Alastair Victor Ferguson

In addition to being recognised for involvement in cardiovascular control and hydromineral balance, the renin‐angiotensin system (RAS) has also been associated with the neuroendocrine control of energy balance. One of the main brain sites for angiotensin II (ANG II)/type 1 receptor (AT₁R) signalling is the subfornical organ (SFO), a circumventricular organ related to the control of autonomic functions, motivated behaviours and energy metabolism. Thus, we hypothesised that circulating ANG II may act on the SFO AT₁R receptors to integrate metabolic and hydromineral balance. We evaluated whether food deprivation can modulate systemic RAS activity and Agrt1a brain expression, and if ANG II/AT₁R signalling influences the hypothalamic expression of mRNAs encoding neuropeptides and food and water ingestion in fed and fasted Wistar rats. We found a significant increase in both ANG I and ANG II plasma levels after 24 and 48 h of fasting. Expression of Agrt1a mRNA in the SFO and paraventricular nucleus (PVN) also increased after food deprivation for 48 h. Treatment of fasted rats with low doses of losartan in drinking water attenuated the decrease in glycemia and meal‐associated water intake without changing the expression in PVN or arcuate nucleus of mRNAs encoding selected neuropeptides related to energy homeostasis control. These findings point to a possible role of peripheral ANG II/SFO‐AT₁R signalling in the control of refeeding‐induced thirst. On the other hand, intracerebroventricular losartan treatment decreased food and water intake over dark time in fed but not in fasted rats.

Phoenixin (PNX) is a neuropeptide shown to play roles in the control of reproduction. The nucleus of the solitary tract (NTS), a critical autonomic integrating centre in the hindbrain, is one of many areas with dense expression of PNX. Using coronal NTS slices obtained from male Sprague‐Dawley rats, the present study characterised the effects of PNX on both spike frequency and membrane potential of NTS neurones. Extracellular recordings demonstrated that bath‐applied 10 nmol L^‐1 PNX increased the firing frequency in 32% of NTS neurones, effects which were confirmed with patch‐clamp recordings showing that 50% of NTS neurones tested depolarised in response to application of the peptide. Surprisingly, the responsiveness to PNX in NTS neurones then declined suddenly to 9% (P < 0.001). This effect was subsequently attributed to stress associated with construction in our animal care facility because PNX responsiveness was again observed in slices from rats delivered and maintained in a construction‐free facility. We then examined whether this loss of PNX responsiveness could be replicated in rats placed on a chronic stress regimen involving ongoing corticosterone (CORT) treatment in the construction‐free facility. Slices from animals treated in this way showed a similar lack of neuronal responsiveness to PNX (9.1 ± 3.9%) within 2 weeks of CORT treatment. These effects were specific to PNX responsiveness because CORT treatment had no effect on the responsiveness of NTS neurones to angiotensin II. These results are the first to implicate PNX with respect to directly controlling the excitability of NTS neurones and also provide intriguing data showing the plasticity of these effects associated with environmental and glucocorticoid stress levels of the animal.

Subfornical organ (SFO) neurons exhibit heterogeneity in current expression and spiking behavior, where the two major spiking phenotypes appear as tonic and burst firing. Insight into the mechanisms behind this heterogeneity is critical for understanding how the SFO, a sensory circumventricular organ, integrates and selectively influences physiological function. To integrate efficient methods for studying this heterogeneity, we built a single-compartment, Hodgkin-Huxley-type model of an SFO neuron that is parameterized by SFO-specific in vitro patch-clamp data. The model accounts for the membrane potential distribution and spike train variability of both tonic and burst firing SFO neurons. Analysis of model dynamics confirms that a persistent Na⁺ and Ca²⁺ currents are required for burst initiation and maintenance and suggests that a slow-activating K⁺ current may be responsible for burst termination in SFO neurons. Additionally, the model suggests that heterogeneity in current expression and subsequent influence on spike afterpotential underlie the behavioral differences between tonic and burst firing SFO neurons. Future use of this model in coordination with single neuron patch-clamp electrophysiology provides a platform for explaining and predicting the response of SFO neurons to various combinations of circulating signals, thus elucidating the mechanisms underlying physiological signal integration within the SFO. NEW & NOTEWORTHY Our understanding of how the subfornical organ (SFO) selectively influences autonomic nervous system function remains incomplete but theoretically results from the electrical responses of SFO neurons to physiologically important signals. We have built a computational model of SFO neurons, derived from and supported by experimental data, which explains how SFO neurons produce different electrical patterns. The model provides an efficient system to theoretically and experimentally explore how changes in the essential features of SFO neurons affect their electrical activity.

