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CELLULAR AND MOLECULAR
Department of Anesthesiology, Pennsylvania State University College of Medicine, The Milton S. Hershey Medical Center, Hershey, Pennsylvania
Received December 20, 2004; accepted January 26, 2005.
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Abstract |
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; Dampney, 1994; Allen, 2002). In addition to the projection to sympathetic preganglionic neurons located in the intermediolateral cell column (IML) of the spinal cord, the PVN neurons also project to neurons within the rostral ventrolateral medulla (RVLM) (Shafton et al., 1998; Yang and Coote, 1998; Pyner and Coote, 2000; Hardy, 2001). The RVLM is a pivotal structure in maintaining tonic sympathetic nerve activity and basal blood pressure through projections to the IML. The PVN parvocellular neurons project to both the RVLM and IML (Shafton et al., 1998; Pyner and Coote, 2000). Available functional evidence suggests that the PVN-RVLM pathway is important in regulation of sympathetic outflow originated from the PVN (Tagawa and Dampney, 1999; Allen, 2002). In this regard, microinjection of a GABAA receptor agonist, muscimol, into the PVN decreases the basal blood pressure and sympathetic nerve activity, and this effect is largely eliminated by synaptic blockade of the RVLM in rats (Allen, 2002). However, the cellular mechanisms involved in regulation of the excitability of PVN-RVLM output neurons remain poorly understood.
The renin-angiotensin system plays a critical role in the control of the sympathetic nervous system (Chen and Toney, 2001; Pan, 2004; Wang et al., 2004b). For example, angiotensin II (Ang II) can influence the PVN neurons through activation of receptors in the circumventricular organs, the regions lacking normal blood-brain barrier (Bains and Ferguson, 1995; Zhang et al., 2002). Also, Ang II may act as a neurotransmitter or modulator in several regions of the hypothalamus including the PVN (Li and Ferguson, 1993; de Wardener, 2001). A major source of the angiotensinergic inputs to PVN neurons is circumventricular organs (Li and Ferguson, 1993; Bains and Ferguson, 1995). Furthermore, the immunoreactivity of angiotensin type 1 (AT1) receptors and their mRNA are densely distributed in the PVN (Gehlert et al., 1991; Aguilera et al., 1995; Li et al., 2003). The excitability of PVN sympathetic premotor neurons are finely regulated by both inhibitory GABAergic and excitatory glutamatergic synaptic inputs (Li et al., 2002, 2003, 2004). The excitatory synaptic input to RVLM pressor neurons is important for the action of Ang II in the PVN (Tagawa and Dampney, 1999). We have recently shown that Ang II increases the excitability of spinally projecting PVN neurons by attenuation of GABAergic synaptic inputs (Li et al., 2003). However, the effect of Ang II on the excitability and synaptic inputs to RVLM-projecting PVN neurons is still not clear. Therefore, we determined the effect of Ang II on the excitability and GABAergic and glutamatergic synaptic inputs to RVLM-projecting PVN neurons using a combination of in vivo retrograde tracing and in vitro whole-cell recording techniques.
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Materials and Methods |
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Slice Preparations
Coronal hypothalamic slices (300 µm in thickness) containing the PVN were cut using a vibratome (Technical Product International, St. Louis, MO), as described previously (Li et al., 2002, 2003). Briefly, 2 to 5 days after tracer injection, the rats were rapidly decapitated under halothane anesthesia. The brain was quickly removed and placed in ice-cold artificial cerebral spinal fluid (aCSF) perfusion solution saturated with 95% O2 and 5% CO2 for 1 to 2 min. A tissue block containing the hypothalamus was cut from the brain and glued onto the stage of the vibratome. After the sectioning, the slices were preincubated in the aCSF, which was continuously gassed with 95% O2 and 5% CO2 at 34°C for at least 1 h until used for recording. For recordings, a slice was transferred to a submersion-type recording chamber continuously perfused (3 ml/min) with aCSF perfusion solution containing 124.0 mM NaCl, 3.0 mM KCl, 1.3 mM MgSO4, 2.4 mM CaCl2, 1.4 mM NaH2PO4, 10.0 mM glucose, and 26.0 mM NaHCO3 saturated with a gas mixture of 95% O2 and 5% CO2 at 34°C.
