Designer Receptors Exclusively Activated by Designer Drugs Approach to Treatment of Sleep-disordered Breathing.

Rationale: Obstructive sleep apnea is recurrent upper airway obstruction caused by a loss of upper airway muscle tone during sleep. The main goal of our study was to determine if designer receptors exclusively activated by designer drugs (DREADD) could be used to activate the genioglossus muscle as a potential novel treatment strategy for sleep apnea. We have previously shown that the prototypical DREADD ligand clozapine-N-oxide increased pharyngeal diameter in mice expressing DREADD in the hypoglossal nucleus. However, the need for direct brainstem viral injections and clozapine-N-oxide toxicity diminished translational potential of this approach, and breathing during sleep was not examined.Objectives: Here, we took advantage of our model of sleep-disordered breathing in diet-induced obese mice, retrograde properties of the adeno-associated virus serotype 9 (AAV9) viral vector, and the novel DREADD ligand J60.Methods: We administered AAV9-hSyn-hM3(Gq)-mCherry or control AAV9 into the genioglossus muscle of diet-induced obese mice and examined the effect of J60 on genioglossus activity, pharyngeal patency, and breathing during sleep.Measurements and Main Results: Compared with control, J60 increased genioglossus tonic activity by greater than sixfold and tongue uptake of 2-deoxy-2-[18F]fluoro-d-glucose by 1.5-fold. J60 increased pharyngeal patency and relieved upper airway obstruction during non-REM sleep.Conclusions: We conclude that following intralingual administration of AAV9-DREADD, J60 can activate the genioglossus muscle and improve pharyngeal patency and breathing during sleep.

Chemogenetic stimulation of the hypoglossal neurons improves upper airway patency.

Obstructive sleep apnea (OSA) is characterized by recurrent upper airway obstruction during sleep. OSA leads to high cardiovascular morbidity and mortality. The pathogenesis of OSA has been linked to a defect in neuromuscular control of the pharynx. There is no effective pharmacotherapy for OSA. The objective of this study was to determine whether upper airway patency can be improved using chemogenetic approach by deploying designer receptors exclusively activated by designer drug (DREADD) in the hypoglossal motorneurons. DREADD (rAAV5-hSyn-hM3(Gq)-mCherry) and control virus (rAAV5-hSyn-EGFP) were stereotactically administered to the hypoglossal nucleus of C57BL/6J mice. In 6-8 weeks genioglossus EMG and dynamic MRI of the upper airway were performed before and after administration of the DREADD ligand clozapine-N-oxide (CNO) or vehicle (saline). In DREADD-treated mice, CNO activated the genioglossus muscle and markedly dilated the pharynx, whereas saline had no effect. Control virus treated mice showed no effect of CNO. Our results suggest that chemogenetic approach can be considered as a treatment option for OSA and other motorneuron disorders.

Nicotinic receptors modulate the onset of reactive oxygen species production and mitochondrial dysfunction evoked by glutamate uptake block in the rat hypoglossal nucleus.

In several neurodegenerative diseases, glutamate-mediated excitotoxicity is considered to be a major process to initiate cell degeneration. Indeed, subsequent to excessive glutamate receptor stimulation, reactive oxygen species (ROS) generation and mitochondrial dysfunction are regarded as two major gateways leading to neuron death. These processes are mimicked in an in vitro model of rat brainstem slice when excitotoxicity is induced by DL-threo-ß-benzyloxyaspartate (TBOA), a specific glutamate-uptake blocker that increases extracellular glutamate. Our recent study has demonstrated that brainstem hypoglossal motoneurons, which are very vulnerable to this damage, were neuroprotected from excitotoxicity with nicotine application through the activation of nicotinic acetylcholine receptors (nAChRs) and subsequent inhibition of ROS and mitochondrial dysfunction. The present study examined if endogenous cholinergic activity exerted any protective effect in this pathophysiological model and how ROS production (estimated with rhodamine fluorescence) and mitochondrial dysfunction (measured as methyltetrazolium reduction) were time-related during the early phase of excitotoxicity (0-4h). nAChR antagonists did not modify TBOA-evoked ROS production (that was nearly doubled over control) or mitochondrial impairment (25% decline), suggesting that intrinsic nAChR activity was insufficient to contrast excitotoxicity and needed further stimulation with nicotine to become effective. ROS production always preceded mitochondrial dysfunction by about 2h. Nicotine prevented both ROS production and mitochondrial metabolic depression with a delayed action that alluded to a complex chain of events targeting these two lesional processes. The present data indicate a relatively wide time frame during which strong nAChR activation can arrest a runaway neurotoxic process leading to cell death.

