Susana P. Gaytán

Connections of the rostral ventral respiratory neuronal cell group: an anterograde and retrograde tracing study in the rat

The connections of the rostral ventral respiratory cell group (VRG) were retrogradely and anterogradely determined after discrete injections of a mixture of the fluorescent tracers Fast Blue (FB) and Fluoro Ruby (FR) into the physiologically identified rostral inspiratory cell group. Retrogradely FB-labeled neurons and/or anterogradely FR-labeled fibers and terminal fields were located bilaterally in a variety of brain areas. Both retrograde and anterograde labelings were mainly found in: 1) the deep cerebellar nuclei; 2) the lateral lemniscus and paralemniscal nuclei, deep gray, and white intermediate layers of the superior colliculus, tegmental (laterodorsal and microcellular) nuclei, and central gray; and 3) the septohypothalamic nucleus, and lateral and posterior hypothalamic areas. The FR-labeled terminal-like elements were found in: 1) Crus 2 of the ansiform lobule, and the simple, 2, and 3 cerebellar lobules; 2) the subcoeruleus, deep mesencephalic, and Edinger-Westphal nuclei; and 3) the premammillary, lateral, and medial mammillary nuclei, retrochiasmatic part of the supraoptic nucleus, and the zona incerta. The FB-labeled neurons were found in: 1) the parapedunculopontine tegmental and cuneiform nuclei, caudal linear nucleus of the raphe, and adjacent area of the cerebral peduncle; 2) the thalamic posterior nuclear group and subparafascicular, parafascicular, and gelatinosus thalamic nuclei; 3) the parastrial amygdaloid and subthalamic nuclei; and 4) the olfactory tubercle, granular, and agranular insular cortex, parietal and lateral orbital cortices. The connections of the rostral VRG with several cerebellar, midbrain, diencephalic, and telencephalic regions could provide an anatomical substrate for a role of these regions in the control of respiratory-related functions.

Pontomedullary Efferent Projections of the Ventral Respiratory Neuronal Subsets of the Rat

The pontomedullary trajectories of projections efferent from the ventral respiratory cell group were anterogradely labelled after discrete injections of Fluoro Ruby into three morphophysiologically identified subdivisions (Bötzinger complex, rostral inspiratory, and caudal expiratory cell groups). The anterogradely labelled varicosities were located in a variety of areas involved in cardiorespiratory function: other subdivisions of the ventral respiratory cell group, the parabrachial (medial, central, and external lateral), Kölliker-Fuse, and lateral paragigantocellular nuclei, A5, and perifacial areas. Although the target areas were similar for the three studied subdivisions, some differences of the location and densities of labelled varicosities were found. Anterogradely labelled fibre bundles were found bilaterally after all of the tracer injections. Three caudally efferent bundles passed through the ventral respiratory cell group, dorsal medullary, and paramedian reticular nuclei. A labelled fibre bundle also took an ascending route through the ventral respiratory cell group: it surrounded the facial nucleus, and then followed two different pathways, one coursing towards forebrain areas and the other to the parabrachial and Kölliker-Fuse complex. Bundles of efferent axons decussated mainly at medullary levels and to a lesser extent in the pons. In the contralateral medulla and pons these labelled fibre bundles followed pathways similar to those observed ipsilaterally. The three ventral respiratory neuronal subsets sent axonal projections through similar tracts, but within them they were topographically organized. The present data are discussed with respect to the circuitry involved in the mechanisms of cardiorespiratory and other visceral functions.

Identification of central nervous system neurons innervating the respiratory muscles of the mouse: a transneuronal tracing study.

In recent years, the central control of breathing in mammals has been the subject of numerous studies. The aim of the present one was to characterize the neuronal network projecting to the main respiratory motoneurons, in adult mice. To this end, the morphology and location of the respiratory motoneurons and their sequential connections with other neurons were revealed using a transneuronal tracing technique by means of the rabies virus infection. The injections of the rabies virus in the respiratory muscles resulted in labeling the motoneurons and their serially connected interneurons at multiple levels of the mouse central nervous system: spinal cord, pons and medulla, cerebellum, mesencephalon, diencephalon, and telencephalon. Most of these labeled areas have been previously identified in the control of cardiorespiratory regulation, as well as in other autonomic functions. These anatomical data provide support for the integration of respiratory-related activities in complex behavioral responses. Furthermore, these data suggest similarities in the evolution of central respiratory networks in mammals.

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.

Neonatal caffeine treatment up-regulates adenosine receptors in brainstem and hypothalamic cardio-respiratory related nuclei of rat pups.

