Reciprocal connections between rostral ventrolateral medulla and inspiration-related medullary areas in the cat

Brain Research, 565 (1991) 171-174 (~) 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 000689939124936G

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BRES 24936

Reciprocal connections between rostral ventrolateral medulla and inspiration-related medullary areas in the cat F u l v i a B o n g i a n n i 1, M a r i e C o r d a 1, G i o v a n n i A . F o n t a n a 2 a n d T i t o P a n t a l e o 1 SDipartimento di Scienze Fisiologiche and 2Unita' di Fisiopatologia Respiratoria, Univers';a' di Firenze, Florence (Italy) (Accepted 20 August 1991)

Key words: Respiration; Rostral ventrolaterai medulla; Nucleus paragigantocellularis lateralis; Dorsal respiratory group; Ventral respiratory group; Nucleus of the solitary tract; Control of breathing

We investigated co,nections between the rostral ventrolateral medulla (rVLM) and the two main inspiration-related medullary areas, i.e., the dorsal respiratory group (DRG) and the rostral ventral respiratory group (rVRG) in the cat. Non respiration-related tonically firing units encountered in the rVLM displayed either antidromic or orthodromic responses to DRG or rVRG microstimulation. Some units responded to the stimulation of both regions. We suggest that at least part of rVLM neurons are components of medullary loops operating in the control of breathing.

Several studies9 hint at the possibility that structures located in the rostral ventrolateral medulla (rVLM), corresponding to a large extent to the nucleus paragigantocellularis lateralis (nPGL) 1'2~, might play an important role in the respiratory rhythmogenesis. Budzinska et ~1.6 have shown that deep depression of inspiratory activity or apnoea can be induced by unilateral focal cold blocks in a region of the rVLM situated immediately beneath the ventral medullary surface, These results have led to the hypothesis6'9 that this 'apnoea region', corresponding partly to the nPGL and close neiRhbouring areas (lying between the retrofacial or facial nucleus and the ventral surface of the medulla), may have a function in the integration and mediation of the various chemical and non chemical ventilatory 'drive' inputs of central and peripheral origin. Accordingly, it has recently been reported that neurons in this region display non respiration-related tonic discharge patterns and receive converging afferent inputs from central and peripheral chemoreceptors and/or from somatosensory afferents 3'~7. Furthermore, neuroanatomical investigations7'2° have demonstrated that neurons in the nPGL and in a region proximal to the ventral surface of the rostral medulla at the level of the rostral retrofacial and facial nucleus (referred to as retrotrapezoid nucleus) have prevailing ipsilateral projections to the two main respiration-related neuronal aggregates in the medulla, i.e., the dorsal (DRG) and ventral (VRG) respiratory group9. The present study was undertaken in an attempt to

provide electrophysiological evidence of connections between neurons in the rVLM and the two main inspiration-related (IR) medullary areas corresponding to the DRG and rostral VRG (rVRG) by means of antidromic invasion techniques 1~. Experiments were performed on 16 cats (2.2-3.4 kg) anaesthetized with sodium pentobarbitone (Nembutal, Abbott, 35 mg/kg i.p.', supplemented when necessary), vagotomized, paralyzed (gallamine triethiodide, Sigma, 5 mg/kg/h iv.), and artificially ventilated. The animal was placed in a stereotaxic frame and the medulla was widely exposed by occipital craniotomy and gentle aspiration of the posterior part of the cerebellum. The efferent phrenic activity was recorded using bipolar platinum electrodes from desheathed C5 phrenic roots, amplified, full-wave rectified and 'integrated' (low-pass filter, time constant 100 ms). Extracellular neuronal activity was recorded with t,ngsten microelectrodes (3-5 Mfl impedance, as tested at 1 kHz) which were also used for monopolar cathodal microstimulation (0.05-0.1 ms, 1-60 #A rectangular pulses, 0.5 Hz). Recordings were made from IR neurons of the ve,trolateral aspect of the nucleus of the solitary tract (nTS), i.e., the DRG (0.5-2.0 mm rostral to the obex, ?.0-2.5 mm lateral to the midline, and 1.5-2.0 mm below the dorsal surface of the medulla), from IR neurons of the rVRG (0-3.0 mm rostral to the obex, 2.8-3.5 mm lateral to the midline, and 3.5-5.0 mm below the dorsal medullary surface) and from neurons in an area of the rVLM approx-

