Apelin acts in the subfornical organ to influence neuronal excitability and cardiovascular function

Abstract

Apelin is an adipocyte-derived hormone involved in the regulation of water balance, food intake and the cardiovascular system partially through actions in the CNS. The subfornical organ (SFO) is a circumventricular organ with identified roles in body fluid homeostasis, cardiovascular control and energy balance. The SFO lacks a normal blood–brain barrier, and is thus able to detect circulating signalling molecules such as angiotensin II and leptin. In this study, we investigated actions of apelin-13, the predominant apelin isoform in brain and circulatory system, on the excitability of dissociated SFO neurons using electrophysiological approaches, and determined the cardiovascular consequences of direct administration into the SFO of anaesthetized rats. Whole cell current clamp recording revealed that bath-applied 100 nm apelin-13 directly influences the excitability of the majority of SFO neurons by eliciting either depolarizing (31.8%, mean 7.0 ± 0.8 mV) or hyperpolarizing (28.6%, mean −10.4 ± 1.8 mV) responses. Using voltage-clamp techniques, we also identified modulatory actions of apelin-13 on specific ion channels, demonstrating that apelin-13 activates a non-selective cationic conductance to depolarize SFO neurons while activation of the delayed rectifier potassium conductance underlies hyperpolarizing effects. In anaesthetized rats, microinjection of apelin into SFO decreased both blood pressure (BP) (mean area under the curve −1492.3 ± 357.1 mmHg.s, n= 5) and heart rate (HR) (−32.4 ± 10.39 beats, n= 5). Our data suggest that circulating apelin can directly affect BP and HR as a consequence of the ability of this peptide to modulate the excitability of SFO neurons.

Key points

• Apelin receptor mRNA is expressed in the subfornical organ.

• Apelin influences the excitability of the majority of subfornical organ neurons, with similar proportion showing depolarizing and hyperpolarizing effects.

• Hyperpolarizations appear to result from the activation of a sustained voltage-activated potassium conductance, while depolarizations may result from modulation of a non-selective cationic conductance.

• In vivo microinjection of apelin into the subfornical organ results in decreases in blood pressure.

Introduction

Apelin, an adipocyte-derived hormone (Boucher et al. 2005), was initially isolated from bovine stomach extracts and finally identified as an endogenous ligand of the human orphan G-protein-coupled apelin receptor (APJ) (Tatemoto et al. 1998). Apelin is found in vascular endothelial cells and peripheral tissue, including heart, liver, kidney, lung and mammary glands (Habata et al. 1999; O’Carroll et al. 2000; Kleinz & Davenport, 2004), and CNS (Hosoya et al. 2000; O’Carroll et al. 2000; Kawamata et al. 2001; Reaux et al. 2001; Medhurst et al. 2003). This peptide is derived from a 77 amino acid precursor and then undergoes post-translational processing into a number of different forms, including apelin-36, apelin-17 and apelin-13, all of which share the fully conserved C-terminal sequence (Tatemoto et al. 1998). These products all have high affinity for APJ and similar biological properties, such as promoting the extracellular acidification, suppressing forskolin-stimulated cAMP production, and inducing APJ internalization (Tatemoto et al. 1998; Zou et al. 2000; Medhurst et al. 2003; El Messari et al. 2004; Iturrioz et al. 2010). Apelin-13 is the predominant molecular form of endogenous apelin in brain and plasma (De Mota et al. 2004; Azizi et al. 2008) and exhibits clear biological activity and affinity for the APJ receptor (Tatemoto et al. 1998).