To verify the injection site and diffusion size of FluoSpheres, the brainstem was taken out after sacrificing the rat and immediately sectioned at the injection level. The brainstem slices were viewed and the injection sites of FluoSpheres were identified under a microscope equipped with epifluorescence illumination. The diffusion of the tracer in the RVLM was generally limited to –11.9 ± 0.05 and –12.4 ± 0.05 mm (bregma), and the diffusion size of FluoSpheres around the site of injection was about 0.4 mm in diameter (Fig. 1A). Rats were excluded if the injection site was not located in the RVLM.
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Electrophysiological Recordings
Whole-cell voltage- and current-clamp recordings were performed in a radio frequency-shielded room, as we described previously (Li et al., 2002, 2003). The recording pipettes were triple-pulled from borosilicate capillaries (1.2 mm o.d., 0.68 mm i.d.; World Precision Instruments, Sarasota, FL) using a micropipette puller (P-97; Sutter Instrument Company, Novato, CA). The resistance of the pipette was 3 to 5 M
when it was filled with a solution containing 130.0 mM potassium gluconate, 1.0 mM MgCl2, 10.0 mM HEPES, 10.0 mM EGTA, 1.0 mM CaCl2, 0.5 mM Na-GTP, and 4.0 mM ATP-Mg adjusted to pH 7.25 with 1 M KOH (290–320 mOsm). The slice was placed in a glass-bottomed recording chamber (Warner Instruments, Hamden, CT) and fixed with a grid of parallel nylon threads supported by a U-shaped stainless steel weight. The slice was perfused at 3.0 ml/min at 34°C maintained by an in-line solution heater and a temperature controller (model TC-324, Warner Instruments). It took about 1.5 min to completely exchange the solution inside the recording chamber at the perfusion speed of 3.0 ml/min. Whole-cell recordings from labeled PVN neurons (Fig. 1, B and C) were made under visual control using a combination of epifluoresence illumination and infrared and differential interference contrast optics on an upright microscope (BX50WI; Olympus, Tokyo, Japan). Because the labeled RVLM-projecting neurons are primarily present in the medial one-third of the PVN area between the third ventricle and the fornix, labeled PVN neurons in this site were selected for recording. Recordings were performed using an Axopatch 200B or Multiclamp 700A amplifier (Axon Instruments Inc., Union City, CA). Signals were filtered at 1 to 2 kHz, digitized at 10 kHz using Digidata 1320A (Axon Instruments Inc.). The series resistance was compensated by 60 to 80%. The recording was abandoned if the input resistance (measured with a voltage range of 15–20 mV) changed more than 15% during the recording.
Voltage-Clamp Recordings of Postsynaptic Currents. Miniature GABAergic inhibitory postsynaptic currents (mIPSCs) were recorded at a holding potential of 0 mV and in the presence of 1 µM tetrodotoxin (TTX) and 20 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). The miniature excitatory postsynaptic currents (mEPSCs) were recorded at a holding potential of –70 mV in the presence of 1 µM TTX and 20 µM bicuculline (Li et al., 2002, 2003). A general G protein inhibitor, 1 mM guanosine 5'-O-(2-thiodiphosphate) (GDP-
-S) was added into the recording pipette solution to block the possible postsynaptic response mediated by G proteins coupled to angiotensin AT1 receptors (Li et al., 2003). The effective concentration of GDP--S to block the postsynaptic effect of Ang II has been shown in previous studies (Ohya and Sperelakis, 1991; Oz and Renaud, 2002). To study the evoked IPSCs and EPSCs (eIPSCs/eEPSCs) in labeled PVN neurons, synaptic currents were evoked by electrical stimulation (0.1 ms, 0.3–0.8 mA, and 0.1 Hz) through a bipolar tungsten electrode connected to a stimulator (Grass Instruments, West Warwick, RI). The tip of the stimulating electrode was placed 200 to 600 µm away from the recorded neuron. A Na+ channel blocker, lidocaine N-ethyl bromide (QX-314, 10 mM), was also included in the pipette solution to block the Na+ current in these voltage-clamp experiments. Based on the optimal reversal potentials determined for CNQX-sensitive EPSCs and bicuculline-sensitive IPSCs, the eEPSCs and eIPSCs were recorded at a holding potential of –70 and 0 mV, respectively (Li et al., 2003).
