Journal of the Autonomic Nervous System, 39 (1992) 191-200 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1838/92/$05.00
191
JANS 01289
Synaptic transmission in rat cardiac neurones A.A. Selyanko and V.I. Skok Department of Autonomic Nervous System Physiology, A.A. Bogomoletz Institute of Physiology, Kiev, Ukraine (Received 17 October 1991) (Revision received and accepted 30 March 1992)
Key words: C a r d i a c n e u r o n e ; A u t o n o m i c ganglia; Synaptic t r a n s m i s s i o n
Abstract Intracellular recordings of spontaneous synaptic activity and synaptic responses to fibre tract stimulation were taken from neurones of ganglia isolated from the left atrium and interatrial septum of the rat. In six out of 57 neurones studied, spontaneous fast excitatory postsynaptic potentials (EPSPs) were recorded. Single stimulation of fibre tracts approaching the ganglion resulted in an all-or-none response consisting of an EPSP, from which an action potential abruptly appeared. This response disappeared in Ca2+-free/high-Mg2+ solution, indicating that it was orthodromic in origin. EPSPs were markedly exaggerated and prolonged by neostigmine (1-5 /zM). EPSPs produced by high-frequency (0.1-20 Hz) fibre tract stimulation were markedly attenuated when compared with responses to single fibre tract stimulation, although they usually remained suprathreshold for spike initiation. High concentrations of hexamethonium (1 mM) and d-tubocurarine (300/~M) failed to inhibit responses to single fibre tract stimulation, although they completely abolished responses to high-frequency stimulation. Responses to single fibre tract stimulation were abolished by trimetaphan (> 100 txM). No slow synaptic responses were detected during single or high-frequency fibre tract stimulation. All cardiac neurones that responded orthodromically were highly excitable: they had a short post-spike after-hyperpolarization (AHP) and responded with multiple firing to prolonged membrane depolarization. It is concluded that cardiac neurones, in the region of the heart studied here, receive single 'strong' cholinergic inputs from some fibre tracts approaching the ganglion that elicit EPSPs accompanied by spikes. EPSPs are rather resistant to ganglion-blocking agents and subject to frequency modulation.
Introduction A u t o n o m i c nerves a r e k n o w n to p l a y a signific a n t role in t h e r e g u l a t i o n o f c a r d i a c function: t h e y m o d u l a t e h e a r t rate, systolic c o n t r a c t i l e force a n d a t r i o v e n t r i c u l a r c o n d u c t i o n [3]. E x c i t a t o r y inf l u e n c e s a r e s u p p l i e d to all p a r t s of t h e h e a r t by fibres o r i g i n a t i n g f r o m t h o r a c i c s y m p a t h e t i c ganglia, w h e r e a s inhibitory influences a r e p r o v i d e d
Correspondence to: A.A. Selyanko, Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK.
by p r e g a n g l i o n i c nerve fibres which o r i g i n a t e f r o m the m e d u l l a o b l o n g a t a a n d synapse with n e u r o n e s in t h e c a r d i a c plexus s e n d i n g t h e i r p r o j e c t i o n s to the heart. Synaptic t r a n s m i s s i o n f r o m p r e g a n g l i o n i c nerve fibres to c a r d i a c n e u r o n e s has b e e n well-chara c t e r i z e d in a m p h i b i a n s [8,17,25]. O r t h o d r o m ic s t i m u l a t i o n results in t h e a p p e a r a n c e o f a fast (nicotinic) excitatory p o s t s y n a p t i c p o t e n t i a l ( E P S P ) a n d a slow ( m u s c a r i n i c ) inhibitory postsyn a p t i c p o t e n t i a l (IPSP). B o t h r e s p o n s e s a r e m i m icked by acetylcholine. I n f o r m a t i o n a b o u t synaptic events in m a m m a l i a n c a r d i a c n e u r o n e s r e m a i n s scarce. It has
I t~2
been briefly reported that, in the guinea pig, stimulation of preganglionic nerves gives rise to cholinergic fast and noncholinergic slow EPSPs, a response mimicked by micro-application of substance P [19,23,34,42,43]. Extracellular recordings from the canine cardiac plexus in situ have revealed that stimulation of sympathetic nerves activates ganglion neurones [4,5,13]. Recent studies on guinea pig and rat cardiac neurones maintained in culture indicate that they have nicotinic and muscarinic acetylcholine receptors, ATP receptors and adrenoceptors [1,2,9, 10]. Furthermore, there are neurones a n d / o r nerve fibres in the cardiac plexus that exhibit acetylcholinesterase activity, contain catecholamine and are immunoreactive for certain peptides [6,14,34,40,41 ]. It has been shown in the preceding paper that ganglia dissected with nerve bundles from the heart of the rat are a suitable preparation for intracellular recording [35]. The aim of the present experiments was to study synaptic responses within these ganglia.
Materials and Methods
The experimental methods have been described previously [35]. In brief, ganglia containing nerve bundles were isolated from left atrium and interatrial septum of the rat. Impalements were made under Nomarski differential interference optics using microelectrodes filled with 3 M KC1. It was clearly seen that one or more nerve bundles reached each ganglion. In some experiments, fibre tracts were stimulated with monopofar extracellular electrodes, which are conventionally used for activation of enteric neurones [28], an attempt to activate ganglion neurones orthodromically. However, such stimulation failed. More successful was another approach, in which stimuli (1-15 V; 0.1-0.5 ms) were applied to one of the nerve bundles through a fine suction electrode. However, even with this technique, only a proportion of cardiac neurones could be stimulated synaptically. In some experiments acetylcholine was applied ionophoretically
A -51 mV
i J
\ ~
. . . . . . . . . .
120 mV L2_O FFIS
B -80 mV
Fig. 1. S p o n t a n e o u s synaptic activity recorded from a cardiac neurone. The cell was hyperpolarized from its resting potential of - 51 m V (A) to - 80 m V (B).
through a micropipette filled with 2 M acetylcholine chloride [36].