Apelin, through actions at the APJ receptor, has been shown to influence a variety of physiological systems, including fluid homeostasis (Lee et al. 2000; Taheri et al. 2002; De Mota et al. 2004; Roberts et al. 2009), metabolic control (Taheri et al. 2002; Sunter et al. 2003; Boucher et al. 2005; Wei et al. 2005; Valle et al. 2008; Lv et al. 2011) the immune response (Cayabyab et al. 2000; Horiuchi et al. 2003) and cardiovascular function (Tatemoto et al. 2001; Lee et al. 2005). Interestingly, while intravenous injection of apelin causes drinking and decreases in blood pressure (BP) in rats (Lee et al. 2000; Tatemoto et al. 2001; Cheng et al. 2003), central (intracerebroventricular, i.c.v.) administration of this peptide has either no effect on cardiovascular function (Reaux et al. 2001) or increases mean arterial pressure and heart rate (HR, Seyedabadi et al. 2002; Kagiyama et al. 2005), suggesting a divergence of peripheral and central actions of apelin. The subfornical organ (SFO), one of the circumventricular organs (CVOs) of the brain (structures lacking a normal blood–brain barrier), has been identified as a potential site at which circulating signals involved in the regulation of cardiovascular and metabolic function may influence the brain (Mangiapane & Simpson, 1980a; Smith & Ferguson, 1997; Riediger et al. 1999; Smith & Ferguson, 2012). Adiponectin, amylin, angiotensin II, leptin, vasopressin, orexin-A and relaxin have all been shown to influence central autonomic pathways by modulating the neuronal activity of individual SFO neurons (Anthes et al. 1997; Ferguson et al. 1997; Riediger et al. 1999; Ono et al. 2008; Smith et al. 2009; Alim et al. 2010) through efferent projections of SFO neurons to hypothalamic autonomic control centres including the arcuate, medial preoptic and paraventricular nuclei (PVN) (Gruber et al. 1987; Lind et al. 1982). In addition, direct administration of many of these signalling molecules into the SFO has been shown to influence cardiovascular function (Mangiapane & Simpson, 1980b; Smith & Ferguson, 1997; Smith et al. 2007). Destruction of the SFO has also been shown to abolish both the pressor response and drinking induced by intravenous injection of angiotensin II (Mangiapane & Simpson, 1980a; Collister & Nahey, 2009), highlighting the critical role for this CVO in transmitting sensory information carried by these circulating signals to central control centres protected by the normal blood–brain barrier.

Apelinergic fibres innervating the SFO have been reported (Reaux et al. 2002), while our microarray analysis of the transcriptome of the SFO demonstrated mRNA for the APJ receptor in this CVO (Hindmarch et al. 2008). These observations suggest a functional role for the SFO in monitoring both neurally derived and/or circulating apelin, and this study was therefore undertaken to investigate the physiological and cellular actions of apelin actions in the SFO.

Methods

All experiments and animal protocols conformed to the standards of the Canadian Council on Animal Care and were approved by the Queen's University Animal Care Committee.

SFO neuron preparation

SFO neurons were prepared from brains removed rapidly from male Sprague-Dawley rats (100–150 g) following decapitation. Tissue was placed in ice-cold oxygenated artificial cerebrospinal fluid (aCSF) containing: 124 mm NaCl, 2.5 mm KCl, 1.24 mm KH₂PO₄, 2.27 mm CaCl₂, 1.3 mm MgSO₄, 20 mm NaHCO₃ and 10 mm glucose. Under a dissecting microscope, the SFO was identified and removed from the ventral surface of the fornix and transferred into Hibernate medium (Brain Bits, Springfield, IL, USA) containing 2 mg ml⁻¹ papain (Worthington Biochemical, Lakewood, NJ, USA). After 30 min incubation at 31°C, the SFO was rinsed three times, gently triturated with Hibernate medium supplemented with B27 (Life Technologies Inc., Carlsbad, CA, USA), and dissociated neurons were collected in a pellet by centrifugation at 200 g for 5 min at 4°C. The cells were resuspended in B27 supplemented Neurobasal A medium (Life Technologies) containing 100 U ml⁻¹ penicillin-streptomycin, 0.5 mm l-glutamine (Life Technologies) and 5 mm glucose, and aliquoted to 35 mm glass-bottomed culture dishes (MatTek, Ashland, MA, USA). The cells were maintained in the incubator at 37°C in 5% CO₂. After the neurons adhered to the bottom of the culture dish (approximately 3 h) additional B27 supplemented Neurobasal A was added. These techniques as well as viability of dissociated neurons have been described in depth previously (Ferguson et al. 1997).

RT-PCR

Total RNA was extracted from acutely microdissected SFO tissue using an RNAqueous-Micro kit (Ambion, Austin, TX, USA). After DNase treatment, the total RNA was transcribed to cDNA using a Superscript III first strand synthesis kit (Life Technologies). RT-PCR was carried out with a Qiagen multiplex PCR kit (Qiagen, Valencia, CA, USA) using primers specific for rat APJ (NM_031349.2 forward primer: 5′-TTCCTTCTAGGCACCACAGG-3′; reverse primer: 5′-CCAAAAGGCCAGTCAAACTC-3′, yielding a 179 bp product). The reaction was denatured at 95°C for 15 min and cycled 30 times through a temperature protocol consisting of 20 s at 94°C, 30 s at 60°C and 40 s at 72°C. PCR products were analysed on a 1.5% agarose gel.

