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1Department of Anesthesiology and 2Department of Neural and Behavioral Sciences, The Pennsylvania State University College of Medicine, The Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033
Submitted 23 February 2004; accepted in final form 24 April 2004
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ABSTRACT |
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INTRODUCTION |
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). The PVN is a heterogenous structure containing interneurons and output neurons projecting to brain stem autonomic centers and the sympathetic preganglionic neurons located in the intermediolateral (IML) cell column of the spinal cord (Ranson et al. 1998; Shafton et al. 1998; Toth et al. 1999). There is strong anatomical and electrophysiological evidence showing that the PVN is important in the regulation of sympathetic outflow, especially during stress and certain types of hypertension (Allen 2002; de Wardener 2001; Miyakubo et al. 2002; Ranson et al. 1998). Both GABA and glutamate are two prominent inhibitory and excitatory neurotransmitters in the PVN, and the firing activities of these PVN output neurons are finely tuned by these synaptic inputs (Boudaba et al. 1997; Hermes et al. 1996; Li et al. 2002, 2003). However, the mechanisms governing glutamatergic and GABAergic inputs in the PVN are not fully known.
The capsaicin vanilloid receptor-1 (VR1, also known as TRPV1 channel) in the primary sensory neurons is best known as a molecular sensor for nociception (Caterina et al. 1997). The VR1 receptors are characterized as a nonselective cation channel that belongs to the transient receptor potential family (Gunthorpe et al. 2002). Activation of VR1 receptors produces an inward current carried by nonselective cations with a high permeability for divalent cations such as Ca2+ (Caterina et al. 1997; Liu and Simon 1994). Recent studies have shown that the VR1 mRNA is present in several regions of the CNS (Mezey et al. 2000). Also, the VR1 immunoreactivity and the binding sites of the VR1-selective radioligand, [3H]resiniferatoxin, are located in the brain regions including the hypothalamus and locus coeruleus (Mezey et al. 2000; Szabo et al. 2002).
The potential physiological role of VR1 receptors in the PVN remain unclear. Recent studies suggest that capsaicin enhances glutamatergic synaptic transmission in the locus coeruleus and substantia nigra through activation of VR1 receptors (Marinelli et al. 2002, 2003). However, the role of VR1 receptors in the control of excitatory and inhibitory synaptic inputs and the excitability of PVN output neurons has not been studied. Therefore in this study, we combined in vivo retrograde labeling and in vitro whole cell recording techniques in the hypothalamic slice to test the hypothesis that VR1 receptor activation excites spinally projecting PVN neurons through potentiation of glutamatergic synaptic inputs.
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METHODS |
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Sprague–Dawley rats (6–8 wk old; Harlan, Indianapolis, IN) of either sex 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. The spinal cord at the T1–T4 level was exposed through dorsal laminectomy under halothane anesthesia. A suspension of rhodamine-labeled fluorescent microspheres (FluoSpheres, 0.04 µm; Molecular Probes, Eugene, OR) was pressure-ejected (Picospritzer II; General Valve, Fairfield, NJ) bilaterally into the region of the IML of the spinal cord in 2 or 3 separate 50-nl injections using a glass micropipette (20- to 30-µm tip diameter). The pipette was positioned with a micromanipulator at about 500 µm below the dorsolateral sulcus, and the tracer injection was monitored through a surgical microscope (Li et al. 2002, 2003). The muscles were sutured and the wound was closed after injection. Animals were returned to their cages for 3–7 days, which is sufficient to permit retrograde tracer being transported to the PVN. The rats were inspected daily for motor activity, signs of infection, and food and water intake to assess the health status of the animals.
Slice preparations
Three to 7 days after the tracer injection, the rats were rapidly decapitated under halothane anesthesia. The brain was quickly removed and placed in ice-cold artificial cerebral spinal fluid (aCSF) 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 Products International, St. Louis, MO), as we described previously (Li et al. 2002, 2003). Coronal slices (300 µm in thickness) containing the PVN were cut from the tissue block in the ice-cold aCSF. The slices were preincubated in aCSF, which was continuously gassed with 95% O2 and 5% CO2 at 34°C for 1 h until they were transferred to the recording chamber. The perfusion solution contained (in mM): 124.0 NaCl, 3.0 KCl, 1.3 MgSO4, 2.4 CaCl2, 1.4 NaH2PO4, 10.0 glucose, and 26.0 NaHCO3. For the Ca2+-free solution, CaCl2 was removed and replaced with an equimolar concentration of CoCl2 in the perfusion solution.
