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NEUROPHARMACOLOGY

Presynaptic ₁ Adrenergic Receptors Differentially Regulate Synaptic Glutamate and GABA Release to Hypothalamic Presympathetic Neurons

Departments of Anesthesiology (Q.C., D.-P.L., H.-L.P.) and Neural and Behavioral Sciences (H.-L.P.), Pennsylvania State University College of Medicine, Hershey, Pennsylvania

Received August 28, 2005; accepted October 24, 2005.

	Abstract

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The hypothalamic paraventricular nucleus (PVN) neurons that project to the spinal intermediolateral cell column and brainstem are important for the control of sympathetic outflow. Stimulation of ₁ adrenergic receptors in the PVN increases sympathetic outflow, but the cellular mechanisms remain unclear. In this study, we determined the role of ₁ adrenergic receptors in the regulation of glutamatergic and GABAergic synaptic inputs to spinally projecting PVN neurons. Whole-cell and cell-attached patch-clamp recordings were performed on retrogradely labeled PVN-spinal neurons in rat brain slices. Bath application of 10 to 100 µM phenylephrine, an ₁ adrenergic receptor agonist, significantly increased the frequency of spontaneous excitatory postsynaptic currents in a concentration-dependent manner. This effect was blocked by the ₁adrenergic receptor antagonists prazosin or corynanthine. Phenylephrine also significantly increased the frequency of miniature excitatory postsynaptic currents (mEPSCs) but not the amplitude and decay constant of mEPSCs. Furthermore, activation of ₁ adrenergic receptors with phenylephrine or cirazoline significantly decreased the frequency of spontaneous inhibitory postsynaptic currents and miniature inhibitory postsynaptic currents, and this effect also was blocked by corynanthine. In addition, 50 µM phenylephrine significantly increased the firing rate of 13 labeled PVN neurons from 3.16 ± 0.42 to 5.83 ± 0.65 Hz. However, phenylephrine failed to increase the firing of most labeled PVN neurons in the presence of GABA_A and ionotropic glutamate receptor antagonists. Thus, these data suggest that activation of ₁adrenergic receptors increases the excitability of PVN presympathetic neurons primarily through augmentation of glutamatergic tone and attenuation of GABAergic inputs.

The tonic activity of PVN neurons is regulated by various excitatory and inhibitory neurotransmitters and neuromodulators, including glutamate, GABA, angiotensin II, nitric oxide, and catecholamines (Decavel and van den Pol, 1992; Tasker and Dudek, 1993; Li et al., 2002, 2003). A majority of GABAergic and glutamatergic inputs to the PVN neurons are derived from the suprachiasmatic nucleus, subfornical organ, and other brain regions (Bains and Ferguson, 1995; Cui et al., 2001). In addition, there exist considerable GABAergic and glutamatergic interneurons in the PVN (Decavel and van den Pol, 1992; Tasker and Dudek, 1993). Many important neuromodulators, such as nitric oxide and angiotensin II, can regulate PVN presympathetic neurons by indirect effects on GABAergic transmission (Li et al., 2002, 2003).

The PVN neurons receive dense noradrenergic projections from the brainstem, especially A1, A2, and A6 (locus coeruleus) noradrenergic cell groups (Swanson and Sawchenko, 1983; Cunningham and Sawchenko, 1988) and adrenergic inputs from the C1, C2, and C3 neurons in the rostral medulla (Cunningham et al., 1990). Furthermore, both ₁ and ₂ adrenergic receptors are present in the PVN (Cummings and Seybold, 1988). Microinjection of norepinephrine into the PVN raises the blood pressure in rats, and this effect is because of, in part, the release of arginine vasopressin (Harland et al., 1989). We recently found that stimulation of ₂ adrenergic receptors attenuates GABAergic, but not glutamatergic, inputs to spinally projecting PVN neurons (Li et al., 2005). Because the function of ₁ adrenergic receptors has not been specifically studied in PVN output neurons with known projections, the role of ₁ receptors in the regulation of the excitability of PVN presympathetic neurons remains unclear. In this study, using in vivo retrograde labeling and in vitro brain slice recordings, we determined the role of ₁ adrenergic receptors in the control of glutamatergic and GABAergic inputs to spinally projecting PVN neurons.

