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Benzodiazepine inhibits hypothalamic presympathetic neurons by potentiation of GABAergic synaptic input
Neuropharmacology 52 (2007) 467e475 www.elsevier.com/locate/neuropharm
Benzodiazepine inhibits hypothalamic presympathetic neurons by potentiation of GABAergic synaptic input Matthew R. Zahner a, De-Pei Li b, Hui-Lin Pan a,b,* a
Department of Anesthesiology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA b Department of Anesthesiology and Pain Medicine, University of Texas, M. D. Anderson Cancer Center, 1400 Holcombe Blvd., Unit 409, Houston, TX 77030-4009, USA Received 20 July 2006; received in revised form 15 August 2006; accepted 17 August 2006
Abstract Presympathetic neurons in the paraventricular nucleus (PVN) of the hypothalamus receive inputs from g-aminobutyric acid (GABA)containing neurons, which regulate sympathetic outflow and cardiovascular function. Benzodiazepines can decrease blood pressure and sympathetic nerve activity when used for induction of anesthesia, but the sites and mechanisms of action are uncertain. In this study, we determined the effect of the benzodiazepine agonist diazepam on GABAergic inhibitory postsynaptic currents (IPSCs) and the firing activity of rostral ventrolateral medulla (RVLM)-projecting PVN neurons. RVLM-projecting PVN neurons were retrogradely labeled by fluorescent microspheres injected into the RVLM in rats. Whole-cell and cell-attached recordings were performed on labeled PVN neurons in the hypothalamic brain slice. Bath application of 1e10 mM diazepam significantly increased the decay time constants of the GABAergic miniature IPSCs and evoked IPSCs in a dose-dependent manner. Also, diazepam significantly increased the amplitude of evoked IPSCs but not of miniature IPSCs. Pretreatment with the benzodiazepine antagonist flumazenil completely blocked the diazepam-induced increases in the amplitude and decay time constants of the evoked IPSCs. Furthermore, diazepam significantly decreased the firing activity of PVN-RVLM neurons that responded with increased firing to the GABAA receptor antagonist bicuculline. In contrast, diazepam had no significant effect on the firing activity of bicuculline-unresponsive PVN-RVLM neurons. This study provides new information that the benzodiazepine suppresses the firing activity of PVN presympathetic neurons by potentiation of GABAergic inputs. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Autonomic system; Sympathetic nervous activity; Hypothalamus; Synaptic transmission
1. Introduction The hypothalamic paraventricular nucleus (PVN) is an integrating center for regulation of neuroendocrine, cardiovascular, and other physiological functions. The PVN is one of the five major sympathetic premotor cell groups in the brain (Strack et al., 1989). Presympathetic neurons in the PVN send projections to the rostral ventrolateral medulla (RVLM) as well as * Corresponding author. Department of Anesthesiology and Pain Medicine, University of Texas, M. D. Anderson Cancer Center, 1400 Holcombe Blvd., Unit 409, Houston, TX 77030-4009, USA. Tel.: þ1 713 563 5838; fax: þ1 713 794 4590. E-mail address: [email protected] (H.-L. Pan). 0028-3908/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2006.08.024
the intermediolateral cell column of the spinal cord to regulate sympathetic outflow (Hardy, 2001; Pyner and Coote, 2000; Shafton et al., 1998). The PVN tonically controls sympathetic activity and blood pressure and is an important source of excitatory drive for sympathetic vasomotor tone. For example, activation of the PVN induces a profound increase in sympathetic nerve activity and blood pressure in anesthetized and conscious rats (Kannan et al., 1989; Martin and Haywood, 1993). Conversely, inhibition of the PVN suppresses sympathetic discharges and lowers basal blood pressure (Allen, 2002; Zahner and Pan, 2005). Furthermore, aberrant PVN neuronal activity has been linked to several cardiovascular diseases, such as hypertension and heart failure (Allen, 2002; Li and Pan, 2005b; Zhang et al., 2002). Modification of the GABAergic synaptic
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input to vasomotor neurons in the central nervous system represents a potential target to attenuate the sympathetic outflow. g-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the PVN (Decavel and Van den Pol, 1990). GABA receptors are categorized into two major types, the ionotropic GABAA and GABAC receptors and the metabotropic GABAB receptors. GABAA receptors gate a Cl ionophore and have binding sites for benzodiazepines and barbiturates, which potentiate the inhibitory response to GABA. GABAA receptors are assembled with several polypeptide units (a1e6, b1e4, g1e3, d, r1e2) that combine to form a pentameric chloride channel (Hevers and Luddens, 1998; Macdonald and Olsen, 1994). Activation of the GABAA receptor induces a Cl conductance and causes the membrane potential to move towards the Cl reversal potential, thereby usually suppressing the firing activity of mature neurons (Choh et al., 1977; Macdonald and Barker, 1978; Macdonald and Olsen, 1994). It has been shown that benzodiazepines act at GABAA receptors by binding to a specific allosteric binding site separate from the GABA binding site on the GABAA receptor complex (Mohler and Okada, 1977). Also, benzodiazepines potentiate the GABA-induced neuronal inhibition by increasing the affinity of GABA for the GABAA receptor and increasing the frequency of channel opening, thereby increasing the Cl current (Rogers et al., 1994). Thus, benzodiazepines can shift the GABA concentration-response curve to the left without increasing the maximum current evoked by GABA. Systemic administration of the benzodiazepine agonist diazepam can reduce blood pressure and decreases sympathetic outflow in humans (Kitajima et al., 2004). Furthermore, benzodiazepines reduce the sympathetic tone when they are used for induction of anesthesia in animals and humans (Flacke et al., 1985; Marty et al., 1986). Diazepam also consistently attenuates the pressor response induced by stress and stimulation of the hypothalamus in rats (Conahan and Vogel, 1986; Kawasaki et al., 1979). However, the mechanisms for this inhibitory action of benzodiazepines in the hypothalamus on the sympathetic outflow are not fully known. The PVN is composed of heterogeneous subpopulations of neurons important for the regulation of different autonomic and neuroendocrine functions (Allen, 2002; Swanson and Sawchenko, 1983; Zhang et al., 2002). Therefore, using in vivo retrograde labeling and in vitro brain slice recordings, we determined the effect of the benzodiazepine agonist on the GABAergic input and firing activity of RVLM-projecting PVN neurons. 2. Materials and methods 2.1. Retrograde labeling of RVLM-projecting PVN neurons Male SpragueeDawley rats (200e225 g; Harlan, Indianapolis, IN) were used in this study. The surgical preparations and experimental protocols were approved by the Animal Care and Use Committee of the Pennsylvania State University College of Medicine and conformed to the National Institutes of Health guidelines on the ethical use of animals. All efforts were made to minimize both the suffering and number of animals used. Rats were anesthetized by subcutaneous injection of a ketamine (70 mg/kg) and xylazine (6 mg/ kg) mixture, and the head of the rat was placed in a stereotaxic apparatus.
A burr hole (4 mm in diameter) was made in the occipital bone bilaterally according to the following coordinates (lambda): 2.5e3.0 mm caudal, 1.8e2.2 mm lateral, and 7.5e8.0 mm deep from the surface of the cortex. A rhodamine-labeled fluorescent microsphere suspension (FluoSpheres, 0.04 mm; Molecular Probes, Eugene, OR) was ejected (Nanojector II; Drummond Scientific Co., Broomall, PA) bilaterally into the region of the RVLM (Li and Pan, 2005a). The pipette was positioned with a micromanipulator and the injection of 50 nl FluoSpheres was monitored through a surgical microscope. After injection, the skin was sutured and the wound was closed. Animals were returned to their cages for 3e5 days, which is sufficient time to allow the retrograde tracer to be transported to the PVN.
2.2. Slice preparation Three to 5 days after tracer injection, the rats were rapidly decapitated under halothane anesthesia. The brain was quickly removed and placed in icecold artificial cerebrospinal fluid saturated with 95% O2 and 5% CO2. A tissue block containing the hypothalamus was cut from the brain and glued onto the stage of the vibratome (Technical Product International, St. Louis, MO), as described previously (Li et al., 2002; Li and Pan, 2001). Coronal hypothalamic slices (300 mm in thickness) containing the PVN were cut from the tissue block in ice-cold artificial cerebrospinal fluid. After sectioning, the slices were pre-incubated in the artificial cerebrospinal fluid, which was continuously gassed with 95% O2 and 5% CO2 at 34 C for at least 1 h before being used for the recording. For recordings, a slice was transferred to a submersiontype recording chamber continuously perfused with artificial cerebrospinal fluid 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 removed after sacrificing the rat and stored in 10% formaldehyde (Li and Pan, 2005a). Brainstems were sectioned (50 mm) at the injection level. The brainstem slices and the injection sites of FluoSpheres were identified under a microscope equipped with fluorescence illumination. The diffusion of the tracer was generally limited to the RVLM, and the diffusion size of FluoSpheres around the site of injection was about 0.5 mm in diameter. Rats were excluded if the injection site was not located within the RVLM.