Nicotinic receptor activation contrasts pathophysiological bursting and neurodegeneration evoked by glutamate uptake block on rat hypoglossal motoneurons.

KEY POINTS: Impaired uptake of glutamate builds up the extracellular level of this excitatory transmitter to trigger rhythmic neuronal bursting and delayed cell death in the brainstem motor nucleus hypoglossus. This process is the expression of the excitotoxicity that underlies motoneuron degeneration in diseases such as amyotrophic lateral sclerosis affecting bulbar motoneurons. In a model of motoneuron excitotoxicity produced by pharmacological block of glutamate uptake in vitro, rhythmic bursting is suppressed by activation of neuronal nicotinic receptors with their conventional agonist nicotine. Emergence of bursting is facilitated by nicotinic receptor antagonists. Following excitotoxicity, nicotinic receptor activity decreases mitochondrial energy dysfunction, endoplasmic reticulum stress and production of toxic radicals. Globally, these phenomena synergize to provide motoneuron protection. Nicotinic receptors may represent a novel target to contrast pathological overactivity of brainstem motoneurons and therefore to prevent their metabolic distress and death. ABSTRACT: Excitotoxicity is thought to be one of the early processes in the onset of amyotrophic lateral sclerosis (ALS) because high levels of glutamate have been detected in the cerebrospinal fluid of such patients due to dysfunctional uptake of this transmitter that gradually damages brainstem and spinal motoneurons. To explore potential mechanisms to arrest ALS onset, we used an established in vitro model of rat brainstem slice preparation in which excitotoxicity is induced by the glutamate uptake blocker dl-threo-ß-benzyloxyaspartate (TBOA). Because certain brain neurons may be neuroprotected via activation of nicotinic acetylcholine receptors (nAChRs) by nicotine, we investigated if nicotine could arrest excitotoxic damage to highly ALS-vulnerable hypoglossal motoneurons (HMs). On 50% of patch-clamped HMs, TBOA induced intense network bursts that were inhibited by 1-10 µm nicotine, whereas nAChR antagonists facilitated burst emergence in non-burster cells. Furthermore, nicotine inhibited excitatory transmission and enhanced synaptic inhibition. Strong neuroprotection by nicotine prevented the HM loss observed after 4 h of TBOA exposure. This neuroprotective action was due to suppression of downstream effectors of neurotoxicity such as increased intracellular levels of reactive oxygen species, impaired energy metabolism and upregulated genes involved in endoplasmic reticulum (ER) stress. In addition, HMs surviving TBOA toxicity often expressed UDP-glucose glycoprotein glucosyltransferase, a key element in repair of misfolded proteins: this phenomenon was absent after nicotine application, indicative of ER stress prevention. Our results suggest nAChRs to be potential targets for inhibiting excitotoxic damage of motoneurons at an early stage of the neurodegenerative process.

Developmental nicotine exposure disrupts dendritic arborization patterns of hypoglossal motoneurons in the neonatal rat.

