Blockade Of Glycine Receptors Research Articles

NOVEL ROLE OF GLYCINE IN CONTROL OF SYMPATHETIC OUTFLOW

The arterial baroreflex is one of the essential negative‐feedback mechanisms that compensates rapid changes in blood pressure (BP) through inhibition of presympathetic neurons in the rostral ventrolateral medulla (RVLM). There is general consensus that GABAergic projections originating from the caudal VLM (CVLM) provide the primary inhibitory input that controls the excitability of presympathetic RVLM neurons and sympathetic output. However, glycine, another inhibitory neurotransmitter, is also present in the RVLM. In anesthetized rats, we found that RVLM glycine played a critical role in arterial baroreflex function by controlling the time course of baroreflex mediated inhibition of renal sympathetic nerve activity. Blockade of glycine receptors in the RVLM, following an increase in BP, increased recovery of renal sympathetic nerve activity to baseline levels (5.1 ± 1.0 vs 11.7 ± 1.3 min, N=6, P<0.01) in comparison to control. In addition, tonic glycinergic inhibition of the RVLM was unmasked following blockade of output from the nucleus tractus solitaries and/or during disinhibition of the CVLM. Our whole‐cell, patch‐clamp recordings were obtained from brainstem slices containing presympathetic neurons in the RVLM labeled with PRV‐152. We confirmed that in steady state conditions GABAergic inhibition of RVLM neurons predominated and glycine contributed less than 25% of overall inhibition. In contrast, activation of the network produced saturation of GABAergic inhibition and glycinergic inhibition became prominent. Our findings demonstrated that blockade of glycine receptors in the RVLM decreased duration of baroreflex sympathoinhibition; increase of BP and activation of the network unmasked glycinergic inhibition of the RVLM; and release of glycine in the RVLM depended on the activity of presynaptic inhibitory inputs.Support or Funding InformationNational Institutes of Health R01 HL98602 (Cheryl M. Heesch) and HL122829 (Andrei V. Derbenev). Australian Research Council Future Fellowship FT170100363 and the National Health and Medical Research Council of Australia GNT 1079680 (Song T. Yao). High Blood Pressure Research Council of Australia, the Rebecca L Cooper Medical Foundation, and the Victorian Government through the Operational Infrastructure Scheme (Willian S. Korim).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Remote ischemic conditioning provides humoural cross-species cardioprotection through glycine receptor activation.

Remote ischaemic conditioning (RIC) releases a humoural factor able to exert cross-species cardioprotection when plasma dialysate is applied to isolated hearts. However, the exact chemical nature of this factor is currently unknown. RIC (4 × 5min femoral occlusion/5min reperfusion) was applied to 10 male pigs, and blood was taken before and after the manoeuvre. Discriminant analysis of 1H-NMR spectra (n = 10-12) obtained from plasma dialysates (12-14 kDa cut-off) allowed to demonstrate a different metabolic profile between control and postRIC samples, with lactate (2.671 ± 0.294 vs. 3.666 ± 0.291 μmol/mL, P = 0.020), succinate (0.062 ± 0.005 vs. 0.082 ± 0.008 μmol/mL, P = 0.035) and glycine (0.055 ± 0.009 vs. 0.471 ± 0.151 μmol/mL, P = 0.015) being the main responsible for such differences. Plasma dialysates were then given to isolated mice hearts submitted to global ischaemia (35 min) and reperfusion (60 min), for 30 min before ischaemia or during the first 15 min of reflow. Infarct size was significantly reduced when postRIC dialysate was applied before ischaemia as compared with hearts pretreated with control dialysate (44.81 ± 3.22 vs. 55.55 ± 2.53%, P = 0.012, n = 12). Blockade of glycine receptors with strychnine 10 μM inhibited the protective effect caused by pretreatment with postRIC dialysate (52.76 ± 6.94 vs. 51.92 ± 5.78%, P-NS, n = 5), whereas pretreatment with glycine 3 mmol/L, but not succinate 100 μmol/L, mimicked RIC protection (41.90 ± 4.50% in glycine-treated vs. 61.51 ± 5.16 and 64.73 ± 4.47% in succinate-treated and control hearts, respectively, P < 0.05, n = 4-7). RIC releases glycine and exerts cross-species cardioprotection against infarction through glycine receptor activation.

