Phrenic motor neuron loss in an animal model of early onset hypertonia.

Phrenic motor neuron (PhMN) development in early onset hypertonia is poorly understood. Respiratory disorders are one of the leading causes of morbidity and mortality in individuals with early onset hypertonia, such as cerebral palsy (CP), but they are largely overshadowed by a focus on physical function in this condition. Furthermore, while the brain is the focus of CP research, motor neurons, via the motor unit and neurotransmitter signaling, are the targets in clinical interventions for hypertonia. Furthermore, critical periods of spinal cord and motor unit development also coincide with the timing that the supposed brain injury occurs in CP. Using an animal model of early-onset spasticity (spa mouse [B6.Cg-Glrbspa/J] with a glycine receptor mutation), we hypothesized that removal of effective glycinergic neurotransmitter inputs to PhMNs during development will result in fewer PhMNs and reduced PhMN somal size at maturity. Adult spa (Glrb-/-), and wild-type (Glrb+/+) mice underwent unilateral retrograde labeling of PhMNs via phrenic nerve dip in tetramethylrhodamine. After three days, mice were euthanized, perfused with 4% paraformaldehyde, and the spinal cord excised and processed for confocal imaging. Spa mice had ~30% fewer PhMNs (P = 0.005), disproportionately affecting larger PhMNs. Additionally, a ~22% reduction in PhMN somal surface area (P = 0.019), an 18% increase in primary dendrites (P < 0.0001), and 24% decrease in dendritic surface area (P = 0.014) were observed. Thus, there are fewer larger PhMNs in spa mice. Fewer and smaller PhMNs may contribute to impaired diaphragm neuromotor control and contribute to respiratory morbidity and mortality in conditions of early onset hypertonia.NEW & NOTEWORTHY Phrenic motor neuron (PhMN) development in early-onset hypertonia is poorly understood. Yet, respiratory disorders are a common cause of morbidity and mortality. In spa mice, an animal model of early-onset hypertonia, we found ~30% fewer PhMNs, compared with controls. This PhMN loss disproportionately affected larger PhMNs. Thus, the number and heterogeneity of the PhMN pool are decreased in spa mice, likely contributing to the hypertonia, impaired neuromotor control, and respiratory disorders.

Diaphragm neuromuscular transmission failure in aged rats.

In aging Fischer 344 rats, phrenic motor neuron loss, neuromuscular junction abnormalities, and diaphragm muscle (DIAm) sarcopenia are present by 24 mo of age, with larger fast-twitch fatigue-intermediate (type FInt) and fast-twitch fatigable (type FF) motor units particularly vulnerable. We hypothesize that in old rats, DIAm neuromuscular transmission deficits are specific to type FInt and/or FF units. In phrenic nerve/DIAm preparations from rats at 6 and 24 mo of age, the phrenic nerve was supramaximally stimulated at 10, 40, or 75 Hz. Every 15 s, the DIAm was directly stimulated, and the difference in forces evoked by nerve and muscle stimulation was used to estimate neuromuscular transmission failure. Neuromuscular transmission failure in the DIAm was observed at each stimulation frequency. In the initial stimulus trains, the forces evoked by phrenic nerve stimulation at 40 and 75 Hz were significantly less than those evoked by direct muscle stimulation, and this difference was markedly greater in 24-mo-old rats. During repetitive nerve stimulation, neuromuscular transmission failure at 40 and 75 Hz worsened to a greater extent in 24-mo-old rats compared with younger animals. Because type IIx and/or IIb DIAm fibers (type FInt and/or FF motor units) display greater susceptibility to neuromuscular transmission failure at higher frequencies of stimulation, these data suggest that the age-related loss of larger phrenic motor neurons impacts nerve conduction to muscle at higher frequencies and may contribute to DIAm sarcopenia in old rats. NEW & NOTEWORTHY Diaphragm muscle (DIAm) sarcopenia, phrenic motor neuron loss, and perturbations of neuromuscular junctions (NMJs) are well described in aged rodents and selectively affect FInt and FF motor units. Less attention has been paid to the motor unit-specific aspects of nerve-muscle conduction. In old rats, increased neuromuscular transmission failure occurred at stimulation frequencies where FInt and FF motor units exhibit conduction failures, along with decreased apposition of pre- and postsynaptic domains of DIAm NMJs of these units.

