Oxytocin-mediated milk ejection (ME) is pivotal to effective breastfeeding and reproductive health, yet behaviorally decoding and revealing neural mechanisms of ME remains challenging. Here, we combined in vivo calcium imaging and intramammary pressure recording to uncover the temporal connections between episodic activity of oxytocin neurons and ME in conscious lactating rats. Leveraging the coordinated behavioral responses of dams and pups, we developed a supervised machine learning framework (ME Decoder) to enable automated analyses of ME. Inspired by its interpretable features, we defined the activity-coupled dam-pup interactions (ADPI), manifested by pronounced kyphosis in dams, followed by pup treading and stretching, as the behavioral signatures of ME. By ME Decoder and ADPI analyses, we detected reduced ME but unaffected activity of oxytocinergic neurons after systemic blockade of oxytocin receptors. Our study uncovers the oxytocinergic and behavioral signatures of ME and provides a generalizable approach for further investigation.

Transpharyngeal (ventral) surgery in urethane-anaesthetised rats allows in vivo electrophysiological recording and/or imaging from superficial hypothalamic brain regions and from the pituitary gland. This surgical approach leaves the whole brain intact, providing a stable platform to study single or multiple identified cells over several hours with all central and peripheral inputs intact and the endocrine system functioning, which allows repeated drug application and stimulation of afferent inputs, as has been done for the arcuate nucleus, organum vasculosum of the lamina terminalis (OVLT) and suprachiasmatic nucleus (SCN) inputs to the supraoptic nucleus (SON). Exposing the ventral surface of the brain also allows simultaneous microdialysis for drug administration directly into the SON and for the collection of dialysate samples for measurement of somatodendritic neuropeptide release without disruption of the brain parenchyma. The most recent development using transpharyngeal surgery is two-photon imaging from the vasculature of the ventral surface of the brain, which has given insight into how SON neuronal activity affects cerebral blood flow and has identified a previously unknown SCN to OVLT portal blood system. Here we provide a brief history of the development of transpharyngeal surgery in the rat, instructions to complete the surgery and suggestions for future studies, extrapolating from the most recent developments in its use.

Many maternal adaptations occur during pregnancy to support the metabolic demands of the developing fetus and to prepare for the continued metabolic demands of lactation. Among these maternal adaptations are changes in the hypothalamic areas that regulate energy homeostasis: the paraventricular nucleus (PVN), ventromedial hypothalamic nucleus (VMH) and arcuate nucleus (ARC). The adaptive changes in the PVN, VMH, and ARC are believed to be driven by reduced responsiveness to the satiety hormone, leptin, during pregnancy. However, increased maternal metabolism is supported by elevated circulating glucose levels in pregnancy, and glucose itself can alter cell function by O-linked N-acetylglucosamine (O-GlcNAc) post-translational modification of proteins (O-GlcNAcylation). Therefore, we hypothesized that O-GlcNAcylation would be increased within the hypothalamic brain areas that are involved in the maternal adaptations to the increased metabolic demands of pregnancy: the ARC, VMH, and PVN. We completed immunohistochemistry and western blotting for O-GlcNAc in the ARC, VMH, and PVN from non-pregnant, late-pregnant, and lactating rats. Unexpectedly, we found that the number of O-GlcNAc-expressing cells and the levels of O-GlcNAc protein expression were similar within each area in non-pregnant, late-pregnant, and lactating rats. However, western blot analysis showed that the specific proteins that were O-GlcNAcylated appeared to be different between the reproductive states within each area. Further work will be required to identify the specific proteins that are differentially O-GlcNAcylated in each of the areas during pregnancy and lactation to determine whether this might contribute to the maternal adaptations required to cope with the metabolic demands of pregnancy and lactation.