Recordings of postsynaptic currents and firing activity of labeled-PVN neurons
Recordings of postsynaptic currents and neuronal activity were performed in a radio frequency–shielded room using the whole cell voltage- and current-clamp technique, as we described previously (Li et al. 2002, 2003). The recording pipettes were triple-pulled using borosilicate glass capillaries (1.2 mm OD, 0.86 mm ID; World Precision Instruments, Sarasota, FL). The resistance of the pipette was 4–6 M
when it was filled with the internal solution containing (in mM): 130.0 potassium gluconate, 1.0 MgCl2, 10.0 HEPES, 10.0 EGTA, 1.0 CaCl2, and 4.0 ATP-Mg; adjusted to pH 7.25 with 1 M KOH (280–300 mOsm). 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 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 min to completely exchange the solution inside the recording chamber at the perfusion rate of 3 ml/min. Whole cell recordings from labeled PVN neurons were performed under visual control using a combination of epifluorescence illumination and differential interference contrast (DIC) optics on an upright microscope (BX50WI, Olympus, Tokyo, Japan). 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 (Li et al. 2002, 2003). The labeled neuron was briefly identified with the aid of epifluorescence illumination. A tight giga- seal was subsequently obtained in the labeled neuron viewed with the DIC optics (Fig. 1). Recordings of postsynaptic currents began 5–10 min later after the whole cell access was established and the recording reached a steady state. Signals were processed with an Axopatch 200B amplifier (Axon Instruments, Foster City, CA). A liquid junction potential of –15.3 mV (for the potassium gluconate pipette solution) was corrected during off-line analysis. Signals were filtered at 1–2 kHz, digitized at 10 kHz using Digidata 1322 (Axon Instruments), and saved to a hard drive of a computer. The miniature excitatory postsynaptic currents (mEPSCs) were recorded in the presence of 1 µM tetrodotoxin (TTX) and 20 µM bicuculline (a GABAA receptor antagonist) at a holding potential of –70 mV. The miniature inhibitory postsynaptic currents (mIPSCs) were recorded in the presence of 1 µM TTX, 20 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, a non-NMDA receptor antagonist), and 50 µM 2-amino-5-phosphonopentanoic acid (AP5, an NMDA receptor antagonist) at a holding potential of 0 mV (Li et al. 2002, 2003).
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). To avoid recording of eEPSCs elicited at the supramaximal stimulating intensity, we reduced the initial stimulation current so that the amplitude of eEPSCs was about 70–80% of maximal eEPSCs. A sodium channel blocker, lidocaine N-ethyl bromide quaternary salt (QX-314, 10 mM) was included in the pipette solution to block the Na+ current and spontaneous action potentials in these voltage-clamp experiments. Based on the optimal reversal potentials determined for CNQX-sensitive EPSCs, the eEPSCs was recorded at a holding potential of –70 mV.
The spontaneous firing activity of labeled PVN neurons was recorded using the whole cell current-clamp technique (Li et al. 2002, 2003). The recording procedures were similar to those used for postsynaptic current recordings as described above except that TTX and QX-314 were not used. Recordings of the firing activity of labeled PVN neurons began about 5 min after the whole cell access was established and the firing activity reached a steady state. Signals were processed, recorded, and analyzed as described above.
Capsaicin, iodo-resiniferatoxin (iodo-RTX), ruthenium red, CNQX, and bicuculline were obtained from Sigma (St. Louis, MO). TTX and QX-314 were purchased from Alomone Labs (Jerusalem, Israel). The concentrations of capsaicin, ruthenium red, CNQX, and iodo-RTX were derived from previous studies (Li et al. 2003; Marinelli et al. 2002, 2003; Trudeau et al. 1996) and tested in the pilot experiments. All the drugs were prepared immediately before the experiments and applied to the recording chamber using syringe pumps.
