Genioglossal Motoneurons Research Articles

Development of γ-aminobutyric acid-, glycine-, and glutamate-immunopositive boutons on the rat genioglossal motoneurons.

Detailed information about the development of excitatory and inhibitory synapses on the genioglossal (GG) motoneuron may help to understand the mechanism of fine control of GG motoneuron firing and the coordinated tongue movement during postnatal development. For this, we investigated the development of γ-aminobutyric acid (GABA)-immunopositive (GABA +), glycine + (Gly +), and glutamate + (Glut +) axon terminals (boutons) on the somata of rat GG motoneurons at a postnatal day 2 (P2), P6 and P18 by retrograde labeling of GG motoneurons with horseradish peroxidase, electron microscopic postembedding immunogold staining with GABA, Gly, and Glut antisera, and quantitative analysis. The number of boutons per GG motoneuron somata and the mean length of bouton apposition, measures of bouton size and synaptic covering percentage, were significantly increased from P2/P6 to P18. The number and fraction of GABA + only boutons of all boutons decreased significantly, whereas those of Gly + only boutons increased significantly from P2/P6 to P18, suggesting developmental switch from GABAergic to glycinergic synaptic transmission. The fraction of mixed GABA +/Gly + boutons of all boutons was the highest among inhibitory bouton types throughout the postnatal development. The fractions of excitatory and inhibitory boutons of all boutons remained unchanged during postnatal development. These findings reveal a distinct developmental pattern of inhibitory synapses on the GG motoneurons different from that on spinal or trigeminal motoneurons, which may have an important role in the regulation of the precise and coordinated movements of the tongue during the maturation of the oral motor system.

Distribution of excitatory and inhibitory axon terminals on the rat hypoglossal motoneurons.

Detailed information about the excitatory and inhibitory synapses on the hypoglossal motoneurons may help understand the neural mechanism for control of the hypoglossal motoneuron excitability and hence the precise and coordinated movements of the tongue during chewing, swallowing and licking. For this, we investigated the distribution of GABA-, glycine (Gly)- and glutamate (Glut)-immunopositive (+) axon terminals on the genioglossal (GG) motoneurons by retrograde tracing, electron microscopic immunohistochemistry, and quantitative analysis. Small GG motoneurons (< 400μm2 in cross-sectional area) had fewer primary dendrites, significantly higher nuclear/cytoplasmic ratio, and smaller membrane area covered by synaptic boutons than large GG motoneurons (> 400μm2). The fraction of inhibitory boutons (GABA + only, Gly + only, and mixed GABA +/Gly + boutons) of all boutons was significantly higher for small GG motoneurons than for large ones, whereas the fraction of Glut + boutons was significantly higher for large GG motoneurons than for small ones. Almost all boutons (> 95%) on both small and large GG motoneurons were GABA + , Gly + or Glut + . The frequency of mixed GABA +/Gly + boutons was the highest among inhibitory boutons types for both small and large GG motoneurons. These findings may elucidate the anatomical substrate for precise regulation of the motoneuron firing required for the fine movements of the tongue, and also suggest that the excitability of small and large GG motoneurons may be regulated differently.

Fiber connections of the caudal mesopallium in the pigeon

In the present study, immunohistochemistry combined with retrograde labeling techniques were used to determine if hypoglossal motoneurons (HMNs), retrogradely labeled after cholera toxin B subunit (CTB) injection to the genioglossus muscle in rats, show immunoreactivity for alpha-7 and alpha-4 subunits of nicotinic acetylcholine receptors (nAChRs). CTB-positive HMNs projecting to the genioglossus muscle were consistently labeled throughout the rostrocaudal extent of the hypoglossal nuclei with the greatest labeling at and caudal to area postrema. Alpha-7 subunit immunoreactivity was found in 39.44 ± 5.10% of 870 CTB-labeled motoneurons and the alpha-4 subunit in 51.01 ± 3.71% of 983 CTB-positive neurons. Rostrally, the number of genioglossal motoneurons demonstrating immunoreactivity for the alpha-7 subunit was 45.85 ± 10.04% compared to 34.96 ± 5.11% at and caudal to area postrema (P > 0.1). The number of genioglossal motoneurons that showed immunoreactivity for the alpha-4 subunit was 55.03 ± 4.83% at and caudal to area postrema compared to 42.98 ± 3.90% in rostral areas (P = 0.074). These results demonstrate that nAChR immunoreactivity is present in genioglossal motoneurons and suggest a role for alpha-7 and alpha-4 subunits containing nAChRs in the regulation of upper airway patency.