Electrophysiology

Voltage and current clamp recordings were obtained from SFO neurons 1–4 days after dissociation using a Multiclamp 700B patch-clamp amplifier (Molecular Devices, Sunnyvale, CA, USA). Recording and stimulation were carried out using Signal (version 4) and Spike2 (version 6) software (Cambridge Electronics Design, Cambridge, UK). Membrane potential and whole-cell current data were digitized at 5 kHz and filtered at 1 kHz using a Cambridge Electronics Design Micro1401 interface. Patch-clamp recording in the whole-cell mode was performed using the perforated patch method as previously described (Price et al. 1999). Electrodes were fabricated from borosilicate glass (World Precision Instruments, Sarasota, FL, USA) and fire-polished. When filled with internal solution containing 55 mm KCl, 75 mm potassium acetate, 8 mm MgCl₂ and 10 mm Hepes (pH 7.3 with KOH), electrodes had a resistance of 2–4 MΩ. The external recording solution contained 140 mm NaCl, 5 mm KCl, 1 mm MgCl₂, 2 mm CaCl₂, 10 mm Hepes, 5 mm mannitol and 5 mm glucose, and pH was adjusted to 7.3 with NaOH.

Dissociated SFO neurons were perfused with external recording solution (32°C) at a rate of 1.6 ml min⁻¹. Once whole cell configuration was achieved, cells were defined as neurons by the presence of voltage-gated Na⁺ currents under voltage-clamp configuration and the occurrence of spontaneous or current evoked action potentials (amplitude greater than 50 mV) in current clamp configuration. A calculated junction potential of −14.4 mV was added to the membrane potential of recorded neurons. After a minimum 200 s stable baseline membrane potential was established, various concentrations (from 1 fm to 100 nm) of apelin-13 (Phoenix Pharmaceuticals) was applied followed by a wash with external recording solution. Responsiveness of SFO neurons was determined by comparing membrane potential of neurons before and after apelin-13 application. Membrane potential changes were determined by comparing the average membrane potential during the 100 s control period, with sequential 100 s periods after apelin-13 application. SFO neurons were considered responsive to apelin-13 application if the difference in membrane potential was greater than twice the standard deviation of the control baseline membrane potential and the cell showed a partial recovery toward baseline values following removal of the apelin-13 from the bath. Reported changes in membrane potential represent the 100 s mean with the largest change from the control period.

In voltage-clamp experiments, currents were recorded using a slow voltage ramp protocol (12 mV s⁻¹, from −100 to 0 mV), prior to and after administration of apelin-13 in bath solution to assess the effects of bath-applied apelin-13 on whole cell currents. SFO neurons were clamped at −80 mV, and ramp currents were determined from an average of three ramps. The apelin-13-induced difference current was calculated by subtraction of the control current before administration of apelin-13 from the current obtained after peptide treatment. Linear regression analysis was used to determine reversal potential. Full current–voltage (I–V) relationships were evaluated using voltage step protocols before and after apelin-13 application. Whole cell currents were evoked by voltage steps from −120 to 10 mV in 10 mV increments from a holding potential of −80 mV and the I–V relationship was constructed using the average current recorded during the final 10 ms of each voltage step plotted against the corresponding voltage.

Mean change (± SEM) in membrane potential or currents before and after apelin-13 administration were calculated as described above, and changes were compared using a Student's t test. All statistical analyses were performed using GraphPad Prism (version 5.0).

