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NEUROSCIENCE |
1 Department of Anatomy and Neurobiology, College of Medicine, University of Vermont, Burlington, VT 05405, USA
2 Department of Pharmacology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, USA
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Abstract |
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50% and an enhanced excitability occurred in only 10% of the cells. These observations suggested that PACAP released from preganglionic nerve terminals during tetanic stimulation enhanced neuronal excitability and evoked sEPSPs. After addition of 1 nM PACAP to the bath, 7 of 9 neurones exhibited a tonic firing pattern whereas in untreated preparations, the neurons had a phasic firing pattern. PACAP6-38 (500 nM) diminished the increase in excitability caused by 1 nM PACAP so that only 4 of 13 neurones exhibited a tonic firing pattern and the other 9 cells retained a phasic firing pattern. These findings indicate that PACAP can be released by tetanic neural stimulation in vitro and increase the excitability of intrinsic cardiac neurones. We hypothesize that in vivo PACAP released during preganglionic firing may modulate neurotransmission within the intrinsic cardiac ganglia.
(Received 19 April 2007;
accepted after revision 9 May 2007;
first published online 10 May 2007)
Corresponding author R. L. Parsons: Department of Anatomy and Neurobiology, College of Medicine, University of Vermont, Burlington, VT 05405, USA. Email: rodney.parsons{at}uvm.edu
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Introduction |
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Repetitive stimulation of interganglionic nerve bundles innervating cardiac ganglia can initiate a slow excitatory postsynaptic potential (sEPSP) (Konishi et al. 1985; Hardwick et al. 1995). A slowly developing depolarization can also be elicited by local application of capsaicin onto individual ganglia (Hardwick et al. 1995). Since capsaicin evokes release of substance P from sensory nerve fibres, previous investigators have suggested that the sEPSP might be generated by substance P released from perineuronal afferent nerve fibres (Konishi et al. 1985; Hardwick et al. 1995).
In addition to eliciting depolarization, neuropeptides, particularly PACAP, are known to affect the excitability of cardiac neurones (Braas et al. 1998; DeHaven & Cuevas, 2004; Tompkins et al. 2006). Even though it is established that a brief period of tetanic neural stimulation can elicit the sEPSP in innervated cells, it is not known whether a change in excitability also occurs following tetanic neural stimulation. Consequently, the present study investigated if high frequency stimulation (HFS) of nerve inputs increases cardiac neuron excitability and if so, whether release of either PACAP or substance P contributed to this effect.
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Methods |
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Intracellular recording procedures
Atrial whole mount preparations were pinned in a 2.5 ml Sylgard-lined Petri dish and superfused continuously (2–3 ml min–1) with Krebs solution also containing 10 mM Hepes buffer (32 ± 1°C) (Hardwick et al. 1995, 1997; Braas et al. 1998; Tompkins et al. 2006). Intracardiac ganglia were visualized with an inverted microscope equipped with Hoffman optics and individual cardiac neurons were impaled using high impedance borosilicate microelectrodes (2 M KCl-filled; 60–100 M
). Active and passive membrane properties were recorded from the impaled neurons using an Axoclamp-2A amplifier coupled with a Digidata 1322A data acquisition system and pCLAMP 8 (Axon Instruments, Union City, CA, USA). Hyperpolarizing current was injected through the recording electrode as needed to maintain a resting membrane potential between –50 and –60 mV. Visually identified nerve bundles innervating a ganglion were stimulated with a bipolar electrode using 1 ms pulses with stimulus intensity ranging between 5 and 150 µA.
For brief local application, PACAP27 (at a concentration of 50 µM) or substance P (at a concentration of 100 µM) was applied by pressure application (Picospritzer, General Valve, Fairfield, NJ, USA) through 5 µm-diameter ‘puffer’ pipettes positioned 50–100 µm from the neurone. PACAP27 (1 nM) was also added directly to the bath solution during some experiments. To test for peptide-induced changes in neuronal excitability, 1 s depolarizing current steps (0.1–0.4 nA) were given before and 2–3 min after either exogenous PACAP27 or substance P application or tetanic neural stimulation. Excitability curves were constructed by plotting the number of action potentials generated by increasing stimulus intensities.
Drugs
PACAP27 (referred to simply as PACAP throughout the text) and PACAP6-38 were obtained from American Peptide Co. (Sunnyvale, CA, USA). Substance P was purchased from Sigma (St Louis, MO, USA).
