Locomotion- And Micturition-related Population Cortical Ca²⁺ Signals In Freely Moving Mice

Locomotion is a particularly complex behavior of organism that requires multiple participated brain areas. Among them, motor cortex is a critical center for controlling the movement and plays a key role in the preparation, execution and regulation of locomotion.Primary motor cortex (Ml) is the core area of the whole motor cortex and neurons in layer 5 project a large number of neural connections to the spinal cord. M1 transmits motor commands to spinal cord and directly controls body movement. The visual cortex is another important brain area for movement regulation and connected extensively to the motor areas.Of which, primary visual cortex (V1) receives visual input from the external environment and is essential for orientation and locomotion guiding. However, during the whole process of locomotion, the mechanism of these two cortices on information processing and coding remains unclear.Micturition is not only a basic physiological function, but also a complex social behavior of humans and animals. It involves two processes of bladder: storage and emptying. The micturition reflex is the key part in the process of bladder emptying and requires regulation of multiple regions in the nervous system. The micturition reflex is mediated by a spinobulbospinal pathway that passes through the pontine micturition center (PMC) which is critical for micturition. PMC is currently known to be the central area for controlling micturition. Its activation can cause relaxation of urethral smooth muscle, contraction of bladder detrusor and thus flow of urine. However, as a voluntary behavior, micturition is supposed to be controlled by advanced brain area such as cortex. The current study has not yet found a definite cortical region for voiding control.Traditional approach for studying cortical information processing is to monitor the activity of individual neurons. However, for a long time, recording the activity from population of neurons is another important approach to analyze the working pattern of brain.Numerous studies have shown that neuronal population activity is involved in a variety of brain functions, such as nervous system development, sensory information processing,learning and memory. At present, commonly used approaches for monitoring population activity include local field potential (LFP), functional magnetic resonance imaging (fMRI),two-photon Ca2+ imaging and so on. However, there are still some limitations existing in these methods, such as insufficient spatial resolution, too superficial depth of the imaging or unable to record in the freely moving animals. In recent years, a simple but efficient approach for monitoring population Ca2+ activity has been rapidly developed and widely used. It is the optical fiber-based Ca2+ recording approach that can be used in deep brain regions in freely behaving animals.Based on the above information, here we applied the optical fiber recording approach to monitor the population Ca2+ activity during locomotion or micturition, trying to dig out the neural mechanisms of information processing and coding.The main findings of the study are as follows:1. The effect of different anesthesia levels on cortical population Ca2+ signals in mice.The effect of anesthesia on brain activity involves diverse spatial scales, from single neuron to the whole brain, but little is known about the effect of anesthesia on local neural population. In this study, we chose the primary motor cortex (Ml) and primary visual cortex(V1) as the study areas. We injected the calcium dye OGB-1AM into layer 5 of Ml or V1 and recorded the Ca2+ signals above the injection site with the optical fiber. In M1, we recorded population Ca2+ signals under flve diminishing anesthesia concentrations (2%, 1.5%, 1.2%,1%, 0.8%) while we just recorded at 0.8% in V1.(1) The effect of different anesthesia levels on the frequency of cortical population Ca2+ signals in mice.In Ml, the frequency increased from 0.04 ± 0.01 Hz to 0.45 ± 0.03 Hz with a decrease in the isoflurane concentration from 2% to 0.8%. There was a significant difference in the frequency of Ca2+ signals between adjacent two anesthesia levels (p< 0.05). However, no significant diaiaerence was found in the frequency of Ca2+ signals between M1 and V1 at the same anesthesia level (p = 0.051).(2) The effect of different anesthesia levels on the amplitude of cortical population Ca2+ signals in mice.The change in the amplitude of the Ca2+ signals was opposite to the frequency. With a decrease of anesthesia concentration,the amplitude in Ml decreased gradually from 0.016 ±0.009 ?F/F to 0.052 ± 0.012 ?F/F (p < 0.05, ?F/F indicated fluorescence intensity). There was a significant difference in the amplitude of Ca2+ signals between adjacent two anesthesia levels (p < 0.05). Similarly, no significant difference was found in the amplitude of Ca2+signals between Ml and VI at the same anesthesia level (p = 0.883).