- Email: [email protected]
Synaptic activation of cardiac vagal neurons by capsaicin sensitive and insensitive sensory neurons
Brain Research 979 (2003) 210–215 www.elsevier.com / locate / brainres
Research report
Synaptic activation of cardiac vagal neurons by capsaicin sensitive and insensitive sensory neurons Cory Evans, Sunit Baxi, Robert Neff, Priya Venkatesan, David Mendelowitz* Department of Pharmacology, George Washington University, 2300 Eye Street NW, Washington, DC 20037, USA Accepted 28 April 2003
Abstract Little is known about the central circuitry involved in the sensory activation of cardioinhibitory vagal neurons (CVNs). To study the polysynaptic activation of CVNs from sensory neurons the postsynaptic currents in CVNs in the dorsal motor nucleus of the vagus (DMNX) were evoked by stimulation of the vagus nerve. In addition, the role of afferent A-fiber and C-fiber activation of CVNs was examined. CVNs were identified by a retrograde fluorescent tracer and were studied in an in vitro slice preparation using patch-clamp electrophysiology. Stimulation of the vagus nerve evoked excitatory postsynaptic currents in CVNs that were reversibly blocked by the NMDA antagonist D-2-amino-5-phosphonovalerate (AP5) and the non-NMDA antagonist 6-cyano-7-nitroquionoxaline-2,3-dione (CNQX). Vagal stimulation also evoked inhibitory postsynaptic currents (IPSCs) that were reversibly blocked by the GABAA antagonist gabazine. Capsaicin, which inactivates C-fibers, was used to examine the role of afferent A-fibers and C-fibers in the synaptic activation of CVNs. Capsaicin significantly (P,0.05) reduced the amplitude of evoked glutamatergic and GABAergic postsynaptic currents by 59% and 76%, respectively. The latency of the GABAergic response increased significantly (P,0.05) in the presence of capsaicin from 3661 to 4161 ms while the latency of the glutamatergic response (4463 ms) was unaffected. There are three conclusions from this study. Stimulation of vagal afferents evokes both GABAergic and glutamatergic responses in CVNs, C-type afferent fibers are critical to the afferent stimulation of CVNs, and the A-fiber GABAergic pathway to CVNs may be more complex than the C-fiber GABAergic pathway. 2003 Elsevier B.V. All rights reserved. Theme: Endocrine and autonomic regulation Topic: Cardiovascular regulation Keywords: Baroreflex; Ambiguus; Dorsal motor nucleus; Bezold-Jarisch
1. Introduction The activity of cardioinhibitory vagal neurons (CVNs) is a major determinant of heart rate. Resting heart rate is normally dominated by the tonic activity of cardioinhibitory CVNs and is influenced to a lesser extent by excitatory sympathetic cardiac activity [11]. CVNs have been found to be localized in the dorsal motor nucleus (DMNX) and the nucleus ambiguus (NA) in the brainstem [13,22]. CVNs are intrinsically silent and therefore must rely on synaptic input to become activated [16]. Two major reflex pathways that control CVN activity are the baroreflex and the Bezold-Jarisch reflex. Baro*Corresponding author. Tel.: 11-202-994-3466; fax: 11-202-9942870. E-mail address: [email protected] (D. Mendelowitz). 0006-8993 / 03 / $ – see front matter 2003 Elsevier B.V. All rights reserved. doi:10.1016 / S0006-8993(03)02937-8
receptors, located in the carotid sinus and the aortic arch respond to increases in arterial pressure, and through a polysynaptic pathway, excite CVNs [14,15]. Similarly, CVNs are activated via a polysynaptic pathway upon activation of cardiac afferent neurons originating from the myocardium in the Bezold-Jarisch reflex. This reflex is thought to be responsible for the bradycardia that occurs during cardiac ischemia [19]. Although the physiological activation of CVNs is well established, the full circuitry of these reflexes is unknown. The first synapse of both the Bezold-Jarisch reflex and the baroreflex is located in the nucleus tractus solitarius (NTS). Aortic baroreceptor sensory fibers within the aortic depressor nerve (ADN) merge into the vagus nerve and synapse upon neurons in the NTS [5,9]. Sensory neurons involved with the Bezold-Jarisch reflex originate mostly from the cardiac ventricles and like aortic baroreceptors,
C. Evans et al. / Brain Research 979 (2003) 210–215
their afferent fibers are located within the vagus nerve and synapse in the NTS [2,12,20]. However, the complete circuitry from sensory neurons to NTS neurons, and finally the pathway to CVNs, is unknown. Although previous work has shown that stimulation of the NTS evokes both monosynaptic GABAergic and glutamatergic synaptic pathways in CVNs [17,23,24] there are a number of limitations in these studies. Stimulation of the NTS excites not only postsensory neurons, but also neurons that may not be activated by sensory pathways. Evoking synaptic responses in CVNs upon stimulation of NTS cannot provide information about the number of potential synapses within the NTS in this reflex pathway. Furthermore these studies cannot address whether activation of sensory neurons activates the glutamatergic, GABAergic or both synaptic pathways from the NTS. This work examines the neurotransmitters, receptors, and latencies that are involved in the polysynaptic activation of CVNs upon stimulation of afferent fibers in the vagus nerve as well as the relative roles of A and C-fiber stimulation in the activation of CVNs.
2. Materials and methods
2.1. Labeling of cardiac vagal neurons ( CVNs) An initial surgery was performed in Sprague–Dawley rats (6–10 days old). Animals were anaesthetized with halothane and hypothermia and the heart was exposed with a right thoracotomy between the 2nd and 3rd ribs and rhodamine (XRITC; Molecular Probes, Eugene, OR, USA) was injected into the pericardial sac and applied to the synaptic terminals of CVNs. The rats were allowed to recover for 3–7 days to allow the retrograde fluorescent tracer to label the cell bodies of CVNs in the brainstem. 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 NIH publication Guide for the Care and Use of Laboratory Animals.
