Control of motoneuron function and muscle tone during REM sleep, REM sleep behavior disorder and cataplexy/narcolepsy.

REM sleep triggers a potent suppression of postural muscle tone - i.e., REM atonia. However, motor control during REM sleep is paradoxical because overall brain activity is maximal, but motor output is minimal. The skeletal motor system remains quiescent during REM sleep because somatic motoneurons are powerfully inactivated. Determining the mechanisms triggering loss of motoneuron function during REM sleep is important because breakdown in REM sleep motor control underlies sleep disorders such as REM sleep behavior disorder (RBD) and cataplexy/narcolepsy. For example, RBD is characterized by dramatic REM motor activation resulting in dream enactment and subsequent patient injury. In contrast, cataplexy a pathognomonic symptom of narcolepsy - is caused by the involuntary onset of REM-like atonia during wakefulness. This review highlights recent work from my laboratory that examines how motoneuron function is lost during normal REM sleep and it also identifies potential biochemical mechanisms underlying abnormal motor control in both RBD and cataplexy. First, I show that both GABAB and GABAA/glycine mediated inhibition of motoneurons is required for generating REM atonia. Next, I show that impaired GABA and glycine neurotransmission triggers the cardinal features of RBD in a transgenic mouse model. Last, I show that loss of an excitatory noradrenergic drive onto motoneurons is, at least in part, responsible for the loss of postural muscle tone during cataplexy in narcoleptic mice. Together, this research indicates that multiple transmitters systems are responsible for regulating postural muscle tone during REM sleep, RBD and cataplexy.

Neurotoxic lesions at the ventral mesopontine junction change sleep time and muscle activity during sleep: an animal model of motor disorders in sleep.

There is no adequate animal model of restless legs syndrome (RLS) and periodic leg movements disorder (PLMD), disorders affecting 10% of the population. Similarly, there is no model of rapid eye movement (REM) sleep behavior disorder (RBD) that explains its symptoms and its link to Parkinsonism. We previously reported that the motor inhibitory system in the brainstem extends from the medulla to the ventral mesopontine junction (VMPJ). We now examine the effects of damage to the VMPJ in the cat. Based on the lesion sites and the changes in sleep pattern and behavior, we saw three distinct syndromes resulting from such lesions; the rostrolateral, rostromedial and caudal VMPJ syndromes. The change in sleep pattern was dependent on the lesion site, but was not significantly correlated with the number of dopaminergic neurons lost. An increase in wakefulness and a decrease in slow wave sleep (SWS) and REM sleep were seen in the rostrolateral VMPJ-lesioned animals. In contrast, the sleep pattern was not significantly changed in the rostromedial and caudal VMPJ-lesioned animals. All three groups of animals showed a significant increase in periodic and isolated leg movements in SWS and increased tonic muscle activity in REM sleep. Beyond these common symptoms, an increase in phasic motor activity in REM sleep, resembling that seen in human RBD, was found in the caudal VMPJ-lesioned animals. In contrast, the increase in motor activity in SWS in rostral VMPJ-lesioned animals is similar to that seen in human RLS/PLMD patients. The proximity of the VMPJ region to the substantia nigra suggests that the link between RLS/PLMD and Parkinsonism, as well as the progression from RBD to Parkinsonism may be mediated by the spread of damage from the regions identified here into the substantia nigra.

Respiratory pre-motor control of hypoglossal motoneurons in the rat.

