Brainstem neurons regulating backward locomotion

Locomotion is one of the most important forms of movement for animals, characterized by different parameters such as gait, speed and directionality. Locomotor episodes, defined as the phase in between the initiation and termination point of a locomotor bouts, can occur in other directions than forward, resulting in side-way steps or backward locomotion. While neuronal circuits involved in the initiation and regulation of forward locomotion have been extensively studied, much less is known about the neuronal circuits regulating backward locomotion. In the mouse brainstem, several regions have been identified that connect to motor neurons controlling forelimbs and hindlimbs without a specific bias, suggesting a role in the regulation of full body movement. In this dissertation, we first investigate the organization of one of such brainstem region, the pontine reticular formation. Specifically, we aim to characterize the molecular and anatomical features of a specific region within this area known as SubC (subcoeruleus nucleus). Then, through different perturbation of function experiments, we analyze how different subpopulations of SubC neurons can affect locomotion directionality with a focus on forward and backward direction.
We first apply in situ hybridization to identify different neuronal populations constituting the SubC region. We found that in this area, there are excitatory and inhibitory neurons, which are intermingled and uniformly distributed throughout the SubC. We also found that inhibitory neurons are characterized by the expression of GABAergic and glycinergic markers. Using viral tracing approaches, we show that GABAergic and glycinergic neurons have both ascending and descending projections, with the GABAergic population forming synapses preferentially with local and ascending targets. In addition, we identified synapses in the contralateral SubC. Caudally, we observed that inhibitory neurons project to a nucleus in the caudal medulla referred to as LPGi (lateral paragigantocellular nucleus), which was previously shown to be targeted by excitatory SubC neurons.
We next designed a tunneled runway assay, to evaluate whether the pontine reticular formation, and in particular SubC, play a role in the regulation of directionality of locomotion, with a focus for forward and backward direction. Since laboratory mice do not perform spontaneous backward locomotion, we trained them in a behavioral context where they could perform only backward and forward locomotion, while turning behavior was impeded to reduce the possibility of confounding results. We found that speed and kinematic features for backward were different from forward locomotion and that our results were in line with other studies, suggesting that our assay represented a good entry point for the study of neuronal circuits involved in backward locomotion.
Since previous experiments conducted in the lab showed that the optogenetic activation of glutamatergic SubC neurons reliably induce backward locomotion in mice, we decided to evaluate how the inhibition of the activity of SubC neurons could impact on the performance of locomotion in different directions. Through loss of function experiments, we show that SubC glutamatergic neurons inhibition results in a reduction of backward speed. On the other hand, the optogenetic inhibition of GABAergic SubC neurons halts backward locomotion for the duration of the inhibition. We also show that this effect is specific for backward locomotion since forward locomotion is only transiently or not affected by the inhibition of glutamatergic or GABAergic neurons respectively. However, the optogenetic activation of GABAergic neurons did not elicit any phenotypical difference, suggesting that the activation of these neurons alone is not sufficient to promote backward locomotion.
Since the anatomical experiments showed connectivity of SubC excitatory and inhibitory neurons with LPGi, we evaluated if this nucleus in the caudal medulla could be receiving information relative to directionality of locomotion and regulate the activity of its targets to perform backward or forward locomotion. Therefore, we performed optogenetic inhibition of glutamatergic neurons in the LPGi, assessing the consequences of such perturbation in the tunneled runway assay. We observed that mice stopped locomotion for the time of inhibition both in the backward and forward direction. This result confirms the role of these neurons previously demonstrated in forward locomotion and suggests that at least a subpopulation of these neurons is involved in the transmission of backward signal, most probably derived from the SubC region.
Taken together, the results presented in this dissertation provide insight into the neuronal mechanisms underlying backward locomotion in mammals. We demonstrate that SubC, through the activity of its excitatory and inhibitory neurons, is an important brainstem regulator for the execution of backward locomotion. In addition, the ascending and descending connections of the neuronal subpopulation in SubC suggests that such regulation occurs through the modulation of different targets. Additionally, we propose that the glutamatergic population in LPGi may be a target of SubC neurons and possibly acts to transmit the instruction for the activation of the backward locomoting pattern.
This work only started to unveil possible centers in the brainstem for the control, initiation, and maintenance of backward locomotion, with SubC potentially representing, in the mammalian brain, a command center for walking in the backward direction. Additional experiments, such as recording of specific SubC neuronal populations, will allow us to understand if excitatory and inhibitory neurons are active in different phases of the backward behavior and link these findings to their role in this behavior. Moreover, the identification of synaptic inputs to the different neuronal subpopulations in SubC will help us to understand whether the different populations are activated by different input regions or whether they share the same inputs.