Double-immunofluorescence labeling of VR1 receptors and synaptophysin in the PVN
To determine whether the VR1 receptors are located presynaptically in the PVN, sections from the hypothalamus were immunostained for colocalization of the VR1 receptor and synaptophysin (Li et al. 2003; Pan et al. 2003), a specific marker for presynaptic terminals, in 3 separate rats. Under deep anesthesia with sodium pentobarbital (60 mg/kg, ip), rats were intracardially perfused with 200 ml of ice-cold normal saline containing 1,000 units of heparin followed by 500 ml of 4% paraformaldehyde in 0.1 M PBS (pH 7.4) and then 200 ml of 10% sucrose in 0.1 M PBS (pH 7.4). The brain was removed quickly and postfixed for 2 h in the same fixative solution and cryoprotected in 30% sucrose in PBS for 48 h at 4°C. The sections were cut to a thickness of 25 µm and collected free floating in 0.1 M PBS. For VR1 and synaptophysin double-immunofluorescence labeling, the labeling intensity of the first primary antibody (rabbit anti-VR1 N terminal polyclonal IgG antibody, dilution 1:1000; Neuromics, Minneapolis, MN) was enhanced with TSA (tyramide signal amplification), and conventional immunofluorescent labeling was then performed with the second primary antibody (mouse anti-synaptophysin). To perform the TSA labeling, sections were first incubated in 3% H2O2 with 10% of methanol to quench endogenous peroxidase and blocked in TNB (0.1 M Tris–HCl, 0.15 M NaCl, and 0.5% blocking reagent) buffer. Next, sections were incubated with the primary antibody (rabbit anti-VR1, dilution 1:1000) for 2 h at room temperature and 24 h at 4°C. Subsequently, sections were rinsed and incubated with horseradish peroxidase–conjugated goat anti-rabbit IgG secondary antibody (dilution 1:100; Jackson ImmunoResearch Laboratories, West Grove, PA) for 2 h at room temperature. The sections were incubated with the FITC conjugated to tyramide (TSA direct kit, Perkin–Elmer, Boston, MA) and incubated with the secondary primary antibody (mouse anti-synaptophysin monoclonal IgM, dilution 1:100; Chemicon International, Temecula, CA) for 2 h at room temperature and overnight at 4°C. Then, sections were rinsed and incubated with the second antibody (biotin-SP–conjugated AffiniPure goat anti-mouse IgM, dilution 1:200; Jackson ImmunoResearch Laboratories) for 2 h at room temperature. The sections were incubated with streptavidin-conjugated Alexa Fluor-594 (dilution 5 µg/ml; Molecular Probes) for 1.5 h at room temperature. After rinsing, the sections were mounted on slides, dried, and coverslipped. The sections were then viewed using a confocal microscope (Leica, Wetzlar, Germany), and the areas of interest were photographed. Confocal laser scanning microscopy was used for accurate colocalization of fluorescent markers, because the thin (
0.3 µm) optical sectioning generated by the confocal microscope eliminates the confounding effect of out-of-focus fluorescence. In the higher magnification images, the colocalization was indicated by the color change (yellow) and represents colocalization (Li et al. 2002, 2003).
Data analysis
Data are presented as means ± SE. To determine the amplitude of the eEPSCs, 20 consecutive eEPSCs were averaged and measured using pClamp 9.0 analysis software. The mEPSCs, mIPSCs, and the firing activity were analyzed off-line with a peak detection program (MiniAnalysis, Synaptosoft, Leonia, NJ). Detection of events was accomplished by setting a threshold above the noise level. The distribution cumulative probability of the amplitude and interevent interval of mEPSCs and mIPSCs was compared using the Komogorov–Smirnov test, which estimates the probability that 2 cumulative distributions are similar. The decay time constant of mEPSCs or mIPSCs was obtained by fitting the decay phase of synaptic currents with exponential equation; 
100 mIPSCs and mEPSCs were used in each analysis. For cells displaying intermittent firing activity, the membrane potential was measured when the cell was silent and the membrane potential became stable. For those cells showing tonic activity, the membrane potential was usually estimated 200 ms before initiation of the action potential. The effects of drugs on the firing activity, peak amplitude of eEPSCs, and amplitude and frequency of mIPSCs and mEPSCs were determined by Wilcoxon signed-rank test or nonparametric ANOVA (Kruskal–Wallis) with Dunn's post hoc test. P < 0.05 was considered to be statistically significant.