The goal of this study was to determine the origin and transmission pathway of respiratory drive to hypoglossal motoneurons. First we recorded intracellularly from 28 antidromically activated inspiratory hypoglossal motoneurons (resting membrane potential, -50+/-3 mV), and found that injection of chloride ions had no discernible effect on the shape of their membrane potential trajectories. We concluded that the membrane potential trajectories of these hypoglossal motoneurons were determined primarily by inspiratory excitation. To determine the origin of this excitation we cross-correlated the extracellular discharge of medullary inspiratory neurons, including those in the hypoglossal motor nucleus, with the hypoglossal nerve discharge. We found 27 inspiratory neurons within the hypoglossal motor nucleus that were not antidromically activated from the ipsilateral hypoglossal nerve; their cross-correlograms featured either central peaks (1.7+/-0.2 ms) alone (n=14; 39%), or central peaks (1.3+/-0.2 ms) followed by troughs (1.3+/-0.1 ms) at short latencies (1.1+/-0.4 ms) (n=13; 36%), and suggest that these neurons are hypoglossal interneurons. We recorded from 238 inspiratory neurons throughout the rest of the medulla; the cross-correlograms of 19 neurons (8%), located mostly in the lateral tegmental field, displayed narrow half-amplitude peaks (1.0+/-0.1 ms) at short latencies (0.9+/-0.1 ms), which we interpreted as evidence for monosynaptic excitation of hypoglossal motoneurons.We conclude that the respiratory control of hypoglossal motoneurons originates from inspiratory premotor neurons scattered throughout the lateral tegmental field and interneurons within the hypoglossal motor nucleus.

Nucleus raphé obscurus modulates hypoglossal output of neonatal rat in vitro transverse brain stem slices.

Nucleus raphé obscurus (NRo) modulates hypoglossal (XII) nerve motor output in the in vitro transverse brain stem slice of neonatal rats (1-5 days old); chemical ablation of NRo and its focal CO(2) acidification modulated the bursting rhythm of XII nerves. We microinjected a 4.5 mM solution of kainic acid into the NRo to disrupt cellular activity and observed that XII nerve activity was temporarily abolished (n = 10). We also microinjected CO(2)-acidified (pH = 6.00 +/- 0.01) artificial cerebrospinal fluid (aCSF) into the NRo (n = 6), the pre-Bötzinger complex (PBC) (n = 6), as well as a control region in the lateral tegmental field equidistant to NRo, PBC, and the XII motor nuclei (n = 12). CO(2) acidification of the control region had no effect on XII motor output. CO(2) acidification of the NRo significantly (P < 0.05) increased the burst discharge frequency of XII nerves by 77%; integrated burst amplitude and burst duration increased by 64% and 52%, respectively. CO(2) acidification of the PBC significantly (P < 0.05) increased the burst discharge frequency of XII nerves by 65%, but neither integrated burst amplitude nor burst duration changed. These results demonstrate that chemical ablation of the NRo can abolish XII nerve bursting rhythm and that stimulation of the NRo with CO(2)-acidified aCSF can excite XII nerve bursting activity. From these observations, we conclude that, in transverse brain stem slices, the NRo contains pH/CO(2)-sensitive cells that modulate XII motor output.

Bilaterally independent respiratory rhythms in the decerebrate rat.

In rats, respiratory neurons in the medulla oblongata are arranged in longitudinally distributed groups that are duplicated on each side of the neuraxis. Our aim was to determine whether respiratory rhythm is generated independently by each side. We made a complete mid-sagittal section of the medulla oblongata, 3.5 mm rostral and 3.5 mm caudal to the obex, in decerebrate, vagotomized, and paralysed adult rats. Respiratory rhythm, monitored by recording the activity of both left and right phrenic nerves, was maintained and became asynchronous between the left and right sides. We concluded that in the adult rat each half of the medulla oblongata is capable of generating respiratory rhythm independently.

Synaptic connections to phrenic motoneurons in the decerebrate rat.

Phrenic motoneuron membrane potential trajectories in decerebrate rats exhibit three stages; depolarisation during inspiration, a decreased depolarisation during early expiration and hyperpolarization during late expiration. These trajectories are a result of excitation by ventral-group medullary inspiratory neurons and upper-cervical inspiratory neurons during inspiration and the early part of expiration, and inhibition from Bötzinger-complex expiratory neurons during the late part of expiration.

Attenuation of mania-like behavior in Na(+),K(+)-ATPase α3 mutant mice by prospective therapies for bipolar disorder: melatonin and exercise.

