4.4 CONTRASTACIÓN DE HIPÓTESIS

Chemo RG Rec Bap « 2 Exp

Figure 1.2.2 Schematic representation of brain-stem-mediated central and peripheral chemoreceptor drives to respiratory motoneurones rhythm generator (RG), operative only in right-hand (hatched) panel. Insp and Exp bulbospinal (Bsp) and motoneurones (Mn). See text for details. Reproduced from Sears (1984).

motoneurones. As the bulbospinal inspiratory neurones are inactive during hypocapnic apnoea there would be no reciprocal inhibition of the inspiratory motoneurones, thus allowing the expiratory motoneurones fully to express the prevailing tonic expiratory bulbospinal drive. When CO2 is slowly elevated, but before the onset o f rhythm, tonic activity may occur in both inspiratory and expiratory groups in hypocapnic apnoea (Bainton et al., 1978a; Sears, Berger & Phillipson, 1982).

The above experiments were made in hyperoxic hypocapnic apnoea where the peripheral chemoreceptor input is minimal. If however, a peripheral chemoreceptor input is added (Fig. 1.2.2 centre panel), it produces a tonic inspiratory bias to the system (Sears et al., 1982). With graded hypoxia there is a graded discharge of inspiratory motoneurones and a graded reciprocal inhibition of expiratory motoneurones discharge, thus causing an ‘inspiratory shift’ in the pattern of respiratory motoneurone activation. If O2 is reduced further, rhythm is now seen as a periodic, expiratory phased inhibition of the tonic inspiratory discharge.

Sears et al. (1982) recognised three distinct effects of hypoxia. First, during normoxic normocapnia, hypoxic stimulation of peripheral chemoreceptors causes an increase in peak phrenic and external intercostal muscle activity with a reciprocal decrease in expiratory activity; a similar pattern occurs with deepening anaesthesia. Second, during normoxic hypocapnic apnoea, hypoxic stimulation of the peripheral chemoreceptors increases the tonic discharge of phrenic and external intercostal motoneurones, and there is a graded reciprocal inhibition o f the tonic expiratory discharge. Thus the ‘inspiratory shift’ is expressed independently o f rhythm generation. This state, when there are balanced drives (peripheral and central), giving co-activation of inspiratory and expiratory neurones, is represented schematically in Fig. 1.2.2 (centre panel). If either the PaC02 was increased, or hypoxia added, respiratory rhythm started. The third effect of hypoxia is a dynamic version of the second with the pump switched off.

The right hand panel of Fig. 1.2.2 illustrates the onset of rhythm generation following a further increase in either the hypoxic or peripheral chemoreceptor drive and this is seen to be sculpted from inhibition of the underlying tonic expiratory activity (Sears et al., 1982; Berger, Phillipson & Sears, 1982). If the animal is ‘inspiratory biased’ then increasing hypoxia or hypercapnia stimulation leads to the onset o f rhythm due to periodic expiratory phased inhibition of the tonic inspiratory motoneurone discharge.

concepts and experimental results do not exclude the mechanisms of previous models, but help to enhance their understanding. Conceptually the condition of apnoea is often regarded as being the absence of respiration, both neurally and mechanically. However, it is not possible to ignore the relevance of the these results when the same neurones are involved in both ‘states’. Thus, in terms of chemical drive the apnoea is the respiratory ‘state’ in the absence o f rhythm generation.

Clearly these two major states of respiration are not equivalent. At high levels of CO2, and with the addition of peripheral chemoreceptor inputs the inspiratory neurones provide an overriding drive for oscillation. When CO2 is lowered the expiratory part of the drive is revealed. The marked asymmetry of such an arrangement would guarantee respiratory rhythm generation in all but the most extreme circumstances.

