Plasma pharmacokinetics of alfaxalone after a single intraperitoneal or intravenous injection of Alfaxan(®) in rats.

Alfaxalone (3α-hydroxy-5α-pregnane-11, 20-dione) is a neuroactive steroid with anaesthetic properties and a wide margin of safety. The pharmacokinetic properties of alfaxalone administered intravenously and intraperitoneally in rats (n = 28) were investigated. Mean t(1/2elim) for 2 and 5 mg/kg i.v. was 16.2 and 17.6 min, respectively, but could not be estimated for IP dosing, due to sustained plasma levels for up to 60 min after injection. Clp for i.v. injection was calculated at 57.8 ± 23.6 and 54.3 ± 6.8 mL/min/kg, which were 24.5% and 23% of cardiac output, respectively. The observed C(max) was 3.0 mg/L for IP administration, and 2.2 ± 0.9 and 5.2 ± 1.3 mg/L for 2 and 5 mg/kg i.v. administration, respectively. AUC(0-60) was 96.2 min.mg/L for IP dosing. The relative bioavailability for IP dosing was 26% and 28% compared to i.v. dosing. Differences in t(1/2elim) and Cl(p) from previous pharmacokinetic studies in rats are likely due to variations in alfaxalone formulation rather than sex differences. Alfaxan® given IP caused sustained levels of alfaxalone, no apnoea and longer sleep times than i.v. dosing, although immobilization was not induced in 30% of rats given Alfaxan® IP. A pharmacodynamic study of the effects of combining IP injection of Alfaxan® with other premedication agents is worthwhile, to determine whether improved anaesthesia induction could ultimately provide an alternative anaesthetic regimen for rats.

A novel presynaptic inhibitory mechanism underlies paired pulse depression at a fast central synapse.

Several distinct mechanisms may cause synaptic depression, a common form of short-term synaptic plasticity. These include postsynaptic receptor desensitization, presynaptic depletion of releasable vesicles, or other presynaptic mechanisms depressing vesicle release. At the endbulb of Held, a fast central calyceal synapse in the auditory pathway, cyclothiazide (CTZ) abolished marked paired pulse depression (PPD) by acting presynaptically to enhance transmitter release, rather than by blocking postsynaptic receptor desensitization. PPD and its response to CTZ were not altered by prior depletion of the releasable vesicle pool but were blocked by lowering external calcium concentration, while raising external calcium enhanced PPD. We conclude that a major component of PPD at the endbulb is due to a novel, transient depression of release, which is dependent on the level of presynaptic calcium entry and is CTZ sensitive.

Morphology and electrophysiology of superior laryngeal nerve afferents and postsynaptic neurons in the medulla oblongata of the cat.

Intra-axonal recordings were made from 24 afferent fibres of the superior laryngeal nerve in and around the nucleus tractus solitarius, in 26 pentobarbitone-anaesthetized cats. Conduction velocity ranged from 15 to 38 m/s. Four afferents were injected with horseradish peroxidase. They showed dense terminal arborization in the region of the ventral and ventrolateral subnuclei of the nucleus tractus solitarius, both rostral and caudal to the obex. Six other intra-axonal recordings were thought to originate from axons of neurons postsynaptic to superior laryngeal afferents; one of these was injected with horseradish peroxidase and showed a similar arborization pattern to that of the afferent axons. In the same region, intracellular recordings were made from 124 neurons which responded to superior laryngeal nerve stimulation with excitatory postsynaptic potentials (mean latency 2.7 +/- 1.0 ms). Ninety-nine of these neurons were thought to receive a monosynaptic input. The stimulation threshold evoking these responses was similar to that which inhibited phrenic nerve discharge. Eleven of the monosynaptically excited neurons were injected with horseradish peroxidase. They had fusiform or stellate somata and simple dendritic trees, radiating mainly in the transverse plane. In one experiment, in which both a superior laryngeal nerve afferent fibre and a neuron were labelled, afferent terminal varicosities were found in close apposition with the postsynaptic membrane of the injected neuron. Four of 14 (29%) tested neurons could be antidromically activated from the C3 spinal segment. The stimulus thresholds and onset latencies of the responses of superior laryngeal nerve afferents and medullary neurons to stimulation of the superior laryngeal nerve are consistent with their involvement in the reflex inhibition of respiratory neurons evoked by superior laryngeal nerve stimulation.

