ICV pre-administration of DNQX at 0.3 nmol per rat had no effect on the ICI shortening induced by U-46619 (Fig. 3). In contrast, DNQX at 3 nmol per rat significantly attenuated U-46619-induced ICI shortening during the 0-20 min period (Fig. 3).

TxA₂ is unstable and has a short chemical half-life of about 30 s2, therefore, we used the stable analogue U-46619, a TP receptor agonist. In preliminary experiments, U-46619 showed no effect on ICI at 0.1 nmol per rat, while a trend toward ICI shortening was observed at 1 or 10 nmol per rat. Therefore, we increased the number of rats in the 1 nmol- and 10 nmol-administered groups. The shortened ICI induced by U-46619 at 10 nmol per rat may have been induced not only by increased sensitivity to bladder filling but also by increased residual volume. Consequently, single cystometry was performed to evaluate the RV, which was not increased by U-46619. These results indicate that brain TxA₂ shortened ICI by increasing bladder sensitivity to urine storage in rats.

We previously found that ICV administered U-46619 at 100 or 300 nmol but not 30 nmol per rat dose-dependently increased plasma noradrenaline and adrenaline (catecholamines) in rats. However, in this study, ICV administered U-46619 (10 nmol per rat) shortened the ICI, which was lower than the dose (30 nmol per rat) which did not increase plasma catecholamines. This suggests that brain TxA₂ might promote the reflex through a mechanism independent of peripheral SNS activation. Likewise, we previously found that neuropeptides, bombesin, and angiotensin II, centrally facilitated the rat micturition independently of the SNS. Like U-46619, ICV administered each peptide shortened the ICI in rats even at 1/10th to 1/100th of the dose required to increase plasma catecholamines. Both peptides shortened the ICI at higher doses, even under conditions where these peptide-induced increments in plasma catecholamines were eliminated by acute bilateral adrenalectomy. Therefore, brain molecules that activate the SNS, such as bombesin and angiotensin II, in addition to TxA₂, might have SNS-independent effects on facilitation of the micturition reflex.

S-18886 blocks the effects of TP receptor ligands. In an in vitro study using rabbit venous smooth muscle, the pA₂ value of S-18886 against U-46619-induced contraction was reported to be 8.9. Adult rats have approximately 300 µL of cerebrospinal fluid (CSF). When 300 nmol of S-18886 was ICV administered, the calculated concentration in the CSF was 1 mM. And, when 10 nmol of U-46619 was administered ICV, its concentration in the CSF was calculated to be approximately 33 µM. Therefore, the dose of S-18886 is sufficient to block the effects of U-46619 via TP receptors. Although nonspecific effects may occur if excessive S-18886 is administered, no significant difference in baseline ICI values was observed between the S-18886 + U-46619 group and the vehicle + U-46619 group during the - 20-0 min period, despite S-18886 having been administered as a pretreatment 1 h prior to U-46619. Our present data in the S-18886-pretreated group indicate that the U-46619-induced ICI shortening was mediated by brain TP receptors in rats. Although the exogenously administered TxA₂ analogue shortened the ICI, ICV administered S-18886 alone did not induce ICI prolongation. Therefore, under normal conditions, endogenous TxA₂ in the brain may not affect the micturition reflex.

Next, we examined whether brain TxA₂-induced facilitation of the micturition reflex is mediated by the glutamatergic nervous system, which is modulated by TxA₂. Glutamate is a major excitatory neurotransmitter, and glutamate receptors are broadly expressed in the CNS. Moreover, a study reported that ICV administered glutamate receptor antagonists blocked bladder contractions in rats, suggesting that the brain glutamatergic neurotransmission is essential for voiding function. Glutamate receptors are subdivided into two classes: ionotropic and metabotropic. While the role of brain metabotropic glutamate receptors in controlling the micturition reflex remains unclear, brain ionotropic glutamate receptors (AMPA and N-methyl-D-aspartate [NMDA] receptors) play an important role in inducing the micturition reflex, as evidenced by the ionotropic receptor antagonist-mediated suppression of the reflex. Accordingly, we previously used NMDA and AMPA receptor antagonists and found that the brain glutamatergic nervous system is involved in ICI shortening induced by ICV-administered SIN-1, a nitric oxide donor, and corticotropin-releasing factor. In this study, we preliminary confirmed that centrally pretreated DNQX (AMPA receptor antagonist), but not MK-801 (NMDA receptor antagonist), showed a tendency to suppress U-46619-induced ICI shortening, therefore, we focused on AMPA receptors. Additionally, we previously investigated the effects of the ICV-administered DNQX alone on ICI. ICV administered DNQX at 3 nmol per rat showed no effect on the micturition reflex, while DNQX at higher doses (>3 nmol per rat, ICV) affected the micturition reflex by itself in continuous cystometry. Consequently, we used DNQX at the maximum dose in this study (3 nmol per rat), which alone showed no effect on the micturition reflex. Conversely, in a previous study from another group, ICV administered LY215490, another AMPA receptor antagonist, decreased ICI at lower doses than those we used in this study. This discrepancy in the results may be due to the several differences in experimental conditions between the studies, including rat strain (Wistar vs. Sprague-Dawley), sex (male vs. female), doses of urethane anaesthesia (0.8 vs. 1.2 g kg per rat), drug administration protocol (single administration per rat vs. cumulative dosing per rat), and experimental duration after each antagonist administration (1 h vs. 45 min interval). Therefore, the results of our study cannot be simply compared to their study. Notably, sex differences in glutamatergic neurotransmission have been reported, with females exhibiting more prominent glutamatergic activity than males across various brain regions. Although no reports have been found on sex differences following ICV administration of each antagonist on ICI, it is possible that such differences contributed to the variation in effective doses affecting micturition observed between the two studies. Nevertheless, at least under our experimental conditions, DNQX (3 nmol per rat) showed no effect on baseline ICI values, while significantly inhibited U-46619-induced ICI shortening. These findings suggest that the brain TxA₂-induced facilitation of the rat micturition reflex may be mediated by brain AMPA glutamate receptors.

