Defining inhibitory neurone function in respiratory circuits: opportunities with optogenetics?

Abstract

Pharmacological and mathematical modelling studies support the view that synaptic inhibition in mammalian brainstem respiratory circuits is essential for generating normal and stable breathing movements. GABAergic and glycinergic neurones are known components of these circuits but their precise functional roles have not been established, especially within key microcircuits of the respiratory pre-Bötzinger (pre-BötC) and Bötzinger (BötC) complexes involved in phasic control of respiratory pump and airway muscles. Here, we review briefly current concepts of relevant complexities of inhibitory synapses and the importance of synaptic inhibition in the operation of these microcircuits. We highlight results and limitations of classical pharmacological studies that have suggested critical functions of synaptic inhibition. We then explore the potential opportunities for optogenetic strategies that represent a promising new approach for interrogating function of inhibitory circuits, including a hypothetical wish list for optogenetic approaches to allow expedient application of this technology. We conclude that recent technical advances in optogenetics should provide a means to understand the role of functionally select and regionally confined subsets of inhibitory neurones in key respiratory circuits such as those in the pre-BötC and BötC.

Figure 1

Inhibitory amino acid co-transmission The main precursors for γ-aminobutyric acid (GABA) synthesis in neurones are glucose, pyruvate and glutamine, which are converted to glutamate. The concentration of GABA in the presynaptic terminal depends on the activity of the synthetic enzyme glutamic acid decarboxylase (GAD, converts glutamate to GABA), and re-uptake by the GABA transporter 1 (GAT₁). The major precursor of glycine in the brain is serine, and there is also re-uptake of glycine into the presynaptic terminal by the glycine transporter 2 (GlyT₂), which is preferentially expressed in neurones. Both glycine and GABA are incorporated into synaptic vesicles by the vesicular inhibitory amino acid transporter (VIAAT), also known as vesicular GABA transporter (VGAT). GABA and glycine bind to the GABA_A and glycine receptor (GlyR), respectively, opening chloride (Cl⁻) channels in the postsynaptic terminal. The activity of the K⁺–Cl^– cotransporter 2 (KCC2) maintains Cl⁻ homeostasis regulating the Cl^– equlibrium potential that determines the resulting change in membrane potential (IPSP, inhibitory postsynaptic potential), usually hyperpolarization in mature neurones. Clustering of GlyR and GABA_A receptors at the postsynaptic membrane is determined by interactions with gephyrin scaffolds. The concentration of GABA and glycine in the synaptic cleft is controlled by both neuronal (GAT₁ and GlyT₂, respectively) and astrocyte re-uptake (GAT₃ and GlyT₁, respectively).

Figure 2

Glycinergic neurones in the pre-Bötzinger complex (pre-BötC) and Bötzinger complex (BötC) regions of transgenic mouse medulla oblongata This parasagittal view (single optical plane confocal image) of one side of the ventral medulla (VS, ventral medullary surface) portrays the dense concentrations of glycinergic neurones (red) in these regions as labelled by expression of tdTomato fluorescent protein, obtained by crossing a GlyT₂-Cre driver mouse strain with a Cre-dependent tdTomato reporter strain (J. C. Smith, H. Koizumi, N. Koshiya & R. Zhang, unpublished observations). Subsets of these glycinergic neurones are components of the respiratory network inhibitory connectome. The concentrations of inhibitory neurones reflect their potential role in circuit function of these classically defined respiratory regions. Motoneurones (green) in compact and semi-compact divisions of nucleus ambiguus (NAc and NAsc, respectively) and facial motor nucleus (VII), serving as anatomical landmarks, are labelled with an antibody against choline acetyltransferase (ChAT).

Figure 3

Schematic diagram of a three-phase respiratory cycle and its neuro-mechanical components Top schematic plots and neurograms represent lung volume, sub-glottal pressure (SGP), phrenic (PN), recurrent laryngeal (RLN) and internal intercostal (int IN) nerve activities during the three phases of a respiratory cycle, i.e. inspiration (I), post-inspiration (PI) and late expiration (E2). Note that RLN conveys outputs from both abductor and adductor motoneurones, which fire respectively during inspiration (to dilate the glottis during inhalation) and post-inspiration (to narrow the glottis during exhalation). Bottom overlay plots represent hypothetical time courses of glycine- (Gly, dotted line) and GABA-mediated (continuous line) inhibition to inspiratory (I) neurones during the respiratory cycle. These time courses reflect the activity of medullary post-inspiratory (PI) inhibitory neurones, thought to be predominantly glycinergic, and GABAergic augmenting expiratory (E-AUG) inhibitory neurones active during E2 phase, both of which inhibit inspiratory neurones during expiration. Phasic inhibition of inspiratory neurones is minimal during inspiration, when active inspiratory neurones inhibit expiratory neurones, but rises abruptly during PI to orchestrate the inspiratory–expiratory phase transition and initiate exhalation.

Figure 4

Co-expression of glycine in GABAergic neurones in the pre-Bötzinger complex (pre-BötC) region of GAD67-GFP transgenic mouse A, GAD67-GFP positive neurones distributed within the pre-BötC (coronal plane, ×10 objective). Motoneurones in the semi-compact division of nucleus ambiguus (NAsc) are labelled with antibody against choline acetyltransferase (ChAT) (white). Ba–b, single optical plane confocal images of GAD67-GFP positive (Ba) and glycine (Bb) immunolabelled neurones (red) in the pre-BötC area marked by dashed box in A. Merged image (Bc) shows the heterogeneous population of pre-BötC inhibitory neurones, including GABAergic neurones co-expressing glycine (yellow, white arrowheads) or neurones without glycine (green), and a subpopulation of glycinergic only neurones (red, GAD67-GFP negative). Modified from Koizumi et al. (2013) with permission.