P2X7-like receptor subunits enhance excitatory synaptic transmission at central synapses by presynaptic mechanisms.

Recent studies demonstrate that P2X7 receptor subunits (P2X7RS) are present at central and peripheral synapses and suggest that P2X7RS can regulate transmitter release. In brainstem slices from 15 to 26 day old pentobarbitone-anesthetized mice, we examined the effect of P2X7RS activation on excitatory postsynaptic currents (EPSCs) recorded from hypoglossal motoneurons using whole-cell patch clamp techniques. After blockade of most P2X receptors with suramin (which is inactive at P2X7RS) and of adenosine receptors with 8-phenyltheophylline (8PT), bath application of the P2X receptor agonist 3'-0-(4-benzoyl)ATP (BzATP) elicited a 40.5+/-16.0% (mean+/-S.E.M., n = 8, P = 0.039) increase in evoked EPSC amplitude and significantly reduced paired pulse facilitation of evoked EPSCs. This response to BzATP (with suramin and 8PT present) was completely blocked by prior application of Brilliant Blue G (200 nM or 2 microM), a P2X7RS antagonist. In contrast, BzATP application with suramin and 8PT present did not alter miniature EPSC frequency or amplitude when action potentials were blocked with tetrodotoxin. These electrophysiological results suggest that P2X7RS activation increases central excitatory transmitter release via presynaptic mechanisms, confirming previous indirect measures of enhanced transmitter release. We suggest that possible presynaptic mechanisms underlying enhancement of evoked transmitter release by P2X7RS activation are modulation of action potential width or an increase in presynaptic terminal excitability, due to subthreshold membrane depolarization which increases the number of terminals releasing transmitter in response to stimulation.

Activity-related pH changes in respiratory neurones and glial cells of cats.

Intracellular pH (pHi) and membrane potential (Em) were measured in vivo in expiratory neurones and glial cells in the medulla of anaesthetized cats using double-barrelled H(+)-sensitive microelectrodes. In glial cells, stimulation of spinal pathways evoked a depolarization of up to 12 mV amplitude and an increase of pHi (7.25 +/- 0.15) by maximally 0.1 pH unit. IN expiratory neurones, pHi (7.15 +/- 0.18) fell by up to 0.2 pH unit during inspiratory inhibition. In axons of expiratory neurones, pHi remained unaffected during rhythmic action potential discharges. We suggest that the glial alkalinization is due to activation of Na+/HCO3- cotransport, whereas the neuronal acidification is caused by efflux of HCO3- via receptor-coupled anion channels.

Limitations of the technique of pressure microinjection of excitatory amino acids for evoking responses from localized regions of the CNS.

The aim of this study, performed on anaesthetized cats and rabbits, was to test the assumption that pressure microinjections of excitatory amino acids cause long-lasting excitation of neurones located close to the injection site. Unitary action potentials or antidromic field potentials were recorded from respiratory or 'reticular' neurones in the medulla oblongata and from phrenic motoneurones at different distances from the injection site. Injection of 10-150 nl (5-150 nmol) of L-glutamate or DL-homocysteic acid into these areas resulted in complex and widespread neuronal events. Generally, more distant neurones (500-1300 microns) were excited for variable periods of time (3-15 min), while neurones in the vicinity of the injection site (0-500 microns) showed, after a brief period of excitation time, a long-lasting (up to 30 min) decrease in excitability or silencing of discharge, probably due to a depolarizing block and disturbances in the ionic composition of the extracellular space. These findings show that interpretation of physiological responses following such injections should not be based on an assumption of local neuronal excitation. Some recommendations regarding the use of this technique are made.

Respiratory interneurons in the C5 segment of the spinal cord of the cat.

