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. 2015 Jun 10;114(2):914–926. doi: 10.1152/jn.00080.2015

Enhanced astroglial GABA uptake attenuates tonic GABAA inhibition of the presympathetic hypothalamic paraventricular nucleus neurons in heart failure

Sudip Pandit 1, Ji Yoon Jo 1, Sang Ung Lee 2, Young Jae Lee 2, So Yeong Lee 3, Pan Dong Ryu 3, Jung Un Lee 2, Hyun-Woo Kim 1, Byeong Hwa Jeon 1, Jin Bong Park 1,✉
PMCID: PMC4533111  PMID: 26063771

Abstract

γ-Aminobutyric acid (GABA) generates persistent tonic inhibitory currents (Itonic) and conventional inhibitory postsynaptic currents in the hypothalamic paraventricular nucleus (PVN) via activation of GABAA receptors (GABAARs). We investigated the pathophysiological significance of astroglial GABA uptake in the regulation of Itonic in the PVN neurons projecting to the rostral ventrolateral medulla (PVN-RVLM). The Itonic of PVN-RVLM neurons were significantly reduced in heart failure (HF) compared with sham-operated (SHAM) rats. Reduced Itonic sensitivity to THIP argued for the decreased function of GABAAR δ subunits in HF, whereas similar Itonic sensitivity to benzodiazepines argued against the difference of γ2 subunit-containing GABAARs in SHAM and HF rats. HF Itonic attenuation was reversed by a nonselective GABA transporter (GAT) blocker (nipecotic acid, NPA) and a GAT-3 selective blocker, but not by a GAT-1 blocker, suggesting that astroglial GABA clearance increased in HF. Similar and minimal Itonic responses to bestrophin-1 blockade in SHAM and HF neurons further argued against a role for astroglial GABA release in HF Itonic attenuation. Finally, the NPA-induced inhibition of spontaneous firing was greater in HF than in SHAM PVN-RVLM neurons, whereas diazepam induced less inhibition of spontaneous firing in HF than in SHAM neurons. Overall, our results showed that combined with reduced GABAARs function, the enhanced astroglial GABA uptake-induced attenuation of Itonic in HF PVN-RVLM neurons explains the deficit in tonic GABAergic inhibition and increased sympathetic outflow from the PVN during heart failure.

Keywords: tonic GABAA inhibition, GABA transporters, sympathetic outflow, heart failure

low concentrations of extracellular γ-aminobutyric acid (GABA) generate a persistent tonic inhibitory current (Itonic) via activation of extracellular GABAA receptors (GABAARs) in the brain. GABA plays a fundamental role in reproduction, energy and fluid balance, and autonomic control in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei (Henderson 2007; Li and Patel 2003; van den Pol 2003), where more than 45% of all synapses are immunoreactive for GABA (Decavel and Van den Pol 1990; El Majdoubi et al. 1997). Accumulating evidence suggests that blunted GABAergic inhibition in the PVN significantly contributes to the sympathoexcitation associated with cardiovascular-related disorders such as hypertension, diabetes, and heart failure (Allen 2002; Haywood et al. 2001; Reynolds et al. 1996; Zhang et al. 2002). However, the role of Itonic in the pathophysiology associated with altered autonomic and neuroendocrine homeostasis is not well understood.

Along with the expression and combination of extrasynaptic GABAARs, Itonic is tightly controlled by extracellular concentrations of GABA in specific brain regions. Evidence suggests that GABA is released from glial cells in the brain. Various mechanisms have been proposed to explain this finding including glutamate/GABA exchange, GABA transporter (GAT) reversal, and a debated role for the GABA-releasing anion channel bestrophin-1 (Best-1) (Diaz et al. 2011; Heja et al. 2012; Lee et al. 2010). Despite evidence suggesting a role for GAT reversal under pathological conditions (Raiteri et al. 2002), GATs are generally believed to be involved in the uptake of extracellular GABA into cells. Of the four GATs (GAT-1, GAT-2, GAT-3, and BGT-1) that have been cloned and characterized, the GAT-1 and GAT-3 isoforms are most likely to be expressed in neurons and glia, respectively, and to be responsible for regulating ambient GABA levels in the brain (Dalby 2003). Pharmacological blockade or genetic deletion of GAT-1 causes an increase in Itonic, which is associated with neurological and psychological disorders such as seizures (Pirttimaki et al. 2013) and schizophrenia (Yu et al. 2013). However, the relationship between astroglial GABA clearance by GAT-3 and its effects on Itonic modulation and neuronal activity under pathological conditions are poorly understood.

The rostral ventrolateral medulla (RLVM) is a pivotal center for the control of tonic and reflex sympathetic activity, and PVN projections to the RVLM (PVN-RVLM) play a significant role in autonomic control of the cardiovascular system. PVN-RVLM neurons and sympathetic outflow are tonically restrained by GABAA inhibition, and enhanced sympathetic outflow is commonly observed in conditions such as congestive heart failure (Cohn et al. 1984; Grassi et al. 1995), hypertension, metabolic syndrome, and chronic renal failure (Anderson et al. 1989; Boero et al. 2001; Mancia et al. 2007). Although changes in the expression or activity of GATs occur in various central nervous system (CNS) disorders (Allen et al. 2004), it is not known whether changes in the function of PVN GATs contribute to GABA-dependent sympathoexcitation in cardiovascular diseases. We investigated the pathophysiology associated with astroglial GABA uptake via GAT-3 and other mechanisms regulating Itonic in PVN-RVLM neurons in heart failure (HF) induced by myocardial infarction.

MATERIALS AND METHODS

Experimental animals.

All animal experimentation was conducted under the license (2009-1-21) issued by Animal Ethics Committee of Chungnam National University. Male Sprague-Dawley rats weighing 140–180 g were purchased and housed in a 12:12-h light/dark schedule and allowed free access to food and water. The rats were allowed to acclimatize for 1 wk before coronary ligation. In the 3–4 wk after the cardiac surgery, retrograde dye was injected into the RVLM. Rhodamine-labeled microspheres (Lumaflor, Naples, FL) were microinjected unilaterally (200 nl) in the RVLM at bregma (B) −11.96, L 2.0, D 8.0, and the injection site and extension were confirmed histologically (Park et al. 2007). In the 4–5 wk after the coronary ligation, (1 wk after the retrograde tracing), brain slices were prepared for the electrophysiological recordings in the dye-labeled neurons and heart samples for the confirmation of myocardial infarction.

Myocardial infarction-induced heart failure.

The myocardial infarction (MI) was induced by left descending artery ligation as described previously (Han et al. 2010). Briefly, rats were anesthetized with ketamine and xylazine (75 mg/kg and 10 mg/kg ip, respectively). After left thoracotomy, the heart was exposed and the anterior descending coronary artery was ligated with 7-0 sterile silk suture at the level of tip of the auricle (Zhang et al. 2001). MI-operated rats with infarct sizes smaller than 30% were excluded from all analysis (Han et al. 2010). For sham control, the same operation was applied except for the coronary artery ligation.

To determine whether the MI rats have heart failure, echocardiography was applied in a subgroup of sham-operated (SHAM) and post-MI rats, and the hemodynamic was compared between the two groups.

Electrophysiology and data analysis.

Patch-clamp recordings from identified PVN-RVLM neurons were obtained in hypothalamic slices (290 μm) as previously described (Park et al. 2007). Briefly, rats were anesthetized with ketamine and xylazine, decapitated, and brains rapidly extracted. Slices were perfused with artificial cerebrospinal fluid (aCSF) containing (in mM) 126 NaCl, 2.5 KCl, 1 MgSO4, 26 NaHCO3, 1.25 NaH2PO4, 20 glucose, 0.4 ascorbic acid, 1 CaCl2, 2 pyruvic acid; pH was 7.3–7.4, saturated with 95% O2-5% CO2. Itonic recorded under conditions of added GABA are more “physiological” than those recorded in the absence of exogenously applied GABA (Glykys and Mody 2007a), since GATs activity is frequently dependent on extracellular GABA concentration (Ortinski et al. 2006). Therefore all recordings were obtained in aCSF containing 3 μM GABA at 35°C, using an Axopatch 200B (Axon Instruments, Foster City, CA).