Narcotics such as morphine are known to depress the ventilatory response to CO2 (see later), Howard & Sears (1991) have recently shown in the rabbit that morphine elevates the CO2 threshold for rhythm generation without significantly depressing the inspiratory or expiratory response to increasing F^C02, either in the rhythmic or tonic state. Morphine had no effect on the threshold of the recruitment of fusimotor or a-motoneurones, which with increasing F ^ C0 2 showed a continuous recruitment up to and beyond the pre-morphine CO2 threshold. The primary effect on ventilation was a prolongation of the expiratory duration (Te). Morphine did not significantly alter the amplitude of either the inspiratory output, thus morphine decoupled the mechanisms through which the chemical drives gained access to the motoneurones fi*om those concerned in the process of rhythm generation, further supporting the idea that the ventilatory drive (central and peripheral chemoreceptors) has a relatively direct access to the bulbospinal neurones as opposed to the Euler model in which the central and peripheral chemoreceptor drives can only drive the bulbospinal neurones through the central pattern generator mechanism.

Therefore, morphine can be used in a similar way to the saggital lesions for studying the tonic drives of hypocapnic apnoea over an extended range.

1.3 A utonom ic regulation o f the heart

The neural control of the heart is mediated by both parasympathetic and sympathetic innervation which together control the heart rate and contractile force to meet the current homeostatic needs. Sympathetic changes take longer to affect the heart and last longer, whereas the vagus can alter the heart period within one beat (Warner & Cox, 1962), and

hence contribute principally to respiratory sinus arrhythmia (RSA), which is the acceleration of the heart during inspiration and the slowing of the heart during expiration. RSA is almost completely abolished by atropine or vagotomy, and the cardiomotor fibres responsible have their origin in two possible areas of the brainstem, the dorsal motor vagal nucleus (DMV) and the nucleus ambiguus (nA) (see Loewy & Spyer, 1990 for review).

1.3.1 Anatom y

Dorsal motor nucleus o f the vagus (DMV). The DMV is located dorso-medially in the caudal medulla near the fourth ventricle. In the cat it comprises a 10- 15mm column of cells extending rostrally from the first cervical spinal segment lying dorsal lateral to the central canal (Kalia, 1981b). The DMV is composed o f two types o f neurone, 80% medium sized neurones which are nearly all vagal motoneurones, and 15-20% small cells which either project to other levels of the brainstem, or are intrinsic neurones terminating within the DMV. The DMV projects to the heart, lungs, bronchi, pharynx, stomach and intestines (Kalia & Mesulam, 1980b; Kalia, 1981b and Hopkins, 1987 for reviews). Cells innervating the supradiaphragmatic structures lie within the lateral third of the nucleus whereas the medial two thirds innervate subdiaphragmatic structures, but with extensive overlapping (Kalia,

1981b).

Cardiac Innervation. The role of the DMV in the control of the heart remains unclear and controversial (see Loewy & Spyer, 1990 for review). Early experiments in the dog reported that electrical stimulation of the DMV elicited a bradycardia (Miller & Bowman, 1916) and confirmed later (Gunn, Sevelius, Puiggari & Myers, 1968; Weiss & Priola, 1972); and for the rabbit (Ellenberger, Haselton, Liskowsky & Schneiderman, 1983); and rat (Nosaka, Yamamoto & Yasunaga, 1979); but not the cat (Calaresu & Pearce, 1965; Geiss & Wurster, 1980b; Gunn et al., 1968). However, DMV stimulation in the cat, decreased the strength (dP/dt^aJ of ventricular contraction (Geis, Kozelka & Wurster, 1981). And in both cat and dog stimulation of the DMV and nA showed a decrease in ventricular contraction, but only nA stimulation slowed the heart; cardiac pacing during nA stimulation removed the inotropic effect indicating it was secondary to the bradycardia. In neither group of experiments was the heart paced during DMV stimulation, therefore, a pure inotropic effect of DMV stimulation has not been confirmed. But clouding all of these results is the problem of the inevitable current spread to adjacent sites, especially in the NTS.

sparse axonal degeneration of the cardiac branches of the vagus (Calaresu & Cottle, 1965), whereas injections of HRP into the right myocardium show marked labelling of the nA, and

a sparse labelling of the DMV and the intermediate zone (rat: Stuesse, 1982; cat: Geiss & Wurster, 1980a; Kalia & Musalam, 1990).

