Developmental nicotine exposure alters cholinergic control of respiratory frequency in neonatal rats

Prenatal nicotine exposure with continued exposure through breast milk over the first week of life (developmental nicotine exposure, DNE) alters the development of brainstem circuits that control breathing. Here, we test the hypothesis that DNE alters the respiratory motor response to endogenous and exogenous acetylcholine (ACh) in neonatal rats. We used the brainstem‐spinal cord preparation in the split‐bath configuration, and applied drugs to the brainstem compartment while measuring the burst frequency and amplitude of the fourth cervical ventral nerve roots (C4VR), which contain the axons of phrenic motoneurons. We applied ACh alone; the nicotinic acetylcholine receptor (nAChR) antagonist curare, either alone or in the presence of ACh; and the muscarinic acetylcholine receptor (mAChR) antagonist atropine, either alone or in the presence of ACh. The main findings include: (1) atropine reduced frequency similarly in controls and DNE animals, while curare caused modest slowing in controls but no consistent change in DNE animals; (2) DNE greatly attenuated the increase in C4VR frequency mediated by exogenous ACh; (3) stimulation of nAChRs with ACh in the presence of atropine increased frequency markedly in controls, but not DNE animals; (4) stimulation of mAChRs with ACh in the presence of curare caused a modest increase in frequency, with no treatment group differences. DNE blunts the response of the respiratory central pattern generator to exogenous ACh, consistent with reduced availability of functionally competent nAChRs; DNE did not alter the muscarinic control of respiratory motor output. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1138–1149, 2016     

Developmental nicotine exposure enhances inhibitory synaptic transmission in motor neurons and interneurons critical for normal breathing

Nicotine exposure in utero negatively affects neuronal growth, differentiation, and synaptogenesis. We used rhythmic brainstems slices and immunohistochemistry to determine how developmental nicotine exposure (DNE) alters inhibitory neurotransmission in two regions essential to normal breathing, the hypoglossal motor nucleus (XIIn), and preBötzinger complex (preBötC). We microinjected glycine or muscimol (GABA_A agonist) into the XIIn or preBötC of rhythmic brainstem slices from neonatal rats while recording from XII nerve roots to obtain XII motoneuron population activity. Injection of glycine or muscimol into the XIIn reduced XII nerve burst amplitude, while injection into the preBötC altered nerve burst frequency. These responses were exaggerated in preparations from DNE animals. Quantitative immunohistochemistry revealed a significantly higher GABA_A receptor density on XII motoneurons from DNE pups. There were no differences in GABA_A receptor density in the preBötC, and there were no differences in glycine receptor expression in either region. Nicotine, in the absence of other chemicals in tobacco smoke, alters normal development of brainstem circuits that are critical for normal breathing. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 337–354, 2016     

Developmental alteration of nerve injury induced glial cell line-derived neurotrophic factor (GDNF) receptor expression is crucial for the determination of injured motoneuron fate

Axotomy-induced neuronal death occurs in neonatal motoneurons, but not in adult rat. Here we demonstrated that during the course of postnatal development, nerve injury induced down-regulation of the glial cell line-derived neurotrophic factor (GDNF) receptor GFRalpha1 in axotomized hypoglossal motoneurons of rat are gradually converted to the adult up-regulation pattern of response. The compensatory expression of GFRalpha1 specifically in the injured motoneurons of neonates by adenovirus succeeded in rescuing the injured neurons without an application of growth factors. To the contrary, the nuclear antisense RNA for GFRalpha1 expression accelerates the axotomy-induced neuronal death in pups. These findings suggest that the receptor expression response after nerve injury is critical for the determination of injured motoneuron fate.     

Ionotropic glutamate receptor subunits are differentially regulated in the motoneuronal pools of the rat hypoglossal nucleus in response to axotomy