For voltage-clamp experiments, patch pipettes were filled with a high-Cl− solution containing (in mM) 140 KCl, 10 HEPES, 5 Mg2+ATP, 0.9 MgCl2, and 10 EGTA. Currents were filtered at 1 kHz and digitized at 10 kHz (Digidata 1322A, pClamp 9 software Axon Instruments). Spontaneous inhibitory postsynaptic currents (sIPSCs, recorded at −70 mV) were recorded in the presence of the glutamate AMPA/kainate receptor antagonist, DNQX (5,7-dintroquinoxaline-2,3-dione, 10 μM), the NMDA receptor antagonist AP5 (d,l-2-amino-5-phosphonopentanoic acid, 100 μM), and analyzed using Mini Analysis (Synaptosoft, Decatur, GA). Itonic was defined as the difference in holding currents (Iholding) before and after application of the GABAA receptor blocker bicuculline (BIC, 20 μM) (Park et al. 2009; Park et al. 2007). The series resistance (8.2 ± 0.1 MΩ, n = 76) was monitored at the beginning and end of the experiments and data were discarded if changes >20% were observed. Cell capacitance was obtained by integrating the area under the transient capacitive phase of a 5-mV depolarizing step pulse, in the voltage-clamp mode (Park et al. 2007).

For current-clamp experiments, patch pipettes filled with a more physiological concentration of Cl− were used (in mM): 130 K-gluconate, 10 KCl, 10 HEPES, 5 Mg2+ATP, 0.9 MgCl2, and 10 EGTA. Ten-picoampere hyperpolarizing step pulses were applied to measure input resistance, and the membrane potential change was plotted as a function of the hyperpolarizing step. Input-output relationships were built by plotting the number of evoked spikes vs. injected current in response to depolarizing pulses of varying amplitudes (1.2-s duration), and fitted by a Boltzmann function: y = 1/{1 + exp[(Ih − I50)/k]}, in which y is the number of spikes at a given injected current (Ih), I50 the current at which half of the maximal spikes are evoked, and k the slope factor. Mean values of membrane potential (Vm) were obtained from averaging various recording segments lacking action potentials.

The cell-attached voltage-clamp configuration of the patch-clamp technique was used to study the firing discharge. Patch pipettes were filled with low-Cl− solution (in mM): 135 K-gluconate, 10 KCl, 10 HEPES, 5 Mg2+ATP, 0.9 MgCl2, and 10 EGTA. Firing rate was calculated using Mini Analysis, by counting the number of firing discharges in 10-s bins, for a period of ∼3 min before and after bath application of drugs, and mean values for each condition were then obtained.

Drugs were added to the perfusing solution at known concentrations. The final concentration of dimethyl sulfoxide (DMSO) was less than 0.05%, when used to dissolve drugs. All drugs were purchased from Sigma-Aldrich (St. Louis, MO).

Western blotting.

All proteins from dissected PVN were lysed with 1× passive lysis buffer (Cell Signaling Technology) and quantified using a Coomassie Protein assay kit (BioRad). Approximately 50 μg of protein was electrophoresed on a 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and transferred onto nitrocellulose membranes. The blots were blocked with 1× Tris-buffered saline (TBS)-Tween 20 containing 3% bovine serum albumin (BSA) + 2% heparan sulfate (HS) for 1 h at room temperature (5% TTBS; Gibco). The blots were then incubated at 4°C with primary antibodies against GABAAR δ subunit, GABAAR γ2 subunit, GAT-1, and GAT3 (1:1,000; Millipore) in 5% TTBS, respectively. The next day, the blots were incubated with a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:2,000; Santa Cruz Biotechnology). An enhanced chemiluminescence detection kit (ECL; Pierce) was used to visualize antibody binding, and the intensity of the bands was measured using ImageJ software 1.42q (NIH).

Immunohistochemistry.

Double immunohistochemical fluorescent reactions were used to study the expression of Best-1, and their possible colocalization with astroglial cell (glial fibrillary acid protein, GFAP immunoreactive). For these studies, a group of rats were deeply anesthetized and perfused transcardially with 0.01 M PBS (150 ml) followed by 4% paraformaldehyde (500 ml). Brains postfixed in 4% paraformaldehyde were cryoprotected at 4°C with 0.1 M PBS containing 30% sucrose for a minimum of 48 h. Sections (25 μm) were then cut using a cryostat and incubated in a solution of 0.01 M PBS with 0.01% Triton X-100 and 10% normal goat serum for 1 h.

For immunofluorescence reactions, sections were incubated for 24 h in the presence of a polyclonal rabbit anti Best-1 primary antibody (Abcam, 1:200 dilution) in conjunction with a polyclonal mouse anti GFAP (Biogenex, 1:1,000 dilution) antibody. Incubation in primary antibodies were followed by a 2-h incubation in secondary antibodies (anti-rabbit Cy3 labeled and anti-mouse FITC labeled, 1:200, respectively). Digital microscopy was performed with 40× objective using a Zeiss Axiophot microscope equipped with a digital CCD camera (AxioCam and AxioVision 4.8 software, Zeiss, Germany). A positive colocalization was considered by the appeared yellow (red + green) profiles in merged images, similar in size, shape, and geometry in red and green profiles.

Statistical analysis.

Numerical data are presented as means ± SE. Statistical significance of the data between SHAM and HF was determined by using independent Student's t-test. Analysis of variance with repeated measures (ANOVA-RM), followed by post hoc tests, was used as needed.

RESULTS

Electrophysiological recordings were obtained from labeled 287 PVN-RVLM neurons of 56 SHAM and 58 HF rats first identified under fluorescent light, and electrophysiological activity was then recorded under bright light (Park et al. 2007).

Attenuated tonic GABAA inhibition in the PVN-RVLM neurons of HF rats.

We investigated changes in tonic GABAA receptor-mediated inhibition associated with HF by comparing the Itonic in SHAM and HF PVN-RVLM neurons. The ejection fraction was significantly decreased in HF compared with SHAM animals (P < 0.01; Fig. 1A). Itonic revealed by a BIC-induced outward shift in the holding current (Iholding) was significant in both SHAM and HF PVN neurons (P < 0.01 for both), although bicuculline (BIC) had only marginal effects on Iholding in 4 of 17 HF neurons. The Itonic was significantly attenuated in the HF compared with the SHAM PVN-RVLM neurons (SHAM 9.5 ± 3.6 pA, n = 20 vs. HF 4.0 ± 1.1 pA, n = 17, P < 0.05, Fig. 1B), which was independent of whole cell capacitance (SHAM 40.7 ± 3.6 pF, n = 20 vs. HF 40.4 ± 3.5 pF, n = 17).

Fig. 1.

Fig. 1.

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Attenuated tonic GABAA current (Itonic) of paraventricular nucleus neurons projecting to the rostral ventrolateral medulla (PVN-RVLM) in heart failure rats. Aa: representative echocardiograph images in movement-mode, short-axis view in a SHAM (top panel) and a heart failure (HF; bottom panel) rat. Note the increased left ventricle internal dimension in the HF rat, indicating loss of contractility function. Ab: mean ejection fraction in SHAM (n = 7) and HF (n = 8) rats shown as a bar graph. *P < 0.05 compared with SHAM control. Ba: representative traces of a SHAM (top panel) and HF (bottom panel) PVN-RVLM neuron showing Itonic attenuation following the application of 20 μM bicuculline (BIC). Bb: paired plots of the holding current (Iholding) in SHAM (n = 20) and HF (n = 17) PVN-RVLM neurons before and after the perfusion of BIC. Thick lines and symbols indicate means ± SE. Ca: representative current traces from a SHAM (top panel) and HF (bottom panel) PVN-RVLM neuron showing the effect of 1 μM 4,5,6,7-tetrahydroisothiazolo-[5,4-c]pyridin-3-ol (THIP) on Itonic, which was blocked by BIC. Cb: mean changes in Iholding induced by THIP and additional application of BIC in SHAM (n = 11) and HF (n = 8) neurons. pA, picoamperes; *P < 0.05 compared with SHAM rats.

The Itonic attenuation in HF neurons was confirmed by the application of 4,5,6,7-tetrahydroisothiazolo-[5,4-c]pyridin-3-ol (THIP, 1 μM), which preferentially activates the δ- over the γ2 subunit-containing GABAARs (Adkins et al. 2001; Brown et al. 2002; Drasbek et al. 2007). Application of THIP produced a significant inward shift in the Iholding of SHAM PVN-RVLM neurons (9.7 ± 2.5, n = 11, P < 0.01) and a minimal inward shift in the neurons of HF rats (2.8 ± 0.8, n = 8, P > 0.1). As a result, the Itonic was significantly smaller in the HF than the SHAM neurons in the presence of THIP (Fig. 1C), suggesting that HF altered GABAAR δ subunit function in PVN-RVLM neurons.

Reduced Itonic response to benzodiazepines in HF neurons.