Microinjection

Urethane-anaesthetized (1.4 g kg⁻¹) male Sprague- Dawley rats (150–350 g) were placed on a feedback- controlled heating blanket for the duration of the experiment to maintain body temperature at 37°C. Urethane was elected for use in these studies in association with its ability to maintain long lasting stable levels of anaesthesia. Animals were fitted with an indwelling femoral arterial catheter (Intramedic PE50 Tubing) for the measurement of BP and HR. The animal was then placed in a stereotaxic frame and a midline incision was made through the skin of the skull. A small burr hole was drilled in the skull such that a microinjection cannula (150 μm tip diameter; Rhodes Medical Instruments, Summerland, CA, USA) could be advanced into the region of the SFO according to the coordinates of Paxinos & Watson (1982; Bregma −0.8 mm, ventral 4.5 mm, midline). After a minimum 2 min stable baseline recording was obtained, apelin-13 (0.5 μl of 10⁻⁷ m-50 fmol, dissolved in aCSF) or aCSF (0.5 μl, vehicle control) was microinjected into the region by a pressure-driven 10 μl Hamilton micro-syringe over 10 s and the effects on BP and HR were assessed. At the conclusion of the experiment, animals were overdosed with anaesthetic, perfused through the left ventricle of the heart with 0.9% saline followed by 10% formalin, and the brain removed and placed in formalin for at least 24 h. Coronal sections (100 μm) were cut through the region of the SFO using a vibratome, mounted and stained with cresyl violet. The anatomical location of microinjection sites were verified at the light microscope level by an observer unaware of the experimental protocol or the data obtained.

Animals were assigned to one of two anatomical groups (SFO or non-SFO) according to the location of the microinjection site. Animals with injection sites that were not wholly confined within either of these regions were excluded from further analysis. Animals with confirmed microinjection sites in the SFO were further divided according to whether apelin-13 (50 fmol) or aCSF (vehicle control) was microinjected into the region. Normalized BP and HR data (mean baseline BP and HR data were calculated for 60 s before injection and subtracted from all data points before and after injection) were obtained for each animal 60 s before the time of microinjection (control period) until 200 s after microinjection. The area under the curve (AUC, area between baseline and each BP and HR response) was calculated for each animal for the 200 s immediately following the injection. Mean AUC for BP and HR responses for each group were then calculated. A one-way analysis of variance (one-way ANOVA, followed by Newman-Keuls post hoc analysis) was used to determine whether BP and HR responses observed in response to substance administered (apelin-13 or aCSF) into the SFO or anatomical location of apelin-13 administration (SFO or non-SFO) were statistically significant.

Chemicals

All chemicals were purchased from Sigma (St Louis, MO, USA), unless otherwise specified.

Results

Apelin influences the excitability of SFO neurons

Using RT-PCR, we detected expression of the apelin receptor gene, APJ, in SFO tissue (Fig. 1), and then utilized whole-cell recording techniques to access the effects of bath administration of apelin-13 on membrane potential of SFO neurons. We obtained recordings from 171 SFO neurons with resting membrane potentials between −46 and −62 mV, input resistance between 3.02 and 0.64 GΩ, spike amplitudes between 65 and 85 mV, and spontaneous activity (observed in 51% of recorded neurons) with a mean frequency of 2.41 ± 0.24 Hz. These data are in accordance with our previous recordings from SFO neurons (Ferguson et al. 1997; Desson & Ferguson, 2003).

Figure 1. Agarose gel RT-PCR analysis of SFO cDNA for APJ gene expression.

The PCR product (179 bp) for APJ is clearly present in SFO tissue but absent in the RT(–) reaction in which reverse transcriptase has been omitted from the cDNA synthesis reaction.

We first examined the sensitivity of SFO neurons to bath application of 100 nm apelin-13 (n= 63) which, as illustrated in Fig. 2, significantly changed membrane potential in 60.3% (n= 38) of SFO neurons tested, with approximately equal proportions of apelin-13-sensitive neurons showing depolarizations (31.8%, mean = 7.0 ± 0.80 mV, n= 20; Fig. 2A) or hyperpolarizations (28.6%, mean =−10.4 ± 1.8 mV, n= 18; Fig. 2B). Depolarizing responses showed relatively quick onsets (<250 s), were accompanied by increased spike frequency, as illustrated in Fig. 2A, and often lasted up to 15 min before return to baseline. Apelin-13-induced hyperpolarizations usually occurred within 180 s and were often accompanied by a reduced spontaneous action potential frequency (see Fig. 2B). The remaining 39.7% (n= 25) of tested SFO neurons did not exhibit any significant changes in membrane potential (0.1 ± 0.32 mV, n= 25, Fig. 2C) or spike frequency (−0.09 ± 0.232 mV, n= 25, Fig. 2D) in response to bath administration of apelin-13, suggesting the existence of subpopulations of SFO neurons with differential sensitivity to apelin-13. Importantly, bath administration of 500 nm apelin F13A (an apelin receptor antagonist) inhibited single cell responses to 100 nm apelin-13 as only 1 of 10 cells tested responded (with a small depolarization +5.3 mV). The chi-squared test showed that effects after F13A were significantly different from the normal depolarizing and hyperpolarizing effects of apelin-13 (P < 0.005).