	Materials and Methods

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Retrograde Labeling of Spinally Projecting PVN Neurons. Male Sprague-Dawley rats (3-5 weeks old; Harlan, Indianapolis, IN) were used for 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. The dorsal laminectomy was performed at the T₂-T₄ level of the spinal cord under halothane (2% in O₂) anesthesia. A rhodamine-labeled fluorescence microsphere suspension (FluoSpheres, 0.04 µm; Invitrogen, Carlsbad, CA) was pressure-injected (Nanojector II; Drummond Scientific, Broomall, PA) bilaterally into the IML region of the spinal cord in three or four separate 50-nl injections using a glass micropipette (tip diameter 20-30 µm). Using a manipulator, the pipette was positioned at approximately 500 µm from the midline and 500 µm below the dorsal surface (Li et al., 2002, 2003). After injection, animals were returned to their cages for 3 to 10 days and inspected daily for motor activity, signs of infection as well as food and water intake to assess the health status of the animals. Because the fluorescence tracer can reach the PVN within 48 h and is retained in labeled neurons for more than 10 days, the recording on the brain slice (one rat/day) was done 3 to 10 days after the tracer injection. In our previous studies, we found no difference in EPSCs and IPSCs recorded over a period of 3 to 10 days in these animals (Li et al., 2002, 2003).

Slice Preparations. After 3 to 10 days, the brain of the FluoSphere-injected rats was quickly removed under halothane anesthesia and placed in ice-cold artificial cerebral spinal fluid (aCSF) per-fusion 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 95% O₂,5%CO₂ for 1 to 2 min. A higher glucose concentration was used to better preserve the viability of neurons in brain slices. A brain block containing the hypothalamus was trimmed and glued onto the stage of a vibratome (Technical Products International Inc., St. Louis, MO), as we described previously (Li et al., 2003, 2005). Coronal slices (300 µm in thickness) containing the PVN were cut in the ice-cold aCSF. Before recording, the slices were incubated in the aCSF continuously gassed with 95% O₂, 5% CO₂ at 34°C for at least 1 h.

Recordings of Postsynaptic Currents and Action Currents of PVN Neurons. The slice was placed in a glass-bottomed chamber (Warner Instruments, Hamden, CT) and fixed with a grid of parallel nylon threads supported by a U-shaped stainless steel weight. The aCSF 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). Whole-cell voltage-clamp recordings were performed to record the postsynaptic currents of the labeled PVN, as we described previously (Li et al., 2002, 2003). The recording pipette was pulled using borosilicate capillaries (1.2 mm o.d., 0.86 mm i.d.; WPI, Sarasota, FL). The resistance of the pipette was 5 to 7 M when it was filled with the pipette solution. The PVN is easily distinguishable under the microscope since it has a higher density of somata passing more light than surrounding tissues. The fluorescence-labeled neurons located in the medial one-third of the PVN area between the third ventricle and the fornix were selected for recording using a combination of epifluorescence illumination and differential interference contrast optics on a fixed stage upright microscope (BX51WI; Olympus, Tokyo, Japan). The labeled neuron was briefly identified with the aid of epifluorescence illumination. After forming a tight gigaohm, a brief negative pressure was used to get the whole-cell configuration. Recordings of postsynaptic currents began 5 min later when the current reached a steady state. Signals were processed with an Axopatch 700B amplifier (Molecular Devices, Sunnyvale, CA), filtered at 1 to 2 kHz, digitized at 20 kHz using DigiData 1322 (Molecular Devices), and saved to a hard drive of a computer. A liquid junction potential of -15.3 mV (for the potassium gluconate pipette solution) was corrected. To record EPSCs, the internal solution contained 130.0 mM potassium gluconate, 2.0 mM MgCl₂, 0.1 mM CaCl₂, 1.1 mM EGTA, 10.0 mM HEPES, 2.0 mM Na₂ATP, 0.3 mM Na₂GTP, adjusted to pH 7.25 with 1 M KOH (280-300 mOsM). Bicuculline (20 µM) was applied continuously to block the IPSCs at the holding potential of -70 mV. For recording of IPSCs, 130.0 mM potassium gluconate was replaced by 110.0 mM Cs₂SO₄ in the pipette solution, and 20 µM 6-cyano-7-nitroquinoxa-line-2,3-dione (CNQX, a glutamate non-NMDA receptor antagonist) was used to block EPSCs at the holding potential of 0 mV. The mEPSCs and mIPSCs were recorded in the presence of 1 µM tetrodotoxin (TTX). To block the possible postsynaptic action mediated by G proteins coupled to adrenergic receptors, a general G protein inhibitor, guanosine 5'-O-(2-thiodiphosphate) (1 mM), was added into the pipette solution during the whole-cell recording. A sodium channel blocker, lidocaine N-ethyl bromide (10 mM), was included in the pipette solution to suppress the action potential generation in whole-cell voltage-clamp protocols. The mEPSCs recorded in the presence of TTX represent the quantal release of glutamate (independent of the action potential), whereas sEPSCs reflect action potential-triggered and action potential-independent spontaneous glutamate release.