In addition to the δ-subunit-containing GABAARs, benzodiazepine (BZ)-sensitive GABAARs mediate Itonic in various brain regions (Caraiscos et al. 2004; Gao and Smith 2010; Yamada et al. 2007). Similarly, BZ-sensitive GABAARs mediate Itonic and phasic current (Iphasic) in PVN-RVLM neurons. We investigated the function of BZ-sensitive GABAARs in HF by comparing Itonic and Iphasic sensitivity to BZ in SHAM and HF PVN-RVLM neurons (Fig. 2).

Fig. 2.

Fig. 2.

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Effect of benzodiazepines on the Itonic and Iphasic of PVN-RVLM neurons in SHAM and HF rats. A: representative traces of neurons from SHAM and HF rats showing the inward shift in Iholding induced by diazepam (DZP, 1 μM) and its reversal following the application of bicuculline (BIC). B: mean changes in Iholding induced by DZP or zolpidem (ZPD) and the additional application of BIC in SHAM and HF neurons. C: summarized data showing the DZP- or ZPD-induced portion to the total Itonic uncovered by the additional application of BIC. Note that the percentage are similar in SHAM and HF neurons. D: average spontaneous inhibitory postsynaptic currents (sIPSC) obtained from the same SHAM and HF neurons depicted in A before and during bath application of DZP. E: mean effects of DZP and ZPD on the IPSC decay time in neurons from SHAM and HF rats. Note that the delay in IPSC decay time shown as weighted value was similar between groups. *P < 0.05 compared with SHAM.

Bath application of diazepam (DZP, 1 μM) enhanced Itonic as indicated by a significant inward shift in Iholding in SHAM and HF PVN-RVLM neurons (P < 0.01 for both). This effect was blocked by the additional application of BIC (Fig. 2A), suggesting that DZP enhanced Itonic in the neurons. We observed an ∼65% decrease in the DZP-induced Iholding shift in HF compared with SHAM neurons (13.6 ± 2.8 pA, n = 18 vs. 4.4 ± 1.1 pA, n = 17, P < 0.01; Fig. 2B). However, DZP potentiated Itonic to similar rate in SHAM (204.6 ± 17.1%, n = 18) and HF (185.0 ± 24.0%, n = 17) neurons (Fig. 2C), because the DZP-induced Iholding shift was dependent on the initial Itonic in both groups.

Furthermore, DZP potentiated Iphasic as indicated by prolongation of spontaneous inhibitory postsynaptic current (sIPSC) decay time with no consistent changes in sIPSC frequency or amplitude in the SHAM or HF neurons. sIPSC occurred at a mean frequency of 2.1 ± 0.3 Hz, had a mean amplitude of 73.1 ± 9.0 pA, and decayed with a time course best fitted by a biexponential function (τfast 7.8 ± 0.6 ms; τslow 27.0 ± 3.5 ms) in SHAM neurons (n = 18). In accordance with a previous report (Han et al. 2010), IPSC frequency was reduced in HF neurons (1.1 ± 0.2 Hz, n = 17; P < 0.05) with no significant changes in IPSC amplitude (71.4 ± 8.6 pA) and decay time constants (τfast 6.4 ± 0.8 ms; τslow 25.1 ± 5.4 ms) (P > 0.3 in both cases). DZP prolonged the IPSC decay time to a similar extent in the SHAM and HF rats (Fig. 2, D and E). The decay phase of IPSCs best fitted with a double-exponential function was compared in weighted values (Pandit et al. 2014). DZP increased the weighted decay time constants from 12.2 ± 0.9 to 19.6 ± 1.4 ms in the SHAM and from 10.8 ± 1.4 to 16.0 ± 2.1 ms in HF rats.

Similar results were obtained following the application of zolpidem (ZPD, 1 μM), suggesting that there is a selective change in the BZ-sensitive GABAARs function mediating Itonic over those mediating Iphasic in HF. While the ZPD-induced Iholding shift was significantly smaller in HF than in SHAM neurons (P < 0.05; Fig. 2B), the percent change of Itonic was similar in SHAM (275.2 ± 64.8%, n = 7) and HF (221.7 ± 46.7%, n = 7) rats (Fig. 2C). The drug also prolonged IPSCs to a similar degree in SHAM and HF neurons (Fig. 2E).

To further verify altered GABAARs function mediating Itonic in HF, we compared the expression of GABAAR γ2 and δ subunit in the PVN between SHAM and HF rats. Although Western blot analysis showed a tendency for the expression of GABAAR δ subunit to decrease in HF relative to SHAM PVN, the expression of the δ and γ2 subunit was not different in SHAM and HF PVN (84.9 ± 7.4% and 91.7 ± 5.9% of SHAM, respectively; n = 6, P > 0.1 in both cases).

Blockade of GAT activity reversed the Itonic attenuation in HF neurons.

The decrease in Itonic sensitivity to BZ and reduced basal Itonic in HF neurons suggested that the attenuated BZ-sensitive GABAAR activity was related to a reduction in ambient GABA concentrations in addition to altered GABAARs function in HF neurons. Given that Itonic in PVN-RVLM neurons is tightly controlled by GAT clearance of GABA (Park et al. 2009), enhanced GAT activity may have caused the attenuation in Itonic. To test this hypothesis, we measured the Itonic in SHAM and HF PVN-RVLM neurons in the presence of GAT blockers.

Bath application of the nonselective GAT blocker, nipecotic acid (NPA, 100 and 300 μM), induced an inward shift in Iholding (INPA) in a concentration-dependent manner (Fig. 3A), although INPA was negligible in 3 of 13 SHAM and 2 of 18 HF neurons. Moreover, the application of 100 and 300 μM NPA evoked a significantly larger INPA in the HF neurons than in the SHAM neurons (P < 0.05 for both; Fig. 3B), suggesting that GABA uptake was increased in the neurons of post-MI rats. As a result, total Itonic as measured by BIC-induced Iholding shifts in the presence of 100 μM NPA (INPA + basal Itonic) was similar in SHAM and HF neurons, and the total Itonic in the presence of 300 μM NPA was larger in the HF than in the SHAM PVN-RVLM neurons (P < 0.07, Fig. 3C). These results suggest that Itonic attenuation may be masked or reversed by blocking the increase in GABA clearance in HF rats.

Fig. 3.

Fig. 3.

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GABA transporter blockade reversed the Itonic attenuation in HF neurons. A: representative traces showing that bath application of the nonselective GAT blocker, nipecotic acid (NPA, 100 and 300 μM), produced a significant inward shift in Iholding in a concentration-dependent manner, which was blocked by the GABAA-receptor blocker, bicuculline (BIC). Note that the NPA-induced increase in Itonic was greater in HF than in SHAM neurons. B: mean effect of NPA on Iholding in SHAM (n = 13) and HF (n = 18) rats. C: mean total Itonic unmasked by BIC in the presence of NPA in SHAM and HF neurons. D: representative traces showing that the attenuation in THIP-induced current was masked by the following NPA-induced current in HF neurons. E: mean Iholding changes induced by the sequential application of THIP (1 μM), NPA (300 μM), and BIC in SHAM (n = 6) and HF (n = 6) neurons. *P < 0.05 compared with SHAM control.

We used sequential application of THIP and THIP + NPA to discriminate between the roles of increased GABA clearance and the reduced GABAAR δ subunit function in HF Itonic attenuation (Fig. 3D). As expected, the attenuation in THIP-induced Itonic was efficiently masked by the larger NPA-induced Itonic in HF PVN-RVLM neurons. As a result, the total Itonic in the presence of THIP and NPA (basal Itonic + ITHIP + INPA) was not different in the SHAM and HF neurons (Fig. 3E). These results showed that the reduced GABAAR δ subunit function was efficiently masked by the blockade of enhanced GABA clearance, suggesting that the two distinct mechanisms contribute independently to Itonic attenuation in HF PVN-RVLM neurons.

Astroglial GABA uptake is enhanced in HF neurons.

Given that Itonic is tightly regulated by astroglial GAT-3 activity in PVN-RVLM neurons, GAT-3 may play an important role in attenuation of the Itonic observed in HF neurons. To test this hypothesis, we compared the effect of SNAP-5114 (100 μM), a GAT-3 blocker (Borden et al. 1994), on Itonic in the presence of NO-711 (5 μM), a GAT-1 blocker (Sitte et al. 2002) in the neurons of SHAM and HF rats (Fig. 4). These concentrations theoretically block ∼95% of their respective GATs (Borden et al. 1994; Sitte et al. 2002).

Fig. 4.

Fig. 4.

