Maternal caffeine ingestion during gestation and lactation influences respiratory adaptation to acute alveolar hypoxia in newborn rats and adenosine A2A and GABA A receptor mRNA transcription.

Caffeine is a widely used psychostimulant freely crossing the placental barrier. At the doses usually absorbed, it acts as an antagonist of both A1 and A2A adenosine receptors. Pregnant women are generally not advised to limit their caffeine consumption and thus expose their progeny to the drug during the whole of gestation and lactation. The possibility that such caffeine exposure may have long-term consequences on brain development has led to several behavioral investigations on animal models. Despite the crucial role played by adenosine receptor systems in neonatal breathing control, few studies in vitro have been concerned with the consequences of maternal caffeine absorption on breathing, and none in the unrestrained intact animal. The present investigation analyzed the influence of caffeine exposure via placental and milk transfer on resting ventilation and on the response to moderate alveolar hypoxia of 0 to 2-day-old newborn rat (P0-P2) together with the possible underlying mechanisms. Dams absorbed caffeine (46+/-3 mg/kg/day) via drinking fluid (0.2 g/L) throughout gestation, in conditions mimicking moderate human consumption. Caffeine exposure did not significantly affect basal respiratory parameters. In contrast, it attenuated both the early increase and the secondary decrease in ventilation triggered by moderate alveolar hypoxia (11% O2 inhaled). The abolition of Fos protein expression evoked by hypoxia suggested that caffeine exposure may decrease the activity of O2-sensing peripheral chemoreceptor pathway. From real-time PCR data, those functional alterations were associated to increases in A2A adenosine receptor and alpha2 GABA(A) receptor subunit mRNAs in the medulla. This indicates that, even at moderate doses, maternal caffeine consumption may induce a series of subtle developmental alterations that may affect modulation of breathing control in the neonate in pathological situations such hypoxia.

Effect of postnatal exposure to caffeine on the pattern of adenosine A1 receptor distribution in respiration-related nuclei of the rat brainstem.

Caffeine, which belongs to the methylxantine family of compounds, is commonly ingested in a range of beverages such as coffee, tea, and cola drinks. It is also used therapeutically and is frequently employed in the treatment of respiratory disturbances in human neonates. The aim of the present work has been to examine the ontogeny of the adenosine A1 receptor system in the brainstem of the newborn rat following postnatal treatment with caffeine to mimic the therapeutic administration of caffeine to premature human infants. The effect of this postnatal exposure to caffeine on the gradual appearance of adenosine A1 receptors was analysed by determining immunohistochemically the distribution of the receptors. The main difference between control animals and animals exposed to caffeine was the transient increase (only at postnatal day 6) in the number of immunopositive neurons in two brainstem areas, the ventrolateral medulla and the rostral dorsolateral pons, in caffeine-treated rat pups, or more specifically, the parabrachial and Kölliker-Fuse nuclei, both of which are classically associated with respiratory control. With previous research highlighting the important role played by the rostral pons in respiratory modulation by the adenosine A1 receptor system, it is thus possible that postnatal exposure to caffeine modulates the ontogeny of the adenosine A1 receptor network. This could imply that the role of caffeine to decrease the incidence of neonatal respiratory disturbances may be due to the earlier than normal development of the adenosinergic system in the brain.

Rhythmic activity from transverse brainstem slice of neonatal rat is modulated by nitric oxide.

We investigated the role of nitric oxide (NO) in the modulation of respiratory-like activity recorded from hypoglossal rootlets in brainstem slices of neonatal rats (P0-P8). Sodium nitroprusside (SNP), S-Nitroso-N-acetyl-D,L-penicillamine (SNAP) and diethylamine-NO (DEA-NO), three NO-donors, reversibly increased hypoglossal burst amplitude with inconsistent effects on burst frequency. Similar effects were also obtained with the endogenous substrate of nitric oxide synthase (NOS), L-arginine, whereas the inactive enantiomer D-arginine had no effect. The NO-trap agent methylene blue significantly depressed both the amplitude and frequency of hypoglossal activity while hemoglobin depressed only the amplitude. Furthermore, the addition of NO-trap agents significantly attenuated the excitatory response to SNP. Inhibiting NOS with either N(omega)-Nitro-L-Arginine (L-NNA) or 7-Nitroindazole (7-NI), decreased the amplitude of hypoglossal activity with no effects on frequency. Histochemical analysis of NADPH-diaphorase activity, a marker for NOS, was performed on slices not treated pharmacologically and in brainstem sections of newborn rats, perfused in situ. Comparison between in vitro and in vivo conditions indicated that NOS activity was maintained in slice preparations. Neurons in the ambiguus and hypoglossal nuclei (dorsal division) exhibited a granular staining, suggesting the presence of NADPHd-positive terminals. Neurons with cytoplasmic staining were identified in regions connected to the hypoglossal nucleus (nucleus tractus solitarius, paramedian and gigantocellular reticular nuclei). These neurons might be involved in nitrergic control of hypoglossal activity. Both pharmacological and histochemical data suggest that endogenous NO may reinforce the output activity of the medullary respiratory network.