Figure 2. Current-clamp recordings and changes in membrane potential and action potential frequency for cells treated with apelin-13.

A and B, representative current-clamp recordings from neurons that depolarized (A) or hyperpolarized (B) in response to bath application of 100 nm apelin-13 (black bar). C and D, graphs representing mean changes in membrane potential (C) and action potential frequency (D) for cells treated with 100 nm apelin-13. SFO neurons in which sodium channels inactivated after depolarization and neurons that were quiescent before hyperpolarization were not included in the analysis of the mean change in action potential frequency. Each bar represents the mean ± SEM for group data.

Interestingly, the magnitude of the changes in membrane potential of SFO neurons in response to apelin-13 did not show a typical concentration–response relationship, as depolarizations (between 5.4 ± 1.4 and 7.0 ± 0.8 mV) or hyperpolarizations (between −6.5 ± 1.5 and 9.8 ± 1.4 mV) were similar when 1 pm (n= 13), 1 nm (n= 28), 10 nm (n= 26) or 100 nm (n= 63) apelin-13 was administrated. In addition, the percentage of neurons influenced by apelin-13 was similar at all effective concentrations (between 47.8 and 60.3%). Statistical analysis of these parameters showed that neither the amplitude of membrane potential nor the proportion of responsive cells showed significant differences in response to apelin-13 at concentrations between 1 pm and 100 nm (P > 0.05, one-way ANOVA), suggesting that the maximal response is in fact observed at 1 pm. This observation is in accordance with previous studies showing that the total concentration of circulating apelin (mainly apelin-17 and apelin-13) in rat plasma is around 3.4 ± 0.25 pm (De Mota et al. 2004). We therefore reduced the concentration of apelin-13 applied to 100 fm and, of 16 SFO neurons tested, one-third responded with three cells showing depolarization (7.5 ± 0.7 mV) and two displaying hyperpolarization (−7.9 ± 0.2 mV). A further reduction to 1 fm apelin-13 resulted in 2 of 13 cells tested responding, one showing depolarization (+8.2 mV) and the other a hyperpolarization (−7.7 mV). Although the amplitudes of changes in membrane potential did not show any obvious reduction at these lower concentrations, the percentage of responding neurons was reduced to 31% (100 fm) and 15% (1 fm). The average absolute value of any change in membrane potential for all SFO neurons tested with each concentration, including non-responsive cells, can be calculated and thus provide a mean absolute change in membrane potential for 100 nm (5.2 ± 0.5 mV), 1 pm (4.6 ± 1.0 mV), 100 fm (3.2 ± 0.8 mV) and 1 fm (2.0 ± 0.8 mV), as illustrated in Fig. 3.

Figure 3. Absolute changes in membrane potential of SFO neurons in response to increasing concentrations of apelin-13.

Mean (±SEM) absolute changes in the membrane potential of SFO neurons in response to increasing concentrations (1 fm to 100 nm) of apelin-13. The percentage of apelin-13-responsive SFO neurons and n values are indicated in columns.

Apelin-13 modulates a K⁺ conductance and a non-selective cationic conductance in SFO neurons

We next undertook experiments designed to investigate the potential ion channels underlying the differential depolarizing and hyperpolarizing effects of apelin-13 on SFO neurons. These studies were carried using a recording protocol in which we first obtained voltage clamp recordings to assess whole cell currents in response to slow voltage ramps (−100 to 0 mV, 12 mV s⁻¹) and standard depolarizing pulse protocols (−120 to 10 mV from holding potential of −80 mV in 10 mV increments) under control conditions. Recording configuration was then changed to current clamp and 100 nm apelin-13 was administered, as described above, and initial effects on membrane potential assessed such that the cell could be classified as depolarized, hyperpolarized or unaffected by apelin-13. The recording configuration was then switched back to voltage clamp and the currents in response to slow voltage ramps and step protocols were reassessed such that changes, and thus the apelin-13-evoked current, could be determined by subtraction.

