Chronic Intranasal Oxytocin has Dose-dependent Effects on Central Oxytocin and Vasopressin Systems in Prairie Voles (Microtus ochrogaster).

Oxytocin (Oxt) is a neuropeptide with many functions, including modulation of social behavior(s) and anxiety. Due to its notable pro-social effects, it has been proposed as a treatment in the management of neuropsychiatric disorders, such as autism spectrum disorder (ASD), schizophrenia, and social anxiety; however, effects of long-term daily treatment are still being explored. Previously, we have shown that in male prairie voles (Microtus ochrogaster) exposure to Oxt during the peri-adolescent period impaired adult pair bonding in a dose-dependent fashion. In females, the medium dose used (0.8 IU/kg) appeared to facilitate pair bonding, and the low and medium doses were associated with fewer lines crossed in the open field. In this study, we examined central receptor binding and immunoreactive (IR) protein for Oxt and vasopressin (Avp), a closely related peptide. Voles were treated with saline vehicle, or one of three doses of Oxt (0.08, 0.8, 8.0 IU/kg) for three weeks from postnatal days 21 to 42, and euthanized as adults. We used autoradiography to examine Oxt and Avp receptor binding and immunohistochemistry to examine Oxt and Avp - IR cells in the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus. Females that received the medium dose of Oxt had higher Oxt receptor binding in the nucleus accumbens shell (NAS), while males that received the medium dose had lower Avp-IR cells in the PVN. In summary, we found sex-specific effects of long-term exposure to intranasal Oxt on the Oxt and Avp systems at the weight-adjusted dose currently being used in clinical trials in humans.

Brain derived neurotrophic factor (BDNF) containing neurons in the hypothalamic paraventricular and supraoptic nuclei of juvenile and middle-aged rats after chronic stress.

The type and duration of stress stimulation are postulated to affect the expression of the brain derived neurotrophic factor (BDNF) differentially during ontogenetic life. The aim of our study was to investigate the influence of two different stressors, i.e. chronic (15 min daily for 21 days) exposure to the forced swim (FS) test or the high light open field (HL-OF) test, on the BDNF contained in magnocellular (PVm) and parvocellular (PVp) neurons of the hypothalamic paraventricular (PV) and the supraoptic (SO) nuclei. The immunofluorescence (-ir) method was used to detect BDNF-ir cells. The research showed that only the PVp part of the PV in juvenile (P28; P-postnatal day) control rats had a significantly lower density of BDNF-ir neurons than that in middle-aged (P360) control subjects. After chronic FS, a significant decrease in BDNF-ir cells was observed in the studied hypothalamic nuclei of the juvenile rats, but no changes were noted in the middle-aged individuals. The PV (PVm, PVp) and the SO nuclei in juvenile rats showed a significantly lower density of BDNF-ir neurons than the corresponding area of the hypothalamus in middle-aged rats. However, following the HL-OF test, the density of BDNF-ir neurons remained unaltered both in the P28 and the P360 groups. The data suggest that the type of the stressor applied was the factor that differentiated the number of BDNF-ir cells in the PVm and the SO only in juvenile rats: chronic HL-OF was more severe than FS. The age of the animals was the main factor that conditioned the BDNF hypothalamic PV (PVm, PVp) and the SO response to FS stimulation. The different density of BDNF-ir containing cells in the PVp of juvenile versus middle-aged rats can be explained by a functional, age-related change in the demand of PVp neurons for BDNF.

Morphological effects of isoflavones (daidzein and genistein) on hypothalamic oxytocin neurons in the neonatal mouse brain slice cultures.

In adults, oxytocin (OXT) has various central functions including social behavior and reproduction. Many of these functions are steroid dependent and are also influenced by naturally occurring phytoestrogens, isoflavones (IFs). The aim of this study was, therefore, to clarify the effects of IFs on OXT neurons in the brain. In particular, the influence of IFs on the central OXT system of infant animal needs to be examined, because IFs are increasingly consumed for weaning as well as dietary supplements. We have morphologically analyzed the central OXT neurons in neonatal mice using slice cultures treated with IFs, daidzein (Ddz) and genistein (Gen). In the supraoptic nucleus (SON) of male mice, Gen decreased the size of OXT neurons, but not of female nor in the paraventricular hypothalamic nucleus (PVN) of neither gender. In female PVN, Ddz and 17ß-estradiol (E(2)) increased the frequencies of varicosity on neurites in small OXT neurons (<21µm diameter of cell body). Ddz and Gen, as well as E(2), induced prominent vacuolation of the OXT neurons more frequently in male than in female mice, and in SON than in PVN. Thus, IFs can modulate the hypothalamic OXT neurons and the effects are site-specific and sexually dimorphic, suggesting that neonatal exposure to IFs may modify such a steroid-dependent development of particular neural pathways, including OXT system.