Results
Spontaneous synaptic actiuity It has previously been shown that in three out of 57 rat neurones studied in vitro, regular spontaneous discharges occurred [35]. They were inhibited by moderate membrane hyperpolarization, they were not accompanied by fast EPSPs, and, hence, could be 'intrinsic' in origin. In another six neurones, a spontaneous activity consisting of occasional spikes was observed (Fig. 1A). Each spike appeared abruptly and was followed by a secondary, slower depolarization (EPSP). These spikes were not abolished by strong membrane hyperpolarization (Fig. 1B). Thus, spontaneous EPSPs were large enough to shift the membrane potential to the threshold level even from very negative membrane potentials. In two more neurones, EPSPs subthreshold for the spike initiation were r e c o r d e d in addition to
193
B
A
2 0 mV
5OO pa
hA_ -J"]
I
5 0 ms
(A)
lo ms (B)
1
Fig. 2. Comparison of responses to fibre tract and direct stimulations. A: Records show discharges evoked by single fibre tract stimulation (a) and intrasomal injection of short-duration (10 ms) depolarizing current pulse (b). B: The initial parts of responses illustrated in A are shown on a faster time scale. Note a similar length of after-hyperpolarizations. Resting membrane potential 52 mV. -
suprathreshold ones (not shown). No spontaneously occurring slow potentials were detected. Orthodromic responses to single fibre tract stimulation In 21 neurones, stimulation of an incoming fibre tract produced a spike followed by a secondary, slower depolarization and subsequent hy-
A
perpolarization (Fig. 2A(a), B(a)). The spike produced in the same neurone by direct stimulation (Fig. 2A(b), B(b)) had a greater amplitude, lacked the secondary depolarization and was followed by an after-hyperpolarization. The comparison between responses to nerve and direct stimulations suggests that the former one is an orthodromic response in which the secondary depolarization
B 2.0 mM Ca 2"
C 0 mM Ca 2,
2.0 mM Ca 2"
a4J 20 mV 0.5 nA
20 ms
Crq
_n
_~
Fig. 3. Effect of Ca2+-free/high-Mgz+ solution on responses to fibre tract and direct stimulations. Responses are shown to single fibre tract stimulation (a) and direct stimulation (b) by intrasomal injection of 10-ms depolarizing pulse (c) before (A), during (B) and after (C) perfusion of the solution containing 0 mM Ca 2+ and 6 mM Mg 2+. Resting membrane potential - 4 8 mV in normal solution and - 41 mV in Ca 2+-free/high-Mg2+ solution.
constitutes the fast EPSP. The inhibitory effects of ganglion-blocking agents (d-tubocurarine, hexamethonium and trimetaphan), or a decreased [Ca 2 ~],,/[Mg2+]o ratio, and the facilitatory effect of neostigmine on the nerve-evoked response (see below) support this suggestion. All threshold stimuli had an all-or-none effect; no additional EPSPs were seen when the stimulus strength was increased. The fast EPSP was decreased and increased when the membrane was de- and hyperpolarized, but the spike generation was not inhibited by a membrane hyperpolarization up to - 100 inV. The orthodromic response had a short latency (mean 2.6, S.E. = 0.14 ms, n = 21) indicating it was monosynaptic in origin. The action potential amplitude was always several mV smaller when elicited by the nerve stimulation than by intrasoreal current injection, possibly due to a conductance increase produced by a transmitter. The after-hyperpolarization following synaptically-evoked spike was smaller in amplitude, never lasted longer than that seen following direct stimulation (n = 12) and was unaffected by atropine (1 /~M, n = 7). Thus there were no slow postsynaptic potentials as a result of single fibre tract stimulation. External Ca 2+. In three cells out of three, incubation in Ca2+-free/high-Mg 2+ solution abolished the response to fibre tract stimulation, without removing the spike produced by direct stimulation (Fig. 3). The slight reduction of the latter response was associated with a m e m b r a n e depolarization resulting from the removal of Ca 2 + ions from the external solution [35]. Neostigmine. The cholinesterase inhibitor neostigmine (1-5 /~M, n = 6) increased and markedly prolonged membrane depolarizations obtained by fibre tract stimulation (Fig. 4A) and ionophoretic application of acetylcholine (Fig. 4B).
Responses to repetitive stimulation Fast EPSPs. When repeated stimuli were applied to the fibre tract the configuration of an ticeably changed (Fig. tested the amplitude of
at frequencies > 0.1 Hz, orthodromic response no5A). In all 21 neurones the post-spike depolariza-
f~
I
Control
I
Neostigmine (5 tsM)
mV ms
2•._20
[3 ACh IIf~
Control
il~
Neostigmine(5
pM)
10 r~V 1500 ms
Fig. 4. Effects of neostigmine (5 ~M) on responses obtained from the same neurone to single fibre tract stimulation (A) and brief (100-ms) ionophoretic application of ACh (B). (a) and (b) are responses recorded before and during perfusion of neostigmine, respectively. Resting membrane potential potential - 46 mV. Note the different calibrations in A and B.
tion substantially declined and the height of the orthodromic spike slightly increased, in accordance with a decreased conductance change produced by a transmitter. Such a 'run-down' of the fast EPSP, which can be attributed to the progressive diminution in transmitter release, was more pronounced at higher frequencies, although even at frequencies as high as 10-20 Hz fast EPSPs usually remained suprathreshold for spike initiation for tens of seconds. Interestingly, when two or more spontaneous fast EPSPs appeared in bursts, the second, third, and subsequent ones were likewise diminished (Fig. 1B, middle trace) which is consistent with the suggestion that the cardiac neurones studied here may be singly-innervated.
195
A
Control
5 Hz
B Flexarnethc~iurn (I mM)
C
Wash
d-Tt,Oocurarine (0.3 raM)
E
Wash
Fig. 5, Effects of hexamethonium and d-tubocurarine on responses of the same cell to high-frequency fibre tract stimulation. Four responses were evoked at 5 Hz before (A) and after (B) adding 1 mM hexamethonium, after washing out of hexamethonium (C) and after adding (D) and washing out of 0.3 mM d-tubocurarine (E). Resting membrane potential - 5 4 mV.