Differential age-, gender-, and side-dependency of vagus, spinal accessory, and phrenic nerve calibers detected with precise ultrasonography measures.

INTRODUCTION: The standard ultrasonographic measurement tools (trace, ellipse) of cross-sectional areas (CSAs) of very small nerves typically yield rough measures in full square millimeters. METHODS: In 70 volunteers, the elliptically shaped CSAs of mid-cervical vagus, accessory, and phrenic nerves were estimated with three methods: 2 on-board tools (area tracing, ellipse fitting) and an off-line calculation of the CSA after on-board measuring of its long-axis and short-axis diameters both displayed with 1-2 digits following the decimal point. RESULTS: CSA measures of all mid-cervical nerves obtained with the precise approach were smaller than the two standard measures (each P < 0.001). Larger CSA of right compared to left vagus nerve was detected with all methods. However, decrease of accessory and phrenic nerve CSAs with increasing age and larger size of vagus nerve CSA in women vs. men were evident only with precise measures. DISCUSSION: Small nerve CSA should preferably be estimated with precise measures. Muscle Nerve 59:486-491, 2019.

Anatomical changes of phrenic motoneurons during development.

Although the phrenic motoneurons are relatively well-developed at the time of birth as compared to non-respiratory motoneurons, they show distinct anatomical changes during postnatal development. In the present review we summarize anatomical changes of phrenic motoneurons during pre- and postnatal development. Cell bodies of phrenic motoneurons migrate into the ventromedial region of the ventral horn of C3-C6 by E13-E14 in the rat. During development the sizes and surface areas of phrenic motoneurons are increased with changes in dendritic morphology.

Key aspects of phrenic motoneuron and diaphragm muscle development during the perinatal period.

At the time of birth, respiratory muscles must be activated to sustain ventilation. The perinatal development of respiratory motor units (comprising an individual motoneuron and the muscle fibers it innervates) shows remarkable features that enable mammals to transition from in utero conditions to the air environment in which the remainder of their life will occur. In addition, significant postnatal maturation is necessary to provide for the range of motor behaviors necessary during breathing, swallowing, and speech. As the main inspiratory muscle, the diaphragm muscle (and the phrenic motoneurons that innervate it) plays a key role in accomplishing these behaviors. Considerable diversity exists across diaphragm motor units, but the determinant factors for this diversity are unknown. In recent years, the mechanisms underlying the development of respiratory motor units have received great attention, and this knowledge may provide the opportunity to design appropriate interventions for the treatment of respiratory disease not only in the perinatal period but likely also in the adult.

How to Wire the Diaphragm: Wholemount Staining Methods to Analyze Mammalian Respiratory Innervation.

Direct or indirect impairment of breathing in humans by diseases or environmental factors can either cause long-term disability and pain, or can ultimately result in death. Automatic respiratory centers in the brainstem control the highly structured process of breathing and signal to a specialized group of motor neurons in the cervical spinal cord that constitute the phrenic nerves. In mammals, the thoracic diaphragm separates the thorax from the abdomen and adopts the function of the primary respiratory musculature. Faithful innervation by the phrenic nerves is a prerequisite for correct functionality of this highly specialized musculature and thus, ultimately, the viability of the entire organism.To analyze the effects of diseases and genetic defects responsible for deleterious or lethal respiratory phenotypes, accurate imaging of respiratory innervation during embryonic development, e.g., in genetically modified mouse models enables the characterization of specific marker genes and pathways that underlie appropriate wiring of the diaphragm. Among the different available immunostaining techniques, wholemount staining methods provide the advantage of clear and faithful three-dimensional information about the location of the antigens of interest. In comparison to routine histological techniques, however, the researcher has to deal with technical challenges, such as antibody penetration, the stability and availability of the antigen, and clearing of the relevant tissue, and the need to be equipped with state-of-the-art microscope equipment.In this methodological chapter, we explain and share our expertise concerning wholemount processing of mouse embryos and thoracic diaphragms for the analysis of mammalian respiratory innervation.

Developmental plasticity of phrenic motoneuron and diaphragm properties with the inception of inspiratory drive transmission in utero.