To examine the effect of Ang II on the paired-pulse facilitation, two consecutive synaptic responses (A1 and A2) were evoked by a pair of stimulus given at a short interval (40 ms for EPSCs and 50 ms for IPSCs). Paired-pulse facilitation was expressed as the amplitude ratio of the second synaptic response to the first synaptic response (A2/A1).
Current-Clamp Recordings of Firing Activity. The spontaneous firing activity of labeled PVN neurons was recorded using the whole-cell current-clamp technique (Li et al., 2003). The recording procedures were similar to those used for postsynaptic current recordings as described above, except that TTX and QX-314 were not used. In some experiments, 1 mM GDP--S was included in the recording pipette solution to block the postsynaptic effect of Ang II. Recordings of the firing activity of labeled PVN neurons began about 5 min after the whole-cell access was established and the firing activity reached a steady state. Signals were processed, recorded, and analyzed as described above. The junction potential was corrected during off-line analysis.
Drugs were applied to the recording chamber at final concentrations. Ang II, PD123319, CNQX, bicuculline, and GDP--S were obtained from Sigma-Aldrich (St. Louis, MO). Losartan was a gift from Merck and Co., Inc. (Rahway, NJ). TTX and QX-314 were purchased from Alomone Labs (Jerusalem, Israel). All the drugs were prepared immediately before the experiments and applied to the recording chamber using syringe pumps.
Data Analysis
Data are presented as means ± S.E.M. The mIPSCs and mEPSCs were analyzed off-line with a peak detection program (MiniAnalysis; Synaptosoft Inc., Decatur, GA). Measurements of the averaged firing rate, amplitude, and the decay time constant of postsynaptic currents were performed over a period of 3 min during control, drug application, and recovery. The mIPSCs and mEPSCs were detected by the fast rise time of the signal over an amplitude threshold above the background noise. The amplitude detection threshold was typically 3 to 5 pA. We manually excluded the event when the noise was erroneously identified as the mEPSCs or mIPSCs by the program. The background noise level was typically constant throughout the recording of a single neuron. The cumulative probability of the amplitude and interevent interval of mEPSCs/mIPSCs was compared using the Kolmogorov-Smirnov test, which estimates the probability that two cumulative distributions are similar. At least 100 randomly selected mIPSCs and mEPSCs were used in each analysis. All the decay phases of mIPSCs and mEPSCs were analyzed with one and two exponential functions. Based on the curve fitting R2 values, all mIPSCs were best fitted by two components under all conditions. For cells displaying intermittent firing activity, the membrane potential was measured when the cell was silent and the membrane potential became stable. For those cells showing tonic activity, the membrane potential was usually estimated 200 ms prior to initiation of the action potential (Li et al., 2003). The effects of drugs on the amplitude and frequency of mIPSCs and mEPSCs were determined by the nonparametric Wilcoxon signed rank test or nonparametric ANOVA (Kruskal-Wallis) with Dunn's post hoc test. P < 0.05 was considered to be statistically significant.
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Results |
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, and action potential amplitude of 68.6 ± 3.5 mV.
