F.M. Smith- Extrinsic inputs to intrinsic neurons in the porcine heart in vitro

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276:R455-R467, 1999.Am J Physiol Regul Integr Comp PhysiolF. M. Smithin vitroExtrinsic inputs to intrinsic neurons in the porcine heart

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, November 1, 2001; 281 (5): R1474-R1482.Am J Physiol Regul Integr Comp PhysiolF. M. Smith, A. S. McGuirt, J. Leger, J. A. Armour and J. L. Ardellneurons in vitroEffects of chronic cardiac decentralization on functional properties of canine intracardiac

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Extrinsic input s to intr insic neur ons in th e porcine

heart in vitro

F. M. SMITH

Departm ent of An atomy and N eurobiology, Faculty of Medicine,

Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7

Smith, F. M. Extrinsic inputs to intrinsic neurons in theporci ne h e a rt i n vit ro. A m . J . P h ys iol . 2 76 (Regulatory

Integrative Comp. Physiol. 45): R455R467, 1999.Conver-genc e of i nput s from e xt ri nsic c a rdi a c ne rves [va gus a ndcardiopulmonary (CPN)] on intr insic cardiac n eurons wasinvestigated in the pig (Sus scrofa ). A segment of the rightatrial wall containing epicardial neurons along with attachedstum ps of the right vagus nerve and CPN was mainta ined invitro; intracellular recordings were made from 57 neurons.Three types of neuron were identified by their responses tolong intracellular depolarizing current pulses: phasic [dis-charged 1 action potential (AP); 40%]; accommodating (dis-cha rge d mul t ipl e APs de cre me nt ing i n fre quenc y dur i ngpulse; 33%); and tonic (discharged multiple APs at a highfrequency; 27%). Sixty-six percent of the neurons responded

with excita tory postsynap tic potent ials (EP SP) to vagal nervestimulation; two-thirds of these cells fired APs when EPSPamplitude exceeded thr eshold level. Postsynaptic r esponsesto vagal nerve stimulation were mediated by nicotinic ioncha nne l s; re sponse s we re e li mina t e d by he xa met honium.CPN stimulation produced EPSPs but no APs in 17% of theneur ons.All neur ons responding with postsyna ptic depolariza-tions to CPN stimulation also received vagal inputs. Com-bined stimulation of the vagus nerve a nd CPN pr oduced APsin all but one of th ese neur ons. Timolol elimina ted postsyna p-t i c re sponse s from CPN st i mula t i on, i ndica t i ng t ha t t he seresponses involved -adrenergic receptors and likely resultedfrom activation of sympath etic postganglionic t erminals.T h es e r e su l t s s h ow t h a t s om e i n t r in s ic ca r d ia c n e u r on sreceive convergent inpu ts from th e CPN and vagus nerve. It

i s suggest e d t ha t such ne urons re pre se nt i nt ra ga ngli oni csites for sympathetic-para sympathetic interactions in n euralcontrol of the heart.

i nt ri nsic ca rdi a c ga ngli a ; sympa t he t ic i nne rva t i on; va gusnerve; cardiopulmonary nerve; intracellular recording

T H E MAMMAL IAN H E A RT i s d u a lly in n e r va t e d b y t h esympathetic and parasympathetic l imbs of the auto-nomic nervous system. The cardiac branches of thevagus n erves convey para sympat hetic efferent pregan-glionic axons to t he hear t (29, 30) where some of theseterminate on postganglionic neurons in the intrinsic

cardiac ganglia. The majority ofsympat hetic postgangli-onica xons inner vating th e hear t course in cardiopulmo-nary nerves originating from the middle cervical, stel-late, and, in some species, upper thoracic ganglia of theparavertebral ganglion chain (30, 31, 40). The anatomicseparation of these autonomic pathways has informedana lyses of their functions in cardiac regulation, andthe sympath etic and parasympath etic l imbs are tradi-tionally considered to control the heart in a reciprocalfashion. That is, when vagal cardiodepressant activityis low, sympa th etic cardioaugmen ta tory activity is high,and vice versa (14). However, studies in which activity

of vagal and sympathetic cardiac nerves was recordeds i m u l t a n e o u s l y h a v e s h o wn t h a t t h e s e i n p u t s t o t h ehear t a re coactivated un der a wide ran ge of physiologi-cal conditions, for example during periods of increasedat ria l filling (36).

W h e n p a r a s y m p a t h e t i c a n d s y m p a t h e t i c i n p u t s t othe heart are activated together, significant interac-tions between t hese input s occur, and t he prima ry sitesfor these interactions are considered to be the auto-nomic neuroeffector junctions at the myocardium (24,38, 39, 46, 47, 53). At these sites, interactions occurthrough sympathetic modulation of vagal function orvagal m odificat ion of sympat hetic function, depending

on the prevailing physiological circumsta nces (58).These interactions are believed to be mediated by th ecombined influen ces of tra nsmitt er an d modulator sub-stances released by sympathetic and parasympath etict e r min a ls on t h e m yoca r d iu m (3 9), a s w ell a s b yprejunctional modulatory mechanisms on neuroeffectorterm inals (38, 47). However, an other potential site forsympath etic-para sympath etic interactions is within thei n t r a ca r d i a c g a n g li a a t t h e l ev el of s in g le n e u r on s .These neurons are organized into ganglionated plex-uses, which are conn ected into a network by inter gangli-on i c n e r ve s (5 , 9 , 1 0, 1 2, 1 3, 2 2, 5 1). Cla s s ica l ly,intracardiac neurons have been viewed as simple re-lays between para sympat hetic preganglionic axons and

the myocardium. However, recent ana tomic r eportsindicate that sympathetic terminals exist within intra-cardiac ganglia (22, 43, 44), and it is therefore possiblet h a t s ym p a t h et ic a n d p a r a sy mp a t h et ic in p u t s m a yconverge on single intr acardiac neu rons.

The main objective of this study was to investigatethe intracellular responses of single intrinsic cardiacneur ons of the pig right at rium in vitro to stimulat ion ofa t t a c h ed ca r d i a c v a g a l a n d ca r d i op u lm on a r y n e r vestum ps t o ident ify neu rons potentially capable of inte-grating informat ion from both efferent l imbs of t heau tonomic ner vous system. Recordings ma de from intra -cardiac neurons in t his study sh ow th at some of thesen e u r on s r e ce iv e b ot h p a r a s ym p a t h e t ic a n d s ym p a -

thetic inputs and that interactions between these dualinputs can modify neur onal firing properties.

METHODS

Experiments were done on 22 pigs of both sexes, 2026days old and with a mean body mass of 8.5 0.3 kg (mean SE). Pilot studies were conducted on a range of species (dog,pi g, a n d guine a pi g) t o de t ermi ne t he be st pre pa ra t i on forrecording intr acellularly from intr acardiac neurons with ex-t ri nsi c i nput s. The pi g he a rt wa s c hose n be c a use , i n t hi sspecies, a population of intracardiac neurons with responsesto extrinsic nerve stimulation was located in th e right atrial