Bipolar disorder is a neuropsychiatric disease characterized by states of mania with or without depression. Pharmacological treatments can be inadequate at regulating mood for many individuals. Melatonin therapy and aerobic exercise are independent prospective therapies for bipolar disorder that have shown potential as mood stabilizers in humans. Myshkin mice (Myk/+) carry a heterozygous missense mutation in the neuronal Na(+),K(+)-ATPase α3 and model mania-related symptoms of bipolar disorder including increased activity, risk-taking behavior and reductions in sleep. One cohort of Myk/+ and wild-type littermates (+/+) was treated with melatonin and a separate cohort was treated with voluntary exercise. Mania-related behavior was assessed in both cohorts. The effect of melatonin on sleep and the effect of exercise on brain-derived neurotrophic factor (BDNF) expression in the hippocampus were assayed. Melatonin and voluntary wheel running were both effective at reducing mania-related behavior in Myk/+ but did not affect behavior in +/+. Melatonin increased sleep in Myk/+ and did not change sleep in +/+. Myk/+ showed higher baseline levels of BDNF protein in the hippocampus than +/+. Exercise increased BDNF protein in +/+ hippocampus, while it did not significantly affect BDNF levels in Myk/+ hippocampus. These findings support initial studies in humans indicating that melatonin and exercise are useful independent adjunct therapies for bipolar disorder. Their effects on mood regulation should be further examined in randomized clinical trials. Our results also suggest that hippocampal BDNF may not mediate the effects of exercise on mania-related behavior in the Myk/+ model of mania.

Day-night differences in the respiratory response to hypercapnia in awake adult rats.

We investigated whether resting ventilation and the hypercapnic ventilatory response vary with time of day in awake adult rats. Respiratory frequency (fR), tidal volume (VT), inspired ventilation (VI), inspiratory interval (tI), carbon dioxide production (VCO2) and abdominal temperature (Tb) were measured before and during a hypercapnic stimulus (3.5% CO2 in air) at 10:00 and 22:00 h. VCO2, Tb and mean inspiratory air flow (VT/tI) were significantly higher at 22:00 h in air. VI/VCO2 was similar at 10:00 and 22:00 h. VI was significantly elevated by hypercapnia and the response at 22:00 h was 2.3 times greater than that at 10:00 h. VT/tI was unchanged at 10:00 h but significantly increased by hypercapnia at 22:00 h. VCO2 was significantly depressed at 10:00 h but not at 22:00 h. Tb was unaffected by hypercapnia. We conclude that the metabolic and ventilatory responses to hypercapnia are dependent on the time of day.

Bilateral synchronisation of respiratory motor output in rats: adult versus neonatal in vitro preparations.

The synchronisation of the discharges recorded from left and right phrenic nerves in the adult rat is produced in part by shared excitation from a common premotor neurone population. However, such synchronisation has not been examined for hypoglossal motoneurones in adult rats, or for phrenic and hypoglossal motoneurons in neonatal in vitro preparations. In adult rats, cross-correlograms computed between the inspiratory discharges of the left and right phrenic nerves, and the left and right hypoglossal nerves displayed central peaks with half-amplitude widths of 1.4+/-0.1 and 1.7+/-0.1 ms (mean+/-SE), respectively. We interpret these as evidence for common excitation. However, such central peaks were absent in the same cross-correlograms computed for neonatal in vitro preparations, although central peaks were observed in cross-correlograms computed between the discharges recorded from adjacent phrenic nerve rootlets. We conclude that, in the adult rat, left and right hypoglossal nerve discharges are synchronised by excitation from a common premotor neurone population, as for the phrenic nerves, but this type of synchronisation is undetectable in neonatal in vitro preparations. We speculate that the differences between the adult and neonatal preparations are due to developmental changes in respiratory drive transmission pathways.

Functional synaptic connections among respiratory neurons.

This presentation focuses on the application of methods to determine functional connections between neurons in the respiratory network of adult decerebrate rats. We employ a general network investigation paradigm that first examines the intracellular recordings of a respiratory neuron and then determines which neurons synapse with it to produce the observed membrane potential changes. It is used to pursue the source of respiratory excitation and inhibition from its arrival at phrenic motoneurons to respiratory neurons in the medulla, and then examine some of the interactions among these neurons that shape their patterns of activity. Findings include a demonstration that phrenic motoneuron activity is determined by excitation from medullary inspiratory premotor neurons and inhibition by Bötzinger complex expiratory neurons, and that the latter neurons inhibit both medullary inspiratory premotor neurons and themselves. We conclude that these functional interconnections explain the activity patterns of some respiratory neurons, but the connections between neurons thought to be involved in rhythm generation remain to be demonstrated in adult rats.

