J.A. Armour et al- Differential selectivity of cardiac neurons in separate intrathoracic autonomic ganglia

8/3/2019 J.A. Armour et al- Differential selectivity of cardiac neurons in separate intrathoracic autonomic ganglia

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274:R939-R949, 1998.Am J Physiol Regul Integr Comp PhysiolJ. A. Armour, K. Collier, G. Kember and J. L. Ardellintrathoracic autonomic gangliaDifferential selectivity of cardiac neurons in separate

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Differen t ial select ivity of car diac neu r ons

in separate intrathoracic autonomic ganglia

J . A. ARMOUR,1 K. COLLIER,2 G. KEMBER,2 AND J . L. ARDELL3

Departm ents of1Physiology and Biophysics and 2Physics, Dalhousie University,

Halifax, N ova S cotia, Canada B3H 4H7; and 3 Departm ent of Physiology,

University of S outh Alabama, Mobile, Alabam a 36688

Armour, J. A., K. Collier, G. Kem ber, and J. L. Ardell.

Differential selectivity ofcardiac neur ons in separ ate intr ath o-racic autonomic ganglia. A m. J . P hy s i ol . 2 7 4 (Regulatory

Integrative Com p. Ph ysiol. 43): R939 R949, 1998.Ana lysesof activity gener at ed by neur ons in middle cervical or stellateganglia versus intrinsic cardiac ganglia were performed tode t ermi ne how n e urons i n di ffe re nt i nt ra t hora ci c ga ngli a ,which are involved in card iac regulation, intera ct. Discha rgesof 19% of i nt ra t hora c ic e xt ra c a rdia c ne urons a nd 32% of intrinsic cardiac neurons were r elated to cardiodynam ics.Epicardial touch increased the activity generated by 80% ofintrinsic cardiac neur ons and 60% of extracardiac neurons.Both populations responded similarly to epicardial chemical

stimuli. Activity generated by neurons in intrinsic cardiacganglia demonstrated no consistent short-term r elationshipst o ne urons i n e xt ra c a rdia c ga ngli a . Myoca rdi a l i sche mi ainfluenced extracardiac and intrinsic cardiac neurons simi-l a rly. Ca rot i d a rt e ry ba rore ce pt ors i nflue nce d ne urons i nipsilateral extracardiac ganglia. After decentralization fromt he ce nt ra l ne rvous syst e m, i nt ri nsi c ca rdi a c ne urons re -ceived afferent inputs primarily from cardiac chemosensitiveneurites, whereas middle cervical ganglion neurons receivedafferent inputs primar ily from cardiac mechan osensory neu-ri t e s. I t i s c onc lude d t ha t t he popul a t ions of ne urons i ndifferent intrathoracic ganglia can display differential reflexcont rol of car diac fun ction. Their r edun dan cy in function an dnoncoupled beha vior minimizes cardiac dependency on asingle populat ion of intr ath oracic neu rons.

adenosine; adenosine t riphosphate; cardiac afferent neur on;middle cervical ganglion neuron; myocardial ischemia; stel-late ganglion neur on; substance P; veratridine

A POPULATION OF INTRINSIC cardiacneurons, the parasym-pat hetic p ostgan glionic ones (3, 9, 12), r eceives directinputs from medullary parasympathetic preganglionicneurons (10, 13). Another population of neurons withini n t r a t h or a ci c g a n g li a , i n cl u di n g t h os e on t h e h e a r t ,receives inputs from spinal cord sympa thet ic pregangli-on i c n e u r on s (5 , 6 , 1 0, 1 3). Th a t ca r d i op u l m on a r ysensory neurites can modify the activity generated by

neur ons in int rat horacic ganglia, including th ose on t heheart, after chronic decentralization of su ch gangliafrom the central n ervous system h as been interpreteda s in d ica t in g t h a t a ffe r en t n e u r on s a r e p r es en t inintrath oracic ganglia (2, 7). F urth ermore, i t has beenproposed that intrathoracic ganglia contain local cir-cuit neu rons (5, 6, 10, 13). Thu s int rat horacic ganglia,including those within the heart, contain afferent neu-rons, local circuit neurons, and sympathetic efferentpostganglionic neur ons in addition to those par asympa-thetic efferent postganglionic neurons that are associ-at ed with t he hear t (1, 8).

The classical view of peripheral intrathoracic auto-nomic ganglia proposes that their synaptic junctionsrepresent relay stations from th e centr al nervous sys-tem t o the end effectors on the heart, with l it t le or nointegrative capabilit ies. Recent data, including theidentificat ion of the m ultiple neur ona l subtypes withinth e intr at hora cic gan glia described above (1, 8), suggestinstead that complex neuronal interactions can occurwithin the intrathoracic nervous system, which poten-tially may be critical for the maintenance of regionalcar diac function (1, 8). Yet how t he activity generat edby various peripheral autonomic neur ons is coordi-

nated within and between separat e intrat horacic gan-glia remains a m ajor un an swered question.

Coherence of activity has been shown to occur withrespect to medullary and spinal cord neurons involvedin cardiac regulation (14). Consistent coherence be-tween neurons is indicative of principal an d directsynaptic interconnections between those neurons or,conversely, the sharing by su ch neurons of commonactivation inputs. Whether intrathoracic neurons in-volved in cardiac regulation display coherence of theiractivity rema ins to be established. It also remains to bedetermined whether neurons in different intrathoracicganglia involved in cardiovascular regulation receivesimilar types of cardiac afferent input s. Fur th ermore, itis not known wheth er extra th oracic vascular m echa no-sensory a xons, such as those in t he carotid sinus, canselectively influence different populations of intra tho-ra cic au tonomic neu rons involved in car diac regulat ion.

The present series of experiments were devised toevaluate how neurons in various intrathoracic gangliaresponded to specificcardiovascular pert urba tions. Thiswas done to determine how afferent information arisingfrom cardiac and extracardiac sensory neurites is pro-cessed by various populations of int rat horacic neur ons.The present stu dy also investigated h ow various popu-lations of intrathoracic neurons respond to transientcorona ry ar tery occlusion a nd t he r esponse char acteris-

tics of ischemia-sensitive neurons to discrete cardiacmechan ical or chemical stimuli. The present experi-ments sought to determine how inputs from specificcardiovascular sensory neurites affect different popula-tions of intrat horacic neurons when the intrath oracicnervous system is disconnected from the spinal cordand brain st em. Evalua tion of th e degree of short-termcoordination between extracardiac a nd intrinsic car-diac neurons was performed by coherence analysis. Int h i s m a n n e r we s o u g h t t o c h a r a c t e r i z e t h e p u t a t i v einputs to and intera ctions a mong different populationsof intra th oracic neur ons involved in cardiac r egulation.

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MATERIALS AND METHODS

Adul t mongre l dogs (n 32) of e it he r se x, we ighingbetween 16 and 21 kg, were used in this study. All experi-m e n t s w er e p er for m e d i n a ccor d a n ce w it h t h e N a t ion a lInstitut es of Health Guid e for th e Care and U se of La boratory

Animals [DHEW Pu blication N o. (NIH ) 8523, Revised 1985,Office of Science and Health Reports, DRR/NIH, Bethesda,MD 20205] a nd we re a pprove d by t he i nst it ut i ona l a ni ma l

c a re a nd use c ommi t t e e s of Da l housi e Uni ve rsi t y a nd t heUn iversity of Sout h Alaba ma.

