Plasticity of spinal L3 propriospinal neurons in urination recovery after thoracic SCI

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Abstract Bladder function recovery is a top priority among the SCI patient population, yet so far there are few effective therapies. One major reason is the limited understanding of intraspinal mechanisms for both spontaneous plasticity and for therapeutic functional recovery after SCI. A transection SCI deprives supraspinal control of the external urethral sphincter (EUS) which paralyzes its relaxation function and leads to urine retention. Partial recovery of EUS relaxation and voiding occurs spontaneously only if the neural circuits between lumbar 3 (L3) and lumbar 6 (L6) spinal cord level remain intact. Voiding function can be further improved by electrical stimulation of the L3 spinal cord in a chronic SCI model. However, the cellular mechanism underneath is unclear. In our preliminary study, we applied an anterograde trans-monosynaptic AAV1-GFP-Cre virus and found that a population of L3 propriospinal neurons (PSNs) projected to the L6 dorsal commissure (DCM) and synapsed with EUS-related interneurons (INs). We named these PSNs as PSNsL3-L6 DCM. Optogenetic stimulation of these PSNsL3-L6 DCM induced field potential of EUS Motoneurons (MNEUS) as well as EUS electromyography (EMGEUS) potential. These results indicate a functional connection between PSNsL3-L6 DCM and the EUS. We hypothesize that PSNsL3-L6 DCM participate in a spinal circuit to modulate EUS relaxation and mediate the spontaneous recovery of the EUS relaxation function after complete thoracic SCI. To evaluate the physiological function of PSNsL3-L6 DCM, we will inject recombinant viruses to transduce those neurons and express channelrhodopsin for optogenetic stimulation or hM4di for chemogenetic inhibition of neuronal function. We will evaluate whether manipulation of these neurons affects EUS relaxation and voiding function by bladder cystometry, EMGEUS, and field potential recording of DCM and MNEUS at L6. We will also combine anterograde and retrograde transsynaptic viral tracing and immunostaining to characterize the neurotransmitter phenotype of these PSNsL3-L6 DCM and their postsynaptic neurons. We will then further explore whether a T8 transection SCI will enhance synaptic connections between PSNsL3-L6 DCM and their target cells to mediate the spontaneous functional recovery of the EUS. We will use similar methods as above to verify whether more synaptic connections are established in this circuit at 4 weeks after SCI when the spinal reflex for EUS relaxation spontaneously recovers. Furthermore, chronic inhibition of PSNsL3-L6 DCM will be made immediately after SCI for 4 weeks to determine the causal contribution of neural plasticity to the functional recovery of EUS relaxation and voiding function. This study will be the first to explore the role of PSNs in EUS control in both intact and injured spinal cords, thus extending our understanding of cellular mechanisms of EUS control. This will provide a fundamental basis for some promising future therapies such as electrical stimulation of the spinal cord as a means of recovering bladder function.