Pedro Nunez-Abades

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.

Physiological changes accompanying anatomical remodeling of mammalian motoneurons during postnatal development.

The development of respiratory motoneurons provides unique data that may be generalized to other mammalian motoneuron populations. Like other motoneurons, respiratory motoneurons undergo developmental changes in the shape of the action potential and their repetitive firing. The unique observations concern the postnatal change in the recruitment pattern of cat phrenic motoneurons that is correlated with a halving of mean input resistance, a stasis of growth in the cell membrane and a reduction in the complexity of the dendritic tree. A similar pattern of change was observed for hypoglossal motoneurons studied in rat brainstem slices. Without an increase in total membrane surface area, the decreased resistance must result from a reduced specific membrane resistance. Two mechanisms are proposed to explain this decrease in resistance: proliferation and redistribution of either synaptic inputs and/or potassium channels. Although there was a significant contribution of synaptic input in determining input resistance throughout postnatal development, it was the density of cesium- or barium-sensitive potassium conductances that differentiated low resistance from high resistance motoneurons. Low resistance motoneurons had more cesium- and barium-sensitive channels than their high resistance counterparts. Based on the variations in the relative changes observed in input resistance versus membrane time constant with these two potassium channel blockers (cesium and barium), it is proposed that the distribution of these potassium channels change with age. Initially, their distribution is skewed toward the dendrites but as development progresses, the distribution becomes more uniform across the motoneuron membrane. During postnatal development, the rapid decrease in input resistance results from a proliferation of potassium channels in the membrane and of synaptic inputs converging onto developing respiratory motoneurons while the membrane is being spatially redistributed but not expanded.

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.

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.

Distribution of bulbospinal neurons supplying bilateral innervation to the phrenic nucleus in the rat.

The location of bulbospinal neurons with axon collaterals in both phrenic nuclei were determined by injecting two different fluorescent tracers into the right and left C4 cervical spinal cord. In contrast to single-labeled neurons that were found throughout the rostrocaudal extent of the medulla, the majority of double-labeled neurons were located in the rostral ventral respiratory group. Only a few double-labeled neurons were found in the ventrolateral nucleus of the solitary tract. The role of this bilateral pathway in synchronizing the activity of the phrenic nucleus is discussed.

Afferent connectivity to different functional zones of the optic tectum in goldfish.

This work studies the afferent connectivity to different functionally identified tectal zones in goldfish. The sources of afferents contributed to different degrees to the functionally defined zones. The dorsocentral area of the telencephalon was connected mainly with the ipsilateral anteromedial tectal zone. At diencephalic levels, neurons were found in three different regions: preoptic, thalamic, and pretectal. Preoptic structures (suprachiasmatic and preoptic nuclei) projected mainly to the anteromedial tectal zone, whereas thalamic (ventral and dorsal) and pretectal (central, superficial, and posterior commissure) nuclei projected to all divisions of the tectum. In the mesencephalon, the mesencephalic reticular formation, torus longitudinalis, torus semicircularis, and nucleus isthmi were, in the anteroposterior axis, topographically connected with the tectum. In addition, neurons in the contralateral tectum projected to the injected zones in a symmetrical point-to-point correspondence. At rhombencephalic levels, the superior reticular formation was connected to all studied tectal zones, whereas medial and inferior reticular formations were connected with medial and posterior tectal zones. The present results support a different quantitative afferent connectivity to each tectal zone, possibly based on the sensorimotor transformations that the optic tectum carries out to generate orienting responses.

Connectivity of the goldfish optic tectum with the mesencephalic and rhombencephalic reticular formation