2.2. Brain stem slice preparation Rats were anesthetized with halothane and exposed to hypothermia. The brain stem was removed in a solution of cold (4 8C) N-2-hydroxyethylpiperazine-N9-2-ethanesulfonic acid (HEPES)-buffered perfusate of the following composition (in mM):140 NaCl, 5 KCl, 2 CaCl 2 , 5 glucose, and 10 HEPES, continually gassed with 100% H 2 O. The vagus nerve remained attached to the medulla and was cut 1–2 mm caudal to the nodose ganglion. The brain stem and intact vagus nerve were mounted on a vibratome and a 900-mm slice was cut so as to preserve the
211
NTS, DMNX, and as many vagal nerve fiber rootlets as possible. The slice was submerged in a perfusion chamber mounted on a Zeiss Axioskop microscope. CVNs were identified by fluorescent imaging and visualized using a water immersion 340 objective and differential interference contrast optics, with infrared illumination, and an infrared sensitive camera.
2.3. Drugs, bath and pipette solutions The neurons were continuously perfused (2–3 ml / min) with a perfusate of the following composition (in mM): 125 NaCl, 3 KCl, 2 CaCl 2 , 26 NaHCO 3 , 5 glucose, and 5 HEPES, constantly bubbled with 95% O 2 –5% CO 2 and maintained at pH 7.4. Drugs used in this study were the following: the NMDA antagonist D-2-amino-5-phosphonovalerate (AP5, 50 mM final concentration), the nonNMDA antagonist 6-cyano-7-nitroquionoxaline-2,3-dione (CNQX, 50 mM final concentration), the glycine antagonist strychnine (1 mM final concentration), and the GABAA antagonist gabazine (25 mM final concentration). Drugs were dissolved in perfusate and applied focally to the patched neuron from a patch pipette (approximately 1 mm diameter) located 20–40 mm from the patched CVN neuron. Focal application was applied continuously throughout the duration of the experiment using a PV830 Pneumatic PicoPump from World Precision Instruments. In some experiments, capsaicin (1 mM final bath concentration) was added to the perfusate and allowed to perfuse the entire slice for at least 30 min to inactivate C-fiber sensory axons. To study glutamatergic synaptic events patch pipettes were filled with a solution consisting of (in mM) 135 gluconic acid, 10 HEPES, 10 ethylene glycol-bis(b-aminoethyl ether)-N,N,N9,N9 -tetraacetic acid (EGTA), 1 CaCl 2 , and 1 MgCl 2 . Strychnine and gabazine were applied simultaneously to isolate the glutamatergic currents. To determine the contribution of NMDA and non-NMDA receptors to the synaptic currents AP5 (50 mM) and CNQX (50 mM) were applied sequentially. To examine GABAergic synaptic events patch pipettes were filled with a solution consisting of (in mM): 150 KCl, 4 MgCl 2 , 2 EGTA, 2 adenosine 59-triphosphate (ATP), and 10 HEPES. Strychnine, AP5, and CNQX were applied to isolate the GABAergic postsynaptic currents. Gabazine (25 mM) was applied at the end of these experiments to confirm the isolation of GABAergic postsynaptic currents. CNQX was obtained from Tocris Cookson (Ellisville, MO, USA). All other drugs and reagents were obtained from Sigma–Aldrich (St. Louis, MO, USA).
2.4. Electrophysiological recordings and stimulation A suction pipette was advanced onto the caudal end of the vagus nerve until the nodose ganglion made a tight seal
212
C. Evans et al. / Brain Research 979 (2003) 210–215
with the tip of the pipette. Identified cardiac vagal neurons were studied using patch clamp electrophysiology methods while the vagus nerve was stimulated with a 3-ms pulse at 40–250 mA to obtain orthodromic polysynaptic responses in CVNs. Since this study is focused on orthodromic responses we rejected neurons in which the orthodromic responses could not be isolated from antidromic activation. In many cells the antidromic activation occurred at higher stimulus intensity and orthodromic responses could be selectively activated at lower stimulus intensities. Postsynaptic currents were recorded at 280 mV to avoid contamination from voltage gated currents present at potentials more positive than 250 mV.
3. Results Cardiac vagal neurons responded to vagal stimulation with both excitatory postsynaptic currents (EPSCs) as well as inhibitory postsynaptic currents (IPSCs). To isolate the excitatory glutamatergic synaptic events strychnine (1 mM) and gabazine (25 mM) were focally applied to the patch clamped CVN. Under these conditions, stimulation of the vagus nerve consistently evoked glutamatergic postsynaptic currents that occurred with a latency of 4463 ms and amplitude of 2245631 pA, see Fig. 1. In the presence of AP5 the amplitude of the evoked glutamatergic current
Fig. 1. Stimulation of the vagus nerve, illustrated by the arrows, evoked glutamatergic currents in CVNs (n56). To examine the glutamatergic currents gabazine (25 mM) and strychnine (1 mM) were focally applied and AP5 (50 mM) and CNQX (50 mM) were used to block NMDA and non-NMDA receptors, respectively. Traces shown from one CVN illustrate the glutamatergic response to vagal stimulation, top. The bottom panel illustrates peak EPSC amplitude through the time-course of the experiment. The first data point under each set of conditions represents the summary data for six CVNs (average6S.E). Evoked glutamatergic synaptic events had an average peak amplitude of 2244631 pA. In the presence of AP5, the average peak amplitude was reduced to 2134632 pA. AP5 and CNQX reversibly abolished the glutamatergic postsynaptic currents.
was significantly reduced by 45% to 2134632 pA, P, 0.05 (see Fig. 1). Co-application of AP5 and CNQX abolished the synaptic response. The block resulting from AP5 and CNQX co-application was reversible (see Fig. 1). To determine the role of A- and C-type afferent fibers in the excitatory synaptic activation of CVNs we observed the glutamatergic synaptic responses before and after addition of capsaicin (1 mM) to the perfusate. Periaxonal capsaicin has been shown to selectively eliminate C-fiber conduction while leaving the A-type response intact [7]. Capsaicin reduced the glutamatergic synaptic currents by 59% from 2168629 pA to 270614 pA (see Fig. 2). The latency of the glutamatergic responses was not significantly different. Stimulation of the afferent fibers in the vagus nerve also evoked GABAergic currents in CVNs. These currents were isolated by focal application of strychnine (1 mM), AP5 (50 mM), and CNQX (50 mM). GABAergic IPSCs had an average amplitude of 211476257 pA and occurred at an average latency of 3861 ms following vagal stimulation (n510). The GABAergic response was reversibly blocked by application of gabazine (25 mM, three of three neurons tested). To determine the role of A- and C-type afferent fibers in the inhibitory synaptic activation of CVNs we observed the GABAergic synaptic responses before and after addition of capsaicin (1 mM) to the perfusate.
