CONTROL OF ABDOMINAL MUSCLES

STEVE ISCOE**Department of Physiology, Queens University, Kingston, Ontario, Canada K7L 3N6

(Received 25 March 1998; accepted in revised form April 1998)

AbstractAbdominal muscles serve many roles; in addition to breathing, especially at higher levels ofchemical drive or at increased end-expiratory lung volumes, they are responsible for, or contribute to,such protective reflexes as cough, sneeze, and vomiting, generate the high intra-abdominal pressuresnecessary for defecation and parturition, are active during postural adjustments, and play an essentialrole in vocalization in many species. Despite this widespread involvement, however, their control has,with rare exceptions, received little attention for two major reasons.First, in most anesthetized or decerebrate preparations, they are relatively inactive at rest, in part

because the position of the preparation (supine or prone with abdomen supported), reduces lung volumeand, therefore, their activity.Second, unlike phrenic motoneurons innervating the diaphragm, identification of motoneurons to a

particular abdominal muscle is dicult.At the lumbar level, a given motoneuron may innervate any one of the four abdominal muscles; at the

thoracic level, they are also intermixed with those innervating the intercostals.The two internal muscles, the internal oblique and the transverse abdominis, respond more to

increases in chemical or volume-related drive than the two external muscles, the rectus abdominis andexternal oblique; the basis for this dierential sensitivity is unknown.Segmental reflexes at the thoracic and lumbar levels are sucient to activate abdominal motoneurons

in the absence of descending drive but the basis for these reflex eects is also unknown.Neuroanatomical experiments demonstrate many more inputs to, and outputs from, the nucleus retro-

ambigualis, the brainstem region in which the premotor neurons are located, than can be accounted forby their respiratory role alone. These other connections likely subserve activities other than respiration.Studies of the multifunctional roles of the abdominal muscles, on the basis of recent work, hold considerable

promise for improving our understanding of their control.# 1998 Elsevier Science Ltd. All rights reserved

CONTENTS1. Introduction 4352. The muscles 436

2.1. Anatomy 4362.2. Innervation 4362.3. Location of motoneurons 4382.4. Morphometry 438

3. Premotor neurons 4383.1. Background 4383.2. Discharge patterns and spinal projections 4393.3. Inputs to cVRG (E neurons) 441

3.3.1. Electrophysiological studies 4413.3.1.1. Medulla 441

3.3.2. Upper airway 4433.3.2.1. Nasal aerents 4443.3.2.2. Hypothalamus 444

3.3.3. Neuroanatomical tracers 4453.4. Axonal projections 446

4. Inputs to abdominal motoneurons 4484.1. Neuroanatomical studies 448

4.1.1. Pathways 4494.2. Electrophysiological studies 449

4.2.1. Expiratory neurons of the caudal ventral respiratory group (cVRG) 4494.2.2. Upper cervical inspiratory neurons 4504.2.3. Vestibular 450

4.3. Divergence and convergence 4514.3.1. Expiratory bulbospinal (E-BS) neurons 4514.3.2. Non-ventral respiratory group (VRG) inputs 4514.3.3. Cortical 451

5. Limitations 4525.1. Recording techniques 4525.2. Analysis of activity 4535.3. Upper airway 4545.4. Blood pressure 4545.5. Gender 455

6. State 455

Progress in Neurobiology Vol. 56, pp. 433 to 506, 1998# 1998 Elsevier Science Ltd. All rights reserved

Printed in Great Britain0301-0082/98/$19.00

PII: S0301-0082(98)00046-X

433

CONTENTS (continued)6.1. Consciousness and sleep 455

6.1.1. Motoneurons and muscles 4556.2. Anesthesia 457

6.2.1. Motoneurons and muscles 4576.2.2. Premotor neurons 458

6.3. Local anesthetics 4606.4. Development 460

7. Lung volume and pulmonary slowly adapting receptors 4607.1. Shifts in end-expiratory lung volume 4607.2. Occlusions (no-inflation tests) 4627.3. Inflation in expiration 4647.4. Timing 4667.5. Neural basis for inhibition/disfacilitation of E-BS neurons 466

8. Chemical drive 4678.1. General considerations 4678.2. Carotid chemoreceptors 469

8.2.1. Abdominal responses 4698.2.2. Medullary responses 470

8.3. Locations of chemosensitive neurons 4728.4. Hypoxia 473

8.4.1. Central hypoxia 4738.4.2. Systemic hypoxia (intact peripheral chemoreceptors) 4748.4.3. In vitro responses 4768.4.4. In vivomedullary recordings 477

8.5. CO2 4788.5.1. Distribution of central and peripheral chemoreceptor drives 4788.5.2. Abdominal motoneurons 4788.5.3. Medullary premotor neurons 4798.5.4. Intracellular recordings 480

9. Behavioral aspects 4819.1. Straining 4819.2. Vocalization 482

10. Segmental control 48510.1. Background 48510.2. Receptor aerents and projections 48710.3. Reflex eects 49010.4. Abdominal aerents 490

11. Conclusions 490Acknowledgements 491References 491

ABBREVIATIONS

AUG augmentingBotC Botzinger complexc caudalC cervicalCPAP continuous positive airway pressureCSF cerebrospinal fluidcVRG caudal ventral respiratory groupDLH DL-homocysteic acidDRG dorsal respiratory groupE expiratoryE-BS expiratory bulbospinal neuron(s)EELV end-expiratory lung volumeEMG electromyogram, electromyographicEO external obliqueEPSP excitatory post-synaptic potentialETL expiratory threshold loadDEC decrementingDial diallylbarbituric acid (allobarbital)ECF extracellular fluidETL expiratory threshold loadGABA g-amino butyric acidGTO Golgi tendon organHRP horseradish peroxidasei intermediateI inspiratoryIO internal obliqueIPSP inhibitory post-synaptic potentialKF Kolliker-Fuse nucleusL lumbarMAC minimal anesthetic concentrationNA nucleus ambiguus

NAA not antidromically activatedNMDA N-methyl D-aspartateNPBL nucleus parabrachialis lateralisNPBM nucleus parabrachialis medialisNRA nucleus retroambigualisNREM non-rapid eye movementNTS nucleus of the solitary tractP pump (cells)PAG periaqueductal grayPBN parabrachial nucleiPEEP positive end-expiratory pressurePga gastric pressurePPB positive pressure breathingr rostralRA rectus abdominisRAR pulmonary rapidly adapting receptorsREM rapid eye movementRFN retrofacial nucleusRTN retrotrapezoid nucleusSAR pulmonary slowly adapting receptorsSLN superior laryngeal nerveSTA spike-triggered averagingT thoracicTA transversus abdominisTI duration of inspirationTE duration of expirationTS triangularis sternisTTX tetrodotoxinvl ventrolateralVT tidal volumeVT/TE mean expiratory flowWGA-HRP wheat germ agglutinin conjugated to HRPXCOR cross-correlation.

S. Iscoe434

1. INTRODUCTION

Abdominal muscles contribute to ventilation whenrespiratory drive increases (e.g., exercise, diaphrag-matic fatigue), are critical for protective reflexes suchas coughing, sneezing, and vomiting, and, dependingon ones viewpoint, contribute to one of humanitysbest or worst attributesspeech. Despite their im-portance, however, much less is known about theircontrol compared to that of the intercostal and phre-nic motoneurons. We know virtually nothing aboutthe inputs to their medullary premotor neurons andthe organization, at the spinal level, of the connec-tions between their aerents, interneurons, andmotoneurons within and between segments and thisis reflected in the limited attention they receive inmost recent reviews (Feldman, 1986; Shannon, 1986;Dun et al., 1995; Berger and Bellingham, 1995;Bianchi et al., 1995; Hilaire and Monteau, (1997),including one related to birds (Gleeson and Molony,1989). Four exceptions are those of Monteau andHilaire (1991) and Hilaire and Monteau (1997), arecent, but brief (84 references) review by Bishop(1997), an update of an earlier one (Bishop, 1963) byher, and that by Leevers and Road (1995a) on reflexinfluences on muscles of the chest wall. In contrast,just four of 603 references by Bianchi et al. (1995)refer explicitly to abdominal muscles althoughanother 54 are indirectly related, primarily becauseof results related to pre-motor neurons. In onereview, only the locations of their motoneurons(Berger and Bellingham, 1995) are described; inanother, only the drives to phrenic and intercostal,but not abdominal, motoneurons (Monteau andHilaire, 1991). The neglect of abdominal motor con-trol is typified by a recent review of respiratoryrhythmogenesis; a schematic of the main groups ofrespiratory neurons in mammalian brainstem andspinal cord omits abdominal motoneurons (Dunet al., 1995). Research on proprioceptive inputs fromrespiratory muscles emphasizes those from the dia-phragm and intercostals (e.g. Duron et al., 1978;Duron, 1981; Jammes et al., 1983a; Jammes andSpeck, 1995; Hussain and Roussos, 1995; Reveletteand Davenport, 1995; Jammes, 1995). Shannon,who, with his colleagues, has done much of the workon abdominal aerents (Shannon, 1980; Shannonand Freeman, 1981; Shannon and Lindsey, 1983;Hernandez et al., 1989), devotes eleven pages to thor-acic, but only one to abdominal, receptors in hisreview (Shannon, 1986). A recent review of the res-piratory responses to loads concentrates on the dia-phragm (Bazzy and Feldman, 1991).Contraction of the abdominal muscles contributes

to inspiration by lengthening the diaphragm (orreducing or preventing its shortening at increasedlung volumes, whether caused by loads, changes inposture, or airway obstruction), thereby maintainingthe diaphragm closer to its optimal length for ten-sion generation (contractility). Abdominal tone alsoreduces the compliance of the abdominal compart-ment (Goldman et al., 1986a), enabling the region incontact with rib cage (the area of apposition) to actas a fulcrum for expansion of the lower rib cageduring inspiration. These considerations account forthe use of abdominal muscle binders in quadriple-

gics (e.g. Goldman et al., 1986b). Finally, by forcinglung volume below the passive end-expiratory pos-ition, the onset of the next inspiration is passive,resulting from the outward recoil of the respiratorysystem when abdominal muscles relax. Dogs (DeTroyer et al., 1989) and horses (Koterba et al.,1988) use this breathing pattern at rest; man (Henkeet al., 1988) and dogs (Ainsworth et al., 1989a), butnot ponies (Gutting et al., 1991), use it during exer-cise. The net eect is to distribute the work ofbreathing between the two sets of muscles or, in thecase of human subjects whose use of abdominalmuscles at rest is minimal, to share it at higher ven-tilatory levels. All these points are made by many, ifnot most, researchers studying this aspect of respir-atory function, are covered in a recent paper byAliverti et al. (1997) and recent reviews describingthe complexity of their action (De Troyer andLoring, 1986; Grassino and Goldman, 1986; DeTroyer and Loring, 1995; De Troyer, 1997;Decramer, 1997), and are not presented in moredetail here.Abdominal activity, either measured directly from