Extracellular recordings were made in the C5 segment of the spinal cord of anaesthetised cats from 129 units which showed respiratory phased discharge. The majority of recordings (88%) were thought to arise from the somata of respiratory spinal interneurons. Inspiratory units and expiratory units comprised 42% and 52% of all recorded units. A small number of postinspiratory units were also found (n = 5). Most units did not respond to electrical stimulation of the ipsilateral superior laryngeal (SLN) and phrenic nerves (PN), but a few expiratory (n = 2) and postinspiratory units (n = 1) were excited by SLN stimulation, while 6 inspiratory units had their discharge suppressed by the same stimulus. PN stimulation evoked a long latency (2-7 ms) burst of firing in 2 inspiratory and 1 expiratory interneurons. It is concluded that these respiratory interneurons may provide a segmental input to phrenic motoneurons, in addition to synaptic drives mediated by bulbospinal pathways.

Synaptic inhibition of phrenic motoneurones evoked by stimulation of the superior laryngeal nerve.

Synaptic responses evoked in phrenic motoneurones (PMNs) by stimulation of the superior laryngeal nerve (SLN) were analysed in anaesthetised cats. Stimulation of the SLN was followed by inhibition of ipsilateral phrenic nerve discharge with the latency of 9.5 +/- 2.3 ms (mean +/- S.D.) and hyperpolarizations of ipsilateral PMN membrane potentials (latency, 8.4 +/- 2.1 ms) which were observed after stimuli applied both during inspiration and expiration. During the injection of Cl ions, the hyperpolarizations were either reversed or flattened in all 28 tested PMNs, thus indicating a direct inhibition. The possibility that the inhibitory postsynaptic potentials are produced by segmental respiratory interneurones is discussed.

Pontine respiratory neurons in anesthetized cats.

The pontine respiratory neurons (PRG) in the 'pneumotaxic centre' have been hypothesized to contribute to phase-switching of neural respiratory activity, especially in terminating inspiration. To define the neural elements involved in phase-switching, we recorded respiratory neurons extra- and intracellularly in anesthetized cats with an intact central nervous system. In total, 54 neurons were recorded: 49 neurons with activity modulated by central respiratory rhythm (20 inspiratory, 17 postinspiratory and 12 expiratory) and 5 neurons with activity correlated to tracheal pressure. The recorded neurons were clustered in dorsolateral pontine tegmentum within the Kölliker-Fuse (KF) subnucleus of the parabrachial nuclei. Stable intracellular membrane potential was recorded in 11 of the 49 respiratory neurons (8 postinspiratory, 1 early inspiratory and 2 inspiratory). During continuous injection of chloride ions (n = 6), synaptic noise increased and IPSPs reversed, including a wave of IPSPs during stage-2 expiration in postinspiratory neurons. Further, relative input resistance varied through the respiratory cycle such that the least input resistance occurred during the neuron's (n = 5) quiescent period. No IPSPs nor EPSPs were evoked in pontine respiratory neurons by vagal stimulation. In conclusion, various types of respiratory neurons were recorded in the KF nucleus. Prominent excitatory and inhibitory postsynaptic activities were similar to those described for medullary neurons. These pontine respiratory neurons do not appear to receive a strong afferent input from the vagus. Rather, vagal afferent inputs seem to be directed towards non-respiratory neurons that are located more medially in the dorsal pons.

Adenosine suppresses excitatory glutamatergic inputs to rat hypoglossal motoneurons in vitro.

Short-latency excitatory postsynaptic potentials (EPSPs), evoked by electrical stimulation lateral to the hypoglossal motor nucleus, were recorded from rat hypoglossal motoneurons (HMs) in brainstem slices. EPSPs were markedly suppressed or abolished by kynurenic acid (1 mM), showing that they were glutamatergic. The adenosine receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA, 100 nM) reduced EPSP amplitude to 42% of control, while the agonist 2-chloroadenosine (2-CA, 0.5-50 microM) caused a dose-dependent reduction of the EPSP. The adenosine receptor antagonist 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX, 0.1-1 microM) increased the EPSP amplitude to 124% of control, and blocked EPSP reduction by CCPA or 2-CA. CCPA, 2-CA and DPCPX did not significantly alter HM input resistance or membrane potential. These data indicate that excitatory glutamatergic inputs to rat HMs are modulated by adenosine A1 receptors, most probably at a presynaptic site. This modulation may be especially significant in hypoxic responses of HMs.

