Systemic inhibition of the Na(+)/H (+) exchanger type 3 in intact rats activates brainstem respiratory regions.

Selective inhibition of the Na(+)/H(+) exchanger type 3 (NHE3) increases the firing rate of brainstem ventrolateral CO(2)/H(+) sensitive neurons, resembling the responses evoked by hypercapnic stimuli. In anesthetized animals, NHE3 inhibition has also been shown to stimulate the central chemosensitive drive. We aimed to analyze the respiratory-related brainstem regions affected by NHE3 inhibition in anaesthetized spontaneously-breathing rats with intact peripheral afferents. For that, c-Fos immunopositive cells were counted along the brainstem in rats intravenously infused with the selective NHE3 inhibitor AVE1599. A rostral extension of the ventral respiratory column which includes the pre-Bötzinger complex was activated by the NHE3 inhibitor. In addition, the number of c-Fos positive cells resulted significantly increased in the most rostral extension of the retrotrapezoid nucleus/parapyramidal region. In the pons, the intravenous infusion of AVE1599 activated the lateral parabrachial and Kölliker-Fuse nuclei. Thus, selective NHE3 inhibition in anaesthetized rats activates the respiratory network and evokes a pattern of c-Fos expressing cells similar to that induced by hypercapnia.

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.

Highly H+-sensitive neurons in the caudal ventrolateral medulla of the rat.

The ventral surface of the caudal ventrolateral medulla (cVLM) has been shown to generate intense respiratory responses after surface acid-base stimulation. With respect to their chemosensitive characteristics, cVLM neurons have been less studied than other rostral-most regions of the brainstem. The purpose of these experiments was to determine the bioelectric responses of cVLM neurons to acidic stimuli and to determine their chemosensitive properties. Using extracellular and microiontophoretic techniques, we recorded electrical activities from 117 neurons in an area close to the ventral surface of the cVLM in anaesthetised rats. All neurons were tested for their sensitivity to H+. The fluorescent probe BCECF was used to measure extracellular pH changes produced by the microiontophoretic injection of H+ in brainstem slices. This procedure provided an estimation of the local changes in pH produced by microiontophoretic H+ application in the anaesthetised rat. Neurons coupled to the respiratory cycle, R (n = 51), were not responsive to direct stimulation with H+. Sixty-six neurons that did respond to H+ stimulation were uncoupled from respiration, and identified as NR neurons. These neurons presented distinct ranges of H+ sensitivity. The neuronal sensitivity to H+ was mainly assessed by the slope of the stimulus-response curve, where the steeper the slope, the higher the H+ sensitivity. On this basis, NR neurons were classed as being either weakly or highly sensitive to H+. NR neurons with a high H+ sensitivity (n = 12) showed an average value of 34.17 +/- 7.44 spikes s-1 (100 nC)-1 (mean +/- S.D.) for maximal slope and an EC50 of 126.76 +/- 33 nC. Suprathreshold H+ stimulation of highly sensitive NR neurons elicited bursting pattern responses coupled to the respiratory cycle. The bursting responses, which were synchronised with the inspiratory phase and the early expiratory phase of the respiratory cycle, lasted for several seconds before returning to the steady state firing pattern characteristic of the pre-stimulus condition. These NR neurons, which possess the capacity to detect distinct H+ concentrations in the extracellular microenvironment, are excellent candidates to serve in a chemoreceptor capacity in the caudal medulla.

Altered respiratory activity and respiratory regulations in adult monoamine oxidase A-deficient mice.

The abnormal metabolism of serotonin during the perinatal period alters respiratory network maturation at birth as revealed by comparing the monoamine oxidase A-deficient transgenic (Tg8) with the control (C3H) mice (Bou-Flores et al., 2000). To know whether these alterations occur only transiently or induce persistent respiratory dysfunction during adulthood, we studied the respiratory activity and regulations in adult C3H and Tg8 mice. First, plethysmographic and pneumotachographic analyses of breathing patterns revealed weaker tidal volumes and shorter inspiratory durations in Tg8 than in C3H mice. Second, electrophysiological studies showed that the firing activity of inspiratory medullary neurons and phrenic motoneurons is higher in Tg8 mice and that of the intercostal motoneurons in C3H mice. Third, histological studies indicated abnormally large cell bodies of Tg8 intercostal but not phrenic motoneurons. Finally, respiratory responses to hypoxia and lung inflation are weaker in Tg8 than in C3H mice. dl-p-chlorophenyl-alanine treatments applied to Tg8 mice depress the high serotonin level present during adulthood; the treated mice recover normal respiratory responses to both hypoxia and lung inflation, but their breathing parameters are not significantly affected. Therefore in Tg8 mice the high serotonin level occurring during the perinatal period alters respiratory network maturation and produces a permanent respiratory dysfunction, whereas the high serotonin level present in adults alters the respiratory regulatory processes. In conclusion, the metabolism of serotonin plays a crucial role in the maturation of the respiratory network and in both the respiratory activity and the respiratory regulations.