Inhibitory control of ascending glutamatergic projections to the lamprey respiratory rhythm generator

Neurons within the vagal motoneuron region of the lamprey have been shown to modulate respiratory activity via ascending excitatory projections to the paratrigeminal respiratory group (pTRG), the proposed respiratory rhythm generator. The present study was performed on in vitro brainstem preparations of the lamprey to provide a characterization of ascending projections within the whole respiratory motoneuron column with regard to the distribution of neurons projecting to the pTRG and related neurochemical markers. Injections of Neurobiotin were performed into the pTRG and the presence of glutamate, GABA and glycine immunoreactivity was investigated by double-labeling experiments. Interestingly, retrogradely labeled neurons were found not only in the vagal region, but also in the facial and glossopharyngeal motoneuron regions. They were also present within the sensory octavolateral area (OLA). The results show for the first time that neurons projecting to the pTRG are immunoreactive for glutamate, surrounded by GABA-immunoreactive structures and associated with the presence of glycinergic cells. Consistently, GABAA or glycine receptor blockade within the investigated regions increased the respiratory frequency. Furthermore, microinjections of agonists and antagonists of ionotropic glutamate receptors and of the GABAA receptor agonist muscimol showed that OLA neurons do not contribute to respiratory rhythm generation. The results provide evidence that glutamatergic ascending pathways to the pTRG are subject to a potent inhibitory control and suggest that disinhibition is one important mechanism subserving their function. The general characteristics of inhibitory control involved in rhythmic activities, such as respiration, appear to be highly conserved throughout vertebrate evolution.

Chloride dysregulation and inhibitory receptor blockade yield equivalent disinhibition of spinal neurons yet are differentially reversed by carbonic anhydrase blockade.

Synaptic inhibition plays a key role in processing somatosensory information. Blocking inhibition at the spinal level is sufficient to produce mechanical allodynia, and many neuropathic pain conditions are associated with reduced inhibition. Disinhibition of spinal neurons can arise through decreased GABAA/glycine receptor activation or through dysregulation of intracellular chloride. We hypothesized that these distinct disinhibitory mechanisms, despite all causing allodynia, are differentially susceptible to therapeutic intervention. Specifically, we predicted that reducing bicarbonate efflux by blocking carbonic anhydrase with acetazolamide (ACTZ) would counteract disinhibition caused by chloride dysregulation without affecting normal inhibition or disinhibition caused by GABAA/glycine receptor blockade. To test this, responses to innocuous tactile stimulation were recorded in vivo from rat superficial dorsal horn neurons before and after different forms of pharmacological disinhibition and again after application of ACTZ. Blocking GABAA or glycine receptors caused hyperresponsiveness equivalent to that caused by blocking the potassium chloride cotransporter KCC2, but, consistent with our predictions, only disinhibition caused by KCC2 blockade was counteracted by ACTZ. ACTZ did not alter responses of neurons with intact inhibition. As pathological downregulation of KCC2 is triggered by brain-derived neurotrophic factor, we also confirmed that ACTZ was effective against brain-derived neurotrophic factor-induced hyperresponsiveness. Our results argue that intrathecal ACTZ has antiallodynic effects only if allodynia arises through chloride dysregulation; therefore, behavioral evidence that ACTZ is antiallodynic in nerve-injured animals affirms the contribution of chloride dysregulation as a key pathological mechanism. Although different disinhibitory mechanisms are not mutually exclusive, these results demonstrate that their relative contribution dictates which specific therapies will be effective.

