Deciphering Spinal Endogenous Dopaminergic Mechanisms That Modulate Micturition Reflexes in Rats with Spinal Cord Injury

Hypothesis: spinal TH⁺ neurons are unmasked as a primitive residual response to SCI. During postnatal development, TH⁺ neurons in the lumbosacral spinal cord contribute to DA synthesis and regulate pelvic organ function before diencephalospinal DAergic pathways originating from A11 cell groups develop. As the descending projections mature and dominate regulation of pelvic visceral activity, most spinal TH⁺ neurons become silent because of competitive synapse formation. However, after SCI interrupts descending pathways, spinal TH⁺ neurons undergo plasticity to augment DA synthesis and take over control of visceral activity to compensate for the loss of supraspinal DA control. Accordingly, we postulate that spinal endogenous DAergic mechanisms reemerge and regulate recovered micturition function following SCI.

Representative traces show bladder CMG and EUS EMG recordings during continuous bladder filling (0.1 ml/min) in a neuroaxis intact or SCI female rat. A, The peak amplitude of intravesical pressure (VA) during voiding (*) is low in the spinal intact rat compared with that in the SCI rat three weeks after T10 spinal transection (p < 0.0001, unpaired t test) and NVCs are more prominent during the filling phase in the SCI rat, indicating bladder hyperreflexia. B, Time-stretched recordings during voiding show that spinal intact rats exhibit well-coordinated bursting EUS activity and HFOs in bladder pressure during voiding, while this coordination is absent in SCI rats. Note that the drop in bladder pressure reflects the opening of the urethral outlet. Statistical analysis shows that the duration of EUS bursting activity is shorter (p < 0.001) or masked by tonic EUS activity in SCI rats when compared to spinal intact rats.

Representative traces show that L-DOPA suppresses bladder overactivity and improves detrusor-sphincter coordination following SCI. Three weeks following a complete T10 spinal cord transection, different concentrations of L-DOPA are administered intravenously during bladder CMG and sphincter EMG recordings. A, Time-compressed traces show that following SCI, control recordings after intravenous administration of vehicle (saline) exhibit numerous large amplitude NVCs before the voiding contraction (marked by *) that are accompanied by large amplitude tonic EUS EMG activity. Administration of L-DOPA to the same animal induces dose-dependent changes in reflex LUT function, including decreased amplitude of voiding contractions (VA), a reduction in the number and amplitude of NVCs, and lowered tonic EUS activity. B, Time-expanded traces of bladder contractions and EUS EMG activity show that DSD, which typically manifests as the lack of detrusor HFOs and irregular and short EUS bursting periods (gray lines) occurs in control recordings after intravenous administration of vehicle. However, injection of L-DOPA unmasks HFOs in bladder pressure and induces a longer and more regular EUS bursting period during voiding, as shown in the 0.16-s-scaled expanded view. EUS tonic activity (two-headed arrows) that occurs in the filling phase and after voiding in the control recording is remarkably reduced. Notably, after L-DOPA administration, detrusor and sphincter activity exhibits better coordination during voiding (* indicating voiding contractions). All recordings are from the same experiment.

Blocking spinal DRs in SCI rats diminishes the effects of L-DOPA on bladder and EUS reflexes. Recordings in A, B and C, D are from two different experiments. A, The left side of the traces show repetitive voiding in a SCI rat consisting of low amplitude bladder contractions and synchronized bursts of EUS EMG activity following sequential injections of L-DOPA (0.1, 10, and 30 mg/kg). Injection of SCH 23390 (0.1 mg/kg, down arrow), which blocks DR₁, increases the VA to similar levels as seen during basal recordings and increases the number of NVCs. In parallel, EUS tonic activity during the filling phase is remarkably elevated. B, Time-expanded views of two voiding responses from the recording in panel A. The traces on the left show HFOs in intravesical pressure and EUS bursting (gray lines) after administration of L-DOPA. The top and bottom traces are from the same time base and time expanded sections of these traces are shown in the middle. The traces on the right are organized in the same manner and show that SCH 23390 reverses the effects of L-DOPA by blocking HFOs and EUS bursting and increasing tonic EUS activity and the VA. HFOs and EUS bursting are reduced in duration or disappear after inhibiting DR₁ with SCH 23390 (right traces). C, D, The records are from the same experiment and organized as described in panels A, B. Recordings at a slow time base (C) show that in a SCI rat treated with L-DOPA, administration of remoxipride (3.0 mg/kg), a DR₂ antagonist, increased EUS tonic activity to a level that is similar to what occurred in basal recordings but did not obviously influence bladder contractions, detrusor HFOs, or EUS bursting during voiding as shown in the time expanded view (D). These data indicate that L-DOPA improves LUT performance via mechanisms involving spinal DR₁ and DR₂ in SCI rats (* indicating voiding contractions).