Voltage ramp analysis of 14 SFO neurons which hyperpolarized in response to apelin-13 (see Fig. 4Aa) show a linear apelin-13-induced whole cell current (r²= 0.97) within the voltage range of −100 to −40 mV with a mean reversal potential of −78.6 ± 3.2 mV (Fig. 4Ab), suggesting the involvement of a potassium conductance in mediating the hyperpolarizing response to apelin-13. In addition, these voltage ramps showed that apelin-13 induced a large voltage-dependent outward current which activated at approximately −30 mV (see Fig. 4Ab), currents which suggested effects on voltage-activated sustained K⁺ current (I_K). Analysis of voltage clamp pulse protocols in the 14 hyperpolarized SFO neurons identified a considerable increase in amplitude of sustained K⁺ current in response to voltage steps to 0 mV from a mean of 0.68 ± 0.08 nA in control conditions to 0.87 ± 0.11 nA during apelin-induced hyperpolarization, effects that show a statistically significant increase in current (paired ‘t’ test P < 0.05 n= 14). These changes represent a normalized increase in current at steps to both 0 mV (25.7 ± 9.9%) and 10 mV (17.8 ± 7.7%), as illustrated in Fig. 5. In contrast, SFO neurons that depolarized in response to apelin-13 showed no such effects on amplitude of sustained K⁺ current in response to the same voltage steps (0 mV 2.5 ± 7.3%; 10 mV 5.4 ± 6.8%, n= 7). Interestingly, unlike the increased sustained K⁺ current, the peak value of transient K⁺ (putative but uncharacterized I_A) from hyperpolarized SFO neurons was reduced (0 mV −16.1 ± 4.5%, n= 14), but was unaffected in SFO neurons depolarized by apelin-13 (0 mV +0.7 ± 3.0%, n= 7) as illustrated in Fig. 5 and summarized in Fig. 5C.

Figure 4. Currents recorded from SFO neurons in response to slow voltage ramps before and during apelin-13-induced hyperpolarization or depolarization.

Representative traces of currents recorded from SFO neurons in response to slow voltage ramps (12 mV s⁻¹: Aa inset) before (black traces) and during (red traces) apelin-13-induced hyperpolarization (Aa) or depolarization (Ba). The lower traces shown in Ab and Bb show the net apelin-13-induced (difference) current elicited, which is derived by subtracting control currents (black) from apelin-13-affected current (red) which are recorded from the same neurons as shown in upper panels. The insets in Ab and Bb show the mean I–V curves (±SEM) of net apelin-13-induced current in hyperpolarized (Ab, n= 14) and depolarized (Bb, n= 4) neurons.

Figure 5. Apelin-13 increases I_K in SFO neurons hyperpolarized by this peptide.

A, whole cell K⁺ currents recorded from an SFO neuron hyperpolarized by apelin-13 in response to voltage steps protocol (inset) before peptide application (left) and during apelin-13-induced hyperpolarization (right). B, mean I–V curves showing currents measured at the end of voltage pulses (#– sustained current) in neurons which were hyperpolarized (n= 14 – left panel) or depolarized (n= 7 – right panel) by apelin-13 (black activation curves are control and red indicate I–V curves during apelin-13). The insets show overlaid traces recorded at 0 mV from control (black trace), hyperpolarized or depolarized neurons (red trace). C, bar graph showing the summary (mean ± SEM) changes in normalized amplitudes of the sustained K⁺ (I_K) and transient K⁺ (I_A– peak current measured in the first 50 ms of pulse ##) elicited at the 0 mV voltage step from SFO neurons in which apelin-13 induced depolarization (n= 7, blue bar) and hyperpolarization (n= 14, orange bar).

We next used 30 mm tetraethylammonium to block I_K and determine whether the apelin-13-induced hyperpolarization was still observed in SFO neurons. We tested 10 neurons in tetraethylammonium, of which 3 showed no response to apelin-13 (−0.08 ± 1.17 mV), 5 depolarized (7.96 ± 1.46 mV), and two responded with small (−2.63 and −3.37 mV) hyperpolarizations that were clearly smaller than the mean hyperpolarizing effects observed in aCSF (−10.4 ± 1.8 mV). In contrast, linear regression analysis for the I–V curve of apelin-13-induced currents from depolarized SFO neurons (data were collected successfully from four depolarized cells) revealed that the net apelin-13-induced whole cell current displayed a linear relationship between −100 and −30 mV (r²= 0.96, see Fig. 4Ba), with a mean reversal potential of −25.9 ± 5.3 mV (n= 4) (Fig. 4Bb inset), characteristic of a non-selective cationic conductance (NSCC) (Oliet & Bourque, 1993; Desson & Ferguson, 2003; Pulman et al. 2006).