electromyographic (EMG) recordings or deducedfrom either the pressures (which are related to dis-charge rate and recruitment of abdominal motorunits during voluntary increases in abdominal press-ure; SantAmbrogio et al., 1967) or configuration ofthe abdomen (Grimby et al., 1976; see also Alivertiet al., 1997 for references to related works), is pre-sent under conditions unrelated to such specificbehaviors as coughing, sneezing, vomiting andstraining, and vocalization. Patients with airwayobstruction (or chronic obstructive pulmonary dis-ease) typically have active abdominal muscles(Martin et al., 1980, 1983; Dodd et al., 1984; Lopataet al., 1985b; Vergeret et al., 1987; Ninane et al.,1992; Breslin, 1992), their use depending on thedegree of obstruction (Martinez et al., 1990). Insuch patients, they are recruited during exercise(Dodd et al., 1984) or application of continuouspositive airway pressure (CPAP) (Petrof et al., 1990)when their activity may oset the benefits (reduceddyspnea) resulting from CPAP. Activity is also pre-sent in patients with cystic fibrosis (Cerny et al.,1992), generalized muscle weakness of diverse ori-gins (Grinman and Whitelaw, 1983; Passerini et al.,1985; Rimmer and Whitelaw, 1993), under con-ditions of impaired diaphragmatic function (includ-ing diaphragmatic fatigue) (Yan et al., 1993a,b;Katagiri et al., 1994; Sliwinski et al., 1996), andafter maximal voluntary ventilation (Kyroussis etal., 1996) and thoracic surgery (Simonneau et al.,1983; Duggan and Drummond, 1987, 1989; Coutureet al., 1994; Clergue et al., 1995). Despite their clini-cal significance, isolated examples of which are pro-vided in several recent reviews (Slack and Shucart,1994; Brown, 1994; Teitelbaum and Borel, 1994;Carter and Noseworthy, 1994; Zulueta andFanburg, 1994; Kaplan and Hollander, 1994; Lynnet al., 1994), these will not be discussed furtherbecause they provide little information about theunderlying neurophysiological control mechanismsat the central or spinal level.Some studies document remarkably little eect of

their absence on ventilation, probably because other

Control of Abdominal Muscles 435

respiratory muscles, including the pectorals, com-pensate (Ainsworth et al., 1992a,b). For example,quadriplegics defend ventilation as well as controlsubjects against an expiratory load (ODonnell etal., 1993). Subjects lacking abdominal muscles(prune belly syndrome) have only modest impair-ments of ventilatory performance and exercise ca-pacity (080% of predicted) (Ewig et al., 1996).Anesthetized supine dogs with paralyzed abdominalmuscles still can generate satisfactory tidal volumes(VT) (Warner et al., 1991; Brichant et al., 1993; butsee Schroeder et al., 1991; Farkas and Schroeder,1993); sudden loss of expiratory muscle activity, per-haps because of subtle changes in posture, has noeect on VT, inspiratory flow, or end-tidal CO2 inawake dogs (Saupe et al., 1992). In healthy men,blockade of intercostal nerves T612 with local anes-thetic has little eect on peak expiratory flow andnone at all on the ventilatory response to CO2 orexercise (Hecker et al., 1989), possibly because lum-bar innervation (see below) was not blocked orbecause accessory expiratory muscles such as trian-gularis sternis (TS) were recruited. Nevertheless, thesignificance of the contribution of abdominalmuscles is illustrated by the respiratory dicultiesencountered by patients with spinal cord injuries(Slack and Shucart, 1994), degenerative diseases(Grinman and Whitelaw, 1983; Rimmer andWhitelaw, 1993; Teitelbaum and Borel, 1994; Carterand Noseworthy, 1994; Zulueta and Fanburg, 1994;Kaplan and Hollander, 1994; Lynn et al., 1994), orafter upper abdominal surgery (see Ford et al., 1993for review). Physiotherapy of abdominal musclesimproves exercise capacity and maximal expiratorypressure generation in patients with chronic obstruc-tive pulmonary disease (Vergeret et al., 1987).Recently, magnetic stimulation has been used toactivate abdominal muscles, the resulting pressuresand flows being similar to those observed in naturalcough (Kyroussis et al., 1997; Lin et al., 1998); sucha procedure has an obvious application to individ-uals with disrupted control of expiratory motoneur-ons (e.g. spinal cord injury).The three preceding paragraphs testify to the

many conditions when abdominal muscles are usedbut, as indicated earlier, the control mechanismsoperating under these conditions are unknown. Inthis review, I concentrate on the abdominal musclesand their innervation, the locations and character-istics of their motoneurons, the medullary pre-motorneurons (discharge patterns, projections, inputs), theresponses of both premotor neurons and motoneur-ons to various inputs which aect their dischargepatterns during eupnea, their responses to changesin lung volume and respiratory drive (hypercapniaand hypoxia), and their roles in such specific activi-ties as straining and vocalization. Puckree et al.(1998) have recently described task-specificity of in-dividual abdominal motor units in upright humans;units recruited during a respiratory manoeuvre (anincrease in end-expiratory lung volume) are notrecruited during a postural one (leg lift). Readersinterested in details about their roles in posturalcontrol are directed to the literature on this topic(e.g. Carman et al., 1972; Grillner et al., 1978;de Sousa and Furlani, 1982; De Troyer, 1983;

Thorstensson et al., 1985; Goldman et al., 1987;Oddsson and Thorstensson, 1987, 1990; Cresswell etal., 1994; Hodges et al., 1997) and recent reviews ofthe role of the vestibular system in the control ofexpiratory premotor neurons (Shiba et al., 1996a)and respiratory muscles (e.g. Huang et al., 1991;Yates et al., 1993; Rossiter et al., 1996) by Yatesand Miller (1996, 1997). In addition, the reader isreferred to recent brief reviews of such protectiverespiratory reflexes as cough and sneezing byShannon and colleagues (1996 and 1997) and an ear-lier and exhaustive review by Korpas and Tomori(1979). Vomiting has been the subject of severalrecent reviews (Grelot and Miller, 1994, 1997;Miller, 1995; Miller and Grelot, 1996).

2. THE MUSCLES

2.1. Anatomy

The respiratory abdominal muscles comprise twoouter (external oblique, EO, and rectus abdominis,RA) and two inner (internal oblique, IO, and trans-versus abdominis, TA) muscles. A generic descrip-tion of their anatomical arrangements is providedby Monteau and Hilaire (1991) but the anatomyvaries considerably between species (Rizk, 1980); insome species of bats, for example, the EO is poorlydeveloped (Lancaster and Henson, 1995a). A com-plete description (architecture, fiber type, and inner-vation) of rat RA is available (Hijikata et al., 1992)as is a description of the fiber types of all fourmuscles in man (Haggmark and Thorstensson, 1979;Caix et al., 1984), the contractile properties ofcanine RA and EO (Farkas and Rochester, 1988),and rat EO during development (Watchko et al.,1992). Two studies, both in man (SantAmbrogio etal., 1967; Puckree et al., 1998), indicate that thepeak firing frequencies of abdominal motor unitsare less than 20 s1 even during expulsive ma-noeuvres, suggesting that recruitment accounts formuch of the increment in force generation.Limited data exist concerning fiber type. In man

(Caix et al., 1984), most fibers in all four musclesare slow oxidative, fatigue resistant (type I), some-what fewer are type IIa (fast, fatigue resistant),whereas relatively few are type IIb (fast, fatiguable);RA has the highest percentage of type I fibers (69%based on the presence of ATPase) and the lowestpercentage of type IIa fibers (31%). These percen-tages dier from those reported earlier for RA byJohnson et al. (1973) who classified 46% as type Iand 54% as type II (IIa + IIb). According toPolgar et al. (1973), RA type I fibers average 43 mmin diameter, significantly smaller than type II fibers(56 mm). In contrast, canine TA contains equal per-centages of only type I and IIa fibers (based onmyofibrillar ATPase), the diameters averaging ap-proximately 35 and 44 mm, respectively (Reid et al.,1987).

2.2. Innervation

Anatomical texts indicate that, in man, all fourmuscles receive branches from the lower six inter-

S. Iscoe436

costal nerves, the internal muscles also receiving abranch from L1; none mentions innervation frommore rostral segments. In cat, all four muscles areinnervated by branches from the ventral rami of in-ternal intercostal nerves of thoracic segments T412and, caudally, by branches from the rostral and cra-nial iliohypogastric and ilioinguinal nerves (L13).Miller (1987) and Holstege et al. (1987b) both usedintramuscular injections of horseradish peroxidase(HRP) to label feline motoneurons, the latter alsoapplying HRP to the central end of nerves to var-ious abdominal muscles. Miller found motoneuronsof all four muscles as caudal as L3 but their rostralextents dier between muscles: RA to T4, EO to T6,TA to T9, and IO to T13. Holstege et al. (1987b)also observed HRP-labeled motoneurons of all fourmuscles in L3 but, in their study, motoneurons werefound more rostral (RA and EO in C7, and IO andTA in C8). They acknowledged that the labeled cellsin the cervical spinal cord could represent false posi-tives resulting from leakage of HRP, a possibilitysupported by Millers observations (1987) that (1)microstimulation in the ventral horn of C7 and C8does not elicit contractions of abdominal muscles,(2) section of the phrenic nerve ipsilateral to the siteof HRP injection into RA eliminates labeling ofmotoneurons in the cervical spinal cord (consistentwith interdigitation of RA and diaphragmaticfibers), and (3) twitches elicited in dierent musclesby electrical microstimulation of the ventral horncorrespond to the distribution of labeled motoneur-ons in dierent segments. Recently, Tami et al.(1994) used injections of cholera toxin subunit Bbound to latex beads (which limits spread) to labelmotoneurons innervating feline trunk muscles; thedistribution of labeled cells resembles that found byMiller (EO, T6 to L2; IO T11 to L2; RA T4 to L2).A schematic representation of the arrangement of

nerves from L1, L2, and L3 in cat is provided inFig. 1. In my experience and that of others (Grelotand Fregosi, personal communications), phasicexpiratory activity is recorded typically from thecaudal branches of these nerves, the cranialbranches rarely displaying phasic activity even indecerebrate cats breathing at elevated end-expira-tory lung volumes. Because of the overlap in inner-vation of dierent muscles by motor axons in agiven nerve (Fig. 2; Fregosi et al., 1992) and vari-ations between dierent muscles in levels of activi-ties, particularly as a result of changes in posture(Section 7.1) or chemical drive (Section 8), a betterdescription of the innervation of abdominal musclesis needed.In rat, RA is innervated by branches originating

from T313 (Hijikata et al., 1992). However, injec-tions of a dye into RA and EO labels motoneuronsin the cervical spinal cord; EO motoneurons arefound in the extreme ventrolateral tips of C67 butno labeled RA motoneurons are found in the cervi-cal spinal cord or in T12 (Charlton et al., 1988).Although these workers claimed to have preventedspread of the dye to other muscles, it is unclear ifthe presence of EO motoneurons in C67 representsfalse positives (due to spread of the dye to the over-lying cutaneous maximus) or if the distribution of

abdominal motoneurons in the spinal cord really isdierent in cats and rats.In many studies of thoracic expiratory motoneur-

ons, there is an implicit assumption that they areintercostal motoneurons. Although expiratory (ab-dominal) activity is preferentially suppressed inanesthetized preparations (Merrill, 1974), thisassumption may be invalid if both abdominal andinternal (typically, but not always, expiratory) inter-costal motoneurons are suppressed similarly byanesthetics. Experimental conditions which enhanceexpiratory activity (higher levels of respiratory driveand lung volumes are common interventions in stu-dies of respiratory control) may elicit increased ac-tivity in both pools of motoneurons. However,because the relative sensitivities of abdominal andexpiratory intercostal (including TS) motoneuronalpools to anesthetic agents, changes in lung volume,and respiratory drive (hypoxia or hypercapnia) are