Correspondence: T. Pantaleo, Dipartimento di Scienze Fisiologiche, Universita' di Firenze, Viale G.B. Morgagni 63, 50134 Firenze, Italy.

172 imately corresponding to the 'apnoea region' of Budzinska et al. 6 (2.5-7.0 mm rostral to the obex, 2.54.5 mm lateral to the midline and 6-7.5 mm deep from the dorsal medullary surface, i.e., below the VRG and close to the ventral surface of the medulla. Neuronal activity was processed in the same way as phrenic activity; it was also monitored as 'raw' signals on an oscilloscope and recorded on magnetic tape for subsequent analysis. Recorded signals were displayed on a storage oscilloscope and photographed. The 'integrated' signals of nerve and neuronal activities, as well as the signals of arterial blood pressure, tracheal pressure, tidal volume and end-tidal CO2 were continously monitored on a multichannei pen recorder. End-tidal CO2 was kept at desired levels (4-6%) by allowing the animal to inspire appropriate CO2 concentrations in oxygen. Rectal temperature was maintained at 37 - 0.5 °C by external heating. The histological control of recording sites was performed on serial frozen sections (50/~m thickness) according to the atlas of Berman 4. Details about the methods used have previously been reported 5't9. The results are expressed as ranges and/or means ± S.E.M. A systematic search for units responding to stimulation of the ipsilateral DRG and rVRG, in sites where strong multiunit IR activity was previously recorded, was made in the rVLM with a series of tracks (intervals of about 400 l~m). In a few experiments (n -- 4) contralateral connections were also investigated. The antidromic character of evoked spikes was t'outinely assessed by standard criteria tt't~ in particular, (a) the short and constant latency, (b) the ability of neurons to respond to high-rate (200-300 Hz) stimulation and (c) the collision test. A total of 320 units with non respiration-related tonic

discharge patterns were sampled in the rVLM. Part of them (n = 119) responded to ipsilateral DRG or rVRG stimulation. Antidromic action potentials were evoked in 60 units. Antidromic responses could be confirmed with collision tests in the majority of these units (n = 42). The threshold current for the antidromic activation varied in different rVLM units from 1 to 50 gA, being frequently (n = 28) less than 15/~A. Fourty-six of these neurons responded to DRG stimulation with latencies ranging from 0.5 to 2.7 ms (1.19 -- 0.08), while 14 responded to rVRG stimulation with latencies ranging from 0.5 to 2.2 ms (1.04 -- 0.15). If we take approximate interelectrode distances into account antidromic latencies give conduction velocities varying from 2.2 to 8.8 ntis (4.95 ± 0.26) and from 1.8 to 9.2 m/s (5.22 -- 0.64) for DRG and rVRG stimulation, respectively. Antidromic mapping techniques 1°'I1 were also used, even if not systematically, in an attempt to define the area of terminal arborization of projecting neurons. We observed two or more different minimal threshold currents (~< 3 gA) and different antidromic latency values (variation range 0.2-1.6 ms) for responses evoked in the same unit from different location or by using different stimulation intensities in a given location both in the DRG (6 out of 9 units tested) and in the rVRG (3 out of 5 units tested). These observations indicated branched axons in these IR medullary areas, The remaining 59 responding neurons were orthodromically activated by DRG (n ~ 43) and rVRG (n ~ 16) stimulation. Threshold current varied from 5 to 50 l~A, but was in most cases (n ~ 32) in the range of 5-15 l~A. The evoked responses consisted either of a single spike or a burst of 2-4 spikes; latencies of re;r, onse ranged from 1.2 to 9.5 ms (3.86 ± 0.38) and fro~, 1.5 to 8.5 ms