(3) The effect of different anesthesia levels on the rise time of cortical population Ca2+ signals in mice.The rise time of calcium signal was very stable and was not affected by changes in anesthesia concentration or different brain regions. The results were as follows: in M1, with the anesthetic concentration decreased, the rise time did not change significantly and kept at 0.174s. While the rise time of V1 at 0.8% is 0.170s. Comprehensive analysis of 6 groups of data from Ml and V1, no statistical difference was found (p = 0.320).Above results suggested that the cortical population Ca2+ signals were closely related to the depth of anesthesia. The frequency and amplitude showed obvious concentration-dependent properties, while the rise time remains stable under different levels of anesthesia.Moreover, the population Ca2+ signals of different cortices do not differ significantly at the same level of anesthesia.2. Properties of the cortical population Ca2+ signals in M1 in freely moving mice.Next, we recorded the population Ca2+ signals in freely moving mice in Ml and compared the properties of the Ca2+ signals between moving and resting states. Also, we analyzed the relation between Ca2+ signals and movement.(1) Different properties of cortical population Ca2+ signals during freely moving and resting states.We still selected the layer 5 of Ml as the recording site. After the fiber fixed and the anesthesia effect degraded, the mice were placed into a white rectangular box for synchronous recordings of Ca2+ signals and behavior. After analyzing the motor amplitude and Ca2+ signals,we found that when the mice were in motion,the frequency of Ca2+ signal was higher (0.42±0.04 Hz) and the amplitude was larger (1.51%±0.06?F/F) than that at resting state(frequency: 0.08 ± 0.01 Hz; amplitude: 0.24% ± 0.02% ?F/F). There were significant differences in the frequency and amplitude of Ca2+ signals between moving and resting states(p <0.001). In order to rule out the effects of motion artifacts, we recorded the movement and fluorescence intensity simultaneously in layer 5 of M1 from Thyl-GFP transgenic mice. The results showed that when the mice were moving, the fluorescence intensity changed slightly.Both the fluorescence intensities of OGB-1AM and GFP fit Gaussian distributions and both the goodness of fitting were more than 99%. The mean values were 0.3% ?F/F and 1.5% AF/F,respectively. The above results suggested that the changes of fluorescence intensities we recorded were not motion artifacts but true signals.(2) The cortical population Ca2+ signals in Ml were closely related to movement.Further, we analyzed the correlation between Ca2+ signals and movement. The results showed that each movement was accompanied by the corresponding Ca2+ signal.Cross-correlation analysis revealed that movement was highly correlated with Ca2+ signal and that the onset of the Ca2+ signal was always ahead of that of movement. The preceding time was 149.7±10.3 milliseconds. All preceding times fit the Gaussian distribution and the median was 136 milliseconds.Above results indicated that the Ml was a brain region highly correlated with locomotion. The population Ca2+ signals represented the movement of the animal and preceded the movement.3. Properties of the cortical population Ca2+ signal in V1 in freely moving micePrevious studies have shown that animals required visual information input for guiding movement, and that neurons in the visual cortex were also regulated by locomotion. Therefore,we hypothesized that there may also be movement-related Ca2+ signals in V1.(1) The cortical population Ca2+ signals in V1 were closely related to movement.We also injected OGB-1AM in layer 5 of V1 and recorded the Ca2+ signals. The results showed that in V1 there were also Ca2+ signals that were highly correlated with the movement.However, this type of movement-related Ca2+ signal was later than that in M1 and there were significant differences between these two kinds of signals (n = 8 and 7 mcie, respectively; M1:149.7±10.3 ms versus V1: 50.0 ± 4.7 ms;p< 0.001). At the same time,we analyzed the amplitude of Ca2+ signals in these two brain regions and found no difference (n = 8 and 7 mice, respectively; p = 0.0854). It was worth mentioning that this movement-related Ca2+signal in V1 still existed under dark conditions, but its amplitude and the correlation of movements and Ca2+ signals significantly decreased (n = 6 mice,p < 0.001).