Brain Research 739 1996.

258262

Research report

Voltage-gated currents in identified parasympathetic cardiac neurons in the nucleus ambiguus

Mark Mihalevich, Robert A. Neff, David Mendelowitz
Accepted 2 July 1996
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Department of Physiology and Biophysics, Uniersity of Tennessee, 894 Union Ae., Memphis, TN 38163, USA

Abstract Heart rate is normally dominated by the activity of the cardioinhibitory parasympathetic nervous system, while abnormally low levels of parasympathetic cardiac activity have been implicated in many cardiovascular diseases including hypertension, heart failure and sudden cardiac death. In this study we have examined the voltage-gated currents in parasympathetic cardiac neurons that were identified with a retrograde fluorescent tracer in visualized sections 250 mm. of nucleus ambiguus. Depolarization of parasympathetic cardiac neurons to potentials more positive than y50 mV evoked a rapidly activating and inactivating inward current which could be blocked by tetrodotoxin TTX., although in some neurons up to 10 m M was required for complete block. The voltage-dependent inactivation properties of this Na current showed relatively broad inactivation characteristics, a characteristic of TTX-resistant Na channels. Depolarization also elicited biphasic outward currents, which were separated into a transient IA type K current using the specific channel antagonist 4-aminopyridine and a long-lasting delayed rectified K current. These voltage-gated Na and K currents define the action potential firing patterns of parasympathetic cardiac neurons, such as frequency adaptation and spike delay, and also determine the activity of these neurons in response to depolarizing and hyperpolarizing synaptic innervation.
Keywords: Vagal; Heart; Cardiovascular; A current; Tetrodotoxin; 4-Aminopyridine

1. Introduction Neurons in the nucleus ambiguus NA. play an essential role in maintaining parasympathetic cardiac, gastrointestinal and respiratory activity w10x. Consistent with the diverse physiological roles of NA neurons, the NA is an anatomically heterogeneous nucleus comprised of neurons with different electrophysiological and morphological characteristics w10x. Anatomical studies using retrograde tracers, such as HRP or fluorescent compounds, have shown that preganglionic parasympathetic cardiac neurons are located mostly in the external formation of the NA w6,916,23x, although recent work with viral tracers has revived a controversy regarding the additional presence of parasympathetic cardiac neurons in the dorsal motor nucleus of the vagus DMNX. w22x. Neurons subserving a gastrointestinal function are densely located in the compact formation of the NA, whereas respiratory neurons have been divided into at least 3 groups within the NA w10x.

) Corresponding author. Fax: q1 901. 448-7126; E-mail: dmendel@physio1.utmem.edu

Unfortunately, this anatomical overlap in the distribution of these physiologically diverse neurons has made in-vitro electrophysiological studies of NA neurons difficult to correlate with specific visceral functions. In-vivo work has the advantage of identifying these neurons by antidromic activation from the visceral organ, but these studies have been generally limited to extracellular recordings which cannot provide information concerning the cellular electrophysiological properties of these neurons. More recently, the physiological identification of neurons has been maintained in vitro by using retrograde and anterograde fluorescent tracers to identify neurons which innervate or originate from a particular visceral organ or sensory site w1214x. Electrophysiological techniques, such as whole cell and perforated path clamp methodology, can then be used to characterize the synaptic, calcium, and voltage-gated currents in these identified neurons in vitro. Previous work has shown that identified preganglionic parasympathetic cardiac neurons in the NA are inherently silent in vitro under conditions in which synaptic activation is blocked, but fire with little delay and without spike frequency adaptation in response to maintained depolarizing injection of current from resting membrane potentials

0006-8993r96r$15.00 Copyright q 1996 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 9 6 . 0 0 8 6 8 - 2