Oxytocin is synthesized by hypothalamic supraoptic nucleus (SON) and paraventricular nucleus (PVN) neurons and is released from the posterior pituitary gland to trigger uterine contractions during parturition. In rats, oxytocin neuron innervation by periventricular nucleus (PeN) kisspeptin neurons increases over pregnancy and intra-SON kisspeptin administration excites oxytocin neurons only in late pregnancy. To test the hypothesis that kisspeptin neurons excite oxytocin neurons to trigger uterine contractions during birth in C57/B6J mice, double-label immunohistochemistry for kisspeptin and oxytocin first confirmed that kisspeptin neurons project to the SON and PVN. Furthermore, kisspeptin fibers expressed synaptophysin and formed close appositions with oxytocin neurons in the mouse SON and PVN before and during pregnancy. Stereotaxic viral delivery of caspase-3 into the AVPV/PeN of Kiss-Cre mice before mating reduced kisspeptin expression in the AVPV, PeN, SON and PVN by > 90% but did not affect the duration of pregnancy or the timing of delivery of each pup during parturition. Therefore, it appears that AVPV/PeN kisspeptin neuron projections to oxytocin neurons are not necessary for parturition in the mouse.

Renal water reabsorption increases in pregnancy and lactation to expand maternal blood volume to cope with the cardiovascular demands of the developing fetus and new-born baby. Vasopressin (antidiuretic hormone) promotes renal water reabsorption and its secretion is principally stimulated by body fluid osmolality. Hence, lowered osmolality normally decreases vasopressin secretion. However, despite water retention profoundly reducing osmolality in pregnancy and lactation, vasopressin levels are maintained to drive blood volume expansion. Despite its importance for successful reproduction, the cellular mechanisms that maintain vasopressin secretion in the face of decreased osmolality during pregnancy and lactation are unknown. Vasopressin is secreted by neurons that are intrinsically osmosensitive through expression of N-terminal truncated-transient receptor potential vanilloid-1 channel, ΔN-TRPV1, which is mechanically activated by osmotically-induced cell shrinkage to increase vasopressin neuron activity. Vasopressin neurons also express TRPV4 but the role of TRPV4 in vasopressin neuron function is not well characterised. Here, we summarise our novel evidence showing that TRPV4 forms functional channels with ΔN-TRPV1 that have a greater single-channel conductance compared to channels with ΔN-TRPV1 alone. We propose that upregulation of TRPV4 heteromerisation with ΔN-TRPV1 might maintain vasopressin secretion in pregnancy and lactation to expand blood volume for successful reproduction.

Neurovascular coupling (NVC), the process that links neuronal activity to cerebral blood flow changes, has been mainly studied in superficial brain areas, namely the neocortex. Still, whether the conventional, rapid and spatially restricted NVC response can be generalized to deeper, and functionally diverse brain regions remains unknown. The hypothalamic supraoptic nucleus (SON) is a deep brain region with unique cytoarchitectural properties including a high vascularization. Paradoxically, the dense vascular network is not required to maintain its metabolic demand. This mismatch in metabolic supply and demand suggests that the SON vasculature may serve additional functions beyond the delivery of energy substrates. To address this important gap in our knowledge, we set up the objective to implement a novel experimental approach to enable in vivo, real-time two-photon imaging from the ventral surface of the brain to monitor SON NVC responses to a systemic homeostatic challenge. Using this approach in anaesthetized male and female transgenic rats expressing GFP driven by the vasopressin (VP) promoter (VP-GFP rats), we found that a systemic hypertonic saline (HTS) infusion evoked a gradual and linear increase in the firing rate of VP neurons (p < 0.0001; n = 4). Rather than resulting in a classical vasodilatory NVC, the increased VP neuronal activity was accompanied by a significant intraparenchymal arteriole (but not venule) vasoconstriction (one-way RM ANOVA; p < 0.0001; n = 13 vessels from 5 rats). This "inverse" NVC was prevented when a VP V1a receptor antagonist was locally delivered within the SON (one-way RM ANOVA; p < 0.0001; n = 4 vessels from 4 rats). The HTS infusion also significantly decreased RBC velocity in SON capillaries (one-way RM ANOVA; p < 0.0001; n = 17 vessels from 6 rats) indicative of a decreased SON blood flow. The salt-induced inverse NVC response resulted in a rapid and progressive decrease in SON pO2 (one-way RM ANOVA; p < 0.002; n = 6 rats), along with an increased expression of the hypoxia inducible factor (HIF)-1a mRNA (p < 0.001; one-sample t test n = 6 rats). Finally, we show that a hypoxic SON microenvironment significantly increased the firing activity of VP neurons in ex vivo slices (p < 0.02; paired t test; n = 14 cells from 10 rats). Taken together, we show that the salt-induced inverse NVC in the SON results in a hypoxia-mediated positive feedback mechanism, which we propose contributes to sustain the activity of VP neurons in response to a systemic homeostatic challenge.