The optic tectum of goldfish, as in other vertebrates, plays a major role in the generation of orienting movements, including eye saccades. To perform these movements, the optic tectum sends a motor command through the mesencephalic and rhombencephalic reticular formation, to the extraocular motoneurons. Furthermore, the tectal command is adjusted by a feedback signal arising from the reticular targets. Since the features of the motor command change with respect to the tectal site, the present work was devoted to determining, quantitatively, the particular reciprocal connectivity between the reticular regions and tectal sites having different motor properties. With this aim, the bidirectional tracer, biotin dextran amine, was injected into anteromedial tectal sites, where eye movements with small horizontal and large vertical components were evoked, or into posteromedial tectal sites, where eye movements with large horizontal and small vertical components were evoked. Labeled boutons and somas were then located and counted in the reticular formation. Both were more numerous in the mesencephalon than in the rhombencephalon, and ipsilaterally than contralaterally, with respect to the injection site. Furthermore, the somas showed a tendency to be located in the area containing the most dense labeling of synaptic endings. In addition, labeled boutons were often observed in close association with retrogradely stained neurons, suggesting the presence of a tectoreticular feedback circuit. Following the injection in the anteromedial tectum, most of the boutons and labeled neurons were found in the reticular formation rostral to the oculomotor nucleus. Conversely, following the injection in the posteromedial tectum, most of the boutons and neurons were also located in the caudal mesencephalic reticular formation. Finally, boutons and neurons were found in the rhombencephalic reticular formation surrounding the abducens nucleus. They were more numerous following the injection in the posteromedial tectum. These results demonstrate characteristic patterns of reciprocal connectivity between physiologically different tectal sites and the mesencephalic and rhombencephalic reticular formation. These patterns are discussed in the framework of the neural substratum that underlies the codification of orienting movements in goldfish.

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.

Phasic and tonic firing properties in rat oculomotor nucleus motoneurons, studied in vitro

Alert‐chronic studies show that ocular motoneurons (Mns) exhibit a phasic and tonic firing correlated with eye saccade‐velocity and position (fixation), respectively. Differences in the phasic and tonic firing among Mns depend on synaptic inputs and/or the intrinsic membrane properties. We have used in vitro slice preparation to investigate the contribution of membrane properties to firing properties of Wistar rat oculomotor nucleus Mns. We recorded different discharge patterns and focused on Mns with sustained discharge (type I) because they were the most abundant, and their firing pattern resembles that reported in alert preparations. Various differences divided these Mns into types IA and IB; the afterhyperpolarization (AHP) phase of the spike was monophasic in IA and biphasic in IB; IA Mns showed tonic or phasic‐tonic firing depending on the current intensity, while IB Mns showed phasic‐tonic discharge; the phasic firing was higher in IB than in IA Mns; IA Mns fired in a narrower range than did IB Mns; and IA Mns showed lower maximum frequency than did IB Mns. In conclusion, IA and IB Mns show different phasic firing properties and dynamic range, supported by intrinsic membrane properties. We suggest that IA and IB Mns innervate fast‐twitch muscle fibres with different contraction speeds, and could contribute to generating a fine phasic signal for a graded muscle contraction. Finally, we have demonstrated an inverse relationship between Mn thresholds and tonic firing gain, concluding that intrinsic membrane properties could not support the covariation between tonic firing gain and recruitment thresholds reported in alert studies.

Muscarinic Modulation of Recruitment Threshold and Firing Rate in Rat Oculomotor Nucleus Motoneurons

Above recruitment threshold, ocular motoneurons (Mns) show a firing rate linearly related with eye position. Current hypothesis suggests that synaptic inputs are determinant for establishing the recruitment threshold and firing rate gain in these Mns. We investigated this proposal by studying the cholinergic modulation in oculomotor nucleus Mns by intracellular recordings in rat brain slice preparation. All recorded Mns were silent at their resting membrane potential. Bath application of carbachol (10 microm) produced a depolarization and a sustained firing that was not silenced on returning membrane potential to the precarbachol value via DC injection. In response to similar membrane depolarization or equal-current steps, carbachol-exposed Mns produced a higher firing rate and a shorter spike afterhyperpolarization phase with lower amplitude. The relationship between injected current and firing rate (I-F) was linear in control and carbachol-exposed Mns. The slope of these relationships (I-F gain) decreased with carbachol exposure. Bath application of agonist and antagonist of nicotinic and muscarinic acetylcholine receptors in addition to immunohistochemical studies support the notion that muscarinic receptors are primarily involved in the preceding responses. We conclude that muscarinic inputs play an important role in determining the recruitment threshold and firing rate gain observed in oculomotor Mns in vivo.