Australian Neuroscience Society--20th Annual Meeting. 30 January-2 February 2000, Melbourne, Australia.

This meeting presented a large array of high quality neuroscience research, some of which held possible relevance for therapeutic development. The potential use of multipotent neural stem cells harvested from adult brain for transplantation and regeneration therapy was highlighted, as was the increasingly central role of the p75 neurotrophin receptor in regulating neuronal cell death. Of particular interest were strategies for the prevention of neuronal death by systemic administration of p75 receptor antisense oligonucleotides. Other significant data included a possible synergy between prostaglandin receptors and opioid receptors in cellular responses, thought to underlie pain perception and opioid analgesia. Groups in Melbourne, Brisbane and Bath, UK, have isolated novel alpha-conotoxins from Conus marine snails, and characterized their effects on neuronal nicotinic acetylcholine receptors (nAChRs), highlighting their subunit specificity and effects on synaptic transmission. While none of these findings are close to effective clinical use as yet, they hold great promise for the future, underlining the necessity for basic research as a starting point for novel therapies.

Postnatal development of hypoglossal motoneuron intrinsic properties.

This review has provided evidence that marked changes are occurring in ionic currents present in upper airway motoneurons during the early postnatal period. Our results have shown that the density of the LVA Ca2+ current decreases during this period, and this probably reflects a reduced expression of the Ca2+ channel responsible for this current, the so-called T-type channel. These results help to explain the changes in burst firing behavior of HMs during the early postnatal period. We have shown that the fraction of HMs exhibiting burst firing behavior was the greatest among HMs just at or after birth, and disappeared by 10 days of age (Viana et al, 1993). The LVA Ca2+ current contributes to this firing behavior. In contrast to the reduction in the LVA Ca2+ current density with postnatal development, there is an apparent increase in Ih current density during this period. The increase in Ih provides a basis for a number of differences in the electrophysiological properties of adult versus neonate HMs. These include a striking depolarizing sag and overshoot during and immediately after application of hyperpolarizing current pulses in adult HMs. It is of interest that rebound depolarization following hyperpolarization can be observed in neonatal HMs even though there is little Ih present. This response probably reflects the activation of a LVA Ca2+ current. Other differences in neonate versus adult HMs also are in part probably due to differences in Ih current density. Since Ih is active at normal resting membrane potential (approximately -70 mV), Ih may contribute to the lower input resistance of adult compared with neonatal HMs (Haddad et al, 1990; Núñez-Abades et al, 1993; Viana et al, 1994), and the lower apparent membrane resistivity of older HMs (Viana et al 1994). The larger Ih in the adult may be a factor in the shorter spike afterhyperpolarization observed in adult versus neonatal HMs (Viana, et al, 1994). This may be a consequence of the greater amount of Ih activated during the afterhyperpolarization in adult HMs. The larger Ih in adult HMs may also contribute to differences in how synaptic inputs are integrated. For example, inhibitory inputs which hyperpolarize the membrane potential may have their effect lessened due to Ih activation with hyperpolarization. Thus in adult HMs Ih may weaken prolonged or strong hyperpolarizations that occur in response to inhibitory synaptic inputs, while depolarizing responses arising from excitatory synaptic inputs may not be compromised. In contrast, neonatal HMs, which lack a substantial Ih current, do not have the stabilizing influence upon membrane potential that is due to Ih. Therefore, these cells may be more susceptible to such inhibitions. In conclusion, this chapter has described the changes that take place in two ionic currents during postnatal development, and how they contribute to distinct subthreshold and firing properties of neonatal and adult motoneurons.

Growth hormone secretion is correlated with neuromuscular innervation rather than motor neuron number in early-symptomatic male amyotrophic lateral sclerosis mice.