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Enhanced GAT-3 activity modulates tonic current (Itonic) in HF PVN-RVLM neurons. A: representative traces showing that the GAT-3 blocker, SNAP-5114, induced a slight increase in the Itonic of SHAM neurons and a robust increase in HF neurons in the presence of the GAT-1 blocker, NO-711. B: mean effects of NO-711 and SNAP-5114 on the Itonic in the PVN-RVLM neurons of SHAM and HF neurons (n = 6 in both groups). C: mean total Itonic unmasked by bicuculline (BIC) in the presence of GAT-1 and GAT-3 blockers in SHAM and HF neurons. *P < 0.05 compared with SHAM neurons. D: representative Western blot analysis showing no significant changes in GAT-1 or GAT-3 expression in the SHAM or HF PVN. E: mean GAT-1 and GAT-3 expression in SHAM and HF rats. GAT-1 and GAT-3 expression was normalized to the level detected in SHAM rats (n = 6) and compared with the expression in HF rats (n = 8).

NO-711 induced similar and minimal changes in Iholding (INO-711) in SHAM and HF neurons; however, the application of SNAP-5114 induced a significantly greater inward shift in Iholding (ISNAP-5114) in HF than in SHAM neurons (Fig. 4, A and B). As a result, the total Itonic was greater in HF than in SHAM neurons in the presence of GAT blockers (INO-711 + ISNAP-5114 + basal Itonic; P < 0.05, Fig. 4C), suggesting a major role for GAT-3 in the attenuation of Itonic in HF PVN-RVLM neurons.

In a subset of experiments, we investigated whether a high concentration of NO-711 (30 μM) could reverse the Itonic attenuation in HF neurons. We found no difference in the NO-711-induced shift in the Iholding between HF and SHAM PVN-RVLM neurons (SHAM 9.6 ± 2.2 pA, n = 18 vs. HF 9.0 ± 1.0 pA, n = 5), which did not mask the Itonic difference between the two groups.

To further verify altered GAT activity in HF neurons, we compared the expression of GAT-1 and GAT-3 in the PVN between SHAM and HF rats. We found a tendency for the expression of GAT-1 to decrease and GAT-3 to increase in HF relative to SHAM rats, although the differences did not reach statistical significance (Fig. 4, D and E).

Effect of Best-1 blockade on Itonic in SHAM and HF PVN-RVLM neurons.

Although our results argued against glial GABA release via GAT reversal in PVN neurons, it is possible that inhibition of astrocyte GABA release via an alternative pathway could contribute to the attenuation of Itonic in HF PVN-RVLM neurons. Astroglial GABA is thought to be released via Best-1 channels in certain brain regions (Lee et al. 2010). Thus we investigated the involvement of Best-1 in the attenuation of Itonic in HF neurons by comparing the effect of Best-1 channel blockade using 5-nitro-2(3-phenylpropylamino)-benzoic acid (NPPB) between SHAM and HF PVN-RVLM neurons (Fig. 5).

Fig. 5.

Fig. 5.

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Bestrophin-1 (Best-1) played no role in the attenuation of the Itonic in HF neurons. A: double-stained Best-1 (red) and glial fibrillary acidic protein (GFAP, green) immunoreactivity in the SHAM PVN. Note the high degree of colocalization (yellow). B: representative traces showing minimal Itonic response to blockade of the anion channel by 5-nitro-2(3-phenylpropylamino)-benzoic acid (NPPB) in SHAM and HF PVN-RVLM neurons. C: mean changes in the holding current (Iholding) induced by NPPB and the additional application of bicuculline (BIC) in SHAM (n = 7) and HF neurons (n = 7). *P < 0.05 compared with SHAM rats.

Double immunofluorescence studies revealed a high degree of colocalization between Best-1 and glial fibrillary acidic protein (GFAP) immunoreactivities in the PVN (Fig. 5A). However, NPPB application had a minimal effect on the Iholding in SHAM and HF PVN-RVLM neurons (Fig. 5, B and C), suggesting that GABA release via Best-1 was not involved in the attenuation of the Itonic in HF PVN-RVLM neurons.

Functional significance of Itonic attenuation in HF PVN-RVLM neurons.

We used the current-clamp mode to investigate the functional significance of Itonic attenuation in HF neurons. We compared resting membrane potential (Vm) and input resistance (IR) between SHAM and HF neurons. The Vm did not differ between the SHAM (−57.7 ± 1.7 mV, n = 13) and HF (−57.5 ± 1.4 mV, n = 15, P > 0.7) PVN-RVLM neurons; however, IR was significantly increased in the HF (732.7 ± 43.6 MΩ, n = 12) compared with the SHAM neurons (602.8 ± 52.1 MΩ, n = 12, P < 0.05).

We investigated the association between Itonic attenuation and increased IR in HF neurons using the GABAAR antagonist, gabazine (GBZ, 1 μM), which selectively blocks Iphasic over Itonic in the PVN-RVLM. GBZ and GBZ + BIC were applied sequentially to the preparation. In agreement with the previous report (Park et al. 2007), GBZ caused minimal effects on Iholding (1.4 ± 1.0 pA, P > 0.4), whereas the additional application of BIC caused significant outward shift in Iholding (6.5 ± 1.7 pA, P < 0.01) in the SHAM PVN-RVLM neurons. A one-way ANOVA-RM revealed that IR was dependent on the sequential application of the drugs in SHAM (F = 17.6, P < 0.00001) but not in HF neurons. A post hoc test revealed that GBZ + BIC significantly increased IR in the SHAM neurons (P < 0.01), whereas GBZ had no effect on IR (P < > 0.5; Table 1).

Table 1.

Input resistance of SHAM and HF PVN-RVLM neurons

CTL GBZ GBZ+BIC CTL NPA NPA+BIC
SHAM 630.6 ± 41.9 649.1 ± 34.1 745.0 ± 52.8* 601.5 ± 88.1 586.8 ± 84.6 675.8 ± 95.1*
HF 710.1 ± 72.7 722.0 ± 73.9 730.8 ± 76.8 727.0 ± 45.2 623.6 ± 52.3* 754.5 ± 45.5
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Values are means ± SE. HF, heart failure; CTL, control; GBZ, gabazine; BIC, bicuculline; NPA, nipecotic acid; SHAM, sham operated; PVN, paraventricular nucleus; RVLM, rostral ventrolateral medulla.

*

P < 0.05 compared with respective CTL (n = 6 for each group).

Similar sequential application of NPA and NPA + BIC was used to investigate the association between enhanced GAT activity and the increase in IR in HF PVN-RVLM neurons. A post hoc test revealed that NPA (300 μM) produced a significant decrease in the IR of HF neurons, whereas the change in IR was marginal in the SHAM neurons. The NPA-induced IR increase in HF neurons was blocked by BIC. These results are shown in Table 1.

Altered input-out function in HF PVN-RVLM neurons.

To assess the influence of Itonic attenuation on repetitive firing activity in HF neurons, we compared the input-output (I-O) function of SHAM and HF PVN-RVLM neurons. Depolarizing current steps of increasing amplitude were applied, and the number of evoked spikes was plotted as a function of the depolarizing steps (Fig. 6). A two-way ANOVA-RM revealed that evoked firing was dependent on current injection (F = 25.5, P < 0.0001) and HF (F = 5.8, P < 0.05). The number of action potentials elicited at all levels of current injection was increased in HF neurons (P < 0.05 at all tested currents, Fig. 6, B and D). The I-O function was expressed by the Boltzmann equation with a half-maximal current (I50) of 41.9 ± 3.1 pA and a slope factor (k) of 15.1 ± 1.6 pA (n = 11) in the SHAM neurons. The I-O function was significantly shifted to the left (I50, 32.3 ± 2.5 pA, P < 0.05 compared with SHAM) with moderate increase of k (18.7 ± 1.4 pA, P = 0.087 compared with SHAM) in the HF neurons (n = 11).

Fig. 6.

Fig. 6.

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Effects of the Itonic attenuation on the input-output (I-O) function in HF PVN-RVLM neurons. A: representative examples of action potentials evoked by depolarizing pulses of increasing amplitude before and during the application of gabazine (GBZ) and GBZ + bicuculline (BIC) to SHAM and HF neurons. The membrane potential was held at −70 mV under all conditions. B: plot of the mean number of evoked spikes as a function of depolarizing current amplitude in SHAM and HF neurons (n = 6 in both cases). The solid lines represent the I-O relationship fitted by a Boltzmann function. C: representative examples of action potentials evoked by depolarizing pulses of increasing amplitude before and during the application of nipecotic acid (NPA) and NPA + BIC to SHAM and HF neurons. D: plot of the mean number of evoked spikes as a function of depolarizing current amplitude in SHAM and HF rats. Note that NPA induced the greater shift of the I-O function to the right in HF than SHAM neurons.