Brainstem and hypothalamic areas activated by tissue hypoxia: Fos-like immunoreactivity induced by carbon monoxide inhalation in the rat.

Acute ambient hypoxia interacts with the ventilatory and cardiocirculatory control systems, via the concomitant activation of arterial chemoreceptors and tissue oxygen-sensing mechanisms. Whether these latter mechanisms may trigger a specific pathway had not yet been elucidated. We addressed this issue, mapping Fos expression in adult conscious rats subjected to tissue hypoxia elicited by carbon monoxide inhalation, under conditions of minimal activation of arterial chemoreceptors. Brief stimuli have been delivered (1% carbon monoxide inhaled during 5, 10 or 20 min) to produce steady tissue hypoxia. Compared to normoxia, even the briefest stimuli led to marked neuronal activation within areas involved in ventilatory and cardiocirculatory control. In the brainstem, stimulated rats exhibited enhanced Fos expression in the nucleus of the solitary tract, the area postrema, the dorsal motor nucleus of the vagus nerve, the ventrolateral medulla, the parapyramidal group, the nucleus raphe pallidus, the lateral paragigantocellular nucleus, the locus coeruleus, the dorsal raphe nucleus, the lateral parabrachial area, and the ventrolateral central gray. In the hypothalamus, activated neurons were identified at the ventral border and in the supramamillary, posterior, and dorsomedial nuclei. Fos expression appeared with increasing the severity of tissue hypoxia in the retrotrapezoid nucleus, the ventral tegmental area and the arcuate and paraventricular hypothalamic nuclei. The present data support the idea that inputs related to tissue hypoxia might play a crucial role in patterning the physiological response to hypoxia.

The sneeze: maturation of the reflex in kittens.

This is the first study to compare the influence of nasal afferent stimulation on inspiratory and expiratory muscle activity during sneezing, in kittens and adult cats. In kittens, we demonstrate that nasal afferent stimulation does not reinforce inspiratory activity prior to the expiratory thrust as it normally does in adult cats. These stimulations evoke an active expiration similar to but weaker than the expiratory thrusts observed under the same conditions during sneezing in adult cats. Sneezing can be elicited from three weeks of life. Among the different hypotheses discussed, the most likely explanation appears to be the immaturity of medullary respiratory connections.

Morphometrical study of the cat thoracic dorsal root ganglion cells in relation to muscular and cutaneous afferent innervation.

The sizes of neuronal somata in cat dorsal root ganglia were determined at the different thoracic segmental levels (T1-T13). The intersegmental variations in the average value and class distribution of diameters were analysed. The maximal and minimal average mean cell diameters were 51.1 and 43.3 microns at the T1 and T2 levels, respectively. Caudally, this value gradually increased from T2 to T8 (47.8 microns) and thereafter decreased progressively to T12 (44.7 microns). At T1, large cells (greater than 50 microns in diameter) were 3.3-fold in excess compared to small ones (less than 35 microns in diameter). The proportion of large to small cells strongly decreased to a 0.9 ratio from T1 to T2, then increased again from T2 (0.9) to T8 (2.3). The size distributions of the overall cell populations were compared to those of neurones supplying muscular targets via the external intercostal nerves or cutaneous targets via the lateral branch of the internal intercostal nerves, identified following the retrograde transport of horseradish peroxidase. The size distribution of cells serving cutaneous nerves was similar to that exhibited by the overall population of ganglion cells. In contrast, the size distributions of cells giving rise to muscle afferents tended towards smaller values. In the thoracic dorsal root ganglia, the cell body sizes of the muscular primary afferents were close to those previously reported for the visceral primary afferents.

The cat cervical dorsal root ganglia: general cell-size characteristics and comparative study of neck muscle, neck cutaneous and phrenic afferents.