2.3. Electrophysiological recordings Whole-cell voltage-clamp recordings of postsynaptic currents were performed in a radio frequency-shielded room, as we described previously (Li et al., 2002; Li and Pan, 2005b). The recording pipettes were pulled from borosilicate capillaries (1.2 mm OD, 0.86 mm ID; World Precision Instruments, Sarasota, FL) using a micropipette puller (P-97; Sutter Instrument Company, Novato, CA). The resistance of the pipette was 4 to 6 MU 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, and 4.0 mM adenosine triphosphate-Mg adjusted to pH 7.25 with 1 M KOH (290e320 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 Ushaped 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 this perfusion rate. Whole-cell recordings from labeled PVN neurons were made under visual control using a combination of fluorescence 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 (Li and Pan, 2005b). The labeled neurons were identified with the aid of fluorescence illumination and differential interference contrast optics on an upright microscope. A tight GU seal was obtained on the labeled neuron identified. Recordings began w5 min after the whole-cell access or cell-attached seal was obtained and the current reached a steady state. Recordings were performed using an Axopatch 200B or Multiclamp 700A amplifier (Axon
M.R. Zahner et al. / Neuropharmacology 52 (2007) 467e475 Instruments Inc., Union City, CA). Signals were filtered at 1 to 2 kHz and 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 15e20 mV) changed more than 15% during the recording. To record the evoked inhibitory postsynaptic currents (eIPSCs), synaptic currents were evoked at a sub-maximal stimulation intensity by electrical stimulation (0.1 ms, 0.3e0.8 mA, and 0.1 Hz) through a bipolar tungsten electrode connected to a simulator (Grass Instruments, West Warwick, RI) in the presence of 20 mM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Because the fast excitatory postsynaptic currents in the PVN are mediated only by non-N-methylD-aspartate receptors under our recording conditions (Li et al., 2002, 2003), it is not necessary to block the N-methyl-D-aspartate receptors. The tip of the stimulating electrode was placed 200 to 600 mm away from the recorded neuron. GABAergic miniature inhibitory postsynaptic currents (mIPSCs) in labeled PVN neurons were recorded in the presence of 1 mM tetrodotoxin and 20 mM CNQX. A Naþ channel blocker, lidocaine N-ethyl bromide (QX-314, 10 mM), was included in the pipette solution to block the Naþ current in these voltageclamp experiments. Based on the optimal reversal potentials for bicucullinesensitive inhibitory postsynaptic currents (IPSCs), the IPSCs were recorded at a holding potential of 0 mV (Li et al., 2003; Li and Pan, 2005b). Cell-attached recordings were performed to examine the drug effect on the firing activity of labeled PVN neurons. In these experiments, action currents were measured in the cell-attached configuration as a capacitive current that charges the membrane (Charles and Hales, 1995). The advantage of measuring action currents in the cell-attached configuration, as compared with whole-cell recording of action potentials, is that the intracellular contents of a cell are not disturbed while the action currents are monitored. The patch was voltageclamped to 0 mV relative to the bath potential. The capacitive current that is measured when the cell fires an action potential appears as a brief spike. Data collection was terminated if the seal resistance fell below 1 GU. Signals were processed, recorded, and analyzed as described above. Drugs were applied to the recording chamber at the final concentrations. Diazepam, flumazenil, bicuculline methiodide, and CNQX were obtained from Sigma (St. Louis, MO). Tetrodotoxin and QX-314 were purchased from Alomone Labs (Jerusalem, Israel). Diazepam and flumazenil were initially dissolved in methanol (1 mg/ml) and brought to final concentrations using artificial cerebrospinal fluid on the day of the experiment. All drugs were prepared immediately before the experiments and applied to the recording chamber using syringe pumps.
2.4. Data analysis Data are presented as means S.E.M. To determine the amplitude and decay time of the eIPSCs, at least 15 consecutive eIPSCs were averaged. The amplitude of eIPSCs was measured using pClamp 8.0 analysis software (Axon Instruments). The evoked and mIPSCs were analyzed off-line with a peak detection program (MiniAnalysis; Synaptosoft Inc., Decatur, GA). For action-currents and mIPSCs, the average firing rate and amplitude were recorded and analyzed over a 3-min period during control, drug application, and recovery. The mIPSCs were detected by the fast rise time of the signal over the amplitude threshold
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above the background noise. The amplitude detection threshold was typically 5e10 pA. Based on the characteristics of IPSCs (e.g., rapid rise and slow decay), we manually excluded the event when the noise was erroneously identified as IPSCs by the program. The background noise level was typically constant throughout the recording of a single neuron. At least 100 randomly selected mIPSCs were used in analysis. The decay phases of the IPSCs were analyzed from 90e10% of the peak amplitude using MiniAnalysis (Synaptosoft Inc.). Based on the curve fitting R2 values, all mIPSCs and eIPSCs were best fit with the two-exponential function. The first exponential function of the fit line to the IPSC decay time represents t1, the fast decay time constant. The second exponential function of the fit line denotes t2, the slow decay time constant. The weighted decay time constant (tw) was calculated by the following formula: tw ¼ (t1A1 þ t2 A2)/(A1 þ A2). A1 and A2 are the amplitude of the fast and slow decay component of IPSCs. To determine if diazepam augmented the decay time of the GABAA currents independent of its effect on the IPSC amplitude, we normalized the amplitude of evoked IPSCs to the control prior to fitting the curves and measuring the decay time of evoked IPSCs. The time course of the effect of diazepam on IPSCs/firing activity was determined in our preliminary study, and the maximum effect occurred about 5 min after application of diazepam. Three min of IPSCs/firing activity were recorded and analyzed after the diazepam reached the peak effect. The cells in which there was a 20% or greater reduction in firing activity by diazepam were judged to be responsive to diazepam. If the decrease in firing activity by diazepam was less than 20%, we considered the cell to be unresponsive to diazepam. The effects of drugs on the frequency, amplitude, and decay time constant of IPSCs were determined using repeated measure ANOVA with Dunnett post hoc test. P < 0.05 was considered to be statistically significant.
3. Results Whole-cell and cell-attached recordings were obtained from a total of 102 labeled PVN neurons (n ¼ 56 rats). Five rats were excluded from the data analysis due to misplaced tracer injection. After sacrificing the rat, the brainstem was removed and the site of injection was viewed under a microscope equipped with fluorescence illumination to verify the injection and diffusion of the tracer. In rats included for this study, the injections were primarily located in the RVLM. Fig. 1 shows a representative RVLM microinjection of the Fluosphere tracer in the brainstem (Fig. 1A) and a labeled PVN neuron (Figs. 1B, C). 3.1. Effect of diazepam on eIPSCs of labeled PVN neurons To determine the effect of diazepam on GABAergic synaptic inputs to PVN-RVLM neurons, eIPSCs were recorded from
Fig. 1. Identification of retrogradely labeled paraventricular nucleus (PVN) neurons. A, Photomicrograph depicting the injection site of FluoSpheres in the rostral ventrolateral medulla (RVLM). B, Identification of a retrogradely labeled PVN-RVLM neuron in the brain slice viewed with fluorescence illumination. C, Photomicrograph of the same neuron shown in B viewed with differential interference contrast optics. Arrows indicate the labeled neuron.