Fig. 1. Schematic representation of lumbar abdominalnerves in cat. Phasic expiratory activity is typicallyobserved in caudal branches. Heavy arrow indicates site ofstimulation and recording of activity from TA and IOmotor axons described by Fregosi et al. 1992 (see Fig. 2).

Fig. 2. Response of IO and TA of a spontaneously breath-ing, chloralose-anesthetized cat to positive end-expiratorypressure (PEEP) and electrical stimulation of the transverse(caudal) branch of L1 (arrow in Fig. 1). PEEP elicited ac-tivity of TA and IO, verifying intact innervation of themuscles. Low intensity stimulation (2.5 to 3 times themotor threshold for TA) always elicited tonic activity inTA and usually IO but not EO and RA (not shown), indi-cating that the site of stimulation (arrow in Fig. 1) includesaxons innervating TA and, to a lesser degree, IO but notEO and RA. Modified with permission from Fregosi et al.

1992.

Control of Abdominal Muscles 437

unknown, one cannot assume that expiratory inter-costal, but not abdominal, motoneurons are acti-vated. For example, internal (expiratory)intercostals and abdominal muscles may beuncoupled during particular behaviors (e.g.vomiting; Iscoe and Grelot, 1992). Finally, identifi-cation of motoneurons as intercostal is oftenbased on their antidromic activation followingstimulation of the internal intercostal nerve. But ifthe nerve is stimulated central to the origin ofbranches to the abdominal muscles, both abdominaland internal intercostal motoneurons will be acti-vated. Similar arguments apply to neuroanatomicalstudies of labeling in the thoracic ventral horns(Feldman et al., 1985), electrophysiological studiesof the connections between medullary expiratoryneurons and internal intercostal motoneurons usingcross-correlation (XCOR) (Cohen et al., 1985;Merrill and Lipski, 1987) (see Section 3.3.1), spike-triggered averaging (STA) of membrane potentialsor extracellular field potentials (Merrill and Lipski,1987; Kirkwood, 1995) (see Section 3.3.1), and anti-dromic activation of medullary pre-motor neuronsby spinal cord stimulation (Merrill and Lipski,1987).

2.3. Location of Motoneurons

Abdominal motoneurons innervating dierentmuscles are located in specific regions of the ventralhorn with varying degrees of overlap. In cat, RAmotoneurons occur centrally, EO motoneurons ven-trolateral to them (Rikard-Bell et al., 1985a; Tani etal., 1994), overlapping IO motoneurons whichextend to the lateral edge of the horn (Tani et al.,1994). Holstege et al. (1987b) reported RA moto-neurons as being more lateral in the rostral thoraciccord, assuming a medial position by T8; TA moto-neurons are located in the lateral part of the ventralhorn. Miller (1987) also observed RA motoneuronsin the central part, the motoneurons of the other ab-dominal muscles overlapping in the ventrolateralhorns of T6 to L3, a result similar to that of Rikard-Bell et al. (1985a). The results of Holstege and hiscolleagues (1987b) suggest that internal intercostalmotoneurons do not overlap with those of IO, theformer extending to T10, the latter starting at T11.Motoneurons innervating EO occupy the same seg-ments as those innervating expiratory internal inter-costals but are located more medially in the ventralhorn.The locations of expiratory motoneurons in rat

are similar. EO motoneurons occupy a similar lo-cation to those of cat (Smith and Hollyday, 1983),expiratory (internal intercostal and EO) motoneur-ons of T67 occupying the medial and the ventrolat-eral part of the ventral horn (Saji and Miura, 1990).In monkey, abdominal motoneurons are distribu-

ted like those in cat; retrogradely labeled RA andEO motoneurons are found as far rostrally as T23,TA and IO to T67, all extending caudally to aboutL3 (Schriever and Jurgens, 1989). However, the dis-tributions of motoneurons within dierent spinalsegments are, except for RA, variable; most TAmotoneurons occur between T12 and L2 with a sec-ondary peak between T79, the numbers of IO moto-

neurons increase gradually from T7, peakingbetween T13L2; EO peaks at L12, T10, and T68.This variation, similar to that found in the results ofMiller (1987) and Holstege et al. (1987b), does notappear to be an artifact due to uneven distributionof intramuscularly injected HRP. Motoneurons ofall four muscles are distributed in the ventral hornof the appropriate segments with a tendency for RAto be central, IO and EO lateral, and TA moreevenly distributed. However, in the most caudalthoracic segments, many motoneurons are locatedin the lateral part of the ventral horn, a region alsocontaining internal intercostal motoneurons.Locations of motoneurons, including those inner-

vating the respiratory muscles, in the cat are sum-marized by Holstege (1996). Given the overlapbetween many motoneuronal pools, identification ofa motoneuron as innervating a particular muscle onthe basis of its coordinates, stereotaxic or histologic,is unjustified.

2.4. Morphometry

Miller (1987) briefly described abdominal moto-neurons as having somal diameters ranging between12 and 41 mm with an average diameter of 025 mm.Additional details, such as information about anydierences between motoneurons based on eitherthe muscle it innervated or the segment in which itwas located, are unavailable. Information about themorphometry of thoracic expiratory motoneurons islimited to one study of four HRP-injected rostral(T4) expiratory motoneurons in cat antidromicallyactivated by stimulation of the internal intercostalnerve just distal to its branching from the externalintercostal nerve (Lipski and Martin-Body, 1987).Therefore, they could be either internal intercostal,RA, or TS motoneurons. The somata were orientedlongitudinally, with diameters of 068 and 45 mm inthe sagittal and transverse planes, respectively. Theyhad extensive dendritic arbors, primarily along themedial and lateral borders of the ventral horn.Because only four motoneurons were reconstructed,it is impossible to generalize from this sample aboutthe properties of expiratory motoneurons, not justbecause their projections were not identified but alsobecause motoneurons in dierent segments may nothave the same morphometry even if they innervatethe same muscle. Although the locations of moto-neuronal somata may be related to the arrangementof the respective muscles (Tani et al., 1994), theextent of dendritic arborizations and distributions ofsynaptic inputs may be more important to moto-neuronal, and hence motor unit, function than thelocation of the soma.

3. PREMOTOR NEURONS

3.1. Background

Of recent reviews of respiratory control andrhythmogenesis (Euler, 1986; Feldman, 1986; Ezure,1990; Monteau and Hilaire, 1991; Berger andBellingham, 1995; Dick et al., 1995; Bianchi et al.,1995; Ramirez and Richter, 1996; Bianchi and

S. Iscoe438

Pasaro, 1997; Harper, 1997; Denavit-Saubie andFoutz, 1997; Rekling and Feldman, 1998), Longand Dun (1986) provide the most detailed reviewof E neurons of the caudal ventral respiratory group(cVRG) and the reader is referred to it for most ma-terial more than a decade old. They concluded,based on the evidence then available, all of it fromcats, that: (1) expiratory bulbospinal (EBS) neur-ons of the cVRG usually start firing after post-I(also called early expiratory) cells stop discharging,indicating the presence of inhibitory post-synapticpotentials (IPSPs) during the initial part of expira-tion when residual inspiratory (phrenic) activity ispresent (stage 1 expiration); (2) they have augment-ing (AUG) discharge patterns due to excitatorypost-synaptic potentials (EPSPs) and not decreasinginhibitory inputs (i.e. disinhibition); (3) during earlyI, they are maximally hyperpolarized by IPSPs, theintensity of which diminishes during I; (4) they havelittle or no synaptic interactions with neighboringneurons based on the absence of peaks or troughs inthe XCOR between them; (5) they receive inhibitorymedullary inputs from early I neurons of the rostralnucleus retroambigualis (rNRA, part of the VRG)(with uncertainty about the dominance of ipsi- ver-sus contralateral projections), post-I neurons of thecontralateral rNRA (although tracers indicate astronger ipsilateral projection than that indicated byantidromic mapping), late I neurons of the rNRA,and perhaps late I neurons of the ventrolateralnucleus tractus solitarius (vlNTS). The axonalarborizations of the latter two populations, how-ever, are not very extensive in the cVRG andMerrill (1979), on the basis of prolonged XCOR ofthe activities of late I and cVRG E-BS neurons (tak-ing advantage of the slight temporal overlap of theirdischarges), concluded that the connections areeither non-existent or too weak to be revealed bythis technique; (6) they are driven by inputs fromlate E neurons of the ipsi- and contralateral retrofa-cial nucleus (also called the Botzinger (BotC) com-plex or nucleus), despite the absence of supportingevidence from XCOR between the two neuronalpopulations; (7) they may receive inputs from otherbrainstem areas such as the pons (nuclei parabra-chialis medialis (NPBM) and lateralis (NPBL),Kolliker-Fuse (KF), and locus coeruleus) andmedulla (paragigantocellularis dorsalis and lateralis,reticularis pontis oralis, and reticular formationbetween NTS and nucleus ambiguus (NA); in manystudies, however, the identities of the neurons (e.g.whether or not they are respiratory) are unknown orthe connections are rare and/or weak; (8) theyreceive indirect inputs from pulmonary slowly(SAR) and rapidly (RAR) adapting receptors;inputs from the peripheral chemoreceptors elicitdiverse responses in dierent cVRG E neuronswhereas central chemoreceptor activation (hypercap-nia) uniformly excites all E neurons; they may beless responsive than I neurons to changes in chemi-cal drive; and (9) they project almost exclusively tothe contralateral thoracic and upper lumbar spinalcord with no collaterals at the cervical level despitethe presence of anterograde label in the cervical andupper thoracic cord (Feldman et al., 1985). Since

that review, new data (described below) haveappeared.