While neonatal caffeine treatment is commonly used to alleviate apnea of prematurity in neonates and to improve neurological outcomes, its effects on adenosine A₁ and A(2A) receptors (A₁-R and A(2A)-R) are poorly known. We hypothesized that the central pharmacological action of caffeine is mediated by modification of the postnatal development of the adenosinergic system during a critical period. On postnatal days 2-6 (P2-P6) two groups of newborn rats were orally administered water plus glucose and/or caffeine at therapeutic doses to mimic the clinical use of caffeine in human neonates. Cardio-respiratory parameters were measured and the presence of A₁-R and A(2A)-R and c-Fos protein was identified immunohistochemically in animals sacrificed from P2 to P11. % Haemoglobin saturation, and hearth and breath rates were significantly increased in caffeine-treated group (P5-P6). Significant differences were identified in the relative gene expression of A₁-R and A(2A)-R, with an increase of A₁-R labeling in the anterior hypothalamic area, ventromedial hypothalamic nucleus, parabrachial complex and ventrolateral medulla of the caffeine-treated group at P6. A moderate increase in A(2A)-R labeling was observed in ponto-medullary nuclei and other hypothalamic areas. An increase in c-Fos-positive labeled cells was found in the caffeine-treated group at P5-P6 within the same areas described above, with the most clear-cut increase seen in the arcuate nucleus. Indeed, increased A₁-R and A(2A)-R gene expression was observed in both the brainstem and hypothalamus at P5. Up-regulation of adenosinergic maturation in central cardio-respiratory areas in caffeine-treated neonatal rats could explain the pharmacological effects of caffeine observed in premature infants.

Distribution of NADPH-diaphorase and nitric oxide synthase reactivity in the central nervous system of the goldfish (Carassius auratus).

The nitrergic system has been inferred from cells positive to nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry and/or to the neuronal isoform of nitric oxide synthase (nNOS) immunohistochemistry in different species of vertebrates. The aim of the present work was to systematically study the distribution of cell producing nitric oxide in the goldfish (Carassius auratus) brain. To reach this goal, we firstly studied co-localization for NADPHd and nNOS techniques and demonstrated an extensive double labeling. Then, we studied the distribution through the brain by the two separate methods and found labeled cells widely distributed in brain and spinal cord. In the telencephalon, such cells were in both dorsal and ventral areas. In the diencephalon, the cells were found in some nuclei of the preoptic area and hypothalamus, habenula, pretectum, and dorsal and ventral thalamic regions. In the midbrain, cells were observed in the optic tectum, torus longitudinalis, and tegmental nuclei. In the rhombencephalon, cells were found in the cerebellum, the reticular formation, the locus coeruleus, the raphe nuclei, and the nuclei of the cranial nerves. Labeled cells were also observed in the gray area of the spinal cord. Cognizing that a direct comparison of the present results with those reported in other vertebrates is not clear-cut because of homologies; we conclude that the nitrergic system is roughly similar from fish to mammals.

Co-localization of nitric oxide synthase and choline acetyltransferase in the brain of the goldfish (Carassius auratus)☆

This work investigates the nitrergic and cholinergic systems in the brain and spinal cord of the goldfish (Carassius auratus). We studied the immunohistochemical localization of antibodies against the neuronal nitric oxide synthase (nNOS) and choline acetyltransferase (ChAT) by bright-field and confocal microscopy. Nitrergic and cholinergic cells were segregated within the telencephalon, in both dorsal and ventral areas, and co-distributed in some nuclei of the diencephalon, mesencephalon, rhombencephalon, and spinal cord. Double-labeling experiments revealed nNOS/ChAT-positive cells in (1) the diencephalon: the preoptic and suprachiasmatic nuclei, the habenula, the dorsal thalamus, and the nucleus of the medial longitudinal fasciculus; (2) the mesencephalon: the optic tectum, the mesencephalic portion of the trigeminal nucleus, the oculomotor and trochlear nuclei, and the Edinger-Westphal nucleus; and (3) the rhombencephalon: the secondary gustatory nucleus, the nucleus isthmi, the lateral lemniscus nucleus, the cerebellum, the reticular formation, different nuclei of the octaval column, the motor zone of the vagal lobe, and the trigeminal, facial, abducens, glosso-pharyngeal, vagal, and hypobranchial motor nuclei. Double-labeled cells were also observed in the spinal motor column. The percentage of double-labeled cells was different in each studied nucleus, indicating a selective distribution pattern. Because double-labeled cells were more abundant in those nuclei involved with sensory and motor physiological processes, we suggest the involvement of both nitric oxide and acetylcholine in these neural functions in fish.