Oxytocin is secreted by hypothalamic supraoptic nucleus (SON) and paraventricular nucleus (PVN) oxytocin neurons to induce uterine contractions during parturition. Increased activation of oxytocin neurons at parturition involves a network of afferent inputs that increase oxytocin neuron excitability. Kisspeptin fibre density increases around oxytocin neurons during pregnancy, and central kisspeptin administration excites oxytocin neurons only in late pregnancy. Kisspeptin signals via extracellular regulated kinase 1/2 (ERK1/2) and p38. Therefore, to determine whether kisspeptin excites oxytocin neurons via ERK1/2-p38 signalling in late-pregnant rats, we performed immunohistochemistry for phosphorylated ERK1/2 (pERK1/2) and phosphorylated p38 (p-p38) in oxytocin neurons of non-pregnant and late-pregnant rats. Intracerebroventricular (ICV) kisspeptin administration (2 µg) did not affect pERK1/2 or p-p38 expression in SON and PVN oxytocin neurons of non-pregnant or late-pregnant rats. Furthermore, ICV kisspeptin did not affect pERK1/2 or p-p38 expression in brain areas with major projections to the SON and PVN: the nucleus tractus solitarius, rostral ventrolateral medulla, locus coeruleus, dorsal raphe nucleus, organum vasculosum of the lamina terminalis, median preoptic nucleus, subfornical organ, anteroventral periventricular nucleus, periventricular nucleus and arcuate nucleus. Hence, kisspeptin-induced excitation of oxytocin neurons in late pregnancy does not appear to involve ERK1/2 or p38 activation in oxytocin neurons or their afferent inputs.

Oxytocin is secreted into the periphery by magnocellular neurons of the hypothalamic supraoptic and paraventricular nuclei (SON and PVN) to trigger uterine contraction during birth and milk ejection during suckling. Peripheral oxytocin secretion is triggered by action potential firing, which is regulated by afferent input activity and by feedback from oxytocin secreted into the extracellular space from magnocellular neuron somata and dendrites. A prominent input to oxytocin neurons arises from proopiomelanocortin neurons of the hypothalamic arcuate nucleus that secrete an alpha-melanocyte-stimulating hormone (alpha-MSH), which inhibits oxytocin neuron tiring in non-pregnant rats by increasing somatodendritic oxytocin secretion. However, alpha-MSH inhibition of oxytocin neuron firing is attenuated in mid-pregnancy and somato-dendritic oxytocin becomes auto-excitatory in late-pregnancy and lactation. Therefore. we hypothesized that attenuated alpha-MSH inhibition of oxytocin neuron firing marks the beginning of a transition from inhibition to excitation to facilitate peripheral oxytocin secretion for parturition and lactation. Intra-SON microdialysis administration of alpha-MSH inhibited oxytocin neuron firing rate by 33 +/- 9% in non-pregnant rats but increased oxytocin neuron firing rate by 37 +/- 12% in late-pregnant rats and by 28 +/- 10% in lactating rats. alpha-MSH-induced somato-dendritic oxytocin secretion measured ex vivo with oxytocin receptor-expressing "sniffer" cells, was of similar amplitude in PVN slices from non-pregnant and lactating rats but longer-lasting in slices from lactating rats. Hence, alpha-MSH inhibition of oxytocin neuron activity switches to excitation over pregnancy while somato-dendritic oxytocin secretion is maintained, which might enhance oxytocin neuron excitability to facilitate the increased peripheral secretion that is required for normal parturition and milk ejection.