Mechanisms of spectral and temporal integration in the mustached bat inferior colliculus

This review describes mechanisms and circuitry underlying combination-sensitive response properties in the auditory brainstem and midbrain. Combination-sensitive neurons, performing a type of auditory spectro-temporal integration, respond to specific, properly timed combinations of spectral elements in vocal signals and other acoustic stimuli. While these neurons are known to occur in the auditory forebrain of many vertebrate species, the work described here establishes their origin in the auditory brainstem and midbrain. Focusing on the mustached bat, we review several major findings: (1) Combination-sensitive responses involve facilitatory interactions, inhibitory interactions, or both when activated by distinct spectral elements in complex sounds. (2) Combination-sensitive responses are created in distinct stages: inhibition arises mainly in lateral lemniscal nuclei of the auditory brainstem, while facilitation arises in the inferior colliculus (IC) of the midbrain. (3) Spectral integration underlying combination-sensitive responses requires a low-frequency input tuned well below a neuron's characteristic frequency (ChF). Low-ChF neurons in the auditory brainstem project to high-ChF regions in brainstem or IC to create combination sensitivity. (4) At their sites of origin, both facilitatory and inhibitory combination-sensitive interactions depend on glycinergic inputs and are eliminated by glycine receptor blockade. Surprisingly, facilitatory interactions in IC depend almost exclusively on glycinergic inputs and are largely independent of glutamatergic and GABAergic inputs. (5) The medial nucleus of the trapezoid body (MNTB), the lateral lemniscal nuclei, and the IC play critical roles in creating combination-sensitive responses. We propose that these mechanisms, based on work in the mustached bat, apply to a broad range of mammals and other vertebrates that depend on temporally sensitive integration of information across the audible spectrum.

Masked excitatory crosstalk between the ON and OFF visual pathways in the mammalian retina

A fundamental organizing feature of the visual system is the segregation of ON and OFF responses into parallel streams to signal light increment and decrement. However, we found that blockade of GABAergic inhibition unmasks robust ON responses in OFF α-ganglion cells (α-GCs). These ON responses had the same centre-mediated structure as the classic OFF responses of OFF α-GCs, but were abolished following disruption of the ON pathway with L-AP4. Experiments showed that both GABA(A) and GABA(C) receptors are involved in the masking inhibition of this ON response, located at presynaptic inhibitory synapses on bipolar cell axon terminals and possibly amacrine cell dendrites. Since the dendrites of OFF α-GCs are not positioned to receive excitatory inputs from ON bipolar cell axon terminals in sublamina-b of the inner plexiform layer (IPL), we investigated the possibility that gap junction-mediated electrical synapses made with neighbouring amacrine cells form the avenue for reception of ON signals. We found that the application of gap junction blockers eliminated the unmasked ON responses in OFF α-GCs, while the classic OFF responses remained. Furthermore, we found that amacrine cells coupled to OFF α-GCs display processes in both sublaminae of the IPL, thus forming a plausible substrate for the reception and delivery of ON signals to OFF α-GCs. Finally, using a multielectrode array, we found that masked ON and OFF signals are displayed by over one-third of ganglion cells in the rabbit and mouse retinas, suggesting that masked crossover excitation is a widespread phenomenon in the inner mammalian retina.

Respiratory responses induced by blockades of GABA and glycine receptors within the Bötzinger complex and the pre-Bötzinger complex of the rabbit

The respiratory role of GABA A, GABA B and glycine receptors within the Bötzinger complex (BötC) and the pre-Bötzinger complex (preBötC) was investigated in α-chloralose-urethane anesthetized, vagotomized, paralysed and artificially ventilated rabbits by using bilateral microinjections (30–50 nl) of GABA and glycine receptor agonists and antagonists. GABA A receptor blockade by bicuculline (5 mM) or gabazine (2 mM) within the BötC induced strong depression of respiratory activity up to apnea. The latter was reversed by hypercapnia. Glycine receptor blockade by strychnine (5 mM) within the BötC decreased the frequency and amplitude of phrenic bursts. Bicuculline microinjections into the preBötC caused decreases in respiratory frequency and the appearance of two alternating different levels of peak phrenic activity. Strychnine microinjections into the preBötC increased respiratory frequency and decreased peak phrenic amplitude. GABA A, but not glycine receptor antagonism within the preBötC restored respiratory rhythmicity during apnea due to bicuculline or gabazine applied to the BötC. GABA B receptor blockade by CGP-35348 (50 mM) within the BötC and the preBötC did not affect baseline respiratory activity, though microinjections of the GABA B receptor agonist baclofen (1 mM) into the same regions altered respiratory activity. The results show that only GABA A and glycine receptors within the BötC and the preBötC mediate a potent control on both the intensity and frequency of inspiratory activity during eupneic breathing. This study is the first to provide evidence that these inhibitory receptors have a respiratory function within the BötC.

