Does the Sleep-Related Neurons Modulate the Sensation of Pain under the Use of GA?

General anesthetics (GA) has been discovered for centuries and was often used in surgeries. However, many patients are dying from the usage of GA for different reasons. Although scientists are working on to solve the problems, the mechanism of GA is still a mystery. Recently, scientists from Duke Uni-versity found neurons that are active during sleep can be activated in anesthesia. These neurons are called Anesthetic Activated Neurons (AANs). This is a massive step for us to break the mystery. In this paper, we designed an experiment that aims to reveal one mechanism of GA: the relationship between sleep-related neurons and sensation of pain under the use of GA. The designed experiment involves several control groups that consist of mice with different treatments on their genes and different GA.


Introduction
How to manage pain has long been a question that could potentially affect every human being [1]. The discovery of anesthesia, which employed to control pain during surgical procedures, has been a considerable success in the history of medicine. General anesthesia (GA) uses a combination of intravenous drugs and inhaled gasses (anesthetics) to put patients into an unconscious state [2]. Propofol, sevoflurane, sufentanil, etc. are commonly used GA in the clinics. Although GA is widely applied to surgeries, we do not fully understand the exact mechan-Advances in Bioscience and Biotechnology no research so far has identified the relationship between sleep-related neurons and transmission of pain signals [8]. Besides, related experiments usually use the larval zebrafish model. However, there are limitations because its brain is quite different from the human brain, and its cortex has not been wholly developed [11]. Compare the structure of the zebrafish brain to the mouse's; the latter is more similar to the human brain. Therefore, our experiment will be conducted on laboratory mice, in which we will examine pain sensation via brain-wide activity measurements while manipulating the activity of sleep neurons. Through the experiment, we will reveal whether sleep neuron activity modulates the transmission of pain signals to the cortex.