The hormone, oxytocin, is synthesised by magnocellular neurones of the supraoptic and paraventricular nuclei and is released from the posterior pituitary gland into the circulation to trigger uterine contractions during parturition. Kisspeptin fibre density increases around the supraoptic nucleus over pregnancy and intracerebroventricular kisspeptin excites oxytocin neurones only in late pregnancy. However, the mechanism of this excitation is unknown. Here, we found that microdialysis administration of kisspeptin into the supraoptic nucleus consistently increased the action potential (spike) firing rate of oxytocin neurones in urethane-anaesthetised late-pregnant rats (gestation day 18-21) but not in non-pregnant rats. Hazard analysis of action potential firing showed that kisspeptin specifically increased the probability of another action potential firing immediately after each action potential (post-spike excitability) in late-pregnant rats. Patch-clamp electrophysiology in hypothalamic slices showed that bath application of kisspeptin did not affect action potential frequency or baseline membrane potential in supraoptic nucleus neurones. Moreover, kisspeptin superfusion did not affect the frequency or amplitude of excitatory postsynaptic currents or inhibitory postsynaptic currents in supraoptic nucleus neurones. Taken together, these studies suggest that kisspeptin directly activates oxytocin neurones in late pregnancy, at least in part, via increased post-spike excitability. KEY POINTS: Oxytocin secretion is triggered by action potential firing in magnocellular neurones of the hypothalamic supraoptic and paraventricular nuclei to induce uterine contractions during birth. In late pregnancy, kisspeptin expression increases in rat periventricular nucleus neurones that project to the oxytocin system. Here, we show that intra-supraoptic nucleus administration of kisspeptin increases the action potential firing rate of oxytocin neurones in anaesthetised late-pregnant rats, and that the increased firing rate is associated with increased oxytocin neurone excitability immediately after each action potential. By contrast, kisspeptin superfusion of hypothalamic slices did not affect the activity of supraoptic nucleus neurones or the strength of local synaptic inputs to supraoptic nucleus neurones. Hence, kisspeptin might activate oxytocin neurons in late pregnancy by transiently increasing oxytocin neuron excitability after each action potential.

Neurovascular coupling (NVC), the process that links neuronal activity to cerebral blood flow changes, has been mainly studied in superficial brain areas, namely the neocortex. Whether the conventional, rapid, and spatially restricted NVC response can be generalized to deeper and functionally diverse brain regions remains unknown. Implementing an approach for in vivo two-photon imaging from the ventral surface of the brain, we show that a systemic homeostatic challenge, acute salt loading, progressively increases hypothalamic vasopressin (VP) neuronal firing and evokes a vasoconstriction that reduces local blood flow. Vasoconstrictions are blocked by topical application of a VP receptor antagonist or tetrodotoxin, supporting mediation by activity-dependent, dendritically released VP. Salt-induced inverse NVC results in a local hypoxic microenvironment, which evokes positive feedback excitation of VP neurons. Our results reveal a physiological mechanism by which inverse NVC responses regulate systemic homeostasis, further supporting the notion of brain heterogeneity in NVC responses.

