Supraoptic Neurons Research Articles

Osmotically evoked PLCδ1-dependent translocation of ΔN-TRPV1 channels in rat supraoptic neurons

Osmoregulation is an essential homeostatic process that requires constant release of vasopressin during sustained increases in plasma osmolality. The magnocellular neurosecretory cells (MNCs) respond to increases in external osmolality through increases in the activity of ΔN-TRPV1 channels, which leads to increased action potential firing and vasopressin release. We show that sustained exposure of acutely isolated rat and mouse MNCs to hypertonic solutions (90min) causes a reversible translocation of ΔN-TRPV1 channels from internal stores to the plasma membrane that depends on the activation of phospholipase C and on SNARE-dependent exocytosis. ΔN-TRPV1 channel translocation is absent in MNCs isolated from transgenic mice lacking the PLCδ1 isoform, suggesting that PLCδ1 is essential for triggering this process. The translocation of ΔN-TRPV1 channels to the cell surface could contribute to the maintenance of MNC excitability during sustained increases in osmolality. Our data therefore have important implications for the mechanisms underlying mammalian osmoregulation.

The influence of melatonin on stress-induced ultramicroscopic changes of the neurons of the supraoptic nuclei of the rat hypothalamus

The aim: to study the effect of melatonin on the ultrastructural state of the supraoptic nuclei of the hypothalamus of rats under immobilization stress.Materials and methods. The experiments were performed on non-linear male white rats weighing 200-220 g. The animals were divided into 3 study series, in each of which the biomaterial was collected at 2 p.m. and at 2 a.m. using electron microscopic method. Long immobilization stress was simulated by keeping rats in special plastic penal cages for 6 hours daily for 7 consecutive days. Melatonin (Sigma, USA, 99.5% purification degree) at a dose of 0.5 mg/kg, in 1.0 ml of solvent (0.9% ethanol solution on physiologic saline) was injected daily, intraperitoneally.Results. When the animals were kept under the standard light regime, the ultrastructural organization of the hypothalamic nuclei at 2 p.m. indicated their low functional activity in comparison with the studies carried out at 2 a.m. Prolonged exposure of rats to immobilization stress was reflected in a significant rearrangement of the ultrastructural organization of supraoptic nuclei of the hypothalamus. The established changes can be considered as a manifestation of neurosecretory activity suppression, a decrease in neurosecretase production by hypothalamic neurons. Melatonin injections against the background of immobilization stress resulted in relative normalization of ultrastructural state of neurons of supraoptic nuclei of the hypothalamus of animals. In particular, studies at 2 a.m. revealed light neurosecretory cells containing a large nucleus, it was pyknotically altered. Karyolema invaginations, euchromatin dominance in the nucleus were observed. Heterogeneous changes were observed on the part of mitochondria. Enlarged tubules of granular endoplasmic reticulum were seen. At the same time, a small number of ribosomes and few hormonal granules were noticeable in neuroplasm. The mentioned picture of neurosecretory cells reflects a relative improvement in their electron microscopic state, which is evidenced by the appearance of neurosecretory granules. However, the ultrastructure of other organelles of the studied neurons indicates a depleted state caused by prolonged immobilization.Conclusions. 1. In animals under standard photoperiod conditions, the structural organization of supraoptic neurons of the hypothalamic nuclei during the nighttime of the experiment reflects the intensity of intracellular synthesizing processes (at 2 a.m.). A decrease in the activity of the structures under study is noted during the daytime. 2. Under immobilization stress, the ultrastructural organization of the above neurons indicates a pronounced disturbance of reactive nature with the signs of decreased functional ability of the structures and the phenomena of edema and destruction during the period of observation. 3. Melatonin injections against the background of immobilization stress led to a relative improvement in the ultrastructural state of the animals’ hypothalamic nuclei neurons, which is evidenced by the appearance of neurosecretory granules. However, the ultrastructure of other organelles of the studied neurons indicated a depleted state caused by prolonged immobilization.

Morphometric analysis of supraoptic neurons of the rat hypothalamic nuclei under conditions of prolonged illumination

The article reviews the results of studies of the morphofunctional state of neurons of the supraoptic nuclei of the rat hypothalamus under conditions of different duration of light regime. Under standard light regime in rats, a diurnal rhythm of morphofunctional activity of supraoptic nucleus neurons with maximum activity during daytime (before 2 p.m.) is recorded.
 In animals subjected to prolonged light exposure, more pronounced changes in the morphofunctional state of the supraoptic neurons of the hypothalamus at 2 a.m. than at 2 p.m. were established. Thus, the neuronal nucleus area was 94.08 ± 9.55 μm2 and was significantly greater than that in intact animals. The nucleo-cytoplasmic ratio of supraoptic hypothalamic neuron at 2 a.m. was lower than that in intact animals due to a decrease in specific nucleus volume. In comparison with the day period (2 p.m.), before 2 a.m. there was revealed a decrease of the neuron body area of supraoptic nuclei of hypothalamus due to possible decrease of the area of nucleus and nucleolus of cells. This was the reason for the increase in the nucleo-cytoplasmic ratio in the neurons under observation at night, which was 2.51 ± 0.023 units. Constant light regime did not cause inversion of the rhythm of morphofunctional activity of the neurons under study, the maximum values, as in intact animals, occurred in the daytime observation period.

