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.

Neural substrata underlying tectal eye movement codification in goldfish.

The optic tectum encodes orienting eye saccades in a spatially ordered map. To investigate whether the functional properties of each tectal site are related to a particular pattern of connectivity with downward structures in the brainstem, two sets of experiments were carried out. First, biotinylated dextran amine (BDA) was injected at different tectal sites along the anteroposterior axis. Electrical stimulation at these sites evoked saccades whose horizontal component amplitudes increased with the distance to the rostral pole. In the second experiment, BDA and fluoro-ruby (FR) were injected at different tectal sites along the mediolateral axis. Electrical stimulation here evoked saccades with different upward and downward directions, but similar horizontal component amplitudes. A major finding of the first experiment was that a topographic link of the tectum exists with the mesencephalic reticular formation, but that such a connection was absent or very attenuated for the rhombencephalic reticular formation. In the second set of experiments, the clusters of BDA and FR boutons left by the mediolateral tectal sites were separated in the rostral mesencephalon, at the level of the nucleus of the medial longitudinal fasciculus, but overlapped in the caudal mesencephalon and rhombencephalon. These data provide evidence that decodification of tectal motor commands is based, at least in part, on the connectivity of each tectal locus on downward structures with the brainstem.

Role of synaptic inputs in determining input resistance of developing brain stem motoneurons.

The contribution of synaptic input to input resistance was examined in 208 developing genioglossal motoneurons in 3 postnatal age groups (5-7 day, 13-16 day, and 18-24 day) using sharp electrode recording in a slice preparation of the rat brain stem. High magnesium (Mg(2+); 6 mM) media generated significant increases (21-38%) in both the input resistance (R(n)) and the first time constant (tau(0)) that were reversible. A large percent of the conductance blocked by high Mg(2+) was also sensitive to tetrodotoxin (TTX). Little increase in resistance was attained by adding blockers of specific amino acid (glutamate, glycine, and GABA) transmission over that obtained with the high Mg(2+). Comparing across age groups, there was a significantly larger percent change in R(n) with the addition of high Mg(2+) at postnatal days 13 to 15 (P13-15; 36%) than that found at P5-6 (21%). Spontaneous postsynaptic potentials were sensitive to the combined application of glycine receptor antagonist, strychnine, and the GABA(A) receptor antagonist, bicuculline. Application of either 10 microM strychnine or bicuculline separately produced a reversible increase in both R(n) and tau(0). Addition of 10 microM bicuculline to a strychnine perfusate failed to further increase either R(n) or tau(0). The strychnine/bicuculline-sensitive component of the total synaptic conductance increased with age so that this form of neurotransmission constituted the majority (>60%) of the observed percent decrease in R(n) and tau(0) in the oldest age group. The proportion of change in tau(0) relative to R(n) following strychnine or high magnesium perfusate varied widely from cell to cell and from age to age without pattern. Based on a model from the literature, this pattern indicates a nonselective distribution of the blocked synaptic conductances over the cell body and dendrites. Taken together, the fast inhibitory synapses (glycine, GABA(A)) play a greater role in determining cell excitability in developing brain stem motoneurons as postnatal development progresses. These findings suggest that synaptically mediated conductances effect the membrane behavior of developing motoneurons.

Role of potassium conductances in determining input resistance of developing brain stem motoneurons.

The role of potassium conductances in determining input resistance was studied in 166 genioglossal (GG) motoneurons using sharp electrode recording in brain stem slices of the rats aged 5-7 days, 13-15 days, and 19-24 days postnatal (P). A high magnesium (Mg(2+); 6 mM) perfusate was used to block calcium-mediated synaptic release while intracellular or extracellular cesium (Cs(+)) and/or extracellular tetraethylammonium (TEA) or barium (Ba(2+)) were used to block potassium conductances. In all cases, the addition of TEA to the high Mg(2+) perfusate generated a larger increase in both input resistance (R(n)) and the first membrane time constant (tau(0)) than did high Mg(2+) alone indicating a substantial nonsynaptic contribution to input resistance. With intracellular injection of Cs(+), GG motoneurons with lower resistance (<40 MOmega), on the average, showed a larger percent increase in R(n) than cells with higher resistance (>40 MOmega). There was also a significant increase in the effect of internal Cs(+) on R(n) and tau(0) with age. The largest percent increase (67%) in the tau(0) due to intracellular Cs(+) occurred at P13-15, a developmental stage characterized by a large reduction in specific membrane resistance. Addition of external Cs(+) blocked conductances (further increasing R(n) and tau(0)) beyond those blocked by the TEA perfusate. Substitution of external calcium with 2 mM barium chloride produced a significant increase in both R(n) and tau(0) at all ages studied. The addition of either intracellular Cs(+) or extracellular Ba(2+) created a depolarization shift of the membrane potential. The amount of injected current required to maintain the membrane potential was negatively correlated with the control R(n) of the cell at most ages. Thus low resistance cells had, on the average, more Cs(+)- and Ba(2+)-sensitive channels than their high resistance counterparts. There was also a disproportionately larger percent increase in tau(0) as compared with R(n) for both internal Cs(+) and external Ba(2+). Based on a model by Redman and colleagues, it might be suggested that the majority of these potassium conductances underlying membrane resistance are initially located in the distal dendrites but become more uniformly distributed over the motoneuron surface in the oldest animals.