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w13x. In-vivo extracellular recording of these neurons using choralose anesthesia. also suggests that these neurons are typically silent w6,9x. The tonic ongoing parasympathetic activity that is present in unanesthetized animals rats w5x; dogs w20x; humans w18x. must therefore be initiated in vivo to a large extent by the synaptic innervation of parasympathetic cardiac neurons that also appears to be highly susceptible to anesthesia. One likely such pathway is the excitatory glutamatergic innervation from neurons in the nucleus tractus solitarius w14x, a nucleus which directly receives baroreceptor afferent input w1x. In order to understand the firing characteristics and modulation of these neurons in response to excitatory and inhibitory synaptic activity the voltage-gated currents in these neurons need to be studied. This work focuses on the voltage-gated sodium Na. and potassium K. currents in identified preganglionic parasympathetic cardiac neurons in the NA. These voltage-gated currents determine the depolarization evoked firing patterns of these neurons and, in addition, shape their response to synaptically evoked changes in voltage.

which provided access resistances of 14.9 " 1.2 M V , n s 39. Picrotoxin 100 m M., strychnine 1 m M., and prazosine 10 m M. were added to the bath perfusate in all experiments to prevent GABAergic, glycinergic, and a 1adrenergic post-synaptic currents, respectively. In three additional experiments, AP5 and CNQX were included in the bath perfusate to block glutamatergic post-synaptic currents. Patch pipettes were filled with a solution consisting of: K Gluconate 130 mM., HEPES 10 mM., EGTA 10 mM., CaCl 2 1 mM., and MgCl 2 1 mM.. Pipette resistance and capacitance were compensated ) 90%. prior to gaining intracellular access. Data are presented as mean " S.E.M.

3. Results Membrane resistances were measured in response to hyperpolarizing voltage steps from y80 to y81 mV. Whole cell resistances were 226 " 40 M V , calculated in 39 neurons from 17 animals. In all neurons tested n s 39.,

2. Materials and methods In an initial surgery, the heart was exposed in rats 612 days old. with a right thoracotomy, and rhodamine was injected into the pericardial sac and applied to the terminals of preganglionic parasympathetic cardiac neurons located mostly in the fat pads at the base of the heart, as described in detail previously w12x. 37 days later the animals were anesthetized with methoxyflurane and sacrificed by cervical dislocation. The hindbrain was removed and placed for 1 min in cold 028C. buffer of the following composition: NaCl 140 mM., KCl 5 mM., CaCl 2 2 mM., glucose 5 mM., HEPES 10 mM., and continually gassed with 100% O 2 . The medulla was then cut in 250-m m-thick sections using a vibratome. Slices were mounted in a perfusion chamber and submerged. The composition of the perfusate in mM. was: NaCl 125., KCl 3., CaCl 2 2., NaHCO 3 26., dextrose 5., HEPES 5., constantly bubbled with 95% O 2 , 5% CO 2 , and maintained at pH 7.4. Individual parasympathetic cardiac neurons were identified by the presence of the fluorescent tracer. These identified parasympathetic cardiac neurons were then imaged with differential interference contrast DIC. optics, infrared illumination and infrared-sensitive video detection cameras to gain better spatial resolution and to visually guide and position the patch pipette onto the surface of the identified neuron. The pipette was advanced until obtaining a GV-seal between the pipette tip and the cell membrane of the identified neuron. The membrane under the pipette tip was then ruptured with a brief suction to obtain whole cell patch clamp configuration. To obtain optimal space clamp low resistance pipettes were used 23 M V

Fig. 1. Parasympathetic cardiac neurons were depolarized from a holding potential of y100 mV to voltages from y80 mV to q30 mV in 10 mV increments. At voltages more positive than y50 mV a rapidly activating and inactivating inward current, followed by biphasic outward currents top., were evoked. The inward current was completely abolished by tetrodotoxin TTX, 10 m M. middle.. The Na currents that were blocked are illustrated in the bottom trace.

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Fig. 3. In the presence of TTX 10 m M., depolarization to potentials more positive than y50 mV evoked a biphasic outward current. The transient component was blocked by 4-aminopyridine 4AP., a specific I A type K channel antagonist bottom..