The subfornical organ (SFO) lacks the normal blood‐brain barrier and senses the concentrations of many different circulating signals, including glucose and angiotensin II (ANG II). ANG II has recently been implicated in the control of food intake and body weight gain. The present study assessed whether single SFO neurones sense changes in glucose and ANG II, and also whether changes in glucose concentration alter the responsiveness of these neurones to ANG II. SFO neurones dissociated from male Sprague‐Dawley rats (100‐175 g) were used. We first examined whether glucose concentration modulates AT₁ receptor expression. Similar AT_1a mRNA expression levels were found at glucose concentrations of 1, 5 and 10 mmol L^‐1 in dissociated SFO neurones. Glucose responsiveness of SFO neurones was assessed using perforated current‐clamp recordings and switching between 5 and 10 mmol L^‐1 glucose artificial cerebrospinal fluid to classify single neurones as nonresponsive (nGS), glucose‐excited (GE) or glucose‐inhibited (GI). In total, 26.7% of the SFO neurones were GI (n = 24 of 90), 21.1% were GE (n = 19 of 90) and 52.2% were nGS (n = 47 of 90). Once classified, the effects of 10 nmol L^‐1 ANG II on the excitability of these neurones were tested, with 52% of GE (n = 10 of 19), 71% of GI (n = 17 of 24) and 43% of nGS (n = 20 of 47) neurones being ANG II sensitive. Finally, we tested whether acute changes in glucose concentration modified the response to ANG II and showed that some neurones (4/17) only respond to ANG II at 10 mmol L^‐1 glucose. Our data demonstrate that the same SFO neurone can sense glucose and ANG II and that acute changes in glucose concentration may change ANG II responsiveness.

Inflammation is thought to play a fundamental role in the pathophysiology of hypertension and heart failure, although the mechanisms for this remain unclear. Proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α), influence the subfornical organ (SFO) to modulate sympathetic activity and blood pressure. The pressor effects of TNF-α in the SFO are partially mediated by angiotensin II (ANG II) receptor type 1 (AT₁R), and TNF-α is known to potentiate ANG II-induced hypertension. However, the cellular mechanism of the interaction between TNF-α and ANG II/AT₁R signaling remains unknown. In the present study, we performed Ca²⁺imaging on dissociated SFO neurons in vitro from male Sprague-Dawley rats to determine whether TNF-α modulates ANG II-induced increases in intracellular Ca²⁺in SFO neurons. We first established that a proportion of SFO neurons respond to ANG II, an effect that required AT₁R signaling and extracellular Ca²⁺. We then tested the hypothesis that TNF-α may modulate the effects of ANG II on SFO neurons by examining the effects of TNF-α treatment on the ANG II-induced rise in intracellular Ca²⁺. We discovered that TNF-α potentiated the ANG II-induced rise in intracellular Ca²⁺, an effect that was dependent on the duration of TNF-α treatment. Finally, we determined that this potentiation of ANG II-induced Ca²⁺activity relied on tetrodotoxin-sensitive voltage-gated Na⁺(vgNa⁺) channels. These data suggest that the potentiation of ANG II/AT₁R activity by TNF-α in SFO neurons results from the previously demonstrated ability of this cytokine to modulate the activation threshold of vgNa⁺currents.