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labeled neurons. The eIPSCs were evoked by electrical stimulation at a constant intensity. The eIPSCs were recorded at a holding potential of 0 mV in the presence of 20 mM CNQX. To determine the dose-dependent effect of diazepam, diazepam was applied to the recording chamber in four incremental concentrations, 10 nM, 100 nM, 1 mM, and 10 mM. The decay phase of the eIPSC was best fit by a double exponential function. Bath application of diazepam (1e10 mM) increased the peak amplitude and the fast and slow decay time constants in a dose-dependent manner (Fig. 2). Diazepam application at a concentration of 1 mM significantly increased the peak amplitude of eIPSCs from 217 15 to 316 33 pA (n ¼ 14, Fig. 2C). The tw of eIPSCs during control and application of 1 mM diazepam was 36 3 and 74 6 ms (P < 0.05), respectively. The onset latency after diazepam application was between 2e3 min in all cells tested. The amplitude and decay time of eIPSCs returned to control within 30 min after washout of diazepam. In all 11 cells tested, the eIPSCs were abolished by 20 mM bicuculline. To further determine the specific effect of diazepam on eIPSCs, the highly specific benzodiazepine binding site antagonist flumazenil (Bertz et al., 1995; Hosoi et al., 1999) was used. The effective concentration of flumazenil (10 mM) was determined in our preliminary study. In 13 cells tested, 10 mM flumazenil completely blocked diazepam (1 mM)-induced increases in the amplitude and decay time constants of eIPSCs (Figs. 3A, B). In these 13 cells, bath application of 1 mM diazepam, prior to flumazenil application, significantly increased the peak amplitude of the eIPSCs from 211 19 to 284 23 pA (Fig. 3C), the
fast decay time constant from 14 2 to 21 2 ms (Fig. 3D), and the slow decay time constant from 53 9 to 109 21 ms (Fig. 3E). In the same 13 cells, 1 mM diazepam failed to alter significantly the peak amplitude and decay time constants of eIPSCs in the presence of 10 mM flumazenil (Fig. 3). 3.2. Effect of diazepam on mIPSCs of labeled PVN neurons To examine the effect of diazepam on the IPSCs mediated by synaptic quantal release of GABA in RVLM-projecting PVN neurons, we examined the effect of diazepam on mIPSCs in 12 separate labeled PVN neurons. The mIPSCs were recorded in the presence of 1 mM tetrodotoxin and 10 mM CNQX. Diazepam (1 mM) application had no significant effect on the amplitude and inter-event interval of mIPSCs (Figs. 4AeC). The decay phase of mIPSCs was best fit by a double exponential function. Application of 1 mM diazepam significantly increased the fast and slow decay time constants (Fig. 4). The tw of mIPSCs during control and application of 1 mM diazepam was 27 3 and 72 7 ms (P < 0.05), respectively. Application of 20 mM bicuculline completely blocked the mIPSCs (data not shown). 3.3. Effect of diazepam on the firing activity of labeled PVN neurons To determine the effect of diazepam on the firing frequency of labeled PVN neurons, the firing activity was recorded using
Fig. 2. Effect of diazepam on evoked inhibitory postsynaptic currents (IPSCs) in labeled paraventricular nucleus (PVN) neurons. A, Original tracings showing the dose-response effect of diazepam on evoked IPSCs in one labeled PVN neuron. The evoked IPSCs were recorded at a holding potential of 0 mV. B, Normalized traces to control in A showing that diazepam concentration-dependently increased the decay time constant of evoked IPSCs in the same neurons. C, D, and E, Summary data showing the effect of diazepam (10 nMe10 mM, n ¼ 14 cells) on the amplitude (C), fast decay time constant (D), and slow decay time constant (E) of evoked IPSCs. Data are presented as means S.E.M. *P < 0.05 compared to the control.
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Fig. 3. Flumazenil (FLZ) blocked the diazepam (DZP)-induced increase in the amplitude and decay time constant of evoked inhibitory postsynaptic currents (IPSCs). A, Original tracings showing the effect of 1 mM diazepam on the amplitude and the decay phase of evoked IPSCs before and during bath application of 10 mM flumazenil in a labeled paraventricular nucleus (PVN) neuron. B, Normalized traces to control in A showing the effect of 1 mM diazepam and diazepam plus 10 mM flumazenil on the decay time of evoked IPSCs in the same PVN neuron. C, D, and E, Summary data showing the effect 1 mM diazepam on the amplitude (C), fast decay time constant (D), and slow decay time constant of evoked IPSCs before and during application of 10 mM flumazenil in 13 labeled PVN neurons. Data are presented as means S.E.M. *P < 0.05 compared to the control.
the cell-attached technique. The majority of labeled PVN neurons (52 of 76) had spontaneous activity. We studied 26 spontaneously firing PVN neurons for the dose-response effect of diazepam. In 15 of 26 cells, bath application of 1e10 mM diazepam significantly decreased the firing activity (Fig. 5). However, diazepam had no significant effect on the firing rate of another 11 labeled PVN neurons (Fig. 5B). After washout of diazepam, we subsequently determined the sensitivity of the above 26 cells to the GABAA receptor antagonist bicuculline. In those 15 cells that responded to diazepam, 20 mM bicuculline significantly increased the firing frequency from to 2.66 0.40 to 7.54 2.08 Hz (Fig. 5A). In contrast, in the 11 labeled cells in which diazepam had no significant effect on the firing frequency, 20 mM bicuculline failed to increase significantly the firing activity of these neurons (Fig. 5B). To ensure the specific effect of diazepam on the firing rate, in another group of 26 cells with spontaneous activity, the benzodiazepine binding site antagonist flumazenil (10 mM) was used. Fig. 6 shows the effect of 1 mM diazepam on the spontaneous firing activity of a labeled PVN neuron before and after the application of flumazenil. Prior to flumazenil application, 1 mM diazepam significantly decreased the firing frequency in 11 of 26 cells (from 3.00 0.30 to 1.42 0.32 Hz; Figs. 6A,B). In those 11 cells that responded to diazepam with a decrease in the firing, the firing frequency returned to control within 30 min after washout of diazepam. Application of 10 mM flumazenil alone had no significant effect on the firing rate (from 3.00 0.30 to 2.80 0.33 Hz). However, in the presence of 10 mM flumazenil, bath application of 1 mM diazepam failed to decrease significantly the firing activity of these 11
neurons (Fig. 6B). We also determined the sensitivity of these 11 cells to the GABAA receptor antagonist bicuculline. Bicuculline (20 mM) significantly increased the firing rate of these cells from to 2.70 0.27 to 7.00 1.36 Hz (Figs. 6A,B). In the remaining 15 of 26 cells, 1 mM diazepam had no significant effect on the firing rate (from 3.54 0.52 to 3.57 0.60 Hz). Also, application of 20 mM bicuculline failed to significantly alter the firing activity of these 15 neurons (3.65 0.54 to 5.01 0.88 Hz). 4. Discussion This is the first study determining the effect of the benzodiazepine agonist diazepam on GABAergic IPSCs and the firing activity of PVN-RVLM output neurons. We found that diazepam significantly increased the amplitude and decay time constant of evoked IPSCs and the decay time constant of mIPSCs. Furthermore, diazepam inhibited the spontaneous firing activity in bicuculline-responsive PVN neurons. The effect of diazepam on GABAergic IPSCs and firing activity was abolished by the specific benzodiazepine antagonist. These data suggest that benzodiazepines can strengthen inhibitory neurotransmission mediated by GABAA receptors. Because the PVN presympathetic neurons are critically involved in the forebrain regulation of sympathetic outflow (Allen, 2002; Martin and Haywood, 1993), our study provides new functional evidence that increased GABAergic inputs to PVN presympathetic neurons may play an important role in the inhibitory effect of benzodiazepines on the sympathetic outflow. Nevertheless, benzodiazepines could influence the sympathetic outflow by acting on brain structures other than the PVN.
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Fig. 4. Effect of diazepam on miniature inhibitory postsynaptic currents (mIPSCs) in labeled paraventricular nucleus (PVN) neurons. A, Raw tracings from a labeled PVN neuron showing mIPSCs during control, during application of 1 mM diazepam, and after washout. B & C, Cumulative probability analysis showing the effect of 1 mM diazepam on the amplitude (B) and inter-event interval (C) of the mIPSCs of the same neuron in A. D, Superimposed averages of 100 randomly selected mIPSCs during control, during application of 1 mM diazepam, and after washout. Note that diazepam significantly increased the decay times of the mIPSCs. E and F, Summary data showing the effect of 1 mM diazepam on the fast (E) and slow (F) component of decay time constants (n ¼ 12 neurons). Data presented as means S.E.M. *, P < 0.05 compared with the control.