General method. Canines were tranquilized with thiopen-t a l sodi um (1520 m g/kg i v) a nd t he n a ne st he t iz ed wit hth iopent al sodium (5 mg/kg iv to effect every 5 10 m in for t heduration of the surgical procedures). Noxious stimuli wereapplied to a paw th roughout the experiments to ascertain t headequacy of the anesthesia. After anesthesia induction, theanimal was intubated and positive-pressure ventilation wasi ni t i a t e d a nd ma i nt a i ne d wi t h a Bi rd Ma rk 7A ve nt i l a t orusin g a gas m ixtur e of 95% O2 and 5% CO2 for the duration ofthe experiment. After a ll the sur gery was completed, anesthe-sia was changed to -chloralose (2550 mg/kg iv), which wasfirst a dmi ni st e re d a s a bol us a nd t he n a s re pe a t dose s (25mg/kg iv) every hour or less th roughout th e experiments, as

required. When n euronal a ctivity was recorded, spontaneousactivity was supp ressed for 5 10 min a fter bolus injections of-chloralose becau se of the n eur onal depr essor effects of th isagent. Therefore, at least 10 min was allowed to elapse aftersuch injections before recordings proceeded.

A bilatera l thoracotomy was made in the fourth intercostalspace. Umbilical tape was placed around the inferior venacava as well as the descending thoracic aorta so that thesevessels could be occluded individually later in the experi-men ts. A lead II electr ocar diogra m (ECG) was recorded. Lefta t r i a l a n d l eft v en t r i cu l a r ch a m b er p r es s u r es a n d a or t i cpre ssure we re me a sure d u sing Be nt le y Tra nt e c model 800transducers connected to either a PE-50 catheter placed inthe left atrial cavity, a Cordis no. 7 pig-tail catheter insertedinto the left ventricular chamber via a femoral artery, or a

Cordis no. 6 cath eter inserted int o the ascending aorta via afemoral artery. Miniature solid-state pressure transducers(Konigsberg In stru ments, P asadena , CA, model P190; 5 mmdiamet er, 1.5 mm t hick) were insert ed in the right vent ricularconus and the left ventricular ventral wall to record regionalintramyocardial pressures. These sensing devices were em-ployed because intraventricular pressure is not a sensitiveindex for detecting inotr opiccha nges indu ced in some ventr icu-l a r re gions by a ut onomi c e ffe re nt ne urons (19). Al l da t a ,including neuronal activity, were recorded on an Astro-Med,model MT 9500 eight-chan nel r ectilinear recorder. Threadswere placed around connections between stellate ganglia andthe spinal cord so that these gan glia could be decentra lizedlater in the experiments. Thereafter a midline incision wasma de i n t he ne ck. The ca uda l ce rvica l va gosympa t he t ic

trunks were exposed, and threads were placed around thems o t h a t t h e y c ou l d b e s ev er e d l at e r i n t h e e xp er i m en t s .Umbilical tape was placed around each carotid artery justcauda l to the car otid bulbs. The neck incision was closed un tillater in th e experiments.

Recording neuronal activity. The a ct i vi t y ge ne ra t e d byneurons in separate pairs of intrathoracic ganglia was stud-i ed i n e a ch a n i m al . I n a ll a n i m a ls (n 32) t he a c t i vi t ygenerated by neurons in the ventral intraventricular gangli-onated plexus was recorded concurrently with activity gener-ated by neu rons in eith er th e left middle cervical (n 24 dogs)or l eft s t e ll a t e (n 8 dogs) ga ngl ion. For re cordi ng of neuronal activity from stellate or middle cervical ganglia, a

tungsten microelectrode was inserted into either ganglion asdescribed by us elsewhere (5, 6). The recording microelectrodeh a d a 1 0 - m d i a m e t e r , a n e x p o s e d t i p o f 5 0 m , a n d a nimpedan ce of 9 11 MW at 1,000 Hz. For recordin g of neu rona lactivity from the ventricular neurons described above (n 32; i.e., all anima ls), th e ventr al pericardium was incised andretra cted latera lly to expose th e fat on t he ventr al sur face ofthe ventricles that contains the ventral septal component of

the cranial medial ventricular ganglionated plexus (19). Acircular ring of heavy-gauge wire was gently placed on thesurface of this epicardial fat located on the ventral surface oft he vent ra l i nt e rve nt ri cul a r grove t o mi nimi ze e pica rdi a lmotion. This fat was explored with another tun gsten micro-electrode mounted on a micromanipulator at depths r angingfrom t he surfa c e of t he fa t t o re gi ons a dj a c e nt t o c a rdi a cmusculature (10, 13). Pr oximity to cardiac musculatur e wasindicated by increases in t he a mplitude of the ECG ar tifact.The indifferent electrodes were a ttached t o stru ctur es adja-cent to investigated ganglia. In this man ner, activity gener-ated simultan eously by neurons in intrat horacic intr insic andextrinsic car diac ganglia was r ecorded.

Si gna l s from t he e xt ra c a rdia c a nd i nt ra ca rdi a c i nt ra t ho-racic neur ons were differentially amplified by separat e Prince-ton Applied Research model 113 amplifiers that had band-pass filters set at 300 Hz10 kH z and am plification ran ges of100500. The out put of t he se de vice s, furt he r a mpl ifie d(50200) and filtered (band width 100 Hz2 k Hz) by meansof optically isolated am plifiers (Applied Microelectronics Inst i-tut e, Halifax, NS, Can ada), were led t o a Nicolet m odel 207oscilloscope a nd to a Grass AM8 audio monitor. Activitygenerated by individual n eurons was identified by the ampli-tu de an d sha pe of recorded a ction poten tials. Separ ate loci ine xt ra c a rdia c a nd i nt ra ca rdi a c i nt ra t hora ci c ga ngli a we reidentified from which action potentials with signal-to-noiseratios 3:1 were r ecorded, individual u nits being identifiedby the amplitude and configuration of their action potentials.With the use of these techniques and criteria, th e m icroelec-trode does not record action potentials generated by axons ofpassage, but rather records action potentials generated by

cell bodies and/or dendrites (10, 13). We did not attempt toactivate int rat horacic neur ons by m eans of electrical stimulia ppli ed t o t he ne rves a ssoci a t e d wi t h t he ga ngli a i nve st i-gated, because such stimu li induce long-term changes in theactivity patterns generated by such neurons (5, 6, 13).

Interventions. To determine if altered r espiratory mechan-ics affected neuronal activity, positive-pressure respirationwas discontinu ed for 30 s. Five minut es after r esumption ofpositive-pressure respiration, pulmonary inflation pressurewas elevated (e.g., from 10 to 25 mmHg) for brief periods oft i me a nd t he n re spi ra t ory ra t e wa s a l t e re d, be i ng re duc e dan d th en increased for 60 s. To determine if altered cardiovas-cul a r st a t e s a ffe ct e d i nt ra t hora ci c n e urona l a ct i vi t y, t hei nfe rior vena ca va a nd t he n t he de sce nding t hora ci c a ort awere pa rt ially occluded ind ividually for 5 10 s. Isoproteren ol

(0.2 g/kg iv) was th en adm inistered a s a bolus, and, after th epreparation had returned to a basal state, dobutamine (0.25g/ kg i v) wa s a dmi ni st e re d a s a bol us. Ea c h of t he a boveinterventions was repeated at least once.