Experiment Design
The goal of our experiment is to reveal the relationship with sleep-related neurons and the sensation of pain. Our experiment will be tested on mice. There are two sections in our experiment, and we are going to use different mice in each section. In the first part, our goal is to determine the pain stimulation activated neurons with and without GA injection by using the (Capturing Activated Neuronal Ensembles) CANE technique ( Figure 1). In the second part, our goal is to determine the neuronal circuits of pain stimulation under the effect of GA.
There are one control group and four experimental groups in each part, and each group contains 6 mice for different GA. These different GA are used to judge whether it is extensive of a specific phenomenon in several GA. As for the first part, the experiment begins with the Fos TVA gene knock-in mice during the embryo state. Then GA and pain stimulation are applied to the mice according to their assigned groups and GA. After the mice are sacrificed and stored at 4˚C, the mice brain will be processed with Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY), which is a tissue clearing technology that can transform intact biological tissue into a three-dimensional transparent hydrogel-based structure [12], and will be examined through confocal microscopy. Pain activated neurons can be found and ensured after observation. In the second part, the membrane-bound Thy1-GCaMP transgenic mice will be used. After some manipulations and pain stimulation, mice brain is sliced, stained with anti-vasopressin/dynorphin antibody, and examined through confocal microscopy. The effect of AANs ablation or gene silence on the neuronal circuit of the pain pathway can be discovered ( Figure 2).
There are 30 mice in the first part of the experiment. The embryos of mice are manipulated by Fos TVA knock-in technique. The mice are divided into five groups, with each group containing six mice corresponding to six different GA as Table 1 shows. The trans-genetic adult male Fos TVA mice at age more than eight weeks should be selected and singly housed for at least one day. Then they will be subjected to the following manipulation. First group mice experience no GA administration and no ablation of AANs. Second group mice experience GA  [14]. Fifth group mice experience GA administration and gene silencing of AANs in VLPO by knocking out gamma-aminobutyric acid (GABA) and galanin gene [15] [16] [17] to remove the VLPO function in the regulation of sleep (Table 1). Mice should be administrated with their assigned GA, as shown in Table 2, and the administration process should follow the appropriate method with the correct dosage based on their weight. After the GA administration, all mice should be unconscious.    After the above manipulations, the mice will receive pain stimulation in the same position on their bodies. The stimulus is controlled with the same strength and duration. Then the mice will be sacrificed, and their brains will be processed by detergent. Next, CLARITY will be used to present the pain activated neurons.
We are able to figure out the position of normal pain activated neurons in mice brain from the first group. There should be red Fos + captured by CANE method in pain activated region. In the second group, with the GA administration, it is predicted that there will be no or little amount of red Fos + in pain activated region. We can observe whether the AANs in the corresponding area are activated by the GA as well. If the AANs are activated, but no pain neuron is activated, we can suppose that AANs are able to block the neuronal circuit of the pain pathway. In the third group, with the ablation of neurons in VLPO, SON, and paraSON area, we predict that no neuron (no Fos + expressed) will be activated in pain activated region at cortex somatosensory part that we would find in the first group. If the result matches what we expect, we can conclude that sleep-related neurons can affect the pain pathway transduction and prevent the pain feeling reaching the cortex. After that, we want to find out which part of AANs has the most significant effect on the neuronal circuit of the pain pathway. Thus, we have different control groups in groups 4 and 5. In the fourth group, neurons in SON and paraSON areas are silenced. If Fos only appear in the thalamus but not cortex, it means VLPO neurons might prevent the transduction of pain pathway from thalamus to cortex but not SON and paraSON neurons. If Fos appear in both thalamus and cortex, but the amount is less than the expression in group 1, neurons in SON and paraSON are assumed that they play a significant role in pain releasing in both brain stem to thalamus and thalamus to cortex pathway. In the fifth group, neurons in VLPO are silenced. If Fos only appear in the thalamus but not cortex, AANs in the SON and paraSON might prevent the transduction of pain pathway from thalamus to cortex but not VLPO neurons. If Fos appear in both thalamus and cortex but less than group 1, we will conclude VLPO neurons play a significant role in pain releasing in both the brain stem to thalamus and thalamus to cortex pathway.
Therefore, from the above experiments, we will be able to discover whether sleep-related neurons play a role in pain releasing under the administration of GA. If they do, we can further find which part of neurons play the most significant role in pain releasing under the administration of GA. There are three possible results. The first possible result is neurons in the SON, paraSON are the significant part to block pain pathway. The second chance is VLPO neurons are a considerable part to block the pain pathway. The third one is SON, paraSON, VLPO neurons are indispensable and must promote each other and activated together to have the final pain releasing effect.
Fos expression has provided an extensive picture of populations of neurons activated by noxious stimuli [18] [19] [20], but there is no information about the circuits that underlie Fos activation. Although there are exceptions, the map of the pain pathway intervening circuits in the brain is almost unknown. Thus a method is designed to trace the pain pathway neuronal circuits and find out the exact position on the axons where AANs block, the neuron membrane-bound Thy1-GCaMP. The GCaMP is a Ca 2+ indicator that can show stronger green fluorescence when the neuron is activated and weaker green fluorescence when the activated neouron is blocked.
Then, we can start the second part of the experiment. There are 30 mice in this part, and they are divided into the same groups as the mice in section 1.
First of all, the neuron membrane-bound Thy1-GCaMP transgenic mice should be got. Then the mice would be manipulated using the same ablation and gene silence method mentioned before to make their characteristics match the standard for each group. After GA administration, give the pain stimulation with the same strength and duration. Then the mice will be sacrificed, and their brains should be sliced. Anti vasopressin/dynorphin/galanin antibodies will be used to stain the brain pieces and indicate the position where AANs might affect. In the first group, the group without GA administration, we can find the standard pain pathway axon circuits in mice brains. In the second group, the group without AANs ablation, anti vasopressin/dynorphin/galanin antibodies can be used to stain the brain pieces to find out whether the AANs secret these peptides. Then, these peptides can be judged by observing the position of the colored antibodies.
If the peptides occur in roughly all parts of the pain pathway (which indicates the Fos expression in the group 1 from the first part), we can suppose that the AANs secreted peptides can block the pain pathway synaptic connections. The vasopressin/dynorphin/galanin position will be a criterion to judge the neurons inhibited by AANs. We should find the neurons that have an effect on the pain pathway among AANs after the experiment of group 1 and 2. After that, we can find out a more specific blocking position in this activated axon tracing and peptide location experiment. In the third, fourth, and fifth groups, we will figure out that whether AANs in the SON and paraSON area or VLPO area can block the pain pathway at some position on axons after observing the inactivated axons and peptide position compared to the first group. We suppose the group will have no peptide and almost the same axon color as group 1 because of the ablation of AANs. In the fourth group, if the axon color is the as same strength as group 1, we infer the VLPO neurons can not play a role in pain releasing. If an axon is lighter than group 1 and the peptide vasopressin/dynorphin/galanin appears, we suppose the VLPO neurons have the pain pathway blocking effect. As for group 5, the situation would be vice versa.  [13].