The review outlines data consistent with the hypothesis that inspiratory drive transmission that generates fetal breathing movements (FBMs) is essential for the developmental plasticity of phrenic motoneurons (PMNs) and diaphragm musculature prior to birth. A systematic examination during the perinatal period demonstrated a very marked transformation of PMN and diaphragm properties coinciding with the onset and strengthening of inspiratory drive and FBMs in utero. This included studies of age-dependent changes of: i) morphology, neuronal coupling, passive and electrophysiological properties of PMNs; ii) rhythmic inspiratory activity in vitro; iii) FBMs generated in vivo detected by ultrasonography; iv) contractile and end-plate potential properties of diaphragm musculature. We also propose how the hypothesis can be further evaluated with studies of perinatal hypoglossal motoneuron-tongue musculature and the use of Dbx1 null mice that provide an experimental model lacking descending inspiratory drive transmission in utero.

The effects of hypercapnia on early and later phases of phrenic neurogram during early maturation.

In this paper, we investigate the influence of hypercapnia on the early and late phases of the phrenic neurogram using the matching pursuit (MP) method in the decebrated piglets. The phrenic neurogram was recorded from 8 piglets (4-7 days old) during control (40% O2 with 5% end-tidal CO2), the mild hypercapnia (40% O2 with 7% end-tidal CO2), and the severe hypercapnia (40% O2 with 15% end-tidal CO2). The time-frequency representations, atoms, of the phrenic neurogram are calculated from the 5 consecutive phrenic neurogram burst for each piglet for each condition using the MP method after vagotomy and chemodenervation. Our results show that the energy percentage of atoms representing the nonperiodic neural activities (NPNAs) significantly increased when the CO2 concentration was shifted from 7% to 15% in the early phase (the first half) of the phrenic neurogram. In addition, the energy percentage of atoms representing the periodic neural activities (PNAs) decreased in the late phase (the second half) when the CO2 concentration was shifted from 7% to 15% (p < 0.01). As a summary, our result suggest that hypercapnia results in significant changes in the phrenic neurogram, an output of the respiratory neural networks in the medulla, both in time and frequency domians during early maturation.

Developmental nicotine exposure alters cholinergic control of respiratory frequency in neonatal rats.

Prenatal nicotine exposure with continued exposure through breast milk over the first week of life (developmental nicotine exposure, DNE) alters the development of brainstem circuits that control breathing. Here, we test the hypothesis that DNE alters the respiratory motor response to endogenous and exogenous acetylcholine (ACh) in neonatal rats. We used the brainstem-spinal cord preparation in the split-bath configuration, and applied drugs to the brainstem compartment while measuring the burst frequency and amplitude of the fourth cervical ventral nerve roots (C4VR), which contain the axons of phrenic motoneurons. We applied ACh alone; the nicotinic acetylcholine receptor (nAChR) antagonist curare, either alone or in the presence of ACh; and the muscarinic acetylcholine receptor (mAChR) antagonist atropine, either alone or in the presence of ACh. The main findings include: (1) atropine reduced frequency similarly in controls and DNE animals, while curare caused modest slowing in controls but no consistent change in DNE animals; (2) DNE greatly attenuated the increase in C4VR frequency mediated by exogenous ACh; (3) stimulation of nAChRs with ACh in the presence of atropine increased frequency markedly in controls, but not DNE animals; (4) stimulation of mAChRs with ACh in the presence of curare caused a modest increase in frequency, with no treatment group differences. DNE blunts the response of the respiratory central pattern generator to exogenous ACh, consistent with reduced availability of functionally competent nAChRs; DNE did not alter the muscarinic control of respiratory motor output. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1138-1149, 2016.

CO2 sensitivity of the complexity of phrenic neurograms in the piglet during early maturation.