Fig. 2. Traces shown illustrate glutamatergic EPSCs before and after global application of capsaicin (1 mM) in the presence of focally applied gabazine (25 mM) and strychnine (1 mM), top (n57). The bottom panel is the peak amplitude of EPSCs throughout the time course of one experiment (break.30 min). The first data point in each set of conditions is the summary data for seven CVNs (average amplitude6S.E). Capsaicin reduced the amplitude of glutamatergic synaptic events by 59% from 2168629 to 270614 pA.
C. Evans et al. / Brain Research 979 (2003) 210–215
Capsaicin had a larger effect on GABAergic neurotransmission than glutamatergic neurotransmission and reduced the GABAergic synaptic response by 76% from 214686288 pA to 2286684 pA after capsaicin application (see Fig. 3, n57). Unexpectedly, unlike the glutamatergic EPSCs which did not change in latency after capsaicin application, the latency of GABAergic IPSCs significantly increased in the presence of capsaicin from 3661 to 4161 ms (P,0.05, n57, see Fig. 3, bottom). To determine whether stimulation of vagal afferents also evoked glycinergic pathways to CVNs, the response to vagal stimulation was examined in the absence of strychnine and the presence of AP5, CNQX, and gabazine (n54). Under these conditions no synaptic currents were
Fig. 3. Traces shown illustrate GABAergic IPSCs before and after global application of capsaicin (1 mM) in the presence of focally applied AP5 (50 mM), CNQX (50 mM), and strychnine (1 mM), top (n57). Capsaicin significantly reduced (P,0.05) the amplitude of GABAergic synaptic events by 76% from 214686288 to 2285684 pA. The middle panel is the peak amplitude of IPSCs throughout the time course of one experiment (break.30 min), and the first data point in each set of conditions is the summary data for seven CVNs (average6S.E). The bottom panel is a frequency histogram showing that the latency of the GABAergic responses to vagal stimulation increases significantly (P,0.05) from an average of 3662 ms to an average of 4161 ms in the presence of capsaicin (1 mM).
213
observed, suggesting that stimulation of vagal afferent fibers did not evoke a glycinergic pathway to CVNs.
4. Discussion There are three major findings in this study. Stimulation of the vagus nerve evokes both GABAergic and glutamatergic, but not glycinergic pathways to CVNs in the DMNX. Both GABAergic and glutamatergic synaptic responses were greatly diminished in the presence of 1 mM capsaicin. The latency of glutamatergic synaptic events were not significantly increased by capsaicin, whereas capsaicin significantly increased the latency of the GABAergic pathway to CVNs. Since stimulation of baroreceptors and sensory neurons involved in the Bezold-Jarisch reflex both evoke bradycardia it is not surprising that vagal stimulation excites CVNs. However, the role of the GABAergic pathway activated upon vagal stimulation is less clear. One possibility is that afferent fibers of pulmonary stretch receptors are activated upon stimulation of the vagus nerve leading to CVN inhibition. During inspiration heart rate increases due to inhibition of CVNs, and this inhibition my be at least partially due to pulmonary stretch sensitive neurons inhibiting CVNs [10,21]. However, the GABAergic pathway to CVNs observed in this study is consistent with previous findings that electrical stimulation of the NTS evokes both glutamatergic EPSCs and GABAergic IPSCs in CVNs of the DMNX and NA [17,23,24]. The latencies observed in this study and others provide some useful information concerning the circuitry of the baroreflex. To examine the latency of the entire baroreflex Kunze stimulated the carotid sinus nerve (CSN) while recording the activity of cardiac vagal fibers. She found that the latencies of activity following CSN stimulation were 30–72 ms [14]. McAllen and Spyer, directly recording from the cell bodies of CVNs, observed evoked activity with latencies of 20–50 ms upon stimulation of the CSN [15]. Longer latencies would be expected in Kunze’s results since the latencies she observed would include the additional delay of conduction through cardiac vagal fibers. Consistent with these studies [14,15], in this study we found the latency of orthodromic responses to vagal stimulation was 4468 ms for glutamatergic EPSCs and 3861 ms for GABAergic IPSCs. Studies which have isolated the potential final central pathway to CVNs have demonstrated that stimulation of NTS evokes both IPSCs and EPSCs in CVNs with onset latencies of 11.861.1 ms, and 8–18 ms, respectively [17,23]. Subtracting the time taken for the action potentials to be conducted through either the vagus nerve or CSN (approximately 2 ms, based on an estimate of 2 mm from nodose ganglia to NTS, and a conduction velocity of 1.0 m / s), the delay of the reflex response due to interactions
214
C. Evans et al. / Brain Research 979 (2003) 210–215
within the NTS would be approximately 15 to 42 ms. This result, along with a highly variable response time, suggests that there is a complex polysynaptic circuitry within the NTS between receiving input from the afferent vagal fibers and relaying this signal to CVNs. There are two types of afferent sensory fibers in the vagus nerve. A-fibers are myelinated, have a lower firing threshold, activate uniformly, and are insensitive to capsaicin. C-fibers are unmyelinated and require a higher threshold for activation. They discharge inconsistently, have highly variable conduction velocities, and are initially excited and then become silent upon prolonged capsaicin application [4]. Yet C-fibers make up about 90% of the axons in the aortic depressor nerve (ADN) [1], which transmits baroreceptor signals in the rat [18]. Fan et al. examined the respective contribution of A- and C-fibers to the baroreflex response and reported that A-fibers were comparatively ineffective at producing reflex bradycardia and C-type baroreceptor input was necessary for maximal reflex bradycardia [8]. Our results are consistent with the study by Fan et al. and demonstrate that capsaicin significantly reduced the amplitude of evoked synaptic events in CVNs. GABAergic IPSCs were reduced by 76% and glutamatergic EPSCs reduced by 59% suggesting that the C-fiber afferent pathway plays a significant role in the activation of CVNs involved with reflex pathways. Recent work suggests the A-fiber and C-fiber pathways are segregated into two populations at the level of the NTS. NTS neurons of one type are activated exclusively from C-fibers, and their responses to stimulation of the solitary tract (ST) can be abolished by prolonged capsaicin application, while NTS neurons of a second type are activated exclusively by A-fibers and their response to ST stimulation is unaffected by capsaicin [3,6]. In this study, all CVNs studied maintained some level of response after exposure to capsaicin. This suggests that individual CVNs are not activated exclusively by the NTS neurons in Afiber or C-fiber afferent pathways, but receive inputs from both types of NTS neurons. Surprisingly, however, the latency for the GABAergic response in CVNs was increased with prolonged capsaicin application. One possible explanation for this finding is that the A-fiber pathway for GABAergic activation of CVNs contains more synapses between NTS neurons than the C-fiber pathway. It is also possible that the alterations with capsaicin in this study were due to capsaicin evoked changes in the activity of neurons within the slice in additional to altering the C-fiber pathway in the vagus nerve. Furthermore, since these results were obtained from CVNs in the DMNX it would be interesting to determine whether CVNs in the nucleus ambiguus have similar synaptic responses to vagal stimulation and alterations with capsaicin. In summary, stimulation of the afferent fibers of the vagus nerve evokes both GABAergic and glutamatergic responses in CVNs, but does not activate a glycinergic
pathway to CVNs. Capsaicin reduces the amplitude of evoked responses to vagal stimulation in CVNs, suggesting that C-type afferent fibers are critical to the afferent stimulation of CVNs. Finally, the A-fiber GABAergic pathway to CVNs may be more complex than the C-fiber pathway since capsaicin treatment increases the latency of evoked GABAergic responses in CVNs.