Oxytocin (OT), a neuropeptide produced in the supraoptic (SON) and paraventricular (PVN) nuclei, is not only essential for lactation and maternal behavior but also for normal immunological activity. However, mechanisms underlying OT regulation of maternal behavior and its association with immunity around parturition, particularly under mental and physical stress, remain unclear. Here, we observed effects of OT on maternal behavior in association with immunological activity in rats after cesarean delivery (CD), a model of reproductive stress. CD significantly reduced maternal interests to the pups throughout postpartum day 1-8. On postpartum day 5, CD decreased plasma OT levels and thymic index but increased vasopressin, interleukin (IL)-1β, IL-6 and IL-10 levels. CD had no significant effect on plasma adrenocorticotropic hormone and corticosterone levels. In the hypothalamus, CD decreased corticotropin-releasing hormone contents in the PVN but increased OT contents in the PVN and SON and OT release from hypothalamic implants. CD also increased c-Fos expression, particularly in the cytoplasm of OT neurons. Lastly, CD depolarized resting membrane potential and increased spike width while increasing the variability of the firing rate of OT neurons in brain slices. Thus, CD can increase hypothalamic OT contents and release but reduce pituitary release of OT into the blood, which is associated with depressive-like maternal behavior, increased inflammatory cytokine release and decreased relative weight of the thymus.

Oxytocin (OT) is a key factor for maternal behavior. However, neurochemical regulation of OT neurons, the major source of OT, remains incompletely understood. Here we report the effect of intranasally-applied OT (IAO) on OT neuronal activity in the supraoptic nucleus (SON) and on maternal behavior in a rat model of cesarean delivery (CD) at day 4–5 (stage I) and day 8–9 (stage II) following delivery. We found that at stage I, CD dams exhibited significantly longer latency of pup retrieval, lower number of anogenital licks and smaller acinar area of the mammary glands. In the SON, the number of OT neurons expressing phosphorylated extracellular signal-regulated protein kinase 1/2 (pERK 1/2) decreased significantly. IAO reversed the depressive-like maternal behavior and involution-like change in the mammary glands, and restored the number of pERK1/2-positive OT neurons in CD dams. At stage II, CD did not significantly influence the latency of retrieval and pERK1/2 expression in the SON. However, CD still reduced the number of anogenital licks during suckling, which was reversed by IAO. Notably, IAO but not hypodermic OT application in CD dams significantly increased litter’s body weight gains. In brain slices, CD but not CD plus IAO significantly depolarized membrane potential and increased spike duration in OT neurons. In vasopressin neurons, CD, but not CD plus IAO, significantly depolarized membrane potential and increased the firing rate. Thus, decreased OT neuronal activity and increased vasopressin neuronal activity impair maternal behavior in CD dams, which can be prevented by IAO .

Increased cardiac sympathetic nerve activity in type 2 diabetes mellitus (DM) suggests impaired autonomic control of the heart. However, the central regions that contribute to the autonomic cardiac pathologies in type 2 DM are unknown. Therefore, we tested the hypothesis that neuronal activation would be increased in central sympathoregulatory areas in a pre-clinical type 2 DM animal model. Immunohistochemistry in 20-week-old male Zucker diabetic fatty (ZDF) rats revealed an increased number of neurones expressing ΔFosB (a marker of chronic neuronal activation) in the intermediolateral column (IML) of the spinal cord in DM compared to non-diabetic (non-DM) rats (P < 0.05). Rostral ventrolateral medulla (RVLM) neurones activate IML neurones and receive inputs from the hypothalamic paraventricular nucleus (PVN), as well as the nucleus tractus solitarius (NTS) and area postrema (AP), in the brainstem. We observed more ΔFosB-positive noradrenergic RVLM neurones (P < 0.001) and corticotrophin-releasing hormone PVN neurones (P < 0.05) in DM compared to non-DM rats. More ΔFosB-positive neurones were also observed in the NTS (P < 0.05) and AP (P < 0.01) of DM rats compared to non-DM rats. Finally, because DM ZDF rats are obese, we also expected increased activation of pro-opiomelanocortin (POMC) arcuate nucleus (ARC) neurones in DM rats; however, fewer ΔFosB-positive POMC ARC neurones were observed in DM compared to non-DM rats (P < 0.01). In conclusion, increased neuronal activation in the IML of type 2 DM ZDF rats might be driven by RVLM neurones that are possibly activated by PVN, NTS and AP inputs. Elucidating the contribution of central sympathoexcitatory drive in type 2 DM might improve the effectiveness of pharmacotherapies for diabetic heart disease.