We compared the effects of GBZ and BIC on I-O function in SHAM and HF PVN-RVLM neurons to determine the effect of Itonic attenuation on I-O function (Fig. 6, A and B). A two-way ANOVA-RM revealed that evoked firing was dependent on current injection (F = 99.9, P < 0.0001) and drug treatment (F = 3.8, P < 0.05) in the SHAM neurons. The post hoc test revealed that the evoked firing response was significantly increased by coapplication of GBZ + BIC (P < 0.01, Fig. 6B), whereas GBZ alone did not alter the I-O function in SHAM neurons. I50 was not different before and after the application of GBZ alone (control 41.0 ± 4.0 vs. GBZ 39.3 ± 3.6 pA), whereas GBZ + BIC significantly decreased I50 (34.1 ± 4.2 pA, n = 6, P < 0.001 and P < 0.01, compared with control and GBZ alone, respectively) in SHAM neurons. Furthermore, evoked firing was dependent on current injection (F = 55.3, P < 0.0001), but not on GABAAR antagonists in HF rats (Fig. 6B). I50 was not different in control, GBZ, and GBZ + BIC (29.4 ± 2.3, 30.9 ± 2.9, and 31.8 ± 2.1 pA, respectively, n = 6) in HF neurons.

The effects of NPA on I-O function were compared in SHAM and HF PVN-RVLM neurons (Fig. 6, C and D) to clarify the role of enhanced GAT activity. A two-way ANOVA-RM revealed that evoked firing was dependent on current injection and drug treatment in SHAM and HF rats. The post hoc test revealed that NPA did not affect evoked firing in SHAM neurons (P > 0.2, Fig. 6D), whereas it significantly decreased the evoked firing response in HF neurons (P < 0.05, Fig. 6D). As a result, NPA significantly increased I50 in HF neurons (control 30.0 ± 5.1 pA vs. NPA 40.5 ± 7.8 pA, n = 5, P < 0.05), while the changes did not reach the statistical difference in SHAM neurons (control 40.1 ± 5.9 pA vs. NPA 44.6 ± 6.7, n = 5, P > 0.3). Consistent with the findings for GBZ + BIC, NPA + BIC increased the evoked firing rate above the control level in SHAM but not in HF neurons (Fig. 6C).

Itonic attenuation along with enhanced GAT activity increased spontaneous firing in HF PVN-RVLM neurons.

We further investigated the functional significance of Itonic attenuation by directly comparing ongoing firing activity in SHAM and HF PVN-RVLM neurons using the cell-attached mode. Of the 71 SHAM and HF PVN-RVLM neurons evaluated, 55 showed spontaneous firing, with the greater proportion of those in the post-MI rat tissue (HF, 28/31 cells, 90.3%; SHAM, 27/40 cells, 67.5%; P > 0.05 by Fisher's exact test). The mean spontaneous firing rate was significantly higher in the HF (1.71 ± 0.20 Hz, n = 19) than in the SHAM neurons (0.94 ± 0.19 Hz, n = 21, P < 0.05). Furthermore, BIC produced a significant increase in the spontaneous firing rate of SHAM (from 0.65 ± 0.20 to 1.41 ± 0.21 Hz, n = 9, P < 0.05) but not of HF neurons (from 1.33 ± 0.31 to 1.62 ± 0.41 Hz, n = 11, P > 0.5). We assessed the functional significance of Itonic attenuation on firing activity by comparing the effects of DZP on firing activity in the presence of GBZ (Fig. 7A). We found that in the presence of GBZ, DZP produced a marked decrease in the firing frequency of SHAM PVN-RVLM neurons (1.17 ± 0.39 to 0.66 ± 0.26 Hz, P < 0.05) and a marginal decrease in that of HF neurons (2.10 ± 0.38 to 1.79 ± 0.33 Hz, P > 0.1). As a result, DZP produced significant larger inhibition on the firing rate in SHAM than HF rats (SHAM 60.8 ± 8.6 of control, n = 11 vs. HF 84.2 ± 5.3% of control, n = 9; P < 0.01), suggesting that Itonic attenuation contributes to the overexcitability of HF PVN-RVLM neurons (Fig. 7, A and B).

Fig. 7.

Fig. 7.

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Enhanced inhibitory effect of GAT blockade on firing activity in HF PVN-RVLM neurons. A: representative traces of the firing activity recorded using the cell-attached mode show that the diazepam (DZP)-induced inhibition was greater in the SHAM than in the HF neurons. The phasic current (Iphasic) was blocked by the application of gabazine (GBZ). B: mean firing inhibition by DZP in SHAM (n = 11) and HF (n = 9) neurons. C: representative traces showing that the GAT blocker, nipecotic acid (NPA), diminished spontaneous firing activity in SHAM and HF neurons. D: mean decrease in firing rate in PVN-RVLM neurons evoked by NPA in SHAM (n = 7) and HF (n = 8) neurons. *P < 0.05 compared with SHAM rats.

We compared the inhibitory effect of NPA on spontaneous firing activity between SHAM and HF neurons to assess the role of enhanced GAT activity. NPA decreased the firing frequency in both SHAM (from 0.96 ± 0.21 to 0.79 ± 0.21 Hz, n = 7, P < 0.07) and HF PVN-RVLM neurons (from 1.62 ± 0.19 to 1.07 ± 0.16 Hz, n = 8, P < 0.01) (Fig. 7C). However, the NPA-induced inhibition was greater in the HF than SHAM neurons (SHAM 80.0 ± 4.15% vs. HF 63.3 ± 5.95%, P < 0.05; Fig. 7D). As a result, the firing frequency in the presence of NPA was not different in the two groups (P > 0.3).

DISCUSSION

Our findings indicate that 1) Itonic was attenuated in HF PVN-RVLM neurons; 2) in addition to altered GABAARs function, increased GAT-3 uptake activity is the most likely mediator of the attenuated Itonic; and 3) Itonic attenuation contributes to the hyperexcitability of presympathetic PVN neurons in HF rats. To our knowledge, our data are the first to demonstrate a link between pathophysiology and GAT-3 uptake modulation of GABAAR tonic inhibition in the brain during altered autonomic nerve activity.

Altered GABAARs underlie the attenuation of Itonic in HF PVN-RVLM neurons.

The peri- and/or extrasynaptic GABAA receptors that mediate Itonic are activated by low ambient concentrations of GABA in the extracellular space (Yeung et al. 2003), and the δ subunit-containing receptors are ideally suited to mediate a persistent Itonic. Facilitation of Itonic but not Iphasic by the δ subunit-selective agonist, THIP, is consistent with the functional presence and selective involvement of the GABAAR δ subunit in the Itonic of PVN-RVLM neurons (Park et al. 2007). Furthermore, our finding of a diminished THIP-induced Itonic consistent with HF Itonic attenuation suggests that GABAAR δ subunit function is reduced in the PVN-RVLM neurons of post-MI rats. However, Western blot analysis did not support the reduction of GABAAR δ subunit expression in HF PVN in the present study. Future studies are warranted to determine the exact molecular mechanism responsible for the downregulation of GABAAR δ subunit function in HF PVN-RVLM.

Given that BZ enhancement of GABA currents in mature animals requires the GABAAR γ2 subunit, our finding that the Itonic was enhanced by DZP and ZPD provided further confirmation that, in addition to the δ subunit, the γ2 subunits are involved in the Itonic of PVN-RVLM neurons (Jo et al. 2011). Altered expression and composition of extrasynaptic GABAARs have been shown to change the amplitude and/or pharmacological properties of the Itonic in various brain diseases (Brickley and Mody 2012; Hines et al. 2012; Pandit et al. 2013). However, our finding that BZ-induced potentiation of the Itonic was dependent on the magnitude of the initial Itonic in SHAM and HF neurons suggested that diminished sensitivity to BZ reflects a reduction in the basal Itonic of HF PVN-RVLM neurons. Although we could not exclude changes in BZ-sensitive GABAARs, it is likely that the attenuated Itonic in HF neurons was largely the result of a reduction in ambient GABA concentration. This notion was supported by our finding that the attenuation in Itonic was masked or reversed by GAT blockade. Thus it is likely that, despite reduced GABAAR δ subunit function, the increased impact on GABAAR γ2 subunits mediating Itonic enabled GAT blockade to reverse the Itonic attenuation in HF PVN-RVLM neurons.

Enhanced GABA uptake as the mechanism underlying the HF attenuation of Itonic.