Sizes of neuronal somata in the cat cervical dorsal root ganglia were determined at different levels (C1-C8). The average value and class distribution of mean cell diameter were analyzed. The ganglia from C1 to C5 could be clearly distinguished from those at levels of brachial plexus afferents (C6-C8) with respect to cell size range, distribution and average. The size distribution, most often limited to 70 microns from C1 to C5, skewed to more than 90 microns from C6 to C8. Cells in the 35-50 microns range of diameter constituted the main portion of the cell population (49-52%) at the C1-C5 levels, whereas from C6 to C8 51-77% of the ganglion cell bodies were more than 50 microns in diameter. The cell size distribution of afferents projecting from C1 to C5 and supplying different muscle or cutaneous targets was studied following retrograde labeling with horseradish peroxidase conjugated to wheatgerm agglutinin. Sizes of cell bodies of biventer cervicis (postural muscle), phrenic (purely respiratory muscle) and cutaneous afferents were all similar. The labelled cell bodies were in the majority (51-64%) less than 35 microns in diameter and ranged towards smaller diameters than counterstained cells in the corresponding ganglia. In spite of similarities in cell size distribution it was estimated from the fiber caliber spectra of the labelled afferents that both unmyelinated and myelinated cutaneous afferents originate from larger cell bodies than muscle afferents in the same diameter range.

c-fos-like immunoreactivity in the cat's neuraxis following moderate hypoxia or hypercapnia.

The overall pattern of c-fos immunoreactivity was studied in the brainstem and spinal cord of cats subjected to moderate hypoxia or hypercapnia. In control cats (normoxic, normocapnic), c-fos was expressed mainly in pontine and periaqueductal grey but not in brainstem structures engaged in respiratory control nor in the spinal cord. Both hypoxia and hypercapnia induced c-fos expression in the parabrachial area (pneumotaxic center). In the retrotrapezoid nucleus, a structure involved in respiratory rhythmogenesis and chemoreception, immunoreactivity was detected in hypoxic but not in hypercapnic cats. Neurons in the nucleus raphe pallidus preferentially expressed c-fos in response to hypercapnia. Labelled neurons were concentrated in the dorsal and gelatinosus subnuclei of the solitary tract following hypoxia and hypercapnia, respectively. Our data suggest that some neurons that express c-fos in hypoxic or hypercapnic cats may be involved in coordination of cardiovascular and respiratory function.

Postnatal changes in Fos-like immunoreactivity evoked by hypoxia in the rat brainstem and hypothalamus.

We have recently used Fos expression in adult rats to map neuronal populations activated in the brainstem and hypothalamus during the acute ventilatory response to moderate hypoxia (O(2) 11%). Although present at birth, this response evolves postnatally. The present investigation aimed at a better understanding of these maturational processes by delineating structures that might functionally develop after birth. The developmental pattern Fos expression evoked by hypoxia was analysed in rats aged from 0 to 26 postnatal days. The numbers of Fos positive neurons markedly increased with the age in the medullary areas related to respiratory control during the 2 first postnatal weeks. Thereafter, the response plateaued in the nucleus tractus solitarius and attenuated in the ventral medulla. In the upper brainstem (parabrachial area, central grey) and the hypothalamus (posterior and dorsomedial nuclei, ventral zone), Fos response to hypoxia was absent or weak at birth and increased until late development. The significance of the development of evoked Fos expression in these rostral sites is discussed together with their possible contribution to the maturation of O(2)-sensitive chemoreflex pathways.

C-Fos-like immunoreactivity in the cat brainstem evoked by sneeze-inducing air puff stimulation of the nasal mucosa.

Sneeze is one of the most important protective reflex of the respiratory tract. It is elicited from trigeminal peripheral fields and results in major changes in the discharge patterns of the medullary respiratory-related neurons. The pattern of c-Fos-like immunoreactivity evoked by sneezing was explored as a structural approach to the networks involved in this particular model of trigemino--respiratory interactions. Sneezes were elicited in anaesthetized adult cats by driving air puffs to the superior nasal meatus through a catheter. Additional cats were used as controls for anaesthesia and for catheter insertion into the nostril. In sneezing cats, immunoreactivity was evoked in projection areas of the ethmoidal afferents which innervate the superior nasal meatus, e.g. the subnuclei caudalis, interpolaris and in the interstitial islands of the trigeminal sensory complex. Immunoreactivity was also markedly enhanced in the areas devoted to respiratory control in the medulla (solitary complex, nucleus retroambiguus) and in the pontine parabrachial area. C-Fos expression was also evoked in the lateral aspect of the parvicellular reticular formation in sneezing cats. This area might be of major importance in the adaptation of the ventilatory system to expulsive functions.