3.2. Discharge Patterns and Spinal Projections

The cat was the preferred experimental model atthe time of Long and Duns 1986 review and mostwork since then confirms that, in this species, E-BSneurons of the cVRG have AUG discharge patternsin which the neurons start to fire at variable inter-vals after the start of the E phase, reaching a peakin late expiration (Cohen et al., 1985; Miller et al.,1985; Ballantyne and Richter, 1986; Miller et al.,1987; Arita et al., 1987; Jodkowski and Berger,1988; Ballantyne et al., 1988; Hernandez et al., 1989;Anders et al., 1991; Takeda and Haji, 1991;Feldman et al., 1992; Klages et al., 1993; Richter etal., 1993; Oku et al., 1994; Sasaki et al., 1994; Lalleyet al., 1994; Kirkwood, 1995). This is particularlytrue of cVRG neurons with confirmed projections tothe lumbar cord as established by antidromic acti-vation (Miller et al., 1985; Jodkowski and Berger,1988; Ballantyne et al., 1988; Hernandez et al., 1989;Feldman et al., 1992). Indeed, Richter (personalcommunication) states that this discharge pattern isso characteristic of cVRG E-BS neurons that he andhis colleagues discontinued testing for axonal projec-tions.The rat has become a more popular experimental

model but the presence of the equivalent populationhas not been fully established. Neurons are presentin the appropriate location (Saether et al., 1987;Ezure et al., 1988; Zheng et al., 1991, 1992b) withthe anticipated discharge patterns (Schwarzacher etal., 1991; Zheng et al., 1991) or membrane potentialtrajectories (Zheng et al., 1991, 1992b) but either theaxonal projections were not tested (Ezure et al.,1988), the discharge patterns of the E-BS neuronsnot determined (Saether et al., 1987), or they didnot have the expected spinal projections (Zheng etal., 1991, 1992b). Schwarzacher et al. (1991)recorded extracellularly from E-AUG discharge pat-terns rostral to the obex and with axons in thespinal cord but all E neurons from which they laterrecorded intracellularly at the same level had DECdischarge patterns. Since intracellular recordings aremore successful from large cells, smaller neuronswith dierent discharge patterns may have dierentprojections (and functions), but insucient data areavailable to confirm or refute this hypothesis.E-AUG neurons are found in the brainstem-

spinal cord preparation of the neonatal rat but rep-resent only 2% of all respiratory neurons (Smith etal., 1990b). Discharge patterns may, however, reflectconditions specific to this in vitro preparation. Paton(1996a) has shown that perfusion of the heart-brain-stem preparation of the mouse results in a normalAUG discharge pattern of the phrenic nerve, unlikethe DEC pattern typically observed in the super-fused brainstem-spinal cord preparation of the neo-natal rat. However, the discharge pattern in themouse can be converted from AUG to DEC by alow perfusion pressure (Paton, 1996a) or hypoxia(Paton, 1996b). If hypoperfusion results in a low tis-sue Po2, as occurs more than 300 mm from the sur-face in the superfused neonatal rat brainstem

Control of Abdominal Muscles 439

(Okada et al., 1993a), the discharge patterns may beabnormal. The situation is complicated by the pre-sence or absence of pulmonary (SAR) feedbackwhich has marked eects on respiratory activity inneonatal rats (Fedorko et al., 1988) and the degreeof maturation of the animal since discharge patternsdisplay developmental changes (Paton and Richter,1995).In the working heart-brainstem preparation of the

mouse, (late) expiratory neurons with AUG dis-charge patterns are relatively common (16 of 96neurons from which extracellular recordings weremade; Paton, 1996b) but their projections down thespinal cord were not tested. Paton (personal com-munication) indicates that these expiratory neurons,which are seldom if ever reported in the brainstem-spinal cord preparation of the neonatal rat, wererecorded from the cVRG.In dog, E-BS neurons are slightly more caudal

(about 1 mm) and about 0.5 mm more lateral, rela-tive to the obex, than those in the cat (Zuperku andHopp, 1987; Bajic et al., 1992). Almost all recordedneurons are bulbospinal; most have a discharge pat-tern in which peak frequency is reached before mid-expiration, then either plateau or decline, usuallystopping before the onset of the next inspiration(Bajic et al., 1992, 1994; D- ogas et al., 1995).However, neurons with AUG discharge patterns arealso present; these start firing during expiration,reach their peak discharge frequencies before mid-expiration, and then maintain their firing at thislevel until the onset of the next inspiration. It isunclear if this pattern is due to a dierence in sensi-tivity to SAR input (Tonkovic-Capin et al., 1992;see below). I refer to these neurons as plateau neur-ons to distinguish them from traditional AUGneurons which reach peak discharge frequencies atthe end of the discharge phase; Ezure (1990) refersto them as CON (constant) neurons. However, theplateau discharge pattern does not seem to be dueto the experimental preparation (paralyzed and ven-tilated) since E neurons with similar discharge pat-terns have been recorded in spontaneously breathingdog (Adams et al., 1987); although their projectionswere not determined, because many were caudal tothe obex, they were likely bulbospinal.The DEC pattern of some E neurons corresponds

to that of the integrated discharge of L1 reported byFregosi et al. (1987) and Fregosi and Bartlett (1988)using a standard preparation, the decerebrate,paralyzed and ventilated cat with bilateral vagotomyand pneumothorax. This pattern is also observed inspontaneously breathing pentobarbital-anesthetizeddogs with intact vagi (Van Lunteren et al., 1988b).It diers, however, from the AUG-plateau patternof L1 or the integrated EMGs of IO or TA inchloralose- and pentobarbital-anesthetized, spon-taneously breathing dogs (Ledlie et al., 1983; Farkasand De Troyer, 1989; Farkas and Schroeder, 1990;Schroeder et al., 1991), of L1 activity in decerebrateservo-ventilated cats (Fregosi et al., 1990), of EOmotor units in spontaneously breathing anesthetizedcats (Mateika et al., 1996), and the AUG pattern inlightly anesthetized dogs (Estenne et al., 1990a) andconscious ponies (Brice et al., 1991), all with intactvagi. Caution should, however, be used in evaluat-

ing these discharge patterns because they oftenexhibit considerable inter-breath variations. Forexample, the discharge pattern of TA varied breath-by-breath between plateau and DEC in pentobarbi-tal-anesthetized spontaneously breathing dogs withintact vagi (Van Lunteren et al., 1988a); in one dog,TA discharged only for the first 15% of expiration,an extreme example of a DEC discharge pattern. Inanother paper from the same group, using the samepreparation, the plateau discharge pattern of TAduring eupnea converted to a DEC pattern duringCO2 rebreathing (Arnold et al., 1988). Motor axonsto TA and IO in decerebrate, paralyzed, ventilatedand vagotomized cats typically have DEC dischargepatterns (Fig. 3; Fregosi et al., 1992) but AUG dis-charge patterns are present in decerebrate, paralyzedand ventilated cats with intact vagi (e.g. Oku et al.,1994; see Fig. 12). The reasons for these dierencesmay be related not just to the presence or absence ofSAR feedback but to such other experimental con-ditions as levels of respiratory drive, anesthetic (ifany) level, posture, feedback from the lung (includ-ing the upper airway) and chest wall, and the actualsite of recording because the abdominal muscles andthe other expiratory muscles of the rib cage (TS,rostral, mid-thoracic, and caudal internal intercos-tals) constitute a heterogeneous group with dierentmechanical actions requiring dierent discharge pat-

Fig. 3. Eect of hypercapnia on discharge of an abdominal(TA or IO) motoneuron in a decerebrate, vagotomized,and ventilated cat. Traces from top down: integrated phre-nic activity (fPhr), activity of whole L1, and activity of anabdominal motor axon from contralateral L1. Hypercapniaincreased the peak discharge frequency at the onset of thedischarge phase. Note decrementing discharge pattern inthis vagotomized preparation, compared with the augment-ing pattern characteristic of preparations with intact vagi(Fig. 12). Reproduced with permission from Fregosi et al.

1992.

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terns. Variations in the relative strengths of IPSPsand EPSPs under dierent experimental conditionslikely contribute to the dierent discharge patterns(e.g. AUG versus DEC) of E-BS neurons(Ballantyne and Richter, 1986) and motoneuronsreported by various investigators. The reasons forthe diverse discharge patterns displayed by ostensi-bly similar preparations are unknown.The organization of the respiratory centers in rab-

bit appears similar to that of cat (Jiang and Shen,1991); all three cVRG E neurons with AUG dis-charge patterns had axons in the spinal cord but thesmall sample size precludes generalizations aboutthe projections and laterality of their axons. E-AUGneurons in the rVRG also project to the spinal cordbut these may belong to the population of BotC E-AUG neurons which inhibit phrenic motoneurons(Merrill and Fedorko, 1984). In an earlier study bySchmid et al. (1985), six of 43 E neurons had theiraxons in the spinal cord but the locations of theirsomata were not provided.One study is available on respiratory neurons of

the cVRG in adult guinea pig (Richerson andGetting, 1992). The organization is similar to that inthe cat. However, recordings were made between 0and 2.5 mm rostral to the obex, corresponding tothe intermediate VRG (iVRG) which, as in cat, con-tains I and E neurons. Thus, definitive studies on E-BS neurons in the cVRG are still needed.The piglet (Lawson et al., 1989b) possesses respir-

atory neurons with discharge patterns identical tothose in the cat but the E-AUG neurons were notapparently tested for projections to the spinal cord.Later studies from the same laboratory (Czyzyk-Krzeska and Lawson, 1991; Lawson et al., 1991)verified that most of these were bulbospinal, havingramp depolarizations starting immediately upon ces-sation of the phrenic burst.

3.3. Inputs to CVRG (E Neurons)

The neurons driving cVRG E-BS neurons are stillnot identified. Comparisons of the discharge pat-terns of dierent neuronal populations indicatepossible candidates. For example, Merrill (1974)suggested that the early-burst I neurons inhibit Eneurons, silencing the latter at the end of the Ephase. Cohen et al. (1985) suggested that early Eneurons of the nucleus of the solitary tract (NTS)(Feldman and Cohen, 1978) with DEC dischargepatterns inhibit E neurons of the cVRG with AUGdischarge patterns (see Section 7.5). Many reviews(e.g. Cohen, 1979; Euler, 1986; Richter et al., 1986;Orem and Trotter, 1994; Dun et al., 1995; Bianchiet al., 1995; Bianchi and Pasaro, 1997) provide pic-torial representations of either the discharge pat-terns or membrane potential trajectories of dierentneuronal populations.