Mechanosensitive neurites in various ventricular epicar-dial loci were activated by touching the ventricular epicar-dium gent l y wi t h a sa l ine -soa ke d cot t on swa b. Once t heextent of the ventricular epicardial region associated with ani de nt i fie d se nsory fie ld wa s de t ermi ne d, che mi ca l s we rea ppl i e d t o i t for 60100 s usi ng 1 1-cm gauze squaressoaked with 0.5 ml of each r espective chemical. Sensory fieldswere washed for 30 s with norma l saline (2 m l/s) after eachchemical was r emoved, with a t least 5 m in elapsing before th e

R940 INTRATHORACIC CARDIAC NEURON FUNCTION


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next intervention. The following chemicals were applied toidentified epicardial sensory fields: veratridine (5 106 g),substance P (1 M), adenosine (1 M), and ATP (1 M). Theorder of their application varied among experiments. Chemi-cals that initially induced neuronal responses were reappliedto the same epicardial locus at least twice to verify reproduc-i bi li t y of i nduce d re sponse s. Ga uz e squa re s soa ke d wit hroom-temperatur e n ormal saline were a lso applied to identi-

fied epicardial sensory fields to determ ine whether neur onalresponses elicited by epicardial chemical application weredue to vehicle effects or the mechan ical effects elicited bygauze squares.

After the interventions described above were completed,the left ventral descending coronary artery was occluded for12 min by means of a silk ligatu re sna re placed around t hea rt e ry 1 cm from its origin. Ten min ut es after th is interven-tion was discontinued, the circumflex coronary artery wasoccluded for 12 min. When these interventions had beencompleted, th e cervical vagosympathetic complexes in theneck were severed. All of th e var ious in ter ventions describedabove tha t h ad elicited responses were t hen repeated. Subse-que nt l y, a l l conne ct i ons be t we en st e ll a t e ga ngli a a nd t hespinal cord were cut, thereby decentralizing the intrathoracicau tonomic ganglia from the cent ra l nervous system. All of th einterventions described immediately above were repeated afinal time. The order of neur onal decentr alization (severingthe vagi or stellate ganglion decentralization) was random-ized among th e animals.

Data acquisition and analysis. The activity generated byintrinsic cardiac an d extracardiac intra thoracic neurons wasrecorded simultaneously along with a lead II ECG, left atrialan d left ventr icular cham ber pressur es, right a nd left ventr icu-lar intramyocardial pressures, and aortic pressure using anAstro-Med model MT 9500 eight-chann el rectilinear recorder(Astro-Med, West Warwick, RI). Data were stored on VHSt a pe (T120 Sc ot c h, 3M Ca na da , London, Ont a ri o) usi ng avideocassette recorder (A. R. Vetter, m odel 820, Rebersbu rg,PA) for later an alysis.

Car diac indexes d erived from 10 consecut ive cardiac cycles

were an alyzed before and during peak responses elicited byeach intervention. The mean s (SE) of these cardiac indexeswere calculated from these data. Spontaneous fluctuations ofcardiodynamics were m inimal du ring control periods, hear tra t e va rying 5 beats/min an d systolic pressure fluctuating5 mm Hg. Thr esholds for classifying ind uced cardiovascularchanges were chosen t o be greater t han these r anges. Actionpotentials generated at a given locus were counted for 30-speriods to establish average activity immediately before andduring maximal responses elicited by each intervention. Ifre quire d, a n a l ysis of a ct i vi t y ge ne ra t e d by t wo or morei n di vi du a l u n i t s w a s p er for m e d b y m e a n s of a w in d owdiscriminator (Hartley Instrumentation Development Labora-tor ies, Baylor College of Medicine, Hou ston , TX).

Fluctuations in the amplitude of action potentials gener-

ated by a un it varied by10 V over severa l minu tes; actionpotentials retained the same configurations over time. Thusaction potentials recorded in a given locus with th e sam econfigurat ion a nd am plitude (10 V) were considered to begenerated by a single u nit. Action potentials with signal-to-noise ratios 3:1 were a nalyzed. The t hreshold for neur onala ct iv it y ch a n g es w a s t a k e n a s a ch a n g e o f 20% frombaseline values. Neuronal activity responses elicited by eachintervention were evaluated by comparing activity generatedimmediately before each intervention with dat a obtained a tthe point of maximum chan ge during th e intervention. Datawere expressed as m eans SE. One-way ana lysis of var ianceand paired t-test with Bonferr oni correction for mu ltiple tests

were u sed for statistical an alysis. A significance value ofP 0.05 was used for these determinations. Coherence ofactivity generated by neurons in two different ganglia wasdeterm ined as a cross-correlation of th e mean corr ected waveform s recorded from the t wo different popula tions of intr ath o-ra cic neur ons (AcKnowledge III for th e MP100WS by BiopacSystems, Goleta, CA). The coherence of neuronal activityre corde d from t wo ga ngli a simul t a ne ousl y wa s e va l ua t e dover 1- to 5-min periods. A continuous analysis of heart ratea nd pe a k l eft vent ri cul a r syst ol ic pre ssure wa s l ike wiseperformed over th e same time periods to compar e the relat ivealterations in activity generated by two populations of intra-thoracic neurons with concomitant changes induced in car-diac indexes.

RESULTS

S pontaneous activity generated by n eurons in differ-ent ganglia. Nineteen percent of spontaneously activeneurons in extracardiac intrathoracic ganglia and 32%of th ose in int rinsic cardiac ganglia displayed a ctivitythat occurred preferentially during specific phases ofthe cardiac cycle. Other intrath oracic n eurons gener-

ated respirat ory-related activity (14% of identified ex-tracardiac and 8% of identified intrinsic cardiac neu-rons), with the remaining neurons generating sporadicactivity.

Coherence (cross correlation) was evaluated for theactivity generated by n eurons in different (intrinsiccardiac versus middle cervical or stellate) ganglia todetermine possible interactions that might occur be-tween n eurons t herein. When coherence was employedto compare t he t iming of activity genera ted by neu ronsin two ganglia, no consistent short-term (ms) relation-ship ofextracardiactointracardiacintrathoracicneuro-nal activity was identified (Figs. 1B and 2). This includedth ose occasions wh en bu rst ing of activity occurr ed after

epicar dial a pplication of a chemical (Fig. 1) or aftersystemic administration of a positive inotropic agent.Sometimes bu rst s of activity with per iodicities of 10 30s were generated by neurons in both studied ganglia.The burst s of activity genera ted by the separat e popula-tions of neurons were usually out of phase with oneanother, not phase shifted (Figs. 1 and 2). Althoughcoherence analysis did identify isolated instances ofcorrelated activity (correlation coefficient more tha n0.5), overall activity patt ern s exhibited minim al corr e-lation, as demonstra ted by corr elation coefficients ofl e s s t h a n 0.3, even when applied t o data recordedcontinuously for up to 5 min (Fig. 1). Thus there wereno consistent short-term in tera ctions between extrinsic

and intrinsic cardiac neurons overall as assessed bycoherence an alysis, even when evaluating neuronalactivity tha t displayed cardiovascular or respiratoryrelated activity.