Unilateral hypoglossal nerve axotomy was used as a model to analyse immunohistochemically the expression of the GluR1, GluR2, GluR3, and GluR4 glutamate receptor subunits of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) subtype and the NR1 subunit of the N-methyl-D-aspartate (NMDA) subtype in the different morphofunctional hypoglossal pools from 1 to 45 days postaxotomy. Following hypoglossal nerve axotomy, the percentage of motoneurons that were GluR1-immunopositive and the labeling intensity for this subunit was increased in some hypoglossal pools. Immunolabeling for the GluR2 subunit was undetectable. These results contrast with the unchanged pattern for these two subunits after sciatic nerve axotomy previously described. Image analysis showed a significant decrease in the intensity of immunohistochemical labeling for the GluR2/3 and GluR4 subunits in motoneurons, although most motoneurons were still immunopositive for these 2 subunits after axotomy. The intensity of immunolabeling for the NR1 subunit was slightly decreased postlesion, whereas the percentage of NR1-immunopositive motoneurons increased. Immunoreactivity returned to basal levels 45 days postlesion. These findings show that in axotomized hypoglossal motoneurons, i) AMPA and NMDA receptor subunits are still expressed, ii) the composition of the ionotropic glutamate receptor subunit pool is subjected to continuous changes during the regeneration process, iii) AMPA receptors, if functional, would have physiological properties different to those in intact motoneurons, and iv) the various AMPA receptor subunits are differentially regulated. The present results also suggest a faster recovery of basal levels of immunoreactivity for caudally localised groups of motoneurons which could reflect a caudo-rostral sequential functional revovery in the hypoglossal nucleus.     

Naturally occurring motoneuron cell death in rat upper respiratory tract motor nuclei: A histological,fast Dil and immunocytochemical study in the hypoglossal nucleus

We have previously reported on our investigation of motoneuron cell death (MCD) in the rat nucleus ambiguus (NA). This article focuses on the other major upper respiratory tract motor nucleus: the hypoglossal. The hypoglossal nucleus (XII) contains motoneurons to the tongue and, as such, plays a critical role in defining patterns of respiration, deglutition, and vocalization. Motoneuron counts were made in XII in a developmental series of rats. In addition, the neural tracer fast DiI was used to ensure that all hypoglossal motoneurons had migrated into the nucleus at the time cell death was assessed. Furthermore, an antibody to γ-aminobutyric acid (GABA) was used to determine the potential effect of inadvertently counting large interneurons on motoneuron counts. Cell death in XII was shown to occur entirely prenatally with a loss of 35% of cells between embryonic day 16 (E16) and birth. Fast DiI tracings of the prenatal hypoglossal nerve indicated that all motoneurons were present in a well-defined nucleus by E15. Immunocytochemical staining for GABA demonstrated considerably fewer interneurons than motoneurons in XII. These findings in XII, in comparison with those previously reported for NA, demonstrate differences in the timing and amount of cell death between upper respiratory tract motor nuclei. These differences establish periods during which one nucleus may be preferentially insulted by environmental or teratogenic factors. Preferential insults may underlie some of the upper respiratory tract incoordination pathologies seen in the newborn such as the sudden infant death syndrome (SIDS). © 1995 John Wiley & Sons, Inc.     

Neuroprotective effects of testosterone metabolites and dependency on receptor action on the morphology of somatic motoneurons following the death of neighboring motoneurons

Partial depletion of spinal motoneuron populations induces dendritic atrophy in neighboring motoneurons, and treatment with testosterone is neuroprotective, attenuating induced dendritic atrophy. In this study we examined whether the protective effects of testosterone could be mediated via its androgenic or estrogenic metabolites. Furthermore, to assess whether these neuroprotective effects were mediated through steroid hormone receptors, we used receptor antagonists to attempt to prevent the neuroprotective effects of hormones after partial motoneuron depletion. Motoneurons innervating the vastus medialis muscles of adult male rats were selectively killed by intramuscular injection of cholera toxin‐conjugated saporin. Simultaneously, some saporin‐injected rats were treated with either dihydrotestosterone or estradiol, alone or in combination with their respective receptor antagonists, or left untreated. Four weeks later, motoneurons innervating the ipsilateral vastus lateralis muscle were labeled with cholera toxin‐conjugated horseradish peroxidase, and dendritic arbors were reconstructed in three dimensions. Compared with intact normal animals, partial motoneuron depletion resulted in decreased dendritic length in remaining quadriceps motoneurons. Dendritic atrophy was attenuated with both dihydrotestosterone and estradiol treatment to a degree similar to that seen with testosterone, and attenuation of atrophy was prevented by receptor blockade. Together, these findings suggest that neuroprotective effects on motoneurons can be mediated by either androgenic or estrogenic hormones and require action via steroid hormone receptors, further supporting a role for hormones as neurotherapeutic agents in the injured nervous system. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 691–707, 2017     