Unraveling the Mechanisms of REM Sleep Atonia

WE THANK THE EDITORS1 FOR THIS FORUM AND THE COMMENTATORS2–6 FOR THEIR DISCUSSION OF OUR WORK.7 WE AGREE WITH THE COMMENTATORS that determining mechanisms of rapid eye movement (REM) sleep motor atonia is of major scientific importance and clinical relevance. The concept that REM atonia is under the control of one physiological mechanism and one neurotransmitter pathway has seduced many of us. The undoubted appeal of the current theory—that glycinergic inhibition of motoneurons is the only mechanism mediating REM atonia—is its inherent simplicity. However, the fact that several laboratories now provide evidence indicating that glycinergic inhibition plays only a minimal role in mediating REM atonia7–9 and that other neurotransmitter pathways contribute to motor suppression in REM sleep10–13 indicates that we need to reevaluate mechanisms of REM atonia. If glycinergic inhibition is indeed the dominating force driving motor inhibition during REM sleep, then why does blockade of glycine receptors at the trigeminal motor pool not prevent REM atonia in masseter muscles?7 Our work demonstrates that antagonism of glycine and GABAA receptors at the trigeminal motor pool causes a profound increase in masseter muscle tone during both wakefulness and non-rapid eye movement (NREM) sleep; however, this same intervention does not prevent or even reverse REM atonia, despite the fact that it potently provokes muscle-twitch activity in REM sleep (Figure 1). In fact, REM atonia persists even when glycine and GABAA receptors are blocked and potent glutamatergic agonists are simultaneously applied to the trigeminal motor pool. We interpret these findings to indicate that (1) glycinergic- and GABAA-mediated inhibition plays a minimal role in triggering REM atonia; (2) inhibition primarily functions to suppress muscle twitches during REM sleep; and, (3) a powerful, yet unidentified, inhibitory mechanism or mechanisms override excitation during REM sleep. Some commentators offer alternative possibilities for our experimental observations; we respond to these discussion points below. Figure 1 Blockade of glycine and GABAA receptors at the trigeminal motor pool does not prevent masseter muscle atonia during REM sleep. A. Typical EMG and EEG traces illustrating that strychnine/bicuculline perfusion into the left trigeminal motor pool increases ...

Local oscillations of spiking activity in organotypic spinal cord slice cultures

The origin of rhythm generation in mammalian spinal cord networks is still poorly understood. We have previously proposed that disinhibition-induced rhythms are based on intrinsic firing, recurrent excitation and several mechanisms to de-activate the network. In order to clarify these mechanisms we here investigated spontaneous spike discharge oscillations in rat spinal cord slice cultures using multi-electrode arrays and patch clamp. Episodes of such oscillations at 8.5 Hz spontaneously appeared in the ventral parts of the cultured slices. The rising phase of their initial cycles was entirely based on AMPA/kainate receptor-dependent recurrent excitation. Initial oscillations were changed into persistent activity by bicuculline and other blockers of GABA A, but not by blockers of glycine receptors, suggesting a role for GABAergic synaptic inhibition in network de-activation during oscillation cycles. Blockade of glycine receptors by strychnine caused a prolongation of oscillations and their spreading in the slice, suggesting that these receptors are mainly involved in the spatial and temporal restriction of oscillations. In most cultures, oscillations reappeared under disinhibition after an initial phase of persistent activity. Both spontaneous and disinhibition-induced oscillations were facilitated by riluzole, which enhances fast sodium current inactivation and thus leads to early cessation of firing during strong depolarization (depolarization block). In single cell recordings, episodes of strong depolarization were mostly seen during oscillations induced by disinhibition, but occasionally also during spontaneous oscillations. We conclude that both GABA A-mediated synaptic inhibition and depolarization block contribute to the de-activation of spinal cord networks during oscillation cycles.