Mutual inhibition between Bötzinger-complex bulbospinal expiratory neurons detected with cross-correlation in the decerebrate rat.

We examined the synaptic connections between pairs of Bötzinger-complex, bulbospinal expiratory neurons in decerebrate rats. All were antidromically activated from the spinal cord at the C2-C3 border. Cross-correlation histograms of 18 ipsilateral pairs showed troughs on both sides of time zero (8) and to one side of time zero (4); most (12) were accompanied by peaks at time zero. Similarly, cross-correlation histograms of the contralateral pairs (12) showed troughs on both sides of time zero (3) and to one side of time zero (3); few (2) were accompanied by peaks at time zero. We considered the troughs in these cross-correlation histograms to be evidence of inhibition between the neurons and sought confirmation of the inhibitory connection. First, using the antidromic activation stimulus, we computed post-stimulus histograms of the extracellularly recorded discharge for six neurons and found that three showed troughs. Then, we continued this approach, computing post-stimulus averages of the membrane potentials recorded intracellularly from these neurons after iontophoresis of chloride to reverse inhibitory synaptic potentials. Depolarising potentials were observed in 15 of 16 of these averages. We interpreted these as reversed inhibitory post-synaptic potentials and concluded that Bötzinger-complex, bulbospinal expiratory neurons inhibit one another in rats as they do in cats.

Bötzinger-complex, bulbospinal expiratory neurones monosynaptically inhibit ventral-group respiratory neurones in the decerebrate rat.

Extracellularly recorded action potentials from 49 Bötzinger-complex, bulbospinal expiratory neurones were used as triggers to compute 162 spike-triggered averages (STAs) of intracellular potentials recorded from 167 respiratory neurones in the ventral respiratory group (VRG) near the obex in 15 vagotomized, paralysed, ventilated and decerebrated rats. All of the Bötzinger-complex expiratory neurones were antidromically activated from the ipsilateral border between the C2/C3 segments of the spinal cord and discharged only during the late part of expiration with an augmenting pattern. We found evidence for monosynaptic inhibitory post-synaptic potentials (IPSPs) in 74 (approximately 44%) of the STAs computed using 34 (approximately 69%) of the trigger neurones. For vagal motoneurones, IPSPs were found in 24 of the 53 STAs of expiratory motoneurones, but in none of the 12 STAs of inspiratory motoneurones. For inspiratory neurones, IPSPs were found in 23 of the 33 STAs of bulbospinal neurones and in 6 of the 26 STAs of not antidromically activated (NAA) neurones. For expiratory neurones, IPSPs were found in one of the two STAs of bulbospinal neurones and in 20 of the 36 STAs of NAA neurones. We conclude that Bötzinger-complex, bulbospinal expiratory neurones monosynaptically inhibit bulbospinal inspiratory neurones, expiratory vagal motoneurones and other unidentified inspiratory and expiratory neurones in the VRG of rats during the late part of expiration.

Bötzinger-complex expiratory neurons monosynaptically inhibit phrenic motoneurons in the decerebrate rat.