Data analysis
Results are expressed as mean ±
S.E.M. In some experiments, control and test results were obtained in the same cell with the results averaged and analysed with Student's paired t test. When results were compared between cells, statistical significance was determined with Student's non-paired t test. Differences with P < 0.05 were considered statistically significant. A 
2 test was used to compare categorical changes in excitability between two groups.
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Results |
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In initial experiments with untreated whole mount cardiac ganglia preparations, we tested whether a 10 s period of HFS could initiate a sEPSP and/or increase excitability of the innervated intrinsic cardiac neurones. Intracellular recordings were made from individual cardiac neurones prior to, during and following a 20 Hz stimulation of an adjacent nerve bundle. To assess cardiac neurone excitability, a series of 1 s intracellular depolarizing current steps were applied before and after the tetanic stimulation of the nerve bundle. Action potentials were produced in the cardiac neurone following each stimulus applied to the nerve bundle, and a slow depolarization developed after termination of HFS in several cells (Fig. 1A). A sEPSP was observed in all cells in which activation followed HFS of a preganglionic nerve; some cells were antidromically activated which did not produce a sEPSP or a change in excitability (data not shown). In several cells, once the sEPSP ended, the number of action potentials produced by the same constant current depolarizing step applied prior to HFS was markedly greater, indicating that cardiac neurone excitability was also enhanced following tetanic stimulation of the nerve bundle (Fig. 1B). The increase in excitability could last for several minutes.
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In subsequent experiments, preparations were continually bathed in solution containing atropine. Following verification that a cell received synaptic input from an adjacent nerve connective, hexamethonium was added to the bathing solution. With nicotinic and muscarinic receptors blocked, HFS consistently caused a sEPSP, which commenced during stimulation and ranged in amplitude between 2 and 13 mV (mean sEPSP amplitude = 7 ± 1 mV, 11 cells). The duration of the sEPSP ranged from 38 to 118 s (mean sEPSP duration = 61 ± 8 s). With the largest sEPSPs multiple action potentials were elicited (Fig. 1E). In the presence of atropine and hexamethonium, HFS also caused an increase in excitability in 5 of 11 cells, tested after the sEPSP had ended (Fig. 1F).
Both PACAP and substance P depolarize the cardiac neurons, but only PACAP causes an increase in excitability
Earlier studies had shown that PACAP and substance P work through distinct receptors to depolarize guinea pig cardiac neurones (Hardwick et al. 1997; Braas et al. 1998). We have confirmed these earlier results. Brief puffer application (500 ms) of either PACAP or substance P elicited a slow depolarization (Fig. 2A and C). The mean amplitude of the depolarization was comparable for both peptides although substance P more consistently initiated depolarization (Fig. 2E). Neuronal excitability was assessed in the same cells before exposure to peptide and again following termination of the agonist-induced depolarization. As illustrated in Fig. 2B and D, excitability was enhanced following application of PACAP, but not after substance P. Excitability curves were determined for multiple cells by intracellular stimulation at several current intensities before and after application of either PACAP or substance P (Fig. 2F). The number of action potentials generated prior to peptide application ranged between one and two regardless of current amplitude (from 0.1 to 0.4 nA). No additional action potentials occurred after application of substance P, whereas following PACAP application, the number of action potentials increased progressively with increased stimulus intensity.
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Based on these results, we postulated that the increase in excitability following HFS could be caused by the release of PACAP from preganglionic nerve terminals, but most likely not by substance P released from afferent fibres. To evaluate this possibility, we tested whether HFS elicited a sEPSP and an increase in excitability when the PAC1 receptor antagonist PACAP6-38 (500 nM) was present in the bath solution along with hexamethonium and atropine. In the presence of PACAP6-38, the mean amplitude of the sEPSP was significantly smaller (3 ± 1 mV, 10 cells) than in the absence of PACAP6-38 (7 ± 1 mV, 11 cells), and no depolarization was evident after HFS in 2 of the 10 cells. Furthermore, excitability was unchanged in 9 out of 10 neurons after HFS in the presence of PACAP6-38 and the remaining cell showed only a small increase. These results provided further evidence that PACAP released from preganglionic nerve terminals by HFS can modulate cardiac neurone excitability and contribute to the generation of the sEPSP.