0363-6119/99 $5.00 Copyright 1999 th e Am er ica n P h ysiologica l Society R455


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wa l l cl ose t o t h e e nt ry poi nt of t h e e xt ri nsic ne rve s. Theoverall size of this segment of atrial wall an d at tached nerves t u m p s w a s s m a ll e n ou g h t o b e m a in t a i n ed in v it r o, a n dintracardiac ganglia receiving extrinsic inputs could be ac-cessed easily for recording. Animals were obtained from alocal livestock supplier an d were mainta ined in the Univer-sity Animal Care Facility for up to 3 days at 20C under a12:12-h light-dark cycle before the experiments. Animals

we re t r e a t e d i n conforma nc e wi t h t he guide li nes of t h eCa na di a n Counc il on Ani ma l Ca re ; t he prot ocol for t he seexperiments was approved by the Dalhousie University Com-mitt ee on Anima l Care. Animals were k illed by an overdose ofpentobarbital sodium (1,000 mg) administered intraperitone-ally. The sternum was opened in the midline, and the heartwi t h a t t a c he d st umps of r i ght va ga l a nd c a rdi opul mona rynerves was quickly removed an d furth er dissected in a dishcontaining flowing oxygenated Tyrode solution at room tem-peratu re. This solution contained (in mM) 120 N aCl, 4 KCl,1. 2 KH 2P O4, 1. 2 MgSO4, 11 gl uc ose , 10 HEPES, a nd 1. 9CaCl2; pH wa s ad justed to 7.4 before use. All chemicals wereobt a i ned from Si gma (St . Loui s, MO). The sol ut ion wa ssa t ura t e d wi t h 100% O2 using a gas disperser. A block oftissue including the intercaval right atrial wall along withattached nerves was dissected free of the heart for in vitrorecording from intracardiac n eurons. To ensure inclusion ofa l l r i ght ca rdi a c va ga l bra nche s, se gment s of t h i s n e rveextending cran ially from t he h eart to the level of the m iddlecervical ganglion near the thoracic inlet and caudally fromt h e h e a r t t o t h e d ia p h r a gm w er e i n cl u de d. T h e a t t a c h edsympathetic cardiac nerves included a ll cardiopulmonarybra nc he s runni ng t o t he he a rt from t he ri ght st e l l a t e a ndmiddle cervical gan glia a nd a nsa e subclavia. The tissue blockwas pinned to the silicone rubber-covered bottom of a 30-mlre cordi ng cha mbe r a nd pe rfuse d wit h oxygena t e d Tyrodesolution maintained at 36C by a thermostatically controlledheater.

The stum ps of the vagal and cardiopulmonary n erves weregathered into bipolar suction electrodes filled with perfusatefor stimulation with rectangular current pulses (0.5-ms dura-

tion). These electrodes were driven by a stimulator (S88,Gra ss Inst rume nt , Qui ncy, MA) t hrough const a nt -curre ntstimulus isolation units. Epicardial ganglia were exposed byblunt dissection with the aid of a dissecting microscope. Inthis ar ea of the atr ium, ganglia were embedded in epicardialfat an d were connected togeth er by small nerves in a gan glion-ated plexus. A gan glion selected for recording was lightlysupported on a metal platform (0.2 mm wide, 0.6 mm long)at tached t o a micropositioner. With t his plat form th e ganglionwas h eld slightly away from th e un derlying tissue t o providemechanical stabilization, thu s facilitating penetrat ion of thetough ganglion sheat h by th e r ecording electrode (50). Gan-glion cells an d connecting nerves were not da maged by thisprocedure. Gan glia were explored with a glass micropipetteelectrode filled with 3 M KCl (electrode resistance 50120

M), mounted on a three-axis mechanical micromanipulator.The electrode was connected to an intracellular amplifierequipped with current injection a nd bridge-balancing cir-cuitry (model 1600, A-M Systems, Everett, WA) for nullingthe electrode resistance. Before neurons were impaled, elec-trode potential was nulled with reference to the bath poten-t i a l, a n d t h i s n u l l w a s con fi r m ed a ft e r w it h d r a w in g t h eelectrode from the neuron at the end of a r ecording session.The difference between intracellular and bath potentials wast a ke n a s t he t ra nsme mbra ne pot e nt i a l . The ba t h re fe re nc eelectrode consisted of a silver wire, coated with silver chlo-r i de , i n s er t e d i n t o a ca p ill a r y t u b e fi ll ed w it h 1 % a g a rdi ssol ve d i n t he sa me sol ut ion use d t o fil l t he re cordi ng

electrode, with the tip of the capillary tu be immersed in thebath. To determine th reshold an d ma ximal responses to nervestimulation, stimulus current was increased by incrementsuntil the first response appeared (threshold response), thenfurther increased unt il the response reached peak amplitude(maximal response). Transm embrane potential, intracellularst i mulus wa veforms, a nd re sponse s t o ne rve st i mula t i onwere monitored on an oscilloscope during the experimentsa nd st ore d on vide ot a pe t hrough a di gi t a l da t a conve rt e r(model PCM4, Medical Syst ems, Gr eenvale, NY) connected t oa videocasset te recorder for later a na lysis. Selected r esponseswere played back from the tape into a data-acquisition boardatta ched to a personal computer a nd a nalyzed with pCLAMPsoftwar e (Axon Inst ru ment s, Foster City, CA).

Data Analysis

Whole cell input r esistan ce was estim ated from t he slope ofa line plotting membran e potential displacements from r est-ing potential again st th e amplitu des of a series of hyperp olar-i zi ng curre nt pul se s (200- t o 400-ms dura t i on) de li ve re dthr ough th e recording electrode to clamp membran e currenta t pre set l evel s. Some ne urons displa yed t i me -de pende ntvoltage chan ges at t he sta rt of hyper polarizing pulses, so only

t he l a st 50 ms of re sponse s, whe n pot e nt i a l s ha d re a c he dsteady-state values, were sampled. Plots of curr ent-voltagerelationsh ips were linea r over a ran ge of voltages u p to 3540mV more ne ga t i ve t ha n t he re st i ng pot e nt i a l . Al l ne uronsdisplayed marked nonlinear ity in th e cur rent -voltage relation-ship when depolarizing pulses subthreshold for action poten-tial (AP) generat ion were injected, so t hese r esponses werenot included in estimates of input resistance. Membrane timeconstant was estimated from the time course of responses tosmall (0.1 nA) hyperpolarizing currents. A curve-fitting pro-gram (CLAMPAN in the suite of pCLAMP programs) indi-cated th at t hese resp onses followed a single-exponent ial timecourse, as reported for other autonomic neurons (28), so timeconstant for each cell was calculated as the time for mem-brane potential to change by 11/e of the final steady-statevalue during hyperpolarization. The amplitude of depolariza-tion to threshold for AP generation was taken as the differ-ence between r esting membran e potential and t he voltage atwhich regenerative changes were initiated. AP dura tion wasmeasured at one-half peak amplitude. Afterhyperpolariza-tion (AHP) amplitude was taken as the difference betweenr e st in g m e m br a n e p ot e n t ia l a n d p e ak a m p li tu d e of t h ehyperpolarization following the AP. For estimating AHPdura tion, the time was measu red from th e point at which th erepolarizing waveform crossed the level of the resting mem-b r a n e p o t en t i a l, t o t h e p oi n t a t w h ich t h e p ot e n t ia l h a dreturned to one-half the peak amplitude of the AHP.

Differences am ong mean values of intr acellular variableswere analyzed by ANOVA (P 0.05). Where f values weresignifica nt , compa ri sons be t we e n me a ns we re done usi ngScheffes mu ltiple mea ns compa rison t est (57). Experim ent al

design and statistical analysis of the data were carried out inaccordance with guidelines suggested by Wallenstein et al.(52), Glant z (25), an d Den enberg (20).