Blockade of glycine receptors modifies temporal and spectral characteristics of phrenic nerve discharge in urethane‐anesthetized adult rat in vivo

The neuronal mechanisms that underlie fast oscillatory rhythms in inspiratory‐related discharges are poorly understood; however, accumulating evidence suggests that fast synaptic inhibition may be amongst the mechanisms participating. In support of this idea, recent work from our laboratory in the urethane‐anesthetized adult rat preparation has shown that blockade of GABAA receptors modulates both the power and timing of spectral activity in phrenic nerve bursts. The role of glycine‐mediated synaptic inhibition requires clarification. Therefore, we examined the effects of glycine receptor antagonism, using cumulative increasing doses strychnine hydrochloride (STR; 0.2–10 mg/kg, iv), on phrenic nerve discharge in urethane‐anesthetized vagotomized adult rats (n=5). STR significantly decreased TI (from 325±43 to 208±24 ms; P=0.007) and TE (from 965±88 to 878±88 ms; P=0.031), resulting in an ~17% increase in burst frequency (P&lt;0.004). STR also increased power of the MFO (by 123±61%) and HFO (by 83±65%) spectral peaks albeit this increase was not significant. Inspiratory network complexity (quantified by approximate entropy), however, was significantly reduced from 0.709±0.016 to 0.602±0.0198 (p=0.004). These findings suggest that glycine‐mediated synaptic inhibition may contribute to timing and spectral activity inherent in phrenic discharges in vivo. Supported by NS045321 and NS049310

Characterization and restoration of altered inhibitory and excitatory control of micturition reflex in experimental autoimmune encephalomyelitis in rats

Multiple sclerosis (MS) is characterized by inflammatory lesions throughout the central nervous system. Spinal cord inflammation correlates with many neurological defecits. Most MS patients suffer from micturition dysfunction with urinary incontinence and difficulty in emptying the bladder. In experimental autoimmune encephalomyelitis (EAE) induced in female Lewis rats, a model of MS, we investigated at distinct clinical severity scores the micturition reflex by cystometrograms. All rats presenting symptomatic EAE suffered from micturition reflex alterations with either detrusor areflexia or hyperactivity. During pre-symptomatic EAE, a majority of rats presented with detrusor areflexia, whereas at onset of clinical EAE, detrusor hyperactivity was predominant. During progression of EAE, detrusor areflexia and hyperactivity were equally expressed. Bladder hyperactivity was suppressed by activation of glycine and GABA receptors in the lumbosacral spinal cord with an order of potency: glycine > GABA(B) > GABA(A). Detrusor areflexia was transformed into detrusor hyperactivity by blocking glycine and GABA receptors. Spinalization abolished bladder activity in rats presenting detrusor hyperactivity and failed to induce activity in detrusor areflexia. Altogether, the results reveal an exaggerated descending excitatory control in both detrusor reflex alterations. In detrusor areflexia, a strong segmental inhibition dominates this excitatory control. As in treatment of MS, electrical stimulation of sacral roots reduced detrusor hyperactivity in EAE. Blockade of glycine receptors in the lumbosacral spinal cord suppressed the stimulation-induced inhibitory effect. Our data help to better understand bladder dysfunction and treatment mechanisms to suppress detrusor hyperactivity in MS.