We examined respiratory neurons in the Bötzinger complex of the medulla oblongata in 18 vagotomized, paralyzed, ventilated, and decerebrated rats and tested the hypothesis that bulbospinal expiratory neurons in this region monosynaptically inhibit phrenic motoneurons. First, we surveyed the types of respiratory neurons found in the Bötzinger complex; only 11 of the 98 (approximately 11%) examined were bulbospinal, and all discharged only during late expiration (E2), usually with an augmenting discharge frequency (AUG). Then, we examined the spinal projections of 34 E2-AUG neurons using antidromic activation and found that all projected as far as the C4 or C5 segments of the spinal cord but no further caudally. Most (30, approximately 88%) had only unilateral projections, the majority (25, approximately 83%) ipsilateral, but 4 neurons (approximately 12%) had bilateral projections. Their axons could be antidromically activated at low currents (less than 10 microA) in the dorsal-lateral part of the spinal cord at the C2-3 border; 0.5-1.2 mm (mean+/-SD 0.84+/-0.23 mm) below the dorsal surface and 0.7-1.5 mm (1.19+/-0.25 mm) lateral from the midline. We sought evidence for connections from bulbospinal E2-AUG neurons to 118 phrenic motoneurons by computing spike-triggered averages (STAs) of their intracellular potentials triggered by the action potentials of 38 unilaterally-projecting E2-AUG neurons. Resting phrenic motoneuron membrane potentials ranged from -40 to -75 mV (-56+/-8 mV) and fluctuations with the respiratory cycle from 7 to 20 mV (14+/-4 mV). Of the 118 STAs computed, hyperpolarizations were evident in 18 (approximately 15%) STAs, evoked by 11 of 38 (approximately 29%) E2-AUG neurons. Their amplitudes varied from 35 to 550 microV (105+/-113 microV), 10-90% fall times from 0.4 to 0.9 ms (0.63+/-0.17 ms), and half-amplitude widths from 1.3 to 3.2 ms (2.0+/-0.52 ms). Most (16/95, approximately 17%) of the STAs that displayed hyperpolarizations were associated with ipsilateral trigger neurons but some (2/23, approximately 9%) resulted from contralateral trigger neurons. We conclude that Bötzinger-complex, expiratory neurons project to the C4 and/or C5 segments of the cervical spinal cord but no further caudal. Their axons are located dorsolaterally in the upper cervical segments of the spinal cord, and they monosynaptically inhibit phrenic motoneurons during the late part of expiration.

Temperature and pH affect respiratory rhythm of in-vitro preparations from neonatal rats.

We examined the respiratory rhythm of two in-vitro preparations from neonatal rats, the brainstem-spinal cord and transverse brainstem slice, recording the bursting activity of phrenic and hypoglossal nerves, respectively at 1 degree C intervals from 25 to 35 degrees C at two pH's, 7.4 and 7.1. In both preparations at either pH, burst frequency increased with temperature, burst duration declined and burst amplitude reached a peak at 30 degrees C. The shapes of the bursts changed from a decrementing pattern at low temperatures to a bell-shaped pattern at high temperatures. At reduced pH, frequency increased for temperatures between 25 and 32 degrees C in the brainstem-spinal cord but not in the slice. Burst duration was increased at reduced pH for temperatures between 27 and 29 degrees C in the brainstem-spinal cord, but not in the slice. Burst amplitude only changed with pH at the lower temperatures, decreasing at the lower pH in the brainstem-spinal cord and increasing in the slice. With respect to the effects of temperature, we concluded that both preparations were similarly affected, and that an increase in temperature alters the in-vitro burst pattern towards that observed in-vivo. With respect to the effects of pH, we concluded that effects differ between these in-vitro preparations and from in-vivo preparations, and that the difference between in-vivo and in-vitro preparations in their response to decreasing pH is not due to differences in temperature.

Respiratory control of hypoglossal motoneurones in the rat.

In this study of adult and neonatal rats, we used cross-correlation analysis to detect synchronous neuronal events in hypoglossal and phrenic nerves to infer synaptic connections. We found evidence for the common excitation of medial and lateral hypoglossal motoneurones in 12 anaesthetized adult rats but not in 6 in vitro brainstem-spinal cord preparations. We did not find evidence for the common activation of phrenic and hypoglossal motoneurones in 23 adult and 10 neonatal rat preparations. We confirmed this negative result by demonstrating that 26 medullary inspiratory neurones activating phrenic motoneurones did not activate hypoglossal motoneurones in 23 adult decerebrate rats (except in one case). We also found that 15 Bötzinger expiratory neurones inhibiting phrenic motoneurones did not inhibit hypoglossal motoneurones. We conclude that: (1) motoneurones of the medial and lateral hypoglossal nerve branches receive inspiratory drive from a common premotor population in adult rats, but in neonatal rats adjacent nerve rootlets do not; (2) in both adult and neonatal rats phrenic premotor neurones do not monosynaptically excite hypoglossal motoneurones; (3) Bötzinger expiratory neurones that inhibit phrenic motoneurones do not inhibit hypoglossal motoneurones. We therefore suggest that the respiratory control of hypoglossal motoneurones is separate from that of phrenic motoneurones.