In this paper, we investigate the influence of hypercapnia on the dynamics of the phrenic neurogram in the piglet in two different age groups: 3-7 days (n = 11) and 10-16 days (n = 9). The phrenic neurogram was recorded from 17 piglets (3-16 days old) during control (40% O(2) with 3-5% end-tidal CO(2)), mild hypercapnia (40% O(2) with 7% CO(2)) and severe hypercapnia (40% O(2) with 15% CO(2)) and analyzed using the approximate entropy (ApEn) method. The mean values of the approximate entropy (complexity) of phrenic neurograms during the first 7 days of the postnatal age were 1.56 +/- 0.1 (standard deviation) during normal breathing, 1.51 +/- 0.1 during mild hypercapnia and 1.37 +/- 0.08 during severe hypercapnia. These values for the 10-16 days age group were 1.51 +/- 0.1 during control, 1.49 +/- 0.11 during mild hypercapnia and 1.38 +/- 0.05 during severe hypercapnia. The mean values of phrenic neurogram durations during the first 7 days of the postnatal age were 0.82 +/- 0.03 (standard deviation) s during normal breathing, 0.85 +/- 0.007 s during mild hypercapnia and 0.65 +/- 0.05 s during severe hypercapnia. These values for the 10-16 days age group were 0.97 +/- 0.09 s during control, 1.10 +/- 0.05 during mild hypercapnia and 0.78 +/- 0.05 s during severe hypercapnia. Our results show that the complexity values of the phrenic neurogram were significantly decreased when the CO(2) concentration was shifted from control or mild to severe hypercapnia (p < 0.05) for both the 3-7 days old and the 10-16 days old groups. In addition, the duration of the phrenic neurogram decreased when the concentration was shifted from control or mild to severe hypercapnia (p < 0.05). But no significant changes in the duration of the phrenic neurogram were observed between control and mild hypercapnia concentration. These results suggest that severe hypercapnia can be characterized with a significant decrease of the complexity values and durations of the phrenic neurogram during inspiration during early maturation.

Carotid chemoafferent plasticity in adult rats following developmental hyperoxia.

Developmental hyperoxia impairs carotid chemoreceptor development and induces long-lasting reduction in carotid sinus nerve (CSN) responses to hypoxia in adult rats. Studies were carried out to determine if CSN responses to acute hypoxia would exhibit hypoxia-induced plasticity in adult 3-5-months-old rats previously treated with postnatal hyperoxia (60% O2, PNH) of 1, 2, or 4 weeks duration. CSN responses to acute hypoxia were assessed in adult rats exposed to 1 week of sustained hypoxia (12% O2, SH). In normal adult rats and adult rats treated with 1 week of PNH, CSN responses to acute hypoxia were significantly increased in urethane-anesthetized rats when studied 3-5 h after SH. Apparent increases in CSN responses to hypoxia were not significant in rats treated with 2 weeks of PNH and were clearly absent after 4 weeks of PNH, but exponential analysis suggests a PNH duration-dependent plasticity of the CSN response to acute hypoxia after SH. In a second study rats exposed to 2 weeks of PNH were treated with SH for 1 week as adults and acute hypoxic responses were tested 4-5 months later. CSN responses in these rats were unaffected by SH suggesting a lack of persistent SH-induced functional plasticity. We conclude that rats treated with 1 week of PNH retain the capacity for hypoxia-induced plasticity of carotid chemoafferent function and some potential for plasticity may be present after 2 weeks of PNH, whereas 4 weeks of PNH impairs the capability of rats to exhibit plasticity following 1 week of SH.

Morphometric analysis of phrenic motoneurons in the cat during postnatal development.

The dendritic geometry of 20 phrenic motoneurons from four postnatal ages (2 weeks, 1 and 2 months, and adult) was examined by using intracellular injection of horseradish peroxidase. The number of primary dendrites (approximately 11-12) remained constant throughout postnatal development. In general, postnatal growth of the dendrites resulted from an increase in the branching and in the length and diameter of segments at all orders of the dendritic tree. There was one exception. Between 2 weeks and 1 month, the maximum extent of the dendrites increased in parallel with the growth of the spinal cord; however, there was no increase in either combined dendritic length or total membrane surface area. In addition, there was a significant decrease in the number of dendritic terminals per cell (59.8 +/- 9.3 vs. 46.4 +/- 7.4 for 2 weeks and 1 month, respectively). The distance from the soma, where the peak number of dendritic terminals per cell occurred, ranged from 700-900 microns at 2 weeks and 2 months to 1,300-1,700 microns in the adult. The diameter of dendrites as a function of distance from the soma along the dendritic path increased with age. The process of maturation tended to increase the distance from the soma over which the surface area and dendritic trunk parameter (sigma d1.5/D1.5) remained constant. The three-dimensional distribution of dendrites was analyzed by dividing space into six equal volumes or hexants. This analysis revealed that the postnatal growth in surface area in the rostral and caudal hexants was proportionately larger than that in either the medial, lateral, dorsal, or ventral hexants. Strong linear correlations were found between the diameter of the primary dendrite and the combined length, surface area, volume, and number of terminals of the dendrite at all ages studied.

Phrenic motoneuron morphology in the neonatal rat.