Anti‐homeostatic synaptic plasticity of glycine receptor function after chronic strychnine in developing cultured mouse spinal neurons

In this study, we describe a novel form of anti-homeostatic plasticity produced after culturing spinal neurons with strychnine, but not bicuculline or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Strychnine caused a large increase in network excitability, detected as spontaneous synaptic currents and calcium transients. The calcium transients were associated with action potential firing and activation of gamma-aminobutyric acid (GABA(A)) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors as they were blocked by tetrodotoxin (TTX), bicuculline, and CNQX. After chronic blockade of glycine receptors (GlyRs), the frequency of synaptic transmission showed a significant enhancement demonstrating the phenomenon of anti-homeostatic plasticity. Spontaneous inhibitory glycinergic currents in treated cells showed a fourfold increase in frequency (from 0.55 to 2.4 Hz) and a 184% increase in average peak amplitude compared with control. Furthermore, the augmentation in excitability accelerated the decay time constant of miniature inhibitory post-synaptic currents. Strychnine caused an increase in GlyR current density, without changes in the apparent affinity. These findings support the idea of a post-synaptic action that partly explains the increase in synaptic transmission. This phenomenon of synaptic plasticity was blocked by TTX, an antibody against brain-derived neurotrophic factor (BDNF) and K252a suggesting the involvement of the neuronal activity-dependent BDNF-TrkB signaling pathway. These results show that the properties of GlyRs are regulated by the degree of neuronal activity in the developing network.

Medullary lateral tegmental field neurons influence the timing and pattern of phrenic nerve activity in cats

In an effort to characterize the role of the medullary lateral tegmental field (LTF) in regulating respiration, we tested the effects of selective blockade of excitatory (EAA) and inhibitory amino acid (IAA) receptors in this region on phrenic nerve activity (PNA) of vagus-intact and vagotomized cats anesthetized with dial-urethane. We found distinct patterns of changes in central respiratory rate, duration of inspiratory and expiratory phases of PNA (Ti and Te, respectively), and I-burst amplitude after selective blockade of EAA and IAA receptors in the LTF. First, blockade of N-methyl-D-aspartate (NMDA) receptors significantly (P < 0.05) decreased central respiratory rate primarily by increasing Ti but did not alter I-burst amplitude. Second, blockade of non-NMDA receptors significantly reduced I-burst amplitude without affecting central respiratory rate. Third, blockade of GABAA receptors significantly decreased central respiratory rate by increasing Te and significantly reduced I-burst amplitude. Fourth, blockade of glycine receptors significantly decreased central respiratory rate by causing proportional increases in Ti and Te and significantly reduced I-burst amplitude. These changes in PNA were markedly different from those produced by blockade of EAA or IAA receptors in the pre-Bötzinger complex. We propose that a proper balance of excitatory and inhibitory inputs to several functionally distinct pools of LTF neurons is essential for maintaining the normal pattern of PNA in anesthetized cats.

GABAergic and glycinergic inhibitory mechanisms in the lamprey respiratory control

The specific role of γ-aminobutyric acid (GABA) and glycine receptors in respiratory rhythm generation and pattern formation was investigated in in vitro brainstem preparations from adult lampreys by analyzing the changes in respiratory activity induced by bath application of specific antagonists, agonists, and uptake blockers. GABA A receptor blockade by bicuculline or picrotoxin increased both the frequency and amplitude of respiratory bursts. Similar effects were observed after glycine receptor blockade by strychnine. Combined bath application of bicuculline and strychnine markedly increased the frequency and amplitude of respiratory activity. These responses were associated, especially at the higher concentrations of the two drugs, with the appearance of tonic activity and irregular, high-frequency bursts followed by transient depression of respiratory activity. GABA A and glycine receptor agonists suppressed respiratory activity. These effects were prevented by bath application of the corresponding specific antagonists. GABA B receptor blockade by 2-hydroxysaclofen reduced the respiratory frequency but increased the peak amplitude of respiratory bursts. Activation of GABA B receptors suppressed respiratory activity. These responses were prevented by 2-hydroxysaclofen. Neither GABA C receptor agonist nor antagonist had any effects on respiration. Depression of both the frequency and amplitude of respiratory bursts was induced by blockades of GABA and glycine uptake using, respectively, nipecotic acid and sarcosine. The results suggest that GABA- and glycine-mediated inhibition is not essential for respiratory rhythm generation in the adult lamprey, although it appears to exert potent influences on respiratory activity and to have a role in maintaining a stable and regular breathing pattern.

