Irregular Breathing in Mice following Genetic Ablation of V2a Neurons

Abstract

Neural networks called central pattern generators (CPGs) generate repetitive motor behaviors such as locomotion and breathing. Glutamatergic neurons are required for the generation and inhibitory neurons for the patterning of the motor activity associated with repetitive motor behaviors. In the mouse, glutamatergic V2a neurons coordinate the activity of left and right leg CPGs in the spinal cord enabling mice to generate an alternating gait. Here, we investigate the role of V2a neurons in the neural control of breathing, an essential repetitive motor behavior. We find that, following the ablation of V2a neurons, newborn mice breathe at a lower frequency. Recordings of respiratory activity in brainstem–spinal cord and respiratory slice preparations demonstrate that mice lacking V2a neurons are deficient in central respiratory rhythm generation. The absence of V2a neurons in the respiratory slice preparation can be compensated for by bath application of neurochemicals known to accelerate the breathing rhythm. In this slice preparation, V2a neurons exhibit a tonic firing pattern. The existence of direct connections between V2a neurons in the medial reticular formation and neurons of the pre-Bötzinger complex indicates that V2a neurons play a direct role in the function of the respiratory CPG in newborn mice. Thus, neurons of the embryonic V2a lineage appear to have been recruited to neural networks that control breathing and locomotion, two prominent CPG-driven, repetitive motor behaviors.

Generation of V2a Interneurons from Mouse Embryonic Stem Cells

“…Studies have also shown that RA activates the expression of bHLH transcription factors to control the differentiation of neuronal cell types, such as V2a interneurons [44]. We hypothesized that by decreasing RA concentration we could promote the differentiation of V2a interneurons found rostrally in respiratory columns of the medial reticular formation of the hindbrain [14]. Our experiments showed that decreasing RA concentration increased Chx10 expression.…”

“…V2a interneurons have been shown to be involved in repetitive motor behaviors in the CPGs of the spinal cord and medial reticular formations of the hindbrain and play an important role in left-right coordination of locomotion, skilled reaching movements, and rhythmic patterning of breathing [10,14,26]. Differentiation of V2a interneurons from mESCs has the potential to increase understanding developmental pathways and possibly provide a source for cell therapies in high cervical spinal cord injuries affecting respiratory and motor function.…”

“…Existing protocols for motoneuron differentiation from mouse ESCs (mESCs) use RA and Shh signaling to drive differentiation of cells with a cervical spinal identity [2,36]. Since V2a interneuron pools lay more rostral in respiratory columns in the medial reticular formation of the hindbrain [14], we hypothesize that a lower RA concentration could promote differentiation of ESCs into V2a interneurons. We explored the effect of RA concentration on the expression of p2 progenitor and V2a markers.…”

“…Recently, V2a interneurons in the medial reticular formation of the hindbrain have been shown to stimulate excitatory signals to produce regular breathing patterns. Mice with genetic ablation of V2a interneurons display irregular and less frequent breathing patterns, leading to decreased survival rates of newborns [14].…”

Complexe de pré-Bötzinger et automatisme respiratoire

“…Ainsi, les souris transgéniques déficientes pour le gène Vglut2 ne survivent pas à la naissance et leur CPB est inactif in vitro [35]. L'ablation génétique d'une sous-population de ces neurones (les neurones V2a) n'abolit pas le rythme du CPB, mais le rend irrégulier [36]. Par ailleurs, il existe une sous-population de neurones glutamatergiques du CPB qui exprime la somatostatine et semble critique pour la respiration de l'adulte [24].…”