Down‐regulation of striatin,a neuronal calmodulin‐binding protein,impairs rat locomotor activity

Striatin, an intraneuronal, calmodulin‐binding protein addressed to dendrites and spines, is expressed in the motor system, particularly the striatum and motoneurons. Striatin contains a high number of domains mediating protein–protein interactions, suggesting a role within a dendritic Ca²⁺‐signaling pathway. Here, we explored the hypothesis of a direct role of striatin in the motor control of behaving rats, by using an antisense strategy based on oligodeoxynucleotides (ODN). Rats were treated by intracerebroventricular infusion of a striatin antisense ODN (A‐ODN) or mismatch ODN (M‐ODN) delivered by osmotic pumps over 6 days. A significant decrease in the nocturnal locomotor activity of A‐ODN–treated rats was observed after 5 days of treatment. Hypomotricity was correlated with a 60% decrease in striatin content of the striata of A‐ODN–treated rats sacrificed on day 6. Striatin thus plays a role in the control of motor function. To approach the cellular mechanisms in which striatin is involved, striatin down‐regulation was studied in a comparatively simpler model: purified rat spinal motoneurons which retain their polarity in culture. Treatment of cells by the striatin A‐ODN resulted in the impairement of the growth of dendrites but not axon. The decrease in dendritic growth paralleled the loss of striatin. This model allows analysis of the molecular basis of striatin function in the dynamic changes occurring in growing dendrites, and offers clues to unravel its function within spines. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 234–243, 1999     

Chronic nicotine induces growth retardation in neonatal rat pups

In the United State, 20% of pregnant women smoke. One of the most consistent adverse outcomes is reduced birth weight in the off-spring. Animal studies using chronic nicotine, the major psychoactive tobacco ingredient, have shown conflicting results, questioning the role of nicotine in growth retardation. To evaluate the direct effects of nicotine during a period equivalent to the human third trimester, we developed an oral gastric intubation model using neonatal rat pups. Nicotine (6 mg/kg/day) was dissolve in milk-formula and delivered during three feedings daily from postnatal day (P)1 to P7. Nicotine immediately and significantly [P<0.05] decreased weight gain per day (WGD) by 13.5% (+/-) 1 day after onset of treatment in both genders and throughout the treatment period. This resulted in significantly lower body weight at P4 and P5 in male and female pups, respectively. After nicotine withdrawal, WGD returned to control level within 1 day, whereas total body weight recovered by P18. There were no long-term consequences on body weight or growth pattern in either gender. The nicotinic acetylcholine receptor (nAChR) antagonist dihydro-beta-erythroidine (DHbetaE) reversed nicotine's effects on WGD suggesting an involvement of heteromeric alpha4beta2, whereas methyllycaconitine (MLA) an antagonist for the homomeric alpha7-type receptor was ineffective. The immediate decrease of growth in neonatal pups suggests that nicotine's effect on birth weight results from direct anorexic rather then indirect effects due to placental dysfunction or increased fetal hypoxia. The postnatal oral gastric intubation model seems to accurately reflect the direct effects of nicotine in neonates.     

Endogenous 5-HT2B receptor activation regulates neonatal respiratory activity in vitro

An implication of 5-HT(2B) receptors in central nervous system has not yet been clearly elucidated. We studied the role of different 5-HT(2) receptor subtypes in the medullary breathing center, the pre-B?tzinger complex, and on hypoglossal motoneurons in rhythmically active transversal slice preparations of neonatal rats and mice. Local microinjection of 5-HT(2) receptor agonists revealed tonic excitation of hypoglossal motoneurons. Excitatory effects of the 5-HT(2B) receptor agonist BW723C86 could be blocked by bath application of LY272015, a highly selective 5-HT(2B) receptor antagonist. Excitatory effects of the 5-HT(2A/B/C) receptor agonist alpha-methyl 5-HT could be blocked by the preferential 5-HT(2A) receptor antagonist ketanserin. Therefore, 5-HT-induced excitation of hypoglossal motoneurons is mediated by convergent activation of 5-HT(2A) and 5-HT(2B) receptors. Local microinjection of BW723C86 in the pre-B?tzinger complex increased respiratory frequency. Bath application of LY272015 blocked respiratory activity, whereas ketanserin had no effect. Therefore, endogenous 5-HT appears to support tonic action on respiratory rhythm generation via 5-HT(2B) receptors. In preparations of 5-HT(2B) receptor-deficient mice, respiratory activity appeared unaltered. Whereas BW723C86 and LY272015 had no effects, bath application of ketanserin disturbed and blocked rhythmic activity. This demonstrates a stimulatory role of endogenous 5-HT(2B) receptor activation at the pre-B?tzinger complex and hypoglossal motoneurons that can be taken up by 5-HT(2A) receptors in the absence of 5-HT(2B) receptors. The presence of functional 5-HT(2B) receptors in the neonatal medullary breathing center indicates a potential convergent regulatory role of 5-HT(2B) and -(2A) receptors on the central respiratory network.     