Oxytocin and vasopressin secretion from the posterior pituitary gland are required for normal pregnancy and lactation. Oxytocin secretion is relatively low and constant under basal conditions but becomes pulsatile during birth and lactation to stimulate episodic contraction of the uterus for delivery of the fetus and milk ejection during suckling. Vasopressin secretion is maintained in pregnancy and lactation despite reduced osmolality (the principal stimulus for vasopressin secretion) to increase water retention to cope with the cardiovascular demands of pregnancy and lactation. Oxytocin and vasopressin secretion are determined by the action potential (spike) firing of magnocellular neurosecretory neurons of the hypothalamic supraoptic and paraventricular nuclei. In addition to synaptic input activity, spike firing depends on intrinsic excitability conferred by the suite of channels expressed by the neurons. Therefore, we analysed oxytocin and vasopressin neuron activity in anaesthetised non-pregnant, late-pregnant, and lactating rats to test the hypothesis that intrinsic excitability of oxytocin and vasopressin neurons is increased in late pregnancy and lactation to promote oxytocin and vasopressin secretion required for successful pregnancy and lactation. Hazard analysis of spike firing revealed a higher incidence of post-spike hyperexcitability immediately following each spike in oxytocin neurons, but not in vasopressin neurons, in late pregnancy and lactation, which is expected to facilitate high frequency firing during bursts. Despite lower osmolality in late-pregnant and lactating rats, vasopressin neuron activity was not different between non-pregnant, late-pregnant, and lactating rats, and blockade of osmosensitive ΔN-TRPV1 channels inhibited vasopressin neurons to a similar extent in non-pregnant, late-pregnant, and lactating rats. Furthermore, supraoptic nucleus ΔN-TRPV1 mRNA expression was not different between non-pregnant and late-pregnant rats, suggesting that sustained activity of ΔN-TRPV1 channels might maintain vasopressin neuron activity to increase water retention during pregnancy and lactation.

Chronic stress exerts multiple negative effects on the physiology and health of an individual. In the present study, we examined hypothalamic, pituitary and endocrine responses to 14 days of chronic variable stress (CVS) in male and female C57BL/6J mice. In both sexes, CVS induced a significant decrease in body weight and enhanced the acute corticosterone stress response, which was accompanied by a reduction in thymus weight only in females. However, single-point blood measurements of basal prolactin, thyroid-stimulating hormone, luteinising hormone, growth hormone and corticosterone levels taken at the end of the CVS were not different from those of controls. Similarly, pituitary mRNA expression of Fshb, Lhb, Prl and Gh was unchanged by CVS, although Pomc and Tsh were significantly elevated. Within the adrenal medulla, mRNA for Th, Vip and Gal were elevated following CVS. Avp transcript levels within the paraventricular nucleus of the hypothalamus were increased by CVS; however, levels of Gnrh1, Crh, Oxt, Sst, Trh, Ghrh, Th and Kiss1 remained unchanged. Oestrous cycles were lengthened slightly by CVS and ovarian histology revealed a reduction in the number of preovulatory follicles and corpora lutea. Taken together, these observations indicate that 14 days of CVS induces an up-regulation of the neuroendocrine stress axis and creates a mild disruption of female reproductive function. However, the lack of changes in other neuroendocrine axes controlling anterior and posterior pituitary secretion suggest that most neuroendocrine axes are relatively resilient to CVS.