Although the precise mechanisms that modulate tonic GABAA inhibition in the PVN are not completely understood, it is clear that the strength of tonic inhibition depends on the distribution and activity of GATs and the amount of vesicular GABA release (Park et al. 2009). Similar to other neurotransmitter transporters, GATs can reverse transport direction in the brain, suggesting that GABA release via GAT reversal is integral in maintaining the GABA levels involved in activating Itonic (Richerson and Wu 2003). Indeed, GABA release via GAT-1 may occur in certain pathological conditions. GAT-3 reversal has been described in the striatum of Huntington's disease mice (Raiteri et al. 2002), although pharmacological blockade of GAT-1 and GAT-3 increases ambient GABA in several brain regions, including the striatum (Kirmse et al. 2008; Kirmse et al. 2009; Waldmeier et al. 1992). Our finding that GAT-1 and GAT-3 blockers enhanced the Itonic in the PVN-RLVM of SHAM and HF rats suggests that, as in the naïve PVN (Park et al. 2009), the transporters operate synergistically to promote GABA uptake in sham-operated and post-MI rats.

GABA released from presynaptic terminals is a major source of ambient GABA activating Itonic in the hippocampus (Glykys and Mody 2007b). Although the Na+ channel blocker TTX has no effects on IPSC frequency, and in turn, Itonic of PVN-RVLM neurons, a significant correlation between Itonic magnitude and IPSCs frequency has been found in these neurons (Park et al. 2007). The Itonic attenuation may be the result of reduced ambient GABA concentrations related to decreased IPSC frequency in HF PVN-RVLM neurons (Han et al. 2010). Although we could not completely exclude this possibility, our results showed that GAT blockade reversed the HF Itonic attenuation. Overall, our results suggest that Itonic is tightly regulated by GATs operating in the uptake mode in the PVN-RVLM neurons of HF, SHAM, and naïve rats (Park et al. 2009).

Dominant role of GAT-3 in the attenuation of Itonic in HF PVN-RVLM neurons.

Although GAT-1 and GAT-3 are believed to operate synergistically in the uptake of GABA, the effect of GAT-3 block on the Itonic is minimal in certain brain regions unless GAT-1 is blocked. Microdialysis studies of GABA have shown that GAT-3 blockade increases extracellular GABA concentrations in the hippocampus only when GAT-1 is blocked (Kersante et al. 2013). However, GAT-1 did not alter GABA receptor responses indirectly via modulation of extracellular GABA concentrations in the periaqueductal gray (Bagley et al. 2011). Furthermore, GAT-1 blockade is not essential for GABA uptake inhibition via GAT-3 in the hypothalamic parvocellular preautonomic (Park et al. 2009) and magnocellular neuroendocrine cells (Park et al. 2006). Our finding that blockade of GAT-3, but not GAT-1, reversed the Itonic attenuation suggests that astroglial GABA uptake plays a major role in the physiological and pathophysiological regulation of Itonic in PVN-RVLM neurons.

The dominant role of GAT-3 in regulating Itonic is consistent with the strong GAT-3 and weak GAT-1 expression observed in the hypothalamic nuclei (Park et al. 2009; Park et al. 2006). Interestingly, a similar differential contribution of GAT-1 and GAT-3 to the control of Itonic was reported in cerebellar granule cells (Rossi et al. 2003; Wall and Usowicz 1997). Along with their synaptic inputs in the glomeruli, cerebellar granule cells are enclosed by a glial coat. It is likely that the structure of the local neuronal-glial microenvironment, including the functional expression and distribution of GATs, influences the lifetime and concentration of ambient GABA concentration and, in turn, its ability to activate the extrasynaptic GABAA receptors that mediate Itonic in specific brain regions (Kersante et al. 2013; Park et al. 2006). In the present study, the changes in GATs expression did not support the enhanced GATs activity in HF. Therefore, a change in localization of the transporters rather than total expression would be responsible for the attenuation of Itonic in HF PVN-RVLM neurons. Given that enhanced brain-derived neurotrophic factor (BDNF) in the PVN results in sympathoexcitation (Erdos et al. 2015), it is interesting that BDNF enhances GABA transport by modulating the trafficking of GATs from the plasma membrane in both neurons (Law et al. 2000) and astrocytes (Vaz et al. 2011). Future studies are warranted to delineate an altered local neuronal-glial microenvironment, including the localization of GAT-3, in HF PVN.

Glial GABA release may not play a role in HF-induced Itonic attenuation.

Despite their debated role in modulating Itonic, astrocytes in the hippocampus and cerebellum express Ca2+-activated anion channels (Best-1) that are uniquely permeable to large anions and osmolytes such as GABA and glutamate (Lee et al. 2010; Woo et al. 2012). Our finding of Best-1-ir in astrocytes raised the possibility that glial GABA released via anion channels regulates ambient GABA concentration in the PVN. However, it is unlikely that glial GABA release via Best-1 channels contributes to the inhibition of Itonic in HF PVN-RVLM neurons, because the anion channel blocker NPPB failed to change Iholding in either SHAM or HF PVN-RVLM neurons under our recording conditions. Our finding that PVN Best-1-ir did not differ between the SHAM and HF rats further supported this view. Future studies are warranted to describe the possible roles of Best-1 in normal and altered PVN-RVLM sympathoexcitatory output in various disease conditions, such as hypertension and diabetes. Our results, together with the fact that HF enhances GAT uptake activity in the PVN, suggest that astroglial GABA release is not involved in attenuation of the Itonic in HF PVN-RVLM neurons.

Functional significance of HF Itonic attenuation.

Chronic HF is characterized by exaggerated sympathoexcitation in both humans (Packer 1988) and animal models of HF (Li et al. 2003; Zheng et al. 2009). Despite major advances in therapy, the increased neurohumoral drive causes significant cardiovascular complications that contribute to increased morbidity and mortality. Blunted GABAergic inhibition in the PVN has been identified as a key integrating mechanism that contributes to the sympathoexcitation associated with cardiovascular-related disorders (Allen 2002; Haywood et al. 2001; Reynolds et al. 1996; Zhang et al. 2002). In the present study, we showed the deficient tonic GABAergic inhibition of PVN-RVLM neurons in HF rats, which could increase sympathetic outflow from the PVN during HF. Similarly, decreased phasic and tonic GABAergic inhibition in kidney-related presympathetic PVN neurons has been associated with increased sympathetic outflow in type 1 diabetic conditions (Jiang et al. 2013). Itonic accounts for the dominant proportion of the total GABAAR-mediated current in various brain regions; thus Itonic has a major impact on PVN-RVLM neuronal excitability (Park et al. 2007). Accordingly, we found that Itonic attenuation increased IR and the firing discharge rate in HF PVN-RVLM neurons. The direct impact of Itonic attenuation on membrane IR, and thus the membrane time constant, may affect synaptic efficacy and integration in HF PVN-RVLM neurons (Blomfield 1974; Mitchell and Silver 2003). The leftward shift in I-O function allows subtraction of baseline levels of excitation, suggesting that Itonic attenuation has a significant impact on neuronal sensitivity to incoming excitatory and/or inhibitory synaptic inputs in the HF PVN-RVLM. Thus the increased impact on membrane IR and the I-O function enabled the GAT blockade to correct altered synaptic integration in HF PVN-RVLM neurons. This notion is consistent with our finding that NPA efficiently inhibited the increased spontaneous firing in HF PVN-RVLM neurons.

In summary, our results support the view that GAT-3 uptake is involved in setting the basal and enhanced tone in PVN-RVLM firing activity in normal and altered sympathoexcitatory outflow from the PVN. Enhanced GABA uptake in addition to altered extrasynaptic GABAARs attenuated Itonic and potentiated neuronal excitability in HF PVN-RVLM neurons. This blunted GABAergic inhibition in the PVN could significantly contribute to the sympathoexcitation in cardiovascular-related disorders. Thus our findings suggest that the neuronal-glial interaction plays a significant role in the modulation of CNS control of autonomic activity and homeostasis under normal and disease conditions.

GRANTS

This work was supported by National Research Foundation of Korea funded by the Ministry of Education, Science, and Technology (no. 2007-0054932 and NRF-2012R1A1A4A01004566).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: S.P. and J.Y.J. performed experiments; S.P. and J.Y.J. analyzed data; S.P., J.Y.J., S.U.L., Y.J.L., S.Y.L., P.D.R., J.U.L., H.-W.K., B.H.J., and J.B.P. interpreted results of experiments; S.P. and J.B.P. prepared figures; S.P. drafted manuscript; S.U.L., Y.J.L., S.Y.L., P.D.R., J.U.L., H.-W.K., B.H.J., and J.B.P. conception and design of research; S.U.L., Y.J.L., S.Y.L., P.D.R., J.U.L., H.-W.K., B.H.J., and J.B.P. approved final version of manuscript; J.B.P. edited and revised manuscript.