Slow postsynaptic potentials. Slow postsynaptic potentials were absent, not only after single fibre tract stimulation, but also during prolonged (up to 5-s) high-frequency (20 Hz) fibre tract stimulation (n = 21), even when fast EPSPs were inhibited by d-tubocurarine (300/zM, n = 5) and cholinesterase was inhibited by neostigmine (1-5 /zM, n =6).
markedly changed the configuration of an orthodromic response. Figure 6 shows that 300 IzM TC reduced the amplitude of the post-spike depolarization, increased the amplitude of the spike and both reduced and shortened the after-hyperpolarization (see also Fig. 5). The two former effects are due to a decreased conductance change produced by transmitter in the presence of TC, while the latter effects might result from an inhibitory action of TC upon the potassium conductance underlying post-spike after-hyperpolarization [30]. The diminution of fast EPSP by TC or C 6 (not shown) was also shown by the fact that in the presence of a blocker, unlike in control, an orthodromic spike was readily eliminated by injection of a hyperpolarizing current (Fig. 6A(c), B(c) and C(c)). Another ganglion-blocking agent, trimetaphan, (at high concentrations) readily abolished both the orthodromic spike (at 100/zM) and fast EPSP (at 300 /zM) in all neurones studied (n = 4, Fig. 7). Effects on responses to high-frequency stimulation. Both orthodromic spikes and fast EPSPs elicited by repeated stimulation were abolished by high concentrations of TC (n --- 5) or C 6 (n = 4) (Fig. 5). Electric properties of cardiac neurones responding to fibre tract stimulation On the basis of spike configuration and firing properties two cell categories were identified among rat cardiac neurones in vitro [35]. All the cardiac neurones which responded to fibre tract stimulation had short post-spike after-hyperpolarizations and responded with multiple firing to prolonged membrane depolarization; they were identified as type I neurones.
Discussion
Effects of ganglion-blocking agents on responses to fibre tract stimulation Effects on responses to single stimulation. Even at high concentrations d-tubocurarine (TC, < 300 /zM, n = 5) and hexamethonium (C6, < 1 mM, n = 5) failed to abolish spikes elicited by single fibre tract stimulation. At the same time they
In the present experiments a single type of response, a fast EPSP, was recorded in rat cardiac neurones following synaptic stimulation. This fast EPSP is presumed to be cholinergic in origin and mediated via nicotinic acetylcholine receptors since it is exaggerated and prolonged by
52 mV
8.
Control
I d [ubocurarine
a
Wash a
b
-80 mV L C
L C~
_
50 ms (Aa-Ca:Ac-Cc) 10 m'~s('-Ab-cb)
C
Fig. 6. Effect of d-tubocurarine on the response to single fibre tract stimulation. Responses were evoked before (A) and after (B) adding 0.3 m M d-tubocurarine and after washing it out (C). Records in (a) and (b) show entire responses and their initial parts evoked at the resting m e m b r a n e potential level of - 5 2 mV. Records in (c) show responses evoked when the cell was hyperpolarized to - 8 0 mV by intrasomal current injection. Note the different time scales in a, c (50 ms) and b (10 ms).
neostigmine and depressed by ganglion-blocking agents. The fast EPSP in rat cardiac neurones can be mimicked by focal micro-ionophoretic application of acetylcholine [36]. The rote of acetylcholine as a transmitter in cardiac ganglia is also suggested by the fact that these ganglia are a part of the vagal efferent pathway where preganglionic fibres are cholinergic [12]. However, since cardiac neurones and many of the fibres in the cardiac plexus are cholinergic, we could not ascertain whether the orthodromic response was due to extrinsic or intrinsic fibres. The latter possibility is consistent with the results of extracellular recording from cat [31] and dog [4,5,13] heart ganglia, where neurones fired in response to mechanical stimulation. In the present experiments no muscarinic slow potentials accompanied the nicotinic fast EPSP during either single or repetitive stimulation. This finding contrasts with the evidence that neurones of the rat heart [15] and guinea pig cardiac neurones in culture [2,16] are endowed with muscarinic acetylcholine receptors and that all guinea pig cardiac neurones maintained in culture [2] and rat cardiac neurones in vitro [36] respond to
muscarinic agonists, with slow de- or hyperpolarizations. The reason for this difference is unclear. One possible explanation is that more muscarinic acetylcholine receptors are activated by exogenously applied acetylcholine or muscarine than by synaptically released acetylcholine. This suggestion is consistent with the finding that muscarinic acetylcholine receptors are present over the entire surface of cultured cardiac neurones [16]. Interestingly, muscarinic slow postsynaptic potentials were also not detected in cardiac neurones of the guinea pig [23] and the neonate rat [34]. In latter studies, however, slow depolarizing potentials were noted, not seen in the present experiments. They appeared in response to highfrequency orthodromic stimulation and were unaffected by nicotinic and muscarinic antagonists; hence, it was suggested that they were peptidergic in origin. Besides a muscarinic response, one might also expect a slow adrenergic synaptic response in mammalian cardiac neurones. Firstly, in a number of mammalian species, including the rat, the cardiac plexus contains terminal varicosities of adrenergic fibres impinging upon ganglionic neurones [20,27]. Secondly, canine cardiac neurones
197
A Control
13 Trimetaphan 100 IJM
a 4j----__
300 #M
. . . . . . . .