The morphology of neonatal rat phrenic motoneurons was studied following retrograde labeling with horseradish peroxidase, which resulted in Golgi-like fills of phrenic motoneuron somata and dendrites. At birth, these neurons have well-developed dendritic trees with many characteristics described for phrenic motoneurons in the adult rat. The dendrites form tightly fasciculated bundles that emerge from the phrenic nucleus primarily along four axes: ventromedial, ventrolateral, dorsolateral, and rostral/caudal, with smaller and more variable projections directly lateral and ventral. Although sparse, some dendritic appendages were also present, and in a few animals, somata clustering was apparent. The most significant difference between adult and neonatal rat phrenic motoneurons is in the extent to which medially and laterally projecting dendrites extend beyond the borders of the ipsilateral gray matter. In the neonate, unlike the adult, these dendrites project extensively past the gray/white border to the edge of the hemicord. Ventromedial dendrites occasionally cross to the contralateral ventral horn in the ventral white commissure and laterally projecting dendrites could be seen reaching the edge of the cord, turning and traveling rostrally or caudally for up to 100 microns. Phrenic motoneurons are not unique in having long dendrites at birth. A brief comparative study showed that neonatal cervical, thoracic, and lumbar motoneurons also have long dendrites that project to the medial and lateral borders of the hemicord.

An overview of phrenic nerve and diaphragm muscle development in the perinatal rat.

In this overview, we outline what is known regarding the key developmental stages of phrenic nerve and diaphragm formation in perinatal rats. These developmental events include the following. Cervical axons emerge from the spinal cord during embryonic (E) day 11. At approximately E12.5, phrenic and brachial axons from the cervical segments merge at the brachial plexi. Subsequently, the two populations diverge as phrenic axons continue to grow ventrally toward the diaphragmatic primordium and brachial axons turn laterally to grow into the limb bud. A few pioneer axons extend ahead of the majority of the phrenic axonal population and migrate along a well-defined track toward the primordial diaphragm, which they reach by E13.5. The primordial diaphragmatic muscle arises from the pleuroperitoneal fold, a triangular protrusion of the body wall composed of the fusion of the primordial pleuroperitoneal and pleuropericardial tissues. The phrenic nerve initiates branching within the diaphragm at approximately E14, when myoblasts in the region of contact with the phrenic nerve begin to fuse and form distinct primary myotubes. As the nerve migrates through the various sectors of the diaphragm, myoblasts along the nerve's path begin to fuse and form additional myotubes. The phrenic nerve intramuscular branching and concomitant diaphragmatic myotube formation continue to progress up until E17, at which time the mature pattern of innervation and muscle architecture are approximated. E17 is also the time of the commencement of inspiratory drive transmission to phrenic motoneurons (PMNs) and the arrival of phrenic afferents to the motoneuron pool. During the period spanning from E17 to birth (gestation period of approximately 21 days), there is dramatic change in PMN morphology as the dendritic branching is rearranged into the rostrocaudal bundling characteristic of mature PMNs. This period is also a time of significant changes in PMN passive membrane properties, action-potential characteristics, and firing properties.

Axons grow in the aging rat but fast transport and acetylcholinesterase content remain unchanged.

Caliber and microtubular density of myelinated fibers, acetylcholinesterase (AChE) content and its accumulation at a ligature were studied in the phrenic nerve of mature (3-4 months) and aging (2-year-old) rats. The number of axons remained constant. The cross-sectional area of the nerve was 67% greater in the older group; the axoplasm, though, constituted about 20% of the nerve tissue irrespective of age. The mean cross-sectional area of myelinated axons was twice as big in aging compared to mature rats. All axons grew in the same proportion irrespective of their original caliber. The microtubular density of 3-microns axons was about 22 microtubules/micron2 in mature and aging rats. The AChE activity of aging rats was half as much as that of mature rats if it was expressed per wet weight of nerve tissue but did not change if it was expressed per nerve fiber. Twenty-four hours after ligation of the nerve, total AChE activity rose in mature and aging rats by ca. 168%; the molecular forms--asymmetric and globular--accumulated in the same proportion in both age groups. We conclude that myelinated axons grow in the adult stage of life but the structure of axoplasm, content of AChE per axon, and rate of fast transport remain lifelong features of nerve fibers.

Efferent phrenic nerve and respiratory neuron activities in the developing kitten: spontaneous discharges and hypoxic responses.