Cell-attached recordings were performed to examine the drug effect on the firing activity of 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, compared with whole-cell recording of action potentials, is that the intracellular contents of a cell are not disturbed while action currents are monitored. The patch was voltage-clamped to 0 mV relative to the bath potential. The capacitive current that is measured when the cell fires an action potential occurs as a brief spike. Data collection was terminated if the seal resistance fell below 1 G.

Phenylephrine, cirazoline, prazosin, CNQX disodium, bicuculline methiodide, AP5, and guanosine 5'-O-(2-thiodiphosphate were purchased from Sigma-Aldrich (St. Louis, MO); TTX and lidocaine N-ethyl bromide were from Alomone Labs (Jerusalem, Israel); and corynanthine hydrochloride was from Tocris Cookson Inc. (Ellisville, MO). Prazosin and corynanthine were initially dissolved in dimethyl sulfoxide to make the stock solution before being diluted in aCSF. Other drugs and agents, including phenylephrine, cirazoline, CNQX, bicuculline, and AP5, were dissolved in aCSF. All the drugs and solutions were freshly prepared before the experiment and delivered using syringe pumps. The reported selectivity value (₁/₂ adrenergic receptors) for corynanthine, prazosin, and cirazoline is 100, 1000 to 5000, and 50 to 100, respectively (Cavero et al., 1982; Doxey et al., 1984; Angel et al., 1995; Ireland et al., 1997).

Data Analysis. Data are presented as means ± S.E.M. The amplitude and frequency of EPSCs and IPSCs, and the firing activity were analyzed off-line using a peak detection program (MiniAnalysis; Synaptosoft, Leonia, NJ). Detection of events was accomplished by setting a threshold above the noise level. The representative tracings were extracted from original recordings over a 3-min period during control, during drug application, and washout. The mean amplitude and frequency of sEPSCS/sIPSCS and mEPSCs/mIPSCs were first determined for each recording and then averaged together according to the experimental group. The cumulative probability of the amplitude and interevent interval of EPSCs and IPSCs was compared using the Kolmogorov-Smirnov test, which estimates the probability that two cumulative distributions are similar. At least 100 EPSCs and 100 IPSCs were used in each analysis. The cumulative probability is a function used to analyze a random (discrete or continuous) variable. Specifically, cumulative probability plots rank the data in a simulation from the highest to the lowest value, and graph each point with its corresponding percentile (1 = 100%). The sEPSCS/sIPSCS and mEPSCs/mIPSCs recorded in this study are random events. Thus, to compare the drug effect on these variables, it is appropriate to analyze these data using cumulative probability. Neurons were considered to be responsive to drugs if the frequency of EPSCs or IPSCs was altered >20%. The decay phase of mIPSCs and mEPSCs was obtained by fitting the decaying phase of mIPSCs and mEPSCs using one or two exponential functions based on the curve-fitting R² values. The effect of drugs on the amplitude and frequency of EPSCs and IPSCs was 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.

	Results

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Whole-cell voltage-clamp recordings were performed on a total of 137 labeled PVN cells. The spinal cord around the level of T₂-T₄ was taken out after killing the rat and sectioned into 30-µm slices. The slices were viewed under the fluorescence microscope to verify the injection and diffusion sites of the tracer, as we described previously (Li et al., 2002, 2003). These sites were largely located in and around the IML of the spinal cord. The labeled PVN neurons displayed a membrane potential between -55 and -75 mV and an input resistance in a range of 500 to 800 M.

View larger version (36K): [in this window] [in a new window]   Fig. 1. Effect of phenylephrine on the frequency of sEPSCs in labeled PVN neurons. A, original recordings showing sEPSCs during control; application of 10, 50, and 100 µM phenylephrine; washout; and perfusion of 20 µM CNQX in a labeled PVN neuron. B, cumulative probability plot of the same neuron showing the distribution of interevent interval and amplitude of sEPSCs during control, perfusion of 50 and 100 µM phenylephrine, and washout. C, summary data showing effect of phenylephrine on the frequency and amplitude of sEPSCs (n = 19). Data are presented as means ± S.E.M. *, P < 0.05, compared with the control (Kruskal-Wallis ANOVA followed by Dunn's post hoc test). phe, phenylephrine.