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RESULTS |
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, 2004). The labeled PVN neurons displayed a resting membrane potential ranging from –72.8 to –55.9 (–60.7 ± 6.8) mV, an input resistance between 360 and 625 (432.7 ± 32.7) M, and an amplitude of action potentials >60 mV. Excitatory effect of capsaicin on glutamatergic EPSCs of labeled PVN neurons
To determine the effect of capsaicin on glutamatergic synaptic inputs to labeled PVN neurons, we examined the effect of capsaicin on the spontaneous mEPSCs. The mEPSCs were recorded in the presence of 1 µM TTX and 20 µM bicuculline. Capsaicin, in a concentration of 1 µM, significantly increased the frequency of mEPSCs from 3.85 ± 0.77 to 11.70 ± 1.30 Hz (398 ± 70% increase from the control, P < 0.05) without altering the amplitude and the decay time constant of mEPSCs in 12 labeled neurons tested (Fig. 2). The capsaicin took effect within 4.98 ± 0.37 min after application, and the effect of capsaicin subsided within 2–5 min as a result of rapid desensitization of VR1 receptors. In each of these 12 cells, the cumulative probability analysis revealed that the distribution pattern of the interevent interval of mEPSCs shifted to the left in response to capsaicin administration, whereas the distribution pattern of the amplitude of mEPSCs was not changed (Fig. 2, B and C). The decay time constant of mEPSCs was best fitted by a single-exponential function (Fig. 2D). The decay phase of mEPSCs during control was not significantly different from that during application of capsaicin (2.67 ± 0.63 vs. 2.83 ± 0.52 ms; P > 0.05; n = 12). The mEPSCs were completely abolished by perfusion of 20 µM CNQX in all 8 cells tested (Fig. 2A).
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Role of endogenous VR1 activation in regulation of glutamatergic EPSCs
To determine whether the glutamatergic inputs to PVN neurons was tonically influenced by endogenous VR1 activation, the effect of a specific VR1 receptor antagonist, iodo-RTX (Wahl et al. 2001), on mEPSCs and evoked EPSCs was tested. In 8 labeled PVN neurons, iodo-RTX (300 nM) alone decreased the frequency of mEPSCs from 3.38 ± 0.5 to 2.11 ± 0.3 Hz (P < 0.05, Fig. 3, A–F) without affecting the amplitude and decay time constant of mEPSCs. The inhibitory effect of iodo-RTX on the frequency of mEPSCs was no longer present after 20–30 min washout. Furthermore, 300 nM iodo-RTX significantly decreased the peak amplitude of evoked EPSCs from 226.1 ± 28.7 to 136.3 ± 22.6 pA (P < 0.05, Fig. 3, G and H) in another 7 PVN neurons.
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To confirm that the effect of capsaicin on mEPSCs was mediated through VR1 receptors, iodo-RTX was applied before perfusion of capsaicin. Capsaicin failed to increase the frequency of mEPSCs in the presence of 300 nM iodo-RTX in 11 labeled PVN neurons (Fig. 4, A–E). Also, to determine whether the effect of capsaicin on mEPSCs was through activation of the VR1 nonselective cation channels, ruthenium red, a dye that exhibits properties of noncompetitive antagonism for VR1 channels (Nagy and Rang 1999), was used. Ruthenium red (10 µM) alone produced a brief increase in the frequency of mEPSCs, which lasted for 3–5 min. This effect of ruthenium red is largely attributed to the rapid and reversible neurotransmitter release induced by a transient rise in intraterminal Ca2+ (Congar and Trudeau 2002; Trudeau et al. 1996). Subsequent capsaicin application failed to increase the frequency of mEPSCs in the presence of ruthenium red in 7 labeled PVN neurons (Fig. 4, F and G).
