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NEUROPHARMACOLOGY

Hypocretin-1 (Orexin-A) Facilitates Inhibitory and Diminishes Excitatory Synaptic Pathways to Cardiac Vagal Neurons in the Nucleus Ambiguus

Department of Pharmacology and Physiology, George Washington University, Washington, DC

Received for publication March 16, 2005
Accepted June 6, 2005.

	Abstract

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Hypocretin-1 is a neuropeptide recently shown to be involved in autonomic regulation. Hypocretin-1 is expressed by hypothalamic neurons, which project to many regions of the central nervous system, including the nucleus ambiguus. One possible site of action of hypocretin-1 could be cardioinhibitory parasympathetic vagal neurons within the nucleus ambiguus. This study examines whether hypocretin-1 modulates inhibitory and excitatory postsynaptic currents in cardiac vagal neurons in the rat nucleus ambiguus. GABAergic, glycinergic, and glutamatergic activity to cardiac vagal neurons was examined using whole-cell patch-clamp recordings in an in vitro brain slice preparation. Hypocretin-1 (1 µM) produced a significant increase in the frequency and amplitude of both GABAergic and glycinergic inhibitory postsynaptic currents and a significant decrease in the frequency of glutamatergic excitatory postsynaptic currents. Application of tetrodotoxin (0.5 µM) blocked all of the responses to hypocretin-1, indicating the changes in neurotransmission with hypocretin-1 do not occur at presynaptic terminals but rather occur at the preceding GABAergic, glycinergic, and glutamatergic neurons that project to cardiac vagal neurons. The increase in GABAergic and glycinergic inhibitory postsynaptic currents, and the decrease in glutamatergic excitatory postsynaptic currents, could be mechanisms by which hypocretin-1 affects heart rate and cardiac function.

Thus, although the majority of evidence suggests hcrt-1 regulates cardiovascular function through sympathetic activation, little is known about the role of hcrt-1 in parasympathetic control of cardiovascular function. Parasympathetic activity regulating heart rate and cardiac function primarily originates from preganglionic cardiac neurons within the nucleus ambiguus (Mendelowitz, 1999; Cheng and Powley, 2000; Wang et al., 2001a,b). Focal electrical or chemical stimulation of the nucleus ambiguus region has been shown to elicit decreases in heart rate mediated by the activation of vagal cardiac neurons (Ciriello and Calaresu, 1982; Ciriello and de Oliveira, 2003). These neurons are intrinsically silent, and recent studies have identified four major synaptic inputs, including glutamatergic excitatory postsynaptic currents (EPSCs), cholinergic inputs, and inhibitory GABAergic and glycinergic inhibitory postsynaptic currents (IPSCs) in cardiac vagal neurons (Mendelowitz, 1998; Neff et al., 1998, 2003; Wang et al., 2001b). The present study was undertaken to test the hypothesis that hcrt-1 may influence cardiovascular function by modulating synaptic neurotransmission to cardiac vagal neurons within the nucleus ambiguus.

	Materials and Methods

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In an initial surgery, Sprague-Dawley rats (postnatal days 2–7) were anesthetized with ketamine/xylazine and hypothermia and received a right thoracotomy. The heart was exposed, and 0.05 ml of rhodamine (XRITC; Molecular Probes, Eugene, OR) was injected into the pericardial sac to retrogradely label cardiac vagal neurons. The selective labeling of cardiac vagal neurons using these procedures has been previously described (Mendelowitz and Kunze, 1991). Specificity of the cardiac vagal labeling has been confirmed by the absence of any labeled neurons in the brainstem when rhodamine is injected into the chest cavity while keeping the pericardial sac intact, or when the injection into the pericardial sac is accompanied by a section of the cardiac branch of the vagus nerve (n = 4). In other control experiments (n = 10), intravenous injection of up to 10 mg of rhodamine failed to label any neurons in the medulla except for rare labeling of neurons in the area postrema, an area with a deficient blood-brain barrier. It is worth noting that the cell bodies of both cardiac sensory and sympathetic cardiac efferent neurons are located in the peripheral nervous system and that the traditional tracers such as rhodamine cannot travel across synapses. On the day of experiment (2–4 days later), the animals were anesthetized with halothane and sacrificed by rapid cervical dislocation. The brain was submerged in cold (4°C) buffer of the following composition: 140 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 5 mM glucose, and 10 mM HEPES, and continually gassed with 100% O₂. Under a dissection microscope, the cerebellum was removed, and the hindbrain was isolated. The brain stem was then secured in the slicing chamber of a vibratome filled with the same buffer; its rostral end was set upward, and the dorsal surface was glued to a wax block facing the razor. Slices of 300-, 500-, and 700-µm thickness for GABAergic, glycinergic, and glutamatergic postsynaptic currents studied, respectively, were taken. All animal procedures were performed in compliance with the institutional guidelines at George Washington University and are in accordance with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association and the National Institutes of Health publication Guide for the Care and Use of Laboratory Animals. Slices were mounted in a perfusion chamber and submerged in the perfusate of following composition: 125 mM NaCl, 3 mM KCl, 2 mM CaCl₂, 26 mM NaHCO₃, 5 mM dextrose, and 5 mM HEPES, constantly bubbled with gas (95% O₂/5% CO₂) and maintained at pH 7.4. In experiments that examined inhibitory or excitatory miniature postsynaptic currents (mIPSCs or mEPSCs, respectively), tetrodotoxin (0.5 µM) was included in the bath, and the concentration of KCl was increased to 23 mM to increase the frequency of mIPSCs. Individual cardiac vagal neurons in the nucleus ambiguus were identified by the presence of the fluorescent tracer using a Zeiss Axioskop upright microscope (Carl Zeiss Inc., Thornwood, NY) using a 40x water immersion objective. These identified cardiac vagal neurons were then imaged with differential interference contrast optics, infrared illumination, and infrared-sensitive video detection cameras to gain better spatial resolution. Cardiac vagal neurons were studied using the whole-cell patch-clamp technique and were voltage clamped at a holding potential of -80 mV.