In Fig. 1, the shortening effect of ICI by U-46619 was observed at 0-20, 40-60 and 60-80 min periods. On the other hand, the significant antagonistic effect of S-18886 and DNQX on the U-46619-induced shortening of ICI was seen only at 0-20 min period (Figs. 2 and 3). Experimental protocols varied across Figs. 1, 2 and 3 in terms of the number of injections (once in Fig. 1; twice in Figs. 2 and 3), duration of CMG (3 h in Figs. 1 and 4h in Figs. 2 and 3), vehicle (DMF in Fig. 2; saline in Fig. 3), and total volume of ICV administration (3 µL in Figs. 1 and 8 µL in Figs. 2 and 3). Therefore, the time-dependent effects of U-46619 observed in Figs. 1, 2 and 3 cannot be directly compared. Although not all changes reached statistical significance, a tendency toward ICI shortening was observed during the 0-20 min and 40-60 min periods in all three figures. Thus, the ICI-shortening effect of U-46619 was consistently observed across all experimental conditions. The ICI values at 40-60 min period in each antagonist-pretreated group was nearly 100% in Figs. 2 and 3. These findings suggest that the U-46619-induced ICI shortening was almost eliminated by each antagonist pretreatment at the period. The lack of a statistically significant difference at 40-60 min period may be due to greater variability between groups compared to 0-20 min period.

This study has several limitations. First, all experiments were conducted under urethane anesthesia, which can modulate central micturition pathways. Therefore, the responses observed here may not fully reflect those in awake animals. Future studies in awake preparations will be necessary to validate the present findings under physiological conditions. Second, because ICV administration was performed, it is unclear in which brain region TP and AMPA receptors facilitates the micturition reflex. We performed ICV administration to engage the supraspinal micturition network (periaqueductal gray-pontine micturition center and associated hypothalamic relays) broadly in this study, as our a priori aim was to test whether brain TP receptor activation can affect the micturition reflex at the systems level rather than to map a single nucleus. Prior central (ICV) administrations of neuromodulators (bombesin, angiotensin II, nitric oxide donor, corticotropin-releasing factor, etc.) have reproducibly modified the micturition reflex in rats, supporting the validity of this approach. However, further studies are necessary to clarify brain specific regions that contributes to TP receptors-mediated facilitation of the micturition. Third, we did not perform immunohistochemical and/or biochemical assays to indicate molecular evidence of the brain TP receptors- and glutamate-mediated facilitation of the micturition reflex (e.g., co-localization of TP receptors and glutamatergic neurons and levels of glutamate and AMPA receptors in brain regions related to micturition). Future studies using site-specific microinjection of drugs and histological confirmation will be warranted to anatomically narrow down the brain regions and to provide molecular corroboration. Finally, the role of brain metabotropic glutamate receptors in the TxA₂-induced facilitation and the relevance of other brain neurotransmitters such as GABA and dopamine to the TxA₂-induced response remain unaddressed. Although spinal ionotropic glutamate receptors have excitatory effects and spinal metabotropic glutamate receptors have inhibitory effects on the micturition reflex, the role of brain metabotropic receptors in the micturition reflex is still unclear. Whether brain TxA₂ facilitates micturition via direct or indirect modulation of the glutamatergic system also requires further investigation, particularly considering its known effects on GABAergic and dopaminergic systems.

In conclusion, we demonstrated that centrally administered the TxA₂ analogue U-46619 acting on brain TP receptors induced facilitation of the micturition reflex via brain AMPA receptors in rats. These results suggest a possible novel function of brain TxA in the micturition regulation.