Hyperosmolarity-induced vasopressin expression and intrinsic excitability of supraoptic neurons of adult offspring are changed by early maternal separation

Adverse early life experiences can produce long-lasting changes in neurocircuits. The aim of this study was to investigate the programming effects of early maternal separation on the adult offspring vasopressin system. We hypothesized that subjecting adult rats to 4.5 h of daily maternal separation between postnatal days 1 – 21 will have altered hyperosmolarity-induced Avp expression and the response of supraoptic (SON) neurons to electrical and osmotic stimulation. We measured Avp mRNA and hn-RNA in the SON and in the paraventricular nucleus (PVN) by quantitative PCR, and assessed the intrinsic excitability of magnocellular SON neurons as well as their osmotic responses by the patch-clamp technique. In maternally-separated rats we found that basal and osmolarity-induced Avp mRNA gene expression was upregulated in the SON, whereas osmolarity-induced Avp hn-RNA gene expression was abolished. Similarly, in the PVN of maternally-separated rats the osmolarity-induced Avp mRNA gene expression was blunted. The supraoptic neurons of separated rats also had greater excitability than those of non-separated rats. Our results indicate that early maternal separation has long-term consequences on basal and hyperosmolarity-induced Avp hypothalamic expression as well as on the intrinsic excitability of magnocellular supraoptic neurons.

PLCδ1 Plays Central Roles in the Osmotic Activation of ΔN-TRPV1 Channels in Mouse Supraoptic Neurons and in Murine Osmoregulation.

The magnocellular neurosecretory cells (MNCs) of the hypothalamus play a vital role in osmoregulation, but the mechanisms underlying MNC osmosensitivity are not fully understood. We showed previously that high osmolality activates phospholipase C (PLC) in rat MNCs in a Ca2+-dependent manner and that PLC activation is necessary for full osmotic activation of an N-terminal variant of the TRPV1 (ΔN-TRPV1) channel. We therefore hypothesized that the Ca2+-dependent δ1 isoform of PLC contributes to ΔN-TRPV1 activation and tested whether MNC function is defective in a transgenic PLCδ1 KO mouse. Water deprivation for 24 h caused greater increases in serum osmolality and losses in body weight in PLCδ1 KO mice than it did in control mice. Action potentials and ΔN-TRPV1 currents were measured in acutely isolated mouse MNCs using whole-cell patch clamp before and after exposure to hypertonic solutions. This treatment elicited a significant activation of ΔN-TRPV1 currents and an increase in firing rate in MNCs isolated from control mice, but not from PLCδ1 KO mice. Submembranous filamentous actin was measured in isolated MNCs before and after treatment with angiotensin II and hypertonic solution. Both treatments caused an increase in filamentous actin fluorescence in MNCs isolated from control mice, but both responses were significantly attenuated in MNCs from PLCδ1 KO mice. Our data demonstrate that the PLCδ1 isoform plays a key role in the activation of ΔN-TRPV1 channels and in osmosensory transduction in MNCs. This study advances our understanding of the molecular mechanisms underlying mammalian osmoregulation.SIGNIFICANCE STATEMENT Magnocellular neurosecretory cells (MNCs) of the hypothalamus play a central role in osmoregulation. We have identified a key role for the PLCδ1 isoform in the activation of ΔN-TRPV1 channels and osmosensory transduction in MNCs. The data indicate that the PLCδ1 isoform is activated by the Ca2+ influx occurring during MNC action potentials and exerts a positive feedback on ΔN-TRPV1 channels to enhance MNC excitability. This study provides evidence that PLCδ1 is a key molecule underlying osmosensory transduction, the regulation of VP release, and osmoregulation.