Efferent projection from the rostral ventrolateral medulla to the area postrema in rats.

The rostral ventrolateral medulla (RVLM) is a region of the brain primarily involved in cardiovascular control. It receives information from several areas of the brainstem, among which the area postrema (AP) and the nucleus of the solitary tract (NTS). The medial subnuclei of the solitary tract (TS) project towards the RVLM, providing cardiopulmonary information, and the AP serves information about circulatory hormones. Although the efferent pathways are well known, it is not the case for the connections from the RVLM towards the AP and the NTS. The present study was designed to examine the efferent connections from the RVLM onto the dorsal structures of the medulla: quantitatively by means of anatomical techniques, and functionally by means of electrophysiological techniques. Morphologically, Biocytin or Biotinylated dextran amine microinjections into the RVLM were followed by labelling of many fibres running towards the bulbar dorsomedial structures, with some pathways lying in the AP itself, or located in its caudal vicinity. Conversely, when microinjections of Fast Blue (FB) were made into the AP, FB-labelled cells could be observed within the RVLM. Electrophysiologically, single shock stimulation carried on AP allowed identification of axonal fibres issuing from somata located into the cardiovascular neuronal pool in the RVLM. From these results, we can assume: (1) the existence of dense efferent projection from RVLM to aspects of the dorsal vagal complex, including the AP and, among this dense projection, (2) the existence of some fibres terminating in, or crossing through the AP, and identified as conveying baroreceptor-related information, in the rat.

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.

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.

Brainstem connections of the rat ventral respiratory subgroups: afferent projections.

Propriobulbar neurons having axonal projections to the Ventral Respiratory Group (VRG) were retrogradely labeled after discrete injections of Fast blue into one of the three physiologically identified subdivisions (Bötzinger Complex, rostral inspiratory and caudal expiratory regions). Neurons that project to these regions were found throughout the rostrocaudal extent of the medulla and the pons in a variety of areas known to have cardio-respiratory function. Labeled somata were located within the nuclei of the solitary tract (commissural, intermediate and ventrolateral), other subdivisions of VRG, parabrachial nuclei (medial, dorsolateral and central lateral), Kölliker-Fuse nucleus, retrotrapezoid nucleus, lateral paragigantocellular nucleus and lateral tegmental field of the pons. Within the nuclei of the solitary tract and the Kölliker-Fuse nucleus, there was a topographical organization with respect to the three subdivisions of the VRG.

A morphological study of the principal and accessory abducens nuclei in the caspian terrapin (Mauremys caspica).

The location of principal and accessory motoneurons and principal interneurons of the nucleus abducens was determined in the caspian terrapin (Mauremys caspica) by means of horseradish peroxidase histochemical tracing. Enzyme injections were made into the ipsilateral lateral rectus and retractor bulbi muscles and into the contralateral oculomotor nucleus. Labeled principal abducens motoneurons formed a cluster of cells in the rhombencephalon, under the IVth ventricle and adjacent to the medial longitudinal fascicle. The accessory abducens motoneurons were located more deeply in the rhombencephalon and more ventrolaterally than the principal motoneurons forming a compact aggregation of neurons. The principal interneurons of abducens nucleus were arranged as a cluster of cells under the floor of the IVth ventricle and more lateral than the principal motoneurons, with no intermingling.

A morphological study of abducens nucleus motoneurons and internuclear neurons in the goldfish (Carassius auratus).