Spontan eous activity genera ted by intra thora cic neu-rons changed after acute decentralization of the intra-thoracic nervous system. In some instances, spontane-ous activity was suppressed (Fig. 3A ), whereas in otherinst an ces it increased (Fig. 3B ) after acute decentr aliza-tion. Thus overall there was no significant change inbasal activity generat ed by int rat horacic neur ons a fterdecentr alization of the intra thora cic nervous system in

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nonstimulated stat es. Moreover, car diac a nd resp ira tory-related a ctivity cont inued to be genera ted by a subpopu-lation of intr ath oracic neur ons after acute decentr aliza-

tion, as were slow cyclic bur sts of neu rona l activity (Fig.3B ).

Carotid sinus stimulation. When mechanical stimuliwere applied to the ipsilatera l car otid bulb, neur ons inthe middle cervical ganglia were activated (16 3 t o28 6 impulses/min; P 0.001) in 16 of 24 dogs,whereas those in stellate ganglia were activated (33 9to 45 15 impulses/min; P 0.05) in 6 of 8 dogs. Incontra st, touching a carotid bulb activated intrinsiccardiac neur ons (12 2 to 35 8 impu lses/min) in only5 of 32 dogs studied; th us t he effects of th is inter vent ionon identified intrinsic cardiac neurons were insignifi-cant overall. In a few instances, touching a carotid bulbactivated pr eviously ina ctive int rinsic card iac neur ons

while concurrently suppressing the ongoing activitygenerat ed by neurons in a n extr acardiac ganglion (Fig.4B ). Investigated intrathoracic neurons were not af-fected when the contralateral carotid bulb or the righta n d l eft ca r o t id a r t e r y b el ow t h e ca r ot i d b u lb wer elightly touched. Acute decentralization of the ipsilat-eral thoracic sympathetic, not parasympathetic, ner-vous system eliminated intrathoracic neur onal resp onseselicited by touching the ipsilateral carotid bulb.

Bilateral carotid artery occlusion, proximal to thecarotid sinu s, reflexly increa sed left (106 6 to 126 8m m Hg ; P 0.001), but not right (28 3 t o 3 0 4

m m Hg ), v en t r i cu l a r i n t r a m y oca r d i a l s y st ol ic p r e s -s u r e s . L eft v en t r i cu l a r ch a m b e r s ys t ol ic p r e ss u r e(126 5 to 135 7 mmHg; P 0.001) also increased

during bilateral carotid occlusion, whereas heart rat eand left atrial systolic pressure did not. This interven-tion simultan eously increased a ctivity generated bymiddle cervical (13 3 t o 3 0 6 impulses/min; P 0 .0 02 ) a n d i n t r in s i c ca r d i a c (1 9 4 t o 5 5 19impulses/min; P 0.04) neurons in 11 animals, sup-pressing neuronal a ctivity in 8 other animals (middlecervical gan glion neu rons: 54 18 to 41 12 impulses/min, P 0.02; intr insic car diac neur ons: 44 11 to 14 13 impulses/min, P 0.01). In the remaining fivean imals, intr insic card iac ganglion an d m iddle cervicalganglion neuronal activity was not affected by t hisintervention.

Epicardial m echanical st imuli . Gentle mechan ical

distortion of epicardial sensory fields modified theactivity generated by neurons in middle cervical andintr insic cardiac ganglia, but not stellate gan glia over-a l l (Ta b le s 1 a n d 2 ). Rep e a t ed m e ch a n i ca l s t im u l iinduced rapid neuronal responses, which waned quicklyafter the stimulus was removed (Fig. 4A ). Most identi-fied epicardial afferent neurites were located on theventra l and latera l surfaces of the left vent ricle (cra nial2/3) and the right ventricular conus, with lesser num-bers being located on the right ventricular sinus. Asubpopulation of investigated neurons was also acti-vated when sensory neurites on the thoracic aorta (4

Fig. 1. Cardiovascular and n euronal varia bles recorded before an d after application of verat ridine to a locus on theright ventricular sinus epicardium (applied between arrows). A : bradycardia and a fall in left ventricular systolicpressure (LVP; top ) were induced initially by this intervention. Activity generated by intrinsic cardiac neurons(m i d d l e) increased, whereas that generated by left middle cervical ganglion neurons (LMCG; bottom ) decreasedimmediately after veratridine application. About 75 s after veratridine application, the activity generated by bothpopulations of neurons started to increase above baseline levels. At that time bursting of activity occurred aboutevery 25 s in each population of neurons, activity being generated in a reciprocal fashion by these two neuronalpopulations. B : cross-correlation of the a ctivity genera ted by these 2 populations of neurons derived from neur onaldat a obtained over the en tire r ecording period. Most of the time, no tight correlat ion of activity generated by t hese 2populations of neu rons occurred (correlation coefficients of less t han 0.3), with occasional and fortuitousrelat ionships occurring r andomly when correlat ion coefficients tra nsiently exceeded 0.5.



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dogs) or inferior vena cava (8 dogs) were touched.Interru pting the connections between the central ner-vous system and intrath oracic ganglia modified t heresponsiveness of intrathoracic neurons to local me-

chanical stimuli. For inst an ce, after acute decentr aliza-t i on of t h e i n t r a t h or a ci c n e r v ou s s ys t e m e p ica r d i a l

touch still increased the activity generated by neuronsin middle cervical ganglia but not the activity gener-ated by intrinsic cardiac neur ons (Fig. 5).

Altered cardiovascular dynamics. Modificat ion of car-

diac preload and afterload alters systemic and cardiachemodynamics. When the inferior vena cava was tem-

Fig. 2. A: cont inuous record (250 s) of ongo-i n g h e a rt r a t e a n d LVP, a s w el l a s t h eactivity generated by intrinsic cardiac andmiddle cervical (MCG) neurons (data de-rived from Fig. 1). These indexes changedw h e n v e r a tr idin e w a s p la ce d o n a r igh tventricular epicardia l locus (between a rrowsabove MCG neuronal activity tracing). B :activity generated by each population of neurons during two 60-s periods was sub-jected t o cross-correlat ion ana lysis (as indi-cated by horizontal lines below the MCGneuronal trace): i ) during the control statea n d ii ) once the veratridine-activated st atehad fully developed. Minimal coherence be-tw e en th e a c t iv ity g en e r a te d b y th e s e 2populations of neurons was detected eitherd u r in g c on tr o l or c h em ica lly s t im u la te dstat es. Note th at, whereas epicardial a pplica-t ion of v er a tr id in e r e s u lte d in e n h a n ce dactivity generated by both populations of neur ons, such a ctivity was out of phase withon e a n oth e r a n d , a s s u ch , f a i le d to s h o wconsistent short-term correlation as evalu-at ed by coherence analysis. bpm, Beats/min.