The Same Magnocellular Neurons Send Axon Collaterals to the Posterior Pituitary and Retina or to the Posterior Pituitary and Autonomic Preganglionic Centers of the Eye in Rats

In rats, some parvocellular paraventricular neurons project to spinal autonomic centers. Using the virus tracing technique, we have demonstrated that some magnocellular paraventricular neurons, but not supraoptic neurons, also project to autonomic preganglionic centers of the mammary gland, gingiva, or lip. A part of these neurons has shown oxytocin immunoreactivity. In the present experiment, we have examined whether the same magnocellular neuron that sends fibers to the retina or autonomic preganglionic centers of the eye also projects to the posterior pituitary. Double neurotropic viral labeling and oxytocin immunohistochemistry were used. After inoculation of the posterior pituitary and the eye with viruses, spreading in a retrograde direction and expressing different fluorescence proteins, we looked for double-labeled neurons in paraventricular and supraoptic nuclei. Double-labeled neurons were observed in non-sympathectomized and cervical-sympathectomized animals. Some double-labeled neurons contained oxytocin. After the optic nerve was cut, the labeling did not appear in the supraoptic nucleus; however, it could still be observed in the paraventricular nucleus. In the paraventricular nucleus, the double-labeled cells may be the origin of centrifugal visual fibers or autonomic premotor neurons. In the supraoptic nucleus, all double-labeled neurons are cells of origin of centrifugal visual fibers.

A fine‐tuned interaction between the ER and mitochondria regulates Ca2+‐dependent after hyperpolarizations in vasopressin supraoptic neurons

It is known that vasopressin magnocellular neurons become hyperexcitable in cardiovascular diseases such as heart failure. This hyperexcitability is mediated by shifts in both synaptic and intrinsic mechanisms. One key intrinsic mechanism is the slow afterhyperpolarization (sAHP), a phenomenon underlain by a calcium‐dependent K+ current (IsAHP) most notable for its role in spike frequency adaptation. While features of the sAHP and its effects on firing are well established, a comprehensive understanding of the underlying mechanisms are still unknown. Previous work demonstrated that sAHP activation requires Ca2+ entry via voltage‐gated Ca2+ channels (VGCCs). However, whether in VP neurons Ca2+ entry via these channels directly activates the sAHP, or alternatively, whether it stimulates Ca2+‐induced Ca2+ release from an internal source such as endoplasmic reticulum (ER) or mitochondria remains unknown. Combining patch clamp electrophysiology with simultaneous Ca2+ imaging in transgenic eGFP‐VP rats, we investigated the role of ER and mitochondrial in shaping the sAHP. We measured sAHPs in voltage clamp by evoking a train of action potentials and measuring the resulting outward current. Pre‐incubation of slices with thapsigargin (3μM), an inhibitor of the sarco/ER Ca2+ ATPase inhibited IsAHPs significantly (96%, p< 0.001) while leaving Ca2+ transients unaffected (p > 0.05). This demonstrates that ER Ca2+ release is necessary for sAHP activation. Conversely, blockade of mitochondrial Ca2+ release via blocking mitochondrial Ca2+ exchangers with TPP (20μM) failed to inhibit the IsAHP (p > 0.05). We next evaluated the role of mitochondrial Ca2+ uptake with bath application of the mitochondrial uncoupler CCCP (1μM) or the mitochondrial Ca2+ uniporter (MCU) blocker Ru360 (20μM) in the patch pipette. These manipulations significantly prolonged the decay time constant of both the IsAHP (p< 0.01) and the underlying Ca2+ transient (p< 0.05), demonstrating that the MCU regulates spatiotemporal dynamics of the sAHP. Finally, combining thapsigargin with Ru360 in the pipette showed an initial block of the sAHP, followed by an enhancement over 15 minutes as Ru360 dialyzed into the cell. These results support the ER (but not mitochondria) as a major source of Ca2+ activating the sAHP, as well as a role for the MCU in regulating the magnitude and time course of the sAHP. Taken together, our studies support a fine‐tuned interaction between VGCCs, ER, and mitochondria in modulating the magnitude and time course of the sAHP in VP neurons. Future studies warrant investigation into whether changes in these interactions contribute to altered VP neuronal activity in cardiovascular disease states.Support or Funding InformationR01HL090948

CIRCADIAN DYNAMICS OF OPTIC DENSITY OF MELATONIN RECEPTORS IN THE NEURONS OF HYPOTHALAMIC SUPRAOPTIC NUCLEUS IN RATS UNDER ALTERED PHOTOPERIOD