In experiments that examined GABAergic and glycinergic IPSCs, the patch pipettes were filled with a solution consisting of 150 mM KCl, 2 mM MgCl₂, 2 mM EGTA, 10 mM HEPES, and 2 mM Mg-ATP, pH 7.35. With this pipette solution, the chloride current induced by activation of GABA or glycine receptors was recorded as an inward current (calculated reversal potential of chloride, +0.3 mV). In experiments that examined glutamatergic EPSCs, the patch pipettes were filled with a solution consisting of 135 mM K-gluconic acid, 10 mM HEPES, 10 mM ethylene glucol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 1 mM CaCl₂, and 1 mM MgCl₂, pH 7.35. Patch pipettes were mounted onto a pipette holder and amplifier head stage (Axopatch 200B; Axon Instruments, Union City, CA), which was positioned using micromanipulators (Narashige, Tokyo, Japan).

To examine whether hcrt-1 modulates either GABAergic, glycinergic, or glutamatergic synaptic activity to cardiac vagal neurons, 1 µM hcrt-1 was applied by inclusion in the perfusate. GABAergic IPSCs and mIPSCs were isolated by the inclusion in the perfusate of strychnine (1 µM), D-2-amino-5-phosphonovalerate (AP5; 50 µM), and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 50 µM) to block glycine, NMDA, and non-NMDA receptors, respectively. Glycinergic IPSCs and mIPSCs were isolated by the inclusion in the perfusate of gabazine (25 µM), AP5 (50 µM), and CNQX (50 µM) to block GABA, NMDA, and non-NMDA receptors, respectively. Glutamatergic EPSCs and mEPSCs were isolated by the inclusion of strychnine (1 µM) and gabazine (25 µM) in the perfusate to block glycine and GABA receptors, respectively. All drugs were purchased from Sigma-Aldrich (St. Louis, MO).

Analysis of action potential-dependent IPSCs and EPSCs and tetrodotoxin-insensitive mIPSCs and mEPSCs was performed using MiniAnalysis (version 4.3.1; Synaptosoft, Decatur, GA) with a minimal acceptable amplitude of GABAergic, glycinergic IPSCs, and glutamatergic EPSCs at 10 to 20 pA and that of the mIPSCs and mEPSCs at 8 to 15 pA. Spontaneous synaptic event frequency and amplitude was analyzed from a 3-min period prior to application of hcrt-1 (control) and a 3-min period after hcrt-1 application. Results are presented as means ± S.E. and statistically compared with the nonparametric Kolmogorov-Smirnov (K-S) test (significant difference was set at p < 0.01).

	Results

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Application of hcrt-1 (1 µM) caused significant increases in both the frequency and amplitude of GABAergic IPSCs. Hcrt-1 (1 µM) increased the frequency of GABAergic IPSCs in 11 of 12 cardiac vagal neurons (mean increase, 126%; from 2.4 ± 0.5 to 5.4 ± 1.2 Hz; p < 0.001, K-S test; n = 12). The change in the amplitude of GABAergic IPSCs was more variable and ranged from 0 to 60% (mean increase, 14%; from 54.0 ± 5.9 to 61.5 ± 5.5 pA; p < 0.001, K-S test; n = 12). The responses to hcrt-1 were reversible; see Fig. 1. The data from a single experiment are shown in Fig. 1, A–C, whereas the summary data are illustrated in Fig. 1D. At the end of experiments, the GABA_A receptor antagonist gabazine (25 µM) blocked the GABAergic IPSCs; see Fig. 1.