RESULTS

Membrane and Firing Propertiesof Intracardiac Neurons

Neurons with resting membrane potentials that didnot settle to values more negative than 40 mV within5 min of impalement were excluded from this study.Mean va lues of resting m embran e potential, whole cell

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input resistance, and whole cell t ime constant, alongwith neu ronal responses to depolarization, ar e summ a-r i z e d i n Ta b l e 1 . Al l n e u r o n s s a m p l e d i n t h i s s t u d yresponded with depolarizations toinward current pulsesof 50- to 60-ms dur ation delivered t hrough the record-

ing electrode, and generated APs when stimulus cur-rent reached t hr eshold (Fig. 1). Thr ee types of responseto prolonged (400- to 600-ms durat ion) depolarizingcurrent pulses permitted classification of intracardiacn e u r on s i n t o t h r e e g r ou p s : 1 ) 40% were phasic (dis-ch a r ge d 1 AP a t t h e s t ar t of t h e s t im u lu s p u ls e,F i g. 2A ); 2 ) 33% were accommodating (dischargedmu ltiple APs tha t decreased in frequency as the st imu-

lus continued, Fig. 2B ); and 3 ) 27% were tonic (dis-charged multiple APs with little or no frequency decre-ment throughout the stimulus pulse, Fig. 2 C). Therewere no statistically significant differences in meanresting membrane potential or mean whole cell t imeconstant among the three types of neuron, but wholecell input resistance was significantly higher in phasicthan in a ccommodating or tonic neurons. Phasic neu-

Table 1. Mem brane properties an d characteristics ofaction p otentia ls for neurons group ed by firin g pattern

Neuron Type

Pha sic (40%)(n23)

Accommodating(33%)

(n19 )Tonic (27%)

(n15)

Resting membrane

potential, mV 494 (23) 475 (19) 495 (15)Whole cell input

resistance, M 728* (23) 337 (19) 397 (15)Whole cell time

con st a nt , m s 5.80 .7 (8 ) 7.21 .1 (9 ) 6 .11.2 (11)Action potent ial

characteristicsThreshold depo-

l a ri za t i on , m V 1 94 (23) 272 (14) 252 (15)Total amplitu de,

m V 735 (23) 754 (19) 725 (15)Over sh oot , m V 244 (23) 274 (19) 242 (15)Du ra tion , m s 1.50.4 (23) 1.90.3 (18) 1.60.2 (15)

After hyperpolar-ization char ac-teristics

Am plit ude, m V 83 (22) 133 (19) 102 (15)

Du r a tion , m s 484 (22) 395 (19) 13011 (15)

Va lu e s a r e e xp r e ss e d a s m e a n s S E . N o. of m e a s u r em e n tscontr ibuting to each mean is shown in parent heses beside each value.Characteristics of a ction potentials were evoked by intr acellulardepolarizing currents delivered through the recording electrode.Significantly different from: * accommodating neurons and tonicneurons; accommodating neurons but not tonic neurons; pha sicneurons and accommodating neurons.

Fig. 1. Response of membra ne potential (top trace) of an intr acardiacneuron t o a depolarizing current pulse (0.2 nA, 60-ms duration;bottom trace) delivered thr ough the int racellular r ecording electrode.Stimulus current intensity was at threshold for evoking an actionpotential (AP) in th is cell. Resting membran e potential before curren tinjection was 48 mV; peak of AP overshoots 0 V (indicated at left).Vertical calibration bar: 20 mV, 1 nA. Horizontal calibration bar:20 ms.

Fig. 2. Examples of responses of 3 types of intracar diac neurons tolong intracellular depolarizing current pulses. A C: top main tracer e p r e s e n ts t r a n s m e m b r a n e p o te n tia l ( 0 V in d ic a te d a t left) , a n db otto m m a in tr a c e r e p r e s en ts s t im u lu s cu r r e n t . S m a lle r t r a c es(insets ) above main voltage traces show AP and afterhyperpolariza-tion (AHP) in response to a brief intracellular depolarizing pulse(5-ms duration; current artifact precedes AP). A: phasic neuron;1.2-nA, 520-ms duration current pulse evoked 1 AP. A , inset: AHPduration was 35 ms. Resting membrane potential before stimulationwa s 67 mV. B : accommodating neuron; 0.7-nA, 540-ms-durationcurrent pulse elicited multiple APs, which decreased in frequencyduring the depolarization pulse. B , inset: AHP dur ation was 29 ms.Resting membrane potential was 42 mV. C: t onic neuron; 0.6-nA,440-ms-duration current pulse produced multiple APs that did notdecrement in frequency over duration of pulse. C, inset:AHP durationwas 118 ms. Resting membrane potential was 57 mV. Tonic neuron(C) ha d longest-duration AHP despite displaying highest firingfrequen cy. Vert ical calibra tion ba r: 30 mV for insettr aces; 20 mV, 1 nAfor main traces. Horizontal calibration bar: 150 ms for insettra ces, 60ms for ma in tra ces.

R457EXTRINSIC INPUTS TO INTRACARDIAC NEURONS


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rons also ha d a significan tly lower depolar ization th resh -old for fir ing APs t han did the other t wo cell types. TheAPs of all neur ons in t his stu dy overshot 0 V, an d APd u r a t i on wa s n ot s ig n ifica n t l y d iffe r e n t a m on g t h eg r ou p s . AHPs wer e of s im i la r a m p l it u d e i n a l l c e lltypes, but the duration of AHP in tonic neurons wassignificantly longer than in phasic or accommodatingneu rons (Table 1; examp les shown in F ig. 2, insets ).

Responses to E xtrinsic Nerve S tim ulation

All intra cardiac neurons in t his stu dy were tested forthe presence of inputs from extrinsic cardiac n erves.Intracellular responses evoked by single-pulse stimula-tion of vagus and cardiopulmonary nerves in the threeclasses of neurons are summar ized by neur on type inTable 2. All phasic neurons except one received inputfrom the vagus nerve, and a subset of these neuronsalso responded to cardiopulmonary nerve stimulation.

Many accommodating neurons also received vagal in-put s, an d one of these cells r esponded to cardiopulmo-nary nerve stimulation as well. Only one tonic neuronresponded to vagal nerve stimulation, and no tonic cellsreceived inputs from cardiopulmonar y nerves. All intra -cellular responses to ner ve stimulat ion were confirm eda s or t h o dr om i c b y t h e ir e li m in a t i on i n a m od ifiedperfusate containing 0 Ca 2 a n d h i g h M g2 (10 mM)(see Figs. 3B , 4B , a n d 5B ). No antidromic responses t oextrinsic nerve stimulat ion wer e recorded in this st udy.All postsyna ptic responses wer e depolar izing; no hyper-polarizing responses were observed. Responses to va-g u s a n d ca r d i op u lm on a r y n e r ve s t im u l a t ion a r e d e -scribed in more d eta il in th e following sections.

Vagus nerve stimu lation . In this st udy, 66% of neu-rons sampled responded to vagus nerve stimulation(Table 2; Figs. 3 and 4). Thresh old n euronal responseswere without exception marked by the appearance of small excitatory postsynaptic potentials (EPSP) at la-t e n c i e s o f u p t o 4 0 m s ( Fi g s . 3 a n d 4 ) ; a s s t i m u l u scurrent intensity was increased, multiple EP SPs wereevoked (Figs. 3 and 4). Graded increases in stimulationproduced corresponding increases in the amplitude ofthe EPSPs (Figs. 3 and 4). In some neurons additionalEPSPs frequently occurred at latencies of 70 to 10 0ms a fter th e stimu lus pulse (e.g., Fig. 4A , 2.1-mA tr ace

from top). One-third of the neurons responding to vagalstimulat ion generated EPSP s that did not reach thr esh-old for AP initiation (Fig. 3) even when the intensity ofnerve stimulation was maximal. Some of these EPSPswere followed by small AHPs (e.g., Fig. 4A , 2.5-mAtrace). The durat ion of EPSP s evoked by vagal nervestimulation was similar in all three cell types, so datafrom all groups have been combined; the mean value of

EPSP du ration was 15 2 ms. In eight phasic cells andfive accommodating cells responsive to vagal nervestimulation, EPSP s reached thr eshold for AP genera-tion. The mean amplitud e of EP SPs r eaching thresh oldfor AP generation was 17 3 mV in phasic and 23 4mV in accommodat ing neurons; these values are consis-tent with threshold values for intracellularly evokedAPs in these two groups (Table 1). All orthodromicallymedia ted APs overshot 0 V, an d, for ph asic an d accom-m od a t i n g n e u r on s , p e a k AP a m p l it u d e wa s wit h i n afew millivolts of the values shown in Table 1 for directly

Table 2. Patterns of extrinsic inp ut to physiologicallyidentified intracardiac neurons

Response toExtrinsic Nerve

Stimulation

Neuron Type

TotalsP h a s ic Accom m od a t in g Ton i c

No extrinsic inputn 1 4 14 19% of t ot a l 2 7 24 33

Vagal input onlyn 22 15 1 38% of t ot a l 38 26 2 66

Vagal an d CPN inpu tn 9 1 0 10% of t ot a l 15 2 0 17

CPN, cardiopulmonar y nerve; n , no. of neurons of each type withineach category of response to extrinsic nerve st imulat ion.