Introduction. Melatonin production is considerably suppressed by light and affects the ability to transfer daily rhythm information from the hypothalamus to other neural target sites and thus alters the expression of some biological rhythms. The hormone controls the state of the hypothalamic-pituitary system and endocrine gland activity through melatonin receptors (membrane, cytosolic and nuclear ones). In addition, using a mechanism of the feedback, it interferes with the activity of supraoptic nucleus of the hypothalamus, which regulates water-salt metabolism and responses to stress. Objective: to provide quantitative circadian characteristics of melatonin receptors density in the neurons of the hypothalamic supraoptic nucleus of rats being under light stimulation as well as the correction of changes after injecting exogenous melatonin. Material and methods. The experiments were conducted on 60 white mongrel mature male rats weighing 150 – 180 g. The test animals were divided into 3 parts each with 2 groups, kept under the conditions of standard light regime, hyperilluminated and the injection of exogenous melatonin and day-round lighting within 7 days. To perform immunohistochemical methods, we used polyclonal antibodies to melatonin 1A receptors produced by Abcam and streptavidin biotin visualization system LSAB2 (peroxidase mark + diaminobenzidine) produced by Chemicon International Inc. We adhered to protocol standardization of methods for all sections. Additional staining of nuclei was performed with Mayer hematoxylin. Results. The indices of optical density of specific melatonin 1A receptors of supraoptic neurocytes staining obtained in the intact group (at 02.00 AM- 0,488 ± 0,0024, at 02.00 P.M. - 0,464 ± 0,0023, p = 0.002) and in animals subjected to light stress (at 02.00 AM-0,295 ± 0, 0019, at 02.00 P.M.- 0,286 ± 0,0018, p = 0,012) had a probable value and were characterized by a clear diurnal periodicity. In the group of animals with pineal gland hypofunction modulation (at 02.00 A.M.- 0,216 ± 0,0017, at 02.00 P.M. - 0,214 ± 0,0021, p> 0,05). Conclusions The density of 1A melatonin receptors in rat’s hypothalamic supraoptic neurons are normally characterized by an accurate circadian rhythm. The highest density of receptors is observed at 02.00 AM, and at 02.00 PM it is significantly lower (p = 0.002). Immunohistochemical studies revealed that under inhibition of pineal gland activity the circadian rhythm of melatonin receptors density in neurons of supraoptic nuclei of the hypothalamus gets disturbed, which is characterized by an incredible difference of indices in the tested periods of the day.

Specificity in the interaction of high-voltage-activated Ca2+ channel types with Ca2+-dependent afterhyperpolarizations in magnocellular supraoptic neurons.

Magnocellular oxytocin (OT) and vasopressin (VP) neurons express an afterhyperpolarization (AHP) following spike trains that attenuates firing rate and contributes to burst patterning. This AHP includes contributions from an apamin-sensitive, medium-duration AHP (mAHP) and from an apamin-insensitive, slow-duration AHP (sAHP). These AHPs are Ca2+ dependent and activated by Ca2+ influx through voltage-gated Ca2+ channels. Across central nervous system neurons that generate Ca2+-dependent AHPs, the Ca2+ channels that couple to the mAHP and sAHP differ greatly, but for magnocellular neurosecretory cells this relationship is unknown. Using simultaneous whole cell recording and Ca2+ imaging, we evaluated the effect of specific high-voltage-activated (HVA) Ca2+ channel blockers on the mAHP and sAHP. Block of all HVA channels via 400 μM Cd2+ inhibited almost the entire AHP. We tested nifedipine, conotoxin GVIA, agatoxin IVA, and SNX-482, specific blockers of L-, N-, P/Q-, and R-type channels, respectively. The N-type channel blocker conotoxin GVIA (1 μM) was the only toxin that inhibited the mAHP in either OT or VP neurons although the effect on VP neurons was weaker by comparison. The sAHP was significantly inhibited by N-type block in OT neurons and by R-type block in VP neurons although neither accounted for the entirety of the sAHP. Thus the mAHP appears to be elicited by Ca2+ from mostly N-type channels in both OT and VP neurons, but the contributions of specific Ca2+ channel types to the sAHP in each cell type are different. Alternative sources to HVA channels may contribute Ca2+ for the sAHP. NEW & NOTEWORTHY Despite the importance of afterhyperpolarization (AHP) mechanisms for regulating firing behavior of oxytocin (OT) and vasopressin (VP) neurons of supraoptic nucleus, which types of high-voltage-activated Ca2+ channels elicit AHPs in these cells was unknown. We found that N-type channels couple to the medium AHP in both cell types. For the slow AHP, N-type channels contribute in OT neurons, whereas R-type contribute in VP neurons. No single Ca2+ channel blocker abolished the entire AHP, suggesting that additional Ca2+ sources are involved.

Sushi repeat-containing protein X-linked 2: A novel phylogenetically conserved hypothalamo-pituitary protein.