Maternal smoking or use of other products containing nicotine during pregnancy can have significant adverse consequences for respiratory function in neonates. We have shown, in previous studies, that developmental nicotine exposure (DNE) in a model system compromises the normal function of respiratory circuits within the brainstem. The effects of DNE include alterations in the excitability and synaptic interactions of the hypoglossal motoneurons, which innervate muscles of the tongue. This study was undertaken to test the hypothesis that these functional consequences of DNE are accompanied by changes in the dendritic morphology of hypoglossal motoneurons. Hypoglossal motoneurons in brain stem slices were filled with neurobiotin during whole-cell patch clamp recordings and subjected to histological processing to reveal dendrites. Morphometric analysis, including the Sholl method, revealed significant effects of DNE on dendritic branching patterns. In particular, whereas within the first five postnatal days there was significant growth of the higher-order dendritic branches of motoneurons from control animals, the growth was compromised in motoneurons from neonates that were subjected to DNE. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1125-1137, 2016.

Influence of developmental nicotine exposure on spike-timing precision and reliability in hypoglossal motoneurons.

Smoothly graded muscle contractions depend in part on the precision and reliability of motoneuron action potential generation. Whether or not a motoneuron generates spikes precisely and reliably depends on both its intrinsic membrane properties and the nature of the synaptic input that it receives. Factors that perturb neuronal intrinsic properties and/or synaptic drive may compromise the temporal precision and the reliability of action potential generation. We have previously shown that developmental nicotine exposure (DNE) alters intrinsic properties and synaptic transmission in hypoglossal motoneurons (XIIMNs). Here we show that the effects of DNE also include alterations in spike-timing precision and reliability, and spike-frequency adaptation, in response to sinusoidal current injection. Current-clamp experiments in brainstem slices from neonatal rats show that DNE lowers the threshold for spike generation but increases the variability of spike-timing mechanisms. DNE is also associated with an increase in spike-frequency adaptation and reductions in both peak and steady-state firing rate in response to brief, square wave current injections. Taken together, our data indicate that DNE causes significant alterations in the input-output efficiency of XIIMNs. These alterations may play a role in the increased frequency of obstructive apneas and altered suckling strength and coordination observed in nicotine-exposed neonatal humans.

Chronic nicotine and ethanol exposure both disrupt central ventilatory responses to hypoxia in bullfrog tadpoles.

The central hypoxic ventilatory response (HVR) comprises a reduction in ventilatory activity that follows a peripherally mediated ventilatory augmentation. Chronic early developmental exposure to nicotine or ethanol are both known to impair the peripherally mediated HVR, and nicotine impairs the central HVR, but the effect of ethanol on the central HVR has not been investigated. Additionally, chronic nicotine and ethanol exposure are known to impair ventilatory responses to hypercapnia in bullfrog tadpoles but HVRs have not been tested. Here early and late metamorphic tadpoles were exposed to either 30 µg/L nicotine or 0.15-0.05 g/dL ethanol for 10 wk. Tadpole brainstems were then isolated and the neurocorrelates of ventilation were monitored in vitro over 180 min of hypoxia (PO2=5.05±1.04 kPa). Both nicotine and ethanol exposure disrupted central HVRs. Nicotine impairments were dependent on development. Central HVRs were impaired only in early metamorphic nicotine-exposed tadpoles. Both early and late metamorphic ethanol-exposed tadpoles failed to exhibit central HVRs. Thus, central HVRs are impaired following both nicotine and ethanol exposure. Such failure to decrease ventilatory activity during hypoxia indicates that central hypoxic ventilatory depression is an active suppression of neural activity in response to hypoxia rather than a metabolic consequence of O2 limitation, and that exposure to ethanol (across development) or nicotine (during early development) disrupts mechanisms that normally induce active ventilatory depression.

Developmental nicotine exposure alters AMPA neurotransmission in the hypoglossal motor nucleus and pre-Botzinger complex of neonatal rats.