The PVN plays an important role in maintaining the vasomotor tone and is important for blood pressure regulation. In this regard, inhibition of the PVN with the GABAA agonist muscimol lowers blood pressure and inhibits sympathetic
nerve discharges (Allen, 2002; Zahner and Pan, 2005). Benzodiazepines can enhance the GABAA receptor function and have been safely used to treat patients with anxiety disorders for many years. It has been shown that intravenous
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Fig. 5. Effect of diazepam and bicuculline (Bic) on the firing activity of labeled paraventricular nucleus (PVN) neurons. A, Summary data showing the effects of 100 nMe10 mM diazepam and 20 mM bicuculline on the firing activity of labeled PVN neurons (n ¼ 15 cells). B, Summary data showing the lack of significant effect of diazepam and 20 mM bicuculline on the firing activity of another 11 labeled PVN neurons. C, A line-scatter plot showing the firing rate of 26 individual neurons during control and application of 1 mM diazepam. Data presented as means S.E.M. *, P < 0.05 compared with the control.
administration of benzodiazepines decreases blood pressure and sympathetic nerve discharges (Kitajima et al., 2004). However, the sites and mechanisms of this action remain to be established. While the PVN sends projections to the spinal intermediolateral cell column and RVLM (Coote et al., 1998; Hardy, 2001; Shafton et al., 1998), the functional significance of the divergent pathways is not fully known. Because inactivation of the RVLM attenuates the depressor response elicited by inhibition of the PVN (Allen, 2002), we used a combination of in vivo retrograde labeling and whole-cell recording techniques in anesthetic-free brain slices to determine the mechanism of action of diazepam on RVLM-projecting PVN neurons.
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Fig. 6. Cell-attached recordings showing that flumazenil blocks the diazepaminduced decrease in the firing activity of labeled paraventricular nucleus (PVN) neurons. A, Original recordings showing the effect of 1 mM diazepam on the firing activity of a labeled PVN neuron before and after application of 10 mM flumazenil. Note that 20 mM bicuculline (Bic) caused a large increase in the firing activity. B, Summary data showing the effect of 1 mM diazepam (DZP), diazepam plus 10 mM flumazenil (FLZ), and 20 mM bicuculline on the firing activity of 11 labeled PVN neurons. Data presented as means S.E.M. *, P < 0.05 compared with the control.
Because GABA is the main inhibitory neurotransmitter in the PVN, we hypothesized that one mechanism by which benzodiazepines can suppress sympathetic outflow is through potentiation of GABAergic inhibition. In the present study, diazepam significantly increased both the amplitude and the decay time constant of the evoked IPSCs. We also observed that diazepam increased the decay time constant of mIPSCs without a significant effect on the amplitude of mIPSCs. This finding is consistent with the study showing that midazolam increases the decay time constant but not the amplitude of mIPSCs in hippocampal neurons (Poncer et al., 1996). The mIPSCs are mediated by the spontaneous quantal release of GABA and GABAA receptor activation. Since the postsynaptic
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GABAA receptor binding sites are fully saturated by each quanta released, this may explain why diazepam had no significant effect on the amplitude of mIPSCs. However, the slow binding rate of the two GABA binding sites and the rapid diffusion of GABA from the synaptic cleft means that not all the postsynaptic GABAA receptors will be activated (Farrant and Nusser, 2005). Nevertheless, diazepam causes an increase in the frequency of GABAA receptor channel opening (Study and Barker, 1981), which probably accounts for its effect on the decay time constant of mIPSCs. Although these data do not rule out the possibility that diazepam may increase the affinity of GABA for its binding site, it is possible that benzodiazepine increases the duration and frequency of channel opening of the postsynaptic GABAA receptors in PVNRVLM projection neurons. In the present study, diazepam increased both the amplitude and the decay time constant of evoked IPSCs in all labeled PVN neurons. This finding is in accord with other studies showing both the decay time and amplitude of evoked IPSCs of hippocampal neurons are augmented by diazepam (Segal and Barker, 1984; Zhang et al., 1993). On the other hand, it has been shown that midazolam only increases the decay time constant but not the amplitude of spontaneous IPSCs in hippocampal neurons (Otis and Mody, 1992). In our study on RVLM-projecting PVN neurons, diazepam increased the amplitude of evoked IPSCs but not of mIPSCs. It is possible that the majority of the quantal released GABA (mIPSCs) is bound to the GABAA receptors. However, for the evoked IPSCs, a much greater amount of GABA is released to the synaptic cleft. Importantly, release of GABA vesicles is not completely synchronous in the case of evoked IPSCs, and separate events (IPSCs) can converge and summate (Hefft and Jonas, 2005; Williams et al., 1998). Diazepam can prolong the decay time constant of each individual IPSCs associated with synchronous and asynchronous GABA release. As a result, diazepam can increase the amplitude of evoked IPSCs by prolonging the individual time constants of IPSCs. Because a two-exponential function best fit the IPSCs, it is possible that there are two individual processes occurring to rectify the inhibitory current. It has been shown that the fast and slow components of the GABAA receptor may represent two separate Cl conductance levels (Kapur et al., 1997). In our study, both the fast and the slow component of decay time constants of evoked IPSCs and mIPSCs were increased by diazepam. This suggests that the two components of the decay kinetics of GABAergic IPSCs are indifferent to diazepam sensitivity. While diazepam prolonged the decay time of GABAergic IPSCs in all labeled PVN neurons, we found unexpectedly that diazepam only selectively inhibited the firing activity of some labeled PVN neurons. Because diazepam had no significant effect on the firing activity of a subpopulation of PVN neurons, we examined the sensitivity of labeled PVN neurons to the GABAA receptor antagonist bicuculline. Bicuculline increases the firing activity of PVN neurons through disinhibition, allowing the cell to fire at an increased frequency (Li et al., 2003; Li and Pan, 2005b). In all of the cells that
responded to diazepam, bicuculline induced a larger increase in the firing frequency. By contrast, bicuculline had no significant effect on the firing activity of PVN neurons insensitive to diazepam. Thus, it appears that there is inadequate GABAAmediated tonic inhibition in those neurons that are unresponsive to diazepam. It is important to note that many GABAergic inputs to PVN neurons may be removed in this thin slice preparation. This may lead to an underestimate of the effect of diazepam on the neuronal firing in vivo. It has been shown that bicuculline methiodide has an effect on the small conductance Ca2þ-activated Kþ channels (Debarbieux et al., 1998; Khawaled et al., 1999). However, we have shown that the specific small conductance Ca2þ-activated Kþ blocker, apamin, has no significant effect on the firing activity of PVN-RVLM neurons (Li and Pan, 2005b). Thus, it is less likely that the bicuculline-induced increase in the firing activity of PVN neurons is due to its effect on small conductance Ca2þ-activated Kþ channels. To ensure that the effect of diazepam on labeled PVN neurons was mediated by benzodiazepine receptors, we used a highly specific benzodiazepine receptor antagonist, flumazenil (Bertz et al., 1995; Hosoi et al., 1999). The diazepam-induced increases in the decay of IPSCs and the decrease in the firing activity of labeled PVN neurons were abolished by flumazenil. Because the IPSCs in the PVN were completed blocked by bicuculline and the inhibitory effect of diazepam on the firing activity was limited to those PVN neurons sensitive to bicuculline, benzodiazepines likely inhibits the firing activity of PVN presympathetic neurons through augmentation of the GABAergic inputs. Additionally, since many PVN neurons also project to the pituitary, the inhibitory effect of benzodiazepines on the excitability of PVN neurons may explain their effect on hypothalamic-pituitary-adrenocortical cortex axis activity. Disturbances in the hypothalamic-pituitary-adrenocortical axis are well known in depression and anxiety in humans and animal models (Arborelius et al., 1999; Heuser et al., 2000). It has been shown that administration of benzodiazepines suppresses the abnormal hyper-secretion of cortisol that occurs in sleep disturbances and anxiety (De Boer et al., 1990). Acknowledgments This study was supported by grants HL60026 and HL04199 from the National Heart, Lung, and Blood Institute. M.R.Z. was supported by a predoctoral fellowship from the American Heart Association-Pennsylvania Affiliate. References Allen, A.M., 2002. Inhibition of the hypothalamic paraventricular nucleus in spontaneously hypertensive rats dramatically reduces sympathetic vasomotor tone. Hypertension 39, 275e280. Arborelius, L., Owens, M.J., Plotsky, P.M., Nemeroff, C.B., 1999. The role of corticotropin-releasing factor in depression and anxiety disorders. J. Endocrinol. 160, 1e12. Bertz, R.J., Reynolds, I.J., Kroboth, P.D., 1995. Effect of neuroactive steroids on [3H]flumazenil binding to the GABAA receptor complex in vitro. Neuropharmacology 34, 1169e1175.