3.3.1. Electrophysiological Studies

More direct evidence of the presence and proper-ties of connections between putative pre-motorneurons and abdominal motoneurons is obtainedusing XCOR, STA, and antidromic stimulation/mapping. XCOR uses extracellularly recorded ac-

tivities whereas STA uses the cells membrane poten-tial or, less often, the terminal or focal synapticpotentials; these techniques, and their limitations,have been reviewed (Kirkwood, 1979; Berger andBellingham, 1995). XCOR describes the probabilityof discharge of a neuron B (or pool of neurons) fol-lowing a trigger (reference) event (e.g. an action po-tential of neuron A). The presence of significantpeaks (or troughs) in the XCOR (Sears and Stagg,1976; Graham and Dun, 1981; Dun and Iscoe,1996), indicated by the ratio of the peak (or trough)to the mean bin count away from the region ofinterest (the k ratio; Sears and Stagg, 1976), indi-cates if A and B are connected or share a commoninput. A peak (or trough) with a rapid rise- (or fall-)time and a narrow width indicates a monosynapticprojection. STA allows one to extract post-synapticpotentials (PSP) from neuron B following a refer-ence event (e.g., action potential of neuron A) fromthe synaptic noise associated with other, presum-ably random, events. The strength of the putativeconnection is given by the amplitude of the PSP. Asis the case in XCOR, a rapid change in the averagedpotential indicates a monosynaptic projection.Antidromic mapping involves stimulating discreteregions in the spinal cord containing a putative pro-jection (stem axon or terminal) to elicit an antidro-mic spike and then plotting the intensity ofstimulation as a function of space; this provides in-formation about the trajectory of the axon and thedistribution of axonal terminals (e.g. Merrill, 1970;1974; Merrill and Lipski, 1987; Dun and Lipski,1987; Hoskin et al., 1988; Jiang and Lipski, 1990;Kirkwood, 1995).

3.3.1.1. Medulla

Models of respiratory rhythmogenesis (Richter etal., 1986; Botros and Bruce, 1990; Ogilvie et al.,1992; Rybak et al., 1997a,b) typically indicate thatrespiratory, particularly E, neurons receive tonic ex-citatory input from the reticular activating systemand chemoreceptors. This is consistent with thetonic discharge of E neurons during hypocapnicapnea (see below) but we know much less aboutthese excitatory inputs compared to the inhibitoryones to cVRG E-BS neurons (for reviews, see Euler,1986; Feldman, 1986; Ezure, 1990; Richter et al.,1992; Dun et al., 1995; Bianchi et al., 1995).Bianchi et al. (1995), for example, list five neuronalpopulations supplying inhibitory inputs: pre-I, earlyI, I-AUG, post-I neurons, and, surprisingly consid-ering the similarity of their discharge patterns, E-AUG of the BotC. The models of Rybak et al.(1997b) show inhibitory synaptic connections butalso indicate a tonic excitatory input from other,undefined, neurons. Ezure (1990) indicates an excit-atory connection to the cVRG from I neurons withplateau discharge patterns in the rVRG (Otake etal., 1990). Aside from an obvious problemtheneurons fire in opposite phasesSTA between theseneurons and cVRG E-BS neurons reveals onlymonosynaptic IPSPs (Ezure et al., 1990). Both E-AUG and E-DEC of BotC inhibit cVRG E neuronsbut two of 49 cVRG E-BS neurons received mono-synaptic EPSPs from BotC E-AUG neurons (Jiang

Control of Abdominal Muscles 441

and Lipski, 1990). Interestingly, many BotC E-DECneurons are excited, but E-AUG neurons sup-pressed, by lung inflation (Manabe and Ezure,1988), a response relevant to the responses of E-BSneurons to changes in lung volume (see Section 7).The putative excitatory projection(s) responsible

for the AUG discharging patterns of cVRG E-BSneurons have not been discovered despite assertionsto the contrary (Euler, 1983, 1986). The idea thatBotC E-AUG drive cVRG E-BS neurons is basednot just on the similarity of their discharge patternsbut the presence of axonal projections from the for-mer to the ipsilateral and, to a lesser degree, thecontralateral cVRG (Bystrzycka, 1980; Bianchi andBarillot, 1982; Fedorko and Merrill, 1984; Jiang andLipski, 1990). Although BotC E-AUG neurons canbe antidromically activated by shocks to the ipsi-and contralateral cVRG (Fedorko and Merrill,1984), I know of no studies reporting peaks in theXCOR, consistent with monosynaptic projections(or common excitation) or EPSPs, between the twopopulations; indeed, XCOR between eight pairs ofBotC E and E-BS neurons were all flat (Hilaire etal., 1984). Jiang and Lipski (1990), using STA,observed 12 IPSPs but only two EPSPs in 58 pairsof BotC E-AUG and cVRG E-BS neurons. Lindseyand colleagues (1987) found evidence only of inhibi-tory interactions between four of 11 pairs of E-AUG (and not antidomically activated (NAA) E)neurons in caudal medulla. Although antidromicmicrostimulation and STA indicates that BotC E-DEC project to the contralateral VRG, none of theVRG neurons was bulbospinal (Ezure and Manabe,1988); monosynaptic IPSPs were, however, obtainedin I-BS, I-NAA, and vagal motoneurons. Moreover,XCOR between 22 pairs of cVRG E neurons (separ-ation 250 to 350 mm) were all flat (Graham andDun, 1981); similar results were obtained in astudy of 31 pairs (separation 100 to 1000 mm)(Merrill, 1978). Both results are consistent with anabsence of shared inputs and, in accord with theabsence of axonal collaterals (Arita et al., 1987), noor very weak recurrent excitation (Bianchi et al.,1995), at least over these inter-electrode distances.Although other workers have reported a high inci-dence of peaks in the XCOR between neuronsrecorded from the same electrode (Feldman et al.,1980), a result consistent with recurrent excitation(or shared inputs), XCOR derived from a singleelectrode are susceptible to false positives (Grahamand Dun, 1981). While excitatory connectionsmay have been missed for technical reasons (e.g.inputs to inactive neurons or small active neuronsfrom which recordings are dicult), the source(s) ofexcitation to E-BS neurons of the cVRG remainspeculative, including those from chemoreceptorsand the reticular activating system. Finally, E-AUGof BotC of conscious cats do not discharge duringsneeze when abdominal muscles are active (Oremand Brooks, 1986), suggesting they are not drivingcVRG E-BS neurons. This result is consistent withother studies by Orem (1988, 1989, 1990, 1994b) in-dicating that medullary respiratory neurons are sub-jected to control by neurons not involved in thecontrol of respiratory timing and amplitude

observed in the typical anesthetized or decerebratepreparation.The only known interaction of an identified

cVRG E-BS AUG neuron with another medullaryneurons is an apparent inhibition of two NAArVRG E neurons (Lindsey et al., 1987), a result con-trary to Merrills and Ezures claims that E-BS neur-ons of the cVRG project only to the spinal cord.Until, however, the results of Lindsey and col-leagues (1987) are substantiated by analysis frommore E-BS neurons, the belief that they project onlyto the spinal cord (Merrill, 1974; Ezure, 1990)should be retained (e.g. for modeling).Despite the absence of evidence based on XCOR

and STA of projections from other medullary neur-ons to cVRG E-BS neurons, other evidence, in ad-dition to that provided by antidromic activation(Fedorko and Merrill, 1984), suggests that BotCneurons project to E-BS neurons. Unilateral coolingof BotC (and the para-ambigual region, the DRG,and the rostral ventrolateral medulla at a site wherecooling causes apnea) can increase both I (phrenicand external intercostal) and E (abdominal) activity(Budzinska et al., 1985a); these increases can beboth tonic and phasic, the tonic activity being unaf-fected by CO2 (Budzinska et al., 1985b). Althoughcooling aects both neurons and axons of passage,these results suggest that various brainstem struc-tures suppress E activity, either by blocking excit-atory input or by inhibiting intrinsically activeneurons. However, electrical stimulation in a regionof BotC containing E-AUG neurons causes apnea(bilateral suppression of phrenic and external inter-costal activity) and elicits tonic bilateral activity ofabdominal muscles (Bongianni et al., 1988b).Microinjection of DL-homocysteic acid (DLH), anexcitatory amino acid, elicits the same responses, in-dicating that the eects are due to activation ofneurons and not axons of passage. Bongianni et al.(1990) later showed that the same stimuli applied tothe region of BotC containing E-AUG neurons acti-vates cVRG E neurons (not tested for projections tothe spinal cord but their discharge patterns and lo-cation are consistent with them being bulbospinal);single shocks activated E neurons bilaterally, evenduring hypocapnic apnea, at latencies of 3.0 to 8.4ms (similar to the latencies of IPSPs determined bySTA between I neurons of the retrofacial nucleus(RFN; close to E neurons of BotC) and cVRG E-BS neurons (Anders et al., 1991), and many neuronscould follow stimulus frequencies exceeding 40 s1.Injection of DLH caused inspiratory apnea andtonic activation of the same neurons; moreover,injections of DLH more than 0.5 mm from the orig-inal injection site were without eect, indicating thatthe eect was not due to stimulation of structuresdistant from the site of injection (Lipski et al.,1988). During fictive cough and swallowing inducedby stimulation of the superior laryngeal nerve (SLN)in decerebrate ventilated cats, abdominal (L1) motornerve, cVRG E-BS and BotC E-AUG neurons dis-charge similarly (Shannon et al., 1997; Grelot andBianchi, 1997), indicating excitatory projectionsfrom BotC E-AUG neurons to cVRG E-BS neur-ons. A recent study by Bongianni et al. (1998) of theresponses of Botzinger E-AUG neurons reveals a

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linear relation between their peak neuronal dis-charge frequencies and peak integrated EMG ac-tivity of EO (Fig. 4). In decerebrate, ventilated cats,systemic administration of codeine blocks comple-tely the development of fos-like immunoreactivity inmultiple brainstem regions, including the NRA andRFN, during fictive coughing elicited by stimulationof the SLN (Gestreau et al., 1997). It would be ofinterest to determine the eects of microinjections ofcodeine into selected regions, including BotC, on theactivation of E-BS neurons in NRA. Takentogether, these results indicate that, despite theabsence of supporting evidence from XCOR orSTA, some neurons in BotC excite cVRG E-BSneurons. However, conflicting resultssilence ofBotC E-AUG neurons during active expiration inconscious cats (Orem and Brooks, 1986) and inhi-bition of BotC E-AUG neurons following singleshocks or short tetani to SLN that evoke excitatoryresponses in cVRG E-BS neurons (Bongianni et al.,1988a)indicate that these connections need moredetailed examination.