Fig. 3. Excitatory response of an intr acardiac neur on to single-pulsestimulation (0.5-ms duration) of cardiac branches of r ight vagusnerve (stimulus indicated by artifacts at start of traces). Restingmembrane potential of cell was 49 mV. A : responses to intensity-graded stimulation (current increasing from top t o bottom traces;intensity indicated at left of each trace). Note multiple excitatorypostsynaptic potentials (EPSP ) and a reduction of laten cy to onset ofresponse at greatest current intensity. No AP could be evoked bynerve stimulation in this neuron. B : response to nerve stimulationwas eliminated by 5-min exposure to modified, Ca 2-free perfusatecontaining 10 mM Mg2. C: exposure to hexamethonium (HEX; 100 M for 6 m in in p e r fu s a te ) a ls o e l im in a te d r e s p on s e to n e r v es tim u la tion , d e m on s tr a t in g th a t th is r e s pon s e w a s m e d ia te d b ynicotinic channels in postsynaptic membrane. Stimulus intensity in

B a n d C was 5 mA. All stimulus artifacts have been t runcated forclarit y. Vertical calibrat ion bar: 10 mV. Horizonta l calibration bar:10 ms.



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evoked APs. Within th e group of neurons r esponding t ov a gu s n e r ve s t im u l a t ion , m e a n AHP a m p l it u d e a n d

du ra tion were, res pectively, 9 2 mV and 54 6 ms forphasic and 15 4 mV and 47 11 ms for a ccommodat -i n g n e u r o n s. I n t h e on l y t o n ic n e u r o n r e s pon d in g t ovagal nerve stimulation, the E PSP did not reach thr esh-old for AP gener at ion.

Cardiopulm onary nerve stimu lation . Graded cardio-pulmonar y nerve stimulat ion evoked EPSPs with corre-spondingly graded amplitudes in 17% of the n eurons inthis stu dy, at latencies up to 15 ms a fter the st imuluspulse (Fig. 5). Nine of these cells were phasic and onewas accommodating (Table 2). These neurons consti-tu ted a subset of those responsive to vagal stimu lation;

n o n e u r o n s i m p a l e d i n t h i s s t u d y r e s p o n d e d o n l y t ocardiopulmonary nerve stimulation. As shown in the

example in Fig. 5, ma ximal cardiopulmonary nervestimulation was not, in itself, sufficient to evoke an APin an y of these neur ons. EP SPs evoked in accommodat-i n g a n d p h a s ic n e u r on s b y ca r d i op u lm on a r y n e r vestimulation had similar amplitudes and durations, soEP SP da ta from all neur ons were combined for compar i-son with t he pr operties of EPSPs evoked in t hese cellsb y v a gu s n e r ve s t im u la t ion , a s s h ow n in Ta b le 3 .EPSPs evoked by cardiopulmonary nerve stimulationwere significantly smaller in amplitude and signifi-cant ly shorter in dur ation tha n those evoked by vagalnerve stimulat ion in th e same neu rons. In none of these

F ig. 4 . I n tr a ca r d ia c n e u r on g en e r a tin g E P S P sand AP in response to vagus nerve stimulation(experimental protocol similar to that in Fig. 3).Re stin g m e m b r a n e p ote n tia l w a s 51 mV. A:intensity-graded single-pulse nerve stimulationevoked multiple EPSPs that summed to exceedthreshold for AP generation at the highest inten-sity (bottom trace; 0-V level indicated at left). Ba n d C: a t s a m e s t im u lu s in ten s ity a s in bottomtrace of A , neuronal responses t o nerve stimula-tion were blocked in 0-Ca 2, high-Mg2 (lo Ca 2,hi Mg2) perfusate and in 100 M bath-appliedhexamethonium, respectively (both 5-min expo-sure). As in Fig. 3, neuronal responses to nervestimulation were mediated by nicotinic postsyn-aptic receptors. D: timolol (TIM; 10 M, 5 min inperfusate) ha d no effect on membrane potentialor responses to vagus nerve stimulation. AP wastruncated for clarity. Vertical calibration bar: 10mV. Horizontal calibration bar: 20 ms.



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dually innervated neurons did vagal nerve stimulation

alone evoke E PSP s a bove thr eshold for AP genera tion.Costimulation of vagus and cardiopulmonary nerves.

There were two types of response to combined vagaland sympathetic nerve stimulation. Two cells, bothpha sic, displayed responses of the t ype designat ed type1, illustr ated in F ig. 6. These neur ons produced EPSP sand APs in response to maximal vagal nerve stimula-tion alone (Fig. 6A ) but did not show any postsynapticresponses to cardiopulmonary nerve stimulation alone(Fig. 6B ). H owever, simulta neous st imulation of vagaland car diopulmonar y ner ves produced a n AP of longerduration and an AHP with a slightly greater amplitude

an d of longer dur ation (Fig. 6C) compared with t he APand AHP generated by vagal nerve stimulation alone.Th e se d iffe r en ce s a r e r e a d il y a p p a r e n t i n Fi g. 6C,where t he AP resulting from vagus nerve st imulationhas been juxtaposed with the response to combined

Fig. 6. Example oftype 1 response of an intr acardiac neur on to vagusnerve and cardiopulmonary nerve (CPN) stimulation. Resting mem-brane potential was 43 mV. A : AP produced by maximal (5.0 mA)single-pulse st imulat ion of vagus nerve (0-V level indicat ed a t left oftrace). B : CPN stim ulat ion pr oduced no direct postsynaptic response,even at highest intensity available (10 mA shown). C: costimulat ionof vagus nerve and CPN with sa me stimulus para meters as in A a n d

B produced prolonged AP and AHP duration. In C, a portion oftrace Ahas been inset at right of main tra ce for compar ison. In both, dashedlin e s in dica te lev el of r e s t in g m e m br a n e p ote n tia l, a n d a r r o wsindicat e h alf-amplitu de of each AP (for dur at ion compar ison). Verti-cal calibrat ion ba r: 10 mV. Horizonta l calibration bar: 20 m s.

Table 3. Comparison of properties of excitatorypostsynaptic potentials evoked in same neurons bystimu lation of vagus and cardiopulm onary n erves

Vagus NerveStimulation

CardipulmonaryNerve Stimulat ion

Am plitu de, m V 193 71*Du r a tion , m s 172 112*

Values are expressed as means SE ; n 10 neurons. *Significantdifference from value for vagus nerve stimulation (P0.05; t-test).

Fig. 5. Effects of cardiopulmonary nerve stim ulat ion on an int racar-diac neuron. Protocol was similar to tha t of Figs. 3 and 4. Restingmembrane potential was 46 mV. A: intensity-graded nerve stimula-tion evoked an EPSP but no AP in this neuron; this was t rue for a llneurons receiving inputs from the cardiopulmonary nerve. B a n dC: blockade of postsynaptic r esponses by lo Ca 2, hi Mg2 perfusateand by timolol (10 M in bath) showed that these responses wereorthodromic and involved -adrenergic receptors. D: postsynapticresponses were u naffected by 100 M hexamethonium in t he bat h.Vertical calibration bar: 10 mV. Horizontal calibration bar: 10 ms.



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nerve stimulation. As only two neurons displaying atype 1 response were found, no st atistical ana lysis of the effects of combined nerve stimulation was possible.