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To determine the role of voltage-dependent Ca2+ channels in the capsaicin-induced increase in mEPSCs, a general voltage-dependent Ca2+ channel blocker, Cd2+, was used in another 7 labeled PVN neurons. Pretreatment with Cd2+ (50–100 µM) had no significant effect on the frequency and amplitude of mEPSCs of labeled PVN neurons. Capsaicin significantly increased the frequency of mEPSCs from 4.37 ± 0.8 to 9.15 ± 1.9 Hz (213 ± 17% increase from the control, P < 0.05) in the presence of Cd2+ without altering the amplitude and the decay time constant of mEPSCs (Fig. 5, A–E). The effect of capsaicin on increased frequency of mEPSCs in normal aCSF (398 ± 70%) was significantly greater than that in the presence of Cd2+ (213 ± 17%, P < 0.05, Student's unpaired t-test).
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Effect of capsaicin on the GABAergic mIPSCs
The spontaneous mIPSCs were recorded from 7 labeled PVN neurons in the presence of 1 µM TTX and 20 µM CNQX. Neither the frequency nor the amplitude of mIPSCs was affected by bath application of 1 and 10 µM capsaicin (Fig. 6). The effect of 10 µM capsaicin on mIPSCs was further analyzed by measuring the time constant of the decay phase of mIPSCs. The decay phase of mIPSCs was best fitted by a double-exponential function equation (Fig. 6D). Neither the fast (7.68 ± 0.58 vs. 6.39 ± 0.48 ms) nor the slow (27.21 ± 2.87 vs. 28.59 ± 3.02 ms) component of the decay phase of mIPSCs during capsaicin application was significantly different from those during the control. Application of 20 µM bicuculline completely abolished the mIPSCs in these 7 cells (Fig. 6).
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To determine the presynaptic location of VR1 receptors, double-immunofluorescence staining was performed using specific antibodies against synaptophysin and VR1 receptors in the same brain section. All negative controls (omitting primary antibodies) displayed no detectable staining. Almost all of the synaptophysin immunoreactivity in the PVN occurred in the form of fine punctate deposits that often outlined neuronal cell bodies in a basketlike fashion (Fig. 7A). From the confocal images, numerous puncta immunoreactive for VR1 receptor (green) were present extensively in the PVN (Fig. 7B). All VR1 receptor immunoreactivies were colocalized with synaptophysin, as indicated by the color change (yellow, Fig. 7C).
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Because capsaicin preferentially increased the excitatory glutamatergic synaptic inputs to labeled PVN neurons, we reasoned that the excitability of these neurons would be increased by capsaicin. To directly test this hypothesis, the effect of 1 µM capsaicin on the spontaneous firing activity of labeled PVN neurons was determined using whole cell current-clamp recordings. The majority (21/26, 81%) of labeled PVN neurons recorded displayed spontaneous activity. In 11 PVN neurons tested, 1 µM capsaicin significantly increased the firing rate from 2.76 ± 0.82 to 6.28 ± 0.1.4 Hz (P < 0.05, Fig. 8) after an onset latency of 5.22 ± 0.41 min. The membrane potential was slightly, but not significantly, depolarized from –70.3 ± 1.47 to –67.8 ± 1.49 mV by capsaicin.