Vasopressinergic neurons of the supraoptic nucleus in perinatal rats: reaction to osmotic stimulation and its regulation.

Osmotic stimulation (OS) of vasopressin (VP) neurons of the supraoptic nucleus (SON) promotes VP secretion and tyrosine hydroxylase (TH) expression in adult mammals. VP secretion is under a noradrenaline control, whereas the regulation of TH expression remains uncertain. This study was aimed to determine at what period of ontogenesis: (1) VP neurons begin to react to OS by modifying simultaneously VP and TH gene expression and synthesis, (2) the noradrenergic control of VP neurons is established. Rats on the 21st embryonic day (E), third postnatal day (P), P13 were salt loaded or salt loaded and treated with an antagonist (prazosin) or agonist (phenylephrine) of α1-adrenoreceptors. According to our immunocytochemical and in situ hybridization data, OS resulted in an increased amount of VP mRNA in each age group and of VP on E21 and P3. TH gene and synthesis was initially expressed under OS on P3. The number of TH-expressing neurons diminished by threefold in salt loaded rats from P3 to P13. OS combined with prazosin administration resulted in an increased level of VP mRNA on P3 and P13, but not on E21 suggesting the onset of the noradrenaline inhibitory control after birth. OS together with prazosin treatment stimulated TH expression on P3 and P13, whereas phenylephrine provided an opposite effect. Thus, VP neurons begin to react to OS by an increased VP synthesis at the end of fetal life and by the onset of TH expression shortly after birth; the expression of both substances appears to be under the inhibitory control of noradrenaline.

Effects of neonatal handling on social memory, social interaction, and number of oxytocin and vasopressin neurons in rats.

Early-life environmental events can induce profound long-lasting changes in several behavioral and neuroendocrine systems. The neonatal handling procedure, which involves repeated brief maternal separations followed by experimental manipulations, reduces stress responses and sexual behavior in adult rats. The purpose of this study was to analyze the effects of neonatal handling on social behaviors of male and female rats in adulthood, as manifest by the results of social memory and social interaction tests. The number of oxytocin (OT) and vasopressin (VP) neurons in the paraventricular (PVN) and supraoptic (SON) nuclei of hypothalamus were also analyzed. The results did not demonstrate impairment of social memory. Notwithstanding, handling did reduce social investigative interaction and increase aggressive behavior in males, but did not do so in females. Furthermore, in both males and females, handling was linked with reduced number of OT-neurons in the parvocellular region of the PVN, while no differences were detected in the magnocellular PVN or the SON. On the other hand, handled males exhibited increased number of VP-neurons in the magnocellular zone of the PVN. We may conclude that the repeated brief maternal separations can reduce affiliative social behavior in adult male rats. Moreover, the disruption of the mother-infant relationship caused by the handling procedure induced long-lasting morphological changes in critical neuroendocrine areas that are involved in social bonding in mammals.

Nuclear compartmentalization and dynamics of the poly(A)-binding protein nuclear 1 (PABPN1) inclusions in supraoptic neurons under physiological and osmotic stress conditions.

Nuclear aggregation of the expanded polyalanine tract in the poly(A)-binding protein nuclear 1 (PABPN1) is the pathological hallmark of oculopharyngeal muscular dystrophy. However, wild type PABPN1 aggregates into nuclear inclusion in oxytocin-producing neurons under physiological conditions. In this study we have analyzed the nuclear organization and dynamics of PABPN1 inclusions in oxytocin-producing neurons. We demonstrated that PABPN1 inclusions represent a distinct compartment of the interchromatin region. They establish a spatial relationship with nuclear speckles, Cajal bodies and clastosomes. PABPN1 inclusions accumulate poly(A) RNA, but do not concentrate highly expressed mRNAs in oxytocin producing neurons and the mRNA-binding proteins hnRNP C, Y14 and REF. PABPN1 inclusions are dynamic structures that appear during the postnatal period and their number decrease in response to the activation of transcription. Our results support that the RNA retained in the PABPN1 inclusions is a noncoding regulatory RNA involved in some aspects of nuclear RNA metabolism.