The second t ype of response, designat ed type 2, wasobserved in n ine neur ons: eight pha sic and one accom-

modating. In t hese cells, independent vagal and cardio-pulmonary nerve stimulation evoked EPSPs (Fig. 7, Aa n d B , respectively; F ig. 8A ), but neither input alonewa s s t r on g e n ou g h a t m a x im a l s t im u l u s i n t e n s it y t ogenerate an AP. When both nerves were costimulatedat their respective maximal stimulus intensities, theresulting EP SPs sum med an d reached thr eshold for APgeneration (Fig. 7C; Fig. 8A , bottom ).

One pha sic neuron in which separate stimulation of the cardiopulmonar y a nd vagus nerves evoked small-amplitude EPSPs showed no augmentation of postsyn-apt ic effects in r esponse to combined ner ve stimu lation,s o fit n e it h e r of t h e a b ov e c a t eg or i es a n d wa s n otincluded in th e an alysis.

Effects of cholinergic and -adrenergic blockad e. Ofthe 38 neurons having vagal but no cardiopulmonaryinputs, a group of six neurons was tested to determinewhether nicotinic neurotransmission was involved. Inth is group of cells, th e nicotinic chann el blocker h exame-thonium [100 M in the perfusate for at least 5 min;this dose was found to be effective in pig heart in aprevious stu dy (50)]elimina ted all postsyna ptic depolar-izing potent ials evoked by n erve stimu lation (Fig. 3C).I n a n ot h e r gr ou p of s ix n e u r on s t h a t w er e d u a llyinnervated by th e vagus a nd cardiopulmonar y nerves,the postsynaptic responses to vagus nerve stimulation

(Fig. 4A ; Fig. 8A , top ) were also elimina ted by hexam e-thonium (Fig. 4C; Fig.8B , top ). The r esponses t o car dio-p u lm on a r y n e r ve s t im u l a t ion wer e u n ch a n g e d a ft e rhexameth onium a pplication (Fig. 5D; Fig. 8B , m i d d l e),and combined stimulation of the vagus and cardiopul-

Fig. 7. Example oftype 2 response of an intra cardiac neuron to vagusnerve and CPN stimulation. A: maximal vagus ner ve stimulat ion (6.8mA) elicited an EPSP but no AP. B : maximal st imulat ion of CPN (9.0mA) also generated an EPSP but no AP. C: costimulation of vagusnerve and CPN, using the same stimulus para meters as in A a n d B ,produced EPSPs that summed to exceed firing threshold. Restingm e m b r a n e p o ten tia l in th is ce ll w a s 41 mV before stimulation.Vertical calibration bar: 10 mV. Horizontal calibration bar: 10 ms.

Fig. 8. N icotinic and -adrenergic receptors are involved in type 2responses of intra cardiac neuron t o vagus n erve and CPN stimula-tion. A : contr ol responses t o separat e stim ulat ion of vagus nerve (7.0m A) a n d CP N (9 .0 m A) a n d AP g en e r a te d b y c om b in e d n e r vestimulation (vagus CPN). Stimulus parameters were unchangedthroughout this experiment. B : hexamethonium (100 M for 5 min inperfusate) eliminated th e vagally generated EPSP. EPSP resultingfrom CPN st imulat ion was un affected by hexamet honium but n eurondid not fire an AP in r esponse to costimulat ion. C: tim olol (100 M inp e r fu s a te ) a d m in is te r ed for 5 m in in th e con tin u e d p r e s e n ce of hexamethonium eliminated the EPSP due to CPN stimulation. Inthis condition, combined nerve stimulation had no postsynapticeffect. Resting mem bran e potential was 57 mV. Vert ical calibra tionbar: 20 mV. Horizontal calibration bar: 10 ms.



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monar y nerves t hen produced th e sam e effect (Fig. 8B ,bottom ) a s d id ca r d i op u lm on a r y n e r ve s t im u l a t ionalone. To address the possibility that this concentrationof hexamethonium was insufficient to block potentialnicotinic neurotransmission through cardiopulmonaryinputs, the antagonist dose was increased in two trialst o 2 0 0 M , b u t t h is d id n ot a lt e r t h e r e sp on s e t ocardiopulmonar y nerve stimulat ion (data not shown).

To determine th e involvement of-adrenergic recep-tors in responses to nerve stimu lation, th e -adrenergicantagonist timolol (10 M) was applied in the perfus-ate. This agent was used because i t is a nonselective-antagonist and does not have membrane-stabilizingeffects (6). The dose used in this study was within therange found effective in previous studies (6) and waschosen for the present study on the basis of a series ofpreliminar y experiments t o determine optimum concen-tration in atrial tissue. Two protocols were followed int h e s e e x p e r i m e n t s . I n t h e fir s t , t i m o l o l wa s a p p l i e dduring vagus nerve stimulation in four experiments todetermine if -adrenergic receptors were involved inthese responses. -Blockade had no effect on the post-synaptic response to vagal nerve stimulation in t hesetr ials, an exam ple of which is shown in F ig. 4D. I n t h esecond protocol (6 experiments), timolol wa s adm inis-tered dur ing vagus and cardiopulmonary nerve stimu-lation in t he continued p resence of hexameth onium. Asshown in Fig. 8C, this protocol eliminated the EPSPdue to cardiopulmonar y nerve stimulation (Fig. 8C,m i d d l e); combined cardiopulmonary and vagus nervestimulation then had no effect (Fig. 8 C, bottom ).

D IS C U S S ION

Three patterns of innervation of right atrial neuronsby axons in extrinsic card iac nerves ha ve been revealed

i n t h i s s t u d y . On e - t h i r d o f t h e n e u r o n s s a m p l e d r e -ceived no synaptic inputs ofextra cardiac origin, whereast h e r e m a i n i n g t wo - t h i r d s we r e i n n e r v a t e d b y a x o n sr u n n in g in t h e va gu s n e r ve . N on e of t h e n e u r on ss a m p le d i n t h i s s t u d y r e ce iv ed i n pu t s s ol el y f r omcardiopulmona ry nerves; instea d, input s from car diopul-monary axons converged on a subset of vagally inner-vated n eurons. Int racar diac neurons could be classifiedinto th ree ph ysiological t ypes based on their responsesto depolarizing test current pulses, and there appearedto be a r elat ionsh ip between th e physiological class of aneuron and the probability of that neuron receivingextrinsic inputs. The properties of extrinsic inputs tointracardiac neurons, the relationships between neu-

ron type an d specificity ofinp ut , the int egrat ive capabili-t ies conferred by t hese input pattern s a nd by intr insicmembrane and active properties, and the functionalimplications of th e r esults are discussed in t he follow-ing sections.

Extrinsic Innervation of Atrial Neurons

Sixty-six percent of the cells sampled in this studyresponded with depolarizing postsynapt ic potent ials tor i g h t v a g u s n e r v e s t i m u l a t i o n , b u t i n s o m e n e u r o n sthese depolarizations did not reach threshold for AP

generation. None of the neurons capable of generatingAPs in response to vagus nerve stimulation exhibitedstrong postsynaptic a ctivation (i .e., a unitary EPSPlarge enough to exceed threshold for AP generation).Instead, multiple EP SPs were summ ed to generate anAP. Neurons generat ing vagally mediated EP SPs t here-for e m u s t i n t eg r a t e s yn a p t i c i n p u t s fr om m u l t ip lepreganglionic axons to produce a regenerative output, aresult tha t concords with t he observations of Seabrooke t a l . ( 49 ) on v a ga l i n n er v a t ion of n e u r o n s i n t h eneonata l rat h eart . Those neurons responding with APsto vagal stimulation in the present study may in factrepresent classical efferent parasympath etic neuronsinnervating the myocardium. If this is the case, i t isinteresting to note tha t these neur ons did not representthe m ajority of intr acardiac neur ons sampled.