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Benzodiazepine inhibits hypothalamic presympathetic neurons by potentiation of GABAergic synaptic input Matthew R. Zahner a, De-Pei Li b, Hui-Lin Pan a,b,* a
Department of Anesthesiology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA b Department of Anesthesiology and Pain Medicine, University of Texas, M. D. Anderson Cancer Center, 1400 Holcombe Blvd., Unit 409, Houston, TX 77030-4009, USA Received 20 July 2006; received in revised form 15 August 2006; accepted 17 August 2006
Abstract Presympathetic neurons in the paraventricular nucleus (PVN) of the hypothalamus receive inputs from g-aminobutyric acid (GABA)containing neurons, which regulate sympathetic outflow and cardiovascular function. Benzodiazepines can decrease blood pressure and sympathetic nerve activity when used for induction of anesthesia, but the sites and mechanisms of action are uncertain. In this study, we determined the effect of the benzodiazepine agonist diazepam on GABAergic inhibitory postsynaptic currents (IPSCs) and the firing activity of rostral ventrolateral medulla (RVLM)-projecting PVN neurons. RVLM-projecting PVN neurons were retrogradely labeled by fluorescent microspheres injected into the RVLM in rats. Whole-cell and cell-attached recordings were performed on labeled PVN neurons in the hypothalamic brain slice. Bath application of 1e10 mM diazepam significantly increased the decay time constants of the GABAergic miniature IPSCs and evoked IPSCs in a dose-dependent manner. Also, diazepam significantly increased the amplitude of evoked IPSCs but not of miniature IPSCs. Pretreatment with the benzodiazepine antagonist flumazenil completely blocked the diazepam-induced increases in the amplitude and decay time constants of the evoked IPSCs. Furthermore, diazepam significantly decreased the firing activity of PVN-RVLM neurons that responded with increased firing to the GABAA receptor antagonist bicuculline. In contrast, diazepam had no significant effect on the firing activity of bicuculline-unresponsive PVN-RVLM neurons. This study provides new information that the benzodiazepine suppresses the firing activity of PVN presympathetic neurons by potentiation of GABAergic inputs. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Autonomic system; Sympathetic nervous activity; Hypothalamus; Synaptic transmission
1. Introduction The hypothalamic paraventricular nucleus (PVN) is an integrating center for regulation of neuroendocrine, cardiovascular, and other physiological functions. The PVN is one of the five major sympathetic premotor cell groups in the brain (Strack et al., 1989). Presympathetic neurons in the PVN send projections to the rostral ventrolateral medulla (RVLM) as well as * Corresponding author. Department of Anesthesiology and Pain Medicine, University of Texas, M. D. Anderson Cancer Center, 1400 Holcombe Blvd., Unit 409, Houston, TX 77030-4009, USA. Tel.: þ1 713 563 5838; fax: þ1 713 794 4590. E-mail address: [email protected] (H.-L. Pan). 0028-3908/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2006.08.024
the intermediolateral cell column of the spinal cord to regulate sympathetic outflow (Hardy, 2001; Pyner and Coote, 2000; Shafton et al., 1998). The PVN tonically controls sympathetic activity and blood pressure and is an important source of excitatory drive for sympathetic vasomotor tone. For example, activation of the PVN induces a profound increase in sympathetic nerve activity and blood pressure in anesthetized and conscious rats (Kannan et al., 1989; Martin and Haywood, 1993). Conversely, inhibition of the PVN suppresses sympathetic discharges and lowers basal blood pressure (Allen, 2002; Zahner and Pan, 2005). Furthermore, aberrant PVN neuronal activity has been linked to several cardiovascular diseases, such as hypertension and heart failure (Allen, 2002; Li and Pan, 2005b; Zhang et al., 2002). Modification of the GABAergic synaptic
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input to vasomotor neurons in the central nervous system represents a potential target to attenuate the sympathetic outflow. g-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the PVN (Decavel and Van den Pol, 1990). GABA receptors are categorized into two major types, the ionotropic GABAA and GABAC receptors and the metabotropic GABAB receptors. GABAA receptors gate a Cl ionophore and have binding sites for benzodiazepines and barbiturates, which potentiate the inhibitory response to GABA. GABAA receptors are assembled with several polypeptide units (a1e6, b1e4, g1e3, d, r1e2) that combine to form a pentameric chloride channel (Hevers and Luddens, 1998; Macdonald and Olsen, 1994). Activation of the GABAA receptor induces a Cl conductance and causes the membrane potential to move towards the Cl reversal potential, thereby usually suppressing the firing activity of mature neurons (Choh et al., 1977; Macdonald and Barker, 1978; Macdonald and Olsen, 1994). It has been shown that benzodiazepines act at GABAA receptors by binding to a specific allosteric binding site separate from the GABA binding site on the GABAA receptor complex (Mohler and Okada, 1977). Also, benzodiazepines potentiate the GABA-induced neuronal inhibition by increasing the affinity of GABA for the GABAA receptor and increasing the frequency of channel opening, thereby increasing the Cl current (Rogers et al., 1994). Thus, benzodiazepines can shift the GABA concentration-response curve to the left without increasing the maximum current evoked by GABA. Systemic administration of the benzodiazepine agonist diazepam can reduce blood pressure and decreases sympathetic outflow in humans (Kitajima et al., 2004). Furthermore, benzodiazepines reduce the sympathetic tone when they are used for induction of anesthesia in animals and humans (Flacke et al., 1985; Marty et al., 1986). Diazepam also consistently attenuates the pressor response induced by stress and stimulation of the hypothalamus in rats (Conahan and Vogel, 1986; Kawasaki et al., 1979). However, the mechanisms for this inhibitory action of benzodiazepines in the hypothalamus on the sympathetic outflow are not fully known. The PVN is composed of heterogeneous subpopulations of neurons important for the regulation of different autonomic and neuroendocrine functions (Allen, 2002; Swanson and Sawchenko, 1983; Zhang et al., 2002). Therefore, using in vivo retrograde labeling and in vitro brain slice recordings, we determined the effect of the benzodiazepine agonist on the GABAergic input and firing activity of RVLM-projecting PVN neurons. 2. Materials and methods 2.1. Retrograde labeling of RVLM-projecting PVN neurons Male SpragueeDawley rats (200e225 g; Harlan, Indianapolis, IN) were used in this study. The surgical preparations and experimental protocols were approved by the Animal Care and Use Committee of the Pennsylvania State University College of Medicine and conformed to the National Institutes of Health guidelines on the ethical use of animals. All efforts were made to minimize both the suffering and number of animals used. Rats were anesthetized by subcutaneous injection of a ketamine (70 mg/kg) and xylazine (6 mg/ kg) mixture, and the head of the rat was placed in a stereotaxic apparatus.
A burr hole (4 mm in diameter) was made in the occipital bone bilaterally according to the following coordinates (lambda): 2.5e3.0 mm caudal, 1.8e2.2 mm lateral, and 7.5e8.0 mm deep from the surface of the cortex. A rhodamine-labeled fluorescent microsphere suspension (FluoSpheres, 0.04 mm; Molecular Probes, Eugene, OR) was ejected (Nanojector II; Drummond Scientific Co., Broomall, PA) bilaterally into the region of the RVLM (Li and Pan, 2005a). The pipette was positioned with a micromanipulator and the injection of 50 nl FluoSpheres was monitored through a surgical microscope. After injection, the skin was sutured and the wound was closed. Animals were returned to their cages for 3e5 days, which is sufficient time to allow the retrograde tracer to be transported to the PVN.
2.2. Slice preparation Three to 5 days after tracer injection, the rats were rapidly decapitated under halothane anesthesia. The brain was quickly removed and placed in icecold artificial cerebrospinal fluid saturated with 95% O2 and 5% CO2. A tissue block containing the hypothalamus was cut from the brain and glued onto the stage of the vibratome (Technical Product International, St. Louis, MO), as described previously (Li et al., 2002; Li and Pan, 2001). Coronal hypothalamic slices (300 mm in thickness) containing the PVN were cut from the tissue block in ice-cold artificial cerebrospinal fluid. After sectioning, the slices were pre-incubated in the artificial cerebrospinal fluid, which was continuously gassed with 95% O2 and 5% CO2 at 34 C for at least 1 h before being used for the recording. For recordings, a slice was transferred to a submersiontype recording chamber continuously perfused with artificial cerebrospinal fluid 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 removed after sacrificing the rat and stored in 10% formaldehyde (Li and Pan, 2005a). Brainstems were sectioned (50 mm) at the injection level. The brainstem slices and the injection sites of FluoSpheres were identified under a microscope equipped with fluorescence illumination. The diffusion of the tracer was generally limited to the RVLM, and the diffusion size of FluoSpheres around the site of injection was about 0.5 mm in diameter. Rats were excluded if the injection site was not located within the RVLM.