Prolactin (PRL) is a peptide hormone that performs over 300 biological functions, including those that require binding to prolactin receptor (PRL‐R) in neurones within the central nervous system (CNS). To enter theCNS, circulatingPRLmust overcome the blood‐brain barrier. Accordingly, areas of the brain that do not possess a blood‐brain barrier, such as the subfornical organ (SFO), are optimally positioned to interact with systemicPRL. TheSFOhas been classically implicated in energy and fluid homeostasis but has the potential to influence oestrous cyclicity and gonadotrophin release, which are also functions ofPRL. We aimed to confirm and characterise the expression ofPRL‐R in theSFO, as well as identify the effects ofPRLapplication on membrane excitability of dissociatedSFOneurones. Using a quantitative real‐time polymerase chain reaction, we found thatPRL‐RmRNAin theSFOof male and female Sprague Dawley rats did not significantly differ between juvenile and sexually mature rats (P = .34), male and female rats (P = .97) or across the oestrous cycle (P = .54). Patch‐clamp recordings were obtained in juvenile male rats to further investigate the actions ofPRLat theSFO. DissociatedSFOneurones perfused with 1 μmol L^‐1PRLresulted in 2 responsive subpopulations of neurones; 40% depolarised (n = 15/43, 11.3 ± 1.7mV) and 14% hyperpolarised (n = 6/43, −6.7 ± 1.4mV) toPRLapplication. Within the range of 10 pmol L^‐1to 1 μmol L^‐1, the concentrations ofPRLwere not significantly different in either the magnitude (P = .53) or proportion (P = .19) of response. Furthermore,PRLapplication significantly reduced the transient K⁺current in 67% ofSFOneurones in voltage‐clamp configuration (n = 6/9,P = .02). The stability in response toPRLand expression ofPRL‐R in theSFOsuggests thatPRLfunction is conserved across physiological states and circulatingPRLconcentrations, prompting further investigations aiming to clarify the nature ofPRLfunction in the SFO.

Brain‐derived neurotrophic factor (BDNF), a neurotrophin traditionally associated with neural plasticity, has more recently been implicated in fluid balance and cardiovascular regulation. It is abundantly expressed in both the central nervous system (CNS) and peripheral tissue, and is also found in circulation. Studies suggest that circulating BDNF may influence the CNS through actions at the subfornical organ (SFO), a circumventricular organ (CVO) characterized by the lack of a normal blood–brain barrier (BBB). The SFO, well‐known for its involvement in cardiovascular regulation, has been shown to express BDNF mRNA and mRNA for the TrkB receptor at which BDNF preferentially binds. This study was undertaken to determine if: (1) BDNF influences the excitability of SFO neurons in vitro; and (2) the cardiovascular consequences of direct administration of BDNF into the SFO of anesthetized rats. Electrophysiological studies revealed that bath application of BDNF (1 nmol/L) influenced the excitability of the majority of neurons (60%, n = 13/22), the majority of which exhibited a membrane depolarization (13.8 ± 2.5 mV, n = 9) with the remaining affected cells exhibiting hyperpolarizations (−11.1 ± 2.3 mV, n = 4). BDNF microinjections into the SFO of anesthetized rats caused a significant decrease in blood pressure (mean [area under the curve] AUC = −364.4 ± 89.0 mmHg × sec, n = 5) with no effects on heart rate (mean AUC = −12.2 ± 3.4, n = 5). Together these observations suggest the SFO to be a CNS site at which circulating BDNF could exert its effects on cardiovascular regulation.

The newly described hypothalamic peptide, phoenixin, is produced in the hypothalamus and adenohypophysis, where it acts to control reproductive hormone secretion. Both phoenixin and its receptor GPR173 are expressed in the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei, suggesting additional, nonreproductive effects of the peptide to control vasopressin (AVP) or oxytocin (OT) secretion. Hypothalamo-neurohypophysial explants released AVP but not OT in response to phoenixin. Intracerebroventricular administration of phoenixin into conscious, unrestrained male and female rats significantly increased circulating AVP, but not OT, levels in plasma, and it increased immediate early gene expression in the supraoptic nuclei of male rats. Bath application of phoenixin in hypothalamic slice preparations resulted in depolarization of PVN neurons, indicating a direct, neural action of phoenixin in the hypothalamus. Our results suggest that the newly described, hypothalamic peptide phoenixin, in addition to its effects on hypothalamic and pituitary mechanisms controlling reproduction, may contribute to the physiological mechanisms regulating fluid and electrolyte homeostasis.