Perfusion with a modified Tyrode solution containingreduced calcium and high m agnesium levels (blockingr e le a s e of n e u r ot r a n s m i t t er a t n e r ve t e r m in a l s ) orexposure to the nicotinic channel blocker hexametho-nium eliminated the postsynaptic effects of vagal nerve

stimulat ion. These results demonstr at e tha t th e effectsof nerve stimulation were m ediated synaptically andimply tha t such st imulus-elicited depolarizations wereproduced by acetylcholine released from vaga l pr egan-glionic terminals and acting at nicotinic postsynapticreceptors. The latter finding agrees with that of Ed-w a r ds e t a l. (2 1), w h o r e p or t e d t h a t t h e n icot in i creceptor antagonist D-tubocurarine blocked vagal trans-m i ss ion t o i n t r a ca r d ia c n e u r on s i n t h e g u in e a p igatrium. Interestingly, Seabrook et al . (49), using thenicotinic receptor antagonist mecamylamine to blockcholinergic neurotransmission, found that some intra-cardiac neurons had a residual postsynaptic responseto vagal nerve stimulat ion an d suggested tha t a noncho-

linergic neurotransmitter was coreleased from vagalpreganglionic terminals in intracardiac ganglia. How-ever, no evidence for th is effect wa s foun d in t he pr esentstudy.

Seventeen percent of intracardiac neurons in thiss t u d y we r e d u a l ly i n n e r v a t ed b y a x on s i n t h e r i gh tvagus and cardiopulmonary nerves; none of the neu-rons samp led r eceived input s exclusively from cardio-pulmonary nerves. Two types of responses were re-corded in these n eurons. In type 1 responses, illust rat edin Fig. 6, vagus ner ve stimulation alone evoked EPSP sand a n AP wherea s car diopulmonar y nerve stimulationalone had no postsynaptic effect. Stimulation of cardio-pulmonary and vagal nerves together broadened the

duration of both the AP and the AHP compared witht h a t p r od u ce d b y v a ga l s t im u l a t ion a l on e . Beca u s ethere was no direct postsynaptic effect of cardiopulmo-nary nerve stimulation, one possible explanation forthis augmentation is that a neuroactive agent releasedfrom intraganglionic sympathetic terminals (possiblynorepinephrine or a substance coreleased with thistra nsmitt er) could have acted presynapt ically on vagalterminals to potentiate the release of a cetylcholine.Higher a cetylcholine concentra tions within t he syna p-tic cleft could th en h ave been resp onsible for prolongingpostsynaptic channel-opening times, thus leading to



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increased AP and AHP durations. The receptor typesi n vol ve d i n t h i s r e s pon s e r e m a in u n k n o wn . I n t h i sstudy, type 1 responses were in the minority and werefound in only two neurons.

Th e m a j or i t y of n e u r on s i n flu e n ce d b y a x on s i ncardiopulmonar y ner ves displayed t y p e 2 responses:concomitant stimulation of cardiopulmonar y nervesenabled firing of APs dur ing vagal stimu lation (Figs. 7an d 8). In these neu rons, vagus nerve stimulation aloneevoked depolarizations th at were orth odromically medi-at ed (elimina ted by low-calcium, high-magnesium per-fu s a t e ), wer e b lock e d b y h e xa m e t h on i u m , a n d h a delectrophysiological properties (mean EP SP amplitudeand duration, Table 3) similar to those produced bynerve stimulation in neurons innervated only by thev a gu s . EPSPs p r od u ce d b y ca r d iop u lm on a r y n e r vestimulation in dually innervated neurons with type 2responses were significantly smaller in amplitude ands h or t e r i n d u r a t i on t h a n t h os e p r od u ce d i n t h e s a m en e u r on s b y v a ga l i n pu t s , a s s u m m a r i ze d i n Ta b le 3 .Nicotinic receptors were not involved in responses to

cardiopulmonary nerve stimulation, as determined bystimulating these nerves during exposure to hexametho-nium; this antagonist had no effect on the response tocardiopulmonary nerve stimulation either at a concen-tra tion that blocked all vagally m ediated responses(100 M; Fig. 8) or at double this dose. However, the-antagonist timolol blocked postsynaptic responses tostimulation of cardiopulmonary nerves (Fig. 5), a ndexposure to hexameth onium a nd t imolol together com-pletely eliminated postsynaptic responses of th ese neu-rons t o combined nerve st imulation (Fig. 8). Given th edifferences in the properties of EPSPs and the differen-tia l effects of cholinergic and a dren ergic ant agonists onthe postsynaptic responses to vagus and cardiopulmo-

nary nerve stimulation, it is clear that different synap-tic mechan isms were a ctivated by t hese inpu ts. Acetyl-choline, r eleased from vagal pr eganglionic terminalsand acting at nicotinic postsynaptic receptors, l ikelymediated the effects pr oduced by vagus ner ve stimula-tion, whereas the adrenergic neurotransmitter norepi-nephrine, most l ikely r eleased from postganglionicsympatheticterminals and acting at -adrenergicrecep-tors, wa s involved in th e effects of cardiopulmonarynerve stimulation.

There have been several anatomic reports demon-strat ing the presence of sympathetic postganglionicn e r ve t e r m in a l s i n m a m m a l ia n i n t r a ca r d i a c g a n g li a(2 2, 4 3, 4 4), a n d t h e r e su lt s of t h e p r es en t s t u dy

con s t i t u t e fu n ct i on a l e vi de n ce t h a t t h e s e t e r m in a l sma y be capable ofm odulating ganglionic neur otran smis-sion when activated. Endogenously released or exog-e n ou s ly a p p li ed n or e p in e p h r in e h a s b ee n s h own t oaffect ion conductances and thus membrane and firingp r op e r t ie s of p e r ip h e r a l a u t o n om i c n e u r on s , e it h e rfacilitating or inh ibiting ganglionic neu rotra nsmission(1, 3, 17, 18, 45, 50). In th e pig hear t, Sm ith et a l. (50)found that exogenously applied norepinephrine facili-ta ted orth odromically mediated r esponses of some n eu-rons to stimulation of intracardiac plexus nerves. Inth at stu dy, it was pr oposed th at facilitat ion could ha ve

resu lted either from direct postsyna ptic effects of norepi-nephrine or through action at a presynaptic site. Theobservation of type 1 a n d 2 responses in the presentstud y provides evidence th at both of these mechan ismsmay be operative in intr insic cardiac gan glia in th e pighear t. Results of the pr esent in vitro study also supportthe findings of a previous study of the beating caninehear t in vivo showing tha t a drener gic agonists applied

locally to intra cardiac ganglia enha nced th e frequencyof neuronal AP discha rge (33). Clearly th e facilita toryeffects of exogenously applied adrenergic agents in theheart in vivo could have been operating by the samemechanism responsible for the excitatory effects of activating sympathetic inputs to intra cardiac neuronsseen in th e present in vitro stu dy.

The results of the present experiments showing thatthe influences of axons in vagus and cardiopulmonaryn e r ve s a r e m e d ia t e d b y ch ol in e r gi c a n d a d r e n er g icreceptors, resp ectively, provide an insight into th e issueof possible mixing of sympa thet ic an d pa ra sympath eticfibers within individua l extrinsic car diac nerves. Someparasympath etic preganglionic fibers innervating t hemamm alian heart have been reported to diverge fromthe vagus n erve in th e neck to course with sympath eticn e r ve s, joi n in g t h e ce r vi ca l s ym p a t h e t ic t r u n k , t h emiddle cervical or st ellat e ganglia, or th e an sae su bcla-via (29, 31). I t is t herefore possible that intracardiacneu rons activated by stimu lation of vagal and cardiopul-m o n a r y n e r v e s i n t h e p r e s e n t s t u d y m a y h a v e b e e nresponding to parasympath etic preganglionic inputsr u n n i n g t o t h e h e a r t i n b o t h n e r v e s . I f t h i s we r e t h ecase, then the postsynapt ic effect of activating pa rasym-pathetic axons in the cardiopulmonar y n erves shouldha ve been eliminat ed by n icotinic blockade. H owever,becau se hexameth onium did not eliminate r esponses tocardiopulmonary nerve stimulation, these responseswere l ikely n ot mediated by displaced vagal axons.There is also anatomic and functional evidence thatsympathet icfibers, originat ing from postganglionic neu-rons in the sympath etic chain, may r each t he hear t viath e car diac vagus (11, 35, 48)a s well as th e car diopulmo-nary nerves. I t is thu s possible th at stimulation of thevagus nerve in the present study could have activatedsome sympath etic postganglionic as well as para sympa-thetic pr eganglionic fibers innervating intracardiacneur ons. This also does not a ppear to be the case, since-adren ergic blockade ha d no effect on th e postsyna pticresponses ofintracardiac neurons to vagus n erve stimu-lation. Although th e number of neur ons sam pled in thisstud y was limited, th e data indicate tha t stimu lation ofthe vagus ner ve activated only parasympa th etic input sand cardiopulmonary nerve stimulation activated onlys y m p a t h e t i c i n p u t s t o t h e s e n e u r o n s . Th i s m a y n o t ,however, be tr ue for a ll intr acardiac neur ons.