2.3. Electrophysiological recordings Whole-cell voltage-clamp recordings of postsynaptic currents were performed in a radio frequency-shielded room, as we described previously (Li et al., 2002; Li and Pan, 2005b). The recording pipettes were pulled from borosilicate capillaries (1.2 mm OD, 0.86 mm ID; World Precision Instruments, Sarasota, FL) using a micropipette puller (P-97; Sutter Instrument Company, Novato, CA). The resistance of the pipette was 4 to 6 MU 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, and 4.0 mM adenosine triphosphate-Mg adjusted to pH 7.25 with 1 M KOH (290e320 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 Ushaped 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 this perfusion rate. Whole-cell recordings from labeled PVN neurons were made under visual control using a combination of fluorescence 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 (Li and Pan, 2005b). The labeled neurons were identified with the aid of fluorescence illumination and differential interference contrast optics on an upright microscope. A tight GU seal was obtained on the labeled neuron identified. Recordings began w5 min after the whole-cell access or cell-attached seal was obtained and the current reached a steady state. Recordings were performed using an Axopatch 200B or Multiclamp 700A amplifier (Axon
M.R. Zahner et al. / Neuropharmacology 52 (2007) 467e475 Instruments Inc., Union City, CA). Signals were filtered at 1 to 2 kHz and 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 15e20 mV) changed more than 15% during the recording. To record the evoked inhibitory postsynaptic currents (eIPSCs), synaptic currents were evoked at a sub-maximal stimulation intensity by electrical stimulation (0.1 ms, 0.3e0.8 mA, and 0.1 Hz) through a bipolar tungsten electrode connected to a simulator (Grass Instruments, West Warwick, RI) in the presence of 20 mM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Because the fast excitatory postsynaptic currents in the PVN are mediated only by non-N-methylD-aspartate receptors under our recording conditions (Li et al., 2002, 2003), it is not necessary to block the N-methyl-D-aspartate receptors. The tip of the stimulating electrode was placed 200 to 600 mm away from the recorded neuron. GABAergic miniature inhibitory postsynaptic currents (mIPSCs) in labeled PVN neurons were recorded in the presence of 1 mM tetrodotoxin and 20 mM CNQX. A Naþ channel blocker, lidocaine N-ethyl bromide (QX-314, 10 mM), was included in the pipette solution to block the Naþ current in these voltageclamp experiments. Based on the optimal reversal potentials for bicucullinesensitive inhibitory postsynaptic currents (IPSCs), the IPSCs were recorded at a holding potential of 0 mV (Li et al., 2003; Li and Pan, 2005b). Cell-attached recordings were performed to examine the drug effect on the firing activity of labeled PVN neurons. In these experiments, action currents were measured in the cell-attached configuration as a capacitive current that charges the membrane (Charles and Hales, 1995). The advantage of measuring action currents in the cell-attached configuration, as compared with whole-cell recording of action potentials, is that the intracellular contents of a cell are not disturbed while the action currents are monitored. The patch was voltageclamped to 0 mV relative to the bath potential. The capacitive current that is measured when the cell fires an action potential appears as a brief spike. Data collection was terminated if the seal resistance fell below 1 GU. Signals were processed, recorded, and analyzed as described above. Drugs were applied to the recording chamber at the final concentrations. Diazepam, flumazenil, bicuculline methiodide, and CNQX were obtained from Sigma (St. Louis, MO). Tetrodotoxin and QX-314 were purchased from Alomone Labs (Jerusalem, Israel). Diazepam and flumazenil were initially dissolved in methanol (1 mg/ml) and brought to final concentrations using artificial cerebrospinal fluid on the day of the experiment. All drugs were prepared immediately before the experiments and applied to the recording chamber using syringe pumps.
2.4. Data analysis Data are presented as means S.E.M. To determine the amplitude and decay time of the eIPSCs, at least 15 consecutive eIPSCs were averaged. The amplitude of eIPSCs was measured using pClamp 8.0 analysis software (Axon Instruments). The evoked and mIPSCs were analyzed off-line with a peak detection program (MiniAnalysis; Synaptosoft Inc., Decatur, GA). For action-currents and mIPSCs, the average firing rate and amplitude were recorded and analyzed over a 3-min period during control, drug application, and recovery. The mIPSCs were detected by the fast rise time of the signal over the amplitude threshold
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above the background noise. The amplitude detection threshold was typically 5e10 pA. Based on the characteristics of IPSCs (e.g., rapid rise and slow decay), we manually excluded the event when the noise was erroneously identified as IPSCs by the program. The background noise level was typically constant throughout the recording of a single neuron. At least 100 randomly selected mIPSCs were used in analysis. The decay phases of the IPSCs were analyzed from 90e10% of the peak amplitude using MiniAnalysis (Synaptosoft Inc.). Based on the curve fitting R2 values, all mIPSCs and eIPSCs were best fit with the two-exponential function. The first exponential function of the fit line to the IPSC decay time represents t1, the fast decay time constant. The second exponential function of the fit line denotes t2, the slow decay time constant. The weighted decay time constant (tw) was calculated by the following formula: tw ¼ (t1A1 þ t2 A2)/(A1 þ A2). A1 and A2 are the amplitude of the fast and slow decay component of IPSCs. To determine if diazepam augmented the decay time of the GABAA currents independent of its effect on the IPSC amplitude, we normalized the amplitude of evoked IPSCs to the control prior to fitting the curves and measuring the decay time of evoked IPSCs. The time course of the effect of diazepam on IPSCs/firing activity was determined in our preliminary study, and the maximum effect occurred about 5 min after application of diazepam. Three min of IPSCs/firing activity were recorded and analyzed after the diazepam reached the peak effect. The cells in which there was a 20% or greater reduction in firing activity by diazepam were judged to be responsive to diazepam. If the decrease in firing activity by diazepam was less than 20%, we considered the cell to be unresponsive to diazepam. The effects of drugs on the frequency, amplitude, and decay time constant of IPSCs were determined using repeated measure ANOVA with Dunnett post hoc test. P < 0.05 was considered to be statistically significant.
3. Results Whole-cell and cell-attached recordings were obtained from a total of 102 labeled PVN neurons (n ¼ 56 rats). Five rats were excluded from the data analysis due to misplaced tracer injection. After sacrificing the rat, the brainstem was removed and the site of injection was viewed under a microscope equipped with fluorescence illumination to verify the injection and diffusion of the tracer. In rats included for this study, the injections were primarily located in the RVLM. Fig. 1 shows a representative RVLM microinjection of the Fluosphere tracer in the brainstem (Fig. 1A) and a labeled PVN neuron (Figs. 1B, C). 3.1. Effect of diazepam on eIPSCs of labeled PVN neurons To determine the effect of diazepam on GABAergic synaptic inputs to PVN-RVLM neurons, eIPSCs were recorded from
Fig. 1. Identification of retrogradely labeled paraventricular nucleus (PVN) neurons. A, Photomicrograph depicting the injection site of FluoSpheres in the rostral ventrolateral medulla (RVLM). B, Identification of a retrogradely labeled PVN-RVLM neuron in the brain slice viewed with fluorescence illumination. C, Photomicrograph of the same neuron shown in B viewed with differential interference contrast optics. Arrows indicate the labeled neuron.