Retrograde labeling reveals extensive distribution of genioglossal motoneurons possessing 5-HT 2A receptors throughout the hypoglossal nucleus of adult dogs

Inspiratory hypoglossal motoneurons (IHMNs) innervate the muscles of the tongue and play an important role in maintaining upper airway patency. However, this may be reduced during sleep and by sedatives, potent analgesics, and volatile anesthetics. The genioglossal (GG) muscle is the main protruder and depressor muscle of the tongue and contributes to upper airway patency during inspiration. In vitro data suggest that serotonin (5-hydroxytryptamine, 5-HT), via the 5-HT 2A receptor (5-HT 2AR) subtype, plays a key role in controlling the excitability of IHMNs. The distribution of GG motoneurons (GGMNs) within the hypoglossal (XII) nucleus has not been studied in the adult dog. Further, it is uncertain whether the 5-HT 2AR is located on GGMNs in the adult dog. We therefore used the cholera toxin B (CTB) subunit as a retrograde tracer to map the location of GGMNs in combination with immunofluorescent labeling to determine the presence and colocalization of 5-HT 2AR within the XII nucleus in adult mongrel dogs. Injection of CTB into the GG muscle resulted in retrogradely labeled cells in a compact column throughout the XII nucleus, extending from 0.75 mm caudal to 3.45 mm rostral to the obex. Fluorescence immunohistochemistry revealed extensive 5-HT 2AR labeling on CTB-labeled GGMNs. Identification of the 5-HT 2AR on GGMNs in the XII nucleus of the adult dog supports in vitro data and suggests a physiological role for this receptor subtype in controlling the excitability of GGMNs, which contribute to the maintenance of upper airway patency.

Alpha-7 and alpha-4 nicotinic receptor subunit immunoreactivity in genioglossus muscle motoneurons

In the present study, immunohistochemistry combined with retrograde labeling techniques were used to determine if hypoglossal motoneurons (HMNs), retrogradely labeled after cholera toxin B subunit (CTB) injection to the genioglossus muscle in rats, show immunoreactivity for alpha-7 and alpha-4 subunits of nicotinic acetylcholine receptors (nAChRs). CTB-positive HMNs projecting to the genioglossus muscle were consistently labeled throughout the rostrocaudal extent of the hypoglossal nuclei with the greatest labeling at and caudal to area postrema. Alpha-7 subunit immunoreactivity was found in 39.44+/-5.10% of 870 CTB-labeled motoneurons and the alpha-4 subunit in 51.01+/-3.71% of 983 CTB-positive neurons. Rostrally, the number of genioglossal motoneurons demonstrating immunoreactivity for the alpha-7 subunit was 45.85+/-10.04% compared to 34.96+/-5.11% at and caudal to area postrema (P>0.1). The number of genioglossal motoneurons that showed immunoreactivity for the alpha-4 subunit was 55.03+/-4.83% at and caudal to area postrema compared to 42.98+/-3.90% in rostral areas (P=0.074). These results demonstrate that nAChR immunoreactivity is present in genioglossal motoneurons and suggest a role for alpha-7 and alpha-4 subunits containing nAChRs in the regulation of upper airway patency.

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.

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.

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.

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)

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)

Anatomical and electrotonic coupling in developing genioglossal motoneurons of the rat

Dye-, tracer- and electronic 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.

Postnatal growth of genioglossal motoneurons.

The postnatal growth of kitten genioglossal motoneurons were examined in six different age groups (newborn, 2, 4, 8, and 12 weeks and adult) using the technique of retrograde transport of horseradish peroxidase (HRP). The cell bodies of 100-150 motoneurons in each age group were analyzed in a transverse plane of section using standard techniques. Somatic genioglossal motoneuron growth occurred primarily along the major axis, which increased from 25.2 microns to 41.3 microns between birth and 8 weeks of postnatal age, after which time there was no further increase in either major or minor dimension of the cell body. The form factor decreased from 0.94 to 0.80 from birth to adulthood indicating an increased eccentricity of the cell body. The number of primary dendrites visible with this technique remained constant throughout the postnatal period. Calculated somal surface area increased in a linear fashion from birth through 8 weeks of postnatal life. There was no further increase in surface area beyond this age. The rate of increase in somal surface area with age was significantly different from both the rate of increase of animal weight and animal surface area with age. The correlations between the demonstrated immature genioglossal morphology and its cellular electrophysiology or integrated respiratory function remain unknown. The recent demonstration of decreased activation of the genioglossus muscle following airway occlusion in premature infants with apnea suggests that the relationships between developing genioglossal motoneuron structure and function warrant further investigation.