ACKNOWLEDGMENTS

The English in this document has been checked by at least two professional editors, both native speakers of English.

REFERENCES

  1. Adkins CE, Pillai GV, Kerby J, Bonnert TP, Haldon C, McKernan RM, Gonzalez JE, Oades K, Whiting PJ, Simpson PB. alpha4beta3delta GABA(A) receptors characterized by fluorescence resonance energy transfer-derived measurements of membrane potential. J Biol Chem 276: 38934–38939, 2001. [DOI] [PubMed] [Google Scholar]
  2. Allen AM. Inhibition of the hypothalamic paraventricular nucleus in spontaneously hypertensive rats dramatically reduces sympathetic vasomotor tone. Hypertension 39: 275–280, 2002. [DOI] [PubMed] [Google Scholar]
  3. Allen NJ, Karadottir R, Attwell D. Reversal or reduction of glutamate and GABA transport in CNS pathology and therapy. Pflügers Arch 449: 132–142, 2004. [DOI] [PubMed] [Google Scholar]
  4. Anderson EA, Sinkey CA, Lawton WJ, Mark AL. Elevated sympathetic nerve activity in borderline hypertensive humans. Evidence from direct intraneural recordings. Hypertension 14: 177–183, 1989. [DOI] [PubMed] [Google Scholar]
  5. Bagley EE, Hacker J, Chefer VI, Mallet C, McNally GP, Chieng BC, Perroud J, Shippenberg TS, Christie MJ. Drug-induced GABA transporter currents enhance GABA release to induce opioid withdrawal behaviors. Nat Neurosci 14: 1548–1554, 2011. [DOI] [PubMed] [Google Scholar]
  6. Blomfield S. Arithmetical operations performed by nerve cells. Brain Res 69: 115–124, 1974. [DOI] [PubMed] [Google Scholar]
  7. Boero R, Pignataro A, Ferro M, Quarello F. Sympathetic nervous system and chronic renal failure. Clin Exp Hypertens 23: 69–75, 2001. [DOI] [PubMed] [Google Scholar]
  8. Borden LA, Dhar TG, Smith KE, Branchek TA, Gluchowski C, Weinshank RL. Cloning of the human homologue of the GABA transporter GAT-3 and identification of a novel inhibitor with selectivity for this site. Recept Channels 2: 207–213, 1994. [PubMed] [Google Scholar]
  9. Brickley SG, Mody I. Extrasynaptic GABA(A) receptors: their function in the CNS and implications for disease. Neuron 73: 23–34, 2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Brown N, Kerby J, Bonnert TP, Whiting PJ, Wafford KA. Pharmacological characterization of a novel cell line expressing human alpha(4)beta(3)delta GABA(A) receptors. Br J Pharmacol 136: 965–974, 2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Caraiscos VB, Elliott EM, You-Ten KE, Cheng VY, Belelli D, Newell JG, Jackson MF, Lambert JJ, Rosahl TW, Wafford KA, MacDonald JF, Orser BA. Tonic inhibition in mouse hippocampal CA1 pyramidal neurons is mediated by alpha5 subunit-containing gamma-aminobutyric acid type A receptors. Proc Natl Acad Sci USA 101: 3662–3667, 2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cohn JN, Levine TB, Olivari MT, Garberg V, Lura D, Francis GS, Simon AB, Rector T. Plasma norepinephrine as a guide to prognosis in patients with chronic congestive heart failure. N Engl J Med 311: 819–823, 1984. [DOI] [PubMed] [Google Scholar]
  13. Dalby NO. Inhibition of gamma-aminobutyric acid uptake: anatomy, physiology and effects against epileptic seizures. Eur J Pharmacol 479: 127–137, 2003. [DOI] [PubMed] [Google Scholar]
  14. Decavel C, Van den Pol AN. GABA: a dominant neurotransmitter in the hypothalamus. J Comp Neurol 302: 1019–1037, 1990. [DOI] [PubMed] [Google Scholar]
  15. Diaz MR, Wadleigh A, Hughes BA, Woodward JJ, Valenzuela CF. Bestrophin1 channels are insensitive to ethanol and do not mediate tonic GABAergic currents in cerebellar granule cells. Front Neurosci 5: 148, 2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Drasbek KR, Hoestgaard-Jensen K, Jensen K. Modulation of extrasynaptic THIP conductances by GABAA-receptor modulators in mouse neocortex. J Neurophysiol 97: 2293–2300, 2007. [DOI] [PubMed] [Google Scholar]
  17. El Majdoubi M, Poulain DA, Theodosis DT. Lactation-induced plasticity in the supraoptic nucleus augments axodendritic and axosomatic GABAergic and glutamatergic synapses: an ultrastructural analysis using the disector method. Neuroscience 80: 1137–1147, 1997. [DOI] [PubMed] [Google Scholar]
  18. Erdos B, Backes I, McCowan ML, Hayward LF, Scheuer DA. Brain-derived neurotrophic factor modulates angiotensin signaling in the hypothalamus to increase blood pressure in rats. Am J Physiol Heart Circ Physiol 308: H612–H622, 2015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gao H, Smith BN. Zolpidem modulation of phasic and tonic GABA currents in the rat dorsal motor nucleus of the vagus. Neuropharmacology 58: 1220–1227, 2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Glykys J, Mody I. Activation of GABAA receptors: views from outside the synaptic cleft. Neuron 56: 763–770, 2007a. [DOI] [PubMed] [Google Scholar]
  21. Glykys J, Mody I. The main source of ambient GABA responsible for tonic inhibition in the mouse hippocampus. J Physiol 582: 1163–1178, 2007b. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Grassi G, Seravalle G, Cattaneo BM, Lanfranchi A, Vailati S, Giannattasio C, Del Bo A, Sala C, Bolla GB, Pozzi M, Mancia G. Sympathetic activation and loss of reflex sympathetic control in mild congestive heart failure. Circulation 92: 3206–3211, 1995. [DOI] [PubMed] [Google Scholar]
  23. Han TH, Lee K, Park JB, Ahn D, Park JH, Kim DY, Stern JE, Lee SY, Ryu PD. Reduction in synaptic GABA release contributes to target-selective elevation of PVN neuronal activity in rats with myocardial infarction. Am J Physiol Regul Integr Comp Physiol 299: R129–R139, 2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Haywood JR, Mifflin SW, Craig T, Calderon A, Hensler JG, Hinojosa-Laborde C. gamma-Aminobutyric acid (GABA)-A function and binding in the paraventricular nucleus of the hypothalamus in chronic renal-wrap hypertension. Hypertension 37: 614–618, 2001. [DOI] [PubMed] [Google Scholar]
  25. Heja L, Nyitrai G, Kekesi O, Dobolyi A, Szabo P, Fiath R, Ulbert I, Pal-Szenthe B, Palkovits M, Kardos J. Astrocytes convert network excitation to tonic inhibition of neurons. BMC Biol 10: 26, 2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Henderson LP. Steroid modulation of GABAA receptor-mediated transmission in the hypothalamus: effects on reproductive function. Neuropharmacology 52: 1439–1453, 2007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hines RM, Davies PA, Moss SJ, Maguire J. Functional regulation of GABAA receptors in nervous system pathologies. Curr Opin Neurobiol 22: 552–558, 2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Jiang Y, Gao H, Krantz AM, Derbenev AV, Zsombok A. Reduced GABAergic inhibition of kidney-related PVN neurons in streptozotocin-treated type 1 diabetic mouse. J Neurophysiol 110: 2192–2202, 2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Jo JY, Jeong JA, Pandit S, Stern JE, Lee SK, Ryu PD, Lee SY, Han SK, Cho CH, Kim HW, Jeon BH, Park JB. Neurosteroid modulation of benzodiazepine-sensitive GABAA tonic inhibition in supraoptic magnocellular neurons. Am J Physiol Regul Integr Comp Physiol 300: R1578–R1587, 2011. [DOI] [PubMed] [Google Scholar]
  30. Kersante F, Rowley SC, Pavlov I, Gutierrez-Mecinas M, Semyanov A, Reul JM, Walker MC, Linthorst AC. A functional role for both aminobutyric acid (GABA) transporter-1 and GABA transporter-3 in the modulation of extracellular GABA and GABAergic tonic conductances in the rat hippocampus. J Physiol 591: 2429–2441, 2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kirmse K, Dvorzhak A, Kirischuk S, Grantyn R. GABA transporter 1 tunes GABAergic synaptic transmission at output neurons of the mouse neostriatum. J Physiol 586: 5665–5678, 2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kirmse K, Kirischuk S, Grantyn R. Role of GABA transporter 3 in GABAergic synaptic transmission at striatal output neurons. Synapse 63: 921–929, 2009. [DOI] [PubMed] [Google Scholar]