Intrinsic N euron Properties and Correlationwith Extrinsic Input Pattern

Properties. Ther e wer e n o significant differences be-tween mean resting membrane potentials of the t hreeneuron types (Table 1). Values reported in this studywere consistent with the mean value of resting poten-



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tial reported in an earlier in vitro study of intra cardiacneur ons in the p ig heart (50), with th e value for R-typecanine intracardiac neurons (55), an d with mean val-ues reported for guinea pig atrial neurons (21, 32).However, mean values of resting potential in the pre-sent study were smaller than those reported for neona-tal ra t intr acardiac neur ons (49), canine S-type intr acar-diac neurons (55), and pha sic neur ons in the gu inea pigleft atrium (27). The mean values for resting potentialin these studies ranged roughly from 50 to 60 mV,and this variation is l ikely to be due at least partly tointerspecies differences and possibly t o d ifferences inrecording conditions (see Ref. 2 for discussion).

Mean wh ole cell inpu t resist an ce of pha sic neu rons inthe present study was double that of accommodatingand tonic neurons (Table 1), but resistance values forall neurons in th is study were within th e ran ge of thosereported for int ra car diac neurons in a previous st udy ofpig atrial neurons (50) and for atrial neurons of otherspecies (21, 27, 49, 55, 56). The higher resistance (orlower m embran e condu cta nce) of pha sic neurons in t he

present stu dy implies th at a smaller current would berequired to depolarize the voltage-gated channels re-sponsible for AP generation to threshold for firing inth ese neurons, compared with curr ents n eeded to depo-larize lower-resistan ce neurons to firing threshold.This characteristic of phasic neurons, coupled with athreshold depolarization voltage for AP generation thatwas significant ly less th an th resh old voltages for a ccom-m od a t i n g a n d t on i c n e u r on s (Ta b le 1 ), m e a n s t h a tphasic neurons may be more excitable than the othertypes of intracardiac neuron. Mean time constants forthe t hree cell types in th e present stu dy were statisti-cally similar (Table 1) and were also similar to thosereported for atr ial neur ons in other species (21, 27, 49,

50, 55, 56).Pr operties of th e APs in duced by int ra cellular stimu-lation in the three classes of neuron (with the exceptionof the significantly lower thr eshold voltage for APgeneration in phasic cells mentioned above) were notsignificantly different. These properties were, more-over, similar to those reported for neurons in porcine(50), ra t (49), an d dog (55, 56) hea rt s.

All neurons in the present study displayed promi-nent AHPs following single APs (Figs. 4 and 6). Whenmean amplitudes and durations of AHPs in the threegroups of neurons wer e compa red, th e only significan tdifference found was that mean AHP duration in toniccells was more than twice the duration of AHPs in the

other two groups of neurons (Table 1; Fig. 2). A similardifference was reported for AHP durat ion betweenS-type (phasically discharging, AHP 50-ms duration)an d SAH an d P-type (repetitively firin g, AHP 20 0ms) intra car diac neurons in th e guinea pig heart (21).

Correlations . In the present stu dy there appears to bea correlation between neuron class (based on electro-physiological criteria) an d pattern of innervation byextracardiac inputs. Data compiled by neuron class inTable 2 show that extrinsic vagal inputs appear to berouted to phasic and to some accommodating intracar-d ia c n e u r on s , wh er e a s e xt r i n s ic s ym p a t h e t ic i n pu t

primarily targeted phasic neurons; most tonic neuronsappeared to receive no extrinsicinput s. Several schemeshave recently been proposed to classify peripheralaut onomic neurons by functiona l criteria (summ arizedin Ref. 2), based either on the time course of the AHPfollowing single intracellularly evoked APs (4, 15, 23,56), firing behavior in response to long depolarizingcurrents injected through the recording electrode (26,54), or a combination of both. In the present study,neurons have been divided into three classes based ontheir firing behavior in response to depolarization, asshown in Fig. 2. This scheme was adopted because ofthe implications of this intrinsic property for the poten-tial roles of the different neuron types in controllingintracardiac neurotransmission.

The existence of correlations between firing proper-ties and patterns of extrinsic innervation of the thr eeclasses of neuron found in this study provides someinsight into the p otent ial roles of these n euron t ypes inregulating th e hear t. Implicit in th e use of th is classifi-cation scheme is the concept that neurons with differ-

ent firing behaviors are involved in different intracar-diac functional pathways. In this respect, autonomicneurons with different types of firing behavior havebeen shown to par ticipate in an atomically and function-ally distinct pathways involved in visceral control (15,23, 41, 42). Firing behavior is an important physiologi-cal char acteristic tha t can at least par tly determ ine theresponses of neurons to ongoing high-frequency synap-tic input sufficient to depolarize the cell membrane tofiring threshold. Thus phasic and accommodating neu-rons would act as low-pass filters in the presence of sustained high-frequency synaptic input. Because themajority ofint racardiac neurons r eceiving vagal pregan-glionic inputs in this study were phasic or accommodat-

ing, high levels of vagal preganglionic activity wouldresult in the generation of only one or twoAPs or a briefdecrementing burst of APs at the start of depolariza-tion, depending on the neuron type. The output path-ways of these neurons are a s yet unk nown, but if theymediate parasympath etic control of t he myocardium,the rat e of tra nsgan glionic tr ansm ission of impulses ofe xt r a c a r di a c or i gi n t o e ffe ct or s it e s wou l d t h u s b esubject to a low cut-off frequ ency even in t he p resen ce ofstr ong, high-frequ ency prega nglionic activity. This cor-r e la t i on i s s t r e n g t h en e d f u r t h e r wh e n t h e p a t t e r n of innervation by axons in cardiopulmonar y n erves isconsidered: n eurons displaying type 2 responses werealmost exclusively phasic, so parasympathetic neuro-

transmission to these neurons would be modulated bysympathetic inputs.

The finding that none of the neurons expressing type2 responses could be induced to fire an AP throughvagal drive alone suggests tha t th ese neurons were noti n t h e p a t h wa y for d ir e ct e ffe r en t p a r a s ym p a t h e t iccontrol of the myocardium. Indeed, inasmuch as theseneurons only generated an output in response to theactivation of both sympathetic and parasympatheticinputs, t hey ma y represent a special cat egory of neur onconcerned with intra cardiac integrat ion of informa tionfrom both efferent l imbs of t he aut onomic n ervous



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system. On t he other h and , some of the a ccommodatingand all but one of the tonically discharging neurons inth is study lacked extrinsic inputs, so presuma bly thesece ll s wou l d b e a ct i va t e d p r im a r i ly b y i n pu t s fr oms o u r c e s i n t r i n s i c t o t h e h e a r t a n d m a y t h e r e f o r e b elocal-circuit neu ron s or in te rn eu ron s (7, 8) involved inintracardiac reflex loops. If th is is t he case, th e high-frequency repetitive firing properties of these cells mayhelp promote strong activation of such reflex loops byfeedback from intr acardiac events.

