Role of endocannabinoid system and acid sensing ion channels on spinal locomotor circuits during physiological and pathological conditions

Background: Mammalian spinal cord can generate well-coordinated locomotor activity called fictive locomotion in the absence of any higher brain center input or of rhythmic sensory feedback. This activity clearly provides evidence for the central pattern generator (CPG) that produces the locomotor rhythm. Such CPG consists of glutamatergic excitatory and glycinergic and GABAergic inhibitory interneuronal connections that finally excite or inhibit the motoneuronal pools. Many factors including fast (ion channels) and slow (modulatory G-protein coupled receptors; GPCRs) processes control these neuronal pools to act rhythmically. These factors are perturbed following spinal cord injury (SCI) in the early and late phases. The present study addresses the role of a few modulatory processes, namely acid sensing ion channels (ASICs) and cannabinoid 1 receptors (CB1Rs) at both initial and the late phases of injury.Objectives: Recent evidence has shown that the deletion of ASICs slows down the progression of disease in ischemic conditions, whereas the same protocol increases seizure severity. CB1R activation or deletion also results in neuroprotective or toxic mechanisms. In order to understand the importance of ASICs and CB1Rs in the spinal locomotor circuits, it is crucial to analyze them in physiological and pathological conditions. To investigate this issue, both organotypic slice culture and an in vitro rat spinal cord model were used. With the latter, fictive locomotion can be recorded from the ventral roots of the lumbar region for a time window of 24 h. Network parameters like synaptic transmission, fictive locomotion and disinhibited bursting provide information to explain the physiological modifications and pathological severity after excitotoxicity caused by transient kainate (KA; glutamate analog) application. Drugs that modulate CB1Rs and ASICs may supply evidence for the role of these processes in fictive locomotion.Results and conclusion: Our results show that the CB1R activation or block for 24 h diminished the locomotor rhythm. In particular, CB1R pharmacological block completely depressed both dorsal root (DR) and chemically evoked fictive locomotion. This depression was amplified following KA treatment. Furthermore, a limited neuroprotection was observed after CB1R agonists (anandamide; AEA or 2-arachidanoyl glycerol; 2AG) and an endogenous cannabinoid uptake inhibitor. These results allow us to propose the innate activity of CB1R (that is well preserved) to be important after KA mediated excitotoxicity, while any neuroprotective role might come in later phases after injury. A low concentration of KA that can induce a borderline injury elicited rapid glutamate release combined with proton discharge (acidification) in the organotypic SCI model. In response to this challenge, the ASIC subtypes (1a, 1b, 2a and 3) mRNA levels were found to be elevated after 24 h. Both neuronal numbers and network activity were highly depressed after application of ASIC pharmacological blockers that intensified the consequences of KA treatment. These results indicate that moderate acidification might be beneficial for the recovery (or limitation) of KA mediated excitotoxicity. Hence, this study demonstrates that both ASICs and CB1Rs activity are important in the early phase of experimental SCI in vitro. Their pharmacological modulation can outline future strategies for neuroprotection.