GH deficiency is thought to be involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). However, therapy with GH and/or IGF-I has not shown benefit. To gain a better understanding of the role of GH secretion in ALS pathogenesis, we assessed endogenous GH secretion in wild-type and hSOD1(G93A) mice throughout the course of ALS disease. Male wild-type and hSOD1(G93A) mice were studied at the presymptomatic, onset, and end stages of disease. To assess the pathological features of disease, we measured motor neuron number and neuromuscular innervation. We report that GH secretion profile varies at different stages of disease progression in hSOD1(G93A) mice; compared with age-matched controls, GH secretion is unchanged prior to the onset of disease symptoms, elevated at the onset of disease symptoms, and reduced at the end stage of disease. In hSOD1(G93A) mice at the onset of disease, GH secretion is positively correlated with the percentage of neuromuscular innervation but not with motor neuron number. Moreover, this occurs in parallel with an elevation in the expression of muscle IGF-I relative to controls. Our data imply that increased GH secretion at symptom onset may be an endogenous endocrine response to increase the local production of muscle IGF-I to stimulate reinnervation of muscle, but that in the latter stages of disease this response no longer occurs.

The relationship between Bayesian motor unit number estimation and histological measurements of motor neurons in wild-type and SOD1(G93A) mice.

OBJECTIVE: To assess the relationship between Bayesian MUNE and histological motor neuron counts in wild-type mice and in an animal model of ALS. METHODS: We performed Bayesian MUNE paired with histological counts of motor neurons in the lumbar spinal cord of wild-type mice and transgenic SOD1(G93A) mice that show progressive weakness over time. We evaluated the number of acetylcholine endplates that were innervated by a presynaptic nerve. RESULTS: In wild-type mice, the motor unit number in the gastrocnemius muscle estimated by Bayesian MUNE was approximately half the number of motor neurons in the region of the spinal cord that contains the cell bodies of the motor neurons supplying the hindlimb crural flexor muscles. In SOD1(G93A) mice, motor neuron numbers declined over time. This was associated with motor endplate denervation at the end-stage of disease. CONCLUSION: The number of motor neurons in the spinal cord of wild-type mice is proportional to the number of motor units estimated by Bayesian MUNE. In SOD1(G93A) mice, there is a lower number of estimated motor units compared to the number of spinal cord motor neurons at the end-stage of disease, and this is associated with disruption of the neuromuscular junction. SIGNIFICANCE: Our finding that the Bayesian MUNE method gives estimates of motor unit numbers that are proportional to the numbers of motor neurons in the spinal cord supports the clinical use of Bayesian MUNE in monitoring motor unit loss in ALS patients.

Impairments to the GH-IGF-I axis in hSOD1G93A mice give insight into possible mechanisms of GH dysregulation in patients with amyotrophic lateral sclerosis.

GH deficiency has been found in subjects with amyotrophic lateral sclerosis (ALS). Disrupted endocrine function could contribute to the progressive muscle loss and hypermetabolism seen in ALS. It is not possible to study all the elements of the GH-IGF-I axis in ALS patients. Consequently, it remains unclear whether dysfunctional GH secretion contributes to disease pathogenesis and why GH and IGF-I directed treatment strategies are ineffective in human ALS. The hSOD1(G93A) transgenic mouse model is useful for the detailed investigation of the pathogenesis of ALS. We report that symptomatic male hSOD1(G93A) transgenic mice exhibit a deficiency in GH secretion similar to that seen in human ALS. Further characterization of the GH-IGF-I axis in hSOD1(G93A) mice reveals central and peripheral abnormalities that are not found in wild-type age-matched controls. Specifically, we observe aberrant endogenous pulsatile GH secretion, reduced pituitary GH content, and decreased circulating levels of IGF-I, indicating global GH deficiency in hSOD1(G93A) mice. Furthermore, a reduction in the expression of the IGF-I receptor α-subunit in skeletal muscle and lumbar spinal cords of hSOD1(G93A) mice suggests impaired IGF-I signaling within these tissues. This is the first account of disrupted GH secretion in a transgenic mouse model of ALS. These observations are essential for the development of effective GH and IGF-I targeted therapies in ALS.

Synaptic inhibition of cat phrenic motoneurons by internal intercostal nerve stimulation.