Effect of Ang II on the Firing Activity of Labeled PVN Neurons. To determine the effect of Ang II on the excitability of labeled PVN neurons, the spontaneous firing activity of these neurons were recorded under current-clamp condition. The majority of the labeled PVN neurons tested had spontaneous activity (38 of 48, 79.1%). Ang II, at a concentration of 2 µM, significantly increased the firing rate from 3.63 ± 0.65 to 6.10 ± 0.75 Hz in all nine cells tested (Fig. 2). However, the membrane potential was not significantly altered by 2 µM Ang II (–64.8 ± 2.01 to –63.5 ± 2.2 mV; P > 0.05; n = 9). Repeat applications of 2 µM Ang II produced a similar increase in firing rate in these labeled PVN neurons (Fig. 2, A and B). To examine whether the excitatory effect of Ang II was mediated through postsynaptic angiotensin receptors, 1 mM GDP--S was included in the pipette internal solution to block the possible postsynaptic effect of Ang II in additional eight labeled PVN neurons. There was no significant alteration of the membrane potential and firing activity during the course of intracellular dialysis of GDP--S (the first 3–5 min after rupturing the cell membrane). Perfusion of 2 µM Ang II still increased the firing rate of these labeled PVN neurons from 1.99 ± 0.33 to 4.6 ± 0.63 Hz (P < 0.05; Fig. 2C). There was no significant difference between the excitatory effect of Ang II on the firing rate of cells recorded with and without GDP--S inside the electrode.
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To further determine the receptor subtype that mediated the effect of Ang II on the firing activity of the PVN neurons, the specific antagonists for the AT1 (losartan) and AT2 (PD123319) receptors were used. The effective concentrations of these antagonists have been determined previously (Li et al., 2003). Ang II (2 µM) failed to increase the firing rate of nine labeled PVN neurons in the presence of 2 µM losartan (4.32 ± 0.91 versus 4.44 ± 0.88 Hz, P > 0.05, Fig. 3, A and B). However, 5 µM PD123319 did not affect the excitatory effect of 2 µM Ang II on another seven labeled PVN neurons. Ang II increased the firing rate from 3.13 ± 0.76 to 5.50 ± 1.08 Hz (P < 0.05) in these seven cells in the presence of PD123319 (Fig. 3C).
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Effect of Ang II on eIPSCs and eEPSCs of Labeled PVN Neurons. Because inclusion of GDP--S in the pipette solution did not affect the excitatory effect of Ang II on labeled PVN cells, we next determined the possible effect of Ang II on the synaptic inputs to the PVN neurons. Both IPSCs and EPSCs were evoked by electrical stimulation at a constant intensity. The eEPSCs were recorded at a holding potential of –70 mV, whereas the eIPSCs were isolated at a holding potential of 0 mV (Li et al., 2003). The eIPSCs were completely eliminated by application of 20 µM bicuculline, and the eEPSCs were abolished by application of 20 µM CNQX (Fig. 4A). Both eEPSCs and eIPSCs were evoked in 10 of 14 labeled PVN neurons, and only eIPSCs were elicited without detectable eEPSCs in the remaining four cells. Ang II (0.5–5 µM) inhibited the peak amplitude of eIPSCs in a concentration-dependent manner (n = 14, Fig. 4A). The peak amplitude of eIPSCs was reduced by Ang II at a concentration of 0.5 µM, and the inhibition reached the maximum (–52.7%) at a concentration of 2 µM (Fig. 4B). In contrast, Ang II had little effect on the amplitude of eEPSCs even at a higher concentration (5 µM, n = 10, Fig. 4). Furthermore, Ang II did not significantly alter the holding current and input resistance in these 14 labeled PVN neurons. The holding current was 78.2 ± 8.5 and 76.1 ± 8.9 pA during control and application of 2 µM Ang II, respectively, at a holding potential of 0 mV. When the cell was voltage-clamped at –70 mV, the holding current was –19.6 ± 3.5 and –20.6 ± 3.8 pA before and during application of 2 µM Ang II, respectively. The input resistance before and during perfusion of 2 µM Ang II was 481.1 ± 23.6 and 473.6 ± 35.9 M, respectively (P > 0.05; n = 14).