Ionotropic glutamate receptor subunit distribution on hypoglossal motoneuronal pools in the rat

The expression of ionotropic glutamate receptor subunits in the motoneuronal pools of the hypoglossal nucleus was studied using specific antibodies against subunits of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), kainate and N-methyl-D-aspartate (NMDA) subtypes. The highest numbers of intensely immunolabelled motoneurons were found in the dorsal tier and caudoventromedial part of the hypoglossal nucleus with all antibodies except that against the GluR1 AMPA subunit. Labelling for the GluR1 subunit was weak except for caudally located groups of motoneurons which innervate tongue muscles related to respiratory activity. By contrast, most motoneurons were intensely immunostained with antibodies against GluR2/3 and GluR4 subunits of the AMPA subtype. The low staining observed using an antibody specific for the GluR2 subunit (which prevents Ca²⁺-entry through AMPA channels) strongly suggests that AMPA receptors in hypoglossal motoneurons are Ca²⁺-permeable. Immunolabelling for the GluR5/6/7 kainate receptor subunits was found in many motoneuronal somata as well as in thin axon-like profiles and puncta that resembled synaptic boutons. Most motoneurons were intensely immunostained for the NMDA receptor subunit NR1. These results show that the hypoglossal nucleus contains five heterogeneous pools of motoneurons which innervate functionally defined groups of tongue muscles. The uneven expression of the different receptor subunits analysed here could reflect diverse phenotypic properties of hypoglossal motoneurons which might be expected to generate different patterns of motor responses under different physiological or pathological conditions.     

Proteins of fast axonal transport in the regenerating hypoglossal nerve of the rat

The composition of proteins conveyed by fast axonal transport in growing or regenerating axons is different from that of intact, mature axons. Consistent alterations have been observed in several different types of neurons, but adult peripheral axons (rabbit hypoglossal motoneurons) seemed to be exceptions because during their regeneration there was no increased labelling of a 23 kilodalton (kD) protein associated with the growth state. We examined the composition of fast-transported proteins, labelled by application of [35S]methionine to the hypoglossal nuclei, in intact and regenerating hypoglossal nerves of the rat. Using one- and two-dimensional electrophoresis we detected both increases and decreases in the labelling of specific polypeptides during regeneration. In particular, there was increased labelling of a 23 kD polypeptide. Changes were maximal 7 days after axotomy and subsided thereafter, coincident with reinnervation of the tongue. We conclude that hypoglossal axons show the same changes in transported protein composition which are characteristic of the growth state in other axons. Thus, we have strengthened the correlation between the growth state and changes in synthesis of a set of polypeptides of unknown function.     

The effects of nicotine on human fetal development

Maternal smoking during pregnancy continues to represent a major public health concern. Nicotine is extremely harmful to the developing fetus through many different mechanisms, and the harms increase with later gestational age at exposure. Pregnancies complicated by maternal nicotine use are more likely to have significant adverse outcomes. Nicotine‐exposed children tend to have several health problems throughout their lives, including impaired function of the endocrine, reproductive, respiratory, cardiovascular, and neurologic systems. Poor academic performance and significant behavioral disruptions are also common, including ADHD, aggressive behaviors, and future substance abuse. To diminish the adverse effects from cigarette smoking, some women are turning to electronic cigarettes, a new trend that is increasing in popularity worldwide. They are largely perceived as being safer to use in pregnancy than traditional cigarettes, although there is not adequate evidence to support this claim. At this time, electronic cigarette use during pregnancy cannot be recommended. Birth Defects Research (Part C) 108:181–192, 2016. © 2016 Wiley Periodicals, Inc.     