Fig. 3. Effects of severing the cervical vagosympat hetic complexes on activity generated by neurons in ventra lintraventricular ganglionated plexus (intrinsic) and LMCG for 2 representative animals. For one animal (A ),severing the right (first arrowhead) and left (second arrowhead) cervical vagosympathetic complexes resulted incessation of activity generated by neurons in intrinsic and LMCG. In another animal (B ) , when the cervicalvagosympathetic complexes were severed, activity generated by neurons in intrinsic cardiac and MCG increased.For the a nimal depicted in B , note the cyclic nat ure of the activity patter ns (0.1 Hz) generat ed by neurons in bothintr at horacic ganglia a fter severing th e cervical vagosympathet ic complexes. For both an imals, LVP increased in atra nsient fashion after cervical vagosympath etic tran section. Calibration bars: vertical bar beside LVP tr ace 10 0mmH g; vertical bar beside neuronal recordings 0.5 mV. Horizonta l bar in B 10 s.



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porar ily occluded, thereby reducing venous return ,right and left vent ricular systolic pressures decreased.The activity generated by vent ral intr aventr icular n eu-rons increa sed in 13 of th e 24 anima ls (24 4 to 37 11impulses/min) and decreased in 2 of th ese anima ls as aconsequence of inferior vena cava occlusion. Intrinsiccardiac neurona l activity cha nges were frequent ly mostpronounced immediately after this occlusion termi-na ted (Fig. 6). The activity generated by middle cervi-cal ganglion neurons changed in 14 of 24 dogs when the

inferior vena cava was occluded. When th e descendingaorta was pa rtially occluded, left vent ricular intr amyo-cardial and cha mber systolic pressur es increased (Table2). This intervention activated most intrinsic cardiacneurons (Table 2). The activity generated by middlecervical ganglion n eurons wa s either enha nced (n 13dogs; 29 7 t o 8 3 23 impulses/min, P 0.1) ordepressed (n 11 dogs; 26 2 to 7 2 impu lses/min;P 0.1), depending on the population studied. In intactpreparations, stellate ganglion neuronal activity wasreduced during partial occlusion of the aorta (Table 2).Modificat ion of car diac preload and afterload a secondtime induced neuronal and cardiovascular responses,that were similar to those induced the first time. After

acute decentralization of t he intrath oracic n ervous

system, intrinsic cardiac but not extracardiac neuronswere st i ll responsive to t he partial occlusion of th einferior vena cava or descendin g aorta (Table 2).

The augmentat ion in cardiac function induced byisoproterenol was accompanied by increases in thea ct i vi t y g en e r a t e d b y i n t r in s ic ca r d ia c a n d m i dd lecervical gan glion neu rons, but not by st ellate ganglionn e u r on s (Ta b le s 1 a n d 2 ). I n con t r a s t , d ob u t a m i n eincreased cardiac indexes as well as th e activity gener-ated by intrinsic cardiac neurons but not extracardiac

neurons (Tables 1 and 2). Readministration of thesepositive inotropic agents in th e inta ct st at e consistent lyinduced similar neuronal and cardiovascular responses.The enh an cement in regiona l cardiac function indu cedby isoproterenol and dobutamine was minimally af-fected by decentr alization of the intra th ora cic nervoussystem; l ikewise, a ugmentation in intrinsic cardiacneuron activity was maintained (Fig. 5, Table 2). Incontr ast , middle cervical gan glion neu rons were virtu-ally un responsive to isoproteren ol after acut e decent ra li-zation of the intrathoracic nervous system (Fig. 5).

Epicardial chemical stim uli. Neuronal r esponses ini-t i a t ed b y e p ica r d i a l c h e m ica l s t im u l i t ook t i m e t odevelop an d lasted , on aver age, 2.8 min a fter rem oval of

the chemical. Although cardiovascular variables were

Fig. 4. Effects of touching the right ventricular conus(A ) or left car otid bulb (B ) on the activity generated byneurons in t he ventral intraventricular ganglionatedplexus (intrinsic) and LMCG of one anima l. A : touchingthe right ventricular outflow tract (arrowhead) resultedin selective activation of intrinsic cardiac neurons. B :when the ipsilateral ( left) carotid bulb was touched(arrowhead), activity generated by intr insic cardiacneurons increased, whereas that generated by LMCG

neur ons decreased. Calibrat ion bar s: vertical bars 0.5mV; horizontal bar 10 s.

Table 1. Responses elicited by various interventions on cardiovascular variables and spontaneouslyactivity generated by intrinsic cardiac and MCG neurons of 24 dogs

InterventionH R,

beats/minLAP,

m m H gRV IMP,m m H g

LV IMP,m m H g

LVP,m m H g

MCG Activit y,impulses/min

Int rins ic Activity,impulses/min

Con t r ol 1233 71 271 1106 1273 204 163E pica r dia l t ou ch 1233 71 271 1106 1273 305* 417*

Con t r ol 1203 71 272 1097 1244 226 122Ver a t r idin e 1203 71 272 1097 1244 419* 357*

Con t r ol 1223 71 262 1105 1263 266 133Su bst a n ce P 1227 71 282 1107 1214 4312 326*

Con t r ol 1223 81 283 1126 1324 203 143

Aden osin e 1223 81 304 1167 1335 4710* 295*

Con t r ol 1193 81 261 1114 1334 315 243Respir at ion off 1193 81 261 1075 1303 285 175

Con t r ol 1194 81 272 1136 1284 217 164Isopr ot er en ol 1706* 101 696* 21013* 1288 4512* 4712 *

Con t r ol 1213 71 263 1106 1254 257 154Dobut a m in e 1454* 101 668* 22414* 1665* 3111 5313*

Va lu e s a r e m e a n s SE (n24 dogs). Chan ges in heart rate (HR), left (LAP) at rial systolic pressures, r ight and left ventricularintramyocardial systolic pressures (RV IMP and LV IMP, respectively), left ventricular chamber systolic pressure (LVP), and neuronal activityare tabu lated. Responses elicited by touching epicardia l sensory fields (epicardia l touch) or sensory field application of veratr idine, substa nceP, or adenosine ar e presented, a s well a s the results obtained during cessation of respiratory (Respiration off) and after systemicadministration of isoproterenol or dobutamine. MCG, middle cervical ganglion. * P 0.05 control vs. intervention.



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not changed overall during epicardial application of chemicals, owing to the small quantities of chemical

ap plied to th e localized r egion of the ep icar dium (Tables1 a nd 2), in a few instances cardiac augmenta tion didoccur (Fig. 1). N eurons in all ganglia studied wereactivated when substance P or purinergic agents wereapplied to epicardial loci (Tables 1 and 2). Most intrin-sic card iac and middle cervical gan glion neur ons stu d-ied were activated by epicardial application of moreth an one chemical (Table 1). Of the chemicals stu died,stellate ganglion neurons responded in a consistentfashion only to epicardial application of purinergicagents (Table 2). Reapplication of chemicals to previ-ously responsive epicardial loci induced similar neuro-na l responses. Epicardial a pplication of gau ze squar essoaked with room-temperature normal saline elicited

no neuronal responses.After acute decentralization, epicardial application

of ch e m i ca l s i n d u ce d n e u r on a l r e s pon s e s l es s fr e -quent ly, the a ctivity generated by extra cardiac neuronsbeing more profoundly suppressed than that of intrin-s ic ca r d i a c n e u r on s . For e xa m p l e, wit h t h e n e r vou ssystem intact, epicardial application of su bstance Pincreased the activity generated by intrinsic cardiacganglion neurons and middle cervical ganglion neuronsby 146 and 59%, respectively (Table 1). After acutedecentr alization, middle cervical gan glion neur ons wereminimally a ffected by epicardial application of sub-

stance P, whereas induced changes in intrinsic cardiacneuronal activity were halved (Fig. 5). Similarly, the

responses indu ced by epicardial application of veratr i-dine (Fig. 5) or puriner gic agents were at tenu ated afteracute decentralization of t he intrath oracic n ervoussystem.