Efferent phrenic nerve and medullary respiratory neuron discharges were examined for age-dependent changes of activities during normocapnic hyperoxia and hypoxia in anesthetized and decerebrate kittens (22-150 days old). In animals less than 39 days of age, phrenic power spectra during hyperoxia were dominated by components in the medium-frequency band (20-50 Hz), whereas spectra of animals of at least 39 days of age were dominated by components in the high-frequency band (50-100 Hz). Such high-frequency oscillations were also observed in the power spectra of some inspiratory neurons in animals of at least 43 days old. In hypoxia, the amplitude of phrenic discharge exhibited an initial facilitation followed by a diminution (i.e. biphasic response) in animals 39 days old or younger. In animals older than 39 days, however, hypoxia elicited a sustained facilitation of phrenic discharge amplitude. In contrast, no such age-dependent change in response pattern to hypoxia was observed for neuronal discharges; rather, responses of most neurons consisted of either decreases of discharge frequency, or complete abolishment of discharges.

Postnatal development of phrenic motoneurons in the cat.

The postnatal growth of phrenic motoneurons in the cat was studied using retrograde transport of horseradish peroxidase (HRP). The mean somal surface area of these developing motoneurons increased 2.5 times from day 3 to adult while the mean somal volume increased four-fold. This change in mean somal surface area during postnatal development was found to be correlated with the change in mean axonal conduction velocity measured from phrenic motoneurons.

Phrenic and recurrent laryngeal motoneuron activities during hyperoxia and hypoxia in piglets.

We hypothesized that synchronization of inspiratory motoneurons may involve inputs from two central pattern generators (CPG): one characterized by medium-frequency (< 50 Hz) and the other by high-frequency oscillations (> or = 50 Hz). We studied phrenic and recurrent laryngeal nerve activities recorded during hyperoxia and hypoxia in Saffan anesthetized, paralyzed, and artificially ventilated piglets. Spectral analyses, derived from the full as well as partitioned halves of inspiration, showed that phrenic and recurrent laryngeal discharges contained peaks in the medium-frequency band, which were indicative of common inputs. The phrenic spectra of many animals had peaks in the high-frequency band; such peaks were uncommon in recurrent laryngeal spectra; consequently, correlated activities corresponding to high-frequency oscillations were not usually observed. Thus, it is likely that acquisition of modulating inputs from a high-frequency CPG may emerge in an age-dependent manner in different motoneuron pools. During hypoxia, both phrenic and recurrent laryngeal discharges were facilitated as shown by increases in both the amplitudes of signal-averaged histograms and the magnitudes of their respective power spectral activities. Also, there was a significant increase in the values of phrenic-recurrent laryngeal coherence estimates in the medium-frequency region. Hence, medium-frequency oscillations are more apparent in early development, perhaps to facilitate synchronization of inspiratory motoneuron activities, especially under conditions of increased chemical drive.

Substance P and central respiratory activity: a comparative in vitro study on foetal and newborn rat.

Experiments were performed in vitro on foetal (embryonic days 18 to 21, E18-21) and newborn rat (postnatal days 0 to 3, P0-3) brainstem spinal cord preparations to analyse the perinatal developmental changes in the effects induced by substance P. Superfusion of the preparations with SP-containing artificial cerebrospinal fluid (aCSF) induced significant increase in the respiratory frequency of newborn rats (10-9 M), whereas concentration up to 10-7 M induced no change in foetal preparations. A whole cell patch clamp approach was used to record intracellularly from phrenic motoneurones. In newborn or E20-21 foetal rats SP-containing aCSF depolarised the phrenic motoneurones, increased their input resistance, reduced the rheobase current and shifted the frequency-intensity curves upward. In E18 foetal rats, no change was evoked by SP. A peptidase inhibitor mixture was used to block the enzymatic degradation of endogenous SP. This mixture was ineffective in changing the respiratory frequency in newborn and foetal preparations. In newborn rat phrenic motoneurones, the peptidase inhibitor mixture induced changes similar to those caused by SP but no change was induced in foetal rats. These results indicate that SP may modulate (i) the activity of the respiratory rhythm generator in newborn but not in foetal rats, and (ii) the activity of phrenic motoneurones at E20, E21 and in newborn rats but not at E18. Results obtained using the peptidase inhibitor mixture suggest that endogenous SP is probably not involved in the control of the respiratory rhythm in the prenatal period, but may influence the activity of the phrenic motoneurones after birth.