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.

Relationship between membrane properties and cell size of developing rat genioglossal motoneurons studied in vitro.

Electrical properties and morphology of 21 genioglossal motoneurons were measured in a slice preparation of the rat brainstem at four different postnatal ages. The motoneurons labeled with neurobiotin were reconstructed and quantified in three-dimensional space. There was no strong correlation found between the input resistance or membrane time constant and the total membrane surface area. We conclude that there is no electrical property of these developing motoneurons that can accurately predict their anatomical size.

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.

Morphology of developing rat genioglossal motoneurons studied in vitro: relative changes in diameter and surface area of somata and dendrites.

This study describes the postnatal change in size of motoneurons in the hypoglossal nucleus that innervate the genioglossus muscle. Such anatomical information is essential for determining the cellular mechanisms responsible for the changes observed in the electrical properties of these motoneurons during postnatal development. The cells analyzed here are part of an earlier study (Núñez-Abades et al. [1994] J. Comp. Neurol. 339:401-420) where 40 genioglossal (GG) motoneurons from four age groups (1-2, 5-6, 13-15, and 19-30 postnatal days) were labeled by intracellular injection of neurobiotin in an in vitro slice preparation of the rat brainstem and their cellular morphology was reconstructed in three-dimensional space. The sequence of postnatal dendritic growth can be described in two phases. The first phase, between birth (1-2 days) and 13-15 days, was characterized by no change in either dendritic diameter (any branch order) or dendritic surface area of GG motoneurons. However, maturation of the dendritic tree produced more surface area at greater distances from the soma by redistributing existing membrane (retracting some terminal branches). During the second phase, between 13-15 days and 19-30 days, the dendritic surface area doubled as a result of an increase in the dendritic diameter across all branch orders and a generation of new terminal branches. In contrast to the growth exhibited by the dendrites, there was little discernible postnatal growth of somata. At all ages, dendrites of GG motoneurons show the largest amount of tapering in the first-and second-order dendrites. The calculated dendritic trunk parameter deviated from a value 1.0, indicating that the dendritic tree of developing GG motoneurons cannot be modeled accurately as an equivalent cylinder. However, the value of this parameter increased with age. Strong correlations were found between the diameter of the first-order dendrite and the dendritic surface area, dendritic volume, combined dendritic length, and, to a lesser extent, the number of terminal dendrites in GG motoneurons. Correlations were also found between somal and dendritic geometry but only when data were pooled across all age groups. These data support earlier studies on kitten phrenic motoneurons, which concluded that postnatal growth of motoneurons was not a continuous process. Based on the fact that there was no growth in the first 2 weeks, the changes in the membrane properties described during this phase of postnatal development (e.g., decrease in input resistance) cannot be attributed to increases in the total membrane surface area of these motoneurons.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Morphology of developing rat genioglossal motoneurons studied in vitro: changes in length, branching pattern, and spatial distribution of dendrites.

The aim of this study is to describe the postnatal change in dendritic morphology of those motoneurons in the hypoglossal nucleus that innervate the genioglossus muscle. Forty genioglossal (GG) motoneurons from four age groups (1-2, 5-6, 13-15, and 19-30 postnatal days) were labeled by intracellular injection of neurobiotin in an in vitro slice preparation of the rat brainstem and were reconstructed in three-dimensional space. The number of primary dendrites per GG motoneuron was approximately 6 and remained unchanged with age. The development of these motoneurons from birth to 13-15 days was characterized by a simplification of the dendritic tree involving a decrease in the number of terminal endings and dendritic branches. Motoneurons lost their 6th-8th order branches, in parallel with an elongation of their terminal dendritic branches maintaining the same combined dendritic length. The elongation of terminal branches was attributed to both longitudinal growth and the apparent lengthening caused by resorption of distal branches. The elimination of dendritic branches tended to increase the symmetry of the tree, as revealed by topological analysis. Later, between 13-15 days and 19-30 days, there was a reelaboration of the dendritic arborization returning to a configuration similar to that found in the newborn. The length of terminal branches was shorter at 19-30 days, while the length of preterminal branches did not change, suggesting that the proliferation of branches at 19-30 days takes place in the intermediate parts of terminal branches. The three-dimensional distribution of dendrites was analyzed by dividing space into six equal volumes (hexants). This analysis revealed that GG motoneurons have major components of their dendritic tree oriented in the lateral, medial, and dorsal hexants. Further two-dimensional polar analysis (consisting of eight sectors) revealed a reconfiguration of the tree from birth up to 5-6 days involving resorption of dendrites in the dorsal, dorsomedial, and medial sectors and growth in the lateral sector. Later in development (between 13-15 days and 19-30 days), there was growth in all sectors, but of a greater magnitude in the dorsomedial, medial, and dorsolateral sectors.