Technique Introduction
To see whether the sleep-related AANs have effects on the pain signal transduction with and without the administration of GA, we will test the Fos expression in manipulated mice. The Fos expression level can indicate the activity of neurons in a different region of the brain after pain stimulation on the trans-genetic Fos TVA knock-in mice. mCherry is a kind of red fluorescence protein, and we will use the EnvA-LV-mCherry to detect Fos expression and evaluate the neuron activity [13].

Axon tracing: Label neuronal circuits with neuron membrane-bound Thy1-GCaMP
The sensation of pain is generated by ascending pain pathways beginning at nociceptors that are activated specifically by painful (noxious) stimuli. Then, the receptors transduce the "noxious" information into an electrical signal and transmit it from the periphery to the central nervous system along axons. In our experiment, we are going to trace axons that transmit pain through the expression of axon membrane-targeted Green Fluorescent Protein (GFP). GCaMP is created from a fusion of green fluorescent protein (GFP), calmodulin, and M13, a peptide sequence from myosin light chain kinase [22]. By applying GCaMP, the generation of action potentials in neurons can be indirectly monitored by detecting calcium influx via voltage-gated calcium channels. The sensitivity of GCaMP and its high expression levels are critical to obtaining an optimal signal-to-noise ratio and, therefore, for the successful detection of neuronal activity [22]. In our experiment, we would construct a neuron membrane-bound GCaMP gene and then transfect into adult mice so that it can show the neuronal circuits during pain stimulation within and without GA. CLARITY Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY), tissue clearing technology, is an advanced technique that can transform intact biological tissue into a three-dimensional transparent hydrogel-based structure with all its essential structures [12]. After CLARITY operation, the membrane-bound GFP and Fos are still in place be-

Designed Procedures
Generating  [14]. Therefore the ablation of some Neurons can be reached by deletion of the functional neuroendocrine substrate gene using homologous knockout.
Trans-genetic adult male Fos TVA mice at ages more than eight weeks will be singly housed for at least one day and then subjected to the manipulation, just as Table 1 shows. We are going to administrate six different kinds of GA (propofol, ketamine, dexmedetomidine, sevoflurane, isoflurane, desflurane) to the mice in each group. Propofol, ketamine, and dexmedetomidine are intravenous GA, while sevoflurane, isoflurane, and desflurane are inhaled GA. Each mouse will be administrated with a certain amount of GA based on their weight to ensure that the effect of GA on them is the same ( Table 2, depending on the real data). Then the virus EnvA-LVs-mCherry should be produced or purchased. Two hours after giving the pain stimulation to these five groups' mice by needle acupuncture, the EnvA-LVs-mCherry will be introduced into the brain of Fos TVA mice to capture pain activated neurons.