Fig. 2. Voltage-dependent inactivation properties of these Na currents were examined by conditioning the neurons at voltages from y110 mV to 0 mV for 2 s, prior to applying a test pulse to 0 mV to activate any remaining non-inactivated channels, as shown in the voltage protocol top.. Inactivation of the Na channels during the conditioning voltage periods are manifested as a decreased amplitude during the subsequent test pulse as shown for one typical experiment labeled with corresponding conditioning voltages middle traces., and the average voltage-dependent inactivation properties from 10 neurons is shown at the bottom. Na channels began to inactivate at y70 mV, but were not fully inactivated until conditioning voltages were positive to y10 mV. In this and all subsequent figures, the error bars are S.E.M.

In some experiments a small 50100 pA. inward current lasting 350 ms with kinetics and voltage-dependent activation properties resembling a high threshold calcium current was initially present after Na channels were blocked with TTX, but this current could not be isolated further due to its small magnitude and rapid rundown. The voltage-dependent inactivation properties of the sodium currents were examined by conditioning the neurons at voltages from y110 mV to 0 mV for 2 s, prior to applying a test pulse to 0 mV to activate any remaining non-inactivated channels. Na channels began to inactivate

there was no evidence for any inwardly rectifying K current in response to hyperpolarizing steps. Depolarization of parasympathetic cardiac neurons to potentials more positive than y50 mV evoked a rapidly activating and inactivating inward current, followed by biphasic outward currents, as shown in Fig. 1, top. To isolate and characterize these voltage-gated currents various specific toxins and voltage-dependent protocols were utilized. As shown in Fig. 1, middle, the inward current was completely abolished by tetrodotoxin TTX.. Surprisingly, however, a concentration of 1 m M TTX inhibited the Na current in these neurons, but in some 3 out of 10 neurons tested with different TTX concentrations., 10 m M TTX was required for complete block. This suggests that the Na currents in some of these neurons may be, at least partly, comprised of a TTX-resistant channel. As has been shown in other neurons with a TTX-resistant current w4,17,19x, 1 m M TTX will partially inhibit Na currents, whereas 10100 m M TTX is required for complete block.

Fig. 4. The I A type K channel fully inactivating within 150 ms as shown in the middle traces. The threshold for activation of the 4AP-sensitive transient current was y50 mV, as shown below ns 7..

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at y70 mV, but did not fully inactivate until conditioning voltages were positive to y10 mV Fig. 2.. This relatively broad voltage-dependent inactivation relationship is also a characteristic of TTX-resistant Na channels w4,19x. In the presence of TTX 10 m M., depolarization of parasympathetic cardiac neurons to potentials more positive than y50 mV evoked a biphasic outward current Fig. 3, top.. This current could be separated into two components using 4-aminopyridine 4AP., which is a specific IA type K channel antagonist w3x. Application of 4AP 5 mM., in each of the 28 neurons tested, blocked a rapidly activating, and fully inactivating within 150 ms. outward current 4AP-sensitive current. while isolating a slowly activating and non-inactivating outward current post-4AP.. The slowly activating and non-inactivating current could be inhibited by tetraethylammonium TEA. which blocks the delayed and rectified K current w3x. The threshold for activation of the 4AP sensitive transient current was y50 mV, as shown in Fig. 4. The voltage-dependent inactivation properties of the IA type K current was examined by conditioning the neurons at voltages from y110 mV to 0 mV for 2 s, prior to applying a test pulse to 0 mV. The IA K current began to inactivate at y110 mV and was fully inactivated at voltage positive to y50 mV, as shown in Fig. 5.