Organization of the motor centres for the innervation of different muscles of the tongue: a neuromorphological study in the frog.

Cobalt labelling studies on the localization and morphology of the frog's hypoglossal nucleus have revealed three subnuclei. The dorsomedial subnucleus innervates the geniohyoid, hyoglossus, genioglossus and the intrinsic tongue muscles. The ventrolateral subnucleus supplies the sternohyoid, geniohyoid, omohyoid and intrinsic tongue muscles. The intermediate subnucleus innervates the omohyoid, geniohyoid and intrinsic tongue muscles. Neurons innervating protractor, retractor and intrinsic tongue muscles differ in their soma surface area and in their dendritic arborization pattern. It is concluded that there exists a musculotopic organization in the frog's hypoglossal nucleus and that motoneurons subserving different function in tongue movements disclose characteristic morphological differences.     

Effect of chronic intermittent hypoxia on noradrenergic activation of hypoglossal motoneurons

In obstructive sleep apnea patients, elevated activity of the lingual muscles during wakefulness protects the upper airway against occlusions. A possibly related form of respiratory neuroplasticity is present in rats exposed to acute and chronic intermittent hypoxia (CIH). Since rats exposed to CIH have increased density of noradrenergic terminals and increased α(1)-adrenoceptor immunoreactivity in the hypoglossal (XII) nucleus, we investigated whether these anatomic indexes of increased noradrenergic innervation translate to increased sensitivity of XII motoneurons to noradrenergic activation. Adult male Sprague-Dawley rats were subjected to CIH for 35 days, with O(2) level varying between 24% and 7% with 180-s period for 10 h/day. They were then anesthetized, vagotomized, paralyzed, and artificially ventilated. The dorsal medulla was exposed, and phenylephrine (2 mM, 10 nl) and then the α(1)-adrenoceptor antagonist prazosin (0.2 mM, 3 × 40 nl) were microinjected into the XII nucleus while XII nerve activity (XIIa) was recorded. The area under integrated XIIa was measured before and at different times after microinjections. The excitatory effect of phenylephrine on XII motoneurons was similar in sham- and CIH-treated rats. In contrast, spontaneous XIIa was more profoundly reduced following prazosin injections in CIH- than sham-treated rats [to 21 ± 7% (SE) vs. 40 ± 8% of baseline, P < 0.05] without significant changes in central respiratory rate, arterial blood pressure, or heart rate. Thus, consistent with increased neuroanatomic measures of noradrenergic innervation of XII motoneurons following exposure to CIH, prazosin injections revealed a stronger endogenous noradrenergic excitatory drive to XII motoneurons in CIH- than sham-treated anesthetized rats.     

Effects of the steroid hormone, 20-hydroxyecdysone, on the growth of neurites by identified insect motoneurons in vitro.

During metamorphosis in the hawkmoth, Manduca sexta, identified larval leg motoneurons survive the degeneration of their larval targets to innervate new muscles of the adult legs. The dendrites and axon terminals of these motoneurons regress at the end of the larval stage and then regrow during adult development. Previous studies have implicated the insect steroid, 20-hydroxyecdysone (20-HE), in similar examples of dendritic reorganization during metamorphosis. The present studies were undertaken to test whether 20-HE acts directly on the leg motoneurons to regulate dendritic growth. Larval leg motoneurons were labeled with a fluorescent dye to permit their identification in culture following the dissociation of thoracic ganglia at later stages of development. Leg motoneurons isolated from early pupal stage animals (just before the normal onset of dendritic regrowth) survived in vitro and grew processes regardless of whether 20-HE was added to the culture medium. The extent of process outgrowth, however, as measured by the total length of all processes and the number of branches, was significantly greater for motoneurons maintained in the presence of 20-HE. The enhancement could be blocked by the addition of a juvenile hormone analog. By contrast, larval leg motoneurons that were isolated just before the normal period of dendritic regression did not show enhanced growth of neurites in the presence of 20-HE. The results suggest that 20-HE acts directly on the leg motoneurons to regulate the growth of processes during metamorphosis.     