View larger version (27K): [in this window] [in a new window]   Fig. 1. In a typical experiment (A), application of 1 µM hcrt-1 reversibly increased both the frequency and the amplitude of GABAergic IPSCs in cardiac vagal neurons. Cumulative fraction plot (B) for this experiment indicates a significant increase (p < 0.001, K-S test) in both the frequency (top) and amplitude (bottom) of GABAergic IPSCs. The time course for this experiment is shown in C, whereas the summary data are illustrated in D. Hcrt-1 (1 µM) significantly increased the GABAergic IPSC frequency and the GABAergic IPSCs amplitude (p < 0.001, K-S test; n = 12). At the end of the experiment, 25 µM gabazine blocked the GABAergic IPSCs.

View larger version (29K): [in this window] [in a new window]   Fig. 2. In the presence of 0.5 µM tetrodotoxin, 1 µM hcrt-1 did not evoke a significant change in either GABAergic mIPSC frequency or amplitude (p > 0.01, K-S test; n = 7), as shown in a single experiment (A and B) and in the summary data (C).

Hcrt-1 (1 µM) also caused significant increases in both the frequency from 2.3 ± 0.4 to 3.9 ± 0.6 Hz (p < 0.001, K-S test; n = 11) and the amplitude from 58.8 ± 4.0 to 77.0 ± 9.1 pA (p < 0.001, K-S test; n = 11) of glycinergic IPSCs in cardiac vagal neurons; see Fig. 3. The increases in frequency were observed in all but one neuron tested (mean increase, 72%; n = 11). The change in the amplitude of glycinergic IPSCs varied from 6 to 78% (mean increase, 31%; n = 11). The effects of hcrt-1 were reversible; see Fig. 3. The data from a single experiment are shown in Fig. 3, A–C, whereas the results from 11 neurons are shown in Fig. 3D. At the end of the experiments the glycine receptor antagonist strychnine (1 µM) blocked the glycinergic IPSCs (data not shown).

View larger version (31K): [in this window] [in a new window]   Fig. 3. Similar to the GABAergic responses, 1 µM hcrt-1 reversibly increased both the frequency and amplitude of glycinergic IPSCs in a typical experiment (A). Cumulative fraction plot (B) for this experiment indicates a significant increase (p < 0.001, K-S test) in both the frequency (top) and amplitude (bottom) of glycinergic IPSCs. The time course from one experiment is shown in C, and the results from 11 neurons are shown in D. Hcrt-1 (1 µM) significantly increased the glycinergic IPSC frequency and amplitude (p < 0.001, K-S test; n = 11).

View larger version (27K): [in this window] [in a new window]   Fig. 4. In a typical experiment (A), in the presence of 0.5 µM tetrodotoxin, 1 µM hcrt-1 had no significant effect on either glycinergic mIPSC frequency or amplitude (p > 0.01, K-S test; n = 6). The time course for this experiment is shown in B, whereas the summary data are demonstrated in C.

Hcrt-1 (1 µM) significantly decreased the frequency of excitatory glutamatergic EPSCs in a majority of cardiac vagal neurons (nine of 13), although in some cardiac vagal neurons hcrt-1 caused either an increase in frequency (three cells) or no change (one cell) (mean decrease in frequency, 44%; from 2.5 ± 0.7 to 1.4 ± 0.5 Hz; p < 0.001, K-S test; n = 13). Application of 1 µM hcrt-1 had no significant effect on the amplitude of glutamatergic EPSCs (33.6 ± 2.2 versus 30.8 ± 1.5 pA; p > 0.01, K-S test; n = 13). The hcrt-1 effects on the frequency of glutamatergic EPSCs were reversible; see Fig. 5. The data from a single experiment are shown in Fig. 5, A–C, whereas the results from 13 neurons are shown in Fig. 5D. At the end of experiments, the glutamate receptor antagonists AP5 (50 µM) and CNQX (50 µM) blocked the EPSCs (data not shown).

View larger version (31K): [in this window] [in a new window]   Fig. 5. The time course from a single glutamatergic EPSC experiment is shown in A and C, whereas a cumulative fraction plot for this experiment is demonstrated in B. Application of hcrt-1 reversibly decreased the frequency of glutamatergic EPSCs (p < 0.001, K-S test). The result from 13 neurons are demonstrated in D. Hcrt-1 (1 µM) significantly decreased the frequency glutamatergic EPSCs (p < 0.001, K-S test; n = 13) but had no significant effect on glutamatergic EPSC amplitude (p > 0.01, K-S test; n = 13).