Developmental nicotine exposure (DNE) impacts central respiratory control in neonates born to smoking mothers. We previously showed that DNE enhances the respiratory motor response to bath application of AMPA to the brainstem, although it was unclear which brainstem respiratory neurons mediated these effects (Pilarski and Fregosi, 2009). Here we examine how DNE influences AMPA-type glutamatergic neurotransmission in the pre-Bötzinger complex (pre-BötC) and the hypoglossal motor nucleus (XIIMN), which are neuronal populations located in the medulla that are necessary for normal breathing. Using rhythmic brainstem slices from neonatal rats, we microinjected AMPA into the pre-BötC or the XIIMN while recording from XII nerve rootlets (XIIn) as an index of respiratory motor output. DNE increased the duration of tonic activity and reduced rhythmic burst amplitude after AMPA microinjection into the XIIMN. Also, DNE led to an increase in respiratory burst frequency after AMPA injection into the pre-BötC. Whole-cell patch-clamp recordings of XII motoneurons showed that DNE increased motoneuron excitability but did not change inward currents. Immunohistochemical studies indicate that DNE reduced the expression of glutamate receptor subunits 2 and 3 (GluR2/3) in the XIIMN and the pre-BötC. Our data show that DNE alters AMPAergic synaptic transmission in both the XIIMN and pre-BötC, although the mechanism by which this occurs is unclear. We suggest that the DNE-induced reduction in GluR2/3 may represent an attempt to compensate for increased cell excitability, consistent with mechanisms underlying homeostatic plasticity.

Increased nicotinic receptor desensitization in hypoglossal motor neurons following chronic developmental nicotine exposure.

Neuronal nicotinic acetylcholine receptors (nAChRs) are expressed on hypoglossal motor neurons (XII MNs) that innervate muscles of the tongue. Activation of XII MN nAChRs evokes depolarizing currents, which are important for regulating the size and stiffness of the upper airway. Although data show that chronic developmental nicotine exposure (DNE) blunts cholinergic neurotransmission in the XII motor nucleus, it is unclear how nAChRs are involved. Therefore, XII MN nAChR desensitization and recovery were examined in tissues from DNE or control pups using a medullary slice preparation and tight-seal whole cell patch-clamp recordings. nAChR-mediated inward currents were evoked by brief pressure pulses of nicotine or the α4ß2 nAChR agonist RJR-2403. We found that, regardless of treatment, activatable nAChRs underwent desensitization, but, following DNE, nAChRs exhibited increased desensitization and delayed recovery. Similar results were produced using RJR-2403, showing that DNE influences primarily the α4ß2 nAChR subtype. These results show that while some nAChRs preserve their responsiveness to acute nicotine following DNE, they more readily desensitize and recover more slowly from the desensitized state. These data provide new evidence that chronic DNE modulates XII MN nAChR function, and suggests an explanation for the association between DNE and the incidence of central and obstructive apneas.

Nociceptive afferents to the premotor neurons that send axons simultaneously to the facial and hypoglossal motoneurons by means of axon collaterals.

It is well known that the brainstem premotor neurons of the facial nucleus and hypoglossal nucleus coordinate orofacial nociceptive reflex (ONR) responses. However, whether the brainstem PNs receive the nociceptive projection directly from the caudal spinal trigeminal nucleus is still kept unclear. Our present study focuses on the distribution of premotor neurons in the ONR pathways of rats and the collateral projection of the premotor neurons which are involved in the brainstem local pathways of the orofacial nociceptive reflexes of rat. Retrograde tracer Fluoro-gold (FG) or FG/tetramethylrhodamine-dextran amine (TMR-DA) were injected into the VII or/and XII, and anterograde tracer biotinylated dextran amine (BDA) was injected into the caudal spinal trigeminal nucleus (Vc). The tracing studies indicated that FG-labeled neurons receiving BDA-labeled fibers from the Vc were mainly distributed bilaterally in the parvicellular reticular formation (PCRt), dorsal and ventral medullary reticular formation (MdD, MdV), supratrigeminal nucleus (Vsup) and parabrachial nucleus (PBN) with an ipsilateral dominance. Some FG/TMR-DA double-labeled premotor neurons, which were observed bilaterally in the PCRt, MdD, dorsal part of the MdV, peri-motor nucleus regions, contacted with BDA-labeled axonal terminals and expressed c-fos protein-like immunoreactivity which induced by subcutaneous injection of formalin into the lip. After retrograde tracer wheat germ agglutinated horseradish peroxidase (WGA-HRP) was injected into VII or XII and BDA into Vc, electron microscopic study revealed that some BDA-labeled axonal terminals made mainly asymmetric synapses on the dendritic and somatic profiles of WGA-HRP-labeled premotor neurons. These data indicate that some premotor neurons could integrate the orofacial nociceptive input from the Vc and transfer these signals simultaneously to different brainstem motonuclei by axonal collaterals.