Dierent responses to stimulation of BotC areobtained from rabbits. Microinjection of DLH inthe region of BotC containing E-AUG neuronsdepresses both phrenic and abdominal motor ac-tivity whereas electrical stimulation suppresses phre-nic activity but increases abdominal activity, evenwhen none is present (Bongianni et al., 1997b).While these latter eects may have been due tostimulation of axons of passage, the reasons for thedierent responses of cats and rabbits to injection ofDLH are unknown. Injection of procaine into themedial RFN, close to BotC, causes apnea andcVRG E neurons (projections unknown) becometonically active but at a lower frequency than whenphasically active (Zhang et al., 1991). As the authorspoint out, the spread of procaine into a relativelylarge area, including ones which, when cooled, resultin apnea in cats (Budzinska et al., 1985a), is consist-ent with block of central chemoreceptor input tobrainstem respiratory neurons.In cats (and, presumably other species), vestibular

nerve stimulation elicits a variety of responses, someexcitatory, others inhibitory or mixed, on cVRGE-BS neurons (Shiba et al., 1996a). Because thelatencies of these responses are typically longer thanthose of the abdominal nerves, vestibular inputs toabdominal motoneurons appear to bypass, at leastin part, medullary premotor neurons. The longerlatencies of the latter may result from vestibularinputs first projecting to BotC, many neurons ofwhich respond to vestibular nerve stimulation(Nakazawa et al., 1997).In rabbit, many E neurons, perhaps in cVRG, are

excited by shocks to pontine nuclei (NPBM or locuscoeruleus) at latencies (up to 11.8 ms) suggestingmultisynaptic pathways (Schmid et al., 1985).Although the results were interpreted in terms ofphase-switching, they may also relate to behavioralaspects such as vocalization, mediated via the peri-aqueductal gray (PAG) (see Section 9.2).Study of the interactions between mid-line (raphe)

respiratory-related neurons in cats indicates fewmono- or paucisynaptic connections to cVRG Eneurons (110 of 2310 pairs; Lindsey et al., 1994), ofwhich only three included a neuron with a spinalprojection. Only four of 83 pairs involving a cVRGE-AUG neuron displayed any short-time scale inter-actions; two had positive time lags, one negative,and one was centered at time zero. These results areconsistent with cVRG E-BS neurons having few ifany interactions with respiratory (or even non-res-piratory) neurons other than those of the BotC.

3.3.2. Upper Airway

As expected from the involvement of abdominalmuscles in vocalization (Section 9.2), aerents fromthe upper airway, principally those carried in theSLN, aect E-BS neurons. Responses, however,cannot be monosynaptic because SLN aerents pro-ject only to the NTS (Lucier et al., 1986; for review,see Kubin and Davies, 1995). Trains of stimuli toSLN, at intensities no greater than twice that of asingle shock which elicits a transient reduction inphrenic activity, suppress phrenic and external inter-costal activity but elicit tonic abdominal (IO or

Fig. 4. Responses of BotC E-AUG neurons and abdominalEMG to mechanical stimulation of tracheobronchial treein a pentobarbitone-anesthetized cat. (A) Traces from topdown: phrenic neurogram (Phr), instantaneous dischargefrequency of a BotC E-AUG neuron, raw activity of theBotC E-AUG neuron, and integrated abdominal (probablyEO) EMG activity (fAbd). (B) Relation between fAbd(expressed as a per cent of maximum fAbd) and peak in-stantaneous discharge frequency (expressed as a per cent ofmaximum) in 12 BotC E-AUG neurons during coughing(n= 101). Modified with permission from Bongianni et al.

(1998).

Control of Abdominal Muscles 443

EO), cVRG E-BS, and BotC E-AUG neuronal ac-tivity (Bongianni et al., 1988a). The level of tonic Eactivity, whether muscle or neuronal, increases withPACO2, consistent with disinhibition at the medul-lary and spinal levels. Single shocks or brief tetaniat low intensity (1.42 times threshold), however,evoked no responses in E muscles while cVRG E-BSneurons were either unaected (n= 20), inhibited(n= 8) or excited (n= 2); all 10 BotC E-AUGneurons were inhibited. At 36 times threshold, theE muscles and 18 of 23 cVRG E-BS were excited(perhaps an expression of the expiratory reflex; seeKorpas and Tomori, 1979) although the responseswere not elicited in I (possibly because of the pento-barbital anesthesia), but BotC E-AUG neurons werestill inhibited. This last result suggests that SLNinputs to cVRG E-BS neurons are not mediated bythis population of BotC E-AUG neurons. All re-sponses were unaected by changes in PACO2, indi-cating that SLN inputs have relatively direct accessto medullary E neurons.In similar experiments, Jodkowski and Berger

(1988) obtained very dierent results. Electricalstimulation of the SLN, sucient to suppress phre-nic activity, activated internal intercostal activitywhile simultaneously reducing or silencing cVRG E-BS neurons. Their results are consistent with disinhi-bition of E neurons and motoneurons during theSLN-induced apnea (see preceding paragraph)which would then, especially under the conditions ofslight hypercapnia employed in their study, becomeactive. This explanation is also consistent with in-ternal intercostal activity decreasing only whenphrenic activity reappeared seconds after termin-ation of SLN stimulation or when brief and smallbursts of phrenic activity occurred during SLNstimulation. The dierences in responses of E-BSneurons to electrical stimulation from those reportedby Bongianni et al. (1988a) may be due to dierentstimulus intensities, since Jodkowski and Bergerused ones which elicited maximal inhibition ofphrenic activity. During instillation of water intothe larynx (which may have activated additionalupper airway aerents), eight of 56 E-BS neuronswere activated, suggesting that electrical and natu-ral activation of SLN aerents are not equivalent.The receptor properties of the aerents responsiblefor these dierent responses are unknown.Stimulation during E of the SLN in decerebrate

cats suppressed the firing of 12 of 14 cVRG E-BSneurons at latencies ranging between 4.8 and 7.0 ms(Oku et al., 1994). These latencies exceeded those ofthe suppression of E-AUG neurons (3.8 to 5.0 ms)elicited by the same stimulation, indicating thatSLN inputs to both neuronal populations are prob-ably indirect. In a survey of the responses of dier-ent medullary neurons to SLN stimulation, Jiangand Lipski (1992) observed IPSPs in 13 of 16 E-BSneurons of the cVRG; the latencies (mean04.2 ms)and rise-times and half-widths of the IPSPs indicatethat they are not monosynaptic. However, the meanlatency of IPSPs in E-AUG neurons of BotC was05.4 ms, indicating that the latter do not mediatethe response of E-BS neurons (i.e. by disfacilitationor withdrawal of synaptic input). Injection of waterinto the larynx of anesthetized cats hyperpolarizes

E-BS neurons and delays their onset of discharge(Ballantyne and Richter, 1986). Similar results, butwith electrical stimulation, were obtained in piglets(Czyzyk-Krzeska and Lawson, 1991); their reversalby Cl or negative current injections indicates theresponses were due to Cl-mediated post-synapticinhibition. The much shorter latencies (03 ms) com-pared to those reported by Oku et al. (1994) andJiang and Lipski (1992) could be due to species butit is nevertheless surprising as the marginally smallersize of the piglets (47 d, 1.12.6 kg) compared toadult cats would presumably be oset by lower con-duction velocities of the aerents in the former.

3.3.2.1. Nasal aerents

Stimulation of nasal receptors evokes sneeze; elec-trical stimulation of their aerents in branches (an-terior ethmoidal, posterior nasal, and infraorbital)of the trigeminal nerve also elicits sneezing (seereview by Shannon et al., 1997). These aerents pro-ject not to brainstem regions containing respiratoryneurons but to the trigeminal nucleus; electricalstimulation of its ventromedial region and adjacentreticular formation evokes sneezes (Nonaka et al.,1990). In sneezing cats, fos-like immunoreactivity, amarker of increased cell activity, is distributed notjust to areas to which nasal aerents project butalso to the NTS, cVRG, the pontine NPBM-KF aswell as the lateral part of the parvicellular reticularformation (Wallois et al., 1995). Electrical stimu-lation of the anterior ethmoidal and/or posteriornasal nerve excited 11 of 14 E-BS neurons, probablyin the cVRG (Wallois et al., 1992). Latencies wereless during expiration than inspiration, but exci-tation in inspiration was possible only in ketamine-,not pentobarbital-, anesthetized cats, clearly indicat-ing a multisynaptic pathway, in accord with thelatencies (013 and 018 ms in expiration and inspi-ration (including data from non-BS neurons), re-spectively). Inhibitory responses had shorterlatencies, mean 6.3 and 7.3 ms for ethmoidal andposterior nasal, respectively. Developmental aspectsinfluence the eectiveness of sneezing; kittens, unlikeadult cats, lack the preparatory inspiratory eort,and thus generate weaker expiratory eorts (Walloiset al., 1993). The authors attribute the dierence toimmaturity of the connections to dierent popu-lations of respiratory neurons but the weaker pre-paratory inspiration could, by reducing the amountof inhibition of E neurons, also reduce post-inhibi-tory rebound (depolarization) of E neurons (seeSection 6.2.2).

3.3.2.2. Hypothalamus

Short trains of pulses to the perifornical region ofthe hypothalamus evoked EPSPs followed by Cl-mediated IPSPs in cVRG E and E-BS neurons atlatencies between 412 ms and 835 ms, respectively(Ballantyne et al., 1988). The amplitudes of theEPSPs were phase-dependent, being greatest duringI when the neurons were hyperpolarized. It isunclear if the EPSPs were mono- or polysynaptic.Because longer trains of stimuli evoked tachypnea,increases in blood pressure, and pupillary dilation,the responses may represent a component of the

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defense reaction and even contribute to vocaliza-tion (Section 9.2).