C

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Fig. 1. Representative antidromic and orthodromic responses of neurons in the rostral ventrolateral medulla (rVLM) to ipsilateral dorsal rcspit'atory group ¢DRG) stimulation. A: 20 superimposed tracings of antidromic responses of a single unit. B: orthodromic responses of a single unit consisting of a burst of 2-3 action potentials (5 superimposed sweeps). In A and B stimulus artefact at the beginning of each tracing. C and D: co)lision test for the same unit showed in A. Spontaneous spikes were used to trigger the oscilloscope and also a stimulator after an adjustable delay. The collision between the spontaneous and the antidromicaUy evoked spike occurred (in D) when the delay of the stimulus was reduced by 0.2 ms. i.e.. below the measured critical delay. Stimuli are indicated by arrows.

173 (4.02 +- 0.54), respectively. Examples of antidromic and orthodromic responses are illustrated in Fig. 1. Interestingly, 3 out of 7 antidromically activated units and 2 out of 10 transsynapticaily evoked neurons proved to respond to both DRG and rVRG stimulation (Fig. 2). We also encountered 26 neurons which responded to contralateral D R G or r V R G stimulation. Part of them (n = 17) displayed antidromic responses to D R G (n = 8) or rVRG (n = 9) stimulation; latencies varied from 0.7 to 1.8 ms (1.28 - 0.23) and from 0.8 to 2.5 ms (1.51 - 0.17), respectively. Calculated conduction velocities were within the same range reported above for ipsilateral rVLM projections. The remaining 9 units were orthodromically evoked by contralateral D R G (n - 4) and rVRG (n = 5) stimulation: latencies varied from 1.4 to 10 ms (4.42 +- 1.91) and from 1.2 to 9.5 (4.72 -+ 1.35), respectively. The general characteristics of both antiand orthodromic responses were similar t~, those described for ipsilateral D R G or rVRG s~hnulation. The anatomical distribution of antidromically and orthodromically activated neurons in the rVLM is shown in Fig. 3. These results provide the first electrophysiological evidence of axonal projections from tonic neurons in the rVLM to the two main aggregates of IR medullary neurons, thus supporting earlier neuroanatomical findings 7' a0. Although the terminal arborization of the axons of tonic rVLM neurons was not extensively studied, present data strongly suggest that at least part of these neurons terminate upon, or in close vicinity of, the IR neurons within the two medullary respiratory groups. Another

A

indication provided by this study is that reciprocal connections may exist between the rVLM and both the DRG and rVRG. In particular, tonic neurons in the rVLM not only may send projections to, but also may receive converging inputs from both these medullary respiration-related areas (Fig. 2). Since functional 3'14a~ and neuroanatomical 2'9'1~''~3 evidence indicates that the rVLM might function as an integrating area, the finding of rVLM projections to the D R G and rVRG is in keeping with previous results obtained with focal cold blocks 6 or microinjections of excitatory amino acids TM in the rVLM. Furthermore, it corroborates the hypothesis that this region plays an important role in the respiratory rhythmogenesis or in the integration and transmission of ventilatory 'drive' inputs of central and peripheral origin 6'9. However, in the light of the results obtained by McAllen ~s and, more recently, by our group (unpublished observations) by

DRG stimulation

VRG stimulation

• Antidromic units Orthodromic units

• Antidromic units OOrthodromic units

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D

P'IO .~4

I.__.',.I.ILI_LILL ...... lY Iv I-

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/

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10 ms

Fig. 2. Responses of neurons in the rVLM to ipsilateral DRG and rVRG stimulation. A and B: antidromic potentials evoked in a sinRle unit by the stimulation of the DRG and rVRG, respectively. Each trace represents 10 superimposed responses. C and D: orthodromic single unit responses (5 superimposed sweeps) to DRG and rVRG stimulation, resp~.ctively. Traces triggered at the onset of the stimulation.