Acknowledgements This work was supported by NIH grants HL59895 and HL49965 to D.M.
References [1] M.C. Andresen, J.M. Krauhs, A.M. Brown, Relationship of aortic wall and baroreceptor properties during development in normotensive and spontaneously hypertensive rats, Circ. Res. 43 (1978) 728–738. [2] D.M. Aviado, D. Guevara Aviado, The Bezold-Jarisch reflex. A historical perspective of cardiopulmonary reflexes, Ann. NY Acad. Sci. 940 (2001) 48–58. [3] T.W. Bailey, Y.H. Jin, M.W. Doyle, M.C. Andresen, Vanilloidsensitive afferents activate neurons with prominent A-type potassium currents in nucleus tractus solitarius, J. Neurosci. 22 (2002) 8230–8237. [4] H.M. Coleridge, J.C. Coleridge, H.D. Schultz, Characteristics of C fibre baroreceptors in the carotid sinus of dogs, J. Physiol. 394 (1987) 291–313. [5] S. Donoghue, R.B. Felder, D. Jordan, K.M. Spyer, The central projections of carotid baroreceptors and chemoreceptors in the cat: a neurophysiological study, J. Physiol. 347 (1984) 397–409. [6] M.W. Doyle, T.W. Bailey, Y.H. Jin, M.C. Andresen, Vanilloid receptors presynaptically modulate cranial visceral afferent synaptic transmission in nucleus tractus solitarius, J. Neurosci. 22 (2002) 8222–8229. [7] W. Fan, M.C. Andresen, Differential frequency-dependent reflex integration of myelinated and nonmyelinated rat aortic baroreceptors, Am. J. Physiol. 275 (1998) H632–640. [8] W. Fan, J.H. Schild, M.C. Andresen, Graded and dynamic reflex summation of myelinated and unmyelinated rat aortic baroreceptors, Am. J. Physiol. 277 (1999) R748–756. [9] J.C. Finley, D.M. Katz, The central organization of carotid body afferent projections to the brainstem of the rat, Brain Res. 572 (1992) 108–116. [10] M.P. Gilbey, D. Jordan, D.W. Richter, K.M. Spyer, Synaptic mechanisms involved in the inspiratory modulation of vagal cardioinhibitory neurones in the cat, J. Physiol. 356 (1984) 65–78. [11] C. Heymans, E. Neil, Reflexogenic Areas of the Cardiovascular System, Churchill, London, 1958. [12] M. Kalia, M.M. Mesulam, Brain stem projections of sensory and motor components of the vagus complex in the cat: II. Laryngeal, tracheobronchial, pulmonary, cardiac, and gastrointestinal branches, J. Comp. Neurol. 193 (1980) 467–508. [13] M. Kalia, J.M. Sullivan, Brainstem projections of sensory and motor components of the vagus nerve in the rat, J. Comp. Neurol. 211 (1982) 248–265. [14] D.L. Kunze, Reflex discharge patterns of cardiac vagal efferent fibres, J. Physiol. 222 (1972) 1–15.
C. Evans et al. / Brain Research 979 (2003) 210–215 [15] R.M. McAllen, K.M. Spyer, The baroreceptor input to cardiac vagal motoneurones, J. Physiol. 282 (1978) 365–374. [16] D. Mendelowitz, Firing properties of identified parasympathetic cardiac neurons in nucleus ambiguus, Am. J. Physiol. 271 (1996) H2609–2614. [17] R.A. Neff, M. Mihalevich, D. Mendelowitz, Stimulation of NTS activates NMDA and non-NMDA receptors in rat cardiac vagal neurons in the nucleus ambiguus, Brain Res. 792 (1998) 277–282. [18] H.N. Sapru, A.J. Krieger, Carotid and aortic chemoreceptor function in the rat, J. Appl. Physiol. 42 (1977) 344–348. [19] H.D. Schultz, Cardiac vagal chemosensory afferents. Function in pathophysiological states, Ann. NY Acad. Sci. 940 (2001) 59–73. [20] L. Silva-Carvalho, J.F. Paton, I. Rocha, G.E. Goldsmith, K.M. Spyer, Convergence properties of solitary tract neurons responsive
[21] [22]
[23]
[24]
215
to cardiac receptor stimulation in the anesthetized cat, J. Neurophysiol. 79 (1998) 2374–2382. K.M. Spyer, M.P. Gilbey, Cardiorespiratory interactions in heart-rate control, Ann. NY Acad. Sci. 533 (1988) 350–357. A. Standish, L.W. Enquist, J.S. Schwaber, Innervation of the heart and its central medullary origin defined by viral tracing, Science 263 (1994) 232–234. J. Wang, M. Irnaten, D. Mendelowitz, Characteristics of spontaneous and evoked GABAergic synaptic currents in cardiac vagal neurons in rats, Brain Res. 889 (2001) 78–83. A. Willis, M. Mihalevich, R.A. Neff, D. Mendelowitz, Three types of postsynaptic glutamatergic receptors are activated in DMNX neurons upon stimulation of NTS, Am. J. Physiol. 271 (1996) R1614–1619.