The location and distribution of abducens (ABD) nucleus motoneurons (Mn) and internuclear neurons (Int) were determined in the goldfish (Carassius auratus) by means of horseradish peroxidase and fluorochrome retrograde labeling. ABD Mn were labeled following tracer injection into the ipsilateral lateral rectus muscle. These Mn were arranged in two ventrolateral clusters along the rostro-caudal axis of the posterior brainstem. Both groups of neurons showed a similar number of cells, and their axons ran ventrally to their respective nerve roots. ABD Int were labeled following the injection of the tracer into the contralateral oculomotor nucleus. They also formed two distinct groups in the rostro-caudal axis. The rostral group of Int formed a dorso-lateral cap around the caudal motoneuronal pool, with little if any intermingling. The caudal group of Int was located at the same position in the dorso-ventral and medio-lateral axis as the rostral group, but 500 microns behind it. Both groups of ABD Int had a similar number of neurons. Int axons ascended dorso-medially, then crossed the midline through the internal arcuate fibers, and entered the contralateral medial longitudinal fasciculus. The soma diameters of both ABD Mn and Int were not significantly different. The relative location of both types of neurons is discussed.

Study of the topographical distribution of different populations of motoneurons within rat's nucleus ambiguus, by means of four different fluorochromes.

The topographical neuronal distribution within the rat nucleus ambiguus has been studied with the simultaneous retrograde labeling technique by means of four different fluorochromes injected within the various muscles and/or nerves of the oro-pharyngeal region. This technique has permitted the identification of several types of neurons along the same coronal plane. Most were motoneurons innervating the various muscles of the upper airway, including pharyngeal constrictor, stylopharyngeal, intrinsic laryngeal and the upper portion of the esophagus. Some neurons may have been preganglionic parasympathetic neurons. No evidence of axonal branching of any of the labeled motoneurons or parasympathetic neurons was found.

Localization of respiratory bulbospinal and propriobulbar neurons in the region of the nucleus ambiguus of the rat.

The location of neurons within the ventral respiratory group (VRG) of rat was mapped following injections of 3 different fluorochrome tracers into different sites known to receive projections from VRG neurons. Injection sites included muscles innervated by the vagus (X) and glossopharyngeal (IX) nerves, and the sites of expiratory activity in the caudal medulla and of inspiratory activity in the spinal cord at the C4 level. Labeling of vagal motoneurons resulting from fluorochrome injections into muscles innervated by X and IX nerves was always ipsilateral to the site of injection. Both propriobulbar and bulbospinal neurons had primarily ipsilateral projections. No double-labeled cell bodies were observed. The cell bodies of the 3 types of neurons, propriobulbar, bulbospinal and vagal/glossopharyngeal, were unevenly distributed along the rostrocaudal axis of the VRG, suggesting a complex mosaic of neurons which regulate respiratory-related functions such as swallowing and vocalization.

Characterisation of afferent projections to the nucleus ambiguus of the rat by means of fluorescent double labelling.

The rat nucleus ambiguus (Amb nucleus) is composed mainly of laryngeal and pharyngeal motoneurons, and can be differentiated into three main subdivisions with respect to the morphology, distribution and physiological function of their neurons. The medial parabrachial and Kölliker-Fuse nuclei (MPB/KF), and the lateral nucleus of the solitary tract (SolL) play a modulator role over some of the physiological functions assigned to the Amb nucleus. The location and characteristics of labelled neurons in the MPB/KF, SolL and Amb nuclei have been studied following the simultaneous and bilateral injections of True blue and Diamidino yellow in each of the three subdivisions of the Amb nucleus. All of the identified axonal projections were mainly ipsilateral. Some of the described pathways also have collaterals which connect both sides of the Amb complex.

Cytoarchitectonic organisation of the abducens nucleus in the pigeon (Columbia livia).

The distribution of abducens nucleus motoneurons and internuclear neurons was determined in the pigeon (Columbia livia) by injecting horseradish peroxidase or fluorochromes into the ipsilateral lateral rectus muscle and/or in the contralateral oculomotor nucleus. A small degree of intermingling of motoneurons and internuclear neurons in the caudal two thirds of the nucleus and an almost complete segregation of both types of neurons in the rostral third was observed. Both labelled populations, motoneurons and internuclear neurons, were more numerous at the central part of the abducens nucleus. Motoneurons were preferentially located nearer the medial longitudinal fasciculus and less numerous at the rostral end of the nucleus. Internuclear neurons were preferentially located further from the medial longitudinal fasciculus and were less abundant at the caudal end of the nucleus. The evolutionary trend of the relative location of both types of neurons is discussed in relation to the degree of conjugate eye movements.