View larger version (38K): [in this window] [in a new window]   Fig. 2. Effect of corynanthine and prazosin on phenylephrine-induced increase in sEPSCs. A, raw tracings showing sEPSCs during control, perfusion of 50 µM phenylephrine, washout, and perfusion of 50 µM phenylephrine plus 100 µM corynanthine in a labeled PVN neuron. B, cumulative probability plot showing the distribution of interevent interval and amplitude of sEPSCs of the same neuron during control, perfusion of 50 µM phenylephrine, washout, and perfusion of 50 µM phenylephrine plus 100 µM corynanthine. C, summary data showing the effect of 100 µM corynanthine on phenylephrine-induced increase in sEPSCs (n = 11). D, summary data showing the effect of 20 µM prazosin on phenylephrine-induced increase in sEPSCs (n = 9). Data are presented as means ± S.E.M. *, P < 0.05, compared with the control (Kruskal-Wallis ANOVA followed by Dunn's post hoc test). phe, phenylephrine; cory, corynanthine; and prz, prazosin.

Phenylephrine (50-100 µM) did not significantly increase the frequency of sEPSCs (from 9.52 ± 2.35 to 9.69 ± 2.34 Hz at 100 µM) in another six cells. Notably, the baseline frequency of sEPSCs of these six nonresponsive cells to phenylephrine varied from 1.23 to 15.14 Hz. For those cells with the frequency of sEPSCs less than 3 Hz, phenylephrine also failed to alter the EPSC frequency. Thus, lack of response of sEPSCs to phenylephrine in these cells is less probably because of the high baseline frequency of sEPSCs.

To further examine whether the effect of phenylephrine is mediated through ₁ adrenergic receptors, the ₁ adrenergic receptor antagonist corynanthine (100 µM) or prazosin (20 µM) was perfused 3 to 4 min before bath application of 50 µM phenylephrine. The concentrations of these ₁ adrenergic receptor antagonists have been determined in previous studies (Medgett and Langer, 1984; Han et al., 2002; Li et al., 2005). Each antagonist alone did not significantly change the frequency of sEPSCs. However, corynanthine or prazosin alone completely blocked the effect of phenylephrine on the frequency of sEPSCs in 20 labeled cells (Fig. 2).

To determine whether phenylephrine increases synaptic glutamate release by acting at presynaptic terminals or a presumed somatodendritic site, we examined the effect of phenylephrine on mEPSCs in the presence of 1 µM TTX. In 15 labeled cells tested, 50 µM phenylephrine significantly increased the frequency of sEPSCs of 13 cells from 6.46 ± 1.26 to 12.70 ± 2.14 Hz. It did not affect the frequency of sEPSCs in another two cells (from 3.45 to 3.86 Hz in one cell and from 4.73 to 5.26 Hz in the other cell). Phenylephrine also significantly increased the frequency of mEPSCs from 6.03 ± 1.35 to 9.77 ± 2.09 Hz in all 13 cells (Fig. 3D), but it had no significant effects on the amplitude and decay constant of mEPSCs (Fig. 3, B and C).

View larger version (33K): [in this window] [in a new window]   Fig. 3. Effect of phenylephrine on the frequency of mEPSCs in labeled PVN neurons. A, raw tracings showing mEPSCs during control, perfusion of 50 µM phenylephrine, and washout in a labeled PVN neuron. B, cumulative distribution probability plot of mEPSCs of the same neuron showing the distribution of interevent interval and amplitude during control, perfusion of 50 µM phenylephrine, and washout. C, superimposed averages of 100 consecutive mEPSCs obtained between control and perfusion of 50 µM phenylephrine. The decay phase of mEPSCs was best fitted with a single exponential function. The decay time constant was similar in control ( = 5.87 ms) and phenylephrine application ( = 5.62 ms). D, summary data showing the effect of 50 µM phenylephrine on the frequency of mEPSCs in 13 labeled PVN neurons. Data are presented as means ± S.E.M. *, P < 0.05, compared with the control; #, P < 0.05, compared with the TTX treatment alone (Kruskal-Wallis ANOVA followed by Dunn's post hoc test). phe, phenylephrine.