Research report
Synaptic activation of cardiac vagal neurons by capsaicin sensitive and insensitive sensory neurons Cory Evans, Sunit Baxi, Robert Neff, Priya Venkatesan, David Mendelowitz* Department of Pharmacology, George Washington University, 2300 Eye Street NW, Washington, DC 20037, USA Accepted 28 April 2003
Abstract Little is known about the central circuitry involved in the sensory activation of cardioinhibitory vagal neurons (CVNs). To study the polysynaptic activation of CVNs from sensory neurons the postsynaptic currents in CVNs in the dorsal motor nucleus of the vagus (DMNX) were evoked by stimulation of the vagus nerve. In addition, the role of afferent A-fiber and C-fiber activation of CVNs was examined. CVNs were identified by a retrograde fluorescent tracer and were studied in an in vitro slice preparation using patch-clamp electrophysiology. Stimulation of the vagus nerve evoked excitatory postsynaptic currents in CVNs that were reversibly blocked by the NMDA antagonist D-2-amino-5-phosphonovalerate (AP5) and the non-NMDA antagonist 6-cyano-7-nitroquionoxaline-2,3-dione (CNQX). Vagal stimulation also evoked inhibitory postsynaptic currents (IPSCs) that were reversibly blocked by the GABAA antagonist gabazine. Capsaicin, which inactivates C-fibers, was used to examine the role of afferent A-fibers and C-fibers in the synaptic activation of CVNs. Capsaicin significantly (P,0.05) reduced the amplitude of evoked glutamatergic and GABAergic postsynaptic currents by 59% and 76%, respectively. The latency of the GABAergic response increased significantly (P,0.05) in the presence of capsaicin from 3661 to 4161 ms while the latency of the glutamatergic response (4463 ms) was unaffected. There are three conclusions from this study. Stimulation of vagal afferents evokes both GABAergic and glutamatergic responses in CVNs, C-type afferent fibers are critical to the afferent stimulation of CVNs, and the A-fiber GABAergic pathway to CVNs may be more complex than the C-fiber GABAergic pathway. 2003 Elsevier B.V. All rights reserved. Theme: Endocrine and autonomic regulation Topic: Cardiovascular regulation Keywords: Baroreflex; Ambiguus; Dorsal motor nucleus; Bezold-Jarisch
1. Introduction The activity of cardioinhibitory vagal neurons (CVNs) is a major determinant of heart rate. Resting heart rate is normally dominated by the tonic activity of cardioinhibitory CVNs and is influenced to a lesser extent by excitatory sympathetic cardiac activity [11]. CVNs have been found to be localized in the dorsal motor nucleus (DMNX) and the nucleus ambiguus (NA) in the brainstem [13,22]. CVNs are intrinsically silent and therefore must rely on synaptic input to become activated [16]. Two major reflex pathways that control CVN activity are the baroreflex and the Bezold-Jarisch reflex. Baro*Corresponding author. Tel.: 11-202-994-3466; fax: 11-202-9942870. E-mail address: [email protected] (D. Mendelowitz). 0006-8993 / 03 / $ – see front matter 2003 Elsevier B.V. All rights reserved. doi:10.1016 / S0006-8993(03)02937-8
receptors, located in the carotid sinus and the aortic arch respond to increases in arterial pressure, and through a polysynaptic pathway, excite CVNs [14,15]. Similarly, CVNs are activated via a polysynaptic pathway upon activation of cardiac afferent neurons originating from the myocardium in the Bezold-Jarisch reflex. This reflex is thought to be responsible for the bradycardia that occurs during cardiac ischemia [19]. Although the physiological activation of CVNs is well established, the full circuitry of these reflexes is unknown. The first synapse of both the Bezold-Jarisch reflex and the baroreflex is located in the nucleus tractus solitarius (NTS). Aortic baroreceptor sensory fibers within the aortic depressor nerve (ADN) merge into the vagus nerve and synapse upon neurons in the NTS [5,9]. Sensory neurons involved with the Bezold-Jarisch reflex originate mostly from the cardiac ventricles and like aortic baroreceptors,
C. Evans et al. / Brain Research 979 (2003) 210–215
their afferent fibers are located within the vagus nerve and synapse in the NTS [2,12,20]. However, the complete circuitry from sensory neurons to NTS neurons, and finally the pathway to CVNs, is unknown. Although previous work has shown that stimulation of the NTS evokes both monosynaptic GABAergic and glutamatergic synaptic pathways in CVNs [17,23,24] there are a number of limitations in these studies. Stimulation of the NTS excites not only postsensory neurons, but also neurons that may not be activated by sensory pathways. Evoking synaptic responses in CVNs upon stimulation of NTS cannot provide information about the number of potential synapses within the NTS in this reflex pathway. Furthermore these studies cannot address whether activation of sensory neurons activates the glutamatergic, GABAergic or both synaptic pathways from the NTS. This work examines the neurotransmitters, receptors, and latencies that are involved in the polysynaptic activation of CVNs upon stimulation of afferent fibers in the vagus nerve as well as the relative roles of A and C-fiber stimulation in the activation of CVNs.
2. Materials and methods
2.1. Labeling of cardiac vagal neurons ( CVNs) An initial surgery was performed in Sprague–Dawley rats (6–10 days old). Animals were anaesthetized with halothane and hypothermia and the heart was exposed with a right thoracotomy between the 2nd and 3rd ribs and rhodamine (XRITC; Molecular Probes, Eugene, OR, USA) was injected into the pericardial sac and applied to the synaptic terminals of CVNs. The rats were allowed to recover for 3–7 days to allow the retrograde fluorescent tracer to label the cell bodies of CVNs in the brainstem. 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 NIH publication Guide for the Care and Use of Laboratory Animals.
2.2. Brain stem slice preparation Rats were anesthetized with halothane and exposed to hypothermia. The brain stem was removed in a solution of cold (4 8C) N-2-hydroxyethylpiperazine-N9-2-ethanesulfonic acid (HEPES)-buffered perfusate of the following composition (in mM):140 NaCl, 5 KCl, 2 CaCl 2 , 5 glucose, and 10 HEPES, continually gassed with 100% H 2 O. The vagus nerve remained attached to the medulla and was cut 1–2 mm caudal to the nodose ganglion. The brain stem and intact vagus nerve were mounted on a vibratome and a 900-mm slice was cut so as to preserve the
211
NTS, DMNX, and as many vagal nerve fiber rootlets as possible. The slice was submerged in a perfusion chamber mounted on a Zeiss Axioskop microscope. CVNs were identified by fluorescent imaging and visualized using a water immersion 340 objective and differential interference contrast optics, with infrared illumination, and an infrared sensitive camera.