Representative traces show that non-selective activation of spinal DR with APO reduces the number of NVCs, lengthens the VI and enhances the duration of EUS EMG bursting in a SCI rat. A, After vehicle (saline) delivery, many NVCs occur during the filling phase and are accompanied by high amplitude tonic EUS activity. EUS activity also increases during and after voiding (marked by *). Administration of APO (10 μg/kg, i.v.) reduces the number of NVCs, decreases tonic EUS activity and increases the VA. The higher dose (0.1 mg/kg) reverses the effects on the VA but maintains suppression of NVCs. B, In a time-expanded view, stimulating DR with APO reduces the amplitude of EUS tonic activity before voiding (two-headed arrows) and increases the duration of EUS bursting during voiding contractions (gray lines), indicating the facilitation of voiding. A higher dose (0.1 mg/kg) of APO elicited slightly larger effects. A time-expanded view of the EUS bursting period is shown in panel B.

Selective stimulation of spinal DR₂ increases bladder capacity and improves voiding reflexes. A, Three weeks after a T10 spinal cord transection, control CMG and EUS EMG recordings during continuous bladder filling and after intravenous saline injections exhibited tonic EUS activity between repeated reflex bladder contractions and voiding (marked by *). Intravenous injections of quinpirole, a DR₂ agonist (10 μg or 0.1 mg/kg), prolonged the VI and reduced EUS tonic activity during bladder filling. B, In a time-expanded view, the phasic bursting of EUS during voiding in SCI rats is short and irregular in the control recording following an injection of saline. However, after administration of quinpirole, EUS bursting is prolonged and more regular although bladder pressure oscillation patterns (HFOs) do not change (* indicating the onset of voiding). C, Following the high dose of quinpirole, administration of remoxipride (3.0 mg/kg) suppresses the large amplitude voiding reflex in bladder activity and masks EUS bursting during voiding.

Inhibition of spinal DR₁ increases tonic EUS activity in SCI rats. A, Traces show that after different doses of SCH 23390 (10 μg and 0.1 mg/kg) were delivered to block spinal DR₁ during bladder CMG and sphincter EMG recordings, EUS tonic activity robustly increases and the VA of bladder contractions slightly elevates (* indicating voiding contractions). B, In a time-expanded view, EUS tonic activity (two-headed arrows) in response to bladder NVCs (#) in the filling phase is greater following SCH 23390 (10 μg/kg) injections than in the control recording after saline injections. C, Blocking DR₁ results in a shorter EUS bursting period compared to vehicle injections. There is still a lack of high-frequency detrusor oscillations during voiding (traces in C are an expanded portion of voiding with bursting in B). Therefore, this suggests that spinal DR₁ regulate the micturition reflex following SCI and that they are mainly involved in the regulation of EUS activity that serves to facilitate urine elimination.

Selective stimulation of spinal DR₁ increases tonic EUS activity in SCI rats. Following administration of saline during a continuous bladder CMG and EUS EMG recordings, voiding occurs at regular intervals and is associated with EUS bursting followed by tonic EUS EMG activity. Low level tonic EUS activity occurs between voids. After administration of either the mid (1.0 mg/kg) or high (3.0 mg/kg) dose of SKF 38393, a DR₁ agonist, EUS tonic activity increases during the period between voids (* indicates voiding contractions). As evident in the representative traces, the agonist has no effect on the VI or VA of voiding contractions.

Effects of drugs that modulate DAergic mechanisms on LUT storage and voiding function in SCI rats. Drugs including L-DOPA (with carbidopa), carbidopa, APO, remoxipride, quinpirole, SKF 38393, and SCH 23390 were administered cumulatively during single CMG recordings in a range of doses that are listed in Table 1. The high dose of L-DOPA plus carbidopa (A) induces a non-significant increase in the voiding volume (VV), significantly reduces the residual volume (RV), and increases the voiding efficiency (VE; both *p < 0.05, Friedman test followed by Dunn’s). Bladder capacity (BC) does not change with any dose. Carbidopa alone (B) does not induce any changes in voiding parameters (p > 0.05). Administration of the high dose of either APO, a non-selective DR agonist (C), or quinpirole (D), a selective DR₂ agonist, elicits effects similar to that of L-DOPA. Blocking DR₁ with the mid or high dose of SCH 23390 (F) significantly reduces voiding efficiency (both *p < 0.05). However, inhibiting DR₂ with remoxipride (E) or stimulating DR₁ with SKF 38393 (G) does not alter voiding efficiency.

Representative traces show volume-frequency patterns of spontaneous micturition in metabolic cage assays in SCI rats during 12-h recordings. In each “step-like” curve (A–C), the horizontal line represents the VI, and the vertical line depicts the volume of urine expelled. A, Statistical analysis demonstrates that within 6 h after subcutaneous delivery of L-DOPA, the VI is prolonged (*p < 0.05, paired t test) when compared with vehicle injections, but the volume of expelled urine does not change. B, Injections of APO, a non-selective DR agonist, does not generate detectable changes in the spontaneous micturition reflex (all p > 0.05). C, Specific stimulation of spinal DR₂ with quinpirole induces a dramatic increase in the amount of urine released per void (**p < 0.01) and the total volume of urine expelled (*p < 0.05) as compared to vehicle delivery within the 6-h period. Additionally, this drug decreases the interval between voids (*p < 0.05) when compared to the control.