C Wash
120 mV 20 ms
Fig. 7. Effect of trimetaphan on the response to single fibre tract stimulation. Responses were evoked before (A) and after
(B) adding 100/~M (a) or 300/zM (b) trimetaphan and after washing the drug out (C). Resting membrane potential -47 mW.
in vivo can be activated by repetitive stimulation of thoracic sympathetic ganglia [4,5,13]. Thirdly noradrenaline produces slow membrane depolarization and discharges in rat cardiac neurones in vitro (A. Selyanko, unpublished observations). However, as mentioned above, slow postsynaptic potentials were not detected in the present experiments. The fast EPSP in cardiac neurones exhibited several peculiarities. First of all, it always appeared in an all-or-none manner and was large enough to trigger a spike. This is an indication that rat cardiac neurones have a single strong synaptic input from successfully stimulated fibre tracts. Consistent with these findings, it has been recently reported that most guinea pig [23] and neonate rat [34] cardiac ganglion neurones re-
ceive single or few synaptic inputs. Interestingly, most amphibian cardiac ganglia neurones are also singly-innervated [8]. In mammals, parasympathetic ganglia contain either singly innervated neurones (e.g. rat submandibular ganglion [24]), both singly and multiply innervated neurones (e.g. rabbit ciliary ganglion [21,22]), or mainly multiply innervated neurones (e.g. guinea pig intestinal [18,28], gastric [33] or gall-bladder [26] ganglia). In mammalian sympathetic ganglia, each neurone can be discharged either by a single strong synaptic input or by a few accessory synaptic inputs [38]. It has been hypothesized [32] that there is a fundamental relationship between the number of inputs to a neurone and the latter's geometry: unipolar neurones are singly innervated whereas multipolar neurones are innervated by two or more axons whose number is proportional to the number of dendrites. If this relationship is extendable to mammalian cardiac ganglia, some of which contain unipolar, bipolar and multipolar neurones [7], then one can expect some mammalian cardiac neurones to be singly innervated, in agreement with the present results. Another feature of the fast EPSP in cardiac neurones is its 'run-down' during repeated stimulation. A similar frequency-dependent decrease in amplitude of fast EPSP was previously noted in amphibian cardiac neurones [8]. In some mammalian ganglia (e.g. guinea pig intestinal plexus [28]), the EPSP amplitude decreases with the frequency of stimulation as low as 0.1 Hz. On the other hand, in other ganglia (e.g. guinea pig gastric [33] or gall-bladder [26] plexuses), the amplitude of the fast EPSP is maintained even at high stimulation frequencies. Run-down of the fast EPSP in intestinal neurones is attributed to inhibition of acetylcholine release, resulting from either autoinhibition by acetylcholine acting on presynaptic muscarinic acetylcholine receptors, or by presynaptic inhibition by other substances released during repetitive stimulation [11]. Rundown of the fast EPSP in rat cardiac neurones might likewise be presynaptic in origin since no indication of a decay of suprathreshold nicotinic acetylcholine depolarization is seen during prolonged (up to several seconds) application of acetylcholine [36].
1~,~
One more feature of the fast EPSP in rat cardiac neurones is its low sensitivity to ganglionblocking agents. It was found that the fast EPSP here remains suprathreshold for triggering the spike in the presence of 300/xM d-tubocurarine, 1 mM hexamethonium, or 100-/xM trimetaphan. This finding is consistent with the earlier observation that spontaneous activity and responses to stimulation of canine cardiac neurones in vivo persist after administration of hexamethonium [4,5,13]. On the other hand, in vitro studies on the same neurones indicate that fast EPSPs are abolished by 100/xM hexamethonium [42,43]. The reason for the latter difference remains to be clarified. One possible explanation is that there are non-cholinergic synaptic mechanisms in cardiac ganglia [4,5,13,43]. Seabrook and co-workers [34] came to a similar conclusion on the basis of their finding that the fast EPSP in neurones of rat neonatal cardiac ganglion persisted in the presence of high concentrations of mecamylamine which abolished the nicotinic acetylcholine response. However, the present results do not allow us to suggest that there is a noncholinergic component in fast EPSP in rat cardiac ganglia neurones: high concentrations of TC and C~, failed to abolish not only the EPSPs but also responses to acetylcholine in these neurones [36]. Thus the high resistance of fast EPSP to ganglion-blockers is probably due to the low sensitivity of nicotinic acetylcholine receptors. Interestingly, the effectiveness of ganglion blockers in synapses of other mammalian ganglia seems to be much higher than in the present experiments on rat cardiac ganglia. For example, 100-400 /xM hexamethonium completely abolished both fast EPSP and fast acetylcholine depolarization in guinea pig intestinal neurones [29,39]. Only in autonomic ganglia of the frog, fast EPSCs are relatively resistant to hexamethonium and trimetaphan, but not to d-tubocurarine [25]. In cats, surprisingly, hexamethonium is more effective in blocking vagus-induced decrease in heart rate than in reducing arterial blood pressure, i.e. in blocking transmission through sympathetic ganglion (see Fig. 5 in ref. 37). The reason for the difference between these and the above-mentioned data remains to be clarified.
