GABAergic Inhibition of Presynaptic Ca2+ Transients in Respiratory PreBötzinger Neurons in Organotypic Slice Cultures

Abstract

GABAergic somatodendritic inhibition in the preBötzinger complex (preBötC), a medullary site for the generation of inspiratory rhythm, is involved in respiratory rhythmogenesis and patterning. Nevertheless, whether GABA acts distally on presynaptic terminals, evoking presynaptic inhibition is unknown. Here, we begin to address this problem by measuring presynaptic Ca²⁺ transients in preBötC neurons, under rhythmic and non-rhythmic conditions, with two variants of genetically encoded Ca²⁺ indicators (GECIs). Organotypic slice cultures from newborn mice, containing the preBötC, were drop-transduced with jGCaMP7s, or injected with jGCaMP7f-labeling commissural preBötC neurons. Then, Ca²⁺ imaging combined with whole-cell patch-clamp or field stimulation was obtained from inspiratory preBötC neurons. We found that rhythmically active neurons expressed synchronized Ca²⁺ transients in soma, proximal and distal dendritic regions, and punctate synapse-like structures. Expansion microscopy revealed morphologic characteristics of bona fide synaptic boutons of the en passant and terminal type. Under non-rhythmic conditions, we found that bath application of the GABA_A receptor agonist muscimol, and local microiontophoresis of GABA, reduced action potential (AP)-evoked and field stimulus-evoked Ca²⁺ transients in presynaptic terminals in inspiratory neurons and commissural neurons projecting to the contralateral preBötC. In addition, under rhythmic conditions, network rhythmic activity was suppressed by muscimol, while the GABA_A receptor antagonist bicuculline completely re-activated spontaneous activity. These observations demonstrate that the preBötC includes neurons that show GABAergic inhibition of presynaptic Ca²⁺ transients, and presynaptic inhibition may play a role in the network activity that underlies breathing.

Keywords: GABAA; GECI; breathing; jGCaMP7; presynaptic inhibition.