3.3.3. Neuroanatomical Tracers

Tracers provide information about connectionsbetween neurons. When injected extracellularly intothe cVRG, the cell bodies of neurons projecting tothe cVRG are stained due to transport of the dyeafter its uptake by their axon terminals (retrogradetracing); the sites to which cVRG neurons projectcan be identified due to the presence of dye in theiraxonal terminals (anterograde tracing). Althoughone cannot assume that neurons that have taken upthe tracer necessarily project to the specific neuronalpopulation of interest (in this case, E-BS neurons),the results do indicate where to record to confirmprojections; this is necessary because some tracers(e.g. HRP) can be taken up by damaged axons ofpassage, resulting in false positives. Intracellularinjection allows one to trace the cells axon and anycollaterals. This technique has the advantage thatthe cells function(s) can often be determined fromits discharge pattern and projection site by antidro-mic stimulation but the process is laborious andbiased to larger cells (somata and, occasionally,axons) which are easier to penetrate and hold.Projections from the VRG to abdominal (and

other respiratory) motoneurons develop early, atleast in marsupials. Farber (1985) indicates that ab-dominal activity is present as early as 30 d inDidelphis virginiana. In Monodelphis domestica,which is born 1415 d post-conception, injections offast blue into the lumbar and cervical spinal cordeven at this early age retrogradely label neurons inmultiple brainstem sites, including the NRA (Wanget al., 1992); functional identification of the project-ing and receiving neurons is not available, nor havethe muscles used for breathing been identified.Injection of HRP into the VRG labels neurons in

the pons (locus coeruleus and subcoeruleus, and thelateral and medial parabrachial and the KF nuclei)and medullary nuclei (reticularispontis oralis, retro-facial, paragigantocellularis lateralis, and the ventro-lateral NTS) (Bystrzycka, 1980) but the size of theinjection site (NA and NRA) makes it impossible tospecify which sites project only to the cVRG.Similar results were obtained by Takeuchi et al.(1980) who observed dense labeling in the ipsilateralparabrachial nuclei (PBN) after injections of HRPinto NA. Injection of wheat germ agglutinin (WGA)conjugated to HRP (WGA-HRP) into the cVRGretrogradely and anterogradely labels neurons inmany regions of the brainstem (J.C. Smith et al.,1989) although electrophysiological mapping indi-cates that feline cVRG E-AUG neurons project onlyto the spinal cord (see Long and Dun, 1986 andEzure, 1990 for reviews). In rat, however, one ofseven cVRG E neurons had extensive local axonalcollaterals (Sun et al., 1996) and some E-BS neuronshad medullary axon collaterals (Zheng et al.,1992a). Therefore, either the cVRG includes other(inactive) neurons projecting to these dierentregions or the injected label spread beyond the bor-ders of the cVRG. The former explanation is plaus-ible, but disparate findings in two cats following

injection of the label into ostensibly the same regionsuggest caution: in one cat, anterograde labeling waspresent in both the ipsi- and contralateral magnocel-lular tegmental fields and the postpyramidal nucleusof the raphe whereas in the other cat no labelingappeared in these regions (Smith et al., 1989b).Gerrits and Holstege (1996) have recently

reported, also in cat, the sites of uptake of WGA-HRP injected into the cVRG (determined fromstereotaxic coordinates and not the presence of E ac-tivity). Label is detected in the PAG (bilateral), theventrolateral PBN and KF (primarily ipsilateral),the retrotrapezoid nucleus (RTN) (ipsilateral), ven-trolateral to the facial (BotC and pre-BotC), theperi-ambigual region (bilateral), the lateral tegmen-tal field (including BotC) and NTS (bilateral), andtwo groups in the medial tegmental field (one at thelevel of the facial and another just rostral to thehypoglossal nuclei) (ipsilateral). Injection of an ante-rograde tracer, tritiated leucine, confirms these pro-jections but not the finding of Smith et al. (1989b)of an input from nuclei raphe pallidus and magnus.Gerrits and Holstege (1996) emphasized the role ofthe PAG, critical in vocalization (Section 9.2), as themost rostral structure with inputs to the VRG, andthat of the two discrete regions of the ventromedialmedullary medial tegmental field as a possible relayfrom rostral limbic structures. Earlier, Holstege(1989) had interpreted the connections to and fromthe cVRG (NRA) determined from retrogradetransport of HRP and anterograde transport of tri-tiated leucine in terms of the structures involved invocalization. Thus, VRG neurons receive input bi-laterally from the lateral part of the caudal PAGfrom which vocalizations can be elicited by electricalstimulation (Section 9.2) and in the tegmentum justlateral to this region. cVRG neurons not only pro-ject down the spinal cord, where he noted consider-able re-crossing of terminal fibers to ipsilateralmotoneurons, but also to neurons innervatingmuscles of the upper airway and mouth, all ofwhich are involved in vocalization. Thus, the exten-sive projections of cVRG neurons to other regionsin the medulla (J.C. Smith et al., 1989; Nunez-Abades et al., 1993; Gaytan et al., 1997) make senseif interpreted in terms of behaviors other thanbreathing. However, this means that the cVRGmust contain (pre-motor)neurons dedicated to beha-vioral rather than just the metabolic aspects of res-piration.More recent studies in rat using tracers like fluor-

oruby, diamino yellow, and fast blue confirm theextensive connections of the cVRG with other brain-stem sites (Nunez-Abades et al., 1993; Morillo et al.,1995; Gaytan et al., 1997); they receive inputs fromrVRG and from BotC (which projects to bothrVRG and cVRG; Morillo et al., 1995), many subdi-visions of the NTS, the area postrema, the PBN andKF, and the nucleus lateral paragigantocellularis(Nunez-Abades et al., 1993; Morillo et al., 1995).The cVRG projects bilaterally, in the medulla, toNA, the periambigual area, the lateral nucleus para-gigantoceullularis, and the dorsal part of the lateralreticular nucleus; in the pons, to the caudal part ofthe PBN and KF (Gaytan et al., 1997). One cannottell, however, if there is a projection from BotC to

Control of Abdominal Muscles 445

cVRG. Even though the injected region contains Eneurons, interpretation must still be cautious as theinjected regions undoubtedly contain neurons whoseprimary function(s) are non-respiratory.These studies illustrate some of the diculties

involved in interpreting results using tracers.Although many sites of retrograde and anterogradelabeling are similar, others are not; it is unclear ifthis reflects spread of the label, small dierences inthe site of injection (particularly in the absence ofrecordings from the desired neurons), or just thenormal biological variation between animals. Theproblem is accentuated by injections of tracers intolarger regions containing dierent neuronal popu-lations. For example, injections of HRP into boththe NA and NRA (Bystrzycka, 1980) cause exten-sive labeling of cells in contralateral vlNTS and inbilateral RFN and nucleus paragigantocellularislateralis, and the pons (locus coeruleus, PBN, andKF). Given the diverse neuronal populations in theVRG (e.g. cranial motoneurons and spinal pre-motor neurons, along with propriobulbar neurons),interpretation is dicult. This point is made particu-larly well by Yajima and Larson (1993); many neur-ons around NA in awake vocalizing monkeysdischarge only in association with vocalization orswallowing or respiration. Disparities between elec-trophysiological and neuroanatomical resultsemphasize the importance of a conservative in-terpretation of the results of experiments using tra-cers until confirmatory electrophysiological data areavailable.In cat, injection of HRP into NA also labels the

pontine regions described above as well as neuronsin the mid-brain (central gray, reticular formation,deep tectum and red nucleus) and diencephalon (lat-eral and peri- and paraventricular hypothalamic)(Rose, 1981). Since many of these latter regionsinclude estrogen-containing neurons, Rose postu-lated that these neurons may be involved in thevocalization associated with estrous behaviors(Section 9.2).In rat, intracellular labeling of BotC E neurons

reveals three major synaptic targets (based on bou-tons): near the cell bodies, neurons of rVRG, andneurons of cVRG (Bryant et al., 1993). These resultsagree with those in cat using antidromic stimulation(Bianchi and Barillot, 1982; Fedorko and Merrill,1984) and STA (Jiang and Lipski, 1990); however,no neuroanatomical data (axonal terminals or bou-tons) in cat yet confirm the electrophysiological evi-dence because eorts to trace the axons as far as thecVRG have either been unsuccessful (Otake et al.,1987) or restricted to more rostral portions of theVRG (Ezure and Manabe, 1988; Otake et al., 1988).These anatomical and electrophysiological results

raise an important question: why do cVRG E-BSneurons fire? Even if their AUG discharge resultsfrom gradually increasing disinhibition during E(Ballantyne and Richter, 1986), this does not answerthe question. They must receive excitatory inputsbecause transverse lesions just rostral to the borderof the cVRG, at about the level of the obex, abolishtheir activity (Merrill, 1979). The source could besupra-medullary because Kalia (1981) reportedlabeling of neurons in the ipsilateral NPBM and KF

nuclei following HRP injections into a region of thecVRG containing E neurons; additional detailsbeyond those presented in her review are unavail-able. Electrical stimulation of the NPBM in anesthe-tized rabbits excites or inhibits E neurons (Schmidet al., 1985) but one cannot determine, from the in-formation provided, the responses of cVRG E-BSneurons. However, E-BS neurons of decerebratecats made apneustic by bilateral cold block of thepneumotaxic region continue to discharge rhythmi-cally, albeit with an altered discharge pattern (St.John and Bianchi, 1983); thus, input from theNPBM and KF is not mandatory. Moreover, Speckand Beck (1989) reported that bilateral lesionsrestricted to the rVRG abolish cVRG E activity.Thus, either neurons of the rVRG excite cVRG Eneurons or inputs from some other source (e.g. che-moreceptors) pass through the rVRG, with or with-out synapsing on intermediary neurons. This lastresult, however, indicates that cVRG E neurons can-not be intrinsically chemosensitive; otherwise, theirdischarge would have persisted. However, the longerdendrites of some feline cVRG E-BS neurons extendtowards the ventral surface of the medulla (Arita etal., 1987), where they could sense changes in thechemical environment. Pilowsky et al. (1990) (rat)and Grelot et al. (1988) (cat) report that some res-piratory neurons in the rVRG also have dendriteswithin 100200 mm of the ventral surface of themedulla. However, proximity to the central surfacemay not be a prerequisite for chemosensitivity. King(1980a, b) has described respiratory neuronsarranged in sheets corresponding to the locations ofblood vessels in the brainstem and Pilowsky et al.(1990) indicate that some neurons have dendriticarbors close to blood vessels. The functional conse-quences of such arrangements, however, remain tobe established.In summary, although neuroanatomical (labeling)

evidence indicates diverse sources of input to cVRG,electrophysiological evidence is less compelling, per-haps because these inputs are related to non-respir-atory behaviors.