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labeled neurons. The eIPSCs were evoked by electrical stimulation at a constant intensity. The eIPSCs were recorded at a holding potential of 0 mV in the presence of 20 mM CNQX. To determine the dose-dependent effect of diazepam, diazepam was applied to the recording chamber in four incremental concentrations, 10 nM, 100 nM, 1 mM, and 10 mM. The decay phase of the eIPSC was best fit by a double exponential function. Bath application of diazepam (1e10 mM) increased the peak amplitude and the fast and slow decay time constants in a dose-dependent manner (Fig. 2). Diazepam application at a concentration of 1 mM significantly increased the peak amplitude of eIPSCs from 217 15 to 316 33 pA (n ¼ 14, Fig. 2C). The tw of eIPSCs during control and application of 1 mM diazepam was 36 3 and 74 6 ms (P < 0.05), respectively. The onset latency after diazepam application was between 2e3 min in all cells tested. The amplitude and decay time of eIPSCs returned to control within 30 min after washout of diazepam. In all 11 cells tested, the eIPSCs were abolished by 20 mM bicuculline. To further determine the specific effect of diazepam on eIPSCs, the highly specific benzodiazepine binding site antagonist flumazenil (Bertz et al., 1995; Hosoi et al., 1999) was used. The effective concentration of flumazenil (10 mM) was determined in our preliminary study. In 13 cells tested, 10 mM flumazenil completely blocked diazepam (1 mM)-induced increases in the amplitude and decay time constants of eIPSCs (Figs. 3A, B). In these 13 cells, bath application of 1 mM diazepam, prior to flumazenil application, significantly increased the peak amplitude of the eIPSCs from 211 19 to 284 23 pA (Fig. 3C), the
fast decay time constant from 14 2 to 21 2 ms (Fig. 3D), and the slow decay time constant from 53 9 to 109 21 ms (Fig. 3E). In the same 13 cells, 1 mM diazepam failed to alter significantly the peak amplitude and decay time constants of eIPSCs in the presence of 10 mM flumazenil (Fig. 3). 3.2. Effect of diazepam on mIPSCs of labeled PVN neurons To examine the effect of diazepam on the IPSCs mediated by synaptic quantal release of GABA in RVLM-projecting PVN neurons, we examined the effect of diazepam on mIPSCs in 12 separate labeled PVN neurons. The mIPSCs were recorded in the presence of 1 mM tetrodotoxin and 10 mM CNQX. Diazepam (1 mM) application had no significant effect on the amplitude and inter-event interval of mIPSCs (Figs. 4AeC). The decay phase of mIPSCs was best fit by a double exponential function. Application of 1 mM diazepam significantly increased the fast and slow decay time constants (Fig. 4). The tw of mIPSCs during control and application of 1 mM diazepam was 27 3 and 72 7 ms (P < 0.05), respectively. Application of 20 mM bicuculline completely blocked the mIPSCs (data not shown). 3.3. Effect of diazepam on the firing activity of labeled PVN neurons To determine the effect of diazepam on the firing frequency of labeled PVN neurons, the firing activity was recorded using
Fig. 2. Effect of diazepam on evoked inhibitory postsynaptic currents (IPSCs) in labeled paraventricular nucleus (PVN) neurons. A, Original tracings showing the dose-response effect of diazepam on evoked IPSCs in one labeled PVN neuron. The evoked IPSCs were recorded at a holding potential of 0 mV. B, Normalized traces to control in A showing that diazepam concentration-dependently increased the decay time constant of evoked IPSCs in the same neurons. C, D, and E, Summary data showing the effect of diazepam (10 nMe10 mM, n ¼ 14 cells) on the amplitude (C), fast decay time constant (D), and slow decay time constant (E) of evoked IPSCs. Data are presented as means S.E.M. *P < 0.05 compared to the control.
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Fig. 3. Flumazenil (FLZ) blocked the diazepam (DZP)-induced increase in the amplitude and decay time constant of evoked inhibitory postsynaptic currents (IPSCs). A, Original tracings showing the effect of 1 mM diazepam on the amplitude and the decay phase of evoked IPSCs before and during bath application of 10 mM flumazenil in a labeled paraventricular nucleus (PVN) neuron. B, Normalized traces to control in A showing the effect of 1 mM diazepam and diazepam plus 10 mM flumazenil on the decay time of evoked IPSCs in the same PVN neuron. C, D, and E, Summary data showing the effect 1 mM diazepam on the amplitude (C), fast decay time constant (D), and slow decay time constant of evoked IPSCs before and during application of 10 mM flumazenil in 13 labeled PVN neurons. Data are presented as means S.E.M. *P < 0.05 compared to the control.
the cell-attached technique. The majority of labeled PVN neurons (52 of 76) had spontaneous activity. We studied 26 spontaneously firing PVN neurons for the dose-response effect of diazepam. In 15 of 26 cells, bath application of 1e10 mM diazepam significantly decreased the firing activity (Fig. 5). However, diazepam had no significant effect on the firing rate of another 11 labeled PVN neurons (Fig. 5B). After washout of diazepam, we subsequently determined the sensitivity of the above 26 cells to the GABAA receptor antagonist bicuculline. In those 15 cells that responded to diazepam, 20 mM bicuculline significantly increased the firing frequency from to 2.66 0.40 to 7.54 2.08 Hz (Fig. 5A). In contrast, in the 11 labeled cells in which diazepam had no significant effect on the firing frequency, 20 mM bicuculline failed to increase significantly the firing activity of these neurons (Fig. 5B). To ensure the specific effect of diazepam on the firing rate, in another group of 26 cells with spontaneous activity, the benzodiazepine binding site antagonist flumazenil (10 mM) was used. Fig. 6 shows the effect of 1 mM diazepam on the spontaneous firing activity of a labeled PVN neuron before and after the application of flumazenil. Prior to flumazenil application, 1 mM diazepam significantly decreased the firing frequency in 11 of 26 cells (from 3.00 0.30 to 1.42 0.32 Hz; Figs. 6A,B). In those 11 cells that responded to diazepam with a decrease in the firing, the firing frequency returned to control within 30 min after washout of diazepam. Application of 10 mM flumazenil alone had no significant effect on the firing rate (from 3.00 0.30 to 2.80 0.33 Hz). However, in the presence of 10 mM flumazenil, bath application of 1 mM diazepam failed to decrease significantly the firing activity of these 11
neurons (Fig. 6B). We also determined the sensitivity of these 11 cells to the GABAA receptor antagonist bicuculline. Bicuculline (20 mM) significantly increased the firing rate of these cells from to 2.70 0.27 to 7.00 1.36 Hz (Figs. 6A,B). In the remaining 15 of 26 cells, 1 mM diazepam had no significant effect on the firing rate (from 3.54 0.52 to 3.57 0.60 Hz). Also, application of 20 mM bicuculline failed to significantly alter the firing activity of these 15 neurons (3.65 0.54 to 5.01 0.88 Hz). 4. Discussion This is the first study determining the effect of the benzodiazepine agonist diazepam on GABAergic IPSCs and the firing activity of PVN-RVLM output neurons. We found that diazepam significantly increased the amplitude and decay time constant of evoked IPSCs and the decay time constant of mIPSCs. Furthermore, diazepam inhibited the spontaneous firing activity in bicuculline-responsive PVN neurons. The effect of diazepam on GABAergic IPSCs and firing activity was abolished by the specific benzodiazepine antagonist. These data suggest that benzodiazepines can strengthen inhibitory neurotransmission mediated by GABAA receptors. Because the PVN presympathetic neurons are critically involved in the forebrain regulation of sympathetic outflow (Allen, 2002; Martin and Haywood, 1993), our study provides new functional evidence that increased GABAergic inputs to PVN presympathetic neurons may play an important role in the inhibitory effect of benzodiazepines on the sympathetic outflow. Nevertheless, benzodiazepines could influence the sympathetic outflow by acting on brain structures other than the PVN.
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Fig. 4. Effect of diazepam on miniature inhibitory postsynaptic currents (mIPSCs) in labeled paraventricular nucleus (PVN) neurons. A, Raw tracings from a labeled PVN neuron showing mIPSCs during control, during application of 1 mM diazepam, and after washout. B & C, Cumulative probability analysis showing the effect of 1 mM diazepam on the amplitude (B) and inter-event interval (C) of the mIPSCs of the same neuron in A. D, Superimposed averages of 100 randomly selected mIPSCs during control, during application of 1 mM diazepam, and after washout. Note that diazepam significantly increased the decay times of the mIPSCs. E and F, Summary data showing the effect of 1 mM diazepam on the fast (E) and slow (F) component of decay time constants (n ¼ 12 neurons). Data presented as means S.E.M. *, P < 0.05 compared with the control.