Changes in Fos-like immunoreactivity evoked by maturation of the sneeze reflex triggered by nasal air puff stimulation in kittens.

The sneeze reflex is a valuable tool for exploring the maturation of the respiratory control in the newborn as it alters both inspiratory and expiratory activities. Air puff stimulation of the superior nasal meatus innervated by ethmoidal afferents consistently evokes sneeze in adult cats. Such stimulation evokes only a reinforcement of expiratory activities in newborn kittens. This study demonstrates that the pattern of Fos-like immunoreactivity evoked by nasal stimulation changes during functional maturation of sneeze. Nasal stimulation evoked immunoreactivity (i) in the trigeminal sensory complex, at the levels where nasal afferents project, (ii) in the reticular formation, (iii) in the solitary complex and (iv) in the parabrachial area of mature kittens. The evoked immunoreactivity was the same in newborn kittens as in mature kittens in the projection areas of the nasal primary afferents. Fos response was less than half that in mature kittens in the reticular formation and absent in the solitary complex or the parabrachial area. Sneeze can be elicited from the time when evoked immunoreactivity in the solitary complex and the parabrachial area is above control levels. These data provide evidence that the maturation of sneeze is dependent on the development of central relays allowing peripheral inputs to be integrated by neurons engaged in respiratory control.

Brainstem and hypothalamic areas involved in respiratory chemoreflexes: a Fos study in adult rats.

The adaptation to hypoxia and hypercapnia requires the activation of several anatomical structures along the neuraxis. In this study, using Fos immunoreactivity, we sought to map neuronal populations involved in chemoreflex networks activated during the responses to moderate hypoxia (O(2) 11%), and hypercapnia (CO(2) 5%) in the brainstem and the hypothalamus of the rat. In the medulla, hypoxia elicited marked and significant staining in the nucleus of the solitary tract (NTS), and in parapyramidal neurons located near the ventral surface, whereas hypercapnia evoked significantly c-fos only near the ventral surface in paraolivar neurons. In contrast, within pontine and suprapontine structures, both hypoxia and hypercapnia evoked similarly Fos immunoreactivity in the lateral parabrachialis area, the central grey, the caudal hypothalamus (dorsomedial and posterior hypothalamic nuclei), and in a ventro-lateral hypothalamic area, extending from the rostral limit of the mammillary nuclei to the retrochiasmatic area. More rostrally, labelling was observed in the paraventricular nucleus of the hypothalamus in response to hypercapnia, and in the supraoptic nucleus in response to hypoxia. These results support the hypothesis that chemoreflexes pathways are not only restricted to medulla and pons but also involved mesencephalic and hypothalamic regions. The parabrachialis area and the central grey may be key relays between caudal and ventral hypothalamic neurons, and medullary neurons involved in the response to hypoxia and hypercapnia.

Postnatal development of small and large dorsal root ganglion neurons in the cat. A study on cervical levels (C5-C6) and on phrenic afferents.

During feline postnatal development, the size of phrenic afferent neurons labelled by horseradish peroxidase was evaluated in comparison to that of the bulk of counterstained neurons located in the same cervical dorsal root ganglia (DRG) (C5-C6). From age 1 week to maturity, small and large cell components were individualized from experimental size distributions using a mathematical approach. The analysis of data in adult indicated a close correspondence between small cells and unmyelinated afferents and between large cells and myelinated afferents, respectively. From age 1 week to adulthood, mean increases in cell diameter ranged between 10 microns (small cells from phrenic afferents) and 29.5 microns (large counterstained cells). In each population, the ratio of small/large cells remained constant during growth. In contrast to data in adults, at 1 week, large phrenic neurons were bigger than the counterstained ones. At 19 weeks, the cat DRG cells had not yet reached their adult size.

Identification of phrenic afferents in the dorsal columns: a fluorescent double-labeling study in the cat.

In this study in the cat, the tracer combination of the anterogradely transported Fast Blue and the retrogradely transported Nuclear Yellow was used to label dorsal root ganglion cells related to phrenic afferents running up through the ipsilateral dorsal column. These afferents are few; some of them leave the dorsal column near their segment of entry. Their localization in the dorsal column suggests that they are related to tendon and muscle receptors, which confirms previous electrophysiological studies.

Identification of phrenic afferents to the external cuneate nucleus: a fluorescent double-labeling study in the cat.