CLARITY observation
Five minutes after pain stimulation, the mice will be sacrificed and stored at 4˚C. Hydrogel polymers have to be grown from inside the tissue to support the mice's brain's structure and molecular content. This can be done with the infusing of 4˚C hydrogel monomers cocktail, formaldehyde, and thermally triggered initiators into the tissue [12]. Formaldehyde serves the dual purposes of cross-linking amine-containing tissue components to each other and covalently binding the hydrogel monomers to native biomolecules including proteins, nucleic acids, and other small molecules but not a membrane. Then, the hydrogel polymerization is triggered by the heat of 37˚C [12]. Because of the formaldehyde binding effect, lipids can now be extracted from the brain without destroying important components of the brain. This can be achieved by using Advances in Bioscience and Biotechnology strong ionic detergent-based clearing solution [borate-buffered 4% (wt/vol) SDS] at 37˚C [12]. After the brain has been cleared, it will be immersed in the refractive index (RI) homogenization solution [12]. Followed by constructing the three-dimensional brain model is the visualization of the brain using confocal microscopy at a high resolution. The previously labeled pain circuitry and the neurons which can express Fos can be detected.

Thy1-GCaMP transgenic mice
GCaMP2.2c gene will be generated by changing the second arginine to valine and serine at 118 to cysteine of GCaMP2.0. All in vitro expression constructs of GCaMPs were connected with the coding sequence of tdTomato via a 2A peptide (P2A) sequence and subcloned into a modified pBluescript plasmid, which contained the CAG promoter (a combination of the cytomegalovirus early enhancer element and chicken beta-actin promoter). When generating the

Section One
By using CLARITY, the brain graphs of each mice group with red Fos + expression can be showed. First of all, through the comparison between group 1 and group 2, whether or not AANs are effective at blocking the neuronal circuit of the pain pathway will be revealed. If the graphs of group 1 and group 2 suggest that AANs can block the neuronal circuitry of the pain pathway, the area of red Fos + expression will be measured and calculated by integral. The results will be compared afterward. According to these results, the effectiveness of the area (VLPO), or the combination of areas (VLPO, SON, paraSON/SON, paraSON), at blocking the neuronal circuit of the pain pathway can be ranked.

Section Two
The images of antibody staining in group 1 and group 2 will be recorded. The comparison between these two groups can demonstrate whether AANs can produce certain neurotransmitters during pain relief. If AANs are proved to be effective at relieving pain, we can continue the analysis for groups 3, 4, and 5.
The color strength of GFP in the last three groups will be recorded by the software Adobe Kular. Then, the images of the experimental groups will be presented. By comparing to the difference between the photos before (which is the image of group 1) and after the change (which are the images of groups 3, 4, and 5), the sites of the pain-sensation pathway can be found. The GFP color intensity of each graph will be calculated by the computer. The data will be recorded, and

Discussion
Due to the limit of equipment, the above is only a proposal for the experiment.
Scientists will have to choose a type of mice for the experiment based on their resources. However, all the mice used in the experiment need to be male, and elder than eight weeks old. In the rest of this section, we are going to discuss the ideal results of the experiment. complications and sequelae that relate to sleep and pain, and may find out a way to prevent these from happening.

Conclusion
Our experiment is designed to test the hypothesis that the sleep neuron activity can modulate the transmission of pain signals to the cortex. By corresponding trans-genetic mice, our experiment is aimed to show the activated neurons and the activated neuronal circuits. In the first part of our experiment, the pain activated neurons can be found. The part of AANs or the combination of multiple parts that has the most significant effect on blocking the pain signal transduction can also be discovered. In the second part of our experiment, we would figure out how and where the AANs block the pain signal transduction. If vasopressin/dynorphin/galanin is detected at pain activated regions, and there is no signal in the mice brain cortex, we can suppose that sleep neurons are activated by GA and that they can secrete peptides which action at pain pathway. After that, by activating axon tracing, the lighter and weaker GFP on axons can tell us what point on the axon where the pain signal disappears. In the end, we can evaluate our hypothesis and find out the significant pain pathway blocking neurons and their preliminary mechanism.