Tongue-muscle-controlling motoneurons in the Japanese toad: topography,morphology and neuronal pathways from the ‘snapping-evoking area’ in the optic tectum

As a step to clarifying the neural bases for the visually-guided prey-catching behavior in the toad, special attention was paid to the flipping movement of the tongue. Tongue-muscle-controlling motoneurons were identified antidromically, and their topographical distribution within the hypoglossal nucleus, the morphology, and the neuronal pathways from the optic tectum including the 'snapping-evoking area' (see below) to these motoneurons were investigated in paralyzed Japanese toads using intracellular recording techniques. The morphology of motoneurons innervating the tongue-protracting or retracting muscles (PMNs or RMNs respectively) was examined by means of intracellular-staining (using HRP/cobaltic lysine) and retrograde-labeling (using cobaltic lysine) methods. Both PMNs and RMNs showed an extensive spread of the branching trees of dendrites; 4 dendritic fields were distinguished: lateral/ventrolateral, dorsal/dorsolateral, medial, and in some motoneurons, contralateral dendritic fields, although there was a tendency for the dorsal/dorsolateral dendritic field to be less extensive in the PMNs than in the RMNs. The axons of both PMNs and RMNs arose from thick dendrites, ran in a ventral direction without any axon-collaterals branching off, and then entered the hypoglossal nerve. The PMNs and RMNs were distributed topographically within the hypoglossal nucleus; the RMNs were located rostrally within the nucleus, whereas the PMNs were located more caudally within it. In about 3/4 of the RMNs tested, depolarizing potentials [presumably the excitatory postsynaptic potentials (EPSPs)], on which action potentials were often superimposed, were evoked by electrical stimuli applied to the nerve branch innervating the tongue protractor. These EPSPs were temporally facilitated when the electrical stimuli were applied at short intervals (10 ms). Both PMNs and RMNs showed hyperpolarizing potentials (IPSPs) in response to single electrical stimuli of various intensities (10-200 microA) applied to the 'snapping-evoking area' (lateral/ventrolateral part of the optic tectum) on either side. These IPSPs were facilitated after repetitive electrical stimulations at short intervals (10 ms) and of weaker intensities (down to 10 microA); i.e., a temporal facilitation of the IPSPs was observed. On the other hand, large and long-lasting EPSPs which prevailed over the underlying IPSPs were evoked after repetitive electrical stimulations (a few pulses or more) at short intervals (10 ms) and of stronger intensities (generally 90 microA or more); thus, a temporal facilitation of the EPSPs was also observed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Neuroplasticity of the central hypercapnic ventilatory response: Teratogen‐induced impairment and subsequent recovery during development

Neuroventilation is highly plastic and exposure to either of two distinct teratogens, nicotine or ethanol, during development results in a similar loss of the neuroventilatory response to hypercapnia in bullfrog tadpoles. Whether this functional deficit is permanent or transient following nicotine or ethanol exposure was unknown. Here, we tested the persistence of hypercapnic neuroventilatory response impairments in tadpoles exposed to either 30 μg/L nicotine or 0.12–0.06 g/dL ethanol for 10 weeks. Brainstem breathing‐related neural activity was assessed in tadpoles allowed to develop teratogen‐free after either nicotine or ethanol exposure. Nicotine‐exposed animals responded normally to hypercapnia after a 3‐week teratogen‐free period but the hypercapnic response in ethanol‐exposed tadpoles remained impaired. Tadpoles allowed to develop for only 1 week nicotine free after chronic exposure were unable to respond to hypercapnia. The hypercapnic response of ethanol‐exposed tadpoles returned by 6 weeks following chronic ethanol exposure. These findings suggest that some nicotine‐ and ethanol‐induced impairments can be resolved during early development. Understanding both the disruptive effects of nicotine and ethanol exposure and how impaired responses return when teratogen exposure stops may offer insight on the function and plasticity of respiratory control. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 726–735, 2010     

Hormonally mediated plasticity of motoneuron morphology in the adult rat spinal cord: A cholera toxin-HRP study