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Effect of Ang II on mIPSCs and mEPSCs of Labeled PVN Neurons. To further determine the presynaptic effect of Ang II in labeled PVN neurons, we tested the effect of 2 µM Ang II on the mIPSCs and mEPSCs. The mIPSCs were recorded in the presence of 1 µM TTX and 20 µM CNQX. The mIPSCs were completely abolished by 20 µM bicuculline (Fig. 5A). Ang II significantly decreased the frequency of mIPSCs from 3.89 ± 0.84 to 2.06 ± 0.45 Hz (P < 0.05; n = 11) without affecting the amplitude and decay time constant of mIPSCs in a concentration of 2 µM (Fig. 5). The cumulative probability analysis of mIPSCs before and during Ang II application revealed that the distribution pattern of the interevent interval of mIPSCs shifted to the right in response to Ang II, whereas the distribution pattern of the amplitude of mIPSCs was not changed (Fig. 5, B and C). The decay phase of mIPSCs was best fitted by a double-exponential function (Fig. 5D). Neither the fast (6.87 ± 0.38 versus 6.89 ± 0.47 ms) nor slow (21.91 ± 1.28 versus 22.32 ± 2.35 ms) components of the decay phase of mIPSCs during Ang II application was significantly different from those during the control. Repeat application of Ang II had a reproducible inhibitory effect on the frequency of mIPSCs in these 11 labeled PVN neurons (data not shown).
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The effect of Ang II on mEPSCs was tested in another nine labeled PVN neurons. The mEPSCs were recorded at a holding potential of –70 mV in the presence of 1 µM TTX and 20 µM bicuculline. In these nine neurons tested, the mEPSCs were completely abolished by 20 µM CNQX (Fig. 6A). Neither the frequency nor the amplitude of mEPSCs was significantly affected by bath application of 2 and 5 µM Ang II (Fig. 6). The effect of Ang II on mEPSCs was further analyzed by measuring the time constant of the decay phase of mEPSCs. The decay phase of mEPSCs was best fitted by a single-exponential function (Fig. 6D). The decay time constants of mEPSCs were similar during control and Ang II application (2.58 ± 0.96 versus 2.46 ± 0.81 ms; P > 0.05; n = 9). To define the receptor subtype mediating the effect of Ang II on mIPSCs, losartan and PD123319 were used. In all 11 labeled PVN neurons, 2 µM losartan completely blocked the inhibitory effect of 2 µM Ang II on the frequency of mIPSCs (Fig. 7). Losartan alone had no effect on mIPSCs of these neurons in the slice preparation. The frequency (2.43 ± 0.55 versus 2.40 ± 0.52 Hz, P > 0.05) and amplitude (40.53 ± 3.6 versus 39.6 ± 3.4 pA, P > 0.05) of mIPSCs were not significantly altered by bath application of Ang II in the presence of losartan. However, 2 µM Ang II still significantly decreased the frequency of mIPSCs from 2.35 ± 0.67 to 1.09 ± 0.26 (P < 0.05) in the presence of 5 µM PD123319 in eight separated labeled PVN neurons tested (Fig. 7, F and G).
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Effect of Ang II on Paired-Pulse Facilitation of Evoked IPSCs and EPSCs. To further determine the presynaptic effect of Ang II, we examined the effect of 2 µM Ang II on paired-pulse ratio (PPR) of evoked IPSCs and EPSCs in nine separate labeled PVN neurons. Ang II increased the PPR (A2/A1) of evoked IPSCs from 1.22 ± 0.06 to 1.56 ± 0.07 in eight of nine labeled PVN neurons (P < 0.05, Fig. 8, A and B). Ang II did not change the PPR of evoked IPSCs in the remaining neuron (1.02 versus 1.03). On the other hand, 2 µM Ang II did not significantly change the PPR of evoked EPSCs in all six labeled PVN neurons tested (Fig. 8C).