Apelin microinjection into the SFO decreases BP and HR

Twenty animals were used in this study, 10 of which had microinjection locations entirely within the SFO (SFO group – baseline BP: 84.97 ± 6.8 mmHg, HR: 417.20 ± 19.9 b.p.m.) while 4 had microinjection locations wholly outside the SFO (non-SFO – baseline BP: 89.34 ± 5.0 mmHg, HR: 440.37 ± 6.3 b.p.m., values which were not significantly different from the SFO group). The remaining animals (n= 6) were excluded from further analysis as sites could not be reliably classified as either SFO or non-SFO sites.

Microinjection of apelin-13 (50 fmol) into the SFO resulted in rapid, long lasting (>5 min) decreases in BP (mean AUC =−1492.3 ± 357.1 mmHg*s, n= 5, P < 0.01 Newman Keuls post hoc analysis vs. non-SFO and aCSF; see Fig. 6A) and HR (mean AUC =−32.4 ± 10.4 beats, n= 5; P < 0.05 Newman–Keuls post hoc analysis vs. non-SFO and aCSF; see Fig. 6B). Apelin-13 microinjection elicited a significant (P < 0.05, one-way ANOVA) maximal decrease in BP (mean =−9.7 ± 3.5 mmHg, n= 5) and HR (mean =−14.2 ± 5.4 b.p.m.) which occurred within 2 min of apelin-13 administration. These effects were due to the administration of apelin-13, as microinjection of aCSF (vehicle) was without effect on BP (mean AUC =−64.2 ± 166.6 mmHg*s, n= 5) or HR (mean AUC = 0.78 ± 3.3 beats, n= 5). In addition, BP effects were shown to be site specific, as apelin-13 microinjection into non-SFO regions was without effect on BP (mean AUC =−276.2 ± 131.3 mmHg*s, n= 4) or HR (mean AUC = 2.6 ± 5.4 beats, n= 4).

Figure 6. Apelin-13 acts in SFO to decrease blood pressure and heart rate.

A and B, normalized mean BP (A, left) and HR (B, left) traces in response to a bolus (0.5 μl) microinjection (time indicated by the arrow) of apelin-13 (50 fmol – blue) or vehicle control (aCSF – grey) into the SFO or apelin-13 into non-SFO (red) locations. The bar graphs on the right show mean AUC for BP and HR. C, the schematic diagram on the left illustrates SFO (blue circles) and non-SFO (red squares) microinjection locations while the photomicrograph to the right shows the anatomical location of an SFO microinjection site (within the circle) in a 100 μm cresyl violet-stained coronal section.

Discussion

The present study provides the first evidence supporting a functional role for the SFO as a CNS target through which apelin may act to influence both cardiovascular, metabolic and body fluid regulation. Our patch clamp recordings demonstrate that apelin-13 directly modulates the excitability of the majority of dissociated SFO cells and thus suggests that these neurons represent a potential site in the CNS at which either centrally produced or circulating apelin may act to influence central autonomic pathways without crossing the blood–brain barrier. These observations are in accordance with studies showing c-Fos expression in SFO neurons in response to intraperitoneal administration of apelin (Takayama et al. 2008), although it should be emphasized that such observations are not in accordance with hyperpolarizing actions. Our recordings do, however, suggest the presence of different subpopulations of SFO neurons which depolarize, hyperpolarize or are unaffected by apelin, observations which are in accordance with previous studies evaluating adiponectin and leptin actions on SFO neurons (Smith et al. 2009; Alim et al. 2010). The functional relevance of these different groups of SFO neurons is not fully understood but may be related to roles either within the SFO (projection vs. interneurons) or to the specific efferent projections (PVN projecting vs. arcuate projecting) of single SFO neurons.

Our voltage clamp recordings in which we measured currents in response to slow voltage ramps in SFO neurons depolarized by apelin-13 revealed an apelin-induced current which was voltage independent and exhibited a reversal potential near −25 mV, indicating that such effects probably result from the activation of an NSCC. These observations are in accordance with a previous study showing that apelin depolarizes hypothalamic magnocellular vasopressin neurons through similar activation of a putative NSCC with reversal potential of −32 ± 2 mV (Tobin et al. 2008). Interestingly, other circulating hormones including angiotensin II (Ono et al. 2001; Washburn & Ferguson, 2001), ghrelin (Pulman et al. 2006) and interleukin 1β (Desson & Ferguson, 2003) have all been shown to influence the excitability of SFO neurons through the modulation of this conductance, which has also been suggested to be a critical site for integration in supraoptic magnocellular neurons (Hiruma & Bourque, 1995).