Altered respiratory dynam ics. Ne u r o n s t h a t g e n e r -ated respiratory-related activity increased their dis-charge frequency when respiratory rate or respiratorypressure increased. The activity generated by theseneur ons decreased when r espiratory rate or respiratorypressure was reduced or respiration ceased (Table 2).The activity of neurons generating respiratory-relatedactivity increased in 5 of 32 dogs when loci in right- orleft-sided pu lmonary t issue were distorted gent ly. Therespiratory-related activity generated by neurons in

the remaining 27 dogs was modified by gentle epicar-dial mechan ical st imuli.

Coronary artery occlusion. Tra nsient coronar y arter yocclusion increased the activity generated by neuronsi n i n t r in s ic ca r d i a c g a n g li a (Ta b le 2 ), wh e r ea s t h eactivity generated by middle cervical ganglion neuronsincreas ed in 12 dogs an d decreased in 7 dogs (no chan geoverall). Stellate ganglion neurons were not affected bytra nsient myocardial ischemia (Table 2). The ma jorityof ischemia-sensitive neurons was sensitive to bothmechanical and chemical stimuli. In general, activitygenerated by ischemia-sensitive neurons was not en-

Table 2. Alterations in cardiac variables and the spontaneous activity generated by intrinsic cardiacand stellate ganglion neurons elicited by various interventions in 8 dogs

InterventionH R,

beats/minLAP,

m m H gRV IMP,m m H g

LV IMP,m m H g

LVP,m m H g

Stellat e Activity,impulses/min

IntrinsicActivity,impulses/min

Con t r ol 1223 101 241 1019 1147 328 204E pica r dia l tou ch 1223 101 241 1019 1147 4711 399*

Con t r ol 1179 102 281 1066 1123 289 296

Su bst a n ce P 1178 102 281 1066 1123 7822 13225*

Con t r ol 1187 91 251 948 1146 3612 2311P u r in es 1187 91 251 948 1147 8327* 9523 *

Con t r ol 1287 111 211 1018 1125 496 213Respir a t ion off 1287 111 211 1018 1125 325* 112*

Con t r ol 1337 92 252 9313 1177 284 278CC Aor ta 11812 102 252 12817* 1819* 133* 11332*

Con t r ol 1169 101 241 8810 1114 334 229CC Aor t a (decen t r a l) 11810 101 282 13416* 1616* 286 11737 *

Cont r ol 12010 101 241 9512 1146 2110 264Isopr ot er enol 1459* 111 701* 17221* 12610 6019 11224*

Con t r ol 11910 111 232 9812 1126 305 262I sop rot er en ol (d ece nt r al) 15011* 111 505* 16717* 12010 335 7412*

Con t r ol 119

8 10

1 25

1 96

10 115

5 33

9 19

3Dobu t a m in e 1289 111 709* 17320* 1469* 326 9724 *

Con tr ol 1119 102 281 1046 1083 368 186Cor on a r y a r t er y Occ 1119 102 281 9013 1062 306 615*

Con t r ol 1077 101 281 1005 1063 294 184Cor on a r y a r te r y O cc (d e ce n tr a l) 1 077 101 282 8511 1042 304 337

Values ar e means SE (n8 dogs/intervention). Changes in HR, LAP, RV IMP and LV IMP, LVP and neuronal activity are tabulated.Responses elicited by touching epicardial sensory fields as well as sensory field application of substance P and purines (adenosine and ATP) aretabulated as the effects of respiratory cessation (Respiration off), cross clamping of the aorta (CC Aorta), and dobutaine and isoproterenolbefore and after decentralization (decentral) of the int rat horacic autonomic ganglia. Effects of brief occlusion of the left ventr al descendingcoronary a rter y (Coronar y art ery Occ) before and a fter decentra lization are a lso presented. * P 0.05 control vs. intervention.



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hanced on reperfusion. Cardiovascular variables wereunaffected overall by the transient myocardial ische-mia employed (Table 2). After acute decentralization,neurons in some animals were modified by transient

coronary occlusion but not with enough frequency toaffect total activity overall (Table 2).

D IS C U S S ION

The results of the present experiments indicate thatcardiodynam ics ar e regulated by a hiera rchy of periph-eral a ut onomic neurons functioning as n ested feedbackloops. In a ddition t o the well-kn own reflex contr ol loopsfor cardiac regulation that use cardiopulmonary andart erial baroreceptor afferent axon inputs to centr aln e u r on s , Fi g. 7 i ll u st r a t e s t h e e m er g in g con ce p t of ma jor int ra thora cic reflex feedback loops th at regulateaut onomic neuronal outflows t o the heart. Within t hish i er a r c h y, ce r t a in a ffe r en t a x on s on t h e h e a r t a n d

major intrathoracic vessels exert predominant effectson t h e ou t flows fr om t h e i n t r in s i c ca r d i a c n e r v ou ssystem, whereas others exert their feedback effects viaascending projections to neur ons in intr ath oracic extra -ca r d i a c g a n gl ia or t h e ce n t r a l n e r vou s s ys t e m . Th ee ffi ca cy of t h e a ffe r en t i n pu t s t o n e u r on s i n t h e s evarious locations depends on where the neurons arelocated, as well as the origins and characteristics of t h e i r s e n s o r y i n p u t s . M a n y o f t h e a f f e r e n t i n p u t s t ointrathoracic neurons arise from intrathoracic cardio-vascular mechanical an d/or chemical sen sory axons. Aseparate and smaller population of intrat horacic neu-

rons receives afferent input s prima rily from pulmonar ymechan osensory endings, t hese neurons generatingrespirat ory-related activity. Other neurons generatesporadic activity, which is not related to cardiac or

respirat ory dynam ics in th e time or frequency domains.A small population of intra thoracic neurons, which areprimarily located in extracardiac ganglia, receives indirectinputs from ipsilateral carotid artery mechanosensoryneur ites mediated via centr al neu ronal interconnections.

