Hypothesis / aims of study
The mechanism of micturition is complicated and especially in the cerebral cortex, micturition-related neural activity remains poorly understood. Two-photon calcium imaging enables to observe individual neural activities with high spatial resolution. In the present study, we observed the neural activities in the cerebral cortex using two-photon calcium imaging and evaluated the relationship with micturition. Our purpose was to reveal the ratio and the localization of micturition-related neurons, and the features of neural activity in various cell-type patterns.
Study design, materials and methods
We genetically expressed red calcium indicators (jRGECO1a) in the anterior cingulate cortex (ACC) or the primary motor cortex (M1) of mice. Two-photon calcium imaging from the ACC or the M1 was performed with bladder perfusion under urethane anesthesia. Micturition-related neurons were extracted according to neural synchrony with micturition (Figure1A).
Cell-type patterns were A) non-selective neurons, B) layer 5 pyramidal neurons, and C) certain projection neurons; the ACC to the periaqueductal gray matter (PAG) or the M1 to the pontine micturition center (PMC) (n=3 or 4 in each group, total n = 22) (Figure 1B).
Results
Micturition-related neural activity was individually identified in every region and pattern (Figure 2A). The rates of micturition-related neurons per all observed neurons were 1) 6.34%, 2) 10.97%, 3) 9.42% in the ACC and 1) 6.61%, 2) 10.33%, 3) 8.50% in the M1.
The hot spot (high density region) of micturition-related neurons in the ACC (posterior and deep region) were more local than in the M1 (Figure 2B). The peak timing histogram of B) layer 5 pyramidal neural activities in the ACC was bimodal (the former mean; 0.72sec, the latter mean; 11.53sec), and the latter peak was analogous with histogram of C) the ACC-PAG projection neural activities (mean 10.75sec). Furthermore, the population of the delayed neurons was located in the hot spot of the ACC (posterior and deep region). On the other hand, the patterns of neural activity were uniform among each variation in the M1 (Figure 2C).
Interpretation of results
The number of micturition-related neurons is not so large population both in the ACC and the M1. ACC may have multi-functional clusters regarding micturition, depending on their location, cell-type, and projection pathway. On the other hand, M1 may have single functional cluster well synchronized with bladder pressure.