View larger version (37K): [in this window] [in a new window]   Fig. 4. Effect of phenylephrine and cirazoline on the frequency of sIPSCs in labeled PVN neurons. A, raw tracings showing sIPSCs during control; application of 10, 50, and 100 µM phenylephrine; washout, and perfusion of 20 µM bicuculline in a labeled PVN neuron. B, cumulative probability plot of sIPSCs of the same neuron showing the distribution of interevent interval and amplitude during control, perfusion of 50 and 100 µM phenylephrine, and washout. C, summary data showing the effect of 1 to 100 µM phenylephrine on the frequency and amplitude of sIPSCs (n = 13). D, summary data showing the effect of 1 to 100 µM cirazoline on the frequency of sIPSCs (n = 7). Data are presented as means ± S.E.M. *, P < 0.05, compared with the control (Kruskal-Wallis ANOVA followed by Dunn's post hoc test). phe, phenylephrine; and Bic, bicuculline.

Phenylephrine produced a different effect on synaptic GABA release in the PVN in the present and previous study (Han et al., 2002). To further determine the effect of ₁ adrenergic receptor activation on the frequency of sIPSCs, cirazoline, another ₁ adrenergic receptor agonist, was used. The concentration of cirazoline has been examined in a previous study (Pudovkina et al., 2001). In another seven labeled PVN cells examined, bath application of 10 to 100 µM cirazoline also caused a significant decrease in the frequency of sIPSCs (Fig. 4D).

To ensure that the effect of phenylephrine on sIPSCs is mediated by ₁ adrenergic receptors, corynanthine (100 µM), an ₁ receptor antagonist, was applied for 3 to 4 min before perfusion of 50 µM phenylephrine. Corynanthine alone did not significantly change the frequency of sIPSCs, but it abolished the phenylephrine-induced decrease in the frequency of sIPSCs in all nine cells tested (Fig. 5).

View larger version (27K): [in this window] [in a new window]   Fig. 5. Effect of corynanthine on the phenylephrine-induced decrease in sIPSCs. A, raw tracings showing control, perfusion of 50 µM phenylephrine, washout, and perfusion of 50 µM phenylephrine plus 100 µM corynanthine in a labeled PVN neuron. B, cumulative probability plot of sIPSCs of the same neuron showing the distribution of interevent interval and amplitude during control, perfusion of 50 µM phenylephrine, washout, and perfusion of 50 µM phenylephrine plus 100 µM corynanthine. C, summary data showing that 100 µM corynanthine completely blocked the phenylephrine-induced decrease in the frequency of sIPSCs (n = 9). Data are presented as means ± S.E.M. *, P < 0.05, compared with the control (Kruskal-Wallis ANOVA followed by Dunn's post hoc test). phe, phenylephrine; and cory, corynanthine.

View larger version (29K): [in this window] [in a new window]   Fig. 6. Effect of phenylephrine on the frequency of mIPSCs in labeled PVN neurons. A, raw tracings showing mIPSCs during control, perfusion of 50 µM phenylephrine, and washout in a labeled PVN neuron. B, cumulative probability plot of mIPSCs of the same neuron showing the distribution of interevent interval and amplitude during control, perfusion of 50 µM phenylephrine, and washout. C, superimposed averages of 100 consecutive mIPSCs obtained during control and perfusion of 50 µM phenylephrine. The decay phase of mIPSCs was best fitted with a double exponential function. The decay time constant was similar during control and phenylephrine application (, 12.79 versus 13.02 ms; slow, 49.10 versus 50.39fastms). D, summary data showing the effect of 50 µM phenylephrine on the frequency of mIPSCs (n = 13). Data are presented as means ± S.E.M. * P < 0.05, compared with the control (Kruskal-Wallis ANOVA followed by Dunn's post hoc test). phe, phenylephrine.

View larger version (37K): [in this window] [in a new window]   Fig. 7. Effect of phenylephrine on the firing rate of labeled PVN neurons in the presence of synaptic blockade. A, original tracings showing action currents of a labeled PVN neuron during control, perfusion of 50 µM phenylephrine, washout, perfusion of 20 µM bicuculline plus 20 µM CNQX plus 20 µM AP5, and 50 µM phenylephrine in the presence of bicuculline, CNQX and AP5. B, summary data showing the effect of 50 µM phenylephrine on the firing rate of 11 labeled PVN neurons before and after application of bicuculline, CNQX, and AP5. C, summary data showing the differential effect of 100 and 500 nM apamin and 20 µM bicuculline on the firing activity of eight labeled PVN neurons. Data are presented as means ± S.E.M. *, P < 0.05, compared with the control (Kruskal-Wallis ANOVA followed by Dunn's post hoc test). phe, phenylephrine; Bic, bicuculline; and apm, apamin.