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Effect of Ang II and Bicuculline on the Firing Activity of Labeled PVN Neurons. Because Ang II selectively reduced inhibitory GABAergic inputs to labeled PVN cells, we subsequently determined whether Ang II-induced disinhibition was responsible for the excitatory effect of Ang II on PVN-RVLM neurons. Thus, the effect of 2 µM Ang II on the firing activity of labeled PVN neurons was tested in the presence of the GABAA receptor antagonist bicuculline. The spontaneous activity of eight labeled PVN neurons was significantly increased by initial application of 2 µM Ang II and following application of 20 µM bicuculline (Fig. 9A). Bicuculline alone significantly increased the firing rate from 2.86 ± 0.54 to 5.54 ± 0.69 Hz and depolarized the cell membrane from –64.8 ± 1.6 to –60.9 ± 2.0 mV (n = 8, P < 0.05). However, subsequent application of 2 µM Ang II failed to further increase the firing rate of these neurons in the presence of 20 µM bicuculline (Fig. 9, A and B). Lack of effect of Ang II in the presence of bicuculline could be possibly due to the fact that bicuculline had maximally increased the firing activity of these neurons. To examine this possibility, capsaicin was further applied in the presence of bicuculline. Capsaicin in a concentration of 1 µM (Li et al., 2004) still significantly increased the firing activity from 4.72 ± 0.42 to 6.09 ± 0.48 Hz (Fig. 9, C and D; P < 0.05; n = 7) in the presence of 20 µM bicuculline (Fig. 9, C and D).
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Discussion |
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; Ranson et al., 1998; Yang and Coote, 1998; Pyner and Coote, 2000; Hardy, 2001). Although the PVN sympathetic premotor neurons send projections to both the RVLM and IML, the relative functional importance of these two pathways in autonomic regulation remains to be established. Our previous studies have focused largely on cellular mechanisms involved in the control of PVN-IML projection neurons (Li et al., 2002, 2003). It has been shown that inhibition of the PVN decreases the basal blood pressure and sympathetic nerve activity, an effect that is largely attenuated by synaptic blockade of the RVLM in rats (Allen, 2002). Thus, the PVN-RVLM pathway appears to be more important for regulation of sympathetic outflow and blood pressure. In this electrophysiological study, we used a combination of retrograde labeling and in vitro brain slice whole-cell recording techniques to specifically determine the cellular mechanisms responsible for the excitatory effect of Ang II on RVLM-projecting PVN neurons.
Both anatomical and functional evidence suggest that the PVN plays an important role in the regulation of autonomic function by Ang II (Pan, 2004). For instance, the sympathoexcitatory response induced by central hyperosmolality is attenuated by blockade of AT1 receptors with losartan in the PVN (Chen and Toney, 2001). We found that Ang II significantly increased the firing rate of labeled PVN neurons, and such an effect was eliminated by an AT1 antagonist, losartan, but not by the AT2 antagonist PD123319 (Li et al., 2003). Thus, the effect of Ang II on RVLM-projecting PVN neurons is mediated by AT1, but not AT2, receptors. These data are consistent with previous studies showing the presence of AT1 receptors and their mRNA in the PVN (Gehlert et al., 1991; Obermuller et al., 1991; Aguilera et al., 1995; Li et al., 2003). Although the AT2 receptor immunoreactivity in the PVN has been reported, there is no binding site for AT2 receptors in the PVN (Gehlert et al., 1991; Song et al., 1992). It is known that the AT1 receptors are G protein-coupled receptors (Richards et al., 1999; Pan, 2004). Surprisingly, however, in the present study the effect of Ang II on the firing activity was not affected by inclusion of a general G protein inhibitor, GDP--S, in the recording pipette solution. Although Ang II induces a small membrane depolarization in the PVN magnocellular neurons by inhibition of a potassium current (Li and Ferguson, 1996), we observed that Ang II had no detectable postsynaptic effect on the RVLM-projecting PVN neurons in this slice preparation. Notably, it has been reported that puff application of Ang II produces an inward current in some RVLM-projecting PVN neurons (Cato and Toney, 2004), although the pre- and postsynaptic effects of Ang II are not differentiated in that study. The absence of postsynaptic action of Ang II cannot be explained by the dose used in this study. We observed that the excitatory effect of 2 µM Ang II on the firing activity was not affected by intracellular application of GDP--S. This suggests that postsynaptic AT1 receptors do not contribute significantly to the excitatory effect of Ang II on the firing activity of RVLM-projecting PVN neurons.