In vitro electrophysiology of developing genioglossal motoneurons in the rat.

1. Experiments were performed to determine the change in membrane properties of genioglossal (GG) motoneurons during development. Intracellular recordings were made in 127 GG motoneurons from rats postnatal ages 1-30 days. 2. The input resistance (R(in)) and the membrane time constant (t(aum)) decreased between 5-6 and 13-15 days from 84.8 +/- 25.4 (SD) to 47.0 +/- 18.9 M omega (P < 0.01) and from 10.0 +/- 4.2 to 7.3 +/- 3.3 ms (P < 0.05), respectively. During this period, the rheobase (Irh) increased (P < 0.01) from 0.13 +/- 0.07 to 0.27 +/- 0.14 nA, and the percentage of cells exhibiting inward rectification increased from 5 to 40%. Voltage threshold (Vthr) of the action potential remained unchanged postnatally. 3. There was also a postnatal change in the shape of the action potential. Specifically, between 1-2 and 5-6 days, there was a decrease (P < 0.05) in the spike half-width from 2.23 +/- 0.53 to 1.45 +/- 0.44 ms, resulting, in part, from a steepening (P < 0.05) of the slope of the falling phase of the action potential from 21.6 +/- 10.1 to 32.9 +/- 13.1 mV/ms. The slope of the rising phase also increased significantly (P < 0.01) between 1-2 and 13-15 days from 68.4 +/- 31.0 to 91.4 +/- 44.3 mV/ms. 4. The average duration of the medium afterhyperpolarization (mAHPdur) decreased (P < 0.05) between 1-2 (193 +/- 53 ms) and 5-6 days (159 +/- 43 ms). Whereas the mAHPdur was found to be independent of membrane potential, there was a linear relationship between the membrane potential and the amplitude of the medium AHP (mAHPamp). From this latter relationship, a reversal potential for the mAHPamp was extrapolated to be -87 mV. No evidence for the existence of a slow AHP was found in these developing motoneurons. 5. All cells analyzed (n = 74) displayed adaptation during the first three spikes. The subsequent firing pattern was classified into two groups, adapting and nonadapting. Cells at birth were all adapting, whereas all cells but two from animals 13 days and older were nonadapting. At the intermediate age (5-6 days), the minority (27%) was adapting and the majority (73%) was nonadapting. 6. The mean slope of primary range for the first interspike interval (1st ISI) was approximately 90 Hz/nA. This value was similar for both adapting and nonadapting cells and did not change postnatally.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Anatomical and electrotonic coupling in developing genioglossal motoneurons of the rat.

Dye-, tracer- and electrotonic coupling were studied independently in genioglossal (GG) motoneurons using intracellular recordings in in vitro brainstem slices from rats postnatal ages 1-30 days. The subpopulation of GG motoneurons were retrogradely labeled after an injection of dextran-rhodamine into the posterior tongue. Dye-coupling was studied with Lucifer yellow injected into 55 motoneurons and tracer-coupling with neurobiotin injected into 89 presumptive GG motoneurons. Of the motoneurons injected with Lucifer yellow, only 6 of 41 cells (16.2%) exhibited dye-coupling; all occurred in animals less than 9 days old. In all but one instance, dye-coupling was restricted to only one other cell. No evidence of dye-coupling was found in the 14 cells injected in animals older than 8 days. Tracer-coupling (neurobiotin) was demonstrated in 12 of 30 cells (40%) from animals 1-2 days old and in 6 of 21 cells (28.6%) from animals 3-8 days old. Of the remaining 38 cells from animals 10 days of age and older, only one cell was found to be tracer-coupled. Cells injected with neurobiotin were coupled to an average of two other cells. Electrotonic coupling, as demonstrated with a short latency depolarization (SLD) in response to stimulation of hypoglossal axons, was found in developing GG motoneurons. These SLDs were revealed in 17 of 40 GG motoneurons (42.5%) examined in 1-8-day-old animals. There were no SLDs recorded in the 10 cells examined from animals of 10 days and older. The significance of coupling relative to patency of the newborn upper airways is discussed.

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.

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.