In contrast to the supraoptic nucleus where administration of apelin induced only depolarizations (Tobin et al. 2008), nearly 30% of SFO neurons were hyperpolarized by 100 nm apelin-13. In these hyperpolarized neurons, currents recorded in response to voltage ramps (−100 and −20 mV) revealed an apelin-induced voltage-independent outward current with a reversal potential of −75 mV, suggesting effects on a potassium channel. At voltages positive to −20 mV, the slope of the difference current increased abruptly, indicating the additional involvement of a voltage-gated conductance. These observations led us to examine currents evoked by standard voltage step protocols, which confirmed apelin activation of the sustained potassium current, I_K. These data therefore suggest that multiple potassium channels may contribute to apelin-mediated hyperpolarization of SFO neurons as has previously been shown for other neuropeptide actions in SFO, arcuate nucleus, PVN, area postrema and nucleus tractus solitarius (Yang & Ferguson, 2003; Fry & Ferguson, 2009; Hoyda & Ferguson, 2010).

The functional relevance of different apelin effects of these subpopulations of SFO neurons remains to be determined. Through extensive efferent projections to hypothalamic autonomic control and neuroendocrine centres, the SFO participates in the regulation of fluid balance, energy homeostasis and cardiovascular control. The heterogeneity of apelin effects on SFO neurons could reflect the association between these subpopulations and their specific connections to different hypothalamic nuclei (e.g. PVN vs. arcuate projecting) where the afferent information will be integrated to determine the final physiological effects.

The effects of apelin on cardiovascular function have been widely studied with the consensus being that intravenous apelin decreases BP in both anaesthetized and conscious rats (Tatemoto et al. 2001; Cheng et al. 2003; El Messari et al. 2004), although effects on HR are more variable. In association with such observations, our microinjection studies showed that direct administration of very low doses (50 fmol) also caused rapid decreases in BP and HR. The fact that BP and HR were influenced in the same manner, i.e. both were decreased by apelin-13 administration, indicates that these responses represent a regulated decrease in BP as no baroreflex-mediated increase in HR was observed. Importantly, these effects were site specific (not observed in non-SFO sites) and not a result of mechanical changes in tissue (vehicle controls were without effect), suggesting specific cardiovascular actions of apelin within the SFO. These effects are consistent with the depressor effects observed in response to peripheral administration of low doses of apelin (Lee et al. 2000; Tatemoto et al. 2001; Cheng et al. 2003) but are in contrast to pressor responses following peripheral administration of higher doses (Kagiyama et al. 2005). Pressor effects to both i.c.v. (Kagiyama et al. 2005) or site-specific microinjections of apelin into nucleus tractus solitarius or rostral ventral lateral medulla (Seyedabadi et al. 2002) suggest differential functional roles for hormonal (depressor) vs. neurotransmitter (pressor) apelin, and perhaps suggest a primary functional role for the SFO is sensing circulating apelin.

In conclusion, we have demonstrated two subpopulations of SFO neurons that respond to apelin-13 with depolarization or hyperpolarization. These effects are mediated through the APJ receptor and appear to result from apelin-13-induced activation of NSCCs or potassium channels, respectively. Furthermore, our demonstration that apelin acts directly in SFO to reduce BP and HR suggests the SFO to be a potential CNS site at which circulating apelin acts to influence central cardiovascular control. The present study clearly establishes a unique and pivotal role for SFO in mediating the central effects of apelin.

Glossary

aCSF

artificial cerebrospinal fluid

AUC

area under the curve

BP

blood pressure

HR

heart rate

PVN

paraventricular nuclei

SFO

subfornical organ

Additional information

Competing interests

None.

Author contributions

All experiments were performed in the laboratory of A.V.F. at Queen's University. Patch clamp recording experiments were carried out by L.D. or M.K. while microinjection studies were carried out by P.M.S. All authors were involved in the conception and design of the experiments, the analysis and interpretation of data, and the initial writing and revising of the article in preparation for publication. All authors approved the final version of the manuscript and all persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.

Funding

This work was supported by the Canadian Institutes for Health Research, grant 12192.