3.4. Axonal Projections

In cat (Merrill, 1970) and rat (Saether et al.,1987), axons of cVRG E neurons cross the medullaat approximately the level of their cell bodies (seeMonteau and Hilaire, 1991 for review) between C1and the obex (Nakayama and von Baumgarten,1964; Merrill, 1974; Miller et al., 1987, 1989; Aritaet al., 1987) and descend in the ventral column ofthe lateral spinal cord (Nakayama and vonBaumgarten, 1964; Merrill, 1970, 1974; Richter etal., 1975; Merrill and Lipski, 1987; Jiang andLipski, 1990; Kirkwood, 1995); spinal projections ofcVRG E neurons in rat appear to be rare (only 5/139, Saether et al., 1987; 0/10 for E-AUG, Zheng etal., 1992b).cVRG E neurons are, compared to other medul-

lary respiratory neurons, unusual in that they donot have collaterals to other respiratory neurons inthe medulla (Merrill, 1974; Arita et al., 1987) andare thus pure pre-motor neurons to internal inter-costal and abdominal motoneurons. Even slightly

S. Iscoe446

more rostral (obex21 mm) E-BS AUG neuronsrarely interact with other neurons. For example,Lindsey et al. (1989), using XCOR, obtained evi-dence for synaptic interactions in only five of 63 E-AUG ipsilateral pairs and 1/13 ipsilateral trios. Ofthe former, only two of 10 neurons were bulbosp-inal; of the latter, none of the neurons displayingconcurrent inhibitory interactions was bulbospinal(two did not have axons in the spinal cord or vagusand one was not tested). An earlier study (Lindseyet al., 1987) of VRG E-AUG neurons yielded simi-lar results. Antidromic activation of cVRG E-BSneurons has no eect on rVRG I neurons, indicatingthe absence of axon collaterals to the latter. ThatcVRG E-BS neurons have no role in respiratoryrhythmogenesis is substantiated by the negligibleeects of transverse sections of the medulla just ros-tral to them; the discharge patterns of I neuronsabove the lesion are unaected (Merrill, 1979) whiletransections of the medulla 2 mm caudal to or at thelevel of the obex have almost no eect on the respir-atory-related discharge of the facial or mylohyoidnerves (Huang and St. John, 1988). Finally, injec-tion of local anesthetic into the cVRG has no eecton respiratory rhythmicity (Zhang et al., 1991).Thus, axons of cVRG E-BS neurons appear to berestricted to the spinal cord.Merrill (1970) tested for projections of E neurons

of the caudal NRA; the vast majority (81 of 83) haddescending projections only to the contralateralspinal cord as determined by antidromic activationat C3, findings later confirmed (Merrill, 1974;Merrill and Lipski, 1987). In cats, injections of HRPinto the lumbar spinal cord label cVRG neurons bi-laterally (Miller et al., 1989); mid-sagittal lesionsbetween C1 and obex prevent labeling of contralat-eral cVRG neurons but no mention is made ofHRP-labeled neurons ipsilateral to the site of injec-tion. Because the injected HRP did not spread tothe other side of the cord, labeling of brainstemneurons ipsilateral to the injection site was due topickup of the HRP by descending axons with term-inals recrossing the spinal cord (Holstege, 1989;Kirkwood, 1995). Thus, identification of an E neur-on as bulbospinal and contralateral based on thepresence of its axon in the cervical or upper thoracicspinal cord (e.g. Merrill, 1970; Bianchi, 1971;Merrill, 1974; Arita et al., 1987; Zheng et al., 1992b)does not necessarily mean that its axon terminalsare restricted to the contralateral side.In contrast to the results of Merrill (preceding

paragraph), none of the 10 cVRG E-AUG (basedon the trajectory of the membrane depolarization)neurons in decerebrate rats studied by Zheng et al.(1992b) was bulbospinal, a finding they attributed tothe failure of the antidromic action potential toinvade a non-spiking soma (28 of 37 neurons werenot spiking at the time of penetration); however,one of the E-all neurons with an axon in the spinalcord was inactive. Thus, the failure to detect spinalcord projections of E-AUG neurons likely rep-resents a false negative (see Ezure, 1990).Dierences in the type of neurons recorded by extra-cellular and intracellular microelectrodes mayexplain the discrepancy between these results andthose of Merrill (1970, 1974). The combination of

extracellular recording and barbiturate anesthesiawould restrict recordings to active E-BS neurons; incontrast, inactive (presumably larger) E-AUG neur-ons with non-bulbospinal (propriobulbar?) projec-tions would be missed when recordingextracellularly but preferentially recorded intracellu-larly. No data, however, indicate if soma size andfunction (projection) are related.Although the axons of E-BS neurons form a rela-

tively discrete bundle in the ventrolateral quadrantof C3, the axons disperse by T1; Merrill (1974), andlater Merrill and Lipski (1987), noted extensive axo-nal arborizations of a given axon, often extendingover as many thoracic segments as were exposed.Similar results have recently been reported for axo-nal projections in the lumbar and sacral spinal cordof cat (Sasaki et al., 1994). In many studies, the siteof antidromic stimulation (often C3) makes itimpossible to determine the precise target(s) of thebulbospinal neurons: motoneurons of dierentabdominal muscles, TS, and internal intercostals, orsome combination. In the absence of identificationof either the motoneurons (by antidromic activationfrom a particular motor nerve) or recordings frommotor nerves to identified muscles, one cannotassume that these projections are distributed uni-formly to all motoneurons in a given segment(s).Indeed, during fictive vomiting (Miller et al., 1987),some VRG E-BS neurons discharge during the inter-val between simultaneous bursts of abdominal andphrenic activity, when some internal intercostals areactive (Iscoe and Grelot, 1992); thus, at least duringthis particular behavior, the discharges of motoneur-ons with ostensibly similar roles can be uncoupled.Such uncoupling could also result from activation ofdierent medullary pre-motor neurons.Ezure (1990) concluded that E-BS neurons of the

cVRG act only as pre-motor neurons to E moto-neurons; the axonal morphology of cVRG E (prob-ably bulbospinal) neurons confirms this assessmentbecause none of the seven stained neurons hadmedullary axon collaterals (Arita et al., 1987).However, Bongianni and colleagues (1994) observedthat microinjections of DLH into the cVRG of cat,at sites containing E-AUG activity, activated con-tralateral E-AUG neurons and abdominal nerve ac-tivities while simultaneously suppressing medullary(rVRG I) and phrenic nerve activity. They arguedthat these eects on respiratory timing weremediated by axon collaterals of cVRG E neuronsrather than, as they acknowledge, other neurons.Anterograde tracing indicates projections of cVRGneurons to other brainstem sites (J.C. Smith et al.,1989) despite the absence, in cat, of medullary axoncollaterals (Merrill, 1974; Arita et al., 1987).Because interpretation of the morphology and func-tions of cVRG are biased by recordings from spon-taneously active cVRG neurons, these findings(projections to other brainstem sites, eects on res-piratory pattern) may result from activation ofquiescent E neurons with dierent properties orfrom other neuronal types within the cVRG. AsBongianni et al. (1994) point out, injection of DLHactivates all neurons, including those recruited onlyduring forceful contractions of the abdominalmuscles or speech. Such recruited neurons may have

Control of Abdominal Muscles 447

properties dierent from those used in normal res-piration.Recently, Shiba et al. (1997b) reported that E-

DEC or plateau neurons of the cVRG both projectto the contralateral NA and receive inputs from thePAG; furthermore, electrical stimulation of the NAelicits spikes of constant latency in silent cells in thecVRG (suggesting an antidromic projection).Stimulation of the PAG also elicits orthodromicspikes in silent cells in the cVRG. In contrast, stimu-lation of NA never elicits antidromic spikes incVRG bulbospinal E-AUG neurons. Thus, thecVRG appears to be made up of at least two popu-lations of E neurons: bulbospinal E-AUG neuronsproviding drive to expiratory motoneurons and E-DEC and plateau neurons involved in vocalization(see Section 9.2) and, perhaps, other motor activitiesrequiring co-ordination of muscles of the upper air-way with expiratory muscles of the trunk.Finally, Vanderhorst and Holstege (1995) report

extensive projections of cVRG neurons to the lum-bosacral cord in cat; these are probably involved inlordosis (Section 9.2) which requires activation ofmuscles of the hindlimbs and trunk. Consequently,projections to the cVRG need not be related to res-piration.

4. INPUTS TO ABDOMINAL MOTONEURONS

In the lumbar spinal cord, all motoneurons withrespiratory-related discharges are abdominal moto-neurons; in the mid- to lower thoracic cord, abdomi-nal motoneurons co-exist with intercostalmotoneurons, the discharges of which vary as afunction of rostral-caudal location and laterality (LeBars and Duron, 1984). Thus, to identify a moto-neuron unambiguously, one has to antidromicallyactivate the motoneuron from a branch of the nerveat its entrance to an identified muscle.

4.1. Neuroanatomical Studies

Retrograde tracers injected into the ventral hornof the spinal cord label neurons in the brainstemand more rostral sites. Such studies cannot prove aparticular functional projection, only that neurons atsite A project to (moto)neurons at site B. Sinceinjected tracers can spread to regions containingnon-respiratory neurons, finding labeled cells inregions not associated with E activity is expected.Moreover, some tracers (e.g. HRP) can be taken upby injured axons and not just by axonal terminals,so the presence of labeled neurons does not necess-arily prove that these neurons project to the site ofaxon terminals. Dierent studies, using dierent tra-cers, provide evidence for projections by variousneuronal populations to the lumbar spinal cord. Allstudies reveal labeled cells in the contralateralcVRG containing E premotor neurons. Section ofthe descending axons by a mid-sagittal lesionbetween C1 and the obex prevents uptake of HRPby neurons in the cVRG (Miller et al., 1989).Dierent studies, however, also reveal uptake of thelabel (typically HRP) by other nuclei.

In cat, Miller et al. (1989) injected HRP into sitescontaining abdominal motoneurons in the ventralhorn of L12 and observed labeled cells in theventromedial reticular formation (containing pre-motor neurons to muscles of the back, the moto-neurons of which are located in the upper lumbarcord). The contributions of reticulospinal and vesti-bulospinal neurons, both involved in postural con-trol, to the control of abdominal motoneurons have,to my knowledge, been investigated only by Miller,Yates and their colleagues (Miller et al., 1995b;Miller and Yates, 1996; Yates and Miller, 1996;Siniaia and Miller, 1996). In another study (Portilloet al., 1986), injection of fast blue into the ventrolat-eral horn of L13 retrogradely labeled cells in thecontralateral cVRG but also, and much more inten-sely, in the ipsilateral NPBM-KF. Labeled neuronswere also detected in the contralateral NPBM-KFand NA, and the ipsilateral BotC. The reasons forthe more extensive labeling in this study, comparedto the results reported by Miller et al. (1989), areunclear but may be related to the dierent tracers orspread of tracers outside the ventral horn. The label-ing in areas other than the cVRG is consistent withspinal projections, at least to the cervical spinal cord(Fedorko and Merrill, 1984), of some neurons ofBotC and the role of the NPBM-KF in strainingand possibly vocalization (Section 9.2).Injection of HRP into T89 at a site containing E

motoneurons retrogradely labels neurons primarilyin the contralateral VRG and, to a lesser degree,DRG and the ipsilateral PBN (Rikard-Bell et al.,1985a). The presence of labeled VRG neurons ipsi-lateral to the injection site can be explained bycrossing over of the terminal axons (see Kirkwood,1995) and of labeled cells in the DRG by spread ofthe injectate into regions containing I motoneurons.Injections of HRP into C2, T1 and S1 cause muchmore labeling in the contralateral than ipsilateralVRG (Holstege, 1989); nevertheless, many cells inthe contralateral VRG are not labeled, a result con-sistent with the widespread connections betweenneurons in the cVRG a