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DISCUSSION |
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Although the VR1 immunoreactivity and mRNA are present in the PVN, its physiological function and precise cellular localization have not been studied. In the present study, we found that capsaicin preferentially increased the frequency of glutamatergic mEPSC without altering the amplitude and decay time constant of mEPSCs in spinally projecting PVN neurons. Our data are consistent with a neurochemical study showing that capsaicin stimulates glutamate release from hypothalamic slices (Sasamura et al. 1998). The effect of capsaicin on glutamatergic synaptic transmission has been shown recently in several brain regions including the nucleus tractus solitarius, locus coeruleus, and the substantia nigra (Doyle et al. 2002; Marinelli et al. 2002, 2003). We observed that capsaicin did not potentiate but even slightly reduced the amplitude of evoked EPSCs in labeled PVN neurons. This finding is consistent with previous studies showing that capsaicin has no consistent effects on evoked EPSCs in the substantia nigra and locus coeruleus (Marinelli et al. 2002, 2003). The reasons for lack of a potentiating effect of capsaicin on evoked EPSCs are not clear. One possibility is that the presynaptic terminals being activated by electrical stimulation are a different population from those being stimulated by capsaicin. We are unable to identify and stimulate specific afferent pathways to labeled PVN neurons in this slice preparation. Another possibility is that an excessive direct depolarization of the presynaptic terminals by capsaicin can interfere with action potential–evoked glutamate release resulting from a "conduction block" (Yang et al. 1999). Our immunocytochemistry experiment provides additional complementary evidence that VR1 receptors are localized at the presynaptic terminals in the PVN. Notably the VR1 immunoreactivity was not systematically examined in the PVN in a previous study (Mezey et al. 2000). We used an enhanced immunolabeling technique in this study, which enabled us to visualize the VR1 immunoreactivity on the presynaptic terminals in the PVN. Previous studies suggest that the glutamatergic innervation of the PVN may originate from multiple sources of the CNS including the subnuclei within the hypothalamus, lateral septum, bed nucleus of the stria terminalis, and amygdala (Boudaba et al. 1997; Csaki et al. 2000; Cui et al. 2001). Our electrophysiological and immuncocytochemistry data strongly suggest that capsaicin increases the synaptic glutamate release probability, and the most likely site of action is at the presynaptic glutamatergic terminals.
The effect of capsaicin on mEPSCs on spinally projecting PVN neurons was mediated byVR1 receptors, given that the specific VR1 receptor antagonist, iodo-RTX, completely abolished the capsaicin-induced increase in the frequency of mEPSCs. Because of its high binding affinity to VR1 receptors, the potent VR1 antagonist, iodo-RTX, was selected in this study (Wahl et al. 2001). The utility of capsazepine, the first known competitive VR1 antagonist, is hindered by its moderate potency (Bevan et al. 1992). At micromolar concentrations necessary to inhibit capsaicin-evoked responses, capsazepine displays nonspecific effects, including block of voltage-gated calcium channels (Docherty et al. 1997) and nicotinic receptors (Wardle et al. 1997). Furthermore, the existence of capsazepine-insensitive VR1 has been reported in rat trigeminal ganglion neurons (Liu et al. 1998). The capsaicin-induced increase in the frequency of mEPSCs was also blocked by ruthenium red, a dye that exhibits properties of noncompetitive antagonism for the nonselective cation channel. Although it has a poor selectivity, ruthenium red can effectively block the transmembrane cation current (Nagy and Rang 1999). These data suggest that the capsaicin-induced glutamate release is through activation of VR1 receptors on presynaptic nerve terminals in the PVN.
In contrast to its action on glutamatergic mEPSCs, capsaicin had no significant effect on the GABAergic mIPSCs in labeled PVN neurons. Also, capsaicin appears to enhance only glutamatergic, but not GABAergic, synaptic transmission in the locus coeruleus and substantia nigra (Marinelli et al. 2002, 2003). The excitatory and inhibitory synaptic inputs to the PVN neurons are finely controlled by many factors. Unlike the effect of capsaicin in the PVN, both nitric oxide and angiotensin II have preferential effects on GABAergic, but not glutamatergic, synaptic inputs in the PVN (Li et al. 2002, 2003). Because activation of VR1 receptors affects only glutamatergic, but not GABAergic, synaptic inputs to PVN neurons, it is likely that VR1 receptors represent a previously unidentified mechanism that selectively regulates glutamatergic synaptic transmission in the PVN.