Sushi repeat-containing protein X-linked 2 (SRPX2) is a novel protein associated with language development, synaptic plasticity, tissue remodeling, and angiogenesis. We investigated the expression and spatial localization of SRPX2 in normal mouse, rat, monkey, and human brain using in situ hybridization and immunohistochemistry. Antibody specificity was determined using in vitro siRNA based silencing of SRPX2. Cell type-specific expression was verified by double-labeling with oxytocin or vasopressin. Western blot was used to detect SRPX2 protein in rat and human plasma and cerebrospinal fluid. Unexpectedly, SRPX2 mRNA expression levels were strikingly higher in the hypothalamus as compared to the cortex. All SRPX2 immunoreactive (ir) neurons were localized in the hypothalamic paraventricular, periventricular, and supraoptic nuclei in mouse, rat, monkey, and human brain. SRPX2 colocalized with vasopressin or oxytocin in paraventricular and supraoptic neurons. Hypothalamic SRPX2-ir positive neurons gave origin to dense projections traveling ventrally and caudally toward the hypophysis. Intense axonal varicosities and terminal arborizations were identified in the rat and human neurohypophysis. SRPX2-ir cells were also found in the adenohypophysis. Light SRPX2-ir projections were observed in the dorsal and ventral raphe, locus coeruleus, and the nucleus of the solitary tract in mouse, rat and monkey. SRPX2 protein was also detected in plasma and CSF. Our data revealed intense phylogenetically conserved expression of SRPX2 protein in distinct hypothalamic nuclei and the hypophysis, suggesting its active role in the hypothalamo-pituitary axis. The presence of SRPX2 protein in the plasma and CSF suggests that some of its functions depend on secretion into body fluids.

Assessing the role of hypothalamic microglia and blood vessel disruption in the development of angiotensin II-dependent hypertension in Cyp1a1-Ren2 rats.

Elevated plasma levels of the hormone vasopressin have been implicated in the pathogenesis of some forms of hypertension. Hypothalamic paraventricular and supraoptic nuclei neurons regulate vasopressin secretion into the circulation. Vasopressin neuron activity is elevated by day 7 in the development of angiotensin II-dependent hypertension in Cyp1a1-Ren2 rats. While microglial activation and blood-brain barrier (BBB) breakdown contribute to the maintenance of well-established hypertension, it is not known whether these mechanisms contribute to the early onset of hypertension. Hence, we aimed to determine whether microglia are activated and/or the BBB is compromised during the onset of hypertension. Here, we used the Cyp1a1-Ren2 rat model of hypertension and showed that ionised calcium-binding adapter molecule 1 staining of microglia does not change in the paraventricular and supraoptic nuclei on day 7 (early onset) and day 28 (well established) of hypertension, compared to the normotensive control. Endothelial transferrin receptor staining, which stains endothelia and reflects blood vessel density, was also unchanged at day 7, but was reduced at day 28, suggesting that breakdown of the BBB begins between day 7 and day 28 in the development of hypertension. Hence, this study does not support the idea that microglial activation or BBB disruption contribute to the onset of angiotensin II-dependent hypertension in Cyp1a1-Ren2 rats, although BBB disruption might contribute to the progression from the early onset to well-established hypertension.

Osmotic activation of a Ca2+-dependent phospholipase C pathway that regulates ∆N TRPV1-mediated currents in rat supraoptic neurons.

The magnocellular neurosecretory cells (MNCs) of the hypothalamus regulate body fluid balance by releasing the hormones vasopressin (VP) and oxytocin (OT) in an osmolality‐dependent manner. Elevations of external osmolality increase MNC firing and hormone release. MNC osmosensitivity is largely due to activation of a mechanosensitive non‐selective cation current that responds to osmotically‐evoked changes in MNC volume and is mediated by an N‐terminal variant of the TRPV1 channel (∆N TRPV1). We report a novel mechanism by which increases in osmolality may modulate ∆N TRPV1‐mediated currents and thus influence MNC electrical behaviour. We showed previously that acute elevations of external osmolality activate the enzyme phospholipase C (PLC) in isolated MNCs. We now show that the osmotic activation of PLC has a time course and dose‐dependence that is consistent with a role in MNC osmosensitivity and that it contributes to the osmotically‐evoked increase in non‐selective cation current in MNCs through a protein kinase C‐dependent pathway. We furthermore show that the mechanism of osmotic activation of PLC requires an increase in internal Ca2+ that depends on influx through L‐type Ca2+ channels. Our data therefore suggest that MNCs possess an osmotically‐activated Ca2+‐dependent PLC that contributes to the osmotic activation of ∆N TRPV1 and may therefore be important in MNC osmosensitivity and in central osmoregulation.

Sodium-calcium exchanger and R-type Ca2+ channels mediate spontaneous [Ca2+]i oscillations in magnocellular neurones of the rat supraoptic nucleus