Testosterone restores respiratory long term facilitation in old male rats by an aromatase-dependent mechanism.

Steroidal sex hormones play an important role in the neural control of breathing. Previous studies in our laboratory have shown that gonadectomy in young male rats (3 months) eliminates a form of respiratory plasticity induced by intermittent hypoxia, known as long term facilitation (LTF). Testosterone replenishment restores LTF in gonadectomized male rats, and this is dependent on the conversion of testosterone to oestradiol by aromatase. By middle age (12 months), male rats no longer exhibit LTF of hypoglossal motor output; phrenic LTF is significantly reduced, and this persists into old age. We tested the hypothesis that LTF can be restored in old male rats by administration of testosterone. Intact Fischer 344 rats (>20 months) were implanted with Silastic tubing containing testosterone (T), T plus an aromatase inhibitor (T+ADT), or 5α-dihydrotestosterone (DHT), a form of testosterone not converted to oestradiol. One week post-surgery, LTF of hypoglossal and phrenic motor output was measured. By comparison with control rats, hypoglossal LTF was increased in testosterone-treated rats, with levels approaching that of normal young rats. LTF was not restored in T+ADT or DHT-treated rats. Aromatase levels in hypoglossal and phrenic nuclei did not change with age. As serum testosterone levels did not decline with age, local bioavailability of testosterone in old rats may be a limiting factor in the expression of this form of respiratory plasticity. Our findings suggest that testosterone supplementation could potentially be used to enhance upper airway control in the elderly.

Developmental nicotine exposure alters neurotransmission and excitability in hypoglossal motoneurons.

Hypoglossal motoneurons (XII MNs) control muscles of the mammalian tongue and are rhythmically active during breathing. Acetylcholine (ACh) modulates XII MN activity by promoting the release of glutamate from neurons that express nicotinic ACh receptors (nAChRs). Chronic nicotine exposure alters nAChRs on neurons throughout the brain, including brain stem respiratory neurons. Here we test the hypothesis that developmental nicotine exposure (DNE) reduces excitatory synaptic input to XII MNs. Voltage-clamp experiments in rhythmically active medullary slices showed that the frequency of excitatory postsynaptic currents (EPSCs) onto XII MNs from DNE animals is reduced by 61% (DNE = 1.7 ± 0.4 events/s; control = 4.4 ± 0.6 events/s; P < 0.002). We also examine the intrinsic excitability of XII MNs to test whether cells from DNE animals have altered membrane properties. Current-clamp experiments showed XII MNs from DNE animals had higher intrinsic excitability, as evaluated by measuring their response to injected current. DNE cells had high-input resistances (DNE = 131.9 ± 13.7 MΩ, control = 78.6 ± 9.7 MΩ, P < 0.008), began firing at lower current levels (DNE = 144 ± 22 pA, control = 351 ± 45 pA, P < 0.003), and exhibited higher frequency-current gain values (DNE = 0.087 ± 0.012 Hz/pA, control = 0.050 ± 0.004 Hz/pA, P < 0.02). Taken together, our data show previously unreported effects of DNE on XII MN function and may also help to explain the association between DNE and the incidence of central and obstructive apneas.