Fig. 3. Series of representative transverse sections of the rVLM showing prevailing location of units antidromically or orthodromically activated by DRG or rVRO stimulation. Horsley.Clarke frontal planes and the approximate distances in mm from the obex are indicated on the left. The outlines of the maps are from Berman's atlas4. Some relevant structures are schematicallyrepresented: FTL, lateral tegmental field; FTM, magnocellular tegmental field; IO, inferior olive; P, pyramidal tract; RFN, retrofacial nucleus; 7, facial nucleus.

174 means of chemical stimulation of neurons in the rVLM, this area seems to be far from homogeneous; rather, it displays both excitatory and inhibitory influences on respiratory activity as well as cardiovascular functions 16. O n the other hand, present results do not allow any interpretation of the excitatory or inhibitory nature of r V L M projections. The presence of bidirectional connections between the rVLM and the two main IR medullary areas suggests that at least part of neurons located in the r V L M may be elements of medullary loops involved in the control of respiratory activity, Likewise, a medullary cardiovascular loop provided by slower conducting axons and

comprising the rVLM and the medial aspect of the nTS has recently been described s. Thus, it seems conceivable that, at least to some extent, segregate circuits operate in the control of respiratory and cardiovascular functions at the m.~dullary level. Nevertheless, the main components of these medullary loops, su,:h as the nTS and in particular the rVLM 16, might be important cardiorespiratory integration sites.

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4 (1981) 1-32. 12 Loewy, A.D. and Burton, H., Nuclei of the solitary tract: efferent projections to the lower brain stem and spinal cord of the cat, J. Comp. NeuroL, 181 (1978) 421-449. 13 Lovick, T.A., Projections from brainstem nuclei to the nucleus paragigantocellularis lateralis in the cat, J. Auton. Nerv. Syst., 16 (1986) 1-11. 14 Lovick, T.A., Convergent afferent inputs to neurones in nucleus paragigantocellularis lateralis in the cat, Brain Research, 456 (1988) 183-187. 15 McAIlen, R,M., Location of neurones with cardiovascular and respiratory actions, at the ventral surface of the cat's medulla, Neuroscience, 18 (1986) 43-49. 16 Millhorn, D.E. and Eldridge, EL., Role of ventrolateral medulla in regulation of respiratory and cardiovascular systems, J. Appl. Physiol., 61 (1986) 1249-1263. 17 Mitra, J., Prabhakar, N., Pantaleo, T., Yamamoto, Y., Runold, M., Lunteren, E. van, Euler, C. yon and Cherniack, N.S., Do structures in the region of nucleus paragigantocellularis (nPO) integrate and mediate ventllatory drive inputs?, Soc. Neuroscl. Abstr., 12 (1986) 304. 18 Pantaleo, T,, Bongianni, F,, Corda, M, and Fontana, G., A search for a respiratory role of neurons located in the region of the nucleus paragig,,,~,~,,eii,laris in the cat, Proceedings of XXX! International Congress of Physiological Sciences, Helsinki 1989, P 2336. 19 Pantaleo, T. and Corda, M., Respiration-related neurons in the medial nuclear complex of the cat, Respir. Physiol., 64 (1986) 135-148. 20 Smith, J.C., Morrison, D.E., Ellenberger, H.H., Otto, M.R. and Feldman, J.L., Brainstem projections to the major respiratory neuron populations in the medulla of the cat, J. Comp. Neurol., 281 (1989)69-96. 21 Taber, E., The cytoarchiteeture of the brain stem of the cat. I. Brain stem nuclei of cat, J. Comp. Neurol., 116 (1961) 27-70.

We wish to thank S. Cammarata and M. Dolfi for technical assistance and A. Vannucchi for the preparation of the illustrations. This work was supported by grants from the Ministero dell' Universita' • della Ricerca Scientifica e Tecnologica of Italy. F.B. was supported by an I.N.R.C.A. fellowship.