Mapping of spinal interneurons involved in regulation of the lower urinary tract in juvenile male rats

Abstract

Coordination between the urinary bladder (BL) and external urethral sphincter (EUS) is necessary for storage and elimination of urine. In rats interneuronal circuits at two levels of the spinal cord (i.e., L6-S1 and L3-L4) play an important role in this coordination. In the present experiments retrograde trans-synaptic transport of pseudorabies virus (PRV) encoding fluorescent markers (GFP and RFP) was used to trace these circuits. To examine the relative localization of EUS-related and BL-related interneuronal populations we injected PRV-GFP into the EUS and PRV-RFP into the BL wall. The PRV infected populations of spinal interneurons were localized primarily in the dorsal commissure (DCM) of L6/S1 and in a hypothesized lumbar spinal coordinating center (LSCC) in L3/L4 above and lateral to central canal (CC). At both sites colocalization of markers occurred in a substantial number of labeled interneurons indicating concomitant involvement of these double-labelled neurons in the EUS- and BL-circuits and suggesting their role in EUS-BL coordination. Intense GFP or RFP fluorescent was detected in a subpopulation of cells at both sites suggesting that they were infected earlier and therefore likely to represent first order, primary interneurons that directly synapse with output neurons. Larger numbers of weakly fluorescent neurons that likely represent second order interneurons were also identified. Within the population of EUS-related first order interneurons only 3-8 % exhibited positive immunoreaction for an early transcription factor Pax2 specific to GABAergic and glycinergic inhibitory neurons suggesting that the majority of interneurons in DCM and LSCC projecting directly to the EUS motoneurons are excitatory.

Keywords: BCM, bulbocavernosus muscle; BL, bladder; B_G, bright green; B_R, bright red; CC -, central canal; DCM, dorsal commissure; DSD, detrusor-sphincter-dyssynergia; EMG, electromyogram; EUS, external urethral sphincter; EUS-MN, motoneuron of the external urethral sphincter; GFP, green fluorescent protein; IML, intermediolateral nucleus; IN, interneuron; LSCC, lumbar spinal coordinating center; LUT, lower urinary tract; Lf, lateral funiculus; PPN, propriospinal neuron; PRV, pseudorabies virus; Pseudorabies virus; RFP, red fluorescent protein; SC, spinal cord; SCI, spinal cord injury; SPPN, spinal parasympathetic preganglionic neuron; Spinal cord; Transsynaptic tracing; VMf, ventro-medial funiculus; W_G, weak/moderate green; W_R, weak/moderate red; pIN, primary interneuron; sIN, secondary interneuron.