Phenylephrine had no significant effect on another six cells (from 6.15 ± 1.04 to 6.96 ± 1.23 Hz). Notably, the baseline frequency of the firing activity of these six nonresponsive cells to 50 µM phenylephrine ranged from 2.79 to 10.29 Hz. For those cells with the firing rate less than 4 Hz, phenylephrine also failed to alter their firing activity. Therefore, lack of response of the firing activity to phenylephrine in these cells is less likely because of the high baseline firing rate.

In addition to blocking GABA_A receptors, bicuculline methiodide may increase the firing activity of PVN neurons through its effect on small conductance Ca²⁺-activated K⁺ channels (SK channels) (Khawaled et al., 1999). Therefore, we determined the effect of apamin, a specific blocker of SK channels, on the firing activity of labeled PVN neurons. Bath application of 20 µM bicuculline methiodide significantly increased the firing rate of eight labeled PVN neurons. However, 100 nM apamin had no significant effect on the firing rate of these eight PVN neurons (Fig. 7C). At 500 nM, apamin also failed to alter the firing activity of these neurons (Fig. 7C).

	Discussion

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In this study, we used retrograde labeling and whole-cell recording techniques in brain slices to investigate the role of ₁ adrenergic receptors in the regulation of synaptic inputs to PVN-spinal projection neurons. We found that phenylephrine significantly increased the frequency of glutamatergic sEPSCs and mEPSCs. On the other hand, phenylephrine significantly decreased the frequency of GABAergic sIPSCs and mIPSCs. These presynaptic effects of phenylephrine on glutamate and GABA release were blocked by specific ₁ adrenergic receptor antagonists. Furthermore, phenylephrine failed to increase the firing of labeled PVN neurons in the presence of GABA_A and ionotropic glutamate receptor antagonists in most of the cells tested. Therefore, our results provide important new evidence showing that activation of presynaptic ₁ adrenergic receptors increases glutamatergic but reduces GABAergic inputs, to augment the excitability of PVN presympathetic neurons.

The hypothalamic PVN neurons are important for autonomic regulation mainly through their projections to the RVLM, NTS, and IML of the spinal cord. The PVN receives dense catecholaminergic afferents from the brainstem neurons, including the A1, A2, and A6 noradrenergic cell groups (Swanson and Sawchenko, 1983; Cunningham and Sawchenko, 1988) and C1, C2, and C3 adrenergic neurons in the rostral medulla (Cunningham et al., 1990). Receptor binding studies have shown that ₁ and ₂ adrenergic receptors are present in the PVN (Cummings and Seybold, 1988). Many studies have shown that ₁ adrenergic receptors in the PVN play an important role in the regulation of neuroendocrine function. For example, norepinephrine increases corticotrophin-releasing hormone release through ₁ adrenergic receptors (Plotsky, 1987). Microinjection of norepinephrine into the PVN causes an increase in blood pressure through ₁ adrenergic receptors (Harland et al., 1989). Increased release of norepinephrine in the PVN also may stimulate the secretion of vasopressin through ₁ adrenergic receptors to increase blood pressure (Bealer and Abell, 1995). However, the cellular and synaptic mechanisms responsible for the effects of norepinephrine and ₁ agonists on PVN presympathetic neurons are not clear. Therefore, in this study we used retrograde labeling to identify the PVN-spinal projection neurons and investigated the role of ₁ adrenergic receptors in regulation of glutamatergic and GABAergic synaptic inputs to labeled PVN neurons.

Glutamate is considered the dominant excitatory neurotransmitter in the PVN (Decavel and van den Pol, 1992). Microinjection of glutamate into the PVN increases renal sympathetic activity (Kannan et al., 1989). The local gluta-matergic synaptic inputs may originate from the dorsomedial hypothalamus and perifornical neurons. In addition, the PVN neurons in the brain slice receive glutamatergic projections from the caudal bed nucleus, the subparaventricular zone, the suprachiasmatic nucleus, the lateral hypothalamus, and the rostral zona incerta (Bains and Ferguson, 1995; Cui et al., 2001). We found that phenylephrine significantly increased the frequency of sEPSCs of labeled PVN neurons. This effect was blocked by either prazosin or corynanthine, the ₁ adrenergic receptor antagonist. Phenylephrine also increased the frequency of mEPSCs, but it did not change the amplitude and decay constant of mEPSCs, suggesting that ₁ adrenergic receptors are located at the presynaptic terminals. Although previous studies have shown that norepinephrine stimulates glutamatergic synaptic inputs to PVN magnocellular and parvocellular neurons through presynaptic ₁ adrenergic receptors (Daftary et al., 1998, 2000; Boudaba et al., 2003), the potential function and projections of these neurons are not defined. Therefore, our results provide new information that activation of presynaptic ₁ adrenergic receptors enhances the glutamatergic synaptic inputs to PVN-spinal output neurons to increase sympathetic outflow.