Intracellular recordings from 65 phrenic motoneurons (PMNs) in the C5 segment and recordings of C5 phrenic nerve activity were made in 27 pentobarbitone-anesthetized, paralyzed, and artificially ventilated adult cats. Inhibition of phrenic nerve activity and PMN membrane potential hyperpolarization (48/55 PMNs tested) was seen after stimulation of the internal intercostal nerve (IIN) at a mean latency to onset of 10.3 +/- 2.7 ms. Reversal of IIN-evoked hyperpolarization (n = 14) by injection of negative current or diffusion of chloride ions occurred in six cases, and the hyperpolarization was reduced in seven others. Stimulation of the IIN thus activates chloride-dependent inhibitory synaptic inputs to most PMNs. The inhibitory phrenic nerve response to IIN stimulation was reduced by ipsilateral transection of the lateral white matter at the C3 level and was converted to an excitatory response by complete ipsilateral cord hemisection at the same level. After complete ipsilateral hemisection of the spinal cord at C3 level, stimulation of the IIN evoked both excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs) in PMNs (n = 10). It was concluded that IIN stimulation can evoke both excitatory and inhibitory responses in PMNs using purely spinal circuitry, but that excitatory responses are normally suppressed by a descending pathway in intact animals. Fifteen PMNs were tested for possible presynaptic convergence of inputs in these reflex pathways, using test and conditioning stimuli. Significant enhancement (>20%) of IPSPs were seen in seven of eight IIN-evoked responses using pericruciate sensorimotor cortex (SMC) conditioning stimuli, but only one of five IIN-evoked responses were enhanced by superior laryngeal nerve (SLN) conditioning stimuli. The IIN-evoked IPSP was enhanced in one of two motoneurons by stimulation of the contralateral phrenic nerve. It was concluded that presynaptic interneurons were shared by the IIN and SMC pathways, but uncommonly by other pathways. These results indicate that PMNs receive inhibitory synaptic inputs from ascending thoracocervical pathways and from spinal interneurons. These inhibitory reflex pathways activated by afferent inputs from the chest wall may play a significant role in the control of PMN discharge, in parallel with disfacilitation following reduced activity in bulbospinal neurons projecting to PMNs.

Driving respiration: the respiratory central pattern generator.

1. The central pattern generator (CPG) for respiration is located in the brainstem and produces rhythmic synaptic drive for motoneurons controlling respiratory muscles. Based on respiratory nerve discharge, the respiratory cycle can be divided into three phases: inspiration, postinspiration and stage 2 expiration. 2. Six basic types of respiratory neuron participate in respiratory rhythmogenesis. Their firing and membrane potential patterns are locked to different phases of the respiratory cycle. 3. In adult mammals, respiratory neurons are subject to excitatory and inhibitory synaptic inputs and show extensive synaptic interconnections that are mainly inhibitory. There are differences in the relative importance of excitatory and inhibitory synaptic drives and the neurotransmitters involved in respiratory rhythmogenesis in neonates compared with adults. 4. Respiratory neurons possess a number of intrinsic membrane currents that may be involved in central pattern generation, including low- and high-voltage-activated calcium, potassium, calcium-dependent potassium, sodium and mixed cationic currents. More quantitative information is needed about the distribution and characteristics of these ionic currents if we are to understand rhythmogenesis. 5. The two main theories for the origin of respiratory rhythm are those of pacemaker neuron-driven and synaptic network-driven CPG. Evidence derived from in vivo and in vitro experiments exists to support both of these theories. There may be a significant switch in the underlying mechanism driving the respiratory CPG during postnatal development.

Presynaptic depression of excitatory synaptic inputs to rat hypoglossal motoneurons by muscarinic M2 receptors.