ANP presence in the hypothalamic suprachiasmatic nucleus of developing rat.

In previous research, we studied both the oxytocin and vasopressin ontogeny in the hypothalamic supraoptic and paraventricular nuclei, and the ANP-ontogeny in the hypothalamic supraoptic nucleus. In this paper we evaluate the ANP-ontogeny in the rat hypothalamic suprachiasmatic nucleus; infact the suprachiasmatic nucleus it is known to synthesize vasopressin, a peptidic hormone involved in the homeostasis of the body fluids by an antagonistic role to ANP. Immunohistochemical techniques show that ANP is present in the hypothalamic suprachiasmatic nucleus of the rat at 18 degrees day of i.u. life and at 0 degrees to 3 degrees day of postnatal life. PCR analysis confirms the ANP-mRNA expression. Thus, it is possible to adfirm that the suprachiasmatic nucleus is a synthesis site of ANP, and ANP appears in both the supraoptic and suprachiasmatic nuclei at the same developmental stage. Moreover, ANP and vasopressin appear at the same developmental stage since both the peptides are involved in the homeostasis of body fluids.

Postnatal developmental changes in Ca2+ homeostasis in supraoptic magnocellular neurons.

Supraoptic magnocellular neurons (SMNs) undergo dramatic changes in morphological and electrical properties during postnatal development. We investigated the developmental change in Ca2+ homeostasis in SMNs. The decay rate of Ca2+ transients markedly increased during the third postnatal week (PW3) to an adult level. This increase in the Ca2+ decay rate was paralleled by hypertrophy of the SMN somata. Activity of Na+/Ca2+ exchanger (Na/CaX) and sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) was quantified as a decrement in the Ca2+ decay rate caused by extracellular [Na+] reduction and that by thapsigargin, respectively. SERCA activity was negligible during PW2, and markedly increased during PW3. SERCA activity and soma size remained stable thereafter. Na/CaX activity was a major Ca2+-clearance mechanism (CCM) during PW2, increased further during PW3, but was negligible in mature SMNs (PW10). In parallel with the decrease in Na/CaX activity, endogenous Ca2+ buffering capacity declined, resulting that the apparent Ca2+ decay rate remained relatively constant between PW4 and PW10. Replacement of intracellular K+ with Li+ had no effect on Na/CaX activity, suggesting that NCX rather than NCKX comprises Na/CaX. These findings indicate a developmental shift in the balance of CCMs from Ca2+ extrusion via NCX toward Ca2+ sequestration into endoplasmic reticulum via SERCA.

A store-operated current (SOC) mediates oxytocin autocontrol in the developing rat hypothalamus.

Oxytocin (OT) and vasopressin (VP) autocontrol their secreting neurons in the supraoptic nucleus (SON) by modulating action potential firing through activation of specific metabotropic receptors. However, the mechanisms linking receptor activation to firing remain unknown. In almost all cell types, activation of plasma membrane metabotropic receptors triggers signalling cascades that induce mobilization of calcium from intracellular stores. In turn, emptying the calcium stores may evoke calcium influx through store-operated channels (SOCs), the functions of which remain largely unknown in neurons. In this study, we show that these channels play a key role in the SON, at least in the response to OT. In isolated rat SON neurons, store depletion by thapsigargin induced an influx of calcium, demonstrating the presence of SOCs in these neurons. This calcium influx was specifically inhibited by 0.2 mM 1-(2-trifluoromethylphenyl-)imidazole (TRIM). At 2 mM, this compound affected neither the resting electrophysiological properties nor the voltage-dependant inward currents. In fresh slices, TRIM (2 mM) did not affect the resting potential of SON neurons, action potential characteristics, spontaneous action potential firing or synaptic activity; this compound thus appears to be a specific blocker of SOCs in SON neurons. TRIM (0.2 mM) specifically reduced the increase in action potential firing triggered by OT but did not affect the VP-induced response. These observations demonstrate that store operated channels exist in hypothalamic neurons and specifically mediate the response to OT in the SON.