Direct application of HRP to the cardiac branches of the vagus shows a clear labelling of the nA and DMV (rat: Nosaka et al., 1979; dog: Bennett, Kidd, Latif & McWilliam, 1981; Hopkins & Armour, 1982; cat: Sugimoto, Itoh, Mizuno, Nomura & Konishi, 1979; Bennett et al., 1981; Jordan, Spyer, Withington- Wray & Wood, 1986). Bennett et al. (1981) found approximately equal distribution of labelling in nA and DMV from both pulmonary and cardiac nerves in the cat, in contrast the dog showed much more labelling in the n A than the DMV, and in the rat also (Nosaka et al., 1979). Injections into the myocardium around the sino atrial node are fraught with difficulty both in accuracy o f placement, and restricting its spread to other structures. Similarly, the use of the cardiac nerve in the cat creates problems because proximally it contains a pulmonary branch (McAllen & Spyer, 1976).

Antidromic Activation. Antidromic mapping techniques have also been used to locate the origin of the cardiac preganglionic neurones. McAllen & Spyer (1976) in the cat found that neurones in the nA could be antidromically activated by electrical stimulation of the right cardiac vagal branches, whose axons had conduction velocities in the B-fibre range (3-15 ms’'). When neurones were sampled in the DMV most were found to send axons down the thoracic vagus below the cardiac branches. Only 3/33 could be antidromically activated with high intensity stimulation, but it was though that these were unlikely to be cardio- inhibitory based upon their thresholds and conduction velocities. In contrast, Cirello & Calaresu (1980, 1982) have antidromically activated neurones in both the nA and DMV regions with latencies corresponding to B-fibre velocities. Bennett, Ford, Kidd & McWilliam (1984a) have also identified neurones in the DMV with non-myelinated axons in the cardiac and pulmonary branches. Only a small number showed spontaneous activity, the rest being silent. Spontaneous and DL-homocysteic acid (DLH) activated neurones showed no relation to the cardiac cycle, increased BP or lung inflation. In an extensive investigation in the cat, 94 neurones in the DMV have been analyzed by antidromic stimulation, for their cardiac (55/94) and pulmonary (39/94) projections (Ford, Bennett, Kidd & McWilliam, 1990). All but a few had C-fibre conduction velocities and were assumed to be non-myelinated fibres. Extracellular recording showed little spontaneous activity, and only one neurone responded to an increase in carotid sinus pressure. lonotophoretic application of DLH to cardiac

projecting neurones had no effect on the HR (cf. with nA, McAllen & Spyer, 1978a). It

would appear the cat cardiac vagus has myelinated and non-myelinated fibres and there is evidence the DMV gives rise to non-myelinated fibres to the cardiac and pulmonary branches. However, stimulation in the DMV has failed to show convincingly a cardiac chronotropic effect in the cat (see above). The lack of response to DLH and carotid sinus inputs contribute to the conclusion that in the cat the DMV has no part in the chronotropic control o f the heart.

In the rabbit the situation is a little clearer. Antidromically activated neurones have been identified in both the DMV and nA by stimulation of the right cervical vagus (Jordan, Khalid, Schneiderman & Spyer, 1982) or right cardiac nerve (Ellenberger et al., 1983). Axons with B-fibre conduction velocities gave a bradycardia upon stimulation. DLH activation showed an increased firing and concomitant fall in heart rate for both DMV and

nA groups of cells (Jordan et al., 1982). Slower conducting fibres were also identified

consistent with non-myelinated fibres, but these were only found in the DMV.

Nucleus Ambiguus (N À ). The nA justifies its name for confusion and ambiguity. It lies

in the ventrolateral part of the medullary reticular formation, extending from the C l level to the level o f the facial nucleus, i.e., about 8mm in the cat (Kalia, 1981b), or 4 - 5mm in the rabbit (Lawn, 1966b). However, the delineation of the borders of this group of nuclei is still unclear.