Neurons identified in most intact intrinsic cardiacand middle cervical ganglia, but not stellate ganglia,were affected by mecha nical stimuli applied to vent ricu-lar epicard ial loci (Tables 1 an d 2). In accord with thefact that the majority of afferent neurons associatedwith cardiac and major vascular mechanosensory neu-rites is slow adapting when exposed to relatively con-stan t m echa nical stimuli (4, 11, 18), intra thora cic neu-rons r esponded rapidly a nd in a relatively consistent

m a n n e r t o s u s t a in e d e p ica r d i a l m e ch a n i ca l s t im u l i(Fig. 4A ). Intrinsic cardiac neu ronal r esponses elicitedby epicardial mecha nical stimuli were du e in par t t o theconnectivity of intra thora cic neurons with centr al n eu-rons, as few of these neu rons responded to such st imuliafter a cute decentr alization (Fig. 5). In contra st, extra -cardiac intrathoracic neurons were still responsive toepicar dial mechanical stimuli after a cute decent raliza-tion of th e intra th oracic nervous system. Tha t a popula-tion of intra th oracic neurons still responded to epicar-dial stimuli after acute decentralization of their ganglias u gg es t s t h a t s om e i de n t ified i n t r a t h or a ci c n e u r o n s

Fig. 5. Graphicrepresentat ion ofneuronal activ-ity data provided in Table 1 and presented as%change from baseline. Dat a obtained whenmechanical stimuli (A ), veratridine (B ), or sub-stance P (C) was applied individually to identi-fied epicardial sensory fields before (open bars)and after (filled bar s) decentralization of thein tr a th o r a cic n e r v ou s s ys te m a r e p r e s en te d .Changes in neuronal activity induced by isopro-terenol (0.2 g/kg iv) before and after decentrali-zation of the intrathoracic nervous system arepresented in D. M CG %change in activitygenerat ed by MCG neurons; intrinsic %changein activity generated by ventral intraventricularganglionat ed plexus neurons.



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were either afferent ones or ones closely associatedwith them . These data also indicat e that some intra th o-racic neurons receive preferential inputs from cardiacmechan osensory neurites when functioning indepen-dently of centr al neu rons.

Intrathoracic neuronal responses elicited by epicar-dial chemical stimuli likewise demonstrated differen-tial effects after a cute decentra lization of th e intr at ho-r a c ic n e r v ou s s ys t e m . I n t r i n s ic ca r d i a c n e u r on a lresponses elicited by epicar dial chemical stimuli wereattenuated and those generated by extracardiac neu-rons were virtua lly eliminat ed after decentralization(Fi g. 5 ). Th e a ct i vi t y g en e r a t e d b y m os t i de n t ifiedi n t r a t h or a ci c n e u r on s i n cr e a s ed wh e n s u b st a n c e P,puriner gica gents, or veratr idine was applied to circum-scribed epicar dial loci in th e inta ct sta te. Because of th erelatively low concentra tions of each chemical em-ployed, neuronal activity changes usually occurredwithout alteration in monitored cardiac indexes (Tables1 and 2). Thus, in confirmation with previous reports (11,18), epicardial application of a chemical did not directly

affect underlying cardiomyocyte contractile behavior in adetectable fashion. That is to say, the neuronal responseselicited by epicardial chemical application were primarilydue to activation of chemosensory axons ra ther tha n beingsecondary to altered r egiona l mecha nics.

Th e m a j or i t y (7 4%) of t h e i n t r a t h or a c ic n e u r on ss t u d ie d wer e a ffe ct e d b y a l t er a t i on s i n r e s pi r a t or ydyna mics (Tables 1 and 2). Respiratory-induced cha ngesin atrial or ventricular systolic pressures can alter theactivity genera ted by cardiac mechan osensory afferentneurons (18). In agreement with that, most intratho-racic neurons that generated respiratory-related activ-ity were influenced by touching the epicardium ratherthan pulmonary t issues. Thus the respiratory-related

activity generated by man y stu died intrath oracic neu-

Fig. 6. Chan ge in neuronal activity in intrat horacic ganglia elicited

by occlusion of t he inferior vena cava (IVC). When the IVC wasoccluded (between arrowheads), right (RV) and left (LV) ventricularintramyocardial systolic pressures (IMP) were reduced. Activitygenerated by ventral interventricular neurons was unaffected duringocclusion of t he IVC; it increased after the IVC occlusion waste r m in a te d. N ote th e t r a n s ien t in cr e a s e in RV a n d LV s y s tolicpressures coincident with increased activity of the intrinsic cardiacneurons. In contrast to intrinsic cardiac neurons, activity generatedby neurons in the LMCG increased during IVC occlusion and thenceased for a short period of time after the occlusion was terminated.Calibrations: vertical bars beside intramyocardial pressures 100mmHg; vertical bars beside activity 0.5 mV; horizontal bar 10 s.

Fig. 7. Schematic representa tion of theputa tive connectivity among various in-tr a th o r a c ic n e u r o n s a n d c e n tr a l n e u -rons that are involved in cardiac regula-tion.Arterial baroreceptors, carotid bulbmechanosensory neurites; nodose, no-dose ganglion afferent neur ons; DRG,dorsal root ganglion afferent neurons;

LCN, local circuit neurons; Sympathefferent, sympat hetic efferent postgan-glionicneur ons;Pa rasym efferent, para -s y m p a th e tic e ff er e n t p os tg a n g lio n icneurons;B1, cardia c myocyte B1-adreno-ceptors; M2, cardiomyocyte M2-musca-rinic receptors.



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rons appear ed to be due to respirat ory-dependent alter-ations in cardiac mechanics. Only 16% of the neuronstha t generated respiratory-related activity receivedinputs from pulmonary mechanosensory neurites, asdeterm ined by touching pulmonary tissues.

When left ventr icular an d ascending aortic pressur eswere elevated by partially occluding the descendingaorta, the activity generated by neurons in each gan-glion studied changed (Table 2). Isoproterenol inducedchan ges in t he activity generated by intrinsic cardiacand middle cervical ganglion neurons, but not stellateganglion neurons. These data are in accord with thefact that more neurons in the former two ganglia asopposed to the latter ganglion received inputs primar-ily from cardiac as opposed to aortic mechanosensoryaxons (2, 6, 7) because isoproterenol au gments leftventricular systolic pressure, but not aortic pressure.Whereas intrinsic cardiac neuronal responses inducedby positive inotropic agents were similar before andafter acute decentralization, few middle cervical andstellate ganglion neurons responded to cardiovascular

chan ges induced by positive inotropic agents afterremoval of central neuronal input. These data indicateth at t he effects exerted on extracardiac neur ons du ringpositive inotropic stat es were due in large part to enha ncedinput from centr al nervous system neurons, which presum-ably were influenced reflexly by arterial mechanosensoryaxons responding to elevations in ar terial pressure.