2.3. Drugs, bath and pipette solutions The neurons were continuously perfused (2–3 ml / min) with a perfusate of the following composition (in mM): 125 NaCl, 3 KCl, 2 CaCl 2 , 26 NaHCO 3 , 5 glucose, and 5 HEPES, constantly bubbled with 95% O 2 –5% CO 2 and maintained at pH 7.4. Drugs used in this study were the following: the NMDA antagonist D-2-amino-5-phosphonovalerate (AP5, 50 mM final concentration), the nonNMDA antagonist 6-cyano-7-nitroquionoxaline-2,3-dione (CNQX, 50 mM final concentration), the glycine antagonist strychnine (1 mM final concentration), and the GABAA antagonist gabazine (25 mM final concentration). Drugs were dissolved in perfusate and applied focally to the patched neuron from a patch pipette (approximately 1 mm diameter) located 20–40 mm from the patched CVN neuron. Focal application was applied continuously throughout the duration of the experiment using a PV830 Pneumatic PicoPump from World Precision Instruments. In some experiments, capsaicin (1 mM final bath concentration) was added to the perfusate and allowed to perfuse the entire slice for at least 30 min to inactivate C-fiber sensory axons. To study glutamatergic synaptic events patch pipettes were filled with a solution consisting of (in mM) 135 gluconic acid, 10 HEPES, 10 ethylene glycol-bis(b-aminoethyl ether)-N,N,N9,N9 -tetraacetic acid (EGTA), 1 CaCl 2 , and 1 MgCl 2 . Strychnine and gabazine were applied simultaneously to isolate the glutamatergic currents. To determine the contribution of NMDA and non-NMDA receptors to the synaptic currents AP5 (50 mM) and CNQX (50 mM) were applied sequentially. To examine GABAergic synaptic events patch pipettes were filled with a solution consisting of (in mM): 150 KCl, 4 MgCl 2 , 2 EGTA, 2 adenosine 59-triphosphate (ATP), and 10 HEPES. Strychnine, AP5, and CNQX were applied to isolate the GABAergic postsynaptic currents. Gabazine (25 mM) was applied at the end of these experiments to confirm the isolation of GABAergic postsynaptic currents. CNQX was obtained from Tocris Cookson (Ellisville, MO, USA). All other drugs and reagents were obtained from Sigma–Aldrich (St. Louis, MO, USA).
2.4. Electrophysiological recordings and stimulation A suction pipette was advanced onto the caudal end of the vagus nerve until the nodose ganglion made a tight seal
212
C. Evans et al. / Brain Research 979 (2003) 210–215
with the tip of the pipette. Identified cardiac vagal neurons were studied using patch clamp electrophysiology methods while the vagus nerve was stimulated with a 3-ms pulse at 40–250 mA to obtain orthodromic polysynaptic responses in CVNs. Since this study is focused on orthodromic responses we rejected neurons in which the orthodromic responses could not be isolated from antidromic activation. In many cells the antidromic activation occurred at higher stimulus intensity and orthodromic responses could be selectively activated at lower stimulus intensities. Postsynaptic currents were recorded at 280 mV to avoid contamination from voltage gated currents present at potentials more positive than 250 mV.
3. Results Cardiac vagal neurons responded to vagal stimulation with both excitatory postsynaptic currents (EPSCs) as well as inhibitory postsynaptic currents (IPSCs). To isolate the excitatory glutamatergic synaptic events strychnine (1 mM) and gabazine (25 mM) were focally applied to the patch clamped CVN. Under these conditions, stimulation of the vagus nerve consistently evoked glutamatergic postsynaptic currents that occurred with a latency of 4463 ms and amplitude of 2245631 pA, see Fig. 1. In the presence of AP5 the amplitude of the evoked glutamatergic current
Fig. 1. Stimulation of the vagus nerve, illustrated by the arrows, evoked glutamatergic currents in CVNs (n56). To examine the glutamatergic currents gabazine (25 mM) and strychnine (1 mM) were focally applied and AP5 (50 mM) and CNQX (50 mM) were used to block NMDA and non-NMDA receptors, respectively. Traces shown from one CVN illustrate the glutamatergic response to vagal stimulation, top. The bottom panel illustrates peak EPSC amplitude through the time-course of the experiment. The first data point under each set of conditions represents the summary data for six CVNs (average6S.E). Evoked glutamatergic synaptic events had an average peak amplitude of 2244631 pA. In the presence of AP5, the average peak amplitude was reduced to 2134632 pA. AP5 and CNQX reversibly abolished the glutamatergic postsynaptic currents.
was significantly reduced by 45% to 2134632 pA, P, 0.05 (see Fig. 1). Co-application of AP5 and CNQX abolished the synaptic response. The block resulting from AP5 and CNQX co-application was reversible (see Fig. 1). To determine the role of A- and C-type afferent fibers in the excitatory synaptic activation of CVNs we observed the glutamatergic synaptic responses before and after addition of capsaicin (1 mM) to the perfusate. Periaxonal capsaicin has been shown to selectively eliminate C-fiber conduction while leaving the A-type response intact [7]. Capsaicin reduced the glutamatergic synaptic currents by 59% from 2168629 pA to 270614 pA (see Fig. 2). The latency of the glutamatergic responses was not significantly different. Stimulation of the afferent fibers in the vagus nerve also evoked GABAergic currents in CVNs. These currents were isolated by focal application of strychnine (1 mM), AP5 (50 mM), and CNQX (50 mM). GABAergic IPSCs had an average amplitude of 211476257 pA and occurred at an average latency of 3861 ms following vagal stimulation (n510). The GABAergic response was reversibly blocked by application of gabazine (25 mM, three of three neurons tested). To determine the role of A- and C-type afferent fibers in the inhibitory synaptic activation of CVNs we observed the GABAergic synaptic responses before and after addition of capsaicin (1 mM) to the perfusate.