Isolated supraoptic neurones generate spontaneous [Ca2+]i oscillations in isolated conditions. Here we report in depth analysis of the contribution of plasmalemmal ion channels (Ca2+, Na+), Na+/Ca2+ exchanger (NCX), intracellular Ca2+ release channels (InsP3Rs and RyRs), Ca2+ storage organelles, plasma membrane Ca2+ pump and intracellular signal transduction cascades into spontaneous Ca2+ activity. While removal of extracellular Ca2+ or incubation with non-specific voltage-gated Ca2+ channel (VGCC) blocker Cd2+ suppressed the oscillations, neither Ni2+ nor TTA-P2, the T-type VGCC blockers, had an effect. Inhibitors of VGCC nicardipine, ω-conotoxin GVIA, ω-conotoxin MVIIC, ω-agatoxin IVA (for L-, N-, P and P/Q-type channels, respectively) did not affect [Ca2+]i oscillations. In contrast, a specific R-type VGCC blocker SNX-482 attenuated [Ca2+]i oscillations. Incubation with TTX had no effect, whereas removal of the extracellular Na+ or application of an inhibitor of the reverse operation mode of Na+/Ca2+ exchanger KB-R7943 blocked the oscillations. The mitochondrial uncoupler CCCP irreversibly blocked spontaneous [Ca2+]i activity. Exposure of neurones to Ca2+ mobilisers (thapsigargin, cyclopiazonic acid, caffeine and ryanodine); 4-aminopyridine (A-type K+ current blocker); phospholipase C and adenylyl cyclase pathways blockers U-73122, Rp-cAMP, SQ-22536 and H-89 had no effect. Oscillations were blocked by GABA, but not by glutamate, apamin or dynorphin. In conclusion, spontaneous oscillations in magnocellular neurones are mediated by a concerted action of R-type Ca2+ channels and the NCX fluctuating between forward and reverse modes.

Activation of Supraoptic Oxytocin Neurons by Secretin Facilitates Social Recognition

BackgroundSocial recognition underlies social behavior in animals, and patients with psychiatric disorders associated with social deficits show abnormalities in social recognition. Oxytocin is implicated in social behavior and has received attention as an effective treatment for sociobehavioral deficits. Secretin receptor–deficient mice show deficits in social behavior. The relationship between oxytocin and secretin concerning social behavior remains to be determined. MethodsExpression of c-Fos in oxytocin neurons and release of oxytocin from their dendrites after secretin application were investigated. Social recognition was examined after intracerebroventricular or local injection of secretin, oxytocin, or an oxytocin receptor antagonist in rats, oxytocin receptor–deficient mice, and secretin receptor–deficient mice. Electron and light microscopic immunohistochemical analysis was also performed to determine whether oxytocin neurons extend their dendrites into the medial amygdala. ResultsSupraoptic oxytocin neurons expressed the secretin receptor. Secretin activated supraoptic oxytocin neurons and facilitated oxytocin release from dendrites. Secretin increased acquisition of social recognition in an oxytocin receptor–dependent manner. Local application of secretin into the supraoptic nucleus facilitated social recognition, and this facilitation was blocked by an oxytocin receptor antagonist injected into, but not outside of, the medial amygdala. In the medial amygdala, dendrite-like thick oxytocin processes were found to extend from the supraoptic nucleus. Furthermore, oxytocin treatment restored deficits of social recognition in secretin receptor–deficient mice. ConclusionsThe results of our study demonstrate that secretin-induced dendritic oxytocin release from supraoptic neurons enhances social recognition. The newly defined secretin-oxytocin system may lead to a possible treatment for social deficits.

Abstract 054: Prorenin Stimulates Firing Activity in Hypothalamic Neurons via Angiotensin II-Dependent and -Independent Mechanisms

The brain RAS plays a central role in cardiovascular homeostasis by modulating sympathetic nerve activity and hormone secretion. Still, the relevance of intrinsic brain RAS function is questioned because renin levels are too low to have an impact on angiotensin II (AngII) formation. The discovery of the prorenin receptor (PRR) brought new support to the brain RAS involvement in neurohumoral regulation. However, the mechanisms underlying PRR-mediated actions remain unknown. We used whole cell patch-clamp electrophysiology in hypothalamic slices to examine the effects of prorenin (PR) on the excitability of magnocellular neurosecretory neurons (MCNs) of the supraoptic nucleus and parvocellular RVLM-projecting presympathetic neurons of the paraventricular nucleus (PVN-RVLM), which play integral roles in cardiovascular control, and have been implicated in the pathophysiology of neurogenic hypertension. PR application (2.5nM) increased the firing activity of both MCNs (basal 1.3±0.4 Hz vs PR 3.3±0.7 Hz; p=0.0086) and parvocellular neurons (basal 0.5±0.1 Hz vs PR 2.1±0.4 Hz; p=0.0006), including a subset of PVN-RVLM neurons (basal 0.5±0.1 Hz vs PR 1.4±0.4 Hz; p=0.048). In MCNs, this effect was blocked by a PRR antagonist ( PRO20 ; 250nM; basal 1.9±0.6 Hz vs PRO20 1.3±0.5 Hz; p=0.017), but persisted in the presence of the AT1 receptor blocker, losartan (50μM; basal 0.8±0.2 Hz vs losartan 2.3±0.5 Hz; p=0.0018). Conversely, PR effects on PVN neurons persisted with PRO20 (basal 0.9±0.2 Hz vs PRO20 1.6±0.3 Hz; p=0.013), but were largely (~75%) blunted with losartan (basal 0.2±0.04 Hz vs losartan 0.6±0.1 Hz; p=0.0193; Δ basal 1.6±0.4 Hz vs Δ losartan 0.4±0.1 Hz; p=0.0175). These results suggest that while PR actions are primarily PRR-mediated in MCNs, they are predominantly AngII-dependent in PVN cells. Lastly, PR effects were abolished in both cell types by dialyzing neurons with the Ca 2+ chelator BAPTA (10mM; MCNs: basal 0.6 ± 0.1 Hz vs BAPTA 0.3 ± 0.1 Hz; p=0.0277; PVN: basal 0.4±0.05 Hz vs BAPTA 0.5±0.2 Hz; p=0.6468). Our results show for the first time that PR stimulates firing activity in both hypothalamic neurosecretory and presympathetic neurons, and support distinct AngII-independent and dependent signaling mechanisms in each neuronal population.

