L-NMMA, 10^− 4 M), or the inhibitor of prostaglandins indomethacin (10^− 6 M).

Key findings

In artery rings under resting tension, vasopressin produced concentration-dependent, endothelium-independent contractions. The vasopressin V₁ receptor antagonist d(CH₂)₅Tyr(Me)AVP (10^− 8 M) displaced the control curve to vasopressin 19-fold to the right in a parallel manner. The contractile response to vasopressin was unaffected by L-NMMA or by indomethacin.

Significance

Vasopressin causes constriction of human thyroid arteries by stimulation of V₁ vasopressin receptors located on smooth muscle cells. These effects are not linked to the presence of an intact endothelium or to the release of nitric oxide or prostaglandins. The constriction of thyroid arteries may be particularly relevant in certain pathophysiological circumstances in which vasopressin is released in amounts that could interfere with the blood supply to the thyroid gland.     

Ca 2+-induced self-assembly in designed peptides with optimally spaced gamma-carboxyglutamic acid residues

We have previously elucidated a new paradigm for the metal ion-induced helix-helix assembly in the natural γ-carboxyglutamic acid (Gla)-containing class of conantokin (con) peptides, typified by con-G and a variant of con-T, con-T[K7Gla], independent of the hydrophobic effect. In these “metallo-zipper” structures, Gla residues spaced at i, i + 4, i + 7, i + 11 intervals, which is similar to the arrangement of a and d residues in typical heptads of coiled-coils, coordinate with Ca²⁺ and form specific antiparallel helical dimers. In order to evaluate the common role of Gla residues in peptide self-assembly, we extend herein the same Gla arrangement to designed peptides: NH₂-(γLSγEAK)₃-CONH₂ (peptide 1) and NH₂-γLSγEAKγLSγQANγLSγKAE-CONH₂ (peptide 2). Peptide 1 and peptide 2 exhibit no helicity alone, but undergo structural transitions to helical conformations in the presence of a variety of divalent cations. Sedimentation equilibrium ultracentrifugation analyses showed that peptide 1 and peptide 2 form helical dimers in the presence of Ca²⁺, but not Mg²⁺. Folding and thiol-disulfide rearrangement assays with Cys-containing peptide variants indicated that the helical dimers are mixtures of antiparallel and parallel dimers, which is different from the strict antiparallel strand orientations of con-G and con-T[K7γGla] dimers. These findings suggest that the Gla arrangement, i, i + 4, i + 7, i + 11, i + 14, plays a key role in helix formation, without a strict adherence to strand orientation of the helical dimer.     

Agrobacterium-mediated transformation of leaf base derived callus tissues of popular indica rice (Oryza sativa L. sub sp. indica cv. ADT 43)

A simple and efficient protocol for the Agrobacterium-mediated transformation of an agronomically useful abiotic sensitive popular indica rice cv. ADT 43 has been developed. Initiation of calli were best achieved from the leaf bases of 4 days old rice seedlings on LS medium supplemented with 2.5 mg/L 2,4-D and 1.0 mg/L thiamine-HCl. Rice calli immersed in Agrobacterium suspension (strain EHA 105, OD₆₀₀ = 0.8) were co-cultured on LS30-AsPC medium for 2 days at 25 ± 2 °C in the dark. Based on GUS expression analysis, 10 min co-cultivation time with 100 μM acetosyringone was found optimum for the delivery of gus gene. Calli were proved to be very sensitive to Agrobacterium infection and we found that the level of necrotic response can be minimized after co-cultivation with 30% LS, 10 g/L PVP, 10% coconut water and 250 mg/L timentin which improved the final transformation efficiency to 9.33%. Molecular and genetic analysis of transgenic plants reveals the integration, expression and inheritance of transgene in the progeny (T₁) of these plants. The copy number of transgenes has been found to vary from 1 to 2 in transgenic plants (T₀ and T₁).     

Change in certain forms of aggressive behavior and the concentration of monoamines in the brain during selection of wild rats for taming

Norway rats have been selected during 20 generations by the absence of aggressive reaction to man (tamed rats). From 7 up to 20th generations of selection, different forms of aggressive behaviour (reaction to glove, intermale, shock-induced aggression and predatory aggression) were studied, and the level of noradrenaline, serotonin and its metabolite 5-hydroxyindoleacetic acid was determined in the brain. In the absence of aggressive reaction to glove in tamed rats, the shock-induced aggression considerably decreased while the predatory aggressiveness (mouse-killing behaviour) and intermale aggressiveness did not change. Beginning from 15-16th generation of selection, a higher level of the 5-hydroxyindoleacetic acid in the hypothalamus was established, in the 20th generation an increased content of serotonin was revealed in the hypothalamus and the midbrain. In some generations of selection an increased level of noradrenaline in the hypothalamus in comparison to wild rats was observed. A conclusion is made that the selection of animals by taming unequally influences different kinds of aggressiveness and is accompanied by inherited consolidated reorganization of the monoamine brain systems.     

Organizational effects of testosterone,estradiol, and dihydrotestosterone on vasopressin mRNA expression in the bed nucleus of the stria terminalis

In adulthood, male rats express higher levels of arginine vasopressin (AVP) mRNA in the bed nucleus of the stria terminalis (BST) than do female rats. We tested whether this sex difference is primarily due to differences in neonatal levels of testosterone. Male and female rats were gonadectomized on the day of birth and treated with testosterone propionate (TP) or vehicle on postnatal days 1, 3, and 5 (P1, P3, and P5). Three months later, all rats were implanted with testosterone‐filled capsules. Two weeks later, brains were processed for in situ hybridization to detect AVP mRNA. We found that neonatal TP treatment significantly increased the number of vasopressinergic cells in the BST over control injections. We then sought to determine the effects of testosterone metabolites, estradiol and dihydrotestosterone, given alone or in combination, on AVP expression in the BST. Rat pups were treated as described above, except that instead of testosterone, estradiol benzoate (EB), dihydrotestosterone propionate (DHTP), a combination of EB and DHTP (EB+DHTP), or vehicle was injected neonatally. Neonatal treatment with either EB or EB+DHTP increased the number of vasopressinergic cells in the BST over that of DHTP or oil treatment. However, treatment with DHTP also significantly increased the number of vasopressinergic cells over that of oil treatment. Hence, in addition to bolstering evidence that estradiol is the more potent metabolite of testosterone in causing sexual differentiation of the brain, these data provide the first example of a masculinizing effect of a nonaromatizable androgen on a sexually dimorphic neuropeptide system. © 2003 Wiley Periodicals, Inc. J Neurobiol 54: 502–510, 2003