We further examined the possible synaptic mechanisms involved in the effect of Ang II on RVLM-projecting PVN neurons. We found that Ang II significantly inhibited the evoked GABAergic IPSCs in a concentration-dependent manner. Furthermore, Ang II increased the paired-pulse ratio of evoked IPSCs and decreased the frequency of GABAergic mIPSCs without affecting the amplitude and decay time constant of mIPSCs, suggesting that the likely site of its action is at the presynaptic GABAergic terminal. The effect of Ang II on mIPSCs of RVLM-projecting PVN neurons was completely blocked by a selective AT1 receptor antagonist, losartan. But the specific AT2 receptor antagonist PD123319 did not alter the effect of Ang II on mIPSCs and the firing activity of the RVLM-projecting PVN neurons. This finding is consistent with our recent study showing that the AT1 receptors are located at the presynaptic terminals in the PVN (Li et al., 2003). In contrast to its action on GABAergic IPSCs, Ang II had no significant effect on evoked glutamatergic EPSCs or mEPSCs of labeled PVN neurons at concentrations that attenuated GABAergic IPSCs. The reason for this selective effect of Ang II on the GABAergic synaptic inputs remains unclear. Both nitric oxide and Ang II also preferentially alter GABAergic, but not glutamatergic, synaptic inputs to spinally projecting PVN neurons (Li et al., 2002, 2003). On the other hand, the vanilloid receptor-1 (TRPV1) agonist capsaicin selectively enhances the glutamatergic, but not GABAergic, synaptic inputs to these PVN neurons (Li et al., 2004). Because Ang II only inhibits GABAergic synaptic inputs to the labeled PVN neurons, it is possible that the glutamatergic terminals, which make synapse with PVN-RVLM output neurons have no sufficient functional AT1 receptors. The signal transduction mechanisms underlying the effect of Ang II on the synaptic GABA release remain largely unidentified. Ang II receptors are coupled to different types of G proteins resulting in activation of many different signaling pathways including phospholipase C, D and A2, protein kinase C, and delayed rectifier and transient A-type K+ currents (Dudley et al., 1990; Pueyo et al., 1996; Sumners et al., 1996; Richards et al., 1999). Recent studies also suggest that the reactive oxygen species may be involved in the action of Ang II in the brain (Zimmerman et al., 2002; Wang et al., 2004a). Further studies are warranted to define the signaling mechanisms of the presynaptic effect of Ang II in the PVN.
To determine whether attenuation of GABAergic synaptic inputs contributes to Ang II-induced excitation of these PVN neurons, we determined the effect of Ang II on the firing activity of labeled PVN neurons following blockade of GABAA receptors with bicuculline. We found that Ang II failed to excite the labeled PVN neurons in the presence of bicuculline, suggesting that the excitatory effect of Ang II on RVLM-projecting PVN neurons is mediated by a disinhibition (reduction of GABAergic inputs) mechanism. Bicuculline alone produced a profound increase in the firing activity in all labeled PVN neurons tested. Hence, these PVN output neurons are under a tonic inhibition by the GABAergic synaptic inputs, and GABA acts as a shunt to maintain a hyperpolarized membrane potential and restrain firing. This tonic GABAergic inhibition of PVN output neurons appears to be similar to modulation of the background firing of thalamocortical neurons by inhibitory sculpturing (Steriade et al., 1993). It is possible that lack of effect of Ang II on the firing activity in the presence of bicuculline may be due to the maximal firing of PVN neurons following bicuculline. Capsaicin excites PVN preautonomic neurons through selective potentiation of glutamatergic inputs (Li et al., 2004). Thus, we applied capsaicin in the presence of bicuculline to examine this possibility. We found that capsaicin still caused a significant increase in the firing activity of labeled PVN neurons in the presence of bicuculline. Therefore, these data provide strong evidence that attenuation of GABAergic synaptic inputs is ultimately involved in the excitatory effect of Ang II on RVLM-projecting PVN neurons. Ang II could excite PVN sympathetic premotor neurons to augment sympathetic outflow by attenuation of synaptic GABA release.