Protein kinase A activators produce a short-term, but not long-term, increase in respiratory-drive transmission at the hypoglossal motor nucleus in vivo.

Synaptic plasticity is an intrinsic and conserved feature of neuronal activity that has been most extensively studied in the context of learning and memory in Aplysia and the mammalian hippocampus. However, the intracellular mechanisms underlying plasticity at motor nuclei, influencing motor behaviour, are less well studied. In vitro experiments in neonatal rodents indicate that protein kinase A (PKA) modulates respiratory-drive transmission at the hypoglossal motor nucleus (HMN), which innervates the genioglossus muscle of the tongue. We hypothesised that PKA activators at the HMN would increase genioglossus activity in vivo, whereas a PKA inhibitor would suppress activity indicative of constitutive PKA activation. Since PKA activators are importantly involved in models of long-term augmentation of neuronal activity following massed stimulation [16], we also hypothesised that application of PKA activators to the HMN would produce long-term facilitation of genioglossus activity. Experiments were performed in 25 isoflurane-anaesthetised, tracheotomised, spontaneously breathing adult rats. Microdialysis perfusion of 8-Br-cAMP (direct PKA activator) into the HMN increased genioglossus activity compared to baseline levels with artificial cerebrospinal fluid (P<0.001). Application of forskolin (indirect PKA activator) had a similar effect (P<0.002). Genioglossus activity progressively decreased back to baseline during a 90-min washout with artificial cerebrospinal fluid, demonstrating a lack of long-term facilitation of genioglossus activity. Similar to massed application of 8-Br-cAMP to the HMN, intermittent application produced a short-term (P<0.001), but not long-term, increase in genioglossus activity in vivo. Application of Rp-8-Cl-cAMPS (PKA inhibitor) did not decrease genioglossus activity, indicating a lack of constitutive PKA activation.

Protein kinase G-dependent mechanisms modulate hypoglossal motoneuronal excitability and long-term facilitation.

Since protein kinase-dependent modulation of motoneuronal excitability contributes to adaptive changes in breathing, we hypothesized that cGMP-dependent pathways activating protein kinase G (PKG) modulate motoneuronal inspiratory drive currents and long-term plasticity. In a medullary slice preparation from neonatal rat (postnatal days 0-4) generating spontaneous respiratory-related rhythm, hypoglossal (XII) motoneuronal inspiratory drive currents and respiratory-related XII nerve activity were recorded. Focal application of a PKG activator, 8-bromoguanosine-3',5'-cyclomonophosphate (8-Br-cGMP), to voltage-clamped XII motoneurones decreased inspiratory drive currents. In the presence of tetrodotoxin (TTX), 8-Br-cGMP decreased the exogenous postsynaptic inward currents induced by focal application of AMPA. Intracellular dialysis of XII motoneurones with an inhibitory peptide to PKG (PKGI) increased endogenous inspiratory-drive currents and exogenous AMPA-induced currents. Application of 8-Br-cGMP with PKGI had no further effect on spontaneous or evoked currents, confirming that the observed effects were induced by PKG. However, PKG differentially increased longer-term plasticity. Three 3 min applications (separated by 5 min) of the α(1)-adrenergic agonist phenylephrine (PE) in combination with 8-Br-cGMP yielded greater in vitro long-term facilitation than PE alone. These data indicate the presence of a cGMP/PKG-dependent signalling pathway in XII motoneurones that modulates inspiratory drive currents and plasticity of XII motoneurones, possibly contributing to their adaptation during physiological challenges, such as sleep and exercise.

Restoration of microglial function by granulocyte-colony stimulating factor in ALS model mice.