Age-Related Changes in the Inhibitory Response Properties of Dorsal Cochlear Nucleus Output Neurons: Role of Inhibitory Inputs

Age-related hearing loss frequently results in a loss in the ability to discriminate speech signals, especially in noise. This is attributable, in part, to a loss in temporal resolving power and ability to adjust dynamic range. Circuits in the adult dorsal cochlear nucleus (DCN) have been shown to preserve signal in background noise. Fusiform cells, major DCN output neurons, receive focused glycinergic inputs from tonotopically aligned vertical cells that also project to the ventral cochlear nucleus. Glycine-mediated inhibition onto fusiform cells results in decreased tone-evoked activity as intensity is increased at frequencies adjacent to characteristic frequency (CF). DCN output is thus shaped by glycinergic inhibition, which can be readily assessed in recordings from fusiform cells. Previous DCN studies suggest an age-related loss of markers for glycinergic neurotransmission. The present study postulated that response properties of aged fusiform cells would show a loss of inhibition, resembling conditions observed with glycine receptor blockade. The functional impact of aging was examined by comparing response properties from units meeting fusiform-cell criteria in young and aged rats. Fusiform cells in aged animals displayed significantly higher maximum discharge rates to CF tones than those recorded from young-adult animals. Fusiform cells of aged rats displayed significantly fewer nonmonotonic CF rate-level functions and an age-related change in temporal response properties. These findings are consistent with an age-related loss of glycinergic input, likely from vertical cells, and with findings from other sensory aging studies suggesting a selective age-related decrement in inhibitory amino acid neurotransmitter function.

Early and late changes in vestibular neuronal excitability after deafferentation

Medial vestibular nucleus neurons develop a sustained increase in electrophysiological excitability after deafferentation, which may be an important component of 'vestibular compensation' (the behavioural recovery that follows peripheral vestibular lesions). We investigated the effects of gamma-aminobutyric acid, glutamate and glycine receptor blockade on the spontaneous activity of deafferented medial vestibular nucleus neurons in slices, to determine whether changes in synaptic inputs contribute to their increased excitability. Soon after deafferentation (4 h after labyrinthectomy) synaptic blockade had no effect on the elevated in-vitro firing of medial vestibular nucleus neurons, while later (48 h, 1 week), synaptic blockade reduced the elevated activity to normal. Intrinsic mechanisms therefore appear to mediate the elevated excitability of medial vestibular nucleus cells initially after deafferentation. Subsequently, changes in the efficacy of synaptic inputs are implicated, presumably involving intranuclear projections that are preserved in slices.

A combined blockade of glycine and calcium-dependent potassium channels abolishes the respiratory rhythm

In order to test whether glycinergic inhibition is essential for the in vivo respiratory rhythm, we analysed the discharge properties of neurones in the medullary respiratory network after blockade of glycine receptors in the in situ perfused brainstem preparation of mature wild type and oscillator mice with a deficient glycine receptor. In wild type mice, selective blockade of glycine receptors with low concentrations of strychnine (0.03–0.3 μM) provoked considerable changes in neuronal discharge characteristics: The cycle phase relationship of inspiratory, post-inspiratory and expiratory specific patterns of membrane potential changes was altered profoundly. Inspiratory, post-inspiratory and expiratory neurones developed a propensity for fast voltage oscillations that were accompanied by multiple burst discharges. These burst discharges were followed by “after-burst” hyperpolarisations that were capable of triggering secondary burst discharges. Blockade of glycine receptors and the “big” Ca 2+-dependent K +-conductance by charybdotoxin (3.3 nM) resulted in loss of the respiratory rhythm, whilst only tonic discharge activity remained. In contrast, rhythmic activity was only weakened, but preserved after the “small” Ca 2+-dependent activated K + conductance was blocked with apamin (8 nM). Also low concentrations of pentobarbital sodium (6 mg/kg) abolished rhythmic respiratory activity after blockade of glycine receptors in the wild type mice and in glycine receptor deficient oscillator mice. The data imply that failure of glycine receptors provokes enhanced bursting behaviour of respiratory neurones, whilst the additional blockade of BKCa channels by charybdotoxin or with pentobarbital abolishes the respiratory rhythm.

