Inhibitions mediated by glycine and GABAA receptors shape the discharge pattern of bulbar respiratory neurons

doi:10.1016/0006-8993(95)01380-6

Brain Research

Volume 710, Issues 1–2, 26 February 1996, Pages 150-160

https://doi.org/10.1016/0006-8993(95)01380-6 Get rights and content

Abstract

Experiments were performed to identify the glycinergic or GABAergic nature, and the timing of discharge, of the neurons which produce chloride-dependent inhibitions on other bulbar respiratory neurons (RNs) during their silent and active phases. RNs recorded extracellularly in pentobarbital-anesthetized or decerebrate cats, were subjected to iontophoretic applications of glutamate, of the glycine antagonist strychnine, and of the GABA_A receptor antagonist bicuculline. Both antagonists induced discharge or increased discharge frequency in restricted parts of the respiratory cycle without affecting the discharge frequency in other parts of the cycle. Strychnine most often elicited activity in late-inspiration and early-expiration, but also in early inspiration and in late expiration. Bicuculline was most often effective throughout the entire discharge period of each neuron with no effect during the silent period, although it also acted selectively during late-inspiration in inspiratory neurons, an effect attributed to GABA_A receptor blockade. The convergence of glycinergic afferent inputs during late inspiration and early expiration suggests that glycinergic neurons may play an important role in the inspiratory to expiratory phase transition.