Age-related changes in oxytocin-, arginine vasopressin- and nitric oxide synthase-expressing neurons in the supraoptic nucleus of the rat.

Many histochemical investigations indicated that the oxytocin (OXY), the arginine vasopressin (AVP) and the nitric oxide synthase (NOS) have been synthesized in the supraoptic nucleus (SON) neurons. The objective of this study was to examine the age-related expression of the OXY, the AVP and the NOS in the SON of the young adult (2-month-old) and the aged (24-month-old) rats. The histochemistry for reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d; marker for the NOS) and the double labeling histochemistry for the OXY/NADPH-d or the AVP/NADPH-d were employed, and the quantitative analysis was performed with a computer-assisted image processing system. In comparison of the young adult and the aged group, the cell number, the cell size and the reactive density of the NOS-expressing neurons showed a significant increase along with age, and these evidences suggested the age-related increase of the nitric oxide (NO) production. The age-related significant increase was not detected in the number of the OXY/NOS-expressing neurons in the dorsal part, but was detected in the number of the AVP/NOS-expressing neurons in the ventral part. Based on our histochemical findings and reports demonstrated by other authors, we attempted to discuss the physiological role of NOS for the secretion of posterior pituitary hormones along with age.

Neurosteroid regulation of oxytocin and vasopressin release from the rat supraoptic nucleus.

In adult rats somato-dendritic release of oxytocin and vasopressin from magnocellular neurones in the supraoptic nucleus of the hypothalamus has important autoregulatory actions on the neuronal electrical activity, and in neonatal rats it plays a role in the development of dendritic arborisation. In the adult, oxytocin effects are modulated by allopregnanolone via an interaction with inhibitory GABAA receptors. This study examined the effects of allopregnanolone, progesterone and 17beta-oestradiol on oxytocin and vasopressin release from intact isolated supraoptic nuclei and from the neurophypophyses in rats of differing ages. In supraoptic nuclei from rats of 3-4 weeks old or less, all three neurosteroids induced oxytocin release from the isolated supraoptic nucleus, but only allopregnanolone induced significant release of vasopressin. Surprisingly, in these very young rats, allopregnanolone-induced oxytocin release was inhibited by GABAA receptor antagonists as well as by an oxytocin receptor antagonist. By contrast, in supraoptic nuclei from adult rats allopregnanolone-induced oxytocin release was much smaller, and was enhanced in the presence of bicuculline. The GABAA receptor agonist muscimol also induced oxytocin release from supraoptic nuclei in young rats, but had no effect in adult rats. Oxytocin cells isolated from young rats showed an increase in [Ca2+]i in response to both allopregnanolone and muscimol. Allopregnanolone had no effect on [Ca2+]i or on the release of oxytocin or vasopressin from neurohypophysial axon terminals in either young or old rats. We conclude that, in very young rats, (i) neurosteroids induce oxytocin release from the supraoptic nucleus by a mechanism that partly depends on the presence of GABA, which in young rats is depolarising to oxytocin cells, and which also partly depends upon endogenous oxytocin, and (ii) the effect of allopregnanolone upon oxytocin release changes with age, as the functional activity of GABAA receptors changes from excitation to inhibition of oxytocin cells.

Space flight affects magnocellular supraoptic neurons of young prepuberal rats: transient and permanent effects.