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30 Nohmi, M. and Kuba, K., (+)Tubocurarine blocks the Ca2+-dependent K+-channel of the bullfrog sympathetic ganglion cells, Brain Res., 301 (1984) 146-148. 31 Nozdrachev, A.D. and Pogorelov, A.G., Extracellular recording of neuronal activity of the cat heart ganglia, J. Auton. Nerv. Syst., 6 (1982) 73-81. 32 Purves, D. and Lichtman, J.W., Principles of Neural Development, Sinauer Associates, Sunderland, MA, 1985, 433 pp. 33 Schemann, M. and Wood, J.D., Synaptic behaviour of myenteric neurones in the gastric corpus of the guinea-pig, J. Physiol. (Lond.), 417 (1989) 519-535. 34 Seabrook, G.R., Fieber, L.A. and Adams, D.J., Neurotransmission in neonatal rat cardiac ganglion in situ, Am. J. Physiol., 259 (1990) H997-H1005. 35 Selyanko, A.A., Membrane properties and firing characteristics of rat cardiac neurones in vitro, J. Auton. Nerv. Syst., 39 (1992) 181-190. 36 Selyanko, A.A. and Skok, V.I., Acetylcholine receptors in rat cardiac neurones, J. Auton. Nerv. Syst., 40 (1992) in press. 37 Skok, V.I., Groisman, S.D. Melnitchenko, L.V., Gersanich, V.V. and Gmiro, V.E., Selective pharmacological blockade of parasympathetic and enteric ganglia, J. Auton. Nerv. Syst, 35 (1991) 211-218. 38 Skok, V.I. and Ivanov, A.Y., What is the ongoing activity of sympathetic neurones?, J. Auton. Nerv. Syst., 7 (1983) 263-270. 39 Vanner, S. and Surprenant, A., Effects of 5-HT 3 receptor antagonists on 5-HT and nicotinic depolarizations in guinea pig submucosal neurones, Br. J. Pharmacol., 99 (1990) 840-844. 40 Weihe, E., McKnight, A.T., Corbett, A.D., Hartschuh, W., Reinecke, M. and Kosterlitz, H.W., Characteristics of opioid peptides in the guinea-pig heart and skin, Life Sci., 33, Suppl., Vol. I (1983) 711-714. 41 Weihe, E., Reinecke, M. and Forssmann, W.G., Distribution of vasoactive intestinal polypeptide-like immunoreactivity in the mammalian heart. (Interrelation with neurotensin and substance P-like immunoreactive nerves.), Cell Tissue Res., 236 (1984) 527-540. 42 Xi, X., Randall, W.C. and Wurster, R.D., Intracellular recording of spontaneous activity of canine intracardiac ganglion cells, Neurosci. Lett., 128 (1991) 129-132. 43 Xi, X., Thomas, J.H., Randall, W.C. and Wurster, R.D., Intracellular recordings from canine intracardiac ganglion cells, J. Auton. Nerv. Syst., 32 (1991) 177-182.
191
JANS 01289
Synaptic transmission in rat cardiac neurones A.A. Selyanko and V.I. Skok Department of Autonomic Nervous System Physiology, A.A. Bogomoletz Institute of Physiology, Kiev, Ukraine (Received 17 October 1991) (Revision received and accepted 30 March 1992)
Key words: C a r d i a c n e u r o n e ; A u t o n o m i c ganglia; Synaptic t r a n s m i s s i o n
Abstract Intracellular recordings of spontaneous synaptic activity and synaptic responses to fibre tract stimulation were taken from neurones of ganglia isolated from the left atrium and interatrial septum of the rat. In six out of 57 neurones studied, spontaneous fast excitatory postsynaptic potentials (EPSPs) were recorded. Single stimulation of fibre tracts approaching the ganglion resulted in an all-or-none response consisting of an EPSP, from which an action potential abruptly appeared. This response disappeared in Ca2+-free/high-Mg2+ solution, indicating that it was orthodromic in origin. EPSPs were markedly exaggerated and prolonged by neostigmine (1-5 /zM). EPSPs produced by high-frequency (0.1-20 Hz) fibre tract stimulation were markedly attenuated when compared with responses to single fibre tract stimulation, although they usually remained suprathreshold for spike initiation. High concentrations of hexamethonium (1 mM) and d-tubocurarine (300/~M) failed to inhibit responses to single fibre tract stimulation, although they completely abolished responses to high-frequency stimulation. Responses to single fibre tract stimulation were abolished by trimetaphan (> 100 txM). No slow synaptic responses were detected during single or high-frequency fibre tract stimulation. All cardiac neurones that responded orthodromically were highly excitable: they had a short post-spike after-hyperpolarization (AHP) and responded with multiple firing to prolonged membrane depolarization. It is concluded that cardiac neurones, in the region of the heart studied here, receive single 'strong' cholinergic inputs from some fibre tracts approaching the ganglion that elicit EPSPs accompanied by spikes. EPSPs are rather resistant to ganglion-blocking agents and subject to frequency modulation.
Introduction A u t o n o m i c nerves a r e k n o w n to p l a y a signific a n t role in t h e r e g u l a t i o n o f c a r d i a c function: t h e y m o d u l a t e h e a r t rate, systolic c o n t r a c t i l e force a n d a t r i o v e n t r i c u l a r c o n d u c t i o n [3]. E x c i t a t o r y inf l u e n c e s a r e s u p p l i e d to all p a r t s of t h e h e a r t by fibres o r i g i n a t i n g f r o m t h o r a c i c s y m p a t h e t i c ganglia, w h e r e a s inhibitory influences a r e p r o v i d e d
Correspondence to: A.A. Selyanko, Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK.
by p r e g a n g l i o n i c nerve fibres which o r i g i n a t e f r o m the m e d u l l a o b l o n g a t a a n d synapse with n e u r o n e s in t h e c a r d i a c plexus s e n d i n g t h e i r p r o j e c t i o n s to the heart. Synaptic t r a n s m i s s i o n f r o m p r e g a n g l i o n i c nerve fibres to c a r d i a c n e u r o n e s has b e e n well-chara c t e r i z e d in a m p h i b i a n s [8,17,25]. O r t h o d r o m ic s t i m u l a t i o n results in t h e a p p e a r a n c e o f a fast (nicotinic) excitatory p o s t s y n a p t i c p o t e n t i a l ( E P S P ) a n d a slow ( m u s c a r i n i c ) inhibitory postsyn a p t i c p o t e n t i a l (IPSP). B o t h r e s p o n s e s a r e m i m icked by acetylcholine. I n f o r m a t i o n a b o u t synaptic events in m a m m a l i a n c a r d i a c n e u r o n e s r e m a i n s scarce. It has
I t~2
been briefly reported that, in the guinea pig, stimulation of preganglionic nerves gives rise to cholinergic fast and noncholinergic slow EPSPs, a response mimicked by micro-application of substance P [19,23,34,42,43]. Extracellular recordings from the canine cardiac plexus in situ have revealed that stimulation of sympathetic nerves activates ganglion neurones [4,5,13]. Recent studies on guinea pig and rat cardiac neurones maintained in culture indicate that they have nicotinic and muscarinic acetylcholine receptors, ATP receptors and adrenoceptors [1,2,9, 10]. Furthermore, there are neurones a n d / o r nerve fibres in the cardiac plexus that exhibit acetylcholinesterase activity, contain catecholamine and are immunoreactive for certain peptides [6,14,34,40,41 ]. It has been shown in the preceding paper that ganglia dissected with nerve bundles from the heart of the rat are a suitable preparation for intracellular recording [35]. The aim of the present experiments was to study synaptic responses within these ganglia.