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The development of the neonatal breathing network, respiratory rhythm generators, and excitatory and inhibitory elements of the pontomedullary respiratory circuitry ontogenically recapitulates the rhombomeric segmentation of the hindbrain as patterned by Hox gene transcription factors (see Chatonnet et al., 2002 for review). More specifically, several studies have extensively evaluated developmental changes occurring in, and the role of, chloride-based neurotransmission in respiratory rhythmogenesis and pattern formation in both adult (Haji et al., 1990, 1992, Paton and Richter, 1995, Schmid et al., 1996, Hayashi and Lipski, 1992, Dogas et al., 1998, Ritter and Zhang, 2000, Bongianni et al., 2010) and neonatal (Paton and Richter, 1995, Shao and Feldman, 1997, Brockhaus and Ballanyi, 1998, Singer et al., 1998, Ritter and Zhang, 2000 , Fujii et al., 2007) mammals. Thus, the role of GABAA- and glycinergic signaling in medullary control of respiration in both the in vivo neonate compared to the in vivo adult remains to be more critically evaluated and resolved at the biomolecular and cellular levels.
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Probing the function of glycinergic neurons in the mouse respiratory network using optogenetics
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Adjacent to the preBötC is the Bötzinger complex (BötC), which has been implicated in the control of expiration and harbors primarily expiratory decrementing (dec-E, also termed post-inspiratory or post-I) and expiratory augmenting neurons (aug-E). Post-I/dec-E neurons are predominantly glycinergic as they expressed glycine transporter-2 (GlyT2) mRNA (Ezure et al., 2003) whereas aug-E neurons may be GABAergic (Champagnat et al., 1982; Haji et al., 1992; Schmid et al., 1996) or glycinergic (Ezure et al., 2003; Fortuna et al., 2008; Schreihofer et al., 1999); this variation may reflect different sub-populations of aug-E neurons or co-expression of glycine and GABA. Most models of the respiratory pattern generator assume an essential role of these neurons located in BötC and preBotC in the generation of three phases breathing rhythm (Del Negro et al., 2018; Feldman and Kam, 2015; Richter, 1982; Richter and Smith, 2014: Ausborn et al., 2018; Rybak et al., 2007).
Glycine is a primary inhibitory transmitter in the ventral medullary respiratory network, but the functional role of glycinergic neurons for breathing remains a matter of debate. We applied optogenetics to selectively modulate glycinergic neuron activity within regions of the rostral ventral respiratory column (VRC). Responses of the phrenic nerve activity to the light-driven stimulation were studied in the working heart-brainstem preparation from adult glycine transporter 2 Cre mice (GlyT2-Cre), which received a unilateral injection of a Cre-dependent AAV virus into Bötzinger and preBötzinger Complex. Sustained light stimulation from the ventral medullary surface resulted in a substantial depression of the phrenic nerve (PN) frequency, which in most cases was compensated by an increase in PN amplitude. Periodic, burst stimulation with variable intervals could alter and reset respiratory rhythm. We conclude that unilateral activation of the rostral VRC glycinergic neurons can significantly affect respiratory pattern by lengthening the expiratory interval and modulating phase transition.
KCC2-mediated regulation of respiration-related rhythmic activity during postnatal development in mouse medulla oblongata
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In the adult central nervous system (CNS), γ-amino butyric acid (GABA) is a major inhibitory neurotransmitter that crucially regulates respiratory rhythmogenesis and the patterning of motor outputs (Johnson et al., 1996; Shao and Feldman, 1997). GABA has been shown to play an essential role in termination of the inspiratory and the expiratory phases (Champagnat et al., 1982; Haji et al., 1992, 2000; Schmid et al., 1996; Yajima and Hayashi, 1999; Yamazaki et al., 2000). In general, synchronous oscillatory firings, including those involved in respiration activities, depend on an intricate interplay among synaptic excitation and inhibition, electrical coupling, and intrinsic membrane properties (Sejnowski and Paulsen, 2006).
GABA acts as inhibitory neurotransmitter in the adult central nervous system but as excitatory neurotransmitter during early postnatal development. This shift in GABA’s action from excitation to inhibition is caused by a decrease in intracellular chloride concentration ([Cl⁻]_i), which in turn is caused by changes in the relative expression levels of the K⁺–Cl⁻ co-transporter (KCC2) and the Na⁺, K⁺–2Cl⁻ co-transporter (NKCC1) proteins. Previous studies have used slices containing the medullary pre-Bötzinger complex (pre-BötC) to record respiration-related rhythmic activity (RRA) from the hypoglossal nucleus (12 N). The role of GABAergic transmission in the regulation of medullary RRA neonatally, however, is yet to be determined. Here, we examined how GABA and chloride co-transporters contribute to RRA during development in the 12 N where inspiratory neurons reside. We recorded extracellular RRA in medullary slices obtained from postnatal day (P) 0–7 mice. RRA was induced by soaking slices in artificial cerebrospinal fluid (aCSF) containing 8 mM-K⁺. Application of GABA significantly increased the frequency of RRA after P3, whereas application of a KCC2 blocker (R (+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-indenyl-5-yl)oxy]acetic acid (DIOA)) significantly decreased the frequency of RRA after P1. In addition, dense KCC2 immunolabeling was seen in the superior longitudinalis (SL) of the 12 N, which is responsible for retraction of the tongue, from P0 and P7. These results indicate that GABA administration can increase RRA frequency during the first week following birth. This in turn suggests that decreasing [Cl⁻]_i levels caused by increasing KCC2 levels in the 12 N could play important roles in regulating the frequency of RRA during development.
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Laryngeal constrictor motoneurons (LCMN) are activated during post-inspiration and act to slow expiratory airflow. However, little is known about how this phasic activity is generated. Here, we investigated the electrophysiological responses of identified LCMN to local application of GABA and bicuculline methiodide (BIC) in 14 anaesthetised Sprague–Dawley rats. During extracellular recordings, GABA iontophoresis (0.5 M) strongly inhibited LCMN (n = 6). Interestingly, BIC iontophoresis (5 mM) reduced, rather than increased, LCMN post-inspiratory activity (5 out of 6). Furthermore, intracellular recording revealed that BIC reduced not only the hyperpolarisation of the LCMN during inspiration (2.5 ± 1.4 mV before and 1.5 ± 0.4 mV after the BIC, P = 0.05, n = 5), but also the depolarisation during post-inspiration (3.0 ± 1.3 mV before and 1.6 ± 0.4 mV after the BIC, P = 0.02, n = 5). Our results demonstrate for the first time that the inspiratory inhibition of LCMN is primarily mediated by GABA_A receptors. A possible involvement of a post-inhibitory rebound mechanism is discussed to explain how blockade of an inspiratory inhibition would affect LCMN excitability during post-inspiration.
Breathing and the Nervous System
2008, Neurology and General Medicine

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Inhibitions mediated by glycine and GABAA receptors shape the discharge pattern of bulbar respiratory neurons

Abstract

Trends Pharmacol. Sci.

Neurosci. Lett.

Brain Res.

Brain Res.

Prog. Neurobiol.

Neurosci. Lett.

Exp. Neurol.

Resp. Physiol.

Brain Res.

Brain Res.

Brain Res.

Neurosci. Lett.

Brain Res.

Neuropharrnacology

Brain Res.

Resp. Physiol.

Comput. Meth. Progr. Biomed.

Strychnine-induced potassium current in isolated dorsal root ganglion cells of the rat

Br. J. Pharmacol.

Inhibition of caudal medullary expiratory neurones by retrofacial inspiratory neurones in the cat

J. Physiol.

Post-synaptic inhibition of bulbar inspiratory neurones in the cat

J. Physiol.

The non-uniform character of expiratory synaptic activity in expiratory bulbospinal neurones of the cat

J. Physiol.

Voltage-dependent block by strychnine of N-methyl-D-aspartic acid-activated cationic channels in rat cortical neurons in culture

Mol. Pharmacol.

Inhibitions mediated by glycine and GABA_A receptors shape the discharge pattern of bulbar respiratory neurons