Data from our study strongly suggest that activation of ₁ adrenergic receptors on presynaptic terminals reduces synaptic GABA release to PVN presympathetic neurons. The local synaptic inputs to PVN neurons are primarily GABAergic (Decavel and van den Pol, 1992; Tasker and Dudek, 1993). Unlike the glutamatergic input, the PVN GABAergic system tonically inhibits the PVN presympathetic neurons (Li et al.,2002, 2003, 2005) and the sympathetic outflow (Martin and Haywood, 1993). In this study, we found that two structurally dissimilar ₁ adrenergic receptor agonists, phenylephrine and cirazoline, both significantly decreased the frequency of GABAergic sIPSCs. This effect was completely blocked by the ₁ adrenergic receptor antagonist corynanthine. In addition, phenylephrine significantly decreased the frequency of mIPSCs but did not affect the amplitude and decay constant of mIPSCs, suggesting that presynaptic ₁ adrenergic receptors alter the release probability of GABA-filled vesicles. Notably, in unlabeled PVN type II (parvocellular) neurons in brain slices, norepinephrine both increases and decreases the IPSC frequency, and norepinephrine-induced decreases in IPSC frequency were blocked by _2A, but not by ₁, receptor antagonists (Chong et al., 2004). In addition, a previous study reports that norepinephrine increases the frequency of sIPSCs of some PVN neurons through ₁ adrenergic receptors, and this effect is sensitive to TTX (Han et al., 2002). This discrepancy may be related to our focus on spinally projecting PVN neurons, whereas the types of PVN neurons (i.e., interneurons versus output neurons) are not clarified in the study by Han et al. (2002). These results suggest a possible anatomical and functional segregation of norepinephrine inputs among different parvocellular PVN subpopulations. Data from our study strongly suggest that activation of ₁ adrenergic receptors on presynaptic terminals reduces synaptic GABA release to PVN presympathetic neurons. We have shown that stimulation of ₂ adrenergic receptors decreases GABAergic (but not glutamatergic) inputs to PVN-spinal neurons (Li et al., 2005). In a given PVN neuron, the net presynaptic effect of norepinephrine on the GABA release depends on the presence and relative density of ₁ and ₂ adrenergic receptors on the presynaptic nerve terminals.

The excitability of PVN neurons critically depends on the balance between the excitatory and inhibitory inputs. Glutamate and GABA are the predominant excitatory and inhibitory neurotransmitters, respectively, in the PVN. We therefore further determined the contribution of the presynaptic effect of phenylephrine to the regulation of the firing activity of PVN-spinal output neurons. We found that combined treatment with bicuculline and CNQX/AP5 had no significant effect on basal discharges, suggesting that GABAergic and glutamatergic tone are in an effective balance. Phenylephrine significantly increased the firing of most labeled PVN neurons. However, the stimulatory effect of phenylephrine on most PVN neurons was abolished when the glutamatergic and GABAergic synaptic inputs were removed. This finding suggests that the excitatory effect of phenylephrine largely depends on its presynaptic effects on GABAergic and glutamatergic inputs to labeled PVN neurons. Bicuculline microinjection into the PVN of intact rats fails to increase sympathetic nerve activity if ionotropic glutamate receptors have been previously blocked (Chen et al., 2003). This suggests that local disinhibition of neurons after bicuculline injection may potentiate glutamate release, and the increase in the firing activity of PVN neurons may be dependent on this process. Thus, it is possible that norepinephrine-induced inhibition of IPSCs is required for the increase in glutamatergic EPSCs. Because bicuculline methiodide may increase the firing activity of PVN neurons through its effect on SK channels (Khawaled et al., 1999), we determined the effect of apamin, a specific blocker of SK channels, on the firing activity of labeled PVN neurons. We found that bicuculline methiodide, but not apamin, significantly increased the firing activity of labeled PVN neurons. Therefore, it is less likely that SK channels are involved in the excitatory effect of bicuculline on spinally projecting PVN neurons. The ₁ adrenergic receptors may affect other neurotransmitters and neuromodulators to regulate the activity of PVN neurons. In this regard, it has been shown that norepinephrine increases the nitric-oxide synthase in PVN magnocellular neurons (Grange-Messent et al., 2004). However, the effect of activation of ₁ adrenergic receptors on the release of catecholamines and adenosine to PVN presympathetic neurons has not been demonstrated. Because some spinally projecting PVN neurons also send collaterals to the RVLM (Pyner and Coote, 2000), the increase in the firing activity of these PVN neurons by the ₁ adrenergic receptors could possibly excite the presympathetic neurons in the RVLM. Hence, stimulation of ₁ adrenergic receptors in the PVN can activate both PVN-IML- and PVN-RVLM-IML descending pathways and could cooperatively augment the sympathetic outflow to increase the blood pressure (Harland et al., 1989).