Materials and Methods
The experimental methods have been described previously [35]. In brief, ganglia containing nerve bundles were isolated from left atrium and interatrial septum of the rat. Impalements were made under Nomarski differential interference optics using microelectrodes filled with 3 M KC1. It was clearly seen that one or more nerve bundles reached each ganglion. In some experiments, fibre tracts were stimulated with monopofar extracellular electrodes, which are conventionally used for activation of enteric neurones [28], an attempt to activate ganglion neurones orthodromically. However, such stimulation failed. More successful was another approach, in which stimuli (1-15 V; 0.1-0.5 ms) were applied to one of the nerve bundles through a fine suction electrode. However, even with this technique, only a proportion of cardiac neurones could be stimulated synaptically. In some experiments acetylcholine was applied ionophoretically
A -51 mV
i J
\ ~
. . . . . . . . . .
120 mV L2_O FFIS
B -80 mV
Fig. 1. S p o n t a n e o u s synaptic activity recorded from a cardiac neurone. The cell was hyperpolarized from its resting potential of - 51 m V (A) to - 80 m V (B).
through a micropipette filled with 2 M acetylcholine chloride [36].
Results
Spontaneous synaptic actiuity It has previously been shown that in three out of 57 rat neurones studied in vitro, regular spontaneous discharges occurred [35]. They were inhibited by moderate membrane hyperpolarization, they were not accompanied by fast EPSPs, and, hence, could be 'intrinsic' in origin. In another six neurones, a spontaneous activity consisting of occasional spikes was observed (Fig. 1A). Each spike appeared abruptly and was followed by a secondary, slower depolarization (EPSP). These spikes were not abolished by strong membrane hyperpolarization (Fig. 1B). Thus, spontaneous EPSPs were large enough to shift the membrane potential to the threshold level even from very negative membrane potentials. In two more neurones, EPSPs subthreshold for the spike initiation were r e c o r d e d in addition to
193
B
A
2 0 mV
5OO pa
hA_ -J"]
I
5 0 ms
(A)
lo ms (B)
1
Fig. 2. Comparison of responses to fibre tract and direct stimulations. A: Records show discharges evoked by single fibre tract stimulation (a) and intrasomal injection of short-duration (10 ms) depolarizing current pulse (b). B: The initial parts of responses illustrated in A are shown on a faster time scale. Note a similar length of after-hyperpolarizations. Resting membrane potential 52 mV. -
suprathreshold ones (not shown). No spontaneously occurring slow potentials were detected. Orthodromic responses to single fibre tract stimulation In 21 neurones, stimulation of an incoming fibre tract produced a spike followed by a secondary, slower depolarization and subsequent hy-
A
perpolarization (Fig. 2A(a), B(a)). The spike produced in the same neurone by direct stimulation (Fig. 2A(b), B(b)) had a greater amplitude, lacked the secondary depolarization and was followed by an after-hyperpolarization. The comparison between responses to nerve and direct stimulations suggests that the former one is an orthodromic response in which the secondary depolarization
B 2.0 mM Ca 2"
C 0 mM Ca 2,
2.0 mM Ca 2"
a4J 20 mV 0.5 nA
20 ms
Crq
_n
_~
Fig. 3. Effect of Ca2+-free/high-Mgz+ solution on responses to fibre tract and direct stimulations. Responses are shown to single fibre tract stimulation (a) and direct stimulation (b) by intrasomal injection of 10-ms depolarizing pulse (c) before (A), during (B) and after (C) perfusion of the solution containing 0 mM Ca 2+ and 6 mM Mg 2+. Resting membrane potential - 4 8 mV in normal solution and - 41 mV in Ca 2+-free/high-Mg2+ solution.
constitutes the fast EPSP. The inhibitory effects of ganglion-blocking agents (d-tubocurarine, hexamethonium and trimetaphan), or a decreased [Ca 2 ~],,/[Mg2+]o ratio, and the facilitatory effect of neostigmine on the nerve-evoked response (see below) support this suggestion. All threshold stimuli had an all-or-none effect; no additional EPSPs were seen when the stimulus strength was increased. The fast EPSP was decreased and increased when the membrane was de- and hyperpolarized, but the spike generation was not inhibited by a membrane hyperpolarization up to - 100 inV. The orthodromic response had a short latency (mean 2.6, S.E. = 0.14 ms, n = 21) indicating it was monosynaptic in origin. The action potential amplitude was always several mV smaller when elicited by the nerve stimulation than by intrasoreal current injection, possibly due to a conductance increase produced by a transmitter. The after-hyperpolarization following synaptically-evoked spike was smaller in amplitude, never lasted longer than that seen following direct stimulation (n = 12) and was unaffected by atropine (1 /~M, n = 7). Thus there were no slow postsynaptic potentials as a result of single fibre tract stimulation. External Ca 2+. In three cells out of three, incubation in Ca2+-free/high-Mg 2+ solution abolished the response to fibre tract stimulation, without removing the spike produced by direct stimulation (Fig. 3). The slight reduction of the latter response was associated with a m e m b r a n e depolarization resulting from the removal of Ca 2 + ions from the external solution [35]. Neostigmine. The cholinesterase inhibitor neostigmine (1-5 /~M, n = 6) increased and markedly prolonged membrane depolarizations obtained by fibre tract stimulation (Fig. 4A) and ionophoretic application of acetylcholine (Fig. 4B).