In Lawn’s classic study (Lawn, 1966a,b) of the rabbit he describes the nA as being formed from a rostral compact formation and a caudal diffuse formation. The rostral compact division can then be subdivided into a lateral ‘principal’ column and a ‘medial’ column. More recent studies in the rat (Bieger & Hopkins, 1987) using HRP labelling have broadly agreed with Lawn’s description. More recently the terms Nucleus Retrofacialis and Nucleus Retroambiguualis have been used to describe the divisions of the nA (see Kalia & Mesulam,

1980a,b).

Retrograde labelling by intracardiac injections of HRP shows a clear column of labelled cells in the nA (Kalia, 1981b). More extensive labelling is seen when HRP is applied to the cut end of the cardiac vagus; Dog: Bennett et al., 1981; Hopkins & Armour, 1982; Rat: Nosaka et al., 1979; and cat: Bennett et al., 1981, Ciriello & Calaresu, 1982; Jordan et al.,

1986). The extent of labelling in the cat is from below the level of the obex (perhaps as much as 2mm, Bennett et al., 1981) to 5mm rostral (Kalia, 1981b; Bennett, et al., 1981). The maximum density of labelled cells tends to be clustered at about 2mm rostral to the obex (cf.

McAllen & Spyer, 1976 (marked neurones); Bennett et a l, 1981; Ciriello & Calresu, 1982); Jordan et al., 1986).

The overall numbers of cells found in the nA following HRP labelling of the cardiac branches o f the cat is variable (range 10 to 341, Bennett et al., 1981) and is approximately the same as in the DMV. Whereas, in the dog a much higher proportion of cardiac efferents have their cell bodies in the nA (Bennett et al., (1981). A small proportion o f HRP labelled

cells are found in the reticular formation between the DMV and nA.

Microstimulation in the nA consistently produces a marked bradycardia in the cat: (Geiss & Wurster, 1980b; Gunn et al., 1968) and dog: (Laubie, Schmitt & Vincent, 1979; Geiss et al., 1981). However, the validity of this technique in such a density populated area, close to the ventrolateral cell groups has been questioned (Loewy & Spyer, 1990). On the other hand, antidromic activation of medullary units is more appropriate in identifying electrophysiologically the location of the cardiac preganglionic neurones (cat: McAllen & Spyer, 1976,1978a; Ciriello & Calaresu, 1982; rabbit: Jordan et al., 1982).

1.3.2 Term ination o f Sinus nerve inputs

The termination of the chemoreceptor and baroreceptor afferent fibres entering the brainstem via the IX and X nerves and terminating in the NTS and its subnuclei has been extensively studied by anatomical tracing and antidromic techniques (see Jordan & Spyer,

1986; Spyer, 1990 for reviews).

Both baroreceptor and chemoreceptor nerves contain a mixture of relatively small diameter non-myelinated and myelinated fibres (Fidone & Sato, 1969), therefore it is difficult to selectively stimulate them electrically, although attempts have been made (De Groat & Lalley, 1974).

Baroreceptor Terminations. The central projections o f the myelinated and non-myelinated fibres of carotid sinus are similar, being restricted to the ipsilateral NTS, most often in its lateral divisions rostral to the obex, and most densely in the dorsolateral and dorsomedial subdivisions, with some innervation of the ventrolateral and commissural NTS (Donoghue, Felder, Jordan & Spyer, 1984; Claps & Torrealba, 1988). Their projection patterns are identical to those of aortic bodies in the cat (Donoghue, Garcia, Jordan & Spyer, 1982) with the exception that whilst myelinated aortic baroreceptors are shown to project bilaterally to include the contralateral commissural nuclei of the NTS, no such projection is seen for myelinated or non-myelinated fibres from the carotid sinus (Donoghue et al., 1984).

In recordings from nTS neurones sinus nerve stimulation evokes responses vvith a

latency compatible with a monosynaptic pathway in only 17% of neurones tested, additional polysynaptic connections have been shovm to the parahypoglossal area and the area o f the

nA (Lipski, McAllen & Spyer, 1975).