Effects of microgravity on postural control and volume of extracellular fluids as well as stress associated with space flight may affect the function of hypothalamic neurosecretory neurons. Since environmental modifications in young animals may result in permanent alterations in neuroendocrine function, the present study was designed to determine the effect of a space flight on oxytocinergic and vasopressinergic magnocellular hypothalamic neurons of prepuberal rats. Fifteen-day-old Sprague-Dawley female rats were flown aboard the Space Shuttle Columbia (STS-90, Neurolab mission, experiment 150) for 16 days. Age-matched litters remained on the ground in cages similar to those of the flight animals. Six animals from each group were killed on the day of landing and eight animals from each group were maintained under standard vivarium conditions and killed 18 weeks after landing. Several signs of enhanced transcriptional and biosynthetic activity were observed in magnocellular supraoptic neurons of flight animals on the day of landing compared to control animals. These include increased c-Fos expression, larger nucleoli and cytoplasm, and higher volume occupied in the neuronal perikaryon by mitochondriae, endoplasmic reticulum, Golgi apparatus, lysosomes and cytoplasmic inclusions known as nematosomes. In contrast, the volume occupied by neurosecretory vesicles in the supraoptic neuronal perikarya was significantly decreased in flight rats. This decrease was associated with a significant decrease in oxytocin and vasopressin immunoreactive levels, suggestive of an increased hormonal release. Vasopressin levels, cytoplasmic volume and c-Fos expression returned to control levels by 18 weeks after landing. These reversible effects were probably associated to osmotic stimuli resulting from modifications in the volume and distribution of extracellular fluids and plasma during flight and landing. However, oxytocin levels were still reduced at 18 weeks after landing in flight animals compared to controls. This indicates that space flight during prepuberal age may induce irreversible modifications in the regulation of oxytocinergic neurons, which in turn may result in permanent endocrine and behavioral impairments.

The hypothalamic magnocellular neurosecretory system in developing rats.

Studies concerning the development of the magnocellular system are scarce and discordant in literature. We carried out an immunohistochemical study on supraotic and paraventricular hypothalamic nuclei using antivasopressin and antioxytocin antibodies in developing rats between the 15th day of intrauterine life and the 6th day of postnatal life. In addition, we performed RT-PCR experiments to establish the stage at which these hormones appear and neurosecretory activity commences. The results showed that supraoptic and paraventricular nuclei appear, respectively, on the 16th and the 18th day of intrauterine life and both immediately synthetize vasopressin neurohormone. By contrast, synthesis of oxytocin takes place from the 2nd day after birth. Probably, these nuclei synthetize oxytocin in conjunction with the decline of placental maternal oxytocin.

Effects of experimental hypothyroidism on the development of the hypothalamo-pituitary-adrenal axis in the rat.

Hypothyroid pups were obtained by adding methimazole in the mother's drinking water from day 15 of gestation and sacrificed at 4, 8 or 15 days. Circulating corticosterone decreased at all ages, while CBG concentrations diminished at day 4, increased at day 8 and did not change at day 15 in hypothyroid rats. As opposed to controls, plasma ACTH concentrations decreased steadily with age while there was an accumulation of ACTH in the anterior pituitary of hypothyroid 15-day-old rats. Anterior pituitary POMC contents were unaffected by the treatment. In the hypothalamic PVN, CRF mRNA levels in the total population of CRF-synthesizing cells and in the CRF+/AVP+ subpopulation were below those of controls whatever the age considered while AVP mRNA in the CRF+/AVP+ subpopulation did not change at day 4 and decreased at day 8 and 15 in hypothyroid animals. Both the number of cell bodies expressing detectable levels of CRF mRNA and the percentage of CRF and AVP colocalization decreased at day 4 and were unchanged thereafter. CRF and AVP immunoreactivity in the zona externa of the median eminence increased with age but was not affected by methimazole treatment. The concentration of AVP mRNA in the magnocellular cell bodies of the PVN and the SON as well as AVP immunoreactivity in the zona interna of the median eminence were not changed by the treatment at days 4 and 8. In hypothyroid 15-day-old rats, SON AVP mRNA increased, AVP immunoreactivity decreased while plasma osmolality was enhanced. In conclusion, our data demonstrate that experimental hypothyroidism impairs specifically the maturation of hypothalamic parvocellular CRF and AVP gene expression during the stress hyporesponsive period. These observations suggest that the physiological peak in plasma thyroxine concentrations that occur between day 8-12 may participate in the maturation of hypothalamic CRF- and AVP-synthesizing cells.

Developmental regulation of a local positive autocontrol of supraoptic neurons.