View larger version (33K): [in this window] [in a new window]   Fig. 6. Hcrt-1 (1 µM) did not evoke a significant change in either glutamatergic mEPSC frequency or amplitude (p > 0.01, K-S test; n = 11, respectively), as shown in a typical experiment (A and B) and the summary data from 11 neurons (C).

	Discussion

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There are two major findings in this work. Application of 1 µM hcrt-1 inhibited cardiac vagal neurons in the nucleus ambiguus via a significant increase in the frequency and amplitude of both inhibitory GABAergic and glycinergic IPSCs as well as a significant decrease in the frequency of excitatory glutamatergic EPSCs. The action of hcrt-1 likely occurs via changing the activity of neurons precedent to cardiac vagal since 1 µM hcrt-1 did not significantly alter either GABAergic and glycinergic mIPSCs or glutamatergic mEPSCs.

In other brain regions, hcrt-1 at concentrations of 0.1 to 10 µM has been shown to alter the excitatory and/or inhibitory spontaneous synaptic currents. Hcrt-1 (1 µM) increased both GABAergic and glutamatergic postsynaptic neurotransmission in hypothalamic cultured cells. The enhanced IPSC frequency appeared to involve peptide binding at presynaptic terminals because it was maintained after blocking action potential-depending neurotransmission release with tetrodotoxin (van den Pol et al., 1998). Hcrt-1 (0.3–1 µM) caused an increase in the frequency of spontaneous IPSCs in neurons in the dorsal motor nucleus of the vagus, and the effect was preserved in the presence of tetrodotoxin, suggesting presynaptic action of the peptide (Davis et al., 2003). The concentration-dependent effect of Hcrt-1 increasing IPSC frequency closely resembles a linear dose-response relationship from 30 nM to 1 µM (Davis et al., 2003). Hcrt-1 increased GABAergic postsynaptic currents in dorsal raphe nucleus neurons at concentrations of 0.1 to 10 µM (including 1 µM) (Liu et al., 2002) and the frequency and amplitude of glutamatergic postsynaptic currents in laterodorsal tegmental neurons at a concentration of 1 µM (Burlet et al., 2002). These changes did not occur in the present of tetrodotoxin (Burlet et al., 2002; Liu et al., 2002). In this study, hcrt-1 was applied at a concentration of 1 µM, consistent with other electrophysiological studies that have examined the effects of hcrt-1 on synaptic neurotransmission (Burlet et al., 2002; Liu et al., 2002; Davis et al., 2003). We found that hcrt-1 increased the frequency of both GABAergic and glycinergic IPSCs of cardiac vagal neurons in the nucleus ambiguus. Since the increases in the frequency of the inhibitory neurotransmission were tetrodotoxin-sensitive, hcrt-1 likely acted on preceding GABAergic and glycinergic neurons to evoke action potential-dependent changes, which increased the IPSC frequency. The amplitude of GABAergic and glycinergic IPSCs also increased with hcrt-1 in cardiac vagal neurons. An increase in amplitude of spontaneous postsynaptic currents is usually thought to be a postsynaptic effect, but in this case the changes did not persist in the present of tetrodotoxin and therefore likely resulted from action of hcrt-1 on preceding GABAergic and glycinergic neurons. Presynaptic action potentials evoked by hcrt-1 may synchronize quanta release across multiple terminals arising from single afferents of preceding neurons or may release multiple quanta from single terminals to produce larger spontaneous postsynaptic currents (Burlet et al., 2002). In addition, hcrt-1 produced a tetrodotoxin-sensitive decrease in the frequency of glutamatergic neurotransmission, indicating hcrt-1 likely also acts on glutamatergic neurons that project to cardiac vagal neurons. To our knowledge this is the first demonstration that hcrt-1 can decrease excitatory neurotransmission.

Although the site of action with bath-applied hcrt-1 in this study is unknown, other work suggests hcrt-1 could act on GABAergic and glutamatergic neurons in the NTS, a medullary area that receives cardiorespiratory sensory inputs. One site of glutamatergic and GABAergic inputs to cardiac vagal neurons within the nucleus ambiguus have recently been demonstrated to originate from the NTS (Neff et al., 1998; Wang et al., 2001a). In addition, the NTS have been reported to receive a direct projection from hcrt-1-containing neurons (de Oliveira et al., 2003). The hypothesis that the hcrt-1-sensitive synaptic pathways originate in the NTS is also supported by the observations that intracerebroventricular injections of hypocretins induced c-fos expression in the NTS neurons (Date et al., 1999), and application of hcrt-1 depolarized 91% of NTS neurons (Yang and Ferguson, 2003). However, it is very possible hcrt-1 could act on other neurons within the slice located inside and/or outside of the nucleus ambiguus.