In the PVN, three ₁ adrenergic receptor subtypes (_1A, _1B, and _1D) have been identified by immunohistochemistry and in situ hybridization techniques (Domyancic and Morilak, 1997; Acosta-Martinez et al., 1999; Day et al., 1999; Sands and Morilak, 1999). The _1A mRNA is mainly detected in the lateral magnocellular region in a lower expression level. The _1D mRNA is primarily in the PVN magnocellular and parvocellular subnuclei, whereas the _1B immunoreactivity is higher in the magnocellular PVN division than in the parvocellular region (Domyancic and Morilak, 1997; Acosta-Martinez et al., 1999; Sands and Morilak, 1999). It is not clear why stimulation of ₁ adrenergic receptors increases synaptic glutamate release but decreases GABA release to spinally projecting PVN neurons. This may be because of the different subtypes of ₁ receptors and signaling pathways present at the glutamatergic and GABAergic synaptic terminals. For example, the effect of _1A is coupled to pertussis toxin-sensitive G proteins, but both _1B and _1D receptors are coupled to pertussis-insensitive G proteins in Chinese hamster ovary cells (Perez et al., 1993). However, highly specific antagonists and agonists for these ₁ receptor subtypes are not available at this time to further define the function of ₁ receptor subtypes. In this study, the firing activity of two of 13 cells was still increased by phenylephrine after the synaptic blockade. This suggests that the excitability of a few PVN-spinal projection neurons is probably controlled by postsynaptic ₁ adrenergic receptors.

In summary, the present study provides new information that stimulation of presynaptic ₁ adrenergic receptors increases the firing of PVN projection neurons through attenuation of tonic GABAergic inhibition and potentiation of excitatory glutamatergic inputs. It has been shown that norepinephrine release in the PVN is increased in hypertensive rats (Qualy and Westfall, 1988; Woo et al., 1993). Furthermore, the firing rate of PVN neurons and the basal norepinephrine level in the PVN are elevated during heart failure (Arabia et al., 2002; Zhang et al., 2002b). Further studies are warranted to determine the role of ₁ adrenergic receptors in the PVN in the regulation of sympathetic outflow under these pathophysiological conditions.

	Acknowledgements

 
We thank C. Y. Yang and P. Myers for technical assistance.

	Footnotes

 
This study was supported by National Institutes of Health Grants HL60026 and HL77400.

doi:10.1124/jpet.105.094797.

ABBREVIATIONS: PVN, paraventricular nucleus; RVLM, rostral ventrolateral medulla; NTS, nucleus of solitary tract; IML, intermediolateral cell column; EPSC, excitatory postsynaptic current; IPSC, inhibitory postsynaptic current; aCSF, artificial cerebrospinal fluid; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; NMDA, N-methyl-D-aspartate; mEPSC, miniature excitatory postsynaptic current; mIPSC, miniature inhibitory postsynaptic current; TTX, tetrodotoxin; AP5, DL-2-amino-5-phosphonovaleric acid; ANOVA, analysis of variance; sEPSC, spontaneous excitatory postsynaptic current; sIPSC, spontaneous inhibitory postsynaptic current; SK, small conductance Ca²⁺-activated K⁺ channel.

Address correspondence to: Dr. Hui-Lin Pan, Department of Anesthesiology, H187, Pennsylvania State University College of Medicine, 500 University Dr., Hershey, PA 17033. E-mail: hpan{at}psu.edu

	References

Top Abstract Materials and Methods Results Discussion References

 

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