Fig. 2. Traces shown illustrate glutamatergic EPSCs before and after global application of capsaicin (1 mM) in the presence of focally applied gabazine (25 mM) and strychnine (1 mM), top (n57). The bottom panel is the peak amplitude of EPSCs throughout the time course of one experiment (break.30 min). The first data point in each set of conditions is the summary data for seven CVNs (average amplitude6S.E). Capsaicin reduced the amplitude of glutamatergic synaptic events by 59% from 2168629 to 270614 pA.
C. Evans et al. / Brain Research 979 (2003) 210–215
Capsaicin had a larger effect on GABAergic neurotransmission than glutamatergic neurotransmission and reduced the GABAergic synaptic response by 76% from 214686288 pA to 2286684 pA after capsaicin application (see Fig. 3, n57). Unexpectedly, unlike the glutamatergic EPSCs which did not change in latency after capsaicin application, the latency of GABAergic IPSCs significantly increased in the presence of capsaicin from 3661 to 4161 ms (P,0.05, n57, see Fig. 3, bottom). To determine whether stimulation of vagal afferents also evoked glycinergic pathways to CVNs, the response to vagal stimulation was examined in the absence of strychnine and the presence of AP5, CNQX, and gabazine (n54). Under these conditions no synaptic currents were
Fig. 3. Traces shown illustrate GABAergic IPSCs before and after global application of capsaicin (1 mM) in the presence of focally applied AP5 (50 mM), CNQX (50 mM), and strychnine (1 mM), top (n57). Capsaicin significantly reduced (P,0.05) the amplitude of GABAergic synaptic events by 76% from 214686288 to 2285684 pA. The middle panel is the peak amplitude of IPSCs throughout the time course of one experiment (break.30 min), and the first data point in each set of conditions is the summary data for seven CVNs (average6S.E). The bottom panel is a frequency histogram showing that the latency of the GABAergic responses to vagal stimulation increases significantly (P,0.05) from an average of 3662 ms to an average of 4161 ms in the presence of capsaicin (1 mM).
213
observed, suggesting that stimulation of vagal afferent fibers did not evoke a glycinergic pathway to CVNs.
4. Discussion There are three major findings in this study. Stimulation of the vagus nerve evokes both GABAergic and glutamatergic, but not glycinergic pathways to CVNs in the DMNX. Both GABAergic and glutamatergic synaptic responses were greatly diminished in the presence of 1 mM capsaicin. The latency of glutamatergic synaptic events were not significantly increased by capsaicin, whereas capsaicin significantly increased the latency of the GABAergic pathway to CVNs. Since stimulation of baroreceptors and sensory neurons involved in the Bezold-Jarisch reflex both evoke bradycardia it is not surprising that vagal stimulation excites CVNs. However, the role of the GABAergic pathway activated upon vagal stimulation is less clear. One possibility is that afferent fibers of pulmonary stretch receptors are activated upon stimulation of the vagus nerve leading to CVN inhibition. During inspiration heart rate increases due to inhibition of CVNs, and this inhibition my be at least partially due to pulmonary stretch sensitive neurons inhibiting CVNs [10,21]. However, the GABAergic pathway to CVNs observed in this study is consistent with previous findings that electrical stimulation of the NTS evokes both glutamatergic EPSCs and GABAergic IPSCs in CVNs of the DMNX and NA [17,23,24]. The latencies observed in this study and others provide some useful information concerning the circuitry of the baroreflex. To examine the latency of the entire baroreflex Kunze stimulated the carotid sinus nerve (CSN) while recording the activity of cardiac vagal fibers. She found that the latencies of activity following CSN stimulation were 30–72 ms [14]. McAllen and Spyer, directly recording from the cell bodies of CVNs, observed evoked activity with latencies of 20–50 ms upon stimulation of the CSN [15]. Longer latencies would be expected in Kunze’s results since the latencies she observed would include the additional delay of conduction through cardiac vagal fibers. Consistent with these studies [14,15], in this study we found the latency of orthodromic responses to vagal stimulation was 4468 ms for glutamatergic EPSCs and 3861 ms for GABAergic IPSCs. Studies which have isolated the potential final central pathway to CVNs have demonstrated that stimulation of NTS evokes both IPSCs and EPSCs in CVNs with onset latencies of 11.861.1 ms, and 8–18 ms, respectively [17,23]. Subtracting the time taken for the action potentials to be conducted through either the vagus nerve or CSN (approximately 2 ms, based on an estimate of 2 mm from nodose ganglia to NTS, and a conduction velocity of 1.0 m / s), the delay of the reflex response due to interactions
214
C. Evans et al. / Brain Research 979 (2003) 210–215
within the NTS would be approximately 15 to 42 ms. This result, along with a highly variable response time, suggests that there is a complex polysynaptic circuitry within the NTS between receiving input from the afferent vagal fibers and relaying this signal to CVNs. There are two types of afferent sensory fibers in the vagus nerve. A-fibers are myelinated, have a lower firing threshold, activate uniformly, and are insensitive to capsaicin. C-fibers are unmyelinated and require a higher threshold for activation. They discharge inconsistently, have highly variable conduction velocities, and are initially excited and then become silent upon prolonged capsaicin application [4]. Yet C-fibers make up about 90% of the axons in the aortic depressor nerve (ADN) [1], which transmits baroreceptor signals in the rat [18]. Fan et al. examined the respective contribution of A- and C-fibers to the baroreflex response and reported that A-fibers were comparatively ineffective at producing reflex bradycardia and C-type baroreceptor input was necessary for maximal reflex bradycardia [8]. Our results are consistent with the study by Fan et al. and demonstrate that capsaicin significantly reduced the amplitude of evoked synaptic events in CVNs. GABAergic IPSCs were reduced by 76% and glutamatergic EPSCs reduced by 59% suggesting that the C-fiber afferent pathway plays a significant role in the activation of CVNs involved with reflex pathways. Recent work suggests the A-fiber and C-fiber pathways are segregated into two populations at the level of the NTS. NTS neurons of one type are activated exclusively from C-fibers, and their responses to stimulation of the solitary tract (ST) can be abolished by prolonged capsaicin application, while NTS neurons of a second type are activated exclusively by A-fibers and their response to ST stimulation is unaffected by capsaicin [3,6]. In this study, all CVNs studied maintained some level of response after exposure to capsaicin. This suggests that individual CVNs are not activated exclusively by the NTS neurons in Afiber or C-fiber afferent pathways, but receive inputs from both types of NTS neurons. Surprisingly, however, the latency for the GABAergic response in CVNs was increased with prolonged capsaicin application. One possible explanation for this finding is that the A-fiber pathway for GABAergic activation of CVNs contains more synapses between NTS neurons than the C-fiber pathway. It is also possible that the alterations with capsaicin in this study were due to capsaicin evoked changes in the activity of neurons within the slice in additional to altering the C-fiber pathway in the vagus nerve. Furthermore, since these results were obtained from CVNs in the DMNX it would be interesting to determine whether CVNs in the nucleus ambiguus have similar synaptic responses to vagal stimulation and alterations with capsaicin. In summary, stimulation of the afferent fibers of the vagus nerve evokes both GABAergic and glutamatergic responses in CVNs, but does not activate a glycinergic
pathway to CVNs. Capsaicin reduces the amplitude of evoked responses to vagal stimulation in CVNs, suggesting that C-type afferent fibers are critical to the afferent stimulation of CVNs. Finally, the A-fiber GABAergic pathway to CVNs may be more complex than the C-fiber pathway since capsaicin treatment increases the latency of evoked GABAergic responses in CVNs.