Prenatal activation of 5‐HT2A receptor induces expression of 5‐HT1B receptor in phrenic motoneurons and alters the organization of their premotor network in newborn mice

In newborn mice of the control [C3H/HeJ (C3H)] and monoamine oxidase A‐deficient (Tg8) strains, in which levels of endogenous serotonin (5‐HT) were drastically increased, we investigated how 5‐HT system dysregulation affected the maturation of phrenic motoneurons (PhMns), which innervate the diaphragm. First, using immunocytochemistry and confocal microscopy, we observed a 5‐HT2A receptor (5‐HT2A‐R) expression in PhMns of both C3H and Tg8 neonates at the somatic and dendritic levels, whereas 5‐HT1B receptor (5‐HT1B‐R) expression was observed only in Tg8 PhMns at the somatic level. We investigated the interactions between 5‐HT2A‐R and 5‐HT1B‐R during maturation by treating pregnant C3H mice with a 5‐HT2A‐R agonist (2,5‐dimethoxy‐4‐iodoamphetamine hydrochloride). This pharmacological overactivation of 5‐HT2A‐R induced a somatic expression of 5‐HT1B‐R in PhMns of their progeny. Conversely, treatment of pregnant Tg8 mice with a 5‐HT2A‐R antagonist (ketanserin) decreased the 5‐HT1B‐R density in PhMns of their progeny. Second, using retrograde transneuronal tracing with rabies virus injected into the diaphragm of Tg8 and C3H neonates, we studied the organization of the premotor network driving PhMns. The interneuronal network monosynaptically connected to PhMns was much more extensive in Tg8 than in C3H neonates. However, treatment of pregnant C3H mice with 2,5‐dimethoxy‐4‐iodoamphetamine hydrochloride switched the premotoneuronal network of their progeny from a C3H‐ to a Tg8‐like pattern. These results show that a prenatal 5‐HT excess affects, via the overactivation of 5‐HT2A‐R, the expression of 5‐HT1B‐R in PhMns and the organization of their premotor network.

Involvement of adenosinergic A1 systems in the occurrence of respiratory perturbations encountered in newborns following an in utero caffeine exposure. a study on brainstem-spinal cord preparations isolated from newborn rats.

Involvement of adenosinergic A1 systems in the occurrence of respiratory perturbations encountered in newborns following an in utero caffeine exposure has been investigated on pontomedullary-spinal cord, caudal pons-medullary-spinal cord and medullary-spinal cord preparations isolated from newborn rats. According to the drinking fluid of dams (tap water or 0.02% caffeine), two groups of preparations were distinguished, no-caffeine and caffeine. In the no-caffeine group, adenosine A1 receptor activation induces a decrease in respiratory frequency (Rf) in caudal pons-medullary-spinal cord and medullary-spinal cord preparations whereas, in presence of the rostral pons, an increase is observed. A parallel Fos detection indicates that this discrepancy may be due to the excitatory action of the medial parabrachial nucleus at the rostral pontine level that surpasses inhibitory influence of the adenosine A1 receptor activation at the medullary level particularly in the ventrolateral reticular nucleus of the medulla. In caffeine group, an increase in the baseline Rf in presence of the pons and no change in medullary-spinal cord preparations have been observed. Depending on Fos detection, we assume that the medial parabrachial nucleus is the main region involved in the exaggeration of Rf. Moreover, adenosine A1 receptor activation was modified by in utero caffeine exposure with an overcharge of the Rf increase in pontomedullary-spinal cord preparations and an exaggeration of the Rf decrease in medullary-spinal cord preparations. Based on Fos detection, we link the overcharge in Rf of pontomedullary spinal cord preparations to an increase in the medial parabrachial nucleus neuronal activity. Similarly, exaggeration of Rf decrease observed without the pons is linked with a decrease in activity of the ventrolateral reticular neurons. This study brings evidence for the involvement of adenosinergic A1 systems in the occurrence of respiratory perturbations in newborns following in utero caffeine exposure and the importance of rostral pons in the adenosinergic A1 modulation of the respiratory control.

Brain stem projections by axonal collaterals to the rostral and caudal ventral respiratory group in the rat

The location of neurons projecting by axonal collaterals to the rostral and caudal ventral respiratory group (VRG) regions was determined after discrete injections of Fast blue and Diamidino yellow into the physiologically identified rostral inspiratory VRG and the caudal expiratory VRG areas, respectively. In contrast with single fluorochrome labeled neurons found throughout the rostro-caudal extent of the medulla and pons (in a variety of areas known to have cardiorespiratory function), double-labeled neurons were located in discrete ponto-medullary regions. The largest number of the double-labeled neurons was counted within the peripheral facial area, lateral paragigantocellular nucleus, and the VRG region, ipsi- and contralaterally to the injected side. Only a few double-labeled neurons were found within the ventrolateral and intermediate subnuclei of the solitary tract, medial parabrachial, and Kölliker-Fuse nuclei. The possible physiological implications of this neuronal network have also been emphasized.