1. Whole cell recordings of glutamatergic excitatory postsynaptic currents (EPSCs) evoked by electrical stimulation in the reticular formation were made from visualized hypoglossal motoneurons (HMs) in rat brain stem slices. 2. Carbachol, muscarine, or physostigmine reduced EPSC amplitude to 50 +/- 3%, 37 +/- 3%, and 54 +/- 7% (mean +/- SE) of control, respectively; effects of carbachol and physostigmine were antagonized by atropine (1-2 microM). EPSC depression was most effectively antagonized by methoctramine, an M2 muscarinic acetylcholine receptor (mAChR) antagonist with a high affinity constant (pKB) of 8.07 for the receptor mediating this response, whereas pirenzepine, an M1 mAChR antagonist, had a pKB of < 7.0, showing that EPSC depression was mediated by the M2 mAChR. 3. Postsynaptic properties of HMs (holding current and input resistance), EPSCs (reversal potential, rise time, half-width, and decay time constant), and postsynaptic glutamate-gated currents (amplitude and waveform) were not altered by carbachol or muscarine. 4. Muscarine did not decrease presynaptic neuron excitability, because the frequency of spontaneous EPSCs in HMs in the absence of tetrodotoxin (TTX) was either unchanged or increased. Leak and action currents of reticular formation neurons were not significantly altered by muscarine. In contrast, with TTX present, the frequency of spontaneous miniature glutamatergic EPSCs in HMs was decreased by both carbachol (mean change = 203 +/- 46%) and muscarine (mean change = 185 +/- 26%), with no change in miniature EPSC amplitude distribution. 5. Muscarinic depression of excitatory transmission to HMs thus occurs at the presynaptic terminal, most probably affecting release mechanisms downstream from calcium entry, and is likely to be significant during rapid eye movement sleep, possibly underlying the loss of tongue tone and inspiratory activity during this state.

Cholinergic modulation of respiratory brain-stem neurons and its function in sleep-wake state determination.

1. Shifts in behavioural state are controlled by reciprocal changes in discharge of cholinergic and aminergic groups of brain-stem/pontine neurons. During rapid eye movement (REM) sleep, cholinergic neurons are most active and aminergic neurons are least active. 2. Significant changes occur in the central control of breathing during REM sleep; respiration rate increases in frequency and variability, brain-stem respiratory neuron discharge is generally enhanced and the outputs of some respiratory motor neuron pools are depressed. 3. Hypoglossal motor neurons (HM) control tongue movement and their depression during REM sleep has been implicated in obstructive sleep apnoea. The cellular basis of HM depression has been investigated in vitro and may be due to enhanced activation of cholinergic receptors or decreased activation of aminergic receptors. 4. In vitro preparations that show respiratory rhythmogenesis possess advantages for the investigation of the neurochemical basis of state-dependent changes in respiration. Cholinergic changes in respiratory modulation of HM recorded in rhythmic brain-stem slices from mice depend on the site of activation of cholinergic receptors.

Late expiratory inhibition of stage 2 expiratory neurons in the cat--a correlate of expiratory termination.

1. Intracellular recordings were made from stage 2 expiratory bulbospinal neurons (E2Ns) in the caudal part of the ventral respiratory group in pentobarbitone-anesthetized cats, to characterize changes in neuronal input resistance (Rn) and synaptic inhibition occurring at the time of the expiratory-inspiratory phase transition of the respiratory cycle. 2. Rn was maximal between 30-90% of stage 2 expiration, but decreased significantly during the last 10% of stage 2 expiration. Mean normalized Rn for 60-90% of stage 2 expiration was 0.9 +/- 0.02, while mean Rn during the last 10% of stage 2 expiration was 0.68 +/- 0.09 (n = 8). This decrease in Rn began 200-300 ms before rapid hyperpolarization of E2N membrane potential and onset of phrenic nerve activity. 3. Under conditions of strong central respiratory drive, constant injection of positive current into E2Ns sometimes revealed a transient membrane hyperpolarization that straddled the expiratory-inspiratory phase transition. During this transient event, Rn was markedly reduced. 4. Intracellular injection of Cl- or NO3- ions into E2Ns produced reversal of chloride-dependent inhibitory synaptic potentials (IPSPs). Comparison of averages of membrane potential pattern over the whole respiratory cycle during control conditions and IPSP reversal revealed several periods of synaptic inhibition: 1) weak but progressively increasing synaptic inhibition during the second half of stage 2 expiration, 2) strong transient synaptic inhibition beginning 200-300 ms before the onset of phrenic nerve activity and ending shortly after the onset of phrenic nerve activity, and 3) strong but progressively decreasing synaptic inhibition throughout inspiration.(ABSTRACT TRUNCATED AT 250 WORDS)