  33. Law RM, Stafford A, Quick MW. Functional regulation of gamma-aminobutyric acid transporters by direct tyrosine phosphorylation. J Biol Chem 275: 23986–23991, 2000. [DOI] [PubMed] [Google Scholar]
  34. Lee S, Yoon BE, Berglund K, Oh SJ, Park H, Shin HS, Augustine GJ, Lee CJ. Channel-mediated tonic GABA release from glia. Science 330: 790–796, 2010. [DOI] [PubMed] [Google Scholar]
  35. Li YF, Cornish KG, Patel KP. Alteration of NMDA NR1 receptors within the paraventricular nucleus of hypothalamus in rats with heart failure. Circ Res 93: 990–997, 2003. [DOI] [PubMed] [Google Scholar]
  36. Li YF, Patel KP. Paraventricular nucleus of the hypothalamus and elevated sympathetic activity in heart failure: the altered inhibitory mechanisms. Acta Physiol Scand 177: 17–26, 2003. [DOI] [PubMed] [Google Scholar]
  37. Mancia G, Bousquet P, Elghozi JL, Esler M, Grassi G, Julius S, Reid J, Van Zwieten PA. The sympathetic nervous system and the metabolic syndrome. J Hypertens 25: 909–920, 2007. [DOI] [PubMed] [Google Scholar]
  38. Mitchell SJ, Silver RA. Shunting inhibition modulates neuronal gain during synaptic excitation. Neuron 38: 433–445, 2003. [DOI] [PubMed] [Google Scholar]
  39. Ortinski PI, Turner JR, Barberis A, Motamedi G, Yasuda RP, Wolfe BB, Kellar KJ, Vicini S. Deletion of the GABA(A) receptor alpha1 subunit increases tonic GABA(A) receptor current: a role for GABA uptake transporters. J Neurosci 26: 9323–9331, 2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Packer M. Neurohormonal interactions and adaptations in congestive heart failure. Circulation 77: 721–730, 1988. [DOI] [PubMed] [Google Scholar]
  41. Pandit S, Jeong JA, Jo JY, Cho HS, Kim DW, Kim JM, Ryu PD, Lee SY, Kim HW, Jeon BH, Park JB. Dual mechanisms diminishing tonic GABAA inhibition of dentate gyrus granule cells in Noda epileptic rats. J Neurophysiol 110: 95–102, 2013. [DOI] [PubMed] [Google Scholar]
  42. Pandit S, Song JG, Kim YJ, Jeong JA, Jo JY, Lee GS, Kim HW, Jeon BH, Lee JU, Park JB. Attenuated benzodiazepine-sensitive tonic GABAA currents of supraoptic magnocellular neuroendocrine cells in 24-h water-deprived rats. J Neuroendocrinol 26: 26–34, 2014. [DOI] [PubMed] [Google Scholar]
  43. Park JB, Jo JY, Zheng H, Patel KP, Stern JE. Regulation of tonic GABA inhibitory function, presympathetic neuronal activity and sympathetic outflow from the paraventricular nucleus by astroglial GABA transporters. J Physiol 587: 4645–4660, 2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Park JB, Skalska S, Son S, Stern JE. Dual GABAA receptor-mediated inhibition in rat presympathetic paraventricular nucleus neurons. J Physiol 582: 539–551, 2007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Park JB, Skalska S, Stern JE. Characterization of a novel tonic gamma-aminobutyric acidA receptor-mediated inhibition in magnocellular neurosecretory neurons and its modulation by glia. Endocrinology 147: 3746–3760, 2006. [DOI] [PubMed] [Google Scholar]
  46. Pirttimaki T, Parri HR, Crunelli V. Astrocytic GABA transporter GAT-1 dysfunction in experimental absence seizures. J Physiol 591: 823–833, 2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Raiteri L, Stigliani S, Zedda L, Raiteri M, Bonanno G. Multiple mechanisms of transmitter release evoked by “pathologically” elevated extracellular [K+]: involvement of transporter reversal and mitochondrial calcium. J Neurochem 80: 706–714, 2002. [DOI] [PubMed] [Google Scholar]
  48. Reynolds AY, Zhang K, Patel KP. Renal sympathetic nerve discharge mediated by the paraventricular nucleus is altered in STZ induced diabetic rats. Nebraska Med J 81: 419–423, 1996. [PubMed] [Google Scholar]
  49. Richerson GB, Wu Y. Dynamic equilibrium of neurotransmitter transporters: not just for reuptake anymore. J Neurophysiol 90: 1363–1374, 2003. [DOI] [PubMed] [Google Scholar]
  50. Rossi DJ, Hamann M, Attwell D. Multiple modes of GABAergic inhibition of rat cerebellar granule cells. J Physiol 548: 97–110, 2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Sitte HH, Singer EA, Scholze P. Bi-directional transport of GABA in human embryonic kidney (HEK-293) cells stably expressing the rat GABA transporter GAT-1. Br J Pharmacol 135: 93–102, 2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. van den Pol AN. Weighing the role of hypothalamic feeding neurotransmitters. Neuron 40: 1059–1061, 2003. [DOI] [PubMed] [Google Scholar]
  53. Vaz SH, Jorgensen TN, Cristovao-Ferreira S, Duflot S, Ribeiro JA, Gether U, Sebastiao AM. Brain-derived neurotrophic factor (BDNF) enhances GABA transport by modulating the trafficking of GABA transporter-1 (GAT-1) from the plasma membrane of rat cortical astrocytes. J Biol Chem 286: 40464–40476, 2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Waldmeier PC, Stocklin K, Feldtrauer JJ. Weak effects of local and systemic administration of the GABA uptake inhibitor, SK&F 89976, on extracellular GABA in the rat striatum. Naunyn Schmiedebergs Arch Pharmacol 345: 544–547, 1992. [DOI] [PubMed] [Google Scholar]
  55. Wall MJ, Usowicz MM. Development of action potential-dependent and independent spontaneous GABAA receptor-mediated currents in granule cells of postnatal rat cerebellum. Eur J Neurosci 9: 533–548, 1997. [DOI] [PubMed] [Google Scholar]
  56. Woo DH, Han KS, Shim JW, Yoon BE, Kim E, Bae JY, Oh SJ, Hwang EM, Marmorstein AD, Bae YC, Park JY, Lee CJ. TREK-1 and Best1 channels mediate fast and slow glutamate release in astrocytes upon GPCR activation. Cell 151: 25–40, 2012. [DOI] [PubMed] [Google Scholar]
  57. Yamada J, Furukawa T, Ueno S, Yamamoto S, Fukuda A. Molecular basis for the GABAA receptor-mediated tonic inhibition in rat somatosensory cortex. Cereb Cortex 17: 1782–1787, 2007. [DOI] [PubMed] [Google Scholar]
  58. Yeung JY, Canning KJ, Zhu G, Pennefather P, MacDonald JF, Orser BA. Tonically activated GABAA receptors in hippocampal neurons are high-affinity, low-conductance sensors for extracellular GABA. Mol Pharmacol 63: 2–8, 2003. [DOI] [PubMed] [Google Scholar]
  59. Yu Z, Fang Q, Xiao X, Wang YZ, Cai YQ, Cao H, Hu G, Chen Z, Fei J, Gong N, Xu TL. GABA transporter-1 deficiency confers schizophrenia-like behavioral phenotypes. PLoS One 8: e69883, 2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Zhang K, Li YF, Patel KP. Blunted nitric oxide-mediated inhibition of renal nerve discharge within PVN of rats with heart failure. Am J Physiol Heart Circ Physiol 281: H995–H1004, 2001. [DOI] [PubMed] [Google Scholar]
  61. Zhang K, Li YF, Patel KP. Reduced endogenous GABA-mediated inhibition in the PVN on renal nerve discharge in rats with heart failure. Am J Physiol Regul Integr Comp Physiol 282: R1006–R1015, 2002. [DOI] [PubMed] [Google Scholar]
  62. Zheng H, Li YF, Wang W, Patel KP. Enhanced angiotensin-mediated excitation of renal sympathetic nerve activity within the paraventricular nucleus of anesthetized rats with heart failure. Am J Physiol Regul Integr Comp Physiol 297: R1364–R1374, 2009. [DOI] [PMC free article] [PubMed] [Google Scholar]

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