Responses to repetitive stimulation Fast EPSPs. When repeated stimuli were applied to the fibre tract the configuration of an ticeably changed (Fig. tested the amplitude of
at frequencies > 0.1 Hz, orthodromic response no5A). In all 21 neurones the post-spike depolariza-
f~
I
Control
I
Neostigmine (5 tsM)
mV ms
2•._20
[3 ACh IIf~
Control
il~
Neostigmine(5
pM)
10 r~V 1500 ms
Fig. 4. Effects of neostigmine (5 ~M) on responses obtained from the same neurone to single fibre tract stimulation (A) and brief (100-ms) ionophoretic application of ACh (B). (a) and (b) are responses recorded before and during perfusion of neostigmine, respectively. Resting membrane potential potential - 46 mV. Note the different calibrations in A and B.
tion substantially declined and the height of the orthodromic spike slightly increased, in accordance with a decreased conductance change produced by a transmitter. Such a 'run-down' of the fast EPSP, which can be attributed to the progressive diminution in transmitter release, was more pronounced at higher frequencies, although even at frequencies as high as 10-20 Hz fast EPSPs usually remained suprathreshold for spike initiation for tens of seconds. Interestingly, when two or more spontaneous fast EPSPs appeared in bursts, the second, third, and subsequent ones were likewise diminished (Fig. 1B, middle trace) which is consistent with the suggestion that the cardiac neurones studied here may be singly-innervated.
195
A
Control
5 Hz
B Flexarnethc~iurn (I mM)
C
Wash
d-Tt,Oocurarine (0.3 raM)
E
Wash
Fig. 5, Effects of hexamethonium and d-tubocurarine on responses of the same cell to high-frequency fibre tract stimulation. Four responses were evoked at 5 Hz before (A) and after (B) adding 1 mM hexamethonium, after washing out of hexamethonium (C) and after adding (D) and washing out of 0.3 mM d-tubocurarine (E). Resting membrane potential - 5 4 mV.
Slow postsynaptic potentials. Slow postsynaptic potentials were absent, not only after single fibre tract stimulation, but also during prolonged (up to 5-s) high-frequency (20 Hz) fibre tract stimulation (n = 21), even when fast EPSPs were inhibited by d-tubocurarine (300/zM, n = 5) and cholinesterase was inhibited by neostigmine (1-5 /zM, n =6).
markedly changed the configuration of an orthodromic response. Figure 6 shows that 300 IzM TC reduced the amplitude of the post-spike depolarization, increased the amplitude of the spike and both reduced and shortened the after-hyperpolarization (see also Fig. 5). The two former effects are due to a decreased conductance change produced by transmitter in the presence of TC, while the latter effects might result from an inhibitory action of TC upon the potassium conductance underlying post-spike after-hyperpolarization [30]. The diminution of fast EPSP by TC or C 6 (not shown) was also shown by the fact that in the presence of a blocker, unlike in control, an orthodromic spike was readily eliminated by injection of a hyperpolarizing current (Fig. 6A(c), B(c) and C(c)). Another ganglion-blocking agent, trimetaphan, (at high concentrations) readily abolished both the orthodromic spike (at 100/zM) and fast EPSP (at 300 /zM) in all neurones studied (n = 4, Fig. 7). Effects on responses to high-frequency stimulation. Both orthodromic spikes and fast EPSPs elicited by repeated stimulation were abolished by high concentrations of TC (n --- 5) or C 6 (n = 4) (Fig. 5). Electric properties of cardiac neurones responding to fibre tract stimulation On the basis of spike configuration and firing properties two cell categories were identified among rat cardiac neurones in vitro [35]. All the cardiac neurones which responded to fibre tract stimulation had short post-spike after-hyperpolarizations and responded with multiple firing to prolonged membrane depolarization; they were identified as type I neurones.
Discussion
Effects of ganglion-blocking agents on responses to fibre tract stimulation Effects on responses to single stimulation. Even at high concentrations d-tubocurarine (TC, < 300 /zM, n = 5) and hexamethonium (C6, < 1 mM, n = 5) failed to abolish spikes elicited by single fibre tract stimulation. At the same time they
In the present experiments a single type of response, a fast EPSP, was recorded in rat cardiac neurones following synaptic stimulation. This fast EPSP is presumed to be cholinergic in origin and mediated via nicotinic acetylcholine receptors since it is exaggerated and prolonged by
52 mV
8.
Control
I d [ubocurarine
a
Wash a
b
-80 mV L C
L C~
_
50 ms (Aa-Ca:Ac-Cc) 10 m'~s('-Ab-cb)
C
Fig. 6. Effect of d-tubocurarine on the response to single fibre tract stimulation. Responses were evoked before (A) and after (B) adding 0.3 m M d-tubocurarine and after washing it out (C). Records in (a) and (b) show entire responses and their initial parts evoked at the resting m e m b r a n e potential level of - 5 2 mV. Records in (c) show responses evoked when the cell was hyperpolarized to - 8 0 mV by intrasomal current injection. Note the different time scales in a, c (50 ms) and b (10 ms).
neostigmine and depressed by ganglion-blocking agents. The fast EPSP in rat cardiac neurones can be mimicked by focal micro-ionophoretic application of acetylcholine [36]. The rote of acetylcholine as a transmitter in cardiac ganglia is also suggested by the fact that these ganglia are a part of the vagal efferent pathway where preganglionic fibres are cholinergic [12]. However, since cardiac neurones and many of the fibres in the cardiac plexus are cholinergic, we could not ascertain whether the orthodromic response was due to extrinsic or intrinsic fibres. The latter possibility is consistent with the results of extracellular recording from cat [31] and dog [4,5,13] heart ganglia, where neurones fired in response to mechanical stimulation. In the present experiments no muscarinic slow potentials accompanied the nicotinic fast EPSP during either single or repetitive stimulation. This finding contrasts with the evidence that neurones of the rat heart [15] and guinea pig cardiac neurones in culture [2,16] are endowed with muscarinic acetylcholine receptors and that all guinea pig cardiac neurones maintained in culture [2] and rat cardiac neurones in vitro [36] respond to
muscarinic agonists, with slow de- or hyperpolarizations. The reason for this difference is unclear. One possible explanation is that more muscarinic acetylcholine receptors are activated by exogenously applied acetylcholine or muscarine than by synaptically released acetylcholine. This suggestion is consistent with the finding that muscarinic acetylcholine receptors are present over the entire surface of cultured cardiac neurones [16]. Interestingly, muscarinic slow postsynaptic potentials were also not detected in cardiac neurones of the guinea pig [23] and the neonate rat [34]. In latter studies, however, slow depolarizing potentials were noted, not seen in the present experiments. They appeared in response to highfrequency orthodromic stimulation and were unaffected by nicotinic and muscarinic antagonists; hence, it was suggested that they were peptidergic in origin. Besides a muscarinic response, one might also expect a slow adrenergic synaptic response in mammalian cardiac neurones. Firstly, in a number of mammalian species, including the rat, the cardiac plexus contains terminal varicosities of adrenergic fibres impinging upon ganglionic neurones [20,27]. Secondly, canine cardiac neurones
197
A Control
13 Trimetaphan 100 IJM
a 4j----__
300 #M
. . . . . . . .