In the cat, C5-C6 dorsal root ganglion cells related to phrenic afferents projecting directly to the ipsilateral external cuneate nucleus (ECN) were submitted to a double-labeling procedure using anterogradely transported Fast Blue and retrogradely transported Nuclear yellow. These afferents, certainly related to muscle spindles and/or Golgi tendon organs, are very few and terminate preferentially in the intermediate and rostral parts of the ECN. Our results confirm previous electrophysiological and histological studies on the participation of phrenic afferents to the spino-cuneo-cerebellar pathway ascending through the dorsal columns.

A comparative HRP study of the neuronal supply to the inferior and superior nasal meatus in the cat.

Neurons supplying the nasal mucosa in the cat were retrogradely labelled with horseradish peroxidase. Sensory trigeminal neurons to the inferior and superior nasal meati are somatotopically organized, according to the ophthalmic or maxillary origin of the afferents studied. Whatever their relative location, the cell bodies from nasal afferents were, on average, smaller than the overall cell population in the ganglion, in keeping with the high proportion of nasal receptors innervated by thin fibers. Postganglionic neurons from parasympathetic origin could be labelled in the sphenopalatine ganglion. These neurons probably supply mucosal secretory glands. They are in the same size range as the bulk of neurons in the same ganglia.

Quantitative morphological changes in phrenic and intercostal motor columns and their respective spinal cord segments during postnatal development in the kitten.

In newborn kittens, the nervous control of breathing appears more mature than that of motricity which follows a cephalo-caudal evolution. In order to determine if the different postnatal evolutions of the respiratory and the motor function have an anatomical support at the spinal cord level, we made morphometric comparisons of the postnatal development of the spinal segments including motor columns sustaining both limb and respiratory movements (cervical and thoracic segments), with the postnatal development of segments containing only motoneurones involved in locomotion (lumbar segments). Furthermore, we used horseradish peroxidase to label cervical and thoracic groups of inspiratory motoneurones, i.e. the phrenic and the intercartilaginous motor nuclei at several postnatal ages. The present study suggests that the development of the white matter is the same at every spinal level and that it is delayed compared to the maturation of the grey matter. Overall evaluations of grey matter areas showed that the thoracic grey matter is more mature at birth but, further, has a slower rate of growth than the cervical and lumbar ones. This observation may be related to the maturity of the respiratory phasic activities within the early postnatal life. The phrenic and intercartilaginous motor nuclei have different patterns of development. These results suggest that the spinal postnatal functional maturation is not strictly related to its quantitative macroscopic changes.

Spinal localization of the intercostal motoneurones innervating the upper thoracic spaces.

At the rostral level of the thorax, the intercostal muscles participate both in postural and respiratory functions to a variable degree depending upon the considered muscle: external intercostal, intercartilaginous, internal intercostal, and triangularis sterni. In order to determine if these physiological properties are related to a special organization at the spinal cord level, we have used the retrograde transport of horseradish peroxidase as a tool for studying the spinal distribution of intercostal motor cells in the adult cat. Results suggest that the intercostal motoneurones could be distributed, in the ventral grey horn, among two areas according to the respiratory or postural muscle specialization.

Somatotopy in the phrenic motor nucleus of the cat as revealed by retrograde transport of horseradish peroxidase.

Cats received unilateral or bilateral horseradish peroxidase (HRP) injections into various portions of the diaphragm. In two experiments one of the cut cervical roots of the phrenic nerve was immersed in HRP. The phrenic motoneurons located in the fifth and occasionally the fourth cervical segment send their axons, via the upper phrenic root, to pars sternalis and pars costalis of the diaphragmatic dome whereas the neurons of the sixth segment innervate preferentially the dorsal portion both crura and dome. No evidence of contralateral innervation of the diaphragm was obtained.

Antenatal environmental stress and maturation of the breathing control, experimental data.

The nervous respiratory system undergoes postnatal maturation and yet still must be functional at birth. Any antenatal suboptimal environment could upset either its building prenatally and/or its maturation after birth. Here, we would like to briefly summarize some of the major stresses leading to clinical postnatal respiratory dysfunction that can occur during pregnancy, we then relate them to experimental models that have been developed in order to better understand the underlying mechanisms implicated in the respiratory dysfunctions observed in neonatal care units. Four sections are aimed to review our current knowledge based on experimental data. The first will deal with the metabolic factors such as oxygen and glucose, the second with consumption of psychotropic substances (nicotine, cocaine, alcohol, morphine, cannabis and caffeine), the third with psychoactive molecules commonly consumed by pregnant women within a therapeutic context and/or delivered to premature neonates in critical care units (benzodiazepine, caffeine). In the fourth section, we take into account care protocols involving extended maternal-infant separation due to isolation in incubators. The effects of this stress potentially adds to those previously described.