The dorsolateral nucleus (DLN) and the spinal nucleus of the bulbocavernosus (SNB) of the rat lumbar spinal cord are sexually dimorphic groups of motoneurons that innervate striated perineal muscles involved in male copulatory behavior. Androgens control the development of these motoneurons and their target muscles, and continue to influence the system in adulthood. Given that several features of SNB motoneuron morphology have been shown to be androgen sensitive in adult male rats, we examined the effects of androgen manipulations on the morphology of motoneurons in the DLN in adult rats. Adult male rats were castrated and implanted with testosterone-filled or blank implants, or were subjected to a sham-castration procedure. Six weeks after treatment, motoneurons in the DLN were retrogradely labeled with cholera toxin-horseradish peroxidase (HRP) after injection into the ischiocavernosus (IC) muscle and their morphology assessed. Measures of the radial extent and coverage of the dendritic arbor of DLN motoneurons projecting to the IC (DLN-IC motoneurons) were similar across the groups, indicating comparable degrees of HRP transport. However, DLN-IC motoneurons in castrates with blank implants possessed both shorter dendritic lengths and smaller somas than those of castrates treated with testosterone. Castrates with testosterone implants had DLN-IC motoneurons that were significantly larger than those of sham castrates in dendritic length and soma area. These results suggest that motoneurons in the DLN, like those in the SNB, possess a significant degree of structural plasticity in adulthood which is influenced by androgens.     

Hormonally mediated plasticity of motoneuron morphology in the adult rat spinal cord: a cholera toxin-HRP study.

The dorsolateral nucleus (DLN) and the spinal nucleus of the bulbocavernosus (SNB) of the rat lumbar spinal cord are sexually dimorphic groups of motoneurons that innervate striated perineal muscles involved in male copulatory behavior. Androgens control the development of these motoneurons and their target muscles, and continue to influence the system in adulthood. Given that several features of SNB motoneuron morphology have been shown to be androgen sensitive in adult male rats, we examined the effects of androgen manipulations on the morphology of motoneurons in the DLN in adult rats. Adult male rats were castrated and implanted with testosterone-filled or blank implants, or were subjected to a sham-castration procedure. Six weeks after treatment, motoneurons in the DLN were retrogradely labeled with cholera toxin-horseradish peroxidase (HRP) after injection into the ischiocavernosus (IC) muscle and their morphology assessed. Measures of the radial extent and coverage of the dendritic arbor of DLN motoneurons projecting to the IC (DLN-IC motoneurons) were similar across the groups, indicating comparable degrees of HRP transport. However, DLN-IC motoneurons in castrates with blank implants possessed both shorter dendritic lengths and smaller somas than those of castrates treated with testosterone. Castrates with testosterone implants had DLN-IC motoneurons that were significantly larger than those of sham castrates in dendritic length and soma area. These results suggest that motoneurons in the DLN, like those in the SNB, possess a significant degree of structural plasticity in adulthood which is influenced by androgens.     

Uncoupling of upper airway motor activity from phrenic bursting by positive end-expired pressure in the rat.

Phasic bursting in the hypoglossal nerve can be uncoupled from phrenic bursting by application of positive end-expired pressure (PEEP). We wished to determine whether similar uncoupling can also be induced in other respiratory-modulated upper airway (UAW) motor outputs. Discharge of the facial, hypoglossal, superior laryngeal, recurrent laryngeal, and phrenic nerves was recorded in anesthetized, ventilated rats during stepwise changes in PEEP with a normocapnic, hyperoxic background. Application of 3- to 6-cmH(2)O PEEP caused the onset inspiratory (I) UAW nerve bursting to precede the phrenic burst but did not uncouple bursting. In contrast, application of 9- to 12-cmH(2)O PEEP uncoupled UAW neurograms such that rhythmic bursting occurred during periods of phrenic quiescence. Single-fiber recording experiments were conducted to determine whether a specific population of UAW motoneurons is recruited during uncoupled bursting. The data indicate that expiratory-inspiratory (EI) motoneurons remained active, while I motoneurons did not fire during uncoupled UAW bursting. Finally, we examined the relationship between motoneuron discharge rate and PEEP during coupled UAW and phrenic bursting. EI discharge rate was linearly related to PEEP during preinspiration, but showed no relationship to PEEP during inspiration. Our results demonstrate that multiple UAW motor outputs can be uncoupled from phrenic bursting, and this response is associated with bursting of EI nerve fibers. The relationship between PEEP and EI motoneuron discharge rate differs during preinspiratory and I periods; this may indicate that bursting during these phases of the respiratory cycle is controlled by distinct neuronal outputs.