Chemoreceptor Terminations. Whilst all baroreceptor afferents appear to show a preference for the lateral aspect of the NTS, although not the ventral and ventrolateral portions, the chemoreceptors showed more extensive terminations in medial regions of the NTS, in contralateral as well as the commissural NTS. Donoghue et al. (1984) point out that even though the sample size was small (12), the results strongly suggest that the two receptor groups innervate distinctly different populations of NTS neurones. All the axons studied had conduction velocities attributed to non-myelinated fibres. Fidone & Sato (1969) have shovm that 66% of the fibres in the sinus nerve distal to the petrosal ganglion are non-myelinated, but the majority of the myelinated fibres had a chemoreceptor function. Therefore, the distribution so far might be a limited picture of the true distribution in the NTS (Spyer, 1990).

It is well knovm that stimulation of the peripheral chemoreceptors has marked short latency excitatory effects on inspiration when timed to occur in the inspiratory phase ( Black & Torrance, 1967,1971; Band, Cameron & Semple, 1970; Eldridge, 1976; Lipski, McAllen & Spyer, 1977). When the stimuli occur in expiration, expiratory activity maybe slightly enhanced, although the response had a much longer latency than the inspiratory response, which suggests a different pathway (Eldridge, 1976).

Intracellular recordings from inspiratory neurones in the VRG have failed to show any subthreshold change in membrane potential when chemoreceptors were stimulated (Mitchell & Herbert, 1974). Therefore, the site of action is in the NTS region which contains the only other group (DRG) of inspiratory neurones. However, there is still some debate as to how the chemoreceptor afferents influence the respiratory activity.

Investigations in the DRG have shown that both categories o f inspiratory neurones, R„ and Rp are excited by chemoreceptor stimuli delivered to the carotid body during inspiration, but not during expiration (Lipski et al., 1977). It is not knovm whether the inspiratory neurones receive an input throughout the respiratory cycle, which becomes subliminal during expiration, or, they only receive an input during inspiration. Lipski et al. (1977) showed that when the R„ and Rp neurones were made to fire tonically during expiration by the ionotophoretic application of DLH, chemoreceptor stimuli were capable of inhibiting this activity. This and other evidence led to the conclusion (Lipski et al., 1977) that

the inspiratory neurones in the DRG do not receive a direct chemoreceptor input, but that the response to chemoreceptor stimulation is due to an enhanced inspiratory drive.

While Lipski et al. (1977) were unable to show a direct excitatory action, Kirkwood, Nisimaru & Sears (1979) have used cross-correlation analysis to demonstrate a monosynaptic excitation of identified inspiratory bulbospinal neurones by single chemoreceptor afferents recorded in the petrosal ganglion. Several other studies have also shown a direct input to neurones in a similar region of the NTS, although these were not respiratory neurones (Lipski, McAllen & Trzebski, 1976; Izzo, Lin, Richter & Spyer, 1988; Mifflin, Spyer & Withington-Wray, 1988a). The chemoreceptor input to these putative intemeurones is not gated as no difference is seen in their excitatory responses during inspiration and expiration.

Intracellular recordings from inspiratory neurones in the DRG (Lipski & Voss, 1990) have been used to demonstrate that peripheral chemoreceptor activation causes excitation during inspiration, and inhibition during expiration, the latter is believed to be due to expiratory neurones in the Botzinger complex. These neurones are known to receive a short latency excitation to chemoreceptor stimulation (Lipski, Trzebski, Chodobska & Kruk, 1984). Anatomical (Kalia et al., 1979; Smith, Morrison, Ellenberger, Otto & Feldman, 1989) and electrophysiological (Kubin & Lipski, 1980; Lipski & Merrill, 1980) studies have demonstrated axonal projections from the Botzinger group to the DRG. Also, intracellular recording and spike-triggered averaging has shown there are mono-synaptic inhibitory connections between the Botzinger neurones and inspiratory neurones in the DRG (Kubin & Lipski, 1980; Merrill, Lipski, Kubin & Fedorko, 1983).