4. Discussion This study has identified the voltage-gated currents that are responsible for action potential generation and which modulate the patterns of activity in parasympathetic cardiac neurons. The major inward current that is activated in these neurons upon depolarization is a rapidly activating and inactivating Na current. This Na current is, at least partly, insensitive to TTX, since in some neurons tested 10 m M TTX was required for complete block. Consistent with these pharmacological properties, the voltage-dependent inactivation properties of the Na current in these neurons is broader than that of TTX-sensitive channels, but is very similar to the voltage-dependent activation and inactivation properties of TTX-resistant channels in dorsal root w2,17,19x, nodose w7x and cardiac ganglia w4x neurons. The factors that determine the relative distribution of TTX-resistant relative to TTX-sensitive channels is unclear. In one study of dorsal root ganglia neurons the relative proportion of TTX-sensitive and -resistant channels changed from young to older animals w19x, whereas in other studies of dorsal root ganglia neurons the relative proportion of TTX-resistant channels did not change with development, but were consistent within morphologically different populations of dorsal root ganglia cells w2,17x. Further work is needed in parasympathetic cardiac neurons to determine the sensitivity of these Na channels to a greater range of TTX concentrations, as well as other blockers of the TTX-resistant current, and the relative proportion of these channels at various ages. The two voltage-gated K currents in parasympathetic cardiac neurons have been identified as the IA type, and delayed rectifier type K currents in this study. The IA type current was transient and inactivated within 150 ms, and was blocked by 4AP. In other neurons the 4AP sensitive K current either inactivates fully within 300 ms w3x, as in these neurons, or is comprised of a transient current which is then followed by a long-lasting component w21x. In neurons with the biphasic 4AP-sensitive component, the transient current is thought to be responsible for the delay before firing is initiated delayed excitation., and the long lasting component is thought to be involved in spike frequency adaptation w21x. Consistent with these hypotheses, in parasympathetic cardiac neurons the 4AP-sensitive current was completely transient, and these neurons exhibit delayed excitation, but have no significant spike frequency adaptation in response to maintained depolarizing current injections w13x. Also consistent with another study w13x, when these neurons are hyperpolarized to y100 mV. the period of delayed excitation increases, as would be expected from the voltage-dependent inactivation properties of the IA current as described in this study. The ionic currents described in this study in identified parasympathetic cardiac neurons are similar to the currents described for one type 31% of the population of the NA neurons examined. of presumptive respiratory neurons in

Fig. 5. The voltage-dependent inactivation properties of the I A type K current was examined by conditioning the neurons at voltages from y110 mV to 0 mV for 2 s, prior to applying a test pulse to 0 mV, as shown in the voltage protocol top.. Inactivation of I A type K current can be seen as a decreased amplitude during the test pulse, as shown for one typical example middle., and the average from 7 neurons is shown at the bottom. The I A K current began to inactivate at y110 mV and was fully inactivated at voltage positive to y50 mV.