(2) The cortical population Ca2+ signals in both Ml and V1 were highly correlated with head movement.To generate goal-directed movements, one must acquire essential information about position and orientation. During the process of generating body movements, head movement is a critical factor in the perception of orientation. Thus,we hypothesized that head movement may be closely associated with neuronal activity in both Ml and V1. Here, we analyzed three kinds of head movements: raising,rotation and withdrawal. We observed that the occurrence of each kind of head movement was always associated with population Ca2- signals. The Ca2+signals had the following properties: this Ca2+ signals were found in both Ml and V1; head movement were always accompanied by Ca2+ signals and the correlation rates were 100%;There was no significant difference in amplitude between these three different head movement-related Ca2+ signals (M1:p=0.066, n=8; V1:p=0.181, n=7).Above results revealed that V1 was another important brain region highly correlated with locomotion. The neural activities in V1 maybe the combination of visual input and motor input. Head movement is a considerable part of the whole motor behavior and correlated the neural activities in both Ml and V1.4. Tracing of the center area for bladder control in central nervous system.Current study shows that the center area for micturition control is located in the pontine micturition center (PMC),but no direct evidence prove that whether there is a higher control center such as the cerebral cortex participating in micturition. To resolve this problem, we injected retrograde transneuronal pseudorabies virus (PRV) into the bladder, aiming to study whether there are cortical regions related to bladder control.(1) Expression of PRV virus in mouse bladder and spinal cordAfter injection with virus for 4-5 days, the mice showed significant symptoms of nervous system toxicity: swinging and biting the tail, shaking and so on. At this time, the bladder,spinal cord and brain tissue of the mice were removed, sliced, imaged and stained. We observed marked nerve fibers both in the entire bladder and on the slices while no obvious neuronal somata were labeled. These results showed that our nerve tracing method definitely labeled the bladder.We confirmed that these neurons were located in sacral parasympathetic nucleus (SPN),which had been proved to be the direct domination of the bladder detrusor. The above results confirmed that our neural tracing method was convincing and effective.(2)Expression of PRV virus in the spinal cord and other subcortical areas of miceAfter that, we also observed the slices from spinal cord and brain. We found that neurons in sacral parasympathetic nucleus (SPN), PMC, locus coeruleus (LC) and hypothalamic paraventricular nucleus (PVN) were also labeled. These nuclei had been reported in previous studies that were related to micturition control. The above results showed that our virus labeling method was available and effective.(3) Expression of PRV in cerebral cortex of miceMore importantly, we also found labeled neurons in cortical regions and these neurons were located in primary motor cortex (Ml) and primary somatosensory cortex (S1) after checking the mouse atlas. Statistical analysis found that the span of these neurons was approximately 1mm and the most intensive neurons were located in 0.94mm behind bregma.The total number of the labeled neurons was approximately 1000 and most of them were vertebral neurons from morphologically observation. More than 80% of them were located in layer 5 while about 20% were in layer 1 and 2/3.5. Fiber recordings of the micturition-related Ca2+ signals in mice Ml.Although we confirmed the neural connections between bladder and Ml or S1, while the functions of these two brain areas remained to be studied. Therefore, we performed optical fiber recording in these two cortices during mice micturition.(1) The cortical population Ca2+ signals in mice M1 were closely related to micturition.We simultaneously recorded the voiding behavior and population Ca2+ activity in freely behaving mice in M1 and S1, respectively. We found that there was always a significant population Ca2+ signal in Ml during mouse micturition and this signal was not found in S1.As a control, we also recorded in Ma which was the front area of Ml and found no micturition-related signal. Further comparisons of the amplitude of Ca2+ activity revealed that M1 was significantly higher than that in S1 and Ma. So we were sure that this micturition-related Ca2+ signal was regional-specific. After that, we compared the onset of Ca2+ signal with that of voiding and found that Ca2+ signal was always ahead of voiding. On average, the latency was approximately 130 milliseconds.(2) Tracing of projection between Ml and PMCAbove results suggested that M1 may be a newly discovered cortical area that was closely related to micturition, but it is still unclear how Ml participates in the classical voiding circuit. In order to clarify the neural mechanism of how the neurons in layer 5 of Ml to regulate the activity of bladder, we injected AAV and VSV tracing viruses in layer 5 of Ml,respectively. In PMC, we found AAV-labeled labeled axons projected from Ml and VSV-labeled neurons. These results showed that the neurons in the Ml region had directly neural connection with PMC. So M1 was possibly participated in the classical voiding circuit by regulating the activity of PMC.