We studied the effects of G-CSF on microglial reactions in mutant SOD1 (mSOD1)-Tg (G93A) ALS model mice. Following hypoglossal axotomy, the numbers of neurons and microglia expressing GDNF were significantly lower in mSOD1-Tg mice than in non-transgenic (NTG) littermates. This decrease in the number of neurons after axotomy and a decrease in the number of large myelinated axons in mSOD1-Tg mice over the disease course were improved by G-CSF, which also increased microglial recruitment. Impaired migration of cultured mSOD1-Tg microglia to MCP-1 was recovered following G-CSF treatment. Restoration of microglial responses by G-CSF may contribute to its neuroprotective effects.

Timing and duration of developmental nicotine exposure contribute to attenuation of the tadpole hypercapnic neuroventilatory response.

The ability for air-breathing vertebrates to adjust ventilation in response to increased CO(2) (hypercapnia) is fundamental to maintaining pH homeostasis. Developmental nicotine exposure has been shown to impair tadpole neuroventilatory responses to hypercapnia following 8-12 weeks of exposure. It is not clear, however, to what extent the timing of exposure during development and/or the duration over which the exposure takes place contribute to this impairment. Here, tadpoles were exposed to 30 microg/L of nicotine for 3- or 10-week durations, either early or late in tadpole development. Correlates of tadpole lung neuroventilation were monitored during normocapnic (1.5% CO(2)) and hypercapnic (5% CO(2)) conditions of isolated brainstems. Preparations derived from early metamorphic tadpoles failed to increase lung neuroventilation in response to hypercapnia whether they had been exposed to nicotine for 3 or 10 weeks. Preparations derived from late metamorphic tadpoles failed to respond to hypercapnia after being exposed to nicotine for 10 weeks. These results suggest that both the developmental timing and duration of exposure are important when considering nicotine's effect on the hypercapnic neuroventilatory response.

Riluzole blocks persistent Na+ and Ca2+ currents and modulates release of glutamate via presynaptic NMDA receptors on neonatal rat hypoglossal motoneurons in vitro.

The neuroprotective agent riluzole is used for the symptomatic treatment of motoneuron disease, which strongly affects the brainstem nucleus hypoglossus. The mechanism of action of riluzole was investigated using, as a model, patch-clamp recording from hypoglossal motoneurons of the neonatal rat brainstem slice preparation. In the presence of riluzole (10 microm), theta-rhythm oscillations evoked by nicotine continued even though the persistent inward current (comprising sodium and calcium components) was halved, but they disappeared when the high frequency of spontaneous glutamatergic currents waned. Riluzole fully inhibited the persistent sodium current and partly depressed a tetrodotoxin (TTX)-insensitive slow current antagonized by Mn(2+) or Cd(2+). Repetitive firing was inhibited by riluzole without changing single action potentials. In the presence of TTX, riluzole depressed miniature glutamatergic currents occurring at high rate. Synaptic transmission with low release probability became sensitive to riluzole if release was stimulated by high potassium solution. Miniature current frequency was depressed by the N-methyl-D-aspartic acid (NMDA) receptor antagonist D-amino-phosphonovaleriate (50 microm), which fully occluded the action of riluzole. As riluzole is a protein kinase C (PKC) inhibitor, the PKC antagonist chelerythrine (2.5 microm) mimicked the effect of riluzole and prevented it. In summary, riluzole blocked the persistent sodium current fully, and the calcium one partly, plus it decreased glutamatergic transmission probably via inhibition of PKC that regulated presynaptic NMDA receptors having a facilitatory effect on glutamate release. Controlling NMDA receptor function and, thus, excitatory transmitter release via modulation of PKC suggests a novel potential target to contrast glutamate excitotoxicity in this motor nucleus.

Developmental exposure to ethanol or nicotine inhibits the hypercapnic ventilatory response in tadpoles.