The increase in neurotransmitter release from presynaptic terminals is typically mediated through a Ca2+-dependent mechanism. The increase in the frequency of mEPSCs after VR1 activation by capsaicin presumably results from an increase in intraterminal Ca2+ concentration through Ca2+ influx into the nerve terminal, in that the effect of capsaicin on mEPSCs was abolished by removal of extracellular Ca2+. However, the general voltage-gated Ca2+ channel blocker, cadmium, failed to abolish the effect of capsaicin on mEPSCs. Thus capsaicin likely increases the glutamate release probability through calcium influx directly through the ionophore of VR1 receptors, but not dependent on voltage-gated Ca2+ channels (Caterina et al. 1997; Gunthorpe et al. 2002). Nevertheless, it should be noted that the effect of capsaicin on mEPSCs in the presence of cadmium was significantly smaller than that in normal aCSF. This finding suggests that activation of VR1 receptors increases the frequency of mEPSCs through calcium influx from both its own ionophore and the voltage-dependent Ca2+ channels.
Based on the observation that capsaicin preferentially increased the excitatory glutamatergic synaptic inputs to labeled PVN neurons, we hypothesized that capsaicin increases the firing activity of labeled PVN neurons through augmented glutamatergic inputs. We found that capsaicin caused a large increase in the firing activity of labeled PVN neurons. Interestingly, a long latency was observed for the effect of capsaicin on both mEPSCs and the firing activity of PVN neurons. This suggests that a second message system is likely involved in the effect of capsaicin on synaptic glutamate release. In addition to the calcium influx directly through the VR1 receptor, a store-operated Ca2+ influx (Liu et al. 2003) may also play a role in the presynaptic action of capsaicin in the PVN. In the presence of blockade of NMDA and non-NMDA receptors with AP5 and CNQX, capsaicin failed to increase the firing activity of PVN neurons. Thus these data indicate that capsaicin indirectly increases the excitability of spinally projecting PVN neurons through potentiation of glutamatergic synaptic inputs. We observed that the glutamate NMDA and non-NMDA receptor antagonists per se did not significantly alter the firing activity in all the PVN neurons tested, suggesting that the basal glutamatergic inputs are probably not sufficient to alter the firing activity of PVN neurons in this slice preparation.
One important finding of the present study is that the specific VR1 antagonist, iodo-RTX, not only abolished the effect of capsaicin on mEPSCs but also reduced both the amplitude of evoked EPSCs and the frequency of mEPSCs. These data suggest that the glutamatergic synaptic input to spinally projecting PVN neurons is tonically regulated by VR1 receptors. Several capsaicin-like substances have been identified to activate or potentiate the activity of VR1 receptors. Besides protons and anandamide (Smart et al. 2000; Zygmunt et al. 1999), 12-hydroperoxyeicosatetraenoic acid and leukotriene B4 (Hwang et al. 2000; Shin et al. 2002) have been considered as endogenous VR1 agonists. Also, N-arachidonoyldopamine and N-oleoyldopamine have been reported to be the endogenous agonists for VR1 receptors (Chu et al. 2003; Huang et al. 2002). At the present time, it remains unclear which of the above substances are present in sufficient concentrations that could function as the endogenous VR1 agonists in the PVN. We recently observed that intracerebroventricular injection of capsaicin caused a profound increase in the blood pressure and renal sympathetic nerve activity in rats (unpublished data). Thus activation of VR1 receptors can increase the excitability of PVN preautonomic neurons and consequently augments the sympathetic output. These results are potentially important because the mechanism studied provides a means for selective modulation of the excitatory input to PVN preautonomic neurons. It should be emphasized that the VR1 immunoreactivity is not restricted to presynaptic terminals synapsing with spinally projecting neurons in the PVN. The VR1 receptors present in the hypothalamus most likely have other important functions including regulation of body temperature, food intake, metabolic balance, and hydromineral homoeostasis. Data from the present study provide important new information about the potential physiological role and mechanisms of VR1 receptors in regulation of neuroendocrine functions.
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GRANTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: H.-L. Pan, Department of Anesthesiology, H187, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033-0850 (E-mail: hpan{at}psu.edu).
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ABSTRACT
INTRODUCTION
) viewed with differential interference contrast optics. Scale bar, 50 µM in A and B.
= 2.58 ms) and capsaicin application (