Acknowledgements This work was supported by NIH grants HL59895 and HL49965 to D.M.
References [1] M.C. Andresen, J.M. Krauhs, A.M. Brown, Relationship of aortic wall and baroreceptor properties during development in normotensive and spontaneously hypertensive rats, Circ. Res. 43 (1978) 728–738. [2] D.M. Aviado, D. Guevara Aviado, The Bezold-Jarisch reflex. A historical perspective of cardiopulmonary reflexes, Ann. NY Acad. Sci. 940 (2001) 48–58. [3] T.W. Bailey, Y.H. Jin, M.W. Doyle, M.C. Andresen, Vanilloidsensitive afferents activate neurons with prominent A-type potassium currents in nucleus tractus solitarius, J. Neurosci. 22 (2002) 8230–8237. [4] H.M. Coleridge, J.C. Coleridge, H.D. Schultz, Characteristics of C fibre baroreceptors in the carotid sinus of dogs, J. Physiol. 394 (1987) 291–313. [5] S. Donoghue, R.B. Felder, D. Jordan, K.M. Spyer, The central projections of carotid baroreceptors and chemoreceptors in the cat: a neurophysiological study, J. Physiol. 347 (1984) 397–409. [6] M.W. Doyle, T.W. Bailey, Y.H. Jin, M.C. Andresen, Vanilloid receptors presynaptically modulate cranial visceral afferent synaptic transmission in nucleus tractus solitarius, J. Neurosci. 22 (2002) 8222–8229. [7] W. Fan, M.C. Andresen, Differential frequency-dependent reflex integration of myelinated and nonmyelinated rat aortic baroreceptors, Am. J. Physiol. 275 (1998) H632–640. [8] W. Fan, J.H. Schild, M.C. Andresen, Graded and dynamic reflex summation of myelinated and unmyelinated rat aortic baroreceptors, Am. J. Physiol. 277 (1999) R748–756. [9] J.C. Finley, D.M. Katz, The central organization of carotid body afferent projections to the brainstem of the rat, Brain Res. 572 (1992) 108–116. [10] M.P. Gilbey, D. Jordan, D.W. Richter, K.M. Spyer, Synaptic mechanisms involved in the inspiratory modulation of vagal cardioinhibitory neurones in the cat, J. Physiol. 356 (1984) 65–78. [11] C. Heymans, E. Neil, Reflexogenic Areas of the Cardiovascular System, Churchill, London, 1958. [12] M. Kalia, M.M. Mesulam, Brain stem projections of sensory and motor components of the vagus complex in the cat: II. Laryngeal, tracheobronchial, pulmonary, cardiac, and gastrointestinal branches, J. Comp. Neurol. 193 (1980) 467–508. [13] M. Kalia, J.M. Sullivan, Brainstem projections of sensory and motor components of the vagus nerve in the rat, J. Comp. Neurol. 211 (1982) 248–265. [14] D.L. Kunze, Reflex discharge patterns of cardiac vagal efferent fibres, J. Physiol. 222 (1972) 1–15.
C. Evans et al. / Brain Research 979 (2003) 210–215 [15] R.M. McAllen, K.M. Spyer, The baroreceptor input to cardiac vagal motoneurones, J. Physiol. 282 (1978) 365–374. [16] D. Mendelowitz, Firing properties of identified parasympathetic cardiac neurons in nucleus ambiguus, Am. J. Physiol. 271 (1996) H2609–2614. [17] R.A. Neff, M. Mihalevich, D. Mendelowitz, Stimulation of NTS activates NMDA and non-NMDA receptors in rat cardiac vagal neurons in the nucleus ambiguus, Brain Res. 792 (1998) 277–282. [18] H.N. Sapru, A.J. Krieger, Carotid and aortic chemoreceptor function in the rat, J. Appl. Physiol. 42 (1977) 344–348. [19] H.D. Schultz, Cardiac vagal chemosensory afferents. Function in pathophysiological states, Ann. NY Acad. Sci. 940 (2001) 59–73. [20] L. Silva-Carvalho, J.F. Paton, I. Rocha, G.E. Goldsmith, K.M. Spyer, Convergence properties of solitary tract neurons responsive
[21] [22]
[23]
[24]
215
to cardiac receptor stimulation in the anesthetized cat, J. Neurophysiol. 79 (1998) 2374–2382. K.M. Spyer, M.P. Gilbey, Cardiorespiratory interactions in heart-rate control, Ann. NY Acad. Sci. 533 (1988) 350–357. A. Standish, L.W. Enquist, J.S. Schwaber, Innervation of the heart and its central medullary origin defined by viral tracing, Science 263 (1994) 232–234. J. Wang, M. Irnaten, D. Mendelowitz, Characteristics of spontaneous and evoked GABAergic synaptic currents in cardiac vagal neurons in rats, Brain Res. 889 (2001) 78–83. A. Willis, M. Mihalevich, R.A. Neff, D. Mendelowitz, Three types of postsynaptic glutamatergic receptors are activated in DMNX neurons upon stimulation of NTS, Am. J. Physiol. 271 (1996) R1614–1619.