Central control of upper airway resistance regulating respiratory airflow in mammals.

This article reviews recent studies on the importance of glycine receptors for both the spontaneous and the reflex respiratory modulation of the laryngeal abductors and adductors. Our findings show that strychnine blockade of glycine receptors within the brainstem changes the eupneic three-phase respiratory pattern into two phases. This has major implications for glottal control: (i) the inspiratory glottic abduction and early expiratory adduction were both compromised--a finding mimicked by 5% hypoxia; (ii) closure of the glottis during defensive upper airway reflexes became intermittent and the reflex apnoea reversed to sustained inspiratory discharge. Based on these data, we predict that periods of prolonged hypoxia, such as those that occur during sleep apnoeas, will constrain inspiratory glottic abduction thereby impeding inhalation.

Glycinergic inhibition is essential for co-ordinating cranial and spinal respiratory motor outputs in the neonatal rat.

Eupnoeic breathing in mammals is dependent on the co-ordinated activity of cranial and spinal motor outputs to both ventilate the lungs and adjust respiratory airflow, which they do by regulating upper-airway resistance. We investigated the role of central glycinergic inhibition in the co-ordination of cranial and spinal respiratory motor outflows. We developed an arterially perfused neonatal rat preparation (postnatal age 0-4 days) to assess the effects of blocking glycine receptors with systemically administered strychnine (0.5-1 microM). We recorded respiratory neurones located within the ventrolateral medulla, inspiratory phrenic nerve activity (PNA) and recurrent laryngeal nerve activity (RLNA), as well as dynamic changes in laryngeal resistance. Central recordings of postinspiratory neurones revealed an earlier onset in firing relative to the onset of inspiratory PNA after exposure to strychnine (260 +/- 38.9 vs. 129 +/- 26.8 ms). After glycine receptor blockade, postinspiratory neurones discharged during the inspiratory phase. Strychnine also evoked a decrease in PNA frequency (from 38.6 +/- 4.7 to 30.7 +/- 2.8 bursts min(-1)), but amplitude was unaffected. In control conditions, RLNA comprised inspiratory and postinspiratory discharges; the amplitude of the latter exceeded that of the former. However, after administration of strychnine, the amplitude of inspiratory-related discharge increased (+65.2 +/- 15.2 %) and exceeded postinspiratory activity. Functionally this change in RLNA caused a paradoxical, inspiratory-related glottal constriction during PNA. We conclude that during the first days of life in the rat, glycine receptors are essential for the formation of the eupnoeic-like breathing pattern as defined by the co-ordinated activity of cranial and spinal motor inspiratory and postinspiratory activities.

Inhibitory synaptic mechanisms regulating upper airway patency.

The breathing cycle of vertebrates comprises three phases (inspiration, postinspiration and expiration) that are apparent in the activities generated in the ponto-medullary respiratory network. A large body of evidence now indicates that in adult mammals generation of this three-phase pattern is based on reciprocal synaptic inhibition between distinct subsets of respiratory neurones. This review summarises our recent experiments focused on the role of glycinergic inhibition in respiratory pattern formation: e.g. in co-ordinating the activity of spinal and cranial motor outputs that drive the ventilatory pump (thoracic and abdominal muscles) and adjust airflow by regulating laryngeal resistance (laryngeal abductors and adductors). We used arterially perfused in situ preparations of neonatal and mature rat and show that specific blockade of glycine receptors within the ponto-medullary network caused a severe disruption of the co-ordination of spinal and cranial motor outputs: postinspiratory neurones lose their characteristic inspiratory inhibition revealing excitatory synaptic drive coincident with inspiratory phrenic nerve activity. The resulting simultaneous discharge of inspiratory and postinspiratory neurones caused co-activation of both glottal abductors and adductors during neural inspiration. The latter resulted in a paradoxical inspiratory adduction of the vocal fold and severe disruption of the eupneic breathing pattern. The effect of blocking glycine receptors was the same in both mature and newborn rats suggesting that glycinergic inhibition is essential for co-ordinating cranial and spinal motor outputs from birth.