The PVN plays an important role in maintaining the vasomotor tone and is important for blood pressure regulation. In this regard, inhibition of the PVN with the GABAA agonist muscimol lowers blood pressure and inhibits sympathetic
nerve discharges (Allen, 2002; Zahner and Pan, 2005). Benzodiazepines can enhance the GABAA receptor function and have been safely used to treat patients with anxiety disorders for many years. It has been shown that intravenous
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Fig. 5. Effect of diazepam and bicuculline (Bic) on the firing activity of labeled paraventricular nucleus (PVN) neurons. A, Summary data showing the effects of 100 nMe10 mM diazepam and 20 mM bicuculline on the firing activity of labeled PVN neurons (n ¼ 15 cells). B, Summary data showing the lack of significant effect of diazepam and 20 mM bicuculline on the firing activity of another 11 labeled PVN neurons. C, A line-scatter plot showing the firing rate of 26 individual neurons during control and application of 1 mM diazepam. Data presented as means S.E.M. *, P < 0.05 compared with the control.
administration of benzodiazepines decreases blood pressure and sympathetic nerve discharges (Kitajima et al., 2004). However, the sites and mechanisms of this action remain to be established. While the PVN sends projections to the spinal intermediolateral cell column and RVLM (Coote et al., 1998; Hardy, 2001; Shafton et al., 1998), the functional significance of the divergent pathways is not fully known. Because inactivation of the RVLM attenuates the depressor response elicited by inhibition of the PVN (Allen, 2002), we used a combination of in vivo retrograde labeling and whole-cell recording techniques in anesthetic-free brain slices to determine the mechanism of action of diazepam on RVLM-projecting PVN neurons.
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Fig. 6. Cell-attached recordings showing that flumazenil blocks the diazepaminduced decrease in the firing activity of labeled paraventricular nucleus (PVN) neurons. A, Original recordings showing the effect of 1 mM diazepam on the firing activity of a labeled PVN neuron before and after application of 10 mM flumazenil. Note that 20 mM bicuculline (Bic) caused a large increase in the firing activity. B, Summary data showing the effect of 1 mM diazepam (DZP), diazepam plus 10 mM flumazenil (FLZ), and 20 mM bicuculline on the firing activity of 11 labeled PVN neurons. Data presented as means S.E.M. *, P < 0.05 compared with the control.
Because GABA is the main inhibitory neurotransmitter in the PVN, we hypothesized that one mechanism by which benzodiazepines can suppress sympathetic outflow is through potentiation of GABAergic inhibition. In the present study, diazepam significantly increased both the amplitude and the decay time constant of the evoked IPSCs. We also observed that diazepam increased the decay time constant of mIPSCs without a significant effect on the amplitude of mIPSCs. This finding is consistent with the study showing that midazolam increases the decay time constant but not the amplitude of mIPSCs in hippocampal neurons (Poncer et al., 1996). The mIPSCs are mediated by the spontaneous quantal release of GABA and GABAA receptor activation. Since the postsynaptic
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GABAA receptor binding sites are fully saturated by each quanta released, this may explain why diazepam had no significant effect on the amplitude of mIPSCs. However, the slow binding rate of the two GABA binding sites and the rapid diffusion of GABA from the synaptic cleft means that not all the postsynaptic GABAA receptors will be activated (Farrant and Nusser, 2005). Nevertheless, diazepam causes an increase in the frequency of GABAA receptor channel opening (Study and Barker, 1981), which probably accounts for its effect on the decay time constant of mIPSCs. Although these data do not rule out the possibility that diazepam may increase the affinity of GABA for its binding site, it is possible that benzodiazepine increases the duration and frequency of channel opening of the postsynaptic GABAA receptors in PVNRVLM projection neurons. In the present study, diazepam increased both the amplitude and the decay time constant of evoked IPSCs in all labeled PVN neurons. This finding is in accord with other studies showing both the decay time and amplitude of evoked IPSCs of hippocampal neurons are augmented by diazepam (Segal and Barker, 1984; Zhang et al., 1993). On the other hand, it has been shown that midazolam only increases the decay time constant but not the amplitude of spontaneous IPSCs in hippocampal neurons (Otis and Mody, 1992). In our study on RVLM-projecting PVN neurons, diazepam increased the amplitude of evoked IPSCs but not of mIPSCs. It is possible that the majority of the quantal released GABA (mIPSCs) is bound to the GABAA receptors. However, for the evoked IPSCs, a much greater amount of GABA is released to the synaptic cleft. Importantly, release of GABA vesicles is not completely synchronous in the case of evoked IPSCs, and separate events (IPSCs) can converge and summate (Hefft and Jonas, 2005; Williams et al., 1998). Diazepam can prolong the decay time constant of each individual IPSCs associated with synchronous and asynchronous GABA release. As a result, diazepam can increase the amplitude of evoked IPSCs by prolonging the individual time constants of IPSCs. Because a two-exponential function best fit the IPSCs, it is possible that there are two individual processes occurring to rectify the inhibitory current. It has been shown that the fast and slow components of the GABAA receptor may represent two separate Cl conductance levels (Kapur et al., 1997). In our study, both the fast and the slow component of decay time constants of evoked IPSCs and mIPSCs were increased by diazepam. This suggests that the two components of the decay kinetics of GABAergic IPSCs are indifferent to diazepam sensitivity. While diazepam prolonged the decay time of GABAergic IPSCs in all labeled PVN neurons, we found unexpectedly that diazepam only selectively inhibited the firing activity of some labeled PVN neurons. Because diazepam had no significant effect on the firing activity of a subpopulation of PVN neurons, we examined the sensitivity of labeled PVN neurons to the GABAA receptor antagonist bicuculline. Bicuculline increases the firing activity of PVN neurons through disinhibition, allowing the cell to fire at an increased frequency (Li et al., 2003; Li and Pan, 2005b). In all of the cells that
responded to diazepam, bicuculline induced a larger increase in the firing frequency. By contrast, bicuculline had no significant effect on the firing activity of PVN neurons insensitive to diazepam. Thus, it appears that there is inadequate GABAAmediated tonic inhibition in those neurons that are unresponsive to diazepam. It is important to note that many GABAergic inputs to PVN neurons may be removed in this thin slice preparation. This may lead to an underestimate of the effect of diazepam on the neuronal firing in vivo. It has been shown that bicuculline methiodide has an effect on the small conductance Ca2þ-activated Kþ channels (Debarbieux et al., 1998; Khawaled et al., 1999). However, we have shown that the specific small conductance Ca2þ-activated Kþ blocker, apamin, has no significant effect on the firing activity of PVN-RVLM neurons (Li and Pan, 2005b). Thus, it is less likely that the bicuculline-induced increase in the firing activity of PVN neurons is due to its effect on small conductance Ca2þ-activated Kþ channels. To ensure that the effect of diazepam on labeled PVN neurons was mediated by benzodiazepine receptors, we used a highly specific benzodiazepine receptor antagonist, flumazenil (Bertz et al., 1995; Hosoi et al., 1999). The diazepam-induced increases in the decay of IPSCs and the decrease in the firing activity of labeled PVN neurons were abolished by flumazenil. Because the IPSCs in the PVN were completed blocked by bicuculline and the inhibitory effect of diazepam on the firing activity was limited to those PVN neurons sensitive to bicuculline, benzodiazepines likely inhibits the firing activity of PVN presympathetic neurons through augmentation of the GABAergic inputs. Additionally, since many PVN neurons also project to the pituitary, the inhibitory effect of benzodiazepines on the excitability of PVN neurons may explain their effect on hypothalamic-pituitary-adrenocortical cortex axis activity. Disturbances in the hypothalamic-pituitary-adrenocortical axis are well known in depression and anxiety in humans and animal models (Arborelius et al., 1999; Heuser et al., 2000). It has been shown that administration of benzodiazepines suppresses the abnormal hyper-secretion of cortisol that occurs in sleep disturbances and anxiety (De Boer et al., 1990). Acknowledgments This study was supported by grants HL60026 and HL04199 from the National Heart, Lung, and Blood Institute. M.R.Z. was supported by a predoctoral fellowship from the American Heart Association-Pennsylvania Affiliate. References Allen, A.M., 2002. Inhibition of the hypothalamic paraventricular nucleus in spontaneously hypertensive rats dramatically reduces sympathetic vasomotor tone. Hypertension 39, 275e280. Arborelius, L., Owens, M.J., Plotsky, P.M., Nemeroff, C.B., 1999. The role of corticotropin-releasing factor in depression and anxiety disorders. J. Endocrinol. 160, 1e12. Bertz, R.J., Reynolds, I.J., Kroboth, P.D., 1995. Effect of neuroactive steroids on [3H]flumazenil binding to the GABAA receptor complex in vitro. Neuropharmacology 34, 1169e1175.
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