Modulation of spike clustering by NMDA receptors and neurotensin in rat supraoptic nucleus neurons.

Magnocellular neurosecretory cells (MNCs) in the rat supraoptic nucleus display clustered firing during hyperosmolality or dehydration. This response is beneficial because this type of activity potentiates vasopressin secretion from axon terminals in the neurohypophysis and thus promotes homoeostatic water reabsorption from the kidney. However, the mechanisms which lead to the generation of clustering activity in MNCs remain unknown. Previous work has shown that clustered firing can be induced in these neurons through the pharmacological activation of NMDA receptors (NMDARs) and that silent pauses observed during this activity are mediated by apamin-sensitive calcium activated potassium (SK) channels. However, it remains unknown if clustered firing can be induced in situ by endogenous glutamate release from axon terminals. Here we show that electrical stimulation of glutamatergic osmosensory afferents in the organum vasculosum lamina terminalis (OVLT) can promote clustering in MNCs via NMDARs and apamin-sensitive channels.We also show that the rate of spike clustering induced by NMDA varies as a bell-shaped function of voltage, and that partial inhibition of SK channels can increase cluster duration and reduce the rate of clustering. Finally,we show that MNCs express neurotensin type 2 receptors, and that activation of these receptors can simultaneously depolarize MNCs and suppress clustered firing induced by bath application of NMDA or by repetitive stimulation of glutamate afferents. These studies reveal that spike clustering can be induced in MNCs by glutamate release from afferent nerve terminals and that that this type of activity can be fine-tuned by neuromodulators such as neurotensin.

Osmotic activation of phospholipase C triggers structural adaptation in osmosensitive rat supraoptic neurons.

The magnocellular neurosecretory cells of the hypothalamus (MNCs) synthesize and secrete vasopressin or oxytocin. A stretch-inactivated cation current mediated by TRPV1 channels rapidly transduces increases in external osmolality into a depolarization of the MNCs leading to an increase in action potential firing and thus hormone release. Prolonged increases in external osmolality, however, trigger a reversible structural and functional adaptation that may enable the MNCs to sustain high levels of hormone release. One poorly understood aspect of this adaptation is somatic hypertrophy. We demonstrate that hypertrophy can be evoked in acutely isolated rat MNCs by exposure to hypertonic solutions lasting tens of minutes. Osmotically evoked hypertrophy requires activation of the stretch-inactivated cation channel, action potential firing, and the influx of Ca(2+). Hypertrophy is prevented by pretreatment with a cell-permeant inhibitor of exocytotic fusion and is associated with an increase in total membrane capacitance. Recovery is disrupted by an inhibitor of dynamin function, suggesting that it requires endocytosis. We also demonstrate that hypertonic solutions cause a decrease in phosphatidylinositol 4,5-bisphosphate in the plasma membranes of MNCs that is prevented by an inhibitor of phospholipase C (PLC). Inhibitors of PLC or protein kinase C (PKC) prevent osmotically evoked hypertrophy, and treatment with a PKC-activating phorbol ester can elicit hypertrophy in the absence of changes in osmolality. These studies suggest that increases in osmolality cause fusion of internal membranes with the plasma membrane of the MNCs and that this process is mediated by activity-dependent activation of PLC and PKC.