During parturition and lactation, oxytocin neurones in the supraoptic and paraventricular nuclei fire high-frequency bursts of action potentials that are coordinated across the entire population. Each burst generates a large pulse of oxytocin release into the circulation to induce uterine contraction for parturition and mammary duct contraction for milk ejection. Bursts are stimulated by cervical stretch during parturition and by suckling during lactation. However, the mechanisms by which these stimuli are translated into episodic bursts are poorly understood, as are the mechanisms that coordinate bursts across the oxytocin neurone population. An elegant series of experiments conducted in the 1980s and 1990s used serial paired recordings to show that oxytocin neurones do not act as a syncytium during bursts; rather, they start each burst within a few hundred milliseconds of each other but with no distinct "leaders" or "followers". In addition to afferent noradrenergic inputs that relay the systemic stimuli to oxytocin neurones, bursts depend on somato-dendritic oxytocin release within the hypothalamus. Hence, bursts are considered to be an emergent property of oxytocin neurones that is bootstrapped by appropriate afferent stimulation. Although much progress was made using traditional electrophysiological recordings in head-fixed anaesthetised animals, research has effectively stalled in the last few decades. However, the emergence of new technologies to monitor neuronal activity in freely-behaving animals has reinvigorated efforts to understand the biology underpinning burst firing in oxytocin neurones. Here, we report the use of fibre photometry to monitor the dynamics of milk ejection bursts in the oxytocin neurone population of freely-behaving mice. This approach will shed light on the neural mechanisms that control the oxytocin bursts underpinning parturition and lactation.

Somato-dendritic secretion was first demonstrated over 30 years ago. However, although its existence has become widely accepted, the function of somato-dendritic secretion is still not completely understood. Hypothalamic magnocellular neurosecretory cells were among the first neuronal phenotypes in which somato-dendritic secretion was demonstrated and are among the neurones for which the functions of somato-dendritic secretion are best characterised. These neurones secrete the neuropeptides, vasopressin and oxytocin, in an orthograde manner from their axons in the posterior pituitary gland into the blood circulation to regulate body fluid balance and reproductive physiology. Retrograde somato-dendritic secretion of vasopressin and oxytocin modulates the activity of the neurones from which they are secreted, as well as the activity of neighbouring populations of neurones, to provide intra- and inter-population signals that coordinate the endocrine and autonomic responses for the control of peripheral physiology. Somato-dendritic vasopressin and oxytocin have also been proposed to act as hormone-like signals in the brain. There is some evidence that somato-dendritic secretion from magnocellular neurosecretory cells modulates the activity of neurones beyond their local environment where there are no vasopressin- or oxytocin-containing axons but, to date, there is no conclusive evidence for, or against, hormone-like signalling throughout the brain, although it is difficult to imagine that the levels of vasopressin found throughout the brain could be underpinned by release from relatively sparse axon terminal fields. The generation of data to resolve this issue remains a priority for the field.

Vasopressin is synthesised by magnocellular neurons in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, and is secreted from the posterior pituitary gland in response to increased plasma osmolality. Vasopressin maintains body fluid balance by promoting renal water retention. Mechanosensitive TRPV channels facilitate vasopressin neuron responses to plasma osmolality and express various TRPV subunits, including ΔN‐TRPV1, TRPV2 and TRPV4. Most TRPV channels express more than one type of TRPV subunit but the subunit composition of the TRPV channels expressed by vasopressin neurons is unknown, as is the potential contribution of any changes in composition to vasopressin neuron function. Here, we tested the hypothesis that a change in TRPV channel composition alters the mechanosensitivity of ΔN‐TRPV1‐containing channels. To test this hypothesis TRPV and vasopressin expression in the SON of female Sprague Dawley rats was visualised using immunohistochemistry and will also be visualised with RNAscope. Xenopus oocytes and HEK293 cells were used for heterologous expression of TRPV channels and membrane current recorded via electrophysiology. TRPV2 was expressed on ~33% of SON vasopressin neurons and TRPV4 was expressed on ~40%. However, the TRPV1 antibody caused non‐specific staining and instead RNAscope will be used. Xenopus oocytes expressing ΔN‐TRPV1, TRPV2 and TRPV4 were recorded via two‐electrode‐voltage‐clamp (TEVC). However, TRPV channels in Xenopus oocytes do not appear to be functional because there was no change in membrane current during application of TRPV antagonists and agonists, independent of injected RNA amount (1–20ng) and incubation time (1–4 days). Transfection of TRPV channels into HEK293 cells with co‐expressed fluorescent protein; ΔN‐TRPV1‐GFP, TRPV2‐mCherry and TRPV4‐CFP was performed. Maximum ΔN‐TRPV1‐GFP expression occurred from 10–20 h, however increased cell death was observed after 24 h. In conclusion, vasopressin neurons express TRPV channels but Xenopus oocytes appear to lack specific cytoskeletal proteins for mechanical gating such as beta‐tubulin. HEK‐293 cells express the required cytoskeletal proteins for TRPV trafficking, membrane tethering and gating and we are in process of testing whether ΔN‐TRPV1‐GFP transfection in HEK‐293 cells forms mechanosensitive channels.