Mature oxytocin (OT) and vasopressin (AVP) magnocellular neurons of the hypothalamic supraoptic nuclei (SON) autocontrol their electrical activity via somatodendritic release of their respective peptides. Because OT and AVP are synthesized early in development and could play an important role in the maturation of these neurons, we checked whether the peptides are released within the SON and act on their secreting neurons during 3 weeks of postnatal development. We used patch-clamp recordings from SON neurons in rat hypothalamic horizontal slices to show that the spontaneous electrical activity of immature SON neurons is blocked by OT or AVP receptor antagonists, demonstrating a basal somatodendritic release of the peptides. Application of OT or AVP depolarizes SON neurons and stimulates activity typical of the corresponding mature neurons. This effect is directly on SON neurons because it is recorded in dissociated neurons. Radioimmunoassays from isolated SON were used to show that each peptide facilitates its own release at a somatodendritic level, exhibiting a self-sustaining positive feedback loop. This autocontrol is not uniform during development because the proportion of neurons depolarized by the peptides, the amplitude of the depolarization, and the propensity of the peptides to facilitate their own release are maximal during the second postnatal week and decrease thereafter. These data are consistent with a role of autocontrol in the maturation of SON neurons because it is maximal during the delimited period of postnatal development that corresponds to the period of major synapse formation.

Development of neuroendocrine lineages requires the bHLH-PAS transcription factor SIM1.

The bHLH-PAS transcription factor SIM1 is expressed during the development of the hypothalamic-pituitary axis in three hypothalamic nuclei: the paraventricular nucleus (PVN), the anterior periventricular nucleus (aPV), and the supraoptic nucleus (SON). To investigate Sim1 function in the hypothalamus, we produced mice carrying a null allele of Sim1 by gene targeting. Homozygous mutant mice die shortly after birth. Histological analysis shows that the PVN and the SON of these mice are hypocellular. At least five distinct types of secretory neurons, identified by the expression of oxytocin, vasopressin, thyrotropin-releasing hormone, corticotropin-releasing hormone, and somatostatin, are absent in the mutant PVN, aPV, and SON. Moreover, we show that SIM1 controls the development of these secretory neurons at the final stages of their differentiation. A subset of these neuronal lineages in the PVN/SON are also missing in mice bearing a mutation in the POU transcription factor BRN2. We provide evidence that, during development of the Sim1 mutant hypothalamus, the prospective PVN/SON region fails to express Brn2. Our results strongly indicate that SIM1 functions upstream to maintain Brn2 expression, which in turn directs the terminal differentiation of specific neuroendocrine lineages within the PVN/SON.

Developmental plasticity of NR1 and NR2B subunit expression in the supraoptic nucleus of the rat hypothalamus.

Vasopressin and oxytocin neuroendocrine cells within the supraoptic nucleus of the adult hypothalamus (SON) display mRNA expression for the NMDA receptor subunits, NR1 and NR2B, NR2C and NR2D. The NR2B subunit confers slow decay kinetics (relative to NR1/NR2A receptors) and high magnesium sensitivity to NMDA receptor responses--properties which may contribute to the NMDA receptor-mediated bursting manifested by these cells. Therefore, we examined NR2B protein expression and its developmental profile in the SON and compared it to that in the cortex and cerebellum--areas which have been studied previously. We performed Western blot analysis on SON homogenates from embryonic, postnatal (PN7, 14, 21), and adult rats using an NR2B-specific antibody. Adult NR2B levels in the SON and PVN were similar but low relative to those of cortex. SON NR2B protein levels rose in the first postnatal week, remained high through PN21, and later declined to significantly lower levels in the adult. A similar profile was observed in cerebellum, where NR2B expression displayed a sharp peak at PN14 and later declined to minimal or undetectable levels in the adult. In contrast, NR2B continued to be overexpressed through adulthood in the cortex. The ontogenic pattern for NR1 expression, which included unregulation during early postnatal life and adulthood, was similar in the SON and cortex. A different pattern was observed for the cerebellum, where NR1 levels increased gradually after ED17 to reach significantly greater adult levels. Of all three areas studied, the SON displayed the earliest developmental rise in NR1 levels. SON explant cultures proved to be a useful preparation, since they contained neurons which synthesized NR1 and NR2B subunits in quantities similar to those of ED17 SON. Our findings suggest that NMDA receptors on SON neuroendocrine cells are assembled using NR1 and NR2B subunits, and that their plastic expression in early postnatal life may play a role during development.