A low concentration of PACAP in the bathing solution causes a sustained increase in the excitability of cardiac neurons
Our results indicate that PACAP can be released from preganglionic terminals during HFS. We hypothesized that low levels of PACAP might be released in vivo during ongoing vagal activity and have a role in regulating cardiac neurone excitability. When the whole mount preparation is prepared for in vitro experiments, the vagal input is severed and the ongoing vagal tone is lost. In these preparations, 90% of the cardiac neurones exhibit a phasic-like pattern of action potential firing when tested with depolarizing current steps (Edwards et al. 1995; Hardwick et al. 1997; Braas et al. 1998; Tompkins et al. 2006). Thus, we hypothesize that in vivo there may be a low level of PACAP present, which could enhance cell excitability. Consequently, we tested in four whole mount preparations whether 1 nM PACAP added to the bathing solution affected the firing pattern of the intrinsic cardiac neurones. In three cells, we tested excitability prior to and following PACAP application. All three neurones were phasic prior to PACAP and two of these three neurons exhibited a tonic-like firing pattern after a few minutes exposure to PACAP. Results from one of these neurones are shown in Fig. 3A. Five of six other neurones tested during a 1–3 h sustained exposure to PACAP also exhibited a tonic-like firing pattern. So in total, 7 of 9 cardiac neurones exhibited a tonic-like firing pattern during sustained exposure to 1 nM PACAP (Fig. 3B).
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2 test). |
Discussion |
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Following nicotinic receptor blockade, HFS generally evoked a hyperpolarization, which was mediated by activation of muscarinic receptors. A neurally evoked IPSP was originally identified in mudpuppy intracardiac neurones (Hartzell et al. 1977) and later also observed in mammalian cardiac neurones (Xi-Moy et al. 1993). For instance, a muscarinic receptor-mediated hyperpolarization or outward current has also been recorded in neonatal rat and guinea pig cultured cardiac neurones (Allen & Burnstock, 1990; Beker et al. 2003).
Exogenous application of PACAP and substance P elicited depolarization, but only PACAP increased cardiac neurone excitability. This observation and the fact that the PAC1 antagonist effectively inhibited the increase in HFS-induced excitability suggested that PACAP released from preganglionic nerve terminals mediated this effect.
Both substance P-IR fibres and PACAP-IR fibres can innervate guinea pig cardiac ganglia (Dalsgaard et al. 1986; Braas et al. 1998). PACAP, which is contained in the cholinergic preganglionic terminals, is present in fibres innervating virtually all guinea pig cardiac neurons (Calupca et al. 2000). Conversely, substance P-IR fibres commonly innervate only a subpopulation of ganglia (Calupca et al. 2000). Thus, it is quite likely that PACAP-IR fibres are present in more nerve connectives and at a greater abundance than substance P-IR fibres. Therefore, we reasoned that PACAP released from preganglionic nerve terminals during HFS was more likely to be responsible for initiation of the sEPSP in most intrinsic cardiac neurones. However, we cannot exclude the possibility that SP released from afferent fibres could contribute to the generation of the sEPSP in some cells under the conditions of these experiments.
PACAP6-38 at a concentration of 500 nM did not always eliminate the sEPSP. We propose three interpretations of this observation. First, the release of substance P from afferent fibres might have contributed to HFS-induced generation of the sEPSP in some cells. Second, given that there is heterogeneity within the population of intrinsic cardiac neurones, not all cells respond similarly to PACAP. Third, at 500 nM, PACAP6-38 might not completely block all PAC1 receptors. The latter observation is consistent with the observation that PACAP6-38 suppressed, but did not eliminate, the PACAP-induced increase in excitability when the peptide was added to the bathing solution.
Application of PACAP can depolarize and increase excitability of guinea pig myenteric neurones (Ermilov et al. 2004). In addition, PACAP released during nerve stimulation or reflex activation of the colon can depolarize these neurones, an effect blunted by PAC1 receptor antagonists. Thus, in the guinea pig, release of endogenous PACAP at different autonomic synapses apparently can alter the properties of the postsynaptic cells.
In conclusion, the present results demonstrate that endogenous PACAP, which is released from preganglionic parasympathetic nerve terminals during repetitive nerve stimulation, contributes to the generation of the sEPSP and can modulate excitability of guinea pig intrinsic cardiac neurones. Furthermore, in the present study, when 1 nM PACAP was included in the bath solution, cardiac neurone excitability was consistently enhanced and this effect was maintained for the duration (1–3 h) of exposure. Thus, we hypothesize that in vivo during ongoing vagal preganglionic fibre firing, low levels of PACAP may be released and could regulate the excitability state of cardiac neurones.