In th e guinea pig heart , an a lterna te, sensory role forintracardiac neurons receiving no extrinsic inputs hasbeen pr oposed (27). The existence of intra cardiac sen-sory neurons h as a lso been postulated in t he h earts of other mammalian species (see Ref. 8 for review). In thepresent study, one-third of the neurons sampled weredevoid of extrinsic inputs, raising the possibility thatthese neurons may be sensory in function. However,solid electrophysiological criteria for identifying intra-cardiac sensory neurons have not yet been establishedat the cellular level. Efferent axons of potential sensory

neurons in the heart tha t might commun icate with th ecentral nervous system or intrathoracic ganglia wouldbe expected t o run in th e extrinsic cardiac nerves andshould have been ant idromically activated by n ervestimulation, but no a ntidromically activated neuronswere detected in th e present st udy.

As a side issue, data on AHP properties of intracar-diac neurons ma y provide some insight int o the relation-ship of AHP chara cteristics to neuronal fir ing behavior.I t h a s b e e n p r o p o s e d t h a t AHP d u r a t i o n m a y h a v e acausal relationship with repetit ive firing behavior inperipheral autonomic neurons (reviewed in Ref. 2). Incells with long-durat ion AHP s, it would be expectedth at repetitive firing in r esponse t o prolonged depolar-ization would be limited to a low frequency because ofth e time n eeded for m embran e potential to recover to av a lu e n e a r t h e r e s t in g l ev el . Ph a s i ca l ly fir in g ce ll swould t herefore be expected t o have longer AHP s t hanrepetitively firing cells. However, this was not t he casefor phasic neurons either in the present stu dy (Fig. 2,insets ) or for phasic (S-type) cells in the guinea pigheart (21). It is thus possible that AHP duration per semay not be directly related to repetitive firing behavior.In r esponse to intra cellular d epolarizing cur rent in th etonic neuron il lustrated in Fig. 2C, t h e i n i t i a l i n t e r -spike int erval was 25 ms, yet the AHP du ra tion of th isneur on was close to th e mean value of 130 ms for t onic

cells (Table 1). In contra st, phasically dischar gingneurons in th is study had a significantly shorter meanAHP du ra tion of 48 ms. The char acteristics of the AHPand the capability for repetit ive firing of a particularneuron type are set by the complement of ionic conduc-t a n c e s p r e s e n t i n t h e c e l l m e m b r a n e ( 2 , 4 ) , b u t t h eresults of the pr esent stu dy and th ose of Edwar ds et al.(21) indicat e tha t in a trial n eurons AHP chara cteristicsand firing behavior may not be closely related. Them e m b r a n e cu r r e n t s u n d e r l yi n g t h e s e p h y si ol og ica lproperties in neurons in the pig heart have not beenestablished.

In conclusion, this study shows that there exists aneuronal substrat e within intra cardiac ganglia for theconvergence of inputs from vagal and cardiopulmonarycar diac nerves onto single int racard iac neurons. Vagalin p u t s t o t h e s e n e u r on s a p p ea r t o b e m e di at e d b ynicotinic postsynaptic receptors, whereas inputs fromcardiopulmonary nerves appear to involve -adrenergicr e ce p t or s . Th e se con v er g en t i n p u t s ca n i n t er a ct t o

modify the firing properties of intracardiac neurons.Evidence is presented to indicate t ha t th ere may a lso bepresynaptic modulation of vagal neurotransmissionthrough intracardiac ganglia, mediated by inputs fromcardiopulmonary nerves. The results presented heresuggest t hat some interactions between sympatheticand pa rasympathetic inputs to the heart can occur at apremyocardial site within intracardiac ganglia. Inputsof extracardiac origin t ar geted pha sic and some a ccom-modating neurons, whereas other accommodating andalmost all tonically firing n eurons lacked extrinsicinputs. Analysis of the correlations between innerva-tion patt ern and electr ophysiological properties of thethree neuron types found in this study suggests thatn e u r on s of e a ch t yp e m a y p la y a d is t in ct r ole inneu rona l contr ol of cardia c fun ction.

Perspectives

Th e m a j or i ss u e a d d r e s s ed i n t h i s s t u d y wa s t h epossibility of vagosympat hetic convergence on singleintracardiac neurons. The identification of such neu-rons now raises further questions about the implica-tions of th is convergence for neu ra l contr ol of th e hea rt .To evoke ort hodromically mediat ed APs in d ua lly inn er-vated neurons, activation of both inputs was requiredbecau se neither in put wa s capa ble on its own of depolar-izing th ese cells t o thr eshold for fir ing. Neurons in th is

group mu st t herefore int egrate signals from both effer-ent limbs of the autonomic nervous system to producean output . I t m ay be tha t sympathetic inputs functionto prim e th ese neu rons for fir ing by increa sing t heirexcitability, acting to increase neuronal sensitivity tosucceeding vagal inputs. Alternatively, vagal inputsmay function t o prime sympat hetically inn ervated n eu-rons for firing. In a ny case, either input pathway m ayfunction as a gat e, cont rolling t ra nsm ission t hr ought h e ot h e r p a t h wa y. I n t h i s wa y, t h e s e n e u r on s m a yrepresent a major site for vagosympathetic interac-tions, a llowing cooperat ion between t hese pa th ways incoordina ting t he neur al r egulation of car diac function.One reflex mechan ism tha t could mak e use of such an

integrat ive pr ocess is t he cardiac r esponse t o increasedatrial filling. In this condition sympathetic activity isreflexly increased to elevate cardiac output by raisingstroke volume and heart rate. However, strong sympa-thetic drive can elevate heart rate to a level too high forefficient ventricular ejection an d it has been pr oposedtha t coactivat ion of vagal preganglionic cardiomotoraxons would ensure that sympathetic overdrive doesnot result in heart rate exceeding the optimal level form a x im i zi n g ca r d i a c o u t p u t d u r i n g i n cr e a s ed a t r i a lfilling (36, 37). Intracardiac neurons that respond toinputs from both limbs of the autonomic nervous sys-



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t e m m a y t h u s a ct a s d u a l-in p u t ga t e s c a pa b le of balancing sympathetic and parasympathetic drive incontr ol of th e hear t. A par allel to th is type of dua l-inputintegration has been described in prevertebral gangliat h a t i n n er v a t e p e lv ic or g a n s (1 6, 1 9, 3 4), i n wh ichp a r a s y m p a t h e t i c a n d s y m p a t h e t i c i n p u t s i n t e r a c t a tthe level of postganglionic n eurons to control theirfiring. In this system parasympathetic preganglionic

input s synapse directly on postganglionic neu rons inner-vating pelvic end organs, while sympathetic inputsreach the same postganglionic neurons either directly(16) or indirectly via interneurons (18). Sympatheticand parasympath etic convergence within the pelvicganglia appears to be a key factor in regulating pelvicorgan fun ction du ring chan ges in relative activity of th etwo limbs of the autonomic nervous system. Evidencein th e presen t st udy for a sim ilar type of convergen ce onintracardiac neurons suggests that sympathetic-para-sympat hetic intera ctions within car diac ganglia, occur-r i n g a t i n t r a ca r d i a c n e u r on s , m a y h e lp b a la n ce t h ecardiac influences of extr insic autonomic inpu ts.

T h is r e s ea r c h w a s s u p por te d b y a n op e r a tin g g r a n t fr om th eMedical Research Council of Canada. The a uthor is grateful to theHeart and Stroke Foundation of Canada for a Research Scholarship.

Address for reprint requests: F. M. Smith, Dept. of Anatomy andNeurobiology, Faculty of Medicine, Dalhousie Univ., Halifax, NovaScotia, Cana da B3H 4H7.

Received 7 July 1997; accepted in final form 16 October 1998.

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F.M. Smith- Extrinsic inputs to intrinsic neurons in the porcine heart in vitro

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