C Wash
120 mV 20 ms
Fig. 7. Effect of trimetaphan on the response to single fibre tract stimulation. Responses were evoked before (A) and after
(B) adding 100/~M (a) or 300/zM (b) trimetaphan and after washing the drug out (C). Resting membrane potential -47 mW.
in vivo can be activated by repetitive stimulation of thoracic sympathetic ganglia [4,5,13]. Thirdly noradrenaline produces slow membrane depolarization and discharges in rat cardiac neurones in vitro (A. Selyanko, unpublished observations). However, as mentioned above, slow postsynaptic potentials were not detected in the present experiments. The fast EPSP in cardiac neurones exhibited several peculiarities. First of all, it always appeared in an all-or-none manner and was large enough to trigger a spike. This is an indication that rat cardiac neurones have a single strong synaptic input from successfully stimulated fibre tracts. Consistent with these findings, it has been recently reported that most guinea pig [23] and neonate rat [34] cardiac ganglion neurones re-
ceive single or few synaptic inputs. Interestingly, most amphibian cardiac ganglia neurones are also singly-innervated [8]. In mammals, parasympathetic ganglia contain either singly innervated neurones (e.g. rat submandibular ganglion [24]), both singly and multiply innervated neurones (e.g. rabbit ciliary ganglion [21,22]), or mainly multiply innervated neurones (e.g. guinea pig intestinal [18,28], gastric [33] or gall-bladder [26] ganglia). In mammalian sympathetic ganglia, each neurone can be discharged either by a single strong synaptic input or by a few accessory synaptic inputs [38]. It has been hypothesized [32] that there is a fundamental relationship between the number of inputs to a neurone and the latter's geometry: unipolar neurones are singly innervated whereas multipolar neurones are innervated by two or more axons whose number is proportional to the number of dendrites. If this relationship is extendable to mammalian cardiac ganglia, some of which contain unipolar, bipolar and multipolar neurones [7], then one can expect some mammalian cardiac neurones to be singly innervated, in agreement with the present results. Another feature of the fast EPSP in cardiac neurones is its 'run-down' during repeated stimulation. A similar frequency-dependent decrease in amplitude of fast EPSP was previously noted in amphibian cardiac neurones [8]. In some mammalian ganglia (e.g. guinea pig intestinal plexus [28]), the EPSP amplitude decreases with the frequency of stimulation as low as 0.1 Hz. On the other hand, in other ganglia (e.g. guinea pig gastric [33] or gall-bladder [26] plexuses), the amplitude of the fast EPSP is maintained even at high stimulation frequencies. Run-down of the fast EPSP in intestinal neurones is attributed to inhibition of acetylcholine release, resulting from either autoinhibition by acetylcholine acting on presynaptic muscarinic acetylcholine receptors, or by presynaptic inhibition by other substances released during repetitive stimulation [11]. Rundown of the fast EPSP in rat cardiac neurones might likewise be presynaptic in origin since no indication of a decay of suprathreshold nicotinic acetylcholine depolarization is seen during prolonged (up to several seconds) application of acetylcholine [36].
1~,~
One more feature of the fast EPSP in rat cardiac neurones is its low sensitivity to ganglionblocking agents. It was found that the fast EPSP here remains suprathreshold for triggering the spike in the presence of 300/xM d-tubocurarine, 1 mM hexamethonium, or 100-/xM trimetaphan. This finding is consistent with the earlier observation that spontaneous activity and responses to stimulation of canine cardiac neurones in vivo persist after administration of hexamethonium [4,5,13]. On the other hand, in vitro studies on the same neurones indicate that fast EPSPs are abolished by 100/xM hexamethonium [42,43]. The reason for the latter difference remains to be clarified. One possible explanation is that there are non-cholinergic synaptic mechanisms in cardiac ganglia [4,5,13,43]. Seabrook and co-workers [34] came to a similar conclusion on the basis of their finding that the fast EPSP in neurones of rat neonatal cardiac ganglion persisted in the presence of high concentrations of mecamylamine which abolished the nicotinic acetylcholine response. However, the present results do not allow us to suggest that there is a noncholinergic component in fast EPSP in rat cardiac ganglia neurones: high concentrations of TC and C~, failed to abolish not only the EPSPs but also responses to acetylcholine in these neurones [36]. Thus the high resistance of fast EPSP to ganglion-blockers is probably due to the low sensitivity of nicotinic acetylcholine receptors. Interestingly, the effectiveness of ganglion blockers in synapses of other mammalian ganglia seems to be much higher than in the present experiments on rat cardiac ganglia. For example, 100-400 /xM hexamethonium completely abolished both fast EPSP and fast acetylcholine depolarization in guinea pig intestinal neurones [29,39]. Only in autonomic ganglia of the frog, fast EPSCs are relatively resistant to hexamethonium and trimetaphan, but not to d-tubocurarine [25]. In cats, surprisingly, hexamethonium is more effective in blocking vagus-induced decrease in heart rate than in reducing arterial blood pressure, i.e. in blocking transmission through sympathetic ganglion (see Fig. 5 in ref. 37). The reason for the difference between these and the above-mentioned data remains to be clarified.
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