Location of bulbospinal neurons and of laryngeal motoneurons within the nucleus ambiguus of the rat and cat by means of retrograde fluorescent labelling.

The location of bulbospinal neurons within the nucleus ambiguus with respect to laryngeal motoneurons has been studied by means of retrograde fluorescent neuronal markers (True Blue and Diamidino Yellow in rats, and Fast Blue and Diamidino Yellow in cats). One marker was injected into the cervical spinal cord, and the other into the intrinsic laryngeal muscles. Afterwards, the precise location of each neuronal pool was observed with the fluorescent microscope. The bulbospinal neurons in the rostral part of the nucleus were located ventrolaterally with respect to motoneurons, both in cats and rats; at more caudal levels of the nucleus the bulbospinal neurons were arranged ventromedially and ventrolaterally with respect to motoneurons in cats, and around the motoneurons in rats.

Location of motoneurons and internuclear neurons within the rat abducens nucleus by means of horseradish peroxidase and fluorescent double labeling.

The distribution of abducens motoneurons and internuclear neurons was determined in the rat by injections of horseradish peroxidase or fluorochromes into the ipsilateral lateral rectus muscle and the contralateral oculomotor nucleus either separately or simultaneously. The labeled somata of abducens internuclear neurons were intermingled with the labeled motoneurons at the medial third of the nucleus, but they were more segregated at the rostral third, where the labeled interneurons were more numerous. Internuclear neurons were preferentially located around and ventral to the central part of the facial genu, while motoneurons were located more dorsomedially, closer to the midline than in other species of mammals. The evolutionary trend of the location of both populations of neurons is also discussed.

Behavioral and morphological effects of oculomotor nucleus lesion on abducens internuclear neurons in the cat.

The activity of identified control and injured abducens internuclear neurons was recorded during spontaneous eye movements in the alert cat. From 2 days following the electrocoagulation of the contralateral oculomotor nucleus, abducens internuclear neurons showed a quick fatigability during eye fixations not observed in controls. Discharge rate during saccades started after the beginning of the eye movement and showed a fast exponential-like decay. These abnormal responses were not further recorded from 20 days following the lesion. Morphological studies indicated that 90% of the abducens internuclear neuron population disappeared 2 months following the lesion and showed no sign of recovery one year later.

Location of motoneurons supplying the intrinsic laryngeal muscles of rats. Horseradish peroxidase and fluorescence double-labeling study.

This paper describes a qualitative and quantitative investigation of the location of the motoneurons innervating the intrinsic laryngeal muscles of rats. Injections of horseradish peroxidase, Diamidino Yellow and True Blue were made either in one or, simultaneously, in three laryngeal muscles. Unlike those in cats and rabbits, the motoneurons that make up the nucleus ambiguus (NA) in rats are not arranged in two separate subgroups, that is one belonging to the cricothyroid (CT) motoneurons and the other to the rest of the intrinsic laryngeal motoneurons. Instead, a superimposition of CT and posterior cricoarytenoid (PCA) motoneurons was observed in the rostral third of the NA. Motoneurons innervating the PCA, thyroarytenoid (TA) and lateral cricoarytenoid (LCA) muscle overlap in the medial third of the NA. Finally, in the region of the NA caudal to the obex, the TA and LCA motoneurons also overlap. Labeled motoneurons were located in the ipsilateral side to the injected muscle in all cases.

Axonal projections to the ventrolateral nucleus of the solitary tract revealed by double labelling of retrograde fluorescent markers in the cat.

The retrofacial nucleus project bilaterally to the ventrolateral nucleus of the tractus solitarius (vlNTS) as revealed by means of retrograde transport of the fluorescent markers, Fast blue (FB) and Diamidino yellow (DY), in the cat. Some of the neurons of the retrofacial nucleus send axonal ramifications to both vlNTS. Extensive projections from other brainstem respiratory related nuclei to the vlNTS were also observed: bilaterally from the nucleus ambiguus, nucleus retroambiguus and nucleus parabrachialis medialis, and ipsilaterally from the Kölliker-Fuse nucleus. Axonal projections from the contralateral vlNTS were also observed.