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M. Mihaleich et al.r Brain Research 739 (1996) 258262 w4x Clark, R.B., Tse, A. and Giles, W.R., Electrophysiology of parasympathetic neurons isolated from the interatrial septum of bull-frog heart, J. Physiol., 427 1990. 89125. w5x Coleman, T.G., Arterial baroreflex control of heart rate in the conscious rat, Am J. Physiol., 238 1980. H515H520. w6x Gilbey, M.P., Jordan, D., Richter, D.W., and Spyer, M.K., Synaptic mechanism involved in the inspiratory modulation of vagal cardioinhibitory neurons in the cat, J. Physiol., 356 1984. 6578. w7x Ikeda, S.R. and Schofield, G.G., Tetrodotoxin-resistant sodium current of rat nodose neurones: monovalent cation selectivity and divalent cation block, J. Physiol., 389 1987. 255270. w8x Johnson, S.M. and Getting, P.A., Electrophysiological properties of neurons within the nucleus ambiguus of adult guinea pigs, J. Neurophysiol., 663. 1991. 744761. w9x Jordan, D., Khalid, M.E.M., Schneiderman, N. and Spyer, K.M., The location and properties of preganglionic vagal cardiomotor neurons in the rabbit, Pflugers Arch., 395 1972. 244250. w10x Loewy, A.D. and Spyer, K.M. Eds.., Central Regulation of Autonomic Functions, Oxford University Press, Oxford, 1990, pp. 7784. w11x McAllen, R.M. and Spyer, K.M., Two types of vagal preganglionic motoneurons projecting to the heart and lungs, J. Physiol., 282 1978. 353364. w12x Mendelowitz, D. and Kunze, D.L., Identification and dissociation of cardiovascular neurons from the medulla for patch clamp analysis, Neurosci. Lett., 132 1991. 217221. w13x Mendelowitz, D., Firing properties of identified parasympathetic cardiac neurons in the nucleus ambiguus recorded with perforated patch clamp techniques, Am. J. Physiol., 1996. in press. w14x Neff, R.A. and Mendelowitz, D., Stimulation of nucleus tractus solitarius evokes both NMDA and non-NMDA post-synaptic currents in vagal nucleus ambiguus neurons, Soc. Neurosci. Abstr., 21 1995. 642. w15x Nosaka, Yasunaga, S.K. and Tamai, S., Localization of vagal cardioinhibitory preganglionic neurons with rat brain stem, J. Comp. Neurol., 186 1979. 7992. w16x Nosaka, Yasunaga, S.K. and Tamai, S., Vagal cardiac preganglionic neurons: distribution, cell types, and reflex discharges, Am, J. Physiol., 243 1982. R92R98. w17x Ogata, N. and Tatebayashi, H., Ontogenic development of the TTX-sensitive and TTX-resistant Na channels in neurons of the rat dorsal root ganglia, De. Brain Res., 65 1992. 93100. w18x Pickering, T.G., Gribbin, B., Peterson, E.S., Cunningham, D.J.C. and Sleight, P., Effects of autonomic blockade on the baroreflex in man at rest and during exercise, Circ. Res., 30 1972. 177185. w19x Roy, M.L. and Narahashi, T., Differential properties of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels in rat dorsal root ganglia neurons, J. Neurosci., 12 1992. 21042111. w20x Scher, A.M. and Young, A.C., Reflex control of heart rate in the unanesthetized dog, Am. J. Physiol., 218 1970. 780789. w21x Schild, J.H., Khushalani, S., Clark, J.W., Andresen, M.C., Kunze, D.L. and Yang, M., An ionic current model for neurons in the rat medial nucleus tractus solitarii receiving sensory afferent input, J. Physiol., 469 1993. 341363. w22x Standish, A., Enquist, L.W., Escardo, J.A. and Schwaber, J.S., Central neuronal circuit innervating the rat heart defined by transneuronal transport of pseudorabies virus, J. Neurosci., 15 1995. 19982012. w23x Stuesse, S.L., Origins of cardiac vagal preganglionic fibers: a retrograde transport study, Brain Res., 236 1982. 1525.

the guinea pig w8x. These neurons possess an IA current which is responsible for delayed excitation. This suggests that either some respiratory neurons in the guinea pig possess similar ionic channels as identified rat parasympathetic cardiac neurons, or this group of neurons in the guinea pig may have been parasympathetic cardiac rather than respiratory neurons. In conclusion, depolarization of identified parasympathetic cardiac neurons in the NA activates a rapidly activating and inactivating Na current that can be blocked by 10 m M TTX. These cardiovascular neurons also possess two K currents, a transient IA current which can be blocked by 4AP, and a delayed rectified K current. The predicted firing patterns of a neuron with a transient IA current has been shown to occur in these parasympathetic cardiac neurons w13x. The IA current introduces a delay before firing can be initiated during depolarizing input, and this delay increases if the neuron is hyperpolarized from resting membrane potentials as the inactivation of the IA current is removed. The highly voltage-dependent inactivation properties of the IA current would also play an important role in shaping the activity of these neurons in response to excitatory and inhibitory synaptic inputs. For example, inhibitory synaptic input would not only directly inhibit these neurons, but would also indirectly inhibit these neurons by removing any inactivation of the IA K current. Further work is needed to determine the synaptic pathways, neurotransmitters and post-synaptic ligand-gated ionic channels, that, together with the voltage-gated currents identified in this study, determine the activity of parasympathetic cardiac neurons.

Acknowledgements Supported by NIH Grant HL 49965 and a Grant-in-Aid from the American Heart Asociation to D.M.

References
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