I n a ccor d wit h p r e vi ou s r e p or t s (5 , 6 , 1 7), b r ie f periods of coronary artery occlusion modified the activ-i t y g e n e r a t e d b y m a n y o f t h e i n t r a t h o r a c i c n e u r o n sstu died. Activity generat ed by su ch ischemia-sensitiveneurons varied, depending on where the neurons werelocated and the location of the sensory neurites thataffected them. As occurs with respect to epicardial

afferent neurons in dorsal root ganglia (18), but notnodose ganglia (11), the majority of ischemia-sensitiveintrathoracic neurons was sensitive to mechanical andchemical stimuli. The activity generated by ischemia-sensitive intrathoracicneurons apparently depended toa gr e a t er e xt e n t on ch e m os en s or y i np u t s t h a n onmechanosensory inputs, because left ventricular end-systolic (Tables 1 and 2) and end-diastolic pressureswere relatively unaffected by the circumscribed andtra nsient coronary artery occlusions employed. Thatischemia-induced neuronal r esponses depended in par ton the connectivity of intrathoracic neurons with cen-tral neurons (Table 2) is in accord with data obtainedwhen specific m echan ical or chemical stimuli were

applied to epicardial sensory fields.The results of the present experiments indicate that

populations of neurons located in separate intrath o-racic ganglia demonstra te minimal short-term (ms)intera ctions. Alth ough sympa thet ic efferent pregangli-onic neurons directly input to a subpopulation of neu-rons in extrinsic (5, 6) and intrinsic (10, 13) cardiacganglia, these neurons do not appear to represent themajor determinant of coordinated neuronal activityamong populations of neurons in various intrathoracicganglia. Similarly, a sma ll population of int rinsic car-

diac neurons receives direct inputs from parasympa-thetic efferent preganglionic n eurons (8, 9, 13). Thelikelihood tha t most int rat horacic neur ons involved incardiac regulation function relatively independent ofone an oth er suggests tha t th ey act as pa rt of a distribu-tive overlapping set of nested neural feedback net-works. The fun ctional selectivity of th e beha vior ex-pressed by n eurons in different intrath oracic gangliamodulating cardiodynamics indicates that there is aredundancy of function among intrath oracic n euronsthat are involved in regulating the heart.

Coordination of autonomic outflows from intratho-racic neur ons to cardiomyocytes depends to a largeextent on sharing of inputs from higher centers alongwith interactions among neurons in and between vari-ous peripheral ganglia. The sharing of cardiopulmo-nar y afferent informa tion acting thr ough both intr at ho-ra cic an d bra in stem /spinal cord feedback loops permitsan overall coordination of effector control that does notdepend on successive synapses fun ctioning solely asdeterministic relay stations. The bursting of neuronal

activity with 10- to 20-s time periodicities, even amongneurons in intrath oracic ganglia disconnected fromcentral neurons (Fig. 3B ), suggests that complex net-work intera ctions can occur am ong th e various popula-t i on s of p e r ip h e r a l a u t o n om i c n e u r o n s , e ve n wh e nfu n c t ion i n g i n de p en d e n t of ce n t r a l n e u r on a l i n pu t .Irrespective of the underlying pa ttern generators thatr e gu l a t e b a s a l a u t o n om i c n e u r a l ou t flow, t h e d a t ap r e se n t e d i n t h i s r e p or t s u p p or t t h e con ce p t of t h ediversity of afferent feedback in card iac contr ol.

Data obtained in t he pr esent experiments also indi-cate the underlying neuronal substrate contained withinthe intrathoracic nervous system, i ts various popula-tions of neurons displaying selective responses t o acti-

vation of specific sensory inputs. Activation of eitherchemo- or m echa nosensory neu rites indu ced brisk n eu-ronal r esponses in all th oracic neur onal populationsinvestigat ed, even when such populations were discon-nected from central neurons. It is proposed th at suchs e n sor y i n pu t s a r e u s e d i n t h e a u t o n om i c n e u r on a lreflex control networks that involve short (intrinsiccar diac nervous system), medium (middle cervical a nds t e ll a t e g a n gl ia ), a n d l on g (s p in a l cor d a n d b r a in )nested feedback loops (Fig. 7). Su ch an arra ngementwould m aximize t he potential for the maintenan ce of coordina ted efferent au tonomic neu ronal outflow whileproviding the flexibility n ecessary for bea t-to-beat regu-lation of efferent outflow to the heart. Therefore, t he

loss of t he long feedback loop mediated by centralneur ons would not jeopar dize cardiac regulat ion, a s isevident in acutely (10, 13) or chronically (2) decentral-ized intr insic car diac nervous system prepar at ions.

Owing to the limited sa mple size of th e populat ions ofneur ons in the var ious gan glia stu died, our inability tofin d coh e r e n ce of a ct i vi t y g en e r a t e d b y n e u r on s i ndistinct and separate intrath oracic ganglia does n otexclude t he possibility that direct neuronal interac-tions exist. Nevertheless, the relative lack of coherencebetween activity genera ted by the separa te an d distinct



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populations of intra thoracic neurons st udied does indi-cate the functional and divergent selectivity of mostintrath oracic neur on populations that are involved incardiac regulation. Our data also indicate the impor-tance of the varied afferent information that acts atm u l t ip le l ev el s of t h e p r op os e d n e t wor k of n e s t edfeedback loops involved in the integrat ive cont rol ofregional cardiac function.

Perspectives

Neurons in different intrathoracic ganglia that areinvolved in car diac regulation r eceive inpu ts primar ilyfrom cardiac mechanosensory and chemosensory neu-rites, with fewer inpu ts a rising from neur ites on m ajorintrathoracic vessels. A small population of intratho-racic extracardiac neurons is influenced by sensoryneurites associated with the ipsilateral carotid bulbm e d ia t e d v ia ce n t r a l n e u r on s . Fe wer i n t r a t h or a ci cn e u r on s a r e i n flu e n ce d b y ca r d ia c a ffe r en t s t im u l iwhen decentralized than in the intact state, indicatingthat many intrathoracic neurons involved in cardiacregulation depend in part on central neuronal connec-t iv it y. O n t h e ot h e r h a n d , t h a t s om e in t r a t h or a cicneurons were sti l l influenced by cardiac sensory neu-r i t e s i n t h e a c u t e l y d e c e n t r a l i z e d s t a t e i m p l i e s t h a tintr ath oracic afferent neur ons, which sense altera tionsin th e mechanical and chemical milieu of the hea rt, caninfluence intra thoracic efferent neur ons independent ofcentral neurons. That neurons in different intr atho-ra cic ganglia primar ily exhibit noncoupled behavior,e ve n w h en t h e y a r e m u t u a lly e n t r a in e d t o ca r d ia cevents by cardiovascular afferent feedback, implies aredun dan cy of cardioregulatory contr ol exerted by dif-ferent populations of intrathoracic neurons. Further-more, that different populations of intrathoracic neu-rons respond different ly to similar cardiac interventionsindicates that selective feedback mechanisms exist atsuccessive levels of the intrathoracic nervous system.That neurons in different ganglia display functionaldissimilarities also implies a minimal reliance of theheart at any time on a ny one population of peripheralaut onomic neurons. The selective influence of eachpopulation of intrathoracic neurons on the heart likelyd e p e n d s o n t h e n a t u r e a n d c o n t e n t o f t h e i r s e n s o r yinputs. I t is concluded that the intrathoracic nervoussystem acts as a distributive processor, using multiplenest ed feedback contr ol loops to modulat e cardia c fun c-tion.

The authors gratefully acknowledge the technical assistance of Richard Livingston.

This work was supported by the Medical Research Council of Canada (MA-10122), the Nova Scotia Heart and Stroke Foundation,the National Heart, Lung, and Blood Institute (Grant HL-58140),and the American Hear t Association.

Address for r eprint requests: J . A.Armour, Dept. of Physiology andBiophysics, Dalhousie University, Halifax, Nova Scotia, Canada B3H4H7.

Received 11 October 1996; accepted in fina l form 8 December 1997.

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J.A. Armour et al- Differential selectivity of cardiac neurons in separate intrathoracic autonomic ganglia