Tadpoles, Lithobates (formerly Rana) catesbeiana, were held for 8-12 weeks in pond water that was either teratogen free or contained 0.15% ethanol or 30 microg/L nicotine. The ventilatory and neuroventilatory consequences of these developmental exposures were assessed. Developmental exposure to ethanol or nicotine blocked the hypercapnia-induced increase in surfacing frequency typically exhibited by tadpoles, as well as the hypercapnia-induced increase in putative lung ventilation exhibited by isolated tadpole brainstems. It was specifically the hypercapnic ventilatory response, previously characterized as an increase in lung activity, that was affected by developmental exposure to these teratogens. Developmental exposure to ethanol or nicotine did not affect the frequency of surfacing or putative lung breaths exhibited by the intact tadpoles or their isolated brainstems when not subjected to a hypercapnic challenge.

Persistent rhythmic oscillations induced by nicotine on neonatal rat hypoglossal motoneurons in vitro.

Patch-clamp recording from hypoglossal motoneurons in neonatal Wistar rat brainstem slices was used to investigate the electrophysiological effects of bath-applied nicotine (10 microm). While nicotine consistently evoked membrane depolarization (or inward current under voltage clamp), it also induced electrical oscillations (3-13 Hz; lasting for >/= 8.5 min) on 40% of motoneurons. Oscillations required activation of nicotinic receptors sensitive to dihydro-beta-erythroidine (0.5 microm) or methyllycaconitine (5 nm), and were accompanied by enhanced frequency of spontaneous glutamatergic events. The slight voltage dependence of oscillations and their block by the gap junction blocker, carbenoxolone, suggest they originate from electrically coupled neurons. Network nicotinic receptors desensitized more slowly than motoneuron ones, demonstrating that network receptors remained active longer to support heightened release of the endogenous glutamate necessary for enhancing the network excitability. The ionotropic glutamate receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and the group I metabotropic receptor antagonist, (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA), suppressed oscillations, while the NMDA receptor antagonist, d-amino-phosphonovaleriate (APV), produced minimal depression. Nicotine-evoked oscillations constrained spike firing at low rates, although motoneurons could still generate high-frequency trains of action potentials with unchanged gain for input depolarization. This is the first demonstration that persistent activation of nicotinic receptors could cause release of endogenous glutamate to evoke sustained oscillations in the theta frequency range. As this phenomenon likely represented a powerful process to coordinate motor output to tongue muscles, our results outline neuronal nicotinic acetylcholine receptors (nAChRs) as a novel target for pharmacological enhancement of motoneuron output in motor dysfunction.

Conversion from testosterone to oestradiol is required to modulate respiratory long-term facilitation in male rats.

Sex hormones modulate plasticity in the central nervous system, including respiratory long-term facilitation (LTF), a form of serotonin-dependent respiratory plasticity induced by intermittent hypoxia. Since gonadectomy (GDX) attenuates LTF in male rats, we tested the hypotheses that: (1) testosterone replenishment restores LTF in gonadectomized male rats, and (2) that the conversion of testosterone to oestradiol (under the influence of aromatase) is required for these effects. Intact and sham operated male F344 rats were compared to gonadectomized rats implanted with Silastic tubing containing testosterone (T), T plus an aromatase inhibitor (ADT), or 5alpha-dihydrotestosterone (DHT), a form of testosterone not converted to oestradiol. Seven days postsurgery, LTF was studied in anaesthetized, neuromuscularly blocked and ventilated rats while monitoring integrated phrenic and hypoglossal (XII) motor output. LTF was elicited by three 5 min hypoxic episodes (P(a,O(2)) = 35 - 45 mmHg). Although significant phrenic and XII LTF were observed in all rat groups, GDX reduced both phrenic and XII LTF, an effect reversed by T. In contrast, LTF was not restored in T + ADT or DHT-treated gonadectomized rats. We conclude that the conversion of testosterone to oestradiol modulates phrenic and XII LTF in male F344 rats.