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References |
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Beker F, Weber M, Fink RHA & Adams DJ (2003). Muscarinic and nicotinic ACh receptor activation differentially mobilize Ca2+ in rat intracardiac ganglion neurons. J Neurophysiol 90, 1956–1964.
Braas KM, May V, Harakall SA, Hardwick JC & Parsons RL (1998). Pituitary adenylate cyclase-activating polypeptide expression and modulation of neuronal excitability in guinea pig cardiac ganglia. J Neurosci 18, 9766–9779.
Brown D (1988). M-currents: an update. Trends Neurosci 11, 294–299.[CrossRef][Medline]
Calupca MA, Vizzard MA & Parsons RL (2000). Origin of pituitary adenylate cyclase-activating polypeptide (PACAP)-immunoreactive fibers innervating guinea pig parasympathetic cardiac ganglia. J Comp Neurol 423, 26–39.[CrossRef][Medline]
Dalsgaard CJ, Franco-Cereceda A, Saria A, Lundberg JM, Theodossson-Norheim E & Hokfelt T (1986). Distribution and origin of substance P- and neuropeptide Y-immunoreactive nerves in the guinea pig heart. Cell Tissue Res 243, 477–485.[Medline]
DeHaven WI & Cuevas J (2004). VPAC receptor modulation of neuroexcitability in intracardiac neurons: dependence on intracellular calcium mobilization and synergistic enhancement by PAC1 receptor activation. J Biol Chem 279, 40609–40621.
Edwards FR, Hirst GDS, Klemm MF & Steele P (1995). Different types of ganglion cells in the cardiac plexus of guinea-pigs. J Physiol 486, 453–471.[Medline]
Ermilov LG, Schmalz PF, Miller SM & Szurszewski JH (2004). PACAP modulation of the colon-inferior mesenteric ganglion reflex in the guinea pig. J Physiol 560, 231–247.
Hardwick JC, Mawe GM & Parsons RL (1995). Evidence for afferent fiber innervation of parasympathetic neurons of the guinea pig cardiac ganglion. J Auton Nerv Syst 53, 166–174.[CrossRef][Medline]
Hardwick JC, Mawe GM & Parsons RL (1997). Tachykinin-induced activation of non-specific cation conductance via NK3 neurokinin receptors in guinea-pig intracardiac neurons. J Physiol 504, 65–74.[CrossRef][Medline]
Hartzell HC, Kuffler SW, Stickgold R & Yoshikami D (1977). Synaptic excitation and inhibition resulting from direct action of acetylcholine on two types of chemoreceptors on individual amphibian parasympathetic neurones. J Physiol 271, 817–846.
Konishi S, Okamoto T & Otsuka M (1985). Substance P as a neurotransmitter released from peripheral branches of primary afferent neurons producing slow synaptic excitation in autonomic ganglion cells. In Substance P. Metabolism and Biological Action, ed. Jordan CC & Oehme P, pp. 121–136. Taylor & Francis, Philadelphia, PA, USA.
Parsons RL (2004). Mammalian cardiac ganglia as local integration centers: histochemical and electrophysiological evidence. In Mechanisms of Cardiovascular Regulation, ed. Dun N, Machado B & Pilowsky P, chap. 15, pp. 335–356. Kluwer Academic Publishers, Norwell, MA, USA.
Tompkins JD, Hardwick JC, Locknar SA, Merriam LA & Parsons RL (2006). Ca2+ influx, but not Ca2+ release from internal stores, is required for the PACAP-induced increase in excitability in guinea pig intracardiac neurons. J Neurophysiol 95, 2134–2142.
Xi-Moy SX, Randall WC & Wurster RD (1993). Nicotinic and muscarinic synaptic transmission in canine intracardiac ganglion cells innervating the sinoatrial node. J Auton Nerv Syst 42, 201–214.[CrossRef][Medline]
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Acknowledgements |
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). In contrast, prior to peptide application or following substance P application (n
= 9 cells, 
), the number of action potentials produced did not change markedly with increasing stimulus strength. The control curve is the average response obtained from 18 cells prior to peptide application (
). The asterisks indicate that the number of action potentials produced was significantly greater following PACAP application than prior to peptide application.