Although PVN neurons project to both the IML and RVLM (Shafton et al., 1998; Pyner and Coote, 2000), there are some major differences as to the proportions of PVN neurons projecting to either the RVLM or IML. When the retrograde tracer is injected into the caudal spinal cord at T13-L1, about one-third of retrogradely labeled PVN neurons project to both the spinal cord and RVLM (Shafton et al., 1998). However, only about 3% PVN neurons have projections to the RVLM and IML if the tracer is injected into the rostral spinal cord at the T2-T4 level (Pyner and Coote, 2000). Notably, we injected the tracer into the T2-T4 level of the spinal cord in the previous study (Li et al., 2003). Based on that, it is likely that at least a large part of the neurons sampled in the present study are different from those studied previously (Li et al., 2003). In general, we found that Ang II has a similar effect on the excitability and GABAergic and glutamatergic synaptic inputs to the two populations of neurons with projections to the spinal cord and RVLM (Li et al., 2003). For example, Ang II increased the firing rate of spinally projecting PVN neurons and RVLM-projecting PVN neurons by 89.2 and 86.6%, respectively. Also, Ang II caused a comparable reduction in the frequency of mIPSCs in spinally projecting and RVLM-projecting PVN neurons (54.5 and 49.8%, respectively). Furthermore, Ang II, in a similar concentration range, decreased the amplitude of eIPSCs in both spinally projecting and RVLM-projecting PVN neurons. Therefore, the present study, together with our recent finding, provides important information that Ang II has a very similar presynaptic effect on these two populations of PVN sympathetic premotor neurons.
In summary, this study provides new information about the synaptic mechanisms underlying the stimulatory effect of Ang II on RVLM-projecting PVN neurons. This information is important for our understanding of the cellular mechanisms of Ang II in regulation of sympathetic outflow in the hypothalamus. The intrinsic brain Ang II or circulating Ang II could influence the sympathetic outflow through its presynaptic effect on sympathetic premotor neurons in the PVN. Thus, Ang II can regulate sympathetic output and hemodynamics through the PVN-IML, PVN-RVLM, or both pathways. The potential functional difference and differential roles of these two descending projections in the regulation of sympathetic drive under physiological and pathophysiological conditions are currently unclear and should be further investigated.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: PVN, paraventricular nucleus; IML, intermediolateral cell column; RVLM, rostral ventrolateral medulla; Ang II, angiotensin II; aCSF, artificial cerebral spinal fluid; IPSCs, inhibitory postsynaptic currents; mIPSCs, miniature IPSCs; TTX, tetrodotoxin; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; EPSCs, excitatory postsynaptic currents; mEPSCs, miniature EPSCs; eIPSCs, evoked IPSCs; eEPSCs, evoked EPSCs; GDP--S, guanosine 5'-O-(2-thiodiphosphate); QX-314, lidocaine N-ethyl bromide; PD123319, S-(+)-1-([4-(dimethylamino)-3-methylphenyl]methyl)-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo(4,5-c)pyridine-6-carboxylic acid; ANOVA, analysis of variance; PPR, paired-pulse ratio.
Address correspondence to: Dr. Hui-Lin Pan, Department of Anesthesiology, H187, Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033. E-mail: hpan{at}psu.edu
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References |
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fast (6.63 versus 6.71 ms) and 