Brain‐derived neurotrophic factor causes chloride gradient collapse in supraoptic neurons inducing vasopressin‐mediated blood pressure increase (LB847)

Vasopressin (VP), an antidiuretic hormone, is also a potent vasoconstrictor and has been found at high levels in salt‐sensitive hypertensives. Hypertension normally reduces VP secretion by MNCs in the SON, by GABA‐dependent synaptic inhibition of their firing activities. However, in rats under chronic hypernatremia, GABAA receptor‐mediated synaptic inhibition is abolished in SON neurons due to a depolarization in chloride reversal potential (ECl), suggesting that continued VP secretion despite BP elevations may underlie the development of salt‐dependent hypertension. In order to test this hypothesis, we measured the mean arterial pressure (MAP) by radio telemetry in freely moving rats made chronically hypernatremic by 2% saline drinking. MAP of these rats rose steadily rise during the course of the 7‐day SL treatment (basal BP: 97.7±1.9 mmHg; day 7: 113.5±2.1 mmHg; n=10; P<0.05), however remained stable in euhydrated rats over the same time period (basal BP: 98.3±2.5 mmHg; day 7: 100.6±2.2 mmHg; n=6; P=0.50). Furthermore, the SL‐mediated increase in MAP was significantly reduced by continuous systemic infusion of a VP receptor type 1 (V1R) antagonist (αday 7; +8.8±2.5 mmHg; n=6) when compared to SL controls (α15.1±1.0 mmHg; n=10; P<0.05). These findings confirm our hypothesis that circulating VP contributes to increases in BP during chronic hypernatremia. Brain‐derived neurotrophic factor (BDNF) is a high‐affinity TrkB agonist previously shown to be responsible for chloride gradient collapse. In order to investigate this possibility in the SON, we induced a specific knockdown of this protein in SL rats in vivo by stereotaxic injections of adeno‐associated virus (AAV) containing BDNF‐shRNA into the SON. ECl measurements via Gramicidin perforated patch‐clamp revealed that this treatment significantly hyperpolarized the ECl of SON neurons (‐56.2±5.4 mV; n=10) when compared to values measured in SL rats injected with control AAV containing a scrambled sequence of shRNA (‐40.1±2.4 mV; n=7; p<0.05). These results strongly suggest that BDNF is the TrkB agonist responsible for producing the chloride gradient collapse in SON neurons under chronic hypernatremia, a phenomenon which potentially underlie the development of salt‐sensitive hypertension. Funding source: Canadian Institutes of Health Research.Grant Funding Source: CIHR

Extending the socio-sexual brain: arginine-vasopressin immunoreactive circuits in the telencephalon of mice

Quantitative analysis of the immunoreactivity for arginine-vasopressin (AVP-ir) in the telencephalon of male (intact and castrated) and female CD1 mice allows us to precisely locate two sexually dimorphic (more abundant in intact than castrated males and females) AVP-ir cell groups in the posterior bed nucleus of the stria terminalis (BST) and the amygdala. Chemoarchitecture (NADPH diaphorase) reveals that the intraamygdaloid AVP-ir cells are located in the intra-amygdaloid BST (BSTIA) rather than the medial amygdala (Me), as previously thought. Then, we have used for the first time tract tracing (combined with AVP immunofluorescence) and fiber-sparing lesions of the BST to analyze the projections of the telencephalic AVP-ir cell groups. The results demonstrate that the posterior BST originates the sexually dimorphic innervation of the lateral septum, the posterodorsal Me and a substance P-negative area in the medioventral striato-pallidum (mvStP).The BSTIA may also contribute to some of these terminal fields. Our material also reveals non-dimorphic AVP-ir processes in two locations of the amygdala. First, the ventral Me shows dendrite-like AVP-ir processes apparently belonging supraoptic neurons, whose possible functions are discussed. Second, the Ce shows sparse, thick AVP-ir axons with high individual variability in density and distribution, whose possible influence on stress coping in relation to the affiliative or agonistic behaviors mediated by the Me are discussed. Finally, we propose that the region of the mvStP showing sexually dimorphic AVP-ir innervation is part of the brain network for socio-sexual behavior, in which it would mediate motivational aspects of chemosensory-guided social interactions.

Variation in sodium current amplitude between vasopressin and oxytocin hypothalamic supraoptic neurons

Biophysical characteristics of tetrodotoxin-sensitive sodium (Na(+)) currents were studied in vasopressin (VP) and oxytocin (OT) supraoptic neurons acutely isolated from rat hypothalamus. Na(+) current density (pA/pF) was significantly greater in VP neurons than in OT neurons. No significant difference between VP and OT neurons was detected regarding the voltage dependence of activation and steady-state inactivation, or rate of recovery from inactivation of Na(+) currents. In both VP and OT neurons, the macroscopic inactivation of the Na(+) currents was best fitted with a double-exponential expression suggesting two rates of inactivation. Also in both types, the time course of recovery from inactivation proceeded with fast and slow time constants averaging around 8 and 350 ms, respectively, suggesting the presence of multiple pathways of recovery from inactivation. The slower time constant of recovery of inactivation may be involved in the decrease in action potential (AP) amplitude that occurs after the first spike during burst firing in both neuronal types. The larger amplitude of Na(+) currents in VP vs. OT neurons may explain the previous observations that VP neurons exhibit a lower AP threshold and greater AP amplitude than OT neurons, and may serve to differently tune the firing properties and responses to neuromodulators of the respective neuronal types.