Somato‐dendritic secretion was first demonstrated over 30 years ago. However, although its existence has become widely accepted, the function of somato‐dendritic secretion is still not completely understood. Hypothalamic magnocellular neurosecretory cells were among the first neuronal phenotypes in which somato‐dendritic secretion was demonstrated and are among the neurones for which the functions of somato‐dendritic secretion are best characterised. These neurones secrete the neuropeptides, vasopressin and oxytocin, in an orthograde manner from their axons in the posterior pituitary gland into the blood circulation to regulate body fluid balance and reproductive physiology. Retrograde somato‐dendritic secretion of vasopressin and oxytocin modulates the activity of the neurones from which they are secreted, as well as the activity of neighbouring populations of neurones, to provide intra‐ and inter‐population signals that coordinate the endocrine and autonomic responses for the control of peripheral physiology. Somato‐dendritic vasopressin and oxytocin have also been proposed to act as hormone‐like signals in the brain. There is some evidence that somato‐dendritic secretion from magnocellular neurosecretory cells modulates the activity of neurones beyond their local environment where there are no vasopressin‐ or oxytocin‐containing axons but, to date, there is no conclusive evidence for, or against, hormone‐like signalling throughout the brain, although it is difficult to imagine that the levels of vasopressin found throughout the brain could be underpinned by release from relatively sparse axon terminal fields. The generation of data to resolve this issue remains a priority for the field.

Determining the effect of ageing on thigh muscle stiffness using magnetic resonance elastography (MRE) and investigate whether fat fraction and muscle cross-sectional area (CSA) are related to stiffness. Six healthy older adults in their eighth and ninth decade and eight healthy young men were recruited and underwent a 3 T MRI protocol including MRE and Dixon fat fraction imaging. Muscle stiffness, fat fraction and muscle CSA were calculated in ROIs corresponding to the four quadriceps muscles (i.e. vastus lateralis (VL), vastus medialis (VM), vastus intermedius (VI), rectus femoris (RF)), combined quadriceps, combined hamstrings and adductors and whole thigh. Muscle stiffness was significantly reduced ( p < 0.05) in the older group in all measured ROIs except the VI ( p = 0.573) and RF ( p = 0.081). Similarly, mean fat fraction was significantly increased ( p < 0.05) in the older group over all ROIs with the exception of the VI ( p = 0.059) and VL muscle groups ( p = 0.142). Muscle CSA was significantly reduced in older participants in the VM ( p = 0.003) and the combined quadriceps ( p = 0.001), hamstrings and adductors ( p = 0.008) and whole thigh ( p = 0.003). Over the whole thigh, stiffness was significantly negatively correlated with fat fraction ( r = − 0.560, p = 0.037) and positively correlated with CSA ( r = 0.749, p = 0.002). Stepwise regression analysis revealed that age was the most significant predictor of muscle stiffness ( p = 0.001). These results suggest that muscle stiffness is significantly decreased in healthy older adults. Muscle fat fraction and muscle CSA are also significantly changed in older adults; however, age is the most significant predictor of muscle stiffness.