Although hcrt-1 evoked significant changes in GABAergic, glycinergic, and glutamatergic neurotransmission to cardiac vagal neurons, there was some heterogeneity to the responses. Similarly, in other in vitro studies, hcrt-1 has been reported to produce heterogeneous responses (van den Pol et al., 1998; Burlet et al., 2002; Liu et al., 2002). It is possible that there may be a number of different groups of hcrt-1-sensitive cells that project to the cardiac vagal neurons that are differently regulated by hcrt-1. It is also possible that the result of hcrt-1 system modulation depends variable functional states of individual neurons. A number of earlier findings are consistent with these possibilities. Microinjection of hcrt-1 into different regions of the NTS caused divergent responses of either increases or decreases in arterial pressure and heart rate (Smith et al., 2002; de Oliveira et al., 2003). The effect of hcrt-1 microinjected 1 mm dorsal to the right lateral ventricle on food intake has been demonstrated to be both dose- and time-dependent (Rodgers et al., 2000). Therefore, hcrt-1-containing neurons can exert complex and opposing effects even within the same site within the central nervous system, and the effect may depend on specific physiological states. Taking these facts together, it is not too surprising that the findings of this in vitro study from neonatal rats are in contrast with the results from Ciriello and de Olivera (2003), who have shown that microinjection of hcrt-1 (0.5–2.5 pmol) into the nucleus ambiguus of anesthetized adult rats elicited a decrease in heart rate. The authors speculated that hcrt-1 either exerted the effect directly on vagal cardiac neurons, or it may have altered the release of a transmitter contained in afferents from the NTS. There are a number of possible explanations for this apparent discrepancy. 1) The cardiac vagal neurons in the slice preparation used in this study are disconnected from pathways originating from outside this preparation, which may contribute and change the hcrt-1 effect in a whole animal. 2) Anesthetics used in vivo could change the responses by altering the balance between different neurons that project to cardiac vagal neurons in the nucleus ambiguus. 3) The age of animals may contribute to the hcrt-1 effect in the nucleus ambiguus. 4) The cardiac effect of microinjection of hcrt-1 into the nucleus ambiguus in a whole animal may include responses from nearby sympathoinhibitory neurons in the caudal ventrolateral medulla as well as sympathoexcitatory neurons of rostral ventrolateral medulla, since the nucleus ambiguus has been shown to overlap these areas (Ciriello et al., 1986; Ciriello and de Oliveira, 2003). 5) Additionally, the discrepancy in the results of two studies could be due to the different concentrations of hcrt-1 used.

In summary, the findings of this work support earlier data that demonstrated physiological effects of hcrt-1 in central control of cardiovascular function (Samson et al., 1999; Smith et al., 2002; Ciriello and de Oliveira, 2003). Moreover, our results suggest that hcrt-1 may control cardiovascular function not only by stimulation of sympathetic function as reported by others (Shirasaka et al., 1999; Chen et al., 2000; Antunes et al., 2001; Matsumura et al., 2001; Machado et al., 2002) but also by diminishing parasympathetic cardiac vagal activity via increased inhibitory and decreased excitatory neurotransmission to cardiac vagal neurons in the nucleus ambiguus. Our results are in accordance with the finding by Kayaba et al. (2003), who have reported that hypocretin knockout mice showed diminished cardiovascular responses to emotional stress. The findings of this study demonstrate mechanisms by which the peptide hcrt-1 may inhibit the cardiac vagal premotor neurons within the nucleus ambiguus and likely diminish cardiac vagal activity.

	Footnotes

 
This work was supported by National Institutes of Health Grants HL 49965, 59895, and 72006 to D.M.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.105.086421.

ABBREVIATIONS: hcrt-1, hypocretin-1; NTS, nucleus tractus solitarius; EPSC, excitatory postsynaptic current; IPSC, inhibitory postsynaptic current; mIPSC, miniature inhibitory postsynaptic current; mEPSC, miniature excitatory postsynaptic current; AP5, D-2-amino-5-phosphonovalerate; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; NMDA, N-methyl-D-aspartate; K-S, Kolmogorov-Smirnov.

Address correspondence to: Dr. David Mendelowitz, Department of Pharmacology, George Washington University, 